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Loop-mediated isothermal amplification assays: Rapid and efficient diagnostics for genetically modified crops
Accepted Manuscript Loop-mediated isothermal amplification assays: Rapid and efficient diagnostics for genetically modified crops Monika Singh, Deepa Pal, Payal Sood, Gurinderjit Randhawa PII:
S0956-7135(19)30348-2
DOI:
https://doi.org/10.1016/j.foodcont.2019.106759
Article Number: 106759 Reference:
JFCO 106759
To appear in:
Food Control
Received Date: 26 April 2019 Revised Date:
15 June 2019
Accepted Date: 7 July 2019
Please cite this article as: Singh M., Pal D., Sood P. & Randhawa G., Loop-mediated isothermal amplification assays: Rapid and efficient diagnostics for genetically modified crops, Food Control (2019), doi: https://doi.org/10.1016/j.foodcont.2019.106759. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Loop-mediated isothermal amplification assays: Rapid and efficient diagnostics for genetically modified crops Monika Singh*, Deepa Palϯ, Payal Soodϯ and Gurinderjit Randhawa Division of Genomic Resources, ICAR-National Bureau of Plant Genetic Resources, New Delhi 110 012, India ϯ
Both the authors contributed equally
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*Corresponding Author
E-mail: [email protected], [email protected]
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Phone: +91 9910067094
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ACCEPTED MANUSCRIPT ABSTRACT
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With the increase in number of globally approved genetically modified (GM) events and
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diversification of traits, efficient GM detection strategies may play key role for regulatory
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compliance. Polymerase chain reaction (PCR) and real-time PCR, targeting different
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components of a transgenic construct, are widely applied analytical methods for GM
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detection. Loop-mediated isothermal amplification (LAMP), an isothermal nucleic acid
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amplification method, has been employed in screening and detection of genetically modified
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organisms (GMOs) since past decade due to user-friendly operation, acceptable specificity,
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less sensitivity to inhibitors and rapid mode of detection. This article reviews the scenario of
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applicability of LAMP in GM detection and potential of LAMP. Amplification of LAMP
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products is done at a constant temperature and no separate steps of denaturation, annealing
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and extension are required. Easy-to-use portable instrumentation has made this technology
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applicable for on-site GMO testing. A series of visual and real-time LAMP assays have been
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developed in recent past for rapid, cost-efficient or on-site detection of GMOs without the
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involvement of time-consuming electrophoretic analysis. Different chemistries and detection
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systems can be used on the basis of practical requirement. Amplification can be detected
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within 30-60 minutes with a limit of detection (LOD) up to 0.005-0.1% GM content
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depending on the target or detection system used. Potential of LAMP for further expanding
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the practical use by modifications/advancement in this technique has also been highlighted in
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this review.
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KEYWORDS
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ACCEPTED MANUSCRIPT Loop Mediated Isothermal Amplification (LAMP), Genetically Modified Organisms
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(GMOs), GM detection, Isothermal nucleic acid amplification technologies (iNAATs), Visual
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Detection, Real-time LAMP, Limit of Detection (LOD), On-site GMO testing
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1. BACKGROUND
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The commercialization of genetically modified (GM) crops has increased globally, with
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respect to the acreage of area under GM crops as well as the diversification of traits/GM
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events (ISAAA, 2017). Area under GM crops has reached 189.8 million hectares in 2017.
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Over 500 GM events covering 30 plant species have got approval in different countries, to be
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used as food and feed (http://www.isaaa.org/gmapprovaldatabase/).
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traits in GM crops include herbicide tolerance, insecticide resistance, either in single or
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stacked form. Stacked GM events account for more than 40% of global area under cultivation
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of GM crops. In India, Bt cotton is the commercialized GM crop with four approved GM
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events, namely, MON531, MON15985, GFM and Event 1. Area under Bt cotton has
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increased to 11.4 million hectares in 2017 (ISAAA, 2017). Several other GM events of
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cotton and other crops have been imported for research purposes through ICAR-National
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Bureau of Plant Genetic Resources, New Delhi or were under field trials (Singh and
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Randhawa, 2016; http://www.moef.nic.in/major-initiatives/geac-clearances).
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Commonly employed
The approval of GM crops as food or feed is regulated in different countries by
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respective regulatory bodies. In some countries, labelling of GM products is voluntary as in
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the United States; whereas it is mandatory in several countries as in the European Union
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(Querci et al., 2010). Most of the countries have implemented labeling regulations for
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genetically modified organisms (GMOs) in food/feed products containing GM content over
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defined threshold. The GMO labeling threshold in European Union is 0.9%, for example, if
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0.9% or more of the ingredient of a food/feed product is GMO, the product needs to be
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labelled as ’containing GMO’ (https://ec.europa.eu/jrc/en/research-topic/gmos). In India, so
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far no labeling threshold is being implemented. For checking the GM status of a sample or to
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check for approved GM events/crops, efficient GM detection methods need to be employed. Reliable GM detection methods play a key role to meet the regulatory requirements.
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Two main methodologies for GM detection target either (i) the components of transgenic
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construct at DNA level, or (ii) the newly expressed recombinant proteins at the protein level.
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DNA-based methods are being commonly employed due to their sensitivity, reproducibility
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and stability. These assays can also be employed for monitoring adventitious presence of
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transgenes in genebanks and for ensuring post-release monitoring of GM events in food and
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supply chain.
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With the increasing number and complexity of GM events (either as single or in
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stacked form) as well as diversification of traits, GM detection has become a challenging
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task. For screening a large number of GM crops/events, it is necessary to develop user-
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friendly, cost-effective and reliable GM detection strategies (Randhawa et al., 2016) Matrix
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approach has been employed as an efficient and cost-effective GMO screening strategy
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(Block et al., 2013; Kralj Novak et al., 2009; Waiblinger et al., 2010; Randhawa et al., 2014;
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Singh et al., 2016). Common screening elements are identified by analysis of GMO matrix,
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which can be used to screen for a range of GM events. This approach could cut down the cost
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of GMO testing by eliminating the need of GM testing methodologies for specific GM
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events. However, for development of more efficient GM detection system, rapid assays
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targeting common screening elements may contribute to an extent.
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Polymerase chain reaction (PCR) and real-time PCR are commonly employed DNA-
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based GM detection methods due to robustness, reliability, sensitivity and reproducibility of
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the assays. Reports of PCR- and real-time PCR-based GM detection were reviewed
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(Randhawa et al., 2016). Validated real-time PCR protocols for over 50 GM events of
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different crops including maize, soybean, cotton and oilseed rape are available at 4
ACCEPTED MANUSCRIPT GMOMETHODS, an European Union Database of Reference Methods for GMO analysis
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based on the Compendium of Reference Methods for GMO Analysis (http://gmo-
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crl.jrc.ec.europa.eu/gmomethods/). In recent past, digital droplet PCR has emerged as a high
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throughput method for GM detection and quantification (Dobnik et al., 2015; Gerdes et al.,
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2016; Košir et al., 2017). However, use of sophisticated equipment and procedures associated
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with PCR/real-time PCR analyses restrict their use for on-site or rapid GMO testing
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(Randhawa et al., 2013).
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Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid
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amplification method where amplification is done at a constant temperature (Notomi et al.,
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2000). Since the introduction of loop-mediated isothermal amplification (LAMP) method in
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2000, it has gained popularity in GM detection for rapid/on-site application (Figure 1). This
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article reviews on the versatility and applicability of LAMP for GM detection.
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2. LOOP-MEDIATED DETECTION
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Isothermal nucleic acid amplification technologies (iNAATs) include the nucleic acid
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amplification assays based on isothermal principles such as strand-displacement
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amplification (SDA), helicase-dependent amplification (HDA), recombinase polymerase
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amplification (RPA) and LAMP (Craw et al., 2012; Walker et al., 1992; Vincent, et al., 2004;
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Piepenburg et al., 2006; Notomi et al., 2000). Among iNAATs, LAMP technology has wider
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applicability in diagnostics including GM detection. Amplification of template DNA is done
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at a constant temperature in LAMP assays using four different primers to recognize six
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distinct regions on the target sequence (Notomi et al., 2000). An outer primer set (F3, B3)
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containing sequences of sense and antisense strands of target DNA initiates LAMP reaction,
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which proceeds at a constant temperature, followed by strand displacement DNA synthesis
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primed by an inner primer set (FIP, BIP). Addition of two loop primers or two stem primers
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further increases the speed and specificity of LAMP assays (Nagamine et al., 2002). Primer designing is crucial for performing LAMP reaction. The PrimerExplorer
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(available at https://primerexplorer.jp/e/) is a software specifically for designing the primer
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sets for LAMP (http://loopamp.eiken.co.jp/e/lamp/primer.html). This software generates the
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primer sets based on the target sequence information, which meets the primer designing
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requirements. The primer regions targeting different regions of the template DNA are shown
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in Figure 2.
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LAMP assay is less sensitive to inhibitors and hence, can be employed for GMO
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testing of crude DNA samples (Francois et al., 2011) LAMP is a user-friendly and reliable
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method for GM detection, which can be performed on a thermal cycler or a heating block or
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in a portable isothermal real-time amplification system. LAMP products can be visualized
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after completion of reactions using nucleic acid staining or fluorescent dyes such as SYBR®
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Green, hydroxylnaphthol blue (Chen et al., 2012; Guan et al., 2010). LAMP products can be
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monitored using turbidometry or by measuring fluorescence using real time LAMP
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(Randhawa et al., 2013; Mori et al., 2004).
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The workflow of LAMP method for GM detection is summarized in Figure 3.
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3. VERSATILITY IN LAMP
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LAMP assay can be performed in different ways for practical use in GMO testing based on
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chemistry, detection method and target to be amplified.
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3.1. Chemistries Employed in LAMP Assays
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Different chemistries and visualization approaches can be used in LAMP depending on the
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practical utility and availability of resources. Several reports of LAMP-based GM detection
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employing different approaches are available (Table 1).
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DNA polymerase: LAMP can amplify DNA isothermally using a simple isothermal
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amplification instrument, based on strand displacement synthesis of DNA. Bst DNA
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polymerase large fragment represents a part of Bacillus stearothermophilus DNA
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polymerase protein having 5’-3’ polymerase activity but lacking 5’-3’ exonuclease
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activity. This enzyme has applicability in strand displacement reactions, thereby
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contributing to development of LAMP-based diagnostics.
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The reaction mixture for conventional LAMP consists of an isothermal buffer,
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MgSO4, dNTP mix, Bst DNA polymerase large fragment; forward (FIP) and
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backward (BIP) inner primers, forward (F3) and backward (B3) primers and DNA
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template. In addition, loop primers may also be used in order to increase specificity of
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assays. The composition of a typical conventional LAMP reaction is given in Table 2.
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Concentration of primers need to be optimized for the specific target. If
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required, concentration of other components may also be optimized for better clarity
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of results. The reaction is incubated at a constant temperature (60-65 ˚C) for 30–60
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minutes depending on the size of targeted region and melting temperature of primers.
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Incubation is performed in a thermal cycler or alternatively in simple instrumentation
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such as a conventional heating block or a dry bath (Randhawa et al., 2013). Due to
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simple instrumentation with low cost resources, conventional LAMP can cut down the cost of GMO testing (Randhawa et al., 2013; Singh et al., 2015).
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3.1.2. Ready-to-use isothermal master mix: For real-time LAMP assays, isothermal master
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mix (OptiGene Limited) is used, which is compatible with the isothermal real-time
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instrument Genei® II. The composition of a typical real-time LAMP reaction is givem
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in Table 3.
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Annealing temperature and time of reaction completion need to be optimized, if
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required. Incubation is performed in a portable isothermal real-time LAMP system
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(Genie® II), hence it can facilitate rapid on-site GM detection when combined with a
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fast DNA extraction method (Zhang et al., 2013; Randhawa et al., 2013; Singh et al.,
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2015)
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3.2. Detection Methods for LAMP Products
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3.2.1. Visual/colorimetric detection:
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Visual LAMP products are detected by adding
SYBR® Green I dye after completion of reaction. SYBR® Green I binds to DNA and
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the resulting DNA-dye-complex absorbs blue light and emits green light. LAMP
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products can be directly visualized as change in color by the naked eye on addition of
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SYBR® green dye. Change in color from orange to green depicts that LAMP
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amplification has occurred (Figure 4a), whereas no color change depicts absence of
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LAMP amplification. Alternatively, other dyes such as calcein, hydroxynaphthol blue
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(HNB), malachite green can be used based on the practical requirement (Tanner et al.,
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2012; Tomita et al., 2008)
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Metal indicators can be added to the pre-reaction solution and the amplification
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products can be detected visually by the change in color of the reaction mixture. Goto
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et al. (2009) have used the HNB as metal indicator for the first time for colorimetric detection of LAMP products that is based on Mg2+ concentration change. HNB changes the color of the reaction solution from violet to sky blue. As both the colors
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are similar, so it is difficult to detect the change with the naked eye, especially when
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the samples contained low levels of the target sequence. Another metal indicator, acid
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chrome blue K (ACBK) has been used for detection of LAMP amplification for P-35S
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by change in colour from red to blue based on a decrease in the Mg2+ concentration in
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the reaction mixture (Wang et al., 2017). 3.2.2. Electrophoretic detection: Specificity of LAMP products can also be checked by
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electrophoresis in 2% agarose gels in 1x TAE (Tris acetate EDTA) running buffer
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stained with ethidium bromide and then visualized under UV light. Positive
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amplification of LAMP products is detected as typical ladder-like pattern in agarose
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gels (Figure 4b) (Randhawa et al., 2013; Lee et al., 2009). In LAMP reactions, dumb-
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bell shaped structures are formed that contains multiple sites for amplification
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initiation. The amplification proceeds from these multiple initiation sites, and long
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concatemers are formed that contain more sites for amplification initiation. The
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annealing between alternate inverted repeats of the target sequence of the same strand
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results in the formation of cauliflower‐like structures (Notomi et al., 2000; Kumar et
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al., 2017). Ladder-like pattern is produced due to formation of concatemers consisting
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of DNA fragments between outer F3/B3 primers.
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3.2.3. Real-time detection:
(a) Turbidometric detection:
Real-time LAMP assays for detection of P-35S using turbidometry has been
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developed by Fukuta et al. (2004). LAMP has high ability to synthesize sufficient
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amount of DNA, therefore, large amount of byproduct (pyrophosphate ion) is produced, yielding a white precipitate of magnesium pyrophosphate in the LAMP reaction mixture (Mori et al., 2001). The turbidity of reaction mixture allows easy
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detection of amplification of target DNA. The turbidity of the reaction mixture can be
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determined by the difference between the absorbance of the sample solution and the
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reference (a reaction buffer of the same concentration) (Fukuta et al., 2004).
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(b) Fluorescence-based detection:
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detection of LAMP products by monitoring fluorescence as amplification and
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annealing curves as well as time of positivity (Tp) during the progress of reaction as
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shown in Figures 4c & 4d (Randhawa et al., 2013; Singh et al., 2017). Tp refers to the
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amplification time at which the fluorescence second derivative reaches its peak above
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the baseline value.
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Reports of fluorescence-based LAMP performed on real-time PCR system
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such as Light Cycler 450, ABI7500 are available, however, this limits the on-site
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application of LAMP (Randhawa et al., 2013; Wang et al., 2016).
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A direct detection approach, Fluorescence of Loop Primer upon Self
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Dequenching-LAMP (FLOS-LAMP), has been recently reported with applicability in
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pathogen detection (Gadkar et al., 2018). In this approach, a labelled loop probe
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quenched in its unbound state, fluoresces only when bound to its target (amplicon),
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allows the sequence-specific detection of LAMP amplicons.
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3.3. Target to be Amplified
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A transgenic construct comprises of various genetic elements: (i) a promoter enabling the
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expression of inserted gene; (ii) the inserted transgene conferring desired trait to the host
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plant; (iii) a marker gene for selection of transformants; and (iv) a terminator sequence acting
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as a stop signal. These genetic elements can be targeted for GM detection as per the practical
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or regulatory requirement depending on the level of specificity. LAMP can be employed for
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GM detection at different levels of specificity: Screening, gene-specific, construct-specific
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and event-specific assays. (Figure 5)
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3.3.1. LAMP-based Screening Assays: Screening methods are used to check the GM status
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of samples. If a sample is GM, only then subsequent tests are required for
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employed control elements, viz., promoter and terminator. These assays would reduce
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the cost of GMO testing by eliminating the need of specific GM testing
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methodologies for each event. LAMP-based screening assay targeting Cauliflower
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Mosaic Virus 35S promoter (P-35) was first reported (Fukuta et al., 2004). LAMP
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assays for other commonly used promoters, namely, P-FMV, P-ract, P-nos and
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terminators such as T-nos, have also been developed for GMO screening (Randhawa
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et al., 2013; Lee et al., 2009; Singh et al., 2017; Fukuta et al., 2004; Kiddle et al.,
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2012; Singh et al., 2018). The reported assays have been summarized in Table 1. A
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collaborative ring trial validation of three established visual LAMP assays targeting
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P-35S, P-FMV and T-nos has been recently reported (Li et al., 2019). Sensitivity was
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found to be 10, 10, and 50 haploid genome equivalents (HGEs) for P-35S, P-FMV,
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and T-nos, respectively. Ten laboratories participated in the ring trial and the results
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demonstrated that these visual LAMP assays are specific, sensitive and time-saving,
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with high application potential for on-site testing and routine screening of GMOs.
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3.3.2. LAMP-based Gene-specific Assays: Gene-specific methods target specific transgenes
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expressed in a GM crop or marker genes used for selection of transgenics. LAMP
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assays targeting commonly employed marker genes/reporter genes including aadA,
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nptII, pat, pmi, uidA were reported (Randhawa et al., 2013; Lee et al., 2009). Genespecific LAMP assays for detection of cry1Ab in GM rice, cry1Ac in GM sugarcane and phytase in GM corn have been developed (Li et al., 2013; Zhou et al., 2014.
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Huang et al., 2014). LAMP assays targeting insecticide resistant cry1Ac and cry2Ab2
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genes and herbicide tolerant, cp4-epsps gene were developed to check for approved
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and unapproved GM events in the country (Singh et al., 2015). Validated LAMP
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protocol for cp4-epsps gene reported by Wang et al. (2015) and Li et al. (2018) is
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international collaborative ring trial with 12 participating laboratories to validate
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gene-specific LAMP assays targeting four common transgenes, namely, bar, pat, cp4-
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epsps and cry1Ac. The results showed high specificity with limit of detection (LOD)
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of 5, 10, 20 and 20 HGE for cp4-epsps, cry1Ac, bar and pat genes, respectively. The
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ring trial results collectively confirmed that these four gene-specific assays can be
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utilized or quick screening of transgenic traits in routine GMO analysis.
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3.3.3. LAMP-based Construct-specific Assays: Construct-specific methods target the
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junction between two components of a transgenic construct, for instance, a region of
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the insert spanning junction between the promoter and transgene. So far, no report of
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construct-specific LAMP assays is available. However, the construct-specific LAMP
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assays may be employed for specific detection of GM events with closely related
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genetic constructs, for example, P35S-cry1Ac construct region can be used to detect a
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range of GM events including Bt cotton events commercialized in India. Construct-
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specific method has been developed for P35S-cry1Ac construct region (unpublished
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data).
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3.3.4. LAMP-based Event-specific Assays: Event-specific methods target the junction
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region at integration locus between recipient genome and inserted DNA, LAMP is
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also being employed for detection of specific GM events. Event-specific LAMP assays for detection GM maize, GM soybean and Bt rice events have been reported (Chen et al., 2012; Guan et al., 2010; Chen et al., 2011; Bhoge et al., 2015). Event-
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specific LAMP for detection of two Bt cotton events, viz., MON531 and MON15985
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commercialized in the country was developed (Randhawa et al., 2015).
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LAMP assays reported for detection of GMOs at screening, gene-specific, construct-specific and event-specific levels are summarized in Table 1. 12
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replication with appropriate positive and negative controls to avoid type I (false
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positives) and type II (false negatives) errors (JRC Technical Report, 2015). Prior to
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conducting LAMP-based GMO testing, amplifiability of test samples need to be
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ensured using endogenous gene specific assays. Though LAMP could work with the
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crude DNA samples but assay for an endogenous gene amplification eliminates the
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chances of type II error.
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4. APPLICABILITY OF LAMP FOR GM DETECTION
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4.1. Specificity and Sensitivity for LAMP Assays
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Specificity of developed assays can be determined at two levels as a part of method
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validation. At the first level, specificity needs to be checked by selecting the primer
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sequences specific to target region. This can be done in silico by performing searches against
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publicly available nucleotide sequence databases such as National Center for Biotechnology
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Information BLAST searches. At the second level, the assays need to be evaluated
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experimentally by testing against reference materials including both the targets as well as
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non-targets for a particular sequence. Based on the published research reports, LAMP assays
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showed acceptable specificity by specifically amplifying respective targets positive for a
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particular transgenic trait (Randhawa et al., 2013; Wang et al., 2015; Singh et al., 2015;
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Singh et al., 2018; Singh et al., 2017). In these studies, set of appropriate targets and non-
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targets for a transgenic element were used to confirm the specificity of an assay.
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Sensitivity of LAMP assay is assessed as the limit of detection (LOD). LOD
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determines the lowest concentration at which all replicates result in positive LAMP reaction
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signals. LAMP assays are sensitive enough to detect the GM content with LOD ranging from
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0.1 to 0.005 depending upon the target and assay as shown in Table 1.
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LAMP-based testing can be completed in comparatively less time as compared to PCR and
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real-time PCR as thermocycling steps and time-consuming electrophoresis analysis is not
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required (Randhawa et al., 2013; Singh et al., 2015). LAMP has been considered as a method
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of choice for rapid GM detection as reaction is completed at a constant temperature (60-65
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˚C) within 30-60 minutes and LAMP products can be visualized by adding SYBR Green dye.
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Conventional LAMP reactions can alternatively be conducted using simple instrument such
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as a heating block or a dry bath, which can reduce the equipment and running costs. Hence,
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conventional LAMP can be utilized by low resource GMO testing laboratories.
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4.3. On-site Application
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Conventional and real-time PCR assays have been widely used for GM detection due to
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efficiency, sensitivity and reproducibility. These assays require expensive and sophisticated
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equipment for thermal cycling or fluorescence determination, which limits their use for on-
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site application because of the lack of portability. PCR assays also require time-consuming
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gel electrophoretic analysis of products. Moreover, Taq DNA polymerase used in PCR assays
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may be inactivated by inhibitors present in crude samples (De Franchis et al., 1988) and thus,
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may not be employed for on-site GM detection.
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Real-time LAMP can be performed on a real-time isothermal system (Genie® II). The
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system is a compact, light-weight and fully portable (http://www.optigene.co.uk/wp-
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content/uploads/2012/06/Genie-II-flier.pdf). Due to portability of system, real-time LAMP
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when combined with a fast DNA extraction protocol can be employed for on-site GM
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detection at port of entry or in farmer fields (Randhawa et al., 2013).
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Zhang et al. (2013) developed a novel GM detection system, which can be utilized to perform
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both on-site as well as routine laboratory GMO testing. In this system, a newly designed
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assay. The DNA extraction device fitted with a silica gel membrane filtration column and a
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modified syringe, could be easily operated without using other laboratory instruments,
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making it applicable for on-site. In the modified LAMP assay, a microcrystalline wax
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encapsulated detection bead containing SYBR green fluorescent dye was added to avoid dye
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inhibition and cross-contaminations.
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4.4. Application and Potential of Multiplex LAMP Assays
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Multiplex real-time LAMP assays are being widely used for detection of pathogens and
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identification of blood-borne viruses (Liu et al., 2017; Kouguchi et al., 2010; Dougbeh-Chris
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et al., 2015). Very little research has been undertaken for development of multiplex LAMP
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assays for detection of GMOs. Chen et al. (2017) developed CALM (Capillary Array-based
352
LAMP for Multiplex visual detection of nucleic acids) where a small, ready-to-use cassette
353
was assembled by capillary array, allowing simultaneous detection of 8 commonly present
354
transgenic elements/genes with high specificity and sensitivity. The capillary array was pre-
355
treated into a hydrophobic and hydrophilic pattern before fixing LAMP primer sets in
356
capillaries. LAMP reaction mixture was added after assembly of the loading adaptor and
357
isolated into each capillary due to capillary force by a single pipetting step. The LAMP
358
reactions were performed in parallel in the capillaries and the results are visually detected
359
with a hand-held UV flashlight.
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LAMP-based detection method s combined with lateral flow dipstick chromatography has
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been reported for detection of GM soybean and maize events targeting P-35S, mannose-6-
362
phosphate isomerase gene, Pisum sativum ribulose 1, 5-bisphosphate carboxylase terminator,
363
a common sequence between the cry1Ab and cry1Ac genes, and a GA21-specific sequence
364
(Takabatake et al., 2018). Development of multiplex LAMP assays can further time-
365
efficiency and would reduce the cost involved in detection and identification of GMOs. 15
ACCEPTED MANUSCRIPT 5. CONCLUSION
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LAMP has emerged as a popular diagnostic tool for being fast, sensitive and user-
368
friendliness. The major advantage of this technique is that the targets are amplified at a
369
constant temperature, thereby eliminating the time-consuming thermocycling steps required
370
for any two- or three-step PCR. With no thermocycling requirements, amplification reactions
371
can be conducted using simple instruments for e.g., a conventional water bath or heat block,
372
thus, drastically reducing equipment costs. Since, real time-LAMP can also be performed in a
373
simple portable machine, it can be employed for on-site detection of GMOs in crop plants
374
when used in combination with a fast DNA extraction method. In the last decade, LAMP has
375
dramatically influenced the research in different areas of diagnostics including GM
376
diagnostics. Development of multiplex LAMP assays for detection of multiple targets in a
377
single reaction can further add to rapid, robust and cost-effective features to this technique.
378
Further refinement and advancement in this technique may be explored to expand its practical
379
utility in the area of diagnostics for GM crops.
380
ACKNOWLEDGMENTS
381
We acknowledge the support provided by Indian Council of Agricultural Research (ICAR),
382
New Delhi, Department of Biotechnology (DBT) and Department of Agriculture,
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Cooperation & Farmers Welfare (DAC&FW). We also thank the Director, ICAR-National
384
Bureau of Plant Genetic Resources for providing necessary facilities.
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Table 1. Summary of LAMP assays reported for GM detection
P-35S
Visual/Real-time LAMP LAMP-BART Real-time turbidometry Visual LAMP
Visual using metal indicator acid chrome blue K
marker genes (genespecific assays)
aadA bar nptII
Visual LAMP Visual LAMP Visual LAMP LAMP-BART Visual LAMP Visual/Real-time LAMP
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P-ract P-nos T-nos
Visual/Real-time LAMP
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P-FMV
limit of detection (LOD) Visual LAMP Real-time LAMP 40 Copies 4 Copies (0.01%) (0.1%) 50 copies (0.1%) 0.5% 5 copies per haploid genome equianlent (HGE) ∼50 copies for GM rice 100 ng of 0.1 % DNA from rice, soybean, rapeseed, and maize 40 Copies 4 Copies (0.01%) (0.1%) 10 copies per HGE 0.05% 0.01% 50 copies (0.1%) 5 copies per HGE 40 Copies 4 Copies (0.01%) (0.1%) 5 copies per HGE
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detection method
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targets/ events
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transgenic element promoter/ terminator sequences (screening assays)
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Visual LAMP
Visual/Real-time LAMP Visual LAMP
uidA
Visual/Real-time LAMP
26
40 Copies (0.1%) 5 copies per HGE
4 Copies (0.01%)
40 Copies (0.1%)
4 Copies (0.01%)
ref Randhawa et al., 2013 Kiddle et al., 2012 Fukuta et al., 2004 Wang et al., 2015
Wang et al., 2017
Randhawa et al., 2013 Wang et al., 2015 Singh et al., 2018 Singh et al., 2018 Kiddle et al., 2012 Wang et al., 2015 Randhawa et al., 2013 Wang et al., 2015 Randhawa et al., 2013 Wang et al., 2015 Randhawa et al., 2013
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constructspecific assays eventspecific sequences
phytase in GM maize bar in GM sugarcane P35S-cry1Ac
Bt Cotton Events (i) MON531 (ii) MON15
10 copies per HGE 43.1 copies
Visual/Real-time LAMP
4 Copies (0.01%)
Visual LAMP Visual LAMP Visual/Real-time LAMP Visual LAMP Visual LAMP
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Wang et al., 2015 Singh et al., 2017 Singh et al., 2017 Singh et al., 2015
Wang et al., 2015 Huang et al., 2014
4 Copies (0.01%)
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Visual LAMP
8 copies (0.01%) 8 copies (0.01%) 2 copies (0.005%)
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Visual LAMP Visual LAMP
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cry1Ab (GM rice) cry3A cp4-epsps (GM cotton, maize) cp4-epsps
10 copies per HGE 44 copies (0.05%) 88 copies (0.1%) 4 Copies (0.01%)
Singh et al., 2015
5 copies
Li et al., 2014
0.5%
Li et al., 2013
5 copies 4 Copies (0.01%)
Li et al., 2014 Singh et al., 2015
4 Copies (0.01%)
Wang et al., 2015 30 copies
Huang et al., 2014
Visual LAMP
10 copies
Zhou et al., 2016
Visual LAMP
0.01%
unpublished data
Visual/Real-time LAMP
2 copies (0.005%)
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transgenes (genespecific assays)
pmi cry1Ac (GM cotton, brinjal , maize) cry1Ac cry1Ac (GM sugarcane) cry2Ab2 (GM cotton, maize) cry2Ab
Visual LAMP Visual/Real-time LAMP Visual/Real-time LAMP Visual/Real-time LAMP
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27
2 copies (0.005%)
Randhawa et al., 2015
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LAMP combined with lateral flow dipstick
2.4 copies
Wang et al., 2013
Visual LAMP
4 copies
Chen et al., 2011
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4 copies (GTS-40-3-2) 8 copies (MON89788)
Visual/Real-time LAMP
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GM Maize Events (i) DAS59122-7 (ii) T25 (iii) Bt176 (iv) TC1507 (v) MON810 (vi) Bt11 (vii) MON863 GM Maize Events (i) Bt11 (ii) GA21 (iii) MON810 (iv) MON8903 4 (v) NK603 (vi) TC1507 GM Maize Event MON88017
Visual LAMP
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GM Soybean Events (i) GTS-40-3-2 (ii) MON89 788 Roundup ready GM Soy event
8 copies (0.01% )
8 copies (0.01% )
Bhoge et al., 2015
73 copies for NK603 (0.1%) 73 copies
Visual LAMP
40 copies
28
Zhen et al., 2016
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0.5%
Xu et al., 2013
Visual LAMP
0.01%–0.005%
Chen et al., 2012
Visual LAMP
6 copies
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Visual LAMP
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GM Maize Event T25 GM Rice Events (i) KMD1 (ii) TT51-1 (iii) KF6 GM Wheat Event B73-6-1
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ACCEPTED MANUSCRIPT Table 2. Composition of a typical 25 µl conventional LAMP reaction Working Concentration 100-200 ng 1X 6 mM 1.4 mM
1 µl
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Template DNA (20-40 ng) 10X ThermoPol Buffer MgSO4 (100 mM) dNTP Mix (10 mM) FIP/BIP Primers (25X) F3/B3 Primers (25X) LoopF/B Primers (25X) Bst DNA Polymerase, Large Fragment/ Bst 2.0 WarmStart® DNA Polymerase (8,000 U/ml) (New England BioLabs) Molecular grade water
25 µl Reaction 5.0 µl 2.5 µl 1.5 µl 3.5 µl
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Component (Stock concentration)
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Table 3. Composition of a typical 25 µl real-time LAMP reaction :Component (Stock concentration) Template DNA (20-40 ng) Isothermal Master Mix (OptiGene Ltd.) Primers – F3, B3, FIP, BIP. loop-F, loop-R
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Working Concentration 100-200 ng 1X
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Molecular grade water (if required)
25 µl Reaction 5.0 µl 15 µl Depending on target To make the volume 25 µl
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Figure 1. Timeline for LAMP-based GM detection
605 606
NA: nucleic acid, LAMP: loop-mediated isothermal amplification, BART: bioluminescent real time reporter
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Figure 2. Location and target regions of LAMP primers
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FIP : Forward Inner Primer (FIP) consists of the F2 region (at the 3' end) that is complementary to the F2c region, and the same sequence as the F1c region at the 5' end. F3 : Forward Outer Primer consists of the F3 region that is complementary to the F3c region. BIP : Backward Inner Primer (BIP) consists of the B2 region (at the 3' end) that is complementary to the B2c region, and the same sequence as the B1c region at the 5' end. B3 : Backward Outer Primer consists of the B3 region that is complementary to the B3c region. Source: http://loopamp.eiken.co.jp/e/lamp/primer.html9
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Figure 3. Workflow of LAMP-based GM detection
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(c) Amplification profile in Real-time LAMP
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Figure 4. Commonly employed GM detection methods for LAMP products
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(d) Annealing curve profile in Real-time LAMP
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(a) Visual LAMP (Change in green colour from orange depicts positive (b) LAMP products on agarose gel with ladderlike pattern amplification)
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Figure 5. Targets for LAMP-based GM detection
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S0956-7135(19)30348-2
DOI:
https://doi.org/10.1016/j.foodcont.2019.106759
Article Number: 106759 Reference:
JFCO 106759
To appear in:
Food Control
Received Date: 26 April 2019 Revised Date:
15 June 2019
Accepted Date: 7 July 2019
Please cite this article as: Singh M., Pal D., Sood P. & Randhawa G., Loop-mediated isothermal amplification assays: Rapid and efficient diagnostics for genetically modified crops, Food Control (2019), doi: https://doi.org/10.1016/j.foodcont.2019.106759. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Loop-mediated isothermal amplification assays: Rapid and efficient diagnostics for genetically modified crops Monika Singh*, Deepa Palϯ, Payal Soodϯ and Gurinderjit Randhawa Division of Genomic Resources, ICAR-National Bureau of Plant Genetic Resources, New Delhi 110 012, India ϯ
Both the authors contributed equally
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*Corresponding Author
E-mail: [email protected], [email protected]
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Phone: +91 9910067094
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ACCEPTED MANUSCRIPT ABSTRACT
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With the increase in number of globally approved genetically modified (GM) events and
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diversification of traits, efficient GM detection strategies may play key role for regulatory
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compliance. Polymerase chain reaction (PCR) and real-time PCR, targeting different
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components of a transgenic construct, are widely applied analytical methods for GM
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detection. Loop-mediated isothermal amplification (LAMP), an isothermal nucleic acid
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amplification method, has been employed in screening and detection of genetically modified
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organisms (GMOs) since past decade due to user-friendly operation, acceptable specificity,
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less sensitivity to inhibitors and rapid mode of detection. This article reviews the scenario of
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applicability of LAMP in GM detection and potential of LAMP. Amplification of LAMP
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products is done at a constant temperature and no separate steps of denaturation, annealing
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and extension are required. Easy-to-use portable instrumentation has made this technology
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applicable for on-site GMO testing. A series of visual and real-time LAMP assays have been
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developed in recent past for rapid, cost-efficient or on-site detection of GMOs without the
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involvement of time-consuming electrophoretic analysis. Different chemistries and detection
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systems can be used on the basis of practical requirement. Amplification can be detected
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within 30-60 minutes with a limit of detection (LOD) up to 0.005-0.1% GM content
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depending on the target or detection system used. Potential of LAMP for further expanding
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the practical use by modifications/advancement in this technique has also been highlighted in
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this review.
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KEYWORDS
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ACCEPTED MANUSCRIPT Loop Mediated Isothermal Amplification (LAMP), Genetically Modified Organisms
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(GMOs), GM detection, Isothermal nucleic acid amplification technologies (iNAATs), Visual
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Detection, Real-time LAMP, Limit of Detection (LOD), On-site GMO testing
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1. BACKGROUND
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The commercialization of genetically modified (GM) crops has increased globally, with
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respect to the acreage of area under GM crops as well as the diversification of traits/GM
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events (ISAAA, 2017). Area under GM crops has reached 189.8 million hectares in 2017.
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Over 500 GM events covering 30 plant species have got approval in different countries, to be
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used as food and feed (http://www.isaaa.org/gmapprovaldatabase/).
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traits in GM crops include herbicide tolerance, insecticide resistance, either in single or
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stacked form. Stacked GM events account for more than 40% of global area under cultivation
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of GM crops. In India, Bt cotton is the commercialized GM crop with four approved GM
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events, namely, MON531, MON15985, GFM and Event 1. Area under Bt cotton has
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increased to 11.4 million hectares in 2017 (ISAAA, 2017). Several other GM events of
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cotton and other crops have been imported for research purposes through ICAR-National
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Bureau of Plant Genetic Resources, New Delhi or were under field trials (Singh and
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Randhawa, 2016; http://www.moef.nic.in/major-initiatives/geac-clearances).
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Commonly employed
The approval of GM crops as food or feed is regulated in different countries by
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respective regulatory bodies. In some countries, labelling of GM products is voluntary as in
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the United States; whereas it is mandatory in several countries as in the European Union
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(Querci et al., 2010). Most of the countries have implemented labeling regulations for
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genetically modified organisms (GMOs) in food/feed products containing GM content over
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defined threshold. The GMO labeling threshold in European Union is 0.9%, for example, if
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0.9% or more of the ingredient of a food/feed product is GMO, the product needs to be
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labelled as ’containing GMO’ (https://ec.europa.eu/jrc/en/research-topic/gmos). In India, so
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far no labeling threshold is being implemented. For checking the GM status of a sample or to
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check for approved GM events/crops, efficient GM detection methods need to be employed. Reliable GM detection methods play a key role to meet the regulatory requirements.
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Two main methodologies for GM detection target either (i) the components of transgenic
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construct at DNA level, or (ii) the newly expressed recombinant proteins at the protein level.
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DNA-based methods are being commonly employed due to their sensitivity, reproducibility
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and stability. These assays can also be employed for monitoring adventitious presence of
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transgenes in genebanks and for ensuring post-release monitoring of GM events in food and
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supply chain.
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With the increasing number and complexity of GM events (either as single or in
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stacked form) as well as diversification of traits, GM detection has become a challenging
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task. For screening a large number of GM crops/events, it is necessary to develop user-
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friendly, cost-effective and reliable GM detection strategies (Randhawa et al., 2016) Matrix
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approach has been employed as an efficient and cost-effective GMO screening strategy
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(Block et al., 2013; Kralj Novak et al., 2009; Waiblinger et al., 2010; Randhawa et al., 2014;
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Singh et al., 2016). Common screening elements are identified by analysis of GMO matrix,
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which can be used to screen for a range of GM events. This approach could cut down the cost
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of GMO testing by eliminating the need of GM testing methodologies for specific GM
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events. However, for development of more efficient GM detection system, rapid assays
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targeting common screening elements may contribute to an extent.
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Polymerase chain reaction (PCR) and real-time PCR are commonly employed DNA-
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based GM detection methods due to robustness, reliability, sensitivity and reproducibility of
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the assays. Reports of PCR- and real-time PCR-based GM detection were reviewed
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(Randhawa et al., 2016). Validated real-time PCR protocols for over 50 GM events of
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different crops including maize, soybean, cotton and oilseed rape are available at 4
ACCEPTED MANUSCRIPT GMOMETHODS, an European Union Database of Reference Methods for GMO analysis
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based on the Compendium of Reference Methods for GMO Analysis (http://gmo-
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crl.jrc.ec.europa.eu/gmomethods/). In recent past, digital droplet PCR has emerged as a high
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throughput method for GM detection and quantification (Dobnik et al., 2015; Gerdes et al.,
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2016; Košir et al., 2017). However, use of sophisticated equipment and procedures associated
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with PCR/real-time PCR analyses restrict their use for on-site or rapid GMO testing
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(Randhawa et al., 2013).
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Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid
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amplification method where amplification is done at a constant temperature (Notomi et al.,
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2000). Since the introduction of loop-mediated isothermal amplification (LAMP) method in
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2000, it has gained popularity in GM detection for rapid/on-site application (Figure 1). This
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article reviews on the versatility and applicability of LAMP for GM detection.
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2. LOOP-MEDIATED DETECTION
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Isothermal nucleic acid amplification technologies (iNAATs) include the nucleic acid
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amplification assays based on isothermal principles such as strand-displacement
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amplification (SDA), helicase-dependent amplification (HDA), recombinase polymerase
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amplification (RPA) and LAMP (Craw et al., 2012; Walker et al., 1992; Vincent, et al., 2004;
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Piepenburg et al., 2006; Notomi et al., 2000). Among iNAATs, LAMP technology has wider
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applicability in diagnostics including GM detection. Amplification of template DNA is done
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at a constant temperature in LAMP assays using four different primers to recognize six
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distinct regions on the target sequence (Notomi et al., 2000). An outer primer set (F3, B3)
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containing sequences of sense and antisense strands of target DNA initiates LAMP reaction,
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which proceeds at a constant temperature, followed by strand displacement DNA synthesis
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primed by an inner primer set (FIP, BIP). Addition of two loop primers or two stem primers
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further increases the speed and specificity of LAMP assays (Nagamine et al., 2002). Primer designing is crucial for performing LAMP reaction. The PrimerExplorer
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(available at https://primerexplorer.jp/e/) is a software specifically for designing the primer
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sets for LAMP (http://loopamp.eiken.co.jp/e/lamp/primer.html). This software generates the
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primer sets based on the target sequence information, which meets the primer designing
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requirements. The primer regions targeting different regions of the template DNA are shown
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in Figure 2.
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LAMP assay is less sensitive to inhibitors and hence, can be employed for GMO
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testing of crude DNA samples (Francois et al., 2011) LAMP is a user-friendly and reliable
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method for GM detection, which can be performed on a thermal cycler or a heating block or
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in a portable isothermal real-time amplification system. LAMP products can be visualized
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after completion of reactions using nucleic acid staining or fluorescent dyes such as SYBR®
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Green, hydroxylnaphthol blue (Chen et al., 2012; Guan et al., 2010). LAMP products can be
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monitored using turbidometry or by measuring fluorescence using real time LAMP
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(Randhawa et al., 2013; Mori et al., 2004).
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The workflow of LAMP method for GM detection is summarized in Figure 3.
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3. VERSATILITY IN LAMP
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LAMP assay can be performed in different ways for practical use in GMO testing based on
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chemistry, detection method and target to be amplified.
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3.1. Chemistries Employed in LAMP Assays
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Different chemistries and visualization approaches can be used in LAMP depending on the
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practical utility and availability of resources. Several reports of LAMP-based GM detection
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employing different approaches are available (Table 1).
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ACCEPTED MANUSCRIPT 3.1.1. Conventional mix with Bst DNA polymerase large fragment or Bst 2.0 Warm Start®
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DNA polymerase: LAMP can amplify DNA isothermally using a simple isothermal
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amplification instrument, based on strand displacement synthesis of DNA. Bst DNA
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polymerase large fragment represents a part of Bacillus stearothermophilus DNA
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polymerase protein having 5’-3’ polymerase activity but lacking 5’-3’ exonuclease
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activity. This enzyme has applicability in strand displacement reactions, thereby
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contributing to development of LAMP-based diagnostics.
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The reaction mixture for conventional LAMP consists of an isothermal buffer,
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MgSO4, dNTP mix, Bst DNA polymerase large fragment; forward (FIP) and
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backward (BIP) inner primers, forward (F3) and backward (B3) primers and DNA
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template. In addition, loop primers may also be used in order to increase specificity of
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assays. The composition of a typical conventional LAMP reaction is given in Table 2.
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Concentration of primers need to be optimized for the specific target. If
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required, concentration of other components may also be optimized for better clarity
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of results. The reaction is incubated at a constant temperature (60-65 ˚C) for 30–60
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minutes depending on the size of targeted region and melting temperature of primers.
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Incubation is performed in a thermal cycler or alternatively in simple instrumentation
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such as a conventional heating block or a dry bath (Randhawa et al., 2013). Due to
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simple instrumentation with low cost resources, conventional LAMP can cut down the cost of GMO testing (Randhawa et al., 2013; Singh et al., 2015).
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3.1.2. Ready-to-use isothermal master mix: For real-time LAMP assays, isothermal master
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mix (OptiGene Limited) is used, which is compatible with the isothermal real-time
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instrument Genei® II. The composition of a typical real-time LAMP reaction is givem
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in Table 3.
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Annealing temperature and time of reaction completion need to be optimized, if
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required. Incubation is performed in a portable isothermal real-time LAMP system
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(Genie® II), hence it can facilitate rapid on-site GM detection when combined with a
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fast DNA extraction method (Zhang et al., 2013; Randhawa et al., 2013; Singh et al.,
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2015)
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3.2. Detection Methods for LAMP Products
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3.2.1. Visual/colorimetric detection:
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Visual LAMP products are detected by adding
SYBR® Green I dye after completion of reaction. SYBR® Green I binds to DNA and
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the resulting DNA-dye-complex absorbs blue light and emits green light. LAMP
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products can be directly visualized as change in color by the naked eye on addition of
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SYBR® green dye. Change in color from orange to green depicts that LAMP
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amplification has occurred (Figure 4a), whereas no color change depicts absence of
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LAMP amplification. Alternatively, other dyes such as calcein, hydroxynaphthol blue
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(HNB), malachite green can be used based on the practical requirement (Tanner et al.,
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2012; Tomita et al., 2008)
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Metal indicators can be added to the pre-reaction solution and the amplification
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products can be detected visually by the change in color of the reaction mixture. Goto
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et al. (2009) have used the HNB as metal indicator for the first time for colorimetric detection of LAMP products that is based on Mg2+ concentration change. HNB changes the color of the reaction solution from violet to sky blue. As both the colors
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are similar, so it is difficult to detect the change with the naked eye, especially when
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the samples contained low levels of the target sequence. Another metal indicator, acid
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chrome blue K (ACBK) has been used for detection of LAMP amplification for P-35S
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by change in colour from red to blue based on a decrease in the Mg2+ concentration in
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the reaction mixture (Wang et al., 2017). 3.2.2. Electrophoretic detection: Specificity of LAMP products can also be checked by
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electrophoresis in 2% agarose gels in 1x TAE (Tris acetate EDTA) running buffer
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stained with ethidium bromide and then visualized under UV light. Positive
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amplification of LAMP products is detected as typical ladder-like pattern in agarose
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gels (Figure 4b) (Randhawa et al., 2013; Lee et al., 2009). In LAMP reactions, dumb-
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bell shaped structures are formed that contains multiple sites for amplification
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initiation. The amplification proceeds from these multiple initiation sites, and long
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concatemers are formed that contain more sites for amplification initiation. The
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annealing between alternate inverted repeats of the target sequence of the same strand
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results in the formation of cauliflower‐like structures (Notomi et al., 2000; Kumar et
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al., 2017). Ladder-like pattern is produced due to formation of concatemers consisting
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of DNA fragments between outer F3/B3 primers.
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3.2.3. Real-time detection:
(a) Turbidometric detection:
Real-time LAMP assays for detection of P-35S using turbidometry has been
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developed by Fukuta et al. (2004). LAMP has high ability to synthesize sufficient
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amount of DNA, therefore, large amount of byproduct (pyrophosphate ion) is produced, yielding a white precipitate of magnesium pyrophosphate in the LAMP reaction mixture (Mori et al., 2001). The turbidity of reaction mixture allows easy
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detection of amplification of target DNA. The turbidity of the reaction mixture can be
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determined by the difference between the absorbance of the sample solution and the
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reference (a reaction buffer of the same concentration) (Fukuta et al., 2004).
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(b) Fluorescence-based detection:
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detection of LAMP products by monitoring fluorescence as amplification and
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annealing curves as well as time of positivity (Tp) during the progress of reaction as
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shown in Figures 4c & 4d (Randhawa et al., 2013; Singh et al., 2017). Tp refers to the
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amplification time at which the fluorescence second derivative reaches its peak above
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the baseline value.
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Reports of fluorescence-based LAMP performed on real-time PCR system
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such as Light Cycler 450, ABI7500 are available, however, this limits the on-site
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application of LAMP (Randhawa et al., 2013; Wang et al., 2016).
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A direct detection approach, Fluorescence of Loop Primer upon Self
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Dequenching-LAMP (FLOS-LAMP), has been recently reported with applicability in
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pathogen detection (Gadkar et al., 2018). In this approach, a labelled loop probe
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quenched in its unbound state, fluoresces only when bound to its target (amplicon),
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allows the sequence-specific detection of LAMP amplicons.
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3.3. Target to be Amplified
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A transgenic construct comprises of various genetic elements: (i) a promoter enabling the
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expression of inserted gene; (ii) the inserted transgene conferring desired trait to the host
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plant; (iii) a marker gene for selection of transformants; and (iv) a terminator sequence acting
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as a stop signal. These genetic elements can be targeted for GM detection as per the practical
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or regulatory requirement depending on the level of specificity. LAMP can be employed for
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GM detection at different levels of specificity: Screening, gene-specific, construct-specific
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and event-specific assays. (Figure 5)
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3.3.1. LAMP-based Screening Assays: Screening methods are used to check the GM status
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of samples. If a sample is GM, only then subsequent tests are required for
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employed control elements, viz., promoter and terminator. These assays would reduce
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the cost of GMO testing by eliminating the need of specific GM testing
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methodologies for each event. LAMP-based screening assay targeting Cauliflower
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Mosaic Virus 35S promoter (P-35) was first reported (Fukuta et al., 2004). LAMP
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assays for other commonly used promoters, namely, P-FMV, P-ract, P-nos and
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terminators such as T-nos, have also been developed for GMO screening (Randhawa
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et al., 2013; Lee et al., 2009; Singh et al., 2017; Fukuta et al., 2004; Kiddle et al.,
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2012; Singh et al., 2018). The reported assays have been summarized in Table 1. A
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collaborative ring trial validation of three established visual LAMP assays targeting
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P-35S, P-FMV and T-nos has been recently reported (Li et al., 2019). Sensitivity was
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found to be 10, 10, and 50 haploid genome equivalents (HGEs) for P-35S, P-FMV,
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and T-nos, respectively. Ten laboratories participated in the ring trial and the results
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demonstrated that these visual LAMP assays are specific, sensitive and time-saving,
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with high application potential for on-site testing and routine screening of GMOs.
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3.3.2. LAMP-based Gene-specific Assays: Gene-specific methods target specific transgenes
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expressed in a GM crop or marker genes used for selection of transgenics. LAMP
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assays targeting commonly employed marker genes/reporter genes including aadA,
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nptII, pat, pmi, uidA were reported (Randhawa et al., 2013; Lee et al., 2009). Genespecific LAMP assays for detection of cry1Ab in GM rice, cry1Ac in GM sugarcane and phytase in GM corn have been developed (Li et al., 2013; Zhou et al., 2014.
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Huang et al., 2014). LAMP assays targeting insecticide resistant cry1Ac and cry2Ab2
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genes and herbicide tolerant, cp4-epsps gene were developed to check for approved
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and unapproved GM events in the country (Singh et al., 2015). Validated LAMP
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protocol for cp4-epsps gene reported by Wang et al. (2015) and Li et al. (2018) is
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international collaborative ring trial with 12 participating laboratories to validate
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gene-specific LAMP assays targeting four common transgenes, namely, bar, pat, cp4-
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epsps and cry1Ac. The results showed high specificity with limit of detection (LOD)
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of 5, 10, 20 and 20 HGE for cp4-epsps, cry1Ac, bar and pat genes, respectively. The
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ring trial results collectively confirmed that these four gene-specific assays can be
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utilized or quick screening of transgenic traits in routine GMO analysis.
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3.3.3. LAMP-based Construct-specific Assays: Construct-specific methods target the
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junction between two components of a transgenic construct, for instance, a region of
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the insert spanning junction between the promoter and transgene. So far, no report of
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construct-specific LAMP assays is available. However, the construct-specific LAMP
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assays may be employed for specific detection of GM events with closely related
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genetic constructs, for example, P35S-cry1Ac construct region can be used to detect a
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range of GM events including Bt cotton events commercialized in India. Construct-
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specific method has been developed for P35S-cry1Ac construct region (unpublished
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data).
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3.3.4. LAMP-based Event-specific Assays: Event-specific methods target the junction
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region at integration locus between recipient genome and inserted DNA, LAMP is
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also being employed for detection of specific GM events. Event-specific LAMP assays for detection GM maize, GM soybean and Bt rice events have been reported (Chen et al., 2012; Guan et al., 2010; Chen et al., 2011; Bhoge et al., 2015). Event-
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specific LAMP for detection of two Bt cotton events, viz., MON531 and MON15985
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commercialized in the country was developed (Randhawa et al., 2015).
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LAMP assays reported for detection of GMOs at screening, gene-specific, construct-specific and event-specific levels are summarized in Table 1. 12
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replication with appropriate positive and negative controls to avoid type I (false
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positives) and type II (false negatives) errors (JRC Technical Report, 2015). Prior to
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conducting LAMP-based GMO testing, amplifiability of test samples need to be
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ensured using endogenous gene specific assays. Though LAMP could work with the
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crude DNA samples but assay for an endogenous gene amplification eliminates the
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chances of type II error.
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4. APPLICABILITY OF LAMP FOR GM DETECTION
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4.1. Specificity and Sensitivity for LAMP Assays
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Specificity of developed assays can be determined at two levels as a part of method
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validation. At the first level, specificity needs to be checked by selecting the primer
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sequences specific to target region. This can be done in silico by performing searches against
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publicly available nucleotide sequence databases such as National Center for Biotechnology
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Information BLAST searches. At the second level, the assays need to be evaluated
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experimentally by testing against reference materials including both the targets as well as
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non-targets for a particular sequence. Based on the published research reports, LAMP assays
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showed acceptable specificity by specifically amplifying respective targets positive for a
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particular transgenic trait (Randhawa et al., 2013; Wang et al., 2015; Singh et al., 2015;
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Singh et al., 2018; Singh et al., 2017). In these studies, set of appropriate targets and non-
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targets for a transgenic element were used to confirm the specificity of an assay.
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Sensitivity of LAMP assay is assessed as the limit of detection (LOD). LOD
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determines the lowest concentration at which all replicates result in positive LAMP reaction
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signals. LAMP assays are sensitive enough to detect the GM content with LOD ranging from
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0.1 to 0.005 depending upon the target and assay as shown in Table 1.
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LAMP-based testing can be completed in comparatively less time as compared to PCR and
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real-time PCR as thermocycling steps and time-consuming electrophoresis analysis is not
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required (Randhawa et al., 2013; Singh et al., 2015). LAMP has been considered as a method
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of choice for rapid GM detection as reaction is completed at a constant temperature (60-65
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˚C) within 30-60 minutes and LAMP products can be visualized by adding SYBR Green dye.
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Conventional LAMP reactions can alternatively be conducted using simple instrument such
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as a heating block or a dry bath, which can reduce the equipment and running costs. Hence,
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conventional LAMP can be utilized by low resource GMO testing laboratories.
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4.3. On-site Application
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Conventional and real-time PCR assays have been widely used for GM detection due to
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efficiency, sensitivity and reproducibility. These assays require expensive and sophisticated
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equipment for thermal cycling or fluorescence determination, which limits their use for on-
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site application because of the lack of portability. PCR assays also require time-consuming
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gel electrophoretic analysis of products. Moreover, Taq DNA polymerase used in PCR assays
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may be inactivated by inhibitors present in crude samples (De Franchis et al., 1988) and thus,
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may not be employed for on-site GM detection.
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Real-time LAMP can be performed on a real-time isothermal system (Genie® II). The
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system is a compact, light-weight and fully portable (http://www.optigene.co.uk/wp-
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content/uploads/2012/06/Genie-II-flier.pdf). Due to portability of system, real-time LAMP
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when combined with a fast DNA extraction protocol can be employed for on-site GM
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detection at port of entry or in farmer fields (Randhawa et al., 2013).
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Zhang et al. (2013) developed a novel GM detection system, which can be utilized to perform
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both on-site as well as routine laboratory GMO testing. In this system, a newly designed
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assay. The DNA extraction device fitted with a silica gel membrane filtration column and a
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modified syringe, could be easily operated without using other laboratory instruments,
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making it applicable for on-site. In the modified LAMP assay, a microcrystalline wax
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encapsulated detection bead containing SYBR green fluorescent dye was added to avoid dye
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inhibition and cross-contaminations.
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4.4. Application and Potential of Multiplex LAMP Assays
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Multiplex real-time LAMP assays are being widely used for detection of pathogens and
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identification of blood-borne viruses (Liu et al., 2017; Kouguchi et al., 2010; Dougbeh-Chris
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et al., 2015). Very little research has been undertaken for development of multiplex LAMP
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assays for detection of GMOs. Chen et al. (2017) developed CALM (Capillary Array-based
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LAMP for Multiplex visual detection of nucleic acids) where a small, ready-to-use cassette
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was assembled by capillary array, allowing simultaneous detection of 8 commonly present
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transgenic elements/genes with high specificity and sensitivity. The capillary array was pre-
355
treated into a hydrophobic and hydrophilic pattern before fixing LAMP primer sets in
356
capillaries. LAMP reaction mixture was added after assembly of the loading adaptor and
357
isolated into each capillary due to capillary force by a single pipetting step. The LAMP
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reactions were performed in parallel in the capillaries and the results are visually detected
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with a hand-held UV flashlight.
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LAMP-based detection method s combined with lateral flow dipstick chromatography has
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been reported for detection of GM soybean and maize events targeting P-35S, mannose-6-
362
phosphate isomerase gene, Pisum sativum ribulose 1, 5-bisphosphate carboxylase terminator,
363
a common sequence between the cry1Ab and cry1Ac genes, and a GA21-specific sequence
364
(Takabatake et al., 2018). Development of multiplex LAMP assays can further time-
365
efficiency and would reduce the cost involved in detection and identification of GMOs. 15
ACCEPTED MANUSCRIPT 5. CONCLUSION
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LAMP has emerged as a popular diagnostic tool for being fast, sensitive and user-
368
friendliness. The major advantage of this technique is that the targets are amplified at a
369
constant temperature, thereby eliminating the time-consuming thermocycling steps required
370
for any two- or three-step PCR. With no thermocycling requirements, amplification reactions
371
can be conducted using simple instruments for e.g., a conventional water bath or heat block,
372
thus, drastically reducing equipment costs. Since, real time-LAMP can also be performed in a
373
simple portable machine, it can be employed for on-site detection of GMOs in crop plants
374
when used in combination with a fast DNA extraction method. In the last decade, LAMP has
375
dramatically influenced the research in different areas of diagnostics including GM
376
diagnostics. Development of multiplex LAMP assays for detection of multiple targets in a
377
single reaction can further add to rapid, robust and cost-effective features to this technique.
378
Further refinement and advancement in this technique may be explored to expand its practical
379
utility in the area of diagnostics for GM crops.
380
ACKNOWLEDGMENTS
381
We acknowledge the support provided by Indian Council of Agricultural Research (ICAR),
382
New Delhi, Department of Biotechnology (DBT) and Department of Agriculture,
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Cooperation & Farmers Welfare (DAC&FW). We also thank the Director, ICAR-National
384
Bureau of Plant Genetic Resources for providing necessary facilities.
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Table 1. Summary of LAMP assays reported for GM detection
P-35S
Visual/Real-time LAMP LAMP-BART Real-time turbidometry Visual LAMP
Visual using metal indicator acid chrome blue K
marker genes (genespecific assays)
aadA bar nptII
Visual LAMP Visual LAMP Visual LAMP LAMP-BART Visual LAMP Visual/Real-time LAMP
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P-ract P-nos T-nos
Visual/Real-time LAMP
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P-FMV
limit of detection (LOD) Visual LAMP Real-time LAMP 40 Copies 4 Copies (0.01%) (0.1%) 50 copies (0.1%) 0.5% 5 copies per haploid genome equianlent (HGE) ∼50 copies for GM rice 100 ng of 0.1 % DNA from rice, soybean, rapeseed, and maize 40 Copies 4 Copies (0.01%) (0.1%) 10 copies per HGE 0.05% 0.01% 50 copies (0.1%) 5 copies per HGE 40 Copies 4 Copies (0.01%) (0.1%) 5 copies per HGE
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detection method
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targets/ events
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transgenic element promoter/ terminator sequences (screening assays)
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Visual LAMP
Visual/Real-time LAMP Visual LAMP
uidA
Visual/Real-time LAMP
26
40 Copies (0.1%) 5 copies per HGE
4 Copies (0.01%)
40 Copies (0.1%)
4 Copies (0.01%)
ref Randhawa et al., 2013 Kiddle et al., 2012 Fukuta et al., 2004 Wang et al., 2015
Wang et al., 2017
Randhawa et al., 2013 Wang et al., 2015 Singh et al., 2018 Singh et al., 2018 Kiddle et al., 2012 Wang et al., 2015 Randhawa et al., 2013 Wang et al., 2015 Randhawa et al., 2013 Wang et al., 2015 Randhawa et al., 2013
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constructspecific assays eventspecific sequences
phytase in GM maize bar in GM sugarcane P35S-cry1Ac
Bt Cotton Events (i) MON531 (ii) MON15
10 copies per HGE 43.1 copies
Visual/Real-time LAMP
4 Copies (0.01%)
Visual LAMP Visual LAMP Visual/Real-time LAMP Visual LAMP Visual LAMP
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Wang et al., 2015 Singh et al., 2017 Singh et al., 2017 Singh et al., 2015
Wang et al., 2015 Huang et al., 2014
4 Copies (0.01%)
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8 copies (0.01%) 8 copies (0.01%) 2 copies (0.005%)
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Visual LAMP Visual LAMP
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cry1Ab (GM rice) cry3A cp4-epsps (GM cotton, maize) cp4-epsps
10 copies per HGE 44 copies (0.05%) 88 copies (0.1%) 4 Copies (0.01%)
Singh et al., 2015
5 copies
Li et al., 2014
0.5%
Li et al., 2013
5 copies 4 Copies (0.01%)
Li et al., 2014 Singh et al., 2015
4 Copies (0.01%)
Wang et al., 2015 30 copies
Huang et al., 2014
Visual LAMP
10 copies
Zhou et al., 2016
Visual LAMP
0.01%
unpublished data
Visual/Real-time LAMP
2 copies (0.005%)
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transgenes (genespecific assays)
pmi cry1Ac (GM cotton, brinjal , maize) cry1Ac cry1Ac (GM sugarcane) cry2Ab2 (GM cotton, maize) cry2Ab
Visual LAMP Visual/Real-time LAMP Visual/Real-time LAMP Visual/Real-time LAMP
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27
2 copies (0.005%)
Randhawa et al., 2015
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LAMP combined with lateral flow dipstick
2.4 copies
Wang et al., 2013
Visual LAMP
4 copies
Chen et al., 2011
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4 copies (GTS-40-3-2) 8 copies (MON89788)
Visual/Real-time LAMP
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GM Maize Events (i) DAS59122-7 (ii) T25 (iii) Bt176 (iv) TC1507 (v) MON810 (vi) Bt11 (vii) MON863 GM Maize Events (i) Bt11 (ii) GA21 (iii) MON810 (iv) MON8903 4 (v) NK603 (vi) TC1507 GM Maize Event MON88017
Visual LAMP
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GM Soybean Events (i) GTS-40-3-2 (ii) MON89 788 Roundup ready GM Soy event
8 copies (0.01% )
8 copies (0.01% )
Bhoge et al., 2015
73 copies for NK603 (0.1%) 73 copies
Visual LAMP
40 copies
28
Zhen et al., 2016
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0.5%
Xu et al., 2013
Visual LAMP
0.01%–0.005%
Chen et al., 2012
Visual LAMP
6 copies
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Visual LAMP
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GM Maize Event T25 GM Rice Events (i) KMD1 (ii) TT51-1 (iii) KF6 GM Wheat Event B73-6-1
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ACCEPTED MANUSCRIPT Table 2. Composition of a typical 25 µl conventional LAMP reaction Working Concentration 100-200 ng 1X 6 mM 1.4 mM
1 µl
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Template DNA (20-40 ng) 10X ThermoPol Buffer MgSO4 (100 mM) dNTP Mix (10 mM) FIP/BIP Primers (25X) F3/B3 Primers (25X) LoopF/B Primers (25X) Bst DNA Polymerase, Large Fragment/ Bst 2.0 WarmStart® DNA Polymerase (8,000 U/ml) (New England BioLabs) Molecular grade water
25 µl Reaction 5.0 µl 2.5 µl 1.5 µl 3.5 µl
30
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Component (Stock concentration)
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Table 3. Composition of a typical 25 µl real-time LAMP reaction :Component (Stock concentration) Template DNA (20-40 ng) Isothermal Master Mix (OptiGene Ltd.) Primers – F3, B3, FIP, BIP. loop-F, loop-R
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Working Concentration 100-200 ng 1X
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Molecular grade water (if required)
25 µl Reaction 5.0 µl 15 µl Depending on target To make the volume 25 µl
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Figure 1. Timeline for LAMP-based GM detection
605 606
NA: nucleic acid, LAMP: loop-mediated isothermal amplification, BART: bioluminescent real time reporter
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Figure 2. Location and target regions of LAMP primers
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FIP : Forward Inner Primer (FIP) consists of the F2 region (at the 3' end) that is complementary to the F2c region, and the same sequence as the F1c region at the 5' end. F3 : Forward Outer Primer consists of the F3 region that is complementary to the F3c region. BIP : Backward Inner Primer (BIP) consists of the B2 region (at the 3' end) that is complementary to the B2c region, and the same sequence as the B1c region at the 5' end. B3 : Backward Outer Primer consists of the B3 region that is complementary to the B3c region. Source: http://loopamp.eiken.co.jp/e/lamp/primer.html9
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Figure 3. Workflow of LAMP-based GM detection
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(c) Amplification profile in Real-time LAMP
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Figure 4. Commonly employed GM detection methods for LAMP products
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(d) Annealing curve profile in Real-time LAMP
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(a) Visual LAMP (Change in green colour from orange depicts positive (b) LAMP products on agarose gel with ladderlike pattern amplification)
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Figure 5. Targets for LAMP-based GM detection
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