- Email: [email protected]
Rapid detection of pork using alkaline lysis- Loop Mediated Isothermal Amplification (AL-LAMP) technique
Journal Pre-proof Rapid detection of pork using alkaline lysis- Loop Mediated Isothermal Amplification (AL-LAMP) technique P.S. Girish, S.B. Barbuddhe, Aparana Kumari, Deepak B. Rawool, Nagappa S. Karabasanavar, M. Muthukumar, S. Vaithiyanathan PII:
S0956-7135(19)30604-8
DOI:
https://doi.org/10.1016/j.foodcont.2019.107015
Reference:
JFCO 107015
To appear in:
Food Control
Received Date: 29 May 2019 Revised Date:
21 November 2019
Accepted Date: 23 November 2019
Please cite this article as: Girish P.S., Barbuddhe S.B., Kumari A., Rawool D.B., Karabasanavar N.S., Muthukumar M. & Vaithiyanathan S., Rapid detection of pork using alkaline lysis- Loop Mediated Isothermal Amplification (AL-LAMP) technique, Food Control (2019), doi: https://doi.org/10.1016/ j.foodcont.2019.107015. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
Author Contribution
Conceived and designed the experiments: GPS, SBB, and SV. Performed the experiments: GPS, MM and AK. Analyzed the data: GPS, AK and NSK. Contributed reagents/materials/ analysis tools: GPS, MM, SBB and DBR. Wrote the manuscript: GPS and NSK. Critical evaluation of manuscript: GPS, SBB and SV.
1
Rapid detection of pork using Alkaline Lysis- Loop Mediated Isothermal Amplification (AL-
2
LAMP) technique
3
Girish, P.S.1*, S.B. Barbuddhe2, Aparana Kumari3, Deepak B. Rawool4, Nagappa S.
4
Karabasanavar5, M. Muthukumar6, S. Vaithiyanathan7
5
ICAR–National Research Centre on Meat
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Chengicherla, Hyderabad, TelanagnaState-500092, India
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*Corresponding author E mail: [email protected] 1. ICAR – National Research Centre on Meat, Chengicherla, Hyderabad, Telangana State - 500 092 India, Phone: +91-9401262522; E mail: [email protected] 2. ICAR – National Research Centre on Meat, Chengicherla, Hyderabad, Telangana State - 500 092 India, E mail: [email protected] 3. ICAR – National Research Centre on Meat, Chengicherla, Hyderabad, Telangana State - 500 092 India, E mail: [email protected] 4. ICAR – Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243 122 India,E-mail: [email protected] 5. Department of Veterinary Public Health & Epidemiology, Veterinary College, Gokula, Vidyanagar, Hassan, Karnataka–573 202, India E-mail: [email protected] 6. ICAR – National Research Centre on Meat, Chengicherla, Hyderabad, Telangana State - 500 092 India, E mail: [email protected] 7. ICAR – National Research Centre on Meat, Chengicherla, Hyderabad, Telangana State - 500 092 India, E mail:[email protected]
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ABSTRACT
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We report a novel, rapid, economical and species-specific DNA-based assay for the authentication
29
of pork. The technique specifically amplified porcine mitochondrial D loop region by combining
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Alkaline Lysis (AL) method of DNA extraction and Loop Mediated Isothermal Amplification
31
(LAMP). Visual detection of the reaction was accomplished by color development in the reaction
32
with the addition of SYBR Green I dye. Dependable amplification was possible in thermally
33
processed meat samples heated up to 121 ºC for 30 min. The assay was able to detect pork in beef
34
up to the level of 0.1% admixture and limit of detection of DNA was at 0.5 ng/µL. Cross-
35
amplification of related species like cattle, buffalo, sheep, goat and chicken was excluded by
36
incorporating their DNA in the reaction assay. The novel approach (AL-LAMP technique) was
37
found to be robust and handy suitable even for resource compromised laboratories engaged in the
38
food analysis.
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Keywords: Pork; Speciation; DNA; D loop; Alkaline lysis; LAMP; Adulteration
41 42 43 44 45 46 47 48 49 50 51 52 2
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1. Introduction
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Adulteration of meat with pig meat is considered as an offence owing to the religious and
55
health concerns (Lubis, Salihah, Hossain, & Ahmed, 2017). Pig meat and products being readily
56
available and cheap sources, are substituted into other species meats (Grundy et al., 2012). Scandals
57
associated with incorporation of pig meat into other species meats have drawn considerable
58
attention in the past (Abdullahi et al., 2017). In addition, risk of acquiring certain diseases and
59
development of allergy among sensitive individuals also poses threat to public health arising due to
60
the consumption of meats adulterated with pork or pig derived tissues. In order to comply with
61
halal authentication, protect consumers’ sentiments, promote fair-trade, implement prompt labeling
62
norms, avoid allergies and prevent disease transmission from pig or pork, there is a need to
63
correctly identify pig derived tissues (Aida, Che, Wong, Raha, & Son, 2005).
64
Origin of pig could be detected using conventional techniques viz., anatomical, histological,
65
chemical, electrophoretic or enzymatic assays (Ballin, Vogensen, & Karlsson, 2009). Nevertheless,
66
these conventional techniques have inherent limitations (lack of discriminating power
67
orrepeatability and cross-reactions); therefore, DNA based techniques particularly PCR-based tools
68
were developed, and conclusively emerged as methods of choice for the purpose of species
69
identification (Ballin, Vogensen, & Karlsson, 2009). The PCR based techniques used for pork
70
detection include sequence analysis of mitochondrial 12S rRNA gene (Girish et al., 2004), species
71
specific PCR targeting mitochondrial D loop region (Karabasanavar, Singh, Kumar, &
72
Shebannavar, 2014), PCR-Restriction Fragment Length Polymorphism of mitochondrial of
73
cytochrome B gene (Chandregowda, Fairoze, Girish, Karabasanavar, & Bagale, 2015), multiplex
74
PCR targeting cytochrome b gene (Ali et al., 2015), real time PCR targeting mitochondrial ND5
75
region (Kesmen, Gulluce, Sahin, & Yetim, 2009) and duplex PCR targeting mitochondrial
76
cytochrome b gene (Di Pinto, Forte, Conversano, & Tantillo, 2005). Nevertheless, the PCR based
77
detection of swine species requires costly instrumentation such as thermocycler, electrophoresis
78
unit and gel documentation system; also, traditional PCR based methods are time consuming, costly 3
79
and require samples to be carried to the laboratory for analysis. On the other hand, quantitative
80
qPCR and sensor based techniques (Lubis et al., 2017) require expensive instruments and
81
sophisticated laboratory setups (Ma, Dai, Fang, Wu, & Zhang, 2016).
82
Loop-mediated Isothermal Amplification (LAMP) is a nucleotide amplification technique
83
(Notomi et al., 2015) that amplifies target DNA at isothermal temperatures and obviates need for
84
thermal cyclersand post-amplification procedures of signal detection (Erwanto, Abidin, Rohman, &
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Sismindari, 2011). As in PCR, LAMP assay could amplify target DNA several folds (109copies in a
86
span of an hour) under isothermal conditions without compromising specificity and having
87
capability of the visual detection of amplified targets using specific dyes (Yang et al., 2014).
88
Consequently, LAMP assay has emerged as an alternative tool to PCR based techniquesfor the
89
purpose of testing food safety hazards including detection of meat adulteration (Abdullahiet al.,
90
2017).
91
Several reports of application of LAMP assay for the detection of pork have been reported
92
which include development of LAMP assay for the visual detection of pork DNA in meat products
93
targeting the mitochondrial DN1 gene sequence (Ran et al., 2016), real-time LAMP assay targeting
94
the Cytb gene for the detection of pork meat in a meat mixture (Yang et al., 2014) and rapid on-site
95
real-time LAMP method for pork species identification in processed meat products by Lee, Kim,
96
Hong, & Kim (2016). Nevertheless, need for a robust, rapid and economic assay involving simple
97
sample preparation methods which can be used by even resource deficient laboratories lacking
98
sophisticated instruments was felt. Therefore, in the present work, a simple method for detection of
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pork using alkaline lysis method of DNA extraction and LAMP assay (AL-LAMP) has been
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described using newly designed set of primers targeting mitochondrial D loop region.
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2. Experimental
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2.1 Collection of samples
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Samples of beef, buffalo meat, mutton and chevon were obtained from Municipal
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slaughterhouse (Chengicherla, Hyderabad, Telangana state, India). Samples of porkwere obtained 4
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from the authentic retail shops located at Hyderabad, Telangana state, India. Samples of chicken meat
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were obtained from experimental abattoir of ICAR – National Research Centre on Meat,
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Chengicherla, Hyderabad, Telangana State, India. Meat samples were collected individually in sterile
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containers, transported to the laboratory under refrigeration temperature and stored at -20ºC until
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further analysis. Pork products viz., kabab, ball, masala sausage, salami, breakfast sausage,cocktail
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sausage and square rolls were collected from specific food stalls of Hyderabad city.
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2.2 Extraction of DNA
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DNA from meat and meat products was extracted usingpreviously described Alkaline Lysis
113
(AL) method (Girish, Haunshi, Vaithiyanathan, Rajitha, & Ramakrishna, 2013). Briefly, one part of
114
sample (500 mg) was triturated with eight volumes (4 mL) of NaOH solution (0.2 mol/L); resultant
115
extract (5 µL) was again mixed with eight volumes (40 µL) of NaOH solution (0.2 mol/L) and heated
116
at 75ºC in dry bath for 20 min. After neutralization of the mixturewith eight volumes (360 µL) of
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Tris-HCl (0.04 mol/L, pH 7) the resultant DNA was used for loop-mediated isothermal amplification
118
(LAMP) reaction.
119
2.3 Designing of primers for the LAMP assay
120
Two sets of primers (F3/B3, FIP/BIP) were designed for the LAMP assay targeting
121
mitochondrial D loop region using Primer Explorer V5 software; the 5′ 3′ primer sequences were F3
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– AGG CCC TAA CAC AGT CAA, B3 – GTT ATA GGG TGT GTA GAG CAT A, FIP - ACT
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GAA TAG CAC CTT GTT TGG ATT TGT AGC TGG ACT TCA TGG and BIP - CGG GAC ATA
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ACG TGC GTA CAA GTT TAA TGG GGG GTA AGG.
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2.4 LAMP reaction mixture preparation
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The LAMP reaction mix consisted of 10X Thermopol buffer (3 µL), 5 M Betaine (3 µL), 50
127
mM MgSo4 (3 µL), 10 mMdNTP mix (4 µL), 10 pmol F3 primer (1 µL),10pmol B3 primer (1 µL), 40
128
pmol FIP primer (4 µL), 40 pmol BIP primer (4 µL) and template DNA (5 µL). The reaction mix was
129
heated at 95ºC for 5 min for denaturation; thereafter, 8 units (1 µL) of BstI enzyme was added and
130
incubated at 65ºC for 1 h followed by heating at 80ºC for 2 min for the inactivation of BstI enzyme. 5
131
After the LAMP reaction, 1 µL of SYBR green I dye (1:10, 10,000X) was added for the visualization
132
of amplification. Positive reaction was indicated by the green colour; whereas, orange color was
133
indicative of the negative reaction. Amplification was confirmed by agarose gel electrophoresis (2%)
134
usingethidium bromide staining.
135
2.5 Validation of AL – LAMP technique in meat admixtures
136
For the detection of pork in beef using AL-LAMP assay, different proportions of pork in beef
137
i.e. 50:50, 40:60, 30:70, 20:80, 10:90, 5:95; 1:90; 0.8:90.2; 0.6:90.4; 0.4:90.6; 0.2; 90.8 and 0.1: 90.9
138
were prepared. Admixture was prepared in total quantity of 500 mg and was used for extraction of
139
DNA by alkaline lysis method. After thorough mixing, meat mixtures were subjected for DNA
140
extraction using AL method.
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2.6 Validation of AL – LAMP technique in heat treated meat
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Pork samples were heated at 60ºC, 80ºC, 100ºC and 121ºC for 30 min in a dry bath.
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Thereafter, DNA was extracted from thermally processed pork samples using alkaline lysis method as
144
described by Girish, Haunshi, Vaithiyanathan, Rajitha, & Ramakrishna (2013).
145
2.7 Sensitivity of the LAMP PCR
146
Sensitivity of developed LAMP assay was assessed by diluting the template pork DNA so as
147
to get concentrations viz., 100, 75, 50, 40, 30, 25, 20, 10 and 5 ng/µL followed by the LAMP assay
148
using the novel set of primers designed.
149
2.8 Fluorometric assay
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Intensity of colour developed after the addition of SYBR Green I dye to the final reaction mix
151
of LAMP product was analyzed in using a fluorometer (Denovix, Model: DS-11FX) at 565–650 nm
152
fluorescence. The final reaction mix of volume 30 µL was diluted with nuclease free water to 200 µL
153
volume and the resultant diluted mix was used for recording fluorescence.
154 155
2.9 Statistical analysis
6
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Average fluorometric readings of AL-LAMP results of non-pig species (cattle, buffalo, goat,
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sheep and chicken) were considered for calculating the cut-off values for pig as described by Goto,
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Honda, Ogura, Nomoto, & Hanaki (2009). Average fluorometric value of non-pig species was added
159
with two standard deviations to calculate the cut-off value of the signal for pig.
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3. Results and discussion
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3.1 Quality and purity of DNA extracted using Alkaline Lysis(AL) method
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The DNA extracted from fresh and heat treated raw meat or meat products using the AL
163
method yielded good quality DNA suitable for LAMP reaction. Average concentrations of DNA
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after extraction by AL method in fresh pork and pork heated at 60°C, 80°C, 100°C and 120°C were
165
71.11, 139.24, 89.71, 80.31 and 59.94 ng/µL, respectively. Average concentration of DNA
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extracted from different processed pork products obtained using AL method was 83.29 ng/µL with
167
OD260:280values in the range of 1.6 to 1.9. Commonly employed methods for extraction of DNA
168
from meat are: Phenol: Chloroform extraction (PC) method and commercial kit based methods
169
(Girish, Haunshi, Vaithiyanathan, Rajitha, & Ramakrishna, 2013). The PC method of DNA
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extraction from meat (Chikuni, Tabata, Kosugiyama, & Monma, 1994) involves lysis of cells using
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proteinase K followed by repeated extraction steps by centrifugation using Phenol, Choloroform
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and Isoamyl alcohol followed by precipitation using ethanol and ammonium acetate. Although pure
173
DNA is obtained by this method, toxicity of phenol and labor-intensity is a drawback. Moreover,
174
the presence of phenol minimizes the quantitation of DNA detected by UV absorbance since phenol
175
shows high extinction coefficient at 260 nm (Javadi et al., 2014). Kit based methods are effective
176
but are costly and involve multiple centrifugation steps. As compared to these routinely used
177
methods, AL method of DNA extraction is simple, involves simple laboratory chemicals, quick and
178
do not require any expensive equipments. Moreover, AL is a single tube technique which reduces
179
the chances of contamination during extraction process. Girish, Haunshi, Vaithiyanathan, Rajitha,
180
& Ramakrishna (2013) also reported alkaline lysis method for extraction of DNA from buffalo meat
181
which was used successfully for species identification by species specific PCR in fresh and 7
182
processed meat products. Ali, Rampazzo, Costa, & Krieger (2017) also reported that Alkaline lysis
183
based methods are fastest, most reliable and relatively easy way to obtain DNA from cells and are
184
suitable for less sensitive applications.
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3.2 SYBR Green I based visual pork specific AL-LAMP assay
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Products of LAMP amplification were mixed with SYBR green I dye and resultant signals
187
were measured fluorometrically (Fig. 3). Fluorometric readings were significantly higher for raw,
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cooked and processed pork samples compared to other species and negative controls as also reported
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by Goto, Honda, Ogura, Nomoto, & Hanaki (2009). In addition to the visual color comparison,
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fluorometry convincingly proved the results of AL-LAMP assay for the purpose of authentication of
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the pork (Fig 1-3).
192
The LAMP assay developed by Notomi and coworker amplified pig specific mitochondrial D
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loop target DNA based on autocycling strand displacement DNA synthesis principle using Bacillus
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stearothermophilus (Bst) DNA polymerase (Notomi et al., 2015). Set of four primers bind to unique
195
sites on the target sequence. LAMP technique amplifies target DNA under isothermal conditions (63
196
to 65 °C) with detection limits comparable to the PCR, eliminating need for the expensive thermal
197
cyclers (Yang et al., 2014). After the isothermal amplification, SYBR Green I dye was added to the
198
reaction tubeand color green was visualized to indicate LAMP amplification and absence of
199
amplification was indicated by orange color (Iwamoto, Sonobe, & Hayashi, 2003). The entire
200
procedure (sample to result) of DNA extraction by alkaline lysis method and LAMP assay took 120
201
minutes. In addition to the PCR, similar DNA based techniques such as FINS (forensically
202
informative nucleotide sequencing), restriction fragment length polymorphism (RFLP) and variants
203
take longer duration for analysis. Also, highly sensitive quantitative techniques such as real time PCR
204
require costly instrumentation and consumables for achieving the same objectives.
205
Variants of LAMP assay such as its coupling with other DNA based techniques has been
206
reported for the purpose of species identifications; Kim and Shin (2017) developed isothermal probe
207
amplification (ITPA) assay targeting mitochondrial 16S rRNA gene using four sets of primers (two 8
208
outer and two inner primers) and fluorescence resonance energy transfer (FRET) that required a
209
heating block and a fluorescence reader. Yang et al. (2014) developed a 45 minutes one-step, SYBR
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Green I real-time, loop-mediated isothermal amplification (RealAmp) assay targeting mtDNA of
211
cytochrome b gene for the specific detection of pork; however, such RealAmp assay required costly
212
Real-Time PCR System.
213
Detection of LAMP signal was undertaken either by gel DNA electrophoresis, visualization of
214
the color of amplified DNA by using DNA staining dyes (SYBR green I), visualization of DNA
215
precipitation of chemicals such as magnesium pyrophosphate (MPP) or visualization of turbidity
216
using MPP (Notomi, Mori, Tomita, & Kanda, 2015). Of these techniques, observation of the
217
amplified LAMP signal by naked eye was the most appreciated method (Lee,Su, Lein, & Sheu,
218
2017). The visual SYBR Green I dye color detection system used in AL-LAMP assay was an easy
219
way of detection of amplification signal. The AL-LAMP assay does not require costly equipment
220
such as real-time PCR system, fluorescence reader, etc as specific amplified signal is visualized by
221
the naked eye. Further, the AL-LAMP assay does not require cumbersome DNA extraction thereby
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making the authentication process easy and economical. Time taken for DNA extraction using
223
alkaline lysis method was 40 min and another 80 min was spent for the LAMP assay. For running a
224
sample at a laboratory having minimum facilities, a maximum of 120 min is required for pork
225
authentication using the novel AL-LAMP assay.
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PCR detection of pig species has been previously accomplished by using FINS targeting
227
mitochondrial 12S rRNA gene (Girish et al., 2004; Chandregowda, Fairoze, Girish, Karabasanavar, &
228
Bagale, 2015); species specific PCR targeting mitochondrial D loop (Karabasanavar, Singh, Kumar,
229
& Shebannavar, 2014) or porcine specific repetitive DNA element (Calvo, Zaragoza, and Osta,
230
2001); duplex PCR (Di Pinto, Forte, Conversano, & Tantillo, 2005); multiplex PCR targeting
231
cytochrome b gene (Ali et al., 2015); DNA barcode (Di Pinto et al., 2013); real time PCR (Kesmen,
232
Gulluce, Sahin, &Yetim, 2009; Lubis, Salihah, Hossain, & Ahmed, 2017) and others. Nevertheless,
233
all these assays require DNA extraction, costly PCR and post PCR processing like costly sequencing 9
234
or electrophoresis. On the other hand, the PCR and restriction fragment length polymorphism (PCR-
235
RFLP) approach used for pork detection targeting mitochondrial D loop sequences such as
236
cytochrome b gene (Ali et al., 2011) also require PCR amplification, restriction digestion of
237
amplicons and electrophoresis making the technique cumbersome with higher false negative reactions
238
(failures in restriction digestions).
239
The PCR based techniques developed for pig species (pork) identification such as qualitative
240
forensically informative nucleotide sequencing (FINS), species specific PCR, multiplex PCR or
241
quantitative real time PCRs require longer reaction times compared to the LAMP. The LAMP does
242
not require denaturation step as in the conventional PCR thereby reducing its reaction time. Also,
243
LAMP primers have higher efficacy than the PCR primers (Chen, Ma, Qiang, & Ma, 2016).
244 245
3.3 Specificity and sensitivity of pork specific AL-LAMP assay
246
Results of AL-LAMP assay were detected by visual comparison of the SYBR Green I dye
247
color; development of green color was indicative of positive reaction and orange color indicated the
248
negative reaction. Specificity of AL-LAMP assay was tested using DNA extracted from closely
249
related species viz., cattle, buffalo, sheep, goat, pig and chicken (Fig1). Pig specific positive
250
amplification signal was demonstrated by the green colorand it was seen only in the pork sample.
251
None of the other related species tested showed any amplification signal; results indicated higher
252
specificity of the designed primers for pig species using the AL-LAMP assay.
253
Sensitivity of AL-LAMP was assessed by diluting the DNA; the lowest concentration of DNA
254
that gave a detectable pork signal was taken as sensitivity. The sensitivity of AL-LAMP assay was
255
0.5 ng/µL (Fig 1). In admixed meat (pork in beef) samples, the AL-LAMP detected 0.1% level
256
adulteration of pork in beef. The LAMP assay developed by Abdullahi et al. (2017) for porcine
257
tRNAlys and ATPase 8 genes detected 0.03 femtograms of pig DNA. Likewise, onsite smart and
258
sealed biosensor based LAMP assay coupled to lateral flow device (LFD) (Xu et al., 2017) detected
259
10 picograms (3~5 copies) of chromosomal gene target. Higher sensitivity of LAMP assay than
260
conventional PCR helps in the detection of target DNA even in those samples supposed to have low 10
261
copies of the target DNA (Erwanto, Abidin, Rohman, & Sismindari, 2011). Higher limits of detection
262
of the AL-LAMP assay indicate its potential applications at the field level for the purpose of
263
authentication of the pork adulterated samples.
264
Specificity and sensitivity of LAMP assay was higher than conventional PCR, this could be
265
attributed to LAMP primers that could detect even a single nucleotide difference and formation of
266
loop-medicated secondary DNA structure (Fukuta, Mizukami, Ishida, & Kanbe, 2006). Lower
267
template was required for LAMP assay than the conventional PCR due to severe deconstruction of
268
the DNA (Lee, Su, Lein, & Sheu, 2017).
269
3.4 Validation of AL-LAMP assay for heated/ thermally processed pork
270
Dependable amplification signal (color change to green) was observed in four types of heat
271
treatments employed viz.,60ºC, 80ºC, 100ºC and 121ºC for 30 min (Fig 1) indicating suitability of the
272
AL-LAMP assay for the detection of pork even in thermally processed samplesheated up to 121°C for
273
30 min. Such detection was without any decline in the color intensity; however, weakening of the
274
amplification signal was observed using conventional PCR heated at 120°C for 30 min (Girish et al.,
275
2004).
276
3.5Validation of AL-LAMP assay for pork products
277
Positive amplification signal marked by the color change (green) using SYBR Green I dye
278
was observed in different pork products viz., kabab, balls, sausage, salami, breakfast sausage, cocktail
279
sausage and square rolls (Fig 1). Different processing conditions and use of meat ingredients in the
280
preparation did not affect the amplification signal or color change.
281
Of the several meat authentication tools, only DNA based methods hold merit for the
282
detection of adulteration of meat products (Barakat, El-Garhy, & Moustafa, 2014). The protein based
283
techniques used previously could not be used for the discrimination of processed products as proteins
284
get denatured by the heat, salt, and pressures used during cooking (Dincer, Spearow, Cassens, &
285
Greaser, 1987). Further, phylogenetically closer species cannot be detected using the protein based
11
286
techniques as such techniques possess considerable cross reactions between the related species
287
(Dooley, Paine, Garrett, & Brown, 2004).
288
Species identification tools targeting both chromosomal and mitochondrial targets have been
289
reported. However, for the detection of adulteration in meat products the mitochondrial targets are
290
preferred over the nuclear targets since the mitochondrial DNA is relatively more resistant to
291
processing and remains undamaged as intact DNA in cooked or processed meat products (Dooley,
292
Paine, Garrett, & Brown, 2004). Further, mitochondrial DNA occurs in multiple copies (~1,000) per
293
cell and even if some majority of copies gets denatured, at least few copies may be left for the
294
diagnostic amplification of the target in processed meat samples (Koh, Kim, Na, Park, & Kim, 2011).
295
3.6 Applicability of AL-LAMP assay for the detection of meat adulteration with pork
296
The AL-LAMP assay was able to detect pork in the adulterated beef to the extent of 0.1% (Fig
297
1). The primary purpose of adulteration of meats with pork linked to the financial gains arising from
298
the price differences in the market (Aida, Che, Wong, Raha, & Son, 2005; Grundy et al., 2012).
299
However, pork adulterated in other species meat has religious and health implications (Rashood,
300
Shaaban, Moety, & Rauf, 1996) requiring prompt labeling and detection of adulterations using robust
301
laboratory tools. Of the several meat speciation tools viz., anatomical, protein based (enzyme assays
302
or electrophoretic), chromatographic, etc (Bottero and Dalmasso, 2011), the DNA based techniques
303
have been construed as best suited methods for the detection of pork due to higher stability of DNA
304
and its presence in each and every cell of an individual (Dooley, Paine, Garrett, & Brown, 2004). The
305
PCR based tools have emerged as the gold standard for the purpose of meat species identification;
306
consequently several PCR based techniques were developed for pork authentication (Tanabe et al.,
307
2007). Nevertheless, PCR requires costly thermocycler or post-PCR processing equipment. The
308
LAMP assay could be set-in using just a simple water bath or heat block (Tomita, Mori, Kanda, &
309
Notomi, 2008).
310
Of the several current alternatives to the PCR for the purpose of species authentication, the
311
LAMP assay appears to be the better option due to its rapid and sensitive detection of the even fewer 12
312
diagnostic target signal under isothermal conditions (Heers et al., 2017) and is well suited for even
313
laboratories lacking sophisticated infrastructure. Nevertheless, the major drawback of the AL-LAMP
314
like any other conventional PCR or variants of LAMP include its inability to quantify the target and
315
identify the extent of adulteration. The real time or qPCR (Köppel, Ruf, Zimmerli, & Breitenmoser,
316
2008), duplex droplet digital PCR (Cai et al., 2017) or real time LAMP (Yang et al., 2014) assays
317
measure extent of adulteration. However, under field conditions such circumstances rarely arise and
318
simple, rapid, easy, economic and on-site tools such as the LAMP described here hold promise.
319
4. Conclusions
320
The present work describes a novel AL-LAMP assay for the specific detection of pig species
321
(pork) in raw, cooked and adulterated meat samples. The 120 min rapid and economic assay involves
322
DNA extraction using alkaline lysis followed by mitochondrial D loop targeted LAMP assay and
323
SYBR Green I dye based visual color comparison for authentication of pork. The assay possessed
324
higher specificity and sensitivity (0.5 ng/µL porcine DNA), able to detect heated (121°C for 30 min)
325
as well as adulterated (0.1% pork in beef) meat and pork products. Owing to its higher specificity and
326
sensitivity, the AL-LAMP assay could be advocated for the purpose of authentication of pork even at
327
laboratories with minimal facilities.
328 329 330
Declaration of interest Authors declare no conflict of interest.
331 332
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18
1
A
Fig. 1. Visualization of LAMP amplification after addition of SYBR Green I dye In different species 1 2 3
4
5
6
7
1) Pig
2) Cattle
3) Buffalo
4) Sheep
5) Goat
6) Chicken
7) Negative control B
In pork heat treated for 30 minutes at different temperatures 1 2 3 4 5 1) 60º C 2) 80º C
3) 100º C
4) 121º C
5) Negative control
C
D
E
Sensitivity of LAMP assay at different DNA concentrations (ng/ µL) 1 2 3 4 5 6 7 8 9 10 11
12
13
14
1)100 2)75 3)50 4)40 5)30 6)25 7)20 8)10 9)5 10)4 11)3 12)2 13)1 14) Negative control In processed pork products 1 2 3 4 5 6 7 8 1) Kabab 2) Balls 3) Masala sausage
4) Salami
5) Breakfast sausage
6) Cocktail sausage
Detection of adulteration of beef with different levels of pork (pork in beef mixture) 1 2 3 4 5 6 7 8 9 10 11 12 13
1)50% 2)40% 3)30% 4)20% 5)15% 6)10% 7)5% 8)1% 9)0.8% 10)0.6% 11)0.4% 12)0.2% 13)0.1% 14)Negative control 2 3 4 5 6
1
14
7
Fig. 2. Visualization of LAMP amplification on 2 % agarose gel M P
8
C
B G S Ch NC
9 10 11 12
M:100 bp DNA ladder; P:Pig; C:Cattle; B:Buffalo; G:Goat; S:Sheep; Ch:Chicken; NC:Negative control
13
Fig. 3. Fluorometric values of LAMP assay obtained using SYBR Green I dye plotted (mean±
14
standard error indicated as bar) for different samples (dashed line indicate cutoff; mean+2x standard
15
deviation of non-pork samples) 160000
140000
120000
100000
80000
55085
60000
40000
20000
2
Negative Control
Pork square roll
Pork cocktail
Pork Salami
Pork masala sausage
Pork balls
Pork kabab
Pork breakfast sausage
16
121º C
100º C
80º C
60º C
Chicken
Mutton
Chevon
Buffalo meat
Beef
Pork
0
Highlights •
Manuscript reports a rapid technique for the species identification of pork
•
Technique uses Alkaline lysis method for the extraction of DNA
•
Target DNA was amplified using novel pork specific LAMP primers
•
Results can be visualized by colour changes after the addition of SYBR Green I dye
•
AL-LAMP assay has potential to be used at field level for pork authentication
S0956-7135(19)30604-8
DOI:
https://doi.org/10.1016/j.foodcont.2019.107015
Reference:
JFCO 107015
To appear in:
Food Control
Received Date: 29 May 2019 Revised Date:
21 November 2019
Accepted Date: 23 November 2019
Please cite this article as: Girish P.S., Barbuddhe S.B., Kumari A., Rawool D.B., Karabasanavar N.S., Muthukumar M. & Vaithiyanathan S., Rapid detection of pork using alkaline lysis- Loop Mediated Isothermal Amplification (AL-LAMP) technique, Food Control (2019), doi: https://doi.org/10.1016/ j.foodcont.2019.107015. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
Author Contribution
Conceived and designed the experiments: GPS, SBB, and SV. Performed the experiments: GPS, MM and AK. Analyzed the data: GPS, AK and NSK. Contributed reagents/materials/ analysis tools: GPS, MM, SBB and DBR. Wrote the manuscript: GPS and NSK. Critical evaluation of manuscript: GPS, SBB and SV.
1
Rapid detection of pork using Alkaline Lysis- Loop Mediated Isothermal Amplification (AL-
2
LAMP) technique
3
Girish, P.S.1*, S.B. Barbuddhe2, Aparana Kumari3, Deepak B. Rawool4, Nagappa S.
4
Karabasanavar5, M. Muthukumar6, S. Vaithiyanathan7
5
ICAR–National Research Centre on Meat
6
Chengicherla, Hyderabad, TelanagnaState-500092, India
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
*Corresponding author E mail: [email protected] 1. ICAR – National Research Centre on Meat, Chengicherla, Hyderabad, Telangana State - 500 092 India, Phone: +91-9401262522; E mail: [email protected] 2. ICAR – National Research Centre on Meat, Chengicherla, Hyderabad, Telangana State - 500 092 India, E mail: [email protected] 3. ICAR – National Research Centre on Meat, Chengicherla, Hyderabad, Telangana State - 500 092 India, E mail: [email protected] 4. ICAR – Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243 122 India,E-mail: [email protected] 5. Department of Veterinary Public Health & Epidemiology, Veterinary College, Gokula, Vidyanagar, Hassan, Karnataka–573 202, India E-mail: [email protected] 6. ICAR – National Research Centre on Meat, Chengicherla, Hyderabad, Telangana State - 500 092 India, E mail: [email protected] 7. ICAR – National Research Centre on Meat, Chengicherla, Hyderabad, Telangana State - 500 092 India, E mail:[email protected]
24 25 26 1
27
ABSTRACT
28
We report a novel, rapid, economical and species-specific DNA-based assay for the authentication
29
of pork. The technique specifically amplified porcine mitochondrial D loop region by combining
30
Alkaline Lysis (AL) method of DNA extraction and Loop Mediated Isothermal Amplification
31
(LAMP). Visual detection of the reaction was accomplished by color development in the reaction
32
with the addition of SYBR Green I dye. Dependable amplification was possible in thermally
33
processed meat samples heated up to 121 ºC for 30 min. The assay was able to detect pork in beef
34
up to the level of 0.1% admixture and limit of detection of DNA was at 0.5 ng/µL. Cross-
35
amplification of related species like cattle, buffalo, sheep, goat and chicken was excluded by
36
incorporating their DNA in the reaction assay. The novel approach (AL-LAMP technique) was
37
found to be robust and handy suitable even for resource compromised laboratories engaged in the
38
food analysis.
39 40
Keywords: Pork; Speciation; DNA; D loop; Alkaline lysis; LAMP; Adulteration
41 42 43 44 45 46 47 48 49 50 51 52 2
53
1. Introduction
54
Adulteration of meat with pig meat is considered as an offence owing to the religious and
55
health concerns (Lubis, Salihah, Hossain, & Ahmed, 2017). Pig meat and products being readily
56
available and cheap sources, are substituted into other species meats (Grundy et al., 2012). Scandals
57
associated with incorporation of pig meat into other species meats have drawn considerable
58
attention in the past (Abdullahi et al., 2017). In addition, risk of acquiring certain diseases and
59
development of allergy among sensitive individuals also poses threat to public health arising due to
60
the consumption of meats adulterated with pork or pig derived tissues. In order to comply with
61
halal authentication, protect consumers’ sentiments, promote fair-trade, implement prompt labeling
62
norms, avoid allergies and prevent disease transmission from pig or pork, there is a need to
63
correctly identify pig derived tissues (Aida, Che, Wong, Raha, & Son, 2005).
64
Origin of pig could be detected using conventional techniques viz., anatomical, histological,
65
chemical, electrophoretic or enzymatic assays (Ballin, Vogensen, & Karlsson, 2009). Nevertheless,
66
these conventional techniques have inherent limitations (lack of discriminating power
67
orrepeatability and cross-reactions); therefore, DNA based techniques particularly PCR-based tools
68
were developed, and conclusively emerged as methods of choice for the purpose of species
69
identification (Ballin, Vogensen, & Karlsson, 2009). The PCR based techniques used for pork
70
detection include sequence analysis of mitochondrial 12S rRNA gene (Girish et al., 2004), species
71
specific PCR targeting mitochondrial D loop region (Karabasanavar, Singh, Kumar, &
72
Shebannavar, 2014), PCR-Restriction Fragment Length Polymorphism of mitochondrial of
73
cytochrome B gene (Chandregowda, Fairoze, Girish, Karabasanavar, & Bagale, 2015), multiplex
74
PCR targeting cytochrome b gene (Ali et al., 2015), real time PCR targeting mitochondrial ND5
75
region (Kesmen, Gulluce, Sahin, & Yetim, 2009) and duplex PCR targeting mitochondrial
76
cytochrome b gene (Di Pinto, Forte, Conversano, & Tantillo, 2005). Nevertheless, the PCR based
77
detection of swine species requires costly instrumentation such as thermocycler, electrophoresis
78
unit and gel documentation system; also, traditional PCR based methods are time consuming, costly 3
79
and require samples to be carried to the laboratory for analysis. On the other hand, quantitative
80
qPCR and sensor based techniques (Lubis et al., 2017) require expensive instruments and
81
sophisticated laboratory setups (Ma, Dai, Fang, Wu, & Zhang, 2016).
82
Loop-mediated Isothermal Amplification (LAMP) is a nucleotide amplification technique
83
(Notomi et al., 2015) that amplifies target DNA at isothermal temperatures and obviates need for
84
thermal cyclersand post-amplification procedures of signal detection (Erwanto, Abidin, Rohman, &
85
Sismindari, 2011). As in PCR, LAMP assay could amplify target DNA several folds (109copies in a
86
span of an hour) under isothermal conditions without compromising specificity and having
87
capability of the visual detection of amplified targets using specific dyes (Yang et al., 2014).
88
Consequently, LAMP assay has emerged as an alternative tool to PCR based techniquesfor the
89
purpose of testing food safety hazards including detection of meat adulteration (Abdullahiet al.,
90
2017).
91
Several reports of application of LAMP assay for the detection of pork have been reported
92
which include development of LAMP assay for the visual detection of pork DNA in meat products
93
targeting the mitochondrial DN1 gene sequence (Ran et al., 2016), real-time LAMP assay targeting
94
the Cytb gene for the detection of pork meat in a meat mixture (Yang et al., 2014) and rapid on-site
95
real-time LAMP method for pork species identification in processed meat products by Lee, Kim,
96
Hong, & Kim (2016). Nevertheless, need for a robust, rapid and economic assay involving simple
97
sample preparation methods which can be used by even resource deficient laboratories lacking
98
sophisticated instruments was felt. Therefore, in the present work, a simple method for detection of
99
pork using alkaline lysis method of DNA extraction and LAMP assay (AL-LAMP) has been
100
described using newly designed set of primers targeting mitochondrial D loop region.
101
2. Experimental
102
2.1 Collection of samples
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Samples of beef, buffalo meat, mutton and chevon were obtained from Municipal
104
slaughterhouse (Chengicherla, Hyderabad, Telangana state, India). Samples of porkwere obtained 4
105
from the authentic retail shops located at Hyderabad, Telangana state, India. Samples of chicken meat
106
were obtained from experimental abattoir of ICAR – National Research Centre on Meat,
107
Chengicherla, Hyderabad, Telangana State, India. Meat samples were collected individually in sterile
108
containers, transported to the laboratory under refrigeration temperature and stored at -20ºC until
109
further analysis. Pork products viz., kabab, ball, masala sausage, salami, breakfast sausage,cocktail
110
sausage and square rolls were collected from specific food stalls of Hyderabad city.
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2.2 Extraction of DNA
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DNA from meat and meat products was extracted usingpreviously described Alkaline Lysis
113
(AL) method (Girish, Haunshi, Vaithiyanathan, Rajitha, & Ramakrishna, 2013). Briefly, one part of
114
sample (500 mg) was triturated with eight volumes (4 mL) of NaOH solution (0.2 mol/L); resultant
115
extract (5 µL) was again mixed with eight volumes (40 µL) of NaOH solution (0.2 mol/L) and heated
116
at 75ºC in dry bath for 20 min. After neutralization of the mixturewith eight volumes (360 µL) of
117
Tris-HCl (0.04 mol/L, pH 7) the resultant DNA was used for loop-mediated isothermal amplification
118
(LAMP) reaction.
119
2.3 Designing of primers for the LAMP assay
120
Two sets of primers (F3/B3, FIP/BIP) were designed for the LAMP assay targeting
121
mitochondrial D loop region using Primer Explorer V5 software; the 5′ 3′ primer sequences were F3
122
– AGG CCC TAA CAC AGT CAA, B3 – GTT ATA GGG TGT GTA GAG CAT A, FIP - ACT
123
GAA TAG CAC CTT GTT TGG ATT TGT AGC TGG ACT TCA TGG and BIP - CGG GAC ATA
124
ACG TGC GTA CAA GTT TAA TGG GGG GTA AGG.
125
2.4 LAMP reaction mixture preparation
126
The LAMP reaction mix consisted of 10X Thermopol buffer (3 µL), 5 M Betaine (3 µL), 50
127
mM MgSo4 (3 µL), 10 mMdNTP mix (4 µL), 10 pmol F3 primer (1 µL),10pmol B3 primer (1 µL), 40
128
pmol FIP primer (4 µL), 40 pmol BIP primer (4 µL) and template DNA (5 µL). The reaction mix was
129
heated at 95ºC for 5 min for denaturation; thereafter, 8 units (1 µL) of BstI enzyme was added and
130
incubated at 65ºC for 1 h followed by heating at 80ºC for 2 min for the inactivation of BstI enzyme. 5
131
After the LAMP reaction, 1 µL of SYBR green I dye (1:10, 10,000X) was added for the visualization
132
of amplification. Positive reaction was indicated by the green colour; whereas, orange color was
133
indicative of the negative reaction. Amplification was confirmed by agarose gel electrophoresis (2%)
134
usingethidium bromide staining.
135
2.5 Validation of AL – LAMP technique in meat admixtures
136
For the detection of pork in beef using AL-LAMP assay, different proportions of pork in beef
137
i.e. 50:50, 40:60, 30:70, 20:80, 10:90, 5:95; 1:90; 0.8:90.2; 0.6:90.4; 0.4:90.6; 0.2; 90.8 and 0.1: 90.9
138
were prepared. Admixture was prepared in total quantity of 500 mg and was used for extraction of
139
DNA by alkaline lysis method. After thorough mixing, meat mixtures were subjected for DNA
140
extraction using AL method.
141
2.6 Validation of AL – LAMP technique in heat treated meat
142
Pork samples were heated at 60ºC, 80ºC, 100ºC and 121ºC for 30 min in a dry bath.
143
Thereafter, DNA was extracted from thermally processed pork samples using alkaline lysis method as
144
described by Girish, Haunshi, Vaithiyanathan, Rajitha, & Ramakrishna (2013).
145
2.7 Sensitivity of the LAMP PCR
146
Sensitivity of developed LAMP assay was assessed by diluting the template pork DNA so as
147
to get concentrations viz., 100, 75, 50, 40, 30, 25, 20, 10 and 5 ng/µL followed by the LAMP assay
148
using the novel set of primers designed.
149
2.8 Fluorometric assay
150
Intensity of colour developed after the addition of SYBR Green I dye to the final reaction mix
151
of LAMP product was analyzed in using a fluorometer (Denovix, Model: DS-11FX) at 565–650 nm
152
fluorescence. The final reaction mix of volume 30 µL was diluted with nuclease free water to 200 µL
153
volume and the resultant diluted mix was used for recording fluorescence.
154 155
2.9 Statistical analysis
6
156
Average fluorometric readings of AL-LAMP results of non-pig species (cattle, buffalo, goat,
157
sheep and chicken) were considered for calculating the cut-off values for pig as described by Goto,
158
Honda, Ogura, Nomoto, & Hanaki (2009). Average fluorometric value of non-pig species was added
159
with two standard deviations to calculate the cut-off value of the signal for pig.
160
3. Results and discussion
161
3.1 Quality and purity of DNA extracted using Alkaline Lysis(AL) method
162
The DNA extracted from fresh and heat treated raw meat or meat products using the AL
163
method yielded good quality DNA suitable for LAMP reaction. Average concentrations of DNA
164
after extraction by AL method in fresh pork and pork heated at 60°C, 80°C, 100°C and 120°C were
165
71.11, 139.24, 89.71, 80.31 and 59.94 ng/µL, respectively. Average concentration of DNA
166
extracted from different processed pork products obtained using AL method was 83.29 ng/µL with
167
OD260:280values in the range of 1.6 to 1.9. Commonly employed methods for extraction of DNA
168
from meat are: Phenol: Chloroform extraction (PC) method and commercial kit based methods
169
(Girish, Haunshi, Vaithiyanathan, Rajitha, & Ramakrishna, 2013). The PC method of DNA
170
extraction from meat (Chikuni, Tabata, Kosugiyama, & Monma, 1994) involves lysis of cells using
171
proteinase K followed by repeated extraction steps by centrifugation using Phenol, Choloroform
172
and Isoamyl alcohol followed by precipitation using ethanol and ammonium acetate. Although pure
173
DNA is obtained by this method, toxicity of phenol and labor-intensity is a drawback. Moreover,
174
the presence of phenol minimizes the quantitation of DNA detected by UV absorbance since phenol
175
shows high extinction coefficient at 260 nm (Javadi et al., 2014). Kit based methods are effective
176
but are costly and involve multiple centrifugation steps. As compared to these routinely used
177
methods, AL method of DNA extraction is simple, involves simple laboratory chemicals, quick and
178
do not require any expensive equipments. Moreover, AL is a single tube technique which reduces
179
the chances of contamination during extraction process. Girish, Haunshi, Vaithiyanathan, Rajitha,
180
& Ramakrishna (2013) also reported alkaline lysis method for extraction of DNA from buffalo meat
181
which was used successfully for species identification by species specific PCR in fresh and 7
182
processed meat products. Ali, Rampazzo, Costa, & Krieger (2017) also reported that Alkaline lysis
183
based methods are fastest, most reliable and relatively easy way to obtain DNA from cells and are
184
suitable for less sensitive applications.
185
3.2 SYBR Green I based visual pork specific AL-LAMP assay
186
Products of LAMP amplification were mixed with SYBR green I dye and resultant signals
187
were measured fluorometrically (Fig. 3). Fluorometric readings were significantly higher for raw,
188
cooked and processed pork samples compared to other species and negative controls as also reported
189
by Goto, Honda, Ogura, Nomoto, & Hanaki (2009). In addition to the visual color comparison,
190
fluorometry convincingly proved the results of AL-LAMP assay for the purpose of authentication of
191
the pork (Fig 1-3).
192
The LAMP assay developed by Notomi and coworker amplified pig specific mitochondrial D
193
loop target DNA based on autocycling strand displacement DNA synthesis principle using Bacillus
194
stearothermophilus (Bst) DNA polymerase (Notomi et al., 2015). Set of four primers bind to unique
195
sites on the target sequence. LAMP technique amplifies target DNA under isothermal conditions (63
196
to 65 °C) with detection limits comparable to the PCR, eliminating need for the expensive thermal
197
cyclers (Yang et al., 2014). After the isothermal amplification, SYBR Green I dye was added to the
198
reaction tubeand color green was visualized to indicate LAMP amplification and absence of
199
amplification was indicated by orange color (Iwamoto, Sonobe, & Hayashi, 2003). The entire
200
procedure (sample to result) of DNA extraction by alkaline lysis method and LAMP assay took 120
201
minutes. In addition to the PCR, similar DNA based techniques such as FINS (forensically
202
informative nucleotide sequencing), restriction fragment length polymorphism (RFLP) and variants
203
take longer duration for analysis. Also, highly sensitive quantitative techniques such as real time PCR
204
require costly instrumentation and consumables for achieving the same objectives.
205
Variants of LAMP assay such as its coupling with other DNA based techniques has been
206
reported for the purpose of species identifications; Kim and Shin (2017) developed isothermal probe
207
amplification (ITPA) assay targeting mitochondrial 16S rRNA gene using four sets of primers (two 8
208
outer and two inner primers) and fluorescence resonance energy transfer (FRET) that required a
209
heating block and a fluorescence reader. Yang et al. (2014) developed a 45 minutes one-step, SYBR
210
Green I real-time, loop-mediated isothermal amplification (RealAmp) assay targeting mtDNA of
211
cytochrome b gene for the specific detection of pork; however, such RealAmp assay required costly
212
Real-Time PCR System.
213
Detection of LAMP signal was undertaken either by gel DNA electrophoresis, visualization of
214
the color of amplified DNA by using DNA staining dyes (SYBR green I), visualization of DNA
215
precipitation of chemicals such as magnesium pyrophosphate (MPP) or visualization of turbidity
216
using MPP (Notomi, Mori, Tomita, & Kanda, 2015). Of these techniques, observation of the
217
amplified LAMP signal by naked eye was the most appreciated method (Lee,Su, Lein, & Sheu,
218
2017). The visual SYBR Green I dye color detection system used in AL-LAMP assay was an easy
219
way of detection of amplification signal. The AL-LAMP assay does not require costly equipment
220
such as real-time PCR system, fluorescence reader, etc as specific amplified signal is visualized by
221
the naked eye. Further, the AL-LAMP assay does not require cumbersome DNA extraction thereby
222
making the authentication process easy and economical. Time taken for DNA extraction using
223
alkaline lysis method was 40 min and another 80 min was spent for the LAMP assay. For running a
224
sample at a laboratory having minimum facilities, a maximum of 120 min is required for pork
225
authentication using the novel AL-LAMP assay.
226
PCR detection of pig species has been previously accomplished by using FINS targeting
227
mitochondrial 12S rRNA gene (Girish et al., 2004; Chandregowda, Fairoze, Girish, Karabasanavar, &
228
Bagale, 2015); species specific PCR targeting mitochondrial D loop (Karabasanavar, Singh, Kumar,
229
& Shebannavar, 2014) or porcine specific repetitive DNA element (Calvo, Zaragoza, and Osta,
230
2001); duplex PCR (Di Pinto, Forte, Conversano, & Tantillo, 2005); multiplex PCR targeting
231
cytochrome b gene (Ali et al., 2015); DNA barcode (Di Pinto et al., 2013); real time PCR (Kesmen,
232
Gulluce, Sahin, &Yetim, 2009; Lubis, Salihah, Hossain, & Ahmed, 2017) and others. Nevertheless,
233
all these assays require DNA extraction, costly PCR and post PCR processing like costly sequencing 9
234
or electrophoresis. On the other hand, the PCR and restriction fragment length polymorphism (PCR-
235
RFLP) approach used for pork detection targeting mitochondrial D loop sequences such as
236
cytochrome b gene (Ali et al., 2011) also require PCR amplification, restriction digestion of
237
amplicons and electrophoresis making the technique cumbersome with higher false negative reactions
238
(failures in restriction digestions).
239
The PCR based techniques developed for pig species (pork) identification such as qualitative
240
forensically informative nucleotide sequencing (FINS), species specific PCR, multiplex PCR or
241
quantitative real time PCRs require longer reaction times compared to the LAMP. The LAMP does
242
not require denaturation step as in the conventional PCR thereby reducing its reaction time. Also,
243
LAMP primers have higher efficacy than the PCR primers (Chen, Ma, Qiang, & Ma, 2016).
244 245
3.3 Specificity and sensitivity of pork specific AL-LAMP assay
246
Results of AL-LAMP assay were detected by visual comparison of the SYBR Green I dye
247
color; development of green color was indicative of positive reaction and orange color indicated the
248
negative reaction. Specificity of AL-LAMP assay was tested using DNA extracted from closely
249
related species viz., cattle, buffalo, sheep, goat, pig and chicken (Fig1). Pig specific positive
250
amplification signal was demonstrated by the green colorand it was seen only in the pork sample.
251
None of the other related species tested showed any amplification signal; results indicated higher
252
specificity of the designed primers for pig species using the AL-LAMP assay.
253
Sensitivity of AL-LAMP was assessed by diluting the DNA; the lowest concentration of DNA
254
that gave a detectable pork signal was taken as sensitivity. The sensitivity of AL-LAMP assay was
255
0.5 ng/µL (Fig 1). In admixed meat (pork in beef) samples, the AL-LAMP detected 0.1% level
256
adulteration of pork in beef. The LAMP assay developed by Abdullahi et al. (2017) for porcine
257
tRNAlys and ATPase 8 genes detected 0.03 femtograms of pig DNA. Likewise, onsite smart and
258
sealed biosensor based LAMP assay coupled to lateral flow device (LFD) (Xu et al., 2017) detected
259
10 picograms (3~5 copies) of chromosomal gene target. Higher sensitivity of LAMP assay than
260
conventional PCR helps in the detection of target DNA even in those samples supposed to have low 10
261
copies of the target DNA (Erwanto, Abidin, Rohman, & Sismindari, 2011). Higher limits of detection
262
of the AL-LAMP assay indicate its potential applications at the field level for the purpose of
263
authentication of the pork adulterated samples.
264
Specificity and sensitivity of LAMP assay was higher than conventional PCR, this could be
265
attributed to LAMP primers that could detect even a single nucleotide difference and formation of
266
loop-medicated secondary DNA structure (Fukuta, Mizukami, Ishida, & Kanbe, 2006). Lower
267
template was required for LAMP assay than the conventional PCR due to severe deconstruction of
268
the DNA (Lee, Su, Lein, & Sheu, 2017).
269
3.4 Validation of AL-LAMP assay for heated/ thermally processed pork
270
Dependable amplification signal (color change to green) was observed in four types of heat
271
treatments employed viz.,60ºC, 80ºC, 100ºC and 121ºC for 30 min (Fig 1) indicating suitability of the
272
AL-LAMP assay for the detection of pork even in thermally processed samplesheated up to 121°C for
273
30 min. Such detection was without any decline in the color intensity; however, weakening of the
274
amplification signal was observed using conventional PCR heated at 120°C for 30 min (Girish et al.,
275
2004).
276
3.5Validation of AL-LAMP assay for pork products
277
Positive amplification signal marked by the color change (green) using SYBR Green I dye
278
was observed in different pork products viz., kabab, balls, sausage, salami, breakfast sausage, cocktail
279
sausage and square rolls (Fig 1). Different processing conditions and use of meat ingredients in the
280
preparation did not affect the amplification signal or color change.
281
Of the several meat authentication tools, only DNA based methods hold merit for the
282
detection of adulteration of meat products (Barakat, El-Garhy, & Moustafa, 2014). The protein based
283
techniques used previously could not be used for the discrimination of processed products as proteins
284
get denatured by the heat, salt, and pressures used during cooking (Dincer, Spearow, Cassens, &
285
Greaser, 1987). Further, phylogenetically closer species cannot be detected using the protein based
11
286
techniques as such techniques possess considerable cross reactions between the related species
287
(Dooley, Paine, Garrett, & Brown, 2004).
288
Species identification tools targeting both chromosomal and mitochondrial targets have been
289
reported. However, for the detection of adulteration in meat products the mitochondrial targets are
290
preferred over the nuclear targets since the mitochondrial DNA is relatively more resistant to
291
processing and remains undamaged as intact DNA in cooked or processed meat products (Dooley,
292
Paine, Garrett, & Brown, 2004). Further, mitochondrial DNA occurs in multiple copies (~1,000) per
293
cell and even if some majority of copies gets denatured, at least few copies may be left for the
294
diagnostic amplification of the target in processed meat samples (Koh, Kim, Na, Park, & Kim, 2011).
295
3.6 Applicability of AL-LAMP assay for the detection of meat adulteration with pork
296
The AL-LAMP assay was able to detect pork in the adulterated beef to the extent of 0.1% (Fig
297
1). The primary purpose of adulteration of meats with pork linked to the financial gains arising from
298
the price differences in the market (Aida, Che, Wong, Raha, & Son, 2005; Grundy et al., 2012).
299
However, pork adulterated in other species meat has religious and health implications (Rashood,
300
Shaaban, Moety, & Rauf, 1996) requiring prompt labeling and detection of adulterations using robust
301
laboratory tools. Of the several meat speciation tools viz., anatomical, protein based (enzyme assays
302
or electrophoretic), chromatographic, etc (Bottero and Dalmasso, 2011), the DNA based techniques
303
have been construed as best suited methods for the detection of pork due to higher stability of DNA
304
and its presence in each and every cell of an individual (Dooley, Paine, Garrett, & Brown, 2004). The
305
PCR based tools have emerged as the gold standard for the purpose of meat species identification;
306
consequently several PCR based techniques were developed for pork authentication (Tanabe et al.,
307
2007). Nevertheless, PCR requires costly thermocycler or post-PCR processing equipment. The
308
LAMP assay could be set-in using just a simple water bath or heat block (Tomita, Mori, Kanda, &
309
Notomi, 2008).
310
Of the several current alternatives to the PCR for the purpose of species authentication, the
311
LAMP assay appears to be the better option due to its rapid and sensitive detection of the even fewer 12
312
diagnostic target signal under isothermal conditions (Heers et al., 2017) and is well suited for even
313
laboratories lacking sophisticated infrastructure. Nevertheless, the major drawback of the AL-LAMP
314
like any other conventional PCR or variants of LAMP include its inability to quantify the target and
315
identify the extent of adulteration. The real time or qPCR (Köppel, Ruf, Zimmerli, & Breitenmoser,
316
2008), duplex droplet digital PCR (Cai et al., 2017) or real time LAMP (Yang et al., 2014) assays
317
measure extent of adulteration. However, under field conditions such circumstances rarely arise and
318
simple, rapid, easy, economic and on-site tools such as the LAMP described here hold promise.
319
4. Conclusions
320
The present work describes a novel AL-LAMP assay for the specific detection of pig species
321
(pork) in raw, cooked and adulterated meat samples. The 120 min rapid and economic assay involves
322
DNA extraction using alkaline lysis followed by mitochondrial D loop targeted LAMP assay and
323
SYBR Green I dye based visual color comparison for authentication of pork. The assay possessed
324
higher specificity and sensitivity (0.5 ng/µL porcine DNA), able to detect heated (121°C for 30 min)
325
as well as adulterated (0.1% pork in beef) meat and pork products. Owing to its higher specificity and
326
sensitivity, the AL-LAMP assay could be advocated for the purpose of authentication of pork even at
327
laboratories with minimal facilities.
328 329 330
Declaration of interest Authors declare no conflict of interest.
331 332
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18
1
A
Fig. 1. Visualization of LAMP amplification after addition of SYBR Green I dye In different species 1 2 3
4
5
6
7
1) Pig
2) Cattle
3) Buffalo
4) Sheep
5) Goat
6) Chicken
7) Negative control B
In pork heat treated for 30 minutes at different temperatures 1 2 3 4 5 1) 60º C 2) 80º C
3) 100º C
4) 121º C
5) Negative control
C
D
E
Sensitivity of LAMP assay at different DNA concentrations (ng/ µL) 1 2 3 4 5 6 7 8 9 10 11
12
13
14
1)100 2)75 3)50 4)40 5)30 6)25 7)20 8)10 9)5 10)4 11)3 12)2 13)1 14) Negative control In processed pork products 1 2 3 4 5 6 7 8 1) Kabab 2) Balls 3) Masala sausage
4) Salami
5) Breakfast sausage
6) Cocktail sausage
Detection of adulteration of beef with different levels of pork (pork in beef mixture) 1 2 3 4 5 6 7 8 9 10 11 12 13
1)50% 2)40% 3)30% 4)20% 5)15% 6)10% 7)5% 8)1% 9)0.8% 10)0.6% 11)0.4% 12)0.2% 13)0.1% 14)Negative control 2 3 4 5 6
1
14
7
Fig. 2. Visualization of LAMP amplification on 2 % agarose gel M P
8
C
B G S Ch NC
9 10 11 12
M:100 bp DNA ladder; P:Pig; C:Cattle; B:Buffalo; G:Goat; S:Sheep; Ch:Chicken; NC:Negative control
13
Fig. 3. Fluorometric values of LAMP assay obtained using SYBR Green I dye plotted (mean±
14
standard error indicated as bar) for different samples (dashed line indicate cutoff; mean+2x standard
15
deviation of non-pork samples) 160000
140000
120000
100000
80000
55085
60000
40000
20000
2
Negative Control
Pork square roll
Pork cocktail
Pork Salami
Pork masala sausage
Pork balls
Pork kabab
Pork breakfast sausage
16
121º C
100º C
80º C
60º C
Chicken
Mutton
Chevon
Buffalo meat
Beef
Pork
0
Highlights •
Manuscript reports a rapid technique for the species identification of pork
•
Technique uses Alkaline lysis method for the extraction of DNA
•
Target DNA was amplified using novel pork specific LAMP primers
•
Results can be visualized by colour changes after the addition of SYBR Green I dye
•
AL-LAMP assay has potential to be used at field level for pork authentication