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Development of duplex fluorescence-based loop-mediated isothermal amplification assay for detection of Mycoplasma bovis and bovine herpes virus 1
Accepted Manuscript Title: Development of Duplex Fluorescence-based Loop-mediated Isothermal Amplification Assay for Detection of Mycoplasma bovis and Bovine Herpes Virus 1 Authors: Qing Fan, Zhixun Xie, Zhiqin Xie, Liji Xie, Jiaoling Huang, Yanfan Zhang, Tingting Zeng, Minxiu Zhang, Sheng Wan, Sisi Luo, Jiabo Liu, Xianwen Deng PII: DOI: Reference:
S0166-0934(18)30170-8 https://doi.org/10.1016/j.jviromet.2018.08.014 VIRMET 13525
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
2-4-2018 3-7-2018 20-8-2018
Please cite this article as: Fan Q, Xie Z, Xie Z, Xie L, Huang J, Zhang Y, Zeng T, Zhang M, Wan S, Luo S, Liu J, Deng X, Development of Duplex Fluorescence-based Loop-mediated Isothermal Amplification Assay for Detection of Mycoplasma bovis and Bovine Herpes Virus 1, Journal of Virological Methods (2018), https://doi.org/10.1016/j.jviromet.2018.08.014 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.
Development of Duplex Fluorescence-based Loop-mediated Isothermal Amplification Assay for Detection of Mycoplasma bovis and Bovine Herpes Virus 1
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Qing Fan, Zhixun Xie*, Zhiqin Xie, Liji Xie, Jiaoling Huang, Yanfan Zhang, Tingting Zeng, Minxiu Zhang, Sheng Wan, Sisi Luo, Jiabo Liu, Xianwen Deng
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Guangxi Veterinary Research Institute, Guangxi Key Laboratory of Veterinary Biotechnology, Nanning 530001, P.R. China
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Qing Fan: [email protected]
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Zhiqin Xie: [email protected]
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Zhixun Xie:[email protected]
Liji Xie: [email protected]
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Jiaoling Huang: [email protected]
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Yanfang Zhang: [email protected]
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Tingting Zeng: [email protected] Minxiu Zhang: [email protected]
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Sheng Wan: [email protected] Sisi Luo: [email protected] Jiabo Liu: [email protected] Xianwen Deng: [email protected] *Corresponding author: [email protected] 1
Highlights
A DLAMP method was developed for simultaneous detection of MB and BHV-1 and applied to clinical samples
The DLAMP results were visualized by the colors of fluorescent-dye-conjugated DLAMP products
The DLAMP assay shows high specificity and sensitivity
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Abstract Mycoplasma bovis (MB) and bovine herpes virus 1 (BHV-1) are two important
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pathogens that cause bovine respiratory disease in the beef feedlot and dairy
industries. The aim of this study was to develop and validate a duplex fluorescence-
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based loop-mediated isothermal amplification (DLAMP) assay for simultaneous
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detection of MB and BHV-1. Two sets of specific primers for each pathogen were
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designed to target the unique sequences of the MB uvrC gene and the BHV-1 gB
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gene. The inner primer for BHV-1 was synthesized with the fluorophore FAM at the 5’ end to detect the BHV-1 gB gene, and the inner primer for MB was synthesized
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with the fluorophore CY5 at the 5’ end to detect the MB uvrC gene. The DLAMP
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reaction conditions were optimized for rapid and specific detection of MB and BHV1. The DLAMP assay developed here could specifically detect MB and BHV-1
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without cross-reaction with other known non-target bovine pathogens. The sensitivity of this DLAMP assay was as low as 2×102 copies for recombinant plasmids
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containing the MB and BHV-1 target genes. In a detection test of 125 clinical samples, the positive rates for MB, BHV-1 and co-infection were 44.8%, 13.6% and 1.6%, respectively. Furthermore, the sensitivity and specificity of DLAMP were determined as 95%-96.6% and 100%, respectively, of those of field sample detection by the real-time polymerase chain reaction (PCR) assay recommended by the World 2
Organisation for Animal Health. Overall, DLAMP provides a rapid, sensitive and specific assay for the identification of MB and BHV-1 in clinical specimens and for epidemiological surveillance.
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Abbreviations
MB: Mycoplasma bovis
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BHV-1: bovine herpes virus 1
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BRD: bovine respiratory disease
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OIE: World Organisation for Animal Health
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DLAMP: duplex fluorescence-based loop-mediated isothermal amplification
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IACUC: Institutional Animal Care and Use Committee
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GVRI: Guangxi Veterinary Research Institution
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YNCIQ: Yunnan Entry-Exit Inspection and Quarantine Bureau
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CVCC: Chinese Veterinary Culture Collection Centre SRSV: bovine respiratory syncytial virus
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BVDV: bovine virus diarrhoea virus BPI3: bovine parainfluenza type 3 FMDV: foot-and-mouth disease virus VSV: vesicular stomatitis virus 3
RPV: rinderpest virus BRV: bovine rotavirus BTV: bluetongue virus
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PPRV: peste des petits ruminants virus
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Key words: MB; BHV-1; DLAMP; Fluorescent detection
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ETEC: Escherichia coli
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1. Introduction
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Bovine respiratory disease (BRD) is one of the major diseases affecting cattle
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health and production, and it causes immense economic losses. Its aetiology is multifactorial; many infectious agents are important in the development of BRD
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including viruses, bacteria and mycoplasmas. Mycoplasma bovis (MB) and bovine
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herpes virus 1 (BHV-1) are two important pathogens of BRD in the cattle industry (Jones and Chowdhury, 2007). Infection with these two pathogens can lead to reduced production, higher levels of morbidity and mortality, and increased veterinary labour
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costs, as well as economic losses. Infections with MB and BHV-1 are related to the occurrence of high fever, pneumonia and polyarthritis, rhinitis, conjunctivitis, genital tract infection, and reproductive problems such as abortion and reduced fertility in cattle (Cernicchiaro et al., 2013; Tomas JD, 2009). MB and BHV-1 are distributed 4
worldwide. Stress and environmental factors such as weaning, temperature, dust, stocking density, humidity, inadequate nutrition and transportation are important factors in the development of MB outbreaks. Furthermore, MB is difficult to remove thoroughly from an infected farm after an outbreak, and infected cattle can carry the
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pathogens for months and even years, remaining a source of infection (Burki et al., 2015; Maunsell et al., 2011). Similar to most other herpesviruses, inapparent infection
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is common in BHV-1 carriers. After BHV-1 infection, the virus can enter neural cells
and establish a latent infection in sensory ganglia. Latent infections also occur in non-
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neural sites, such as the tonsils and lymph nodes (Jones and Chowdhury, 2007). These
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latent pathogens can be reactivated both by stressful conditions and by administration
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of glucocorticoids. BHV-1-infected bulls may shed virus intermittently in the semen
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long after the primary infection (Graham, 2013; Nandi et al., 2009). Irrespective of
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the infectious agent involved, clinical manifestations of BRD may present similarly. Moreover, detection of a bacterial pathogen can mask an underlying viral cause, as
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both bacteria and viruses can co-infect their natural hosts (Caswell et al., 2010). A
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survey of exposure to BHV-1 in Canadian feedlots suggested that respiratory disease caused by MB was associated with previous exposure to BHV-1 (Prysliak et al., 2011). Cattle co-infected with BHV-1 and MB are regarded as lifelong carriers and
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potential shedders of these pathogens, and such infected cattle pose a potential risk to herds in the cattle industry. Therefore, differential diagnosis of MB and BHV-1 infections may lead to a better understanding of the epidemiology and natural distribution of MB and BHV-1 infections in the field, which may provide useful 5
information for the control of BRD. PCR, a highly sensitive and specific test, is considered a routine diagnostic test for MB and BHV-1 infections (Bashiruddin et al., 2005; Moore et al., 2000). LAMP is an alternative molecular genetic method derived from PCR for carrying out
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reactions under isothermal conditions using a single enzyme. LAMP-amplified products can be easily visualized by the naked eye. Compared with the two primers of
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conventional PCR, LAMP has four primers that can recognize 6 locations in the target gene; therefore, the LAMP method has higher reaction specificity than that of a
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conventional PCR assay. LAMP is 102~105 times more sensitive than conventional
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PCR (Ashraf et al., ; Notomi et al., 2000; Xie et al., 2014).
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The aim of the present study was to develop and validate a DLAMP assay for
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simultaneous detection of MB and BHV-1 in a single reaction. The DLAMP assay
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was compared to the World Organisation for Animal Health (OIE) recommended real-time PCR assay to assess its application in routine diagnosis of the aetiological
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agents involved in BRD.
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2. Materials and Methods 2.1. Ethical statement This study was approved by the Institutional Animal Care and Use Committee
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(IACUC) of Guangxi Veterinary Research Institution (GVRI). Sample collection was conducted based on protocol #2016C103 issued by the IACUC of GVRI. All the samples were collected from live cattle by well-trained veterinarians on approved farms. 6
2.2. Pathogens and DNA/RNA extraction The pathogens used in this study are listed in Table 1. Ten nasal swabs, 11 whole blood samples, and 21 semen specimens were collected from healthy cattle, and the extracted DNA samples were used as negative controls. The DNA of mycoplasmas,
DNA Extraction Kit version 5.0 (Takara, Dalian, China) according to the
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bacteria and DNA viruses was extracted by using the MiniBEST Universal Genomic
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manufacturer’s instructions. The RNA of RNA viruses was extracted by using the Universal RNA Extraction Kit (Takara) and subsequently reverse-transcribed to
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cDNA by using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). DNA/cDNA
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2.3. Primer design
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template for the DLAMP reaction.
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was stored at -20°C until use. Finally, 2 μl of each DNA/cDNA solution was used as a
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The selected genes, uvrC of MB and gB of BHV-1, are highly conserved and were
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used to design primers to identify these specific pathogens (Clothier et al., 2010; Thomas et al., 2004; Wang et al., 2007). Published sequences of the MB uvrC gene
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and BHV-1 gB gene were downloaded from GenBank and aligned using MegAlign 7.0 software (DNAStar, USA). Primer selection was supported by the software Primer
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Premier Version 5.0 (Premier Biosoft International, Canada) according to the restricted design rules for LAMP primers. The DLAMP assay included two sets of each of the four specific primers: two outer primers (F3, B3) and two inner primers (FIP = F1c + F2, BIP = B1c + B2). The inner primer FIP was synthesized with a fluorophore at the 5’ end. The BHV-1-FIP primer, labelled with 6-carboxyfluorescein 7
(FAM), showed a green colour at an emission wavelength of 520 nm, whereas the MB-FIP primer, labelled with Cyanine 5 (CY5), showed a red colour at an emission wavelength of 694 nm. A BLAST search program on the GenBank website was used to verify oligonucleotide specificity. All primers were synthesized and HPLC-purified
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2.4. DLAMP assay for simultaneous MB and BHV-1 detection
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by Takara Inc. (Takara). The sequences of the primers are shown in Table 2 and Fig.
The DLAMP reaction was performed by using the DNA Amplification Kit
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(Loopamp, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, 2.5 μl
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10× reaction buffer (200 mM Tris–HCl (pH 8.8), 100 mM KCl, 80 mM MgSO4, 100
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mM (NH4)2SO4, 1% Tween 20, 8 M betaine, and 14 mM dNTPs), 2 μl of extracted
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DNA/cDNA template, 5 pmol F3 and B3 primers, 40 pmol FIP and BIP primers, and
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15 units of Bst DNA polymerase (3.0 version) were mixed, and ddH2O was added to reach a final 25 μl reaction volume. The reaction mixture was incubated at 62°C for 60
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min using a Loopamp real-time turbidimeter (LA-320; Eiken Chemical Co., Ltd.,
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Tokyo, Japan), and the reaction was terminated by incubation at 80°C for 5 min. 2.5. Analysis of DLAMP products
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The DLAMP products were analysed by two methods. The first method was to
visually inspect the turbidity of the samples. DNA amplification generated a large amount of white magnesium pyrophosphate precipitate, a byproduct of the DLAMP reaction, which caused turbidity. The turbidity was monitored by the naked eye or by a Loopamp real-time turbidimeter. This method could only identify infected samples; 8
it was unable to differentiate MB from BHV-1. It can be applied in rural areas. The second method was to run agarose gel electrophoresis. A volume of 5 μl of each DLAMP product was subjected to electrophoresis in a 2% agarose gel at 100 V for 60 min, and the fluorescent-dye-conjugated DLAMP products were examined with an
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image analyser (Universal Hood III, 731BR01622, Bio-Rad, USA) to display different colours of electrophoretic bands. Green bands were considered BHV-1,
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while red bands were considered MB. Since DLAMP products are mixtures of DNA fragments with various lengths, several ladder-like stripes are usually shown on an
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agarose gel after electrophoresis. No DNA marker is necessary for a DLAMP assay.
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2.6. Preparation of standards
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PCR amplicons containing the full-length sequence of each DLAMP target gene
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were cloned into the PMD-18T vector (Takara) separately by using the standard
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procedure, generating the recombinant plasmids PMD-18T-MB and PMD-18T-BHV1. The concentrations of the recombinant plasmids were measured at 260 nm by using a
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NanoDrop 2000 (ThermoFisher Scientific, Waltham, USA). The copy numbers of the
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target DNA were calculated based on the following formula: copies/μl = [concentration of plasmid (ng/μl) × 6.02×1014 / length of plasmid × 660](Xie et al., 2014). Serial 10-
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fold dilutions of each target DNA, ranging from 1×108 copies/μl to 1 copy/μl, were stored at -20°C until use. 2.7. Detection of clinical samples The present study was conducted on a total of 125 swab samples consisting of 70 nasal swabs and 55 vaginal swabs, which were collected from the same dairy farm in 9
Guangxi, southern China, during 2015 to 2017, where recurrent problems with MB infection were reported (Ma C et al., 2015). The swab samples were collected from cows displaying typical respiratory-system-related lesions. Following clinical examination, which included assessment of body temperature and respiratory
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function, the swab samples were eluted in 3.0 ml of PBS. The eluent was centrifuged at 5000 rpm for 5 min at 4°C, and 250 μl supernatant was subjected to DNA
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extraction using the MiniBEST Universal Genomic DNA Extraction Kit version 5.0
(Takara) according to the manufacturer’s instructions. Finally, 2 μl of extracted DNA
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was used as a template for the DLAMP test described above in section 2.4. The
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samples were also tested for MB and BHV-1 infection by using the single real-time
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PCR assays recommended by OIE and conventional PCR as references (Bashiruddin
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et al., 2005; Moore et al., 2000; Wang et al., 2008).
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3. Results
3.1 Specificity of DLAMP
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All five BHV-1 strains and seven MB strains tested positive by DLAMP and showed
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green bands and red bands, respectively, on agarose gels. DLAMP amplification of other common bovine pathogens did not show non-specific products or any inter-assay
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cross-amplification. Only pathogen-specific targets were amplified by DLAMP (Table 1, Fig. 2). To validate the specificity of DLAMP, a total of 42 negative samples including 10 nasal swabs, 11 whole blood samples, and 21 semen samples from cattle free from both BHV-1 and MB were tested. All these control samples tested negative for MB and BHV-1 by the DLAMP assay. 10
3.2 Sensitivity of DLAMP The sensitivity of the DLAMP assay containing two primer sets for each pathogen as well as fluorescent-dye-labelled primers was evaluated by using serial 10-fold dilutions of standards prepared as described in the previous section. The standards
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contain the same copy numbers of each recombinant plasmid, which possesses the specific gene sequence of either BHV-1 or MB. Standards containing equivalent
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amounts of each recombinant plasmid, ranging from 1×108 to 1 copy/μl, were
prepared from stock by using serial 10-fold dilutions with RNase-free H2O. Two
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microliters was used as template in the DLAMP assay for the detection of BHV-1 and
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MB. The detection limit of the DLAMP assay was 200 copies/μl recombinant plasmid
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DNA (Fig. 3). All these results demonstrated that the DLAMP assay is capable of
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3.3 Interference of DLAMP
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simultaneously and sensitively identifying BHV-1 and MB.
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Six artificial samples with various concentrations of plasmid containing the BHV1 or MB gene were prepared and subjected to the DLAMP assay to assess the
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interference between higher concentrations and lower concentrations of nucleic acid templates. The six artificial samples were as follows: sample 1, MB (108 copies/μl) +
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BHV-1 (104 copies/μl); sample 2, MB (104 copies/μl) + BHV-1 (108 copies/μl); sample 3, MB (107 copies/μl) + BHV-1 (103 copies/μl); sample 4, MB (104 copies/μl) + BHV-1 (107 copies/μl); sample 5, MB (106 copies/μl) + BHV-1 (102 copies/μl); sample 6, MB (102 copies/μl) + BHV-1 (106 copies/μl). Fig. 4 shows that the amplified bands with corresponding colours were easily observed in the 11
electrophoretogram. These results demonstrated that, in the presence of higher concentrations of the MB DNA template, the amplification of lower concentrations of BHV-1 DNA template was not inhibited in the DLAMP assay, and vice versa. 3.4 Clinical detection
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The performance of the DLAMP assay in clinical specimens was evaluated on
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125 swab samples. The OIE-recommended real-time PCR and conventional PCR assays were conducted in parallel to assess the accuracy of the DLAMP assay.
Individual test results were summarized in Table 3; the detection results by DLAMP
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were in 100% agreement with those of conventional PCR. Identical results were
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obtained for 122 samples compared by using these three methods. Three discrepant
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samples were found, including two positive results for MB in real-time PCR that were negative in DLAMP and one sample that was positive for BHV-1 in real-time PCR
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but negative in DLAMP. To determine whether the three discrepant samples were true
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positives, the real-time PCR products of the three discrepant samples were sequenced to rule out false positive results. The sensitivity and specificity of DLAMP for
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detection of MB were 96.6% (56/58) and 100% (65/65), and those for BHV-1 detection by DLAMP were 95% (19/20) and 100% (106/106), respectively, compared
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to the real-time PCR assay for detection of field samples. This DLAMP assay could detect and differentiate MB from BHV-1 in infected samples. 4. Discussion In recent years, LAMP has drawn a great deal of attention from researchers due to its ease of manipulation and rapid detection speed. However, the use of LAMP for 12
detection of multiplex targets has met with some difficulty due to the methodological limitations of LAMP results for specific target analysis. Whether turbidity is observed, dye is added, or electrophoresis is performed, the results of single LAMP and multiplex LAMP look the same and cannot be distinguished. This feature is in
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contrast to PCR, in which products of a specific length for each target can be simply distinguished by gel electrophoresis. LAMP products consist of a number of amplified
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DNA bands of different sizes. The results of LAMP assays commonly display as
ladder-like patterns after electrophoresis, making it difficult to distinguish two distinct
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targets in one multiplex reaction (Aonuma et al., 2010; Iseki et al., 2007; Kouguchi et
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al., 2010; Kubota and Jenkins, 2015; Mahony et al., 2013; Nurul Najian et al., 2016;
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Song et al., 2010; Yamazaki et al., 2013).
In this study, a DLAMP assay with fluorescently labelled primers was able to
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identify BHV-1 and MB and to distinguish them from each other in clinical samples
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with BRD symptoms. Theoretically, LAMP can generate upwards of 109 copies from less than one copy of DNA template within an hour. In an experimental study by
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Socha, W. et al., the sensitivity of LAMP for the BHV-1 gD and gE genes was 2×104 copies and 2×105 copies, respectively, which was lower than the sensitivity of the
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DLAMP assay (Socha et al., 2017). One reason for this result might be the use of different target genes. In this study, the DLAMP assay utilized the gB gene, whereas Socha et al. chose the gD and gE genes instead. Another reason might be that the DLAMP assay was conducted with optimized Bst DNA polymerase 3.0, which has better performance than that of Bst DNA polymerase (Zhao et al., 2015). Compared to 13
the OIE-recommended real-time PCR assays for clinical detection, the DLAMP assay missed two MB-positive samples and one BHV-1-positive sample. These three discrepant samples were confirmed by a single-target LAMP assay, which was performed using a set of primers, and the results were consistent with those of the
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real-time PCR. However, no false positive results were detected in the DLAMP assay. These results demonstrated that this DLAMP assay is specific, although with a
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slightly decreased sensitivity. It is likely that the two sets of primers used in the
DLAMP assay could compete for the reagents in the reaction and interfere with other
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amplification, resulting in reduced amplification efficiency. In contrast, in a typical
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real-time PCR, only two primers and one probe are used. This difference might
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explain why the sensitivity of this DLAMP assay is slightly lower than that of real-
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time PCR. DLAMP is cost-effective compared to real-time PCR, in that it can
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simultaneously detect two pathogens without expensive instruments. The DLAMP assay needs only a water bath, and the reaction can be finished within 65 min. As co-
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infection is a common feature of BRD in field outbreaks, the DLAMP assay may be
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useful for surveillance of the different pathogenic organisms causing BRD without false positive detection (Thonur et al., 2012).
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Aside from the detection of multiple pathogens, the DLAMP assay has several
advantages. First, DLAMP has all the merits of conventional single-target LAMP assays, including high sensitivity and specificity, lower cost, smaller sample demand, short detection time and simple processing to suit veterinary field diagnostics in rural areas. Second, this DLAMP assay may decrease the risk of generating false positive 14
results. There is a report that strand displacement in the LAMP reaction, starting from randomly existing nicks in the DNA samples, often results in non-specific amplification (Mitsunaga et al.). In this study, non-specific amplification was observed after a prolonged reaction time of 90 min. Although the real-time
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turbidimeter could monitor the turbidity of the non-specific amplification curve, the electrophoresis bands of non-specific amplification products do not contain
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fluorescent colour, showing neither green nor red. In the DLAMP reaction system, the primers with fluorophores require more energy to incorporate than ordinary primers
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do in the process of amplification. This process can effectively suppress the
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generation of false positive reactions. Finally, the detection results of DLAMP are
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accurate and clear, and the final results are determined by the colours of the DLAMP
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products. This study employed two fluorophores, FAM and CY5, which have
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different excitation and absorption wavelengths. Two different colours are displayed: the FAM emission wavelength is 520 nm, shown as a green colour, whereas the CY5
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emission wavelength is 694 nm, shown as a red colour. Moreover, different
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fluorophores can be observed in only the selected channels. For example, FAM can be observed in only the 520 nm channel, where CY5 cannot be seen. Fluorescent-dyeconjugated fragments in the DLAMP assay are more specifically and accurately
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determined compared to judgement of sediment appearance or observation by the naked eye. In conclusion, the DLAMP assay is a rapid, specific and sensitive duplex method for simultaneous detection of MB and BHV-1. This assay has the potential to be 15
applied to clinical diagnosis and epidemiological screening of MB and BHV-1 coinfection in clinical samples and to monitoring MB and BHV-1 infection status in cattle herds.
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Funding This work was supported by the Guangxi Science and Technology Bureau (grant
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number: AA17204057); and the Special Support Plan for National High Level Talents (grant number: W02060083)
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Ethics approval and consent to participate
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This study was approved by the Institutional Animal Care and Use Committee
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(IACUC) of Guangxi Veterinary Research Institution (GVRI). Sample collection was
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conducted based on protocol #2016C103 issued by the IACUC of GVRI. All the
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veterinarians.
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samples were collected from live cattle on approved farms by well-trained
Competing interests
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The authors declare that they have no competing interests.
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Authors' contributions
QF conceived the study. ZXX provided funding. ZQX, LX, JH and YZ carried out
the experiments. TZ, MZ, SW, SL, JL, and XD collected clinical samples, and QF drafted the manuscript. All authors amended and approved the final version of the manuscript.
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Nurul Najian, A.B., Engku Nur Syafirah, E.A., Ismail, N., Mohamed, M. and Yean, C.Y., 2016. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira.
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Anal Chim Acta 903, 142-8.
Prysliak, T., van der Merwe, J., Lawman, Z., Wilson, D., Townsend, H., van Drunen Littel-van den Hurk, S. and Perez-Casal, J., 2011. Respiratory disease caused by Mycoplasma bovis is
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enhanced by exposure to bovine herpes virus 1 (BHV-1) but not to bovine viral diarrhea virus (BVDV) type 2. Can Vet J 52, 1195-202.
Socha, W., Rola, J., Urban-Chmiel, R. and Zmudzinski, J.F., 2017. Application of loop-mediated isothermal amplification (LAMP) assays for the detection of bovine herpesvirus 1. Pol J Vet Sci 20, 619-622.
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Song, H., Bae, Y., Park, S., Kwon, H., Lee, H. and Joh, S., 2010. Loop-mediated isothermal amplification assay for detection of four immunosuppressive viruses in chicken. J Virol Methods 256, 6-11. Thomas, A., Dizier, I., Linden, A., Mainil, J., Frey, J. and Vilei, E.M., 2004. Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis. Vet J 168, 100-2. Thonur, L., Maley, M., Gilray, J., Crook, T., Laming, E., Turnbull, D., Nath, M. and Willoughby, K., 2012. One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial
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virus, bovine herpesvirus 1 and bovine parainfluenza virus 3. BMC Vet Res 8, 37. Tomas JD, S.F., 2009. Rebhun's Diseases of Dairy Cattle, 2st ed. Elsevier (Singapore) Pte Ltd Press, 91-93.
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Wang, J., O'Keefe, J., Orr, D., Loth, L., Banks, M., Wakeley, P., West, D., Card, R., Ibata, G., Van
Maanen, K., Thoren, P., Isaksson, M. and Kerkhofs, P., 2007. Validation of a real-time PCR assay for the detection of bovine herpesvirus 1 in bovine semen. J Virol Methods 144, 103-
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Wang, J., O'Keefe, J., Orr, D., Loth, L., Banks, M., Wakeley, P., West, D., Card, R., Ibata, G., Van
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Maanen, K., Thoren, P., Isaksson, M. and Kerkhofs, P., 2008. An international interlaboratory ring trial to evaluate a real-time PCR assay for the detection of bovine
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herpesvirus 1 in extended bovine semen. Vet Microbiol 126, 11-9.
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Xie, Z., Luo, S., Xie, L., Liu, J., Pang, Y., Deng, X., Xie, Z., Fan, Q. and Khan, M.I., 2014. Simultaneous typing of nine avian respiratory pathogens using a novel GeXP analyzer-based multiplex
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PCR assay. J Virol Methods 207, 188-95.
Yamazaki, W., Mioulet, V., Murray, L., Madi, M., Haga, T., Misawa, N., Horii, Y. and King, D.P., 2013. Development and evaluation of multiplex RT-LAMP assays for rapid and sensitive detection
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of foot-and-mouth disease virus. J Virol Methods 192, 18-24. Zhao, Y., Chen, F., Li, Q., Wang, L. and Fan, C., 2015. Isothermal Amplification of Nucleic Acids. Chem
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Rev 115, 12491-545.
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Figure legends Fig. 1. Locations of the primers used in DLAMP. The GenBank accession numbers for MB and BHV-1 are CP023663.1 and KU198480.1, respectively. The nucleotide sequences of the primers are underlined.
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Fig. 2. Specificity results of DLAMP for BHV-1 and MB. The DLAMP products on an agarose gel were imaged separately using the 520 nm channel (A), 670 nm
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channel (B) and duplex channel (C). FAM fluorescence (green) indicates amplified DNA products from BHV-1, whereas CY5 fluorescence (red) indicates amplified
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DNA products from MB; lane 1, MB; lane 2, BHV-1; 3, MB + BHV-1; lane 4, SRSV;
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lane 5, BPI3; lane 6, BVDV; lane 7, Mycoplasma agalactiae; lane 8, Mycoplasma
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conjunctivae; lane 9, Mycobacterium bovis; lane 10, Mycoplasma mycoides subsp.
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mycoides; lane 11, Mycoplasma mycoides subsp. capri; lane 12, Pasteurella multocida.
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Fig. 3. Sensitivity results of DLAMP for BHV-1 and MB. A: 520 nm channel; B: 670 nm channel; C: duplex channel. D: Amplification of target sequences with two
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sets of primers monitored by real-time turbidimeter (turbidity at 650 nm); lane 1, 106
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copies/μl (standards of equivalent PMD-18T-MB and PMD-18T-BHV-1); lane 2, 105 copies/μl; lane 3, 104 copies/μl; lane 4, 103 copies/μl; lane 5, 102 copies/μl; lane 6, 10 copies/μl; lane 7, 1 copy/μl; 8: negative control.
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Fig. 4. Interference results of DLAMP for MB and BHV-1. Lane 1, sample 1: MB (108 copies/μl) + BHV-1 (104 copies/μl); lane 2, sample 2: MB (104 copies/μl) + BHV-1 (108 copies/μl); lane 3, sample 3: MB (107 copies/μl) + BHV-1 (103 copies/μl); lane 4, sample 4: MB (104 copies/μl) + BHV-1 (107 copies/μl); lane 5,
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sample 5: MB (106 copies/μl) + BHV-1 (102 copies/μl); lane 6, sample 6: MB (102
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copies/μl) + BHV-1 (106 copies/μl)
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Table 1 Pathogens used and DLAMP assay results Pathogen
Source
DLAMP result
Mycoplasma bovis (MB)
7 isolate strains from Guangxi, GVRI
+
Bovine herpes virus 1 (BHV-1)
2 reference strains (Barta Nu, BK125), CVCC; 3 isolate strains
+
from Guangxi, GVRI 1 isolate strain from Yunnan, YNCIQ
Bovine virus diarrhoea virus (BVDV)
14 isolate strains from Guangxi, GVRI; 3 reference strains
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(NADL, Oregon CV24, BA), CVCC
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Bovine respiratory syncytial virus (SRSV)
-
Bovine parainfluenza type 3 (BPI3)
1 isolate strain from Guangxi, GVRI
-
Mycoplasma agalactiae
2 reference strains (PG2, SI), CVCC; 2 isolate strains from
-
Guangxi, GVRI
1 isolate strain from Guangxi, GVRI
Mycobacterium bovis
1 isolate strain from Guangxi, GVRI
Porcine Mycoplasma hyopneumoniae
1 reference strain (354, PV11), CVCC
A
N
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Mycoplasma conjunctivae
Mycoplasma mycoides subsp. capri
Pasteurella multocida
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Pasteurella haemolytica
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Foot-and-mouth disease virus (FMDV) Vesicular stomatitis virus (VSV)
-
3 reference strains (Y-goat, C88021, PGI), CVCC
-
4 reference strains (C87011, PG3, C87-13, C87001), CVCC
-
1 reference strain (C52-1), CVCC
-
3 reference strains (P19, P-2225, C467), CVCC
-
3 serotypes (A, O, Asia I) of inactivated viruses, YNCIQ
-
2 serotypes (New Jersey, Indiana) of inactivated viruses,
-
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Mycoplasma mycoides subsp. mycoides
-
YNCIQ 1 reference strain (AV1711), CVCC
-
Bovine rotavirus (BRV)
8 isolate strains from Guangxi, GVRI; 2 reference strains
-
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Rinderpest virus (RPV)
5 serotypes (4, 8, 9, 17, 18) of inactivated viruses, YNCIQ
-
Peste des petits ruminants virus (PPRV)
1 inactivated virus, YNCIQ
-
Escherichia coli (ETEC)
3 reference strains (C83919, C83920, C83924), CVCC
-
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Bluetongue virus (BTV)
(NCDV, C486), CVCC
GVRI = Guangxi Veterinary Research Institute. YNCIQ = Yunnan Entry-Exit Inspection and Quarantine Bureau. CVCC = Chinese Veterinary Culture Collection Center. 22
Table 2 Sequences of primers Primers Outer MB
Inner
uvrC
MB-F3 MB-B3 MB-FIP MB-BIP
Outer Inner
BHV-1
BHV-1-F3 BHV-1-B3 BHV-1-FIP
gB BHV-1-BIP
Sequence (5’-3’)
TM (°C)
CCTGTCGGAGTTGCAATTGTT CGGTCAACTTCAACTTGAATTTG CY5TACCGCCATCAGCTATAACTAAGTCATGAGC GCAGTGCTGATGTTG TCCCTGTTATTGGATTAGTAAAAAACATATCT AGGTCAATTAAGGCTTTGG GGACGATGTGTACACGGC CTCGATCTGCTGGAAGCG FAMTCGTACGGGTACACCGAGCGTACCGCACGG GCACCT TACATGTCGCCCTTTTACGGGCCCGGCGAGT AGCTGGT
61 60 60/63* 61/59*
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Target gene
56 59 60/65* 56/68*
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Target pathogen
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* The inner primers are FIP = F2 + F1c, BIP = B2 + B1c
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Table 3 Evaluation of 125 swab samples for MB and BHV-1 by using DLAMP,
Pathogen
DLAMP result
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real-time PCR and PCR
Detection rate
Real-time PCR
PCR
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(%)
MB
BHV-1
MB
BHV-1
56
44.8%
58
0
56
0
BHV-1
17
13.6%
0
18
0
17
2
1.6%
2
2
2
2
75
60%
60
20
58
19
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MB
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MB + BHV-1
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Total
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U
N
A
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Figr-1
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U
N
A
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Figr-2
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U
N
A
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Figr-3
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N
A
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Figr-4
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S0166-0934(18)30170-8 https://doi.org/10.1016/j.jviromet.2018.08.014 VIRMET 13525
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
2-4-2018 3-7-2018 20-8-2018
Please cite this article as: Fan Q, Xie Z, Xie Z, Xie L, Huang J, Zhang Y, Zeng T, Zhang M, Wan S, Luo S, Liu J, Deng X, Development of Duplex Fluorescence-based Loop-mediated Isothermal Amplification Assay for Detection of Mycoplasma bovis and Bovine Herpes Virus 1, Journal of Virological Methods (2018), https://doi.org/10.1016/j.jviromet.2018.08.014 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.
Development of Duplex Fluorescence-based Loop-mediated Isothermal Amplification Assay for Detection of Mycoplasma bovis and Bovine Herpes Virus 1
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Qing Fan, Zhixun Xie*, Zhiqin Xie, Liji Xie, Jiaoling Huang, Yanfan Zhang, Tingting Zeng, Minxiu Zhang, Sheng Wan, Sisi Luo, Jiabo Liu, Xianwen Deng
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Guangxi Veterinary Research Institute, Guangxi Key Laboratory of Veterinary Biotechnology, Nanning 530001, P.R. China
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Qing Fan: [email protected]
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Zhiqin Xie: [email protected]
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Zhixun Xie:[email protected]
Liji Xie: [email protected]
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Jiaoling Huang: [email protected]
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Yanfang Zhang: [email protected]
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Tingting Zeng: [email protected] Minxiu Zhang: [email protected]
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Sheng Wan: [email protected] Sisi Luo: [email protected] Jiabo Liu: [email protected] Xianwen Deng: [email protected] *Corresponding author: [email protected] 1
Highlights
A DLAMP method was developed for simultaneous detection of MB and BHV-1 and applied to clinical samples
The DLAMP results were visualized by the colors of fluorescent-dye-conjugated DLAMP products
The DLAMP assay shows high specificity and sensitivity
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Abstract Mycoplasma bovis (MB) and bovine herpes virus 1 (BHV-1) are two important
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pathogens that cause bovine respiratory disease in the beef feedlot and dairy
industries. The aim of this study was to develop and validate a duplex fluorescence-
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based loop-mediated isothermal amplification (DLAMP) assay for simultaneous
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detection of MB and BHV-1. Two sets of specific primers for each pathogen were
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designed to target the unique sequences of the MB uvrC gene and the BHV-1 gB
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gene. The inner primer for BHV-1 was synthesized with the fluorophore FAM at the 5’ end to detect the BHV-1 gB gene, and the inner primer for MB was synthesized
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with the fluorophore CY5 at the 5’ end to detect the MB uvrC gene. The DLAMP
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reaction conditions were optimized for rapid and specific detection of MB and BHV1. The DLAMP assay developed here could specifically detect MB and BHV-1
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without cross-reaction with other known non-target bovine pathogens. The sensitivity of this DLAMP assay was as low as 2×102 copies for recombinant plasmids
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containing the MB and BHV-1 target genes. In a detection test of 125 clinical samples, the positive rates for MB, BHV-1 and co-infection were 44.8%, 13.6% and 1.6%, respectively. Furthermore, the sensitivity and specificity of DLAMP were determined as 95%-96.6% and 100%, respectively, of those of field sample detection by the real-time polymerase chain reaction (PCR) assay recommended by the World 2
Organisation for Animal Health. Overall, DLAMP provides a rapid, sensitive and specific assay for the identification of MB and BHV-1 in clinical specimens and for epidemiological surveillance.
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Abbreviations
MB: Mycoplasma bovis
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BHV-1: bovine herpes virus 1
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BRD: bovine respiratory disease
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OIE: World Organisation for Animal Health
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DLAMP: duplex fluorescence-based loop-mediated isothermal amplification
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IACUC: Institutional Animal Care and Use Committee
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GVRI: Guangxi Veterinary Research Institution
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YNCIQ: Yunnan Entry-Exit Inspection and Quarantine Bureau
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CVCC: Chinese Veterinary Culture Collection Centre SRSV: bovine respiratory syncytial virus
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BVDV: bovine virus diarrhoea virus BPI3: bovine parainfluenza type 3 FMDV: foot-and-mouth disease virus VSV: vesicular stomatitis virus 3
RPV: rinderpest virus BRV: bovine rotavirus BTV: bluetongue virus
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PPRV: peste des petits ruminants virus
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Key words: MB; BHV-1; DLAMP; Fluorescent detection
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ETEC: Escherichia coli
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1. Introduction
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Bovine respiratory disease (BRD) is one of the major diseases affecting cattle
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health and production, and it causes immense economic losses. Its aetiology is multifactorial; many infectious agents are important in the development of BRD
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including viruses, bacteria and mycoplasmas. Mycoplasma bovis (MB) and bovine
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herpes virus 1 (BHV-1) are two important pathogens of BRD in the cattle industry (Jones and Chowdhury, 2007). Infection with these two pathogens can lead to reduced production, higher levels of morbidity and mortality, and increased veterinary labour
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costs, as well as economic losses. Infections with MB and BHV-1 are related to the occurrence of high fever, pneumonia and polyarthritis, rhinitis, conjunctivitis, genital tract infection, and reproductive problems such as abortion and reduced fertility in cattle (Cernicchiaro et al., 2013; Tomas JD, 2009). MB and BHV-1 are distributed 4
worldwide. Stress and environmental factors such as weaning, temperature, dust, stocking density, humidity, inadequate nutrition and transportation are important factors in the development of MB outbreaks. Furthermore, MB is difficult to remove thoroughly from an infected farm after an outbreak, and infected cattle can carry the
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pathogens for months and even years, remaining a source of infection (Burki et al., 2015; Maunsell et al., 2011). Similar to most other herpesviruses, inapparent infection
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is common in BHV-1 carriers. After BHV-1 infection, the virus can enter neural cells
and establish a latent infection in sensory ganglia. Latent infections also occur in non-
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neural sites, such as the tonsils and lymph nodes (Jones and Chowdhury, 2007). These
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latent pathogens can be reactivated both by stressful conditions and by administration
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of glucocorticoids. BHV-1-infected bulls may shed virus intermittently in the semen
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long after the primary infection (Graham, 2013; Nandi et al., 2009). Irrespective of
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the infectious agent involved, clinical manifestations of BRD may present similarly. Moreover, detection of a bacterial pathogen can mask an underlying viral cause, as
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both bacteria and viruses can co-infect their natural hosts (Caswell et al., 2010). A
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survey of exposure to BHV-1 in Canadian feedlots suggested that respiratory disease caused by MB was associated with previous exposure to BHV-1 (Prysliak et al., 2011). Cattle co-infected with BHV-1 and MB are regarded as lifelong carriers and
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potential shedders of these pathogens, and such infected cattle pose a potential risk to herds in the cattle industry. Therefore, differential diagnosis of MB and BHV-1 infections may lead to a better understanding of the epidemiology and natural distribution of MB and BHV-1 infections in the field, which may provide useful 5
information for the control of BRD. PCR, a highly sensitive and specific test, is considered a routine diagnostic test for MB and BHV-1 infections (Bashiruddin et al., 2005; Moore et al., 2000). LAMP is an alternative molecular genetic method derived from PCR for carrying out
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reactions under isothermal conditions using a single enzyme. LAMP-amplified products can be easily visualized by the naked eye. Compared with the two primers of
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conventional PCR, LAMP has four primers that can recognize 6 locations in the target gene; therefore, the LAMP method has higher reaction specificity than that of a
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conventional PCR assay. LAMP is 102~105 times more sensitive than conventional
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PCR (Ashraf et al., ; Notomi et al., 2000; Xie et al., 2014).
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The aim of the present study was to develop and validate a DLAMP assay for
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simultaneous detection of MB and BHV-1 in a single reaction. The DLAMP assay
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was compared to the World Organisation for Animal Health (OIE) recommended real-time PCR assay to assess its application in routine diagnosis of the aetiological
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agents involved in BRD.
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2. Materials and Methods 2.1. Ethical statement This study was approved by the Institutional Animal Care and Use Committee
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(IACUC) of Guangxi Veterinary Research Institution (GVRI). Sample collection was conducted based on protocol #2016C103 issued by the IACUC of GVRI. All the samples were collected from live cattle by well-trained veterinarians on approved farms. 6
2.2. Pathogens and DNA/RNA extraction The pathogens used in this study are listed in Table 1. Ten nasal swabs, 11 whole blood samples, and 21 semen specimens were collected from healthy cattle, and the extracted DNA samples were used as negative controls. The DNA of mycoplasmas,
DNA Extraction Kit version 5.0 (Takara, Dalian, China) according to the
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bacteria and DNA viruses was extracted by using the MiniBEST Universal Genomic
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manufacturer’s instructions. The RNA of RNA viruses was extracted by using the Universal RNA Extraction Kit (Takara) and subsequently reverse-transcribed to
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cDNA by using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). DNA/cDNA
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2.3. Primer design
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template for the DLAMP reaction.
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was stored at -20°C until use. Finally, 2 μl of each DNA/cDNA solution was used as a
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The selected genes, uvrC of MB and gB of BHV-1, are highly conserved and were
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used to design primers to identify these specific pathogens (Clothier et al., 2010; Thomas et al., 2004; Wang et al., 2007). Published sequences of the MB uvrC gene
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and BHV-1 gB gene were downloaded from GenBank and aligned using MegAlign 7.0 software (DNAStar, USA). Primer selection was supported by the software Primer
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Premier Version 5.0 (Premier Biosoft International, Canada) according to the restricted design rules for LAMP primers. The DLAMP assay included two sets of each of the four specific primers: two outer primers (F3, B3) and two inner primers (FIP = F1c + F2, BIP = B1c + B2). The inner primer FIP was synthesized with a fluorophore at the 5’ end. The BHV-1-FIP primer, labelled with 6-carboxyfluorescein 7
(FAM), showed a green colour at an emission wavelength of 520 nm, whereas the MB-FIP primer, labelled with Cyanine 5 (CY5), showed a red colour at an emission wavelength of 694 nm. A BLAST search program on the GenBank website was used to verify oligonucleotide specificity. All primers were synthesized and HPLC-purified
1.
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2.4. DLAMP assay for simultaneous MB and BHV-1 detection
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by Takara Inc. (Takara). The sequences of the primers are shown in Table 2 and Fig.
The DLAMP reaction was performed by using the DNA Amplification Kit
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(Loopamp, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, 2.5 μl
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10× reaction buffer (200 mM Tris–HCl (pH 8.8), 100 mM KCl, 80 mM MgSO4, 100
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mM (NH4)2SO4, 1% Tween 20, 8 M betaine, and 14 mM dNTPs), 2 μl of extracted
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DNA/cDNA template, 5 pmol F3 and B3 primers, 40 pmol FIP and BIP primers, and
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15 units of Bst DNA polymerase (3.0 version) were mixed, and ddH2O was added to reach a final 25 μl reaction volume. The reaction mixture was incubated at 62°C for 60
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min using a Loopamp real-time turbidimeter (LA-320; Eiken Chemical Co., Ltd.,
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Tokyo, Japan), and the reaction was terminated by incubation at 80°C for 5 min. 2.5. Analysis of DLAMP products
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The DLAMP products were analysed by two methods. The first method was to
visually inspect the turbidity of the samples. DNA amplification generated a large amount of white magnesium pyrophosphate precipitate, a byproduct of the DLAMP reaction, which caused turbidity. The turbidity was monitored by the naked eye or by a Loopamp real-time turbidimeter. This method could only identify infected samples; 8
it was unable to differentiate MB from BHV-1. It can be applied in rural areas. The second method was to run agarose gel electrophoresis. A volume of 5 μl of each DLAMP product was subjected to electrophoresis in a 2% agarose gel at 100 V for 60 min, and the fluorescent-dye-conjugated DLAMP products were examined with an
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image analyser (Universal Hood III, 731BR01622, Bio-Rad, USA) to display different colours of electrophoretic bands. Green bands were considered BHV-1,
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while red bands were considered MB. Since DLAMP products are mixtures of DNA fragments with various lengths, several ladder-like stripes are usually shown on an
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agarose gel after electrophoresis. No DNA marker is necessary for a DLAMP assay.
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2.6. Preparation of standards
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PCR amplicons containing the full-length sequence of each DLAMP target gene
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were cloned into the PMD-18T vector (Takara) separately by using the standard
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procedure, generating the recombinant plasmids PMD-18T-MB and PMD-18T-BHV1. The concentrations of the recombinant plasmids were measured at 260 nm by using a
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NanoDrop 2000 (ThermoFisher Scientific, Waltham, USA). The copy numbers of the
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target DNA were calculated based on the following formula: copies/μl = [concentration of plasmid (ng/μl) × 6.02×1014 / length of plasmid × 660](Xie et al., 2014). Serial 10-
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fold dilutions of each target DNA, ranging from 1×108 copies/μl to 1 copy/μl, were stored at -20°C until use. 2.7. Detection of clinical samples The present study was conducted on a total of 125 swab samples consisting of 70 nasal swabs and 55 vaginal swabs, which were collected from the same dairy farm in 9
Guangxi, southern China, during 2015 to 2017, where recurrent problems with MB infection were reported (Ma C et al., 2015). The swab samples were collected from cows displaying typical respiratory-system-related lesions. Following clinical examination, which included assessment of body temperature and respiratory
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function, the swab samples were eluted in 3.0 ml of PBS. The eluent was centrifuged at 5000 rpm for 5 min at 4°C, and 250 μl supernatant was subjected to DNA
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extraction using the MiniBEST Universal Genomic DNA Extraction Kit version 5.0
(Takara) according to the manufacturer’s instructions. Finally, 2 μl of extracted DNA
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was used as a template for the DLAMP test described above in section 2.4. The
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samples were also tested for MB and BHV-1 infection by using the single real-time
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PCR assays recommended by OIE and conventional PCR as references (Bashiruddin
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et al., 2005; Moore et al., 2000; Wang et al., 2008).
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3. Results
3.1 Specificity of DLAMP
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All five BHV-1 strains and seven MB strains tested positive by DLAMP and showed
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green bands and red bands, respectively, on agarose gels. DLAMP amplification of other common bovine pathogens did not show non-specific products or any inter-assay
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cross-amplification. Only pathogen-specific targets were amplified by DLAMP (Table 1, Fig. 2). To validate the specificity of DLAMP, a total of 42 negative samples including 10 nasal swabs, 11 whole blood samples, and 21 semen samples from cattle free from both BHV-1 and MB were tested. All these control samples tested negative for MB and BHV-1 by the DLAMP assay. 10
3.2 Sensitivity of DLAMP The sensitivity of the DLAMP assay containing two primer sets for each pathogen as well as fluorescent-dye-labelled primers was evaluated by using serial 10-fold dilutions of standards prepared as described in the previous section. The standards
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contain the same copy numbers of each recombinant plasmid, which possesses the specific gene sequence of either BHV-1 or MB. Standards containing equivalent
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amounts of each recombinant plasmid, ranging from 1×108 to 1 copy/μl, were
prepared from stock by using serial 10-fold dilutions with RNase-free H2O. Two
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microliters was used as template in the DLAMP assay for the detection of BHV-1 and
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MB. The detection limit of the DLAMP assay was 200 copies/μl recombinant plasmid
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DNA (Fig. 3). All these results demonstrated that the DLAMP assay is capable of
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3.3 Interference of DLAMP
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simultaneously and sensitively identifying BHV-1 and MB.
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Six artificial samples with various concentrations of plasmid containing the BHV1 or MB gene were prepared and subjected to the DLAMP assay to assess the
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interference between higher concentrations and lower concentrations of nucleic acid templates. The six artificial samples were as follows: sample 1, MB (108 copies/μl) +
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BHV-1 (104 copies/μl); sample 2, MB (104 copies/μl) + BHV-1 (108 copies/μl); sample 3, MB (107 copies/μl) + BHV-1 (103 copies/μl); sample 4, MB (104 copies/μl) + BHV-1 (107 copies/μl); sample 5, MB (106 copies/μl) + BHV-1 (102 copies/μl); sample 6, MB (102 copies/μl) + BHV-1 (106 copies/μl). Fig. 4 shows that the amplified bands with corresponding colours were easily observed in the 11
electrophoretogram. These results demonstrated that, in the presence of higher concentrations of the MB DNA template, the amplification of lower concentrations of BHV-1 DNA template was not inhibited in the DLAMP assay, and vice versa. 3.4 Clinical detection
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The performance of the DLAMP assay in clinical specimens was evaluated on
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125 swab samples. The OIE-recommended real-time PCR and conventional PCR assays were conducted in parallel to assess the accuracy of the DLAMP assay.
Individual test results were summarized in Table 3; the detection results by DLAMP
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were in 100% agreement with those of conventional PCR. Identical results were
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obtained for 122 samples compared by using these three methods. Three discrepant
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samples were found, including two positive results for MB in real-time PCR that were negative in DLAMP and one sample that was positive for BHV-1 in real-time PCR
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but negative in DLAMP. To determine whether the three discrepant samples were true
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positives, the real-time PCR products of the three discrepant samples were sequenced to rule out false positive results. The sensitivity and specificity of DLAMP for
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detection of MB were 96.6% (56/58) and 100% (65/65), and those for BHV-1 detection by DLAMP were 95% (19/20) and 100% (106/106), respectively, compared
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to the real-time PCR assay for detection of field samples. This DLAMP assay could detect and differentiate MB from BHV-1 in infected samples. 4. Discussion In recent years, LAMP has drawn a great deal of attention from researchers due to its ease of manipulation and rapid detection speed. However, the use of LAMP for 12
detection of multiplex targets has met with some difficulty due to the methodological limitations of LAMP results for specific target analysis. Whether turbidity is observed, dye is added, or electrophoresis is performed, the results of single LAMP and multiplex LAMP look the same and cannot be distinguished. This feature is in
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contrast to PCR, in which products of a specific length for each target can be simply distinguished by gel electrophoresis. LAMP products consist of a number of amplified
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DNA bands of different sizes. The results of LAMP assays commonly display as
ladder-like patterns after electrophoresis, making it difficult to distinguish two distinct
U
targets in one multiplex reaction (Aonuma et al., 2010; Iseki et al., 2007; Kouguchi et
N
al., 2010; Kubota and Jenkins, 2015; Mahony et al., 2013; Nurul Najian et al., 2016;
M
A
Song et al., 2010; Yamazaki et al., 2013).
In this study, a DLAMP assay with fluorescently labelled primers was able to
ED
identify BHV-1 and MB and to distinguish them from each other in clinical samples
PT
with BRD symptoms. Theoretically, LAMP can generate upwards of 109 copies from less than one copy of DNA template within an hour. In an experimental study by
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Socha, W. et al., the sensitivity of LAMP for the BHV-1 gD and gE genes was 2×104 copies and 2×105 copies, respectively, which was lower than the sensitivity of the
A
DLAMP assay (Socha et al., 2017). One reason for this result might be the use of different target genes. In this study, the DLAMP assay utilized the gB gene, whereas Socha et al. chose the gD and gE genes instead. Another reason might be that the DLAMP assay was conducted with optimized Bst DNA polymerase 3.0, which has better performance than that of Bst DNA polymerase (Zhao et al., 2015). Compared to 13
the OIE-recommended real-time PCR assays for clinical detection, the DLAMP assay missed two MB-positive samples and one BHV-1-positive sample. These three discrepant samples were confirmed by a single-target LAMP assay, which was performed using a set of primers, and the results were consistent with those of the
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real-time PCR. However, no false positive results were detected in the DLAMP assay. These results demonstrated that this DLAMP assay is specific, although with a
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slightly decreased sensitivity. It is likely that the two sets of primers used in the
DLAMP assay could compete for the reagents in the reaction and interfere with other
U
amplification, resulting in reduced amplification efficiency. In contrast, in a typical
N
real-time PCR, only two primers and one probe are used. This difference might
A
explain why the sensitivity of this DLAMP assay is slightly lower than that of real-
M
time PCR. DLAMP is cost-effective compared to real-time PCR, in that it can
ED
simultaneously detect two pathogens without expensive instruments. The DLAMP assay needs only a water bath, and the reaction can be finished within 65 min. As co-
PT
infection is a common feature of BRD in field outbreaks, the DLAMP assay may be
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useful for surveillance of the different pathogenic organisms causing BRD without false positive detection (Thonur et al., 2012).
A
Aside from the detection of multiple pathogens, the DLAMP assay has several
advantages. First, DLAMP has all the merits of conventional single-target LAMP assays, including high sensitivity and specificity, lower cost, smaller sample demand, short detection time and simple processing to suit veterinary field diagnostics in rural areas. Second, this DLAMP assay may decrease the risk of generating false positive 14
results. There is a report that strand displacement in the LAMP reaction, starting from randomly existing nicks in the DNA samples, often results in non-specific amplification (Mitsunaga et al.). In this study, non-specific amplification was observed after a prolonged reaction time of 90 min. Although the real-time
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turbidimeter could monitor the turbidity of the non-specific amplification curve, the electrophoresis bands of non-specific amplification products do not contain
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fluorescent colour, showing neither green nor red. In the DLAMP reaction system, the primers with fluorophores require more energy to incorporate than ordinary primers
U
do in the process of amplification. This process can effectively suppress the
N
generation of false positive reactions. Finally, the detection results of DLAMP are
A
accurate and clear, and the final results are determined by the colours of the DLAMP
M
products. This study employed two fluorophores, FAM and CY5, which have
ED
different excitation and absorption wavelengths. Two different colours are displayed: the FAM emission wavelength is 520 nm, shown as a green colour, whereas the CY5
PT
emission wavelength is 694 nm, shown as a red colour. Moreover, different
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fluorophores can be observed in only the selected channels. For example, FAM can be observed in only the 520 nm channel, where CY5 cannot be seen. Fluorescent-dyeconjugated fragments in the DLAMP assay are more specifically and accurately
A
determined compared to judgement of sediment appearance or observation by the naked eye. In conclusion, the DLAMP assay is a rapid, specific and sensitive duplex method for simultaneous detection of MB and BHV-1. This assay has the potential to be 15
applied to clinical diagnosis and epidemiological screening of MB and BHV-1 coinfection in clinical samples and to monitoring MB and BHV-1 infection status in cattle herds.
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Funding This work was supported by the Guangxi Science and Technology Bureau (grant
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number: AA17204057); and the Special Support Plan for National High Level Talents (grant number: W02060083)
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Ethics approval and consent to participate
N
This study was approved by the Institutional Animal Care and Use Committee
A
(IACUC) of Guangxi Veterinary Research Institution (GVRI). Sample collection was
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conducted based on protocol #2016C103 issued by the IACUC of GVRI. All the
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veterinarians.
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samples were collected from live cattle on approved farms by well-trained
Competing interests
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The authors declare that they have no competing interests.
A
Authors' contributions
QF conceived the study. ZXX provided funding. ZQX, LX, JH and YZ carried out
the experiments. TZ, MZ, SW, SL, JL, and XD collected clinical samples, and QF drafted the manuscript. All authors amended and approved the final version of the manuscript.
16
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Figure legends Fig. 1. Locations of the primers used in DLAMP. The GenBank accession numbers for MB and BHV-1 are CP023663.1 and KU198480.1, respectively. The nucleotide sequences of the primers are underlined.
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Fig. 2. Specificity results of DLAMP for BHV-1 and MB. The DLAMP products on an agarose gel were imaged separately using the 520 nm channel (A), 670 nm
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channel (B) and duplex channel (C). FAM fluorescence (green) indicates amplified DNA products from BHV-1, whereas CY5 fluorescence (red) indicates amplified
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DNA products from MB; lane 1, MB; lane 2, BHV-1; 3, MB + BHV-1; lane 4, SRSV;
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lane 5, BPI3; lane 6, BVDV; lane 7, Mycoplasma agalactiae; lane 8, Mycoplasma
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conjunctivae; lane 9, Mycobacterium bovis; lane 10, Mycoplasma mycoides subsp.
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mycoides; lane 11, Mycoplasma mycoides subsp. capri; lane 12, Pasteurella multocida.
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Fig. 3. Sensitivity results of DLAMP for BHV-1 and MB. A: 520 nm channel; B: 670 nm channel; C: duplex channel. D: Amplification of target sequences with two
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sets of primers monitored by real-time turbidimeter (turbidity at 650 nm); lane 1, 106
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copies/μl (standards of equivalent PMD-18T-MB and PMD-18T-BHV-1); lane 2, 105 copies/μl; lane 3, 104 copies/μl; lane 4, 103 copies/μl; lane 5, 102 copies/μl; lane 6, 10 copies/μl; lane 7, 1 copy/μl; 8: negative control.
A
Fig. 4. Interference results of DLAMP for MB and BHV-1. Lane 1, sample 1: MB (108 copies/μl) + BHV-1 (104 copies/μl); lane 2, sample 2: MB (104 copies/μl) + BHV-1 (108 copies/μl); lane 3, sample 3: MB (107 copies/μl) + BHV-1 (103 copies/μl); lane 4, sample 4: MB (104 copies/μl) + BHV-1 (107 copies/μl); lane 5,
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sample 5: MB (106 copies/μl) + BHV-1 (102 copies/μl); lane 6, sample 6: MB (102
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A
N
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copies/μl) + BHV-1 (106 copies/μl)
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Table 1 Pathogens used and DLAMP assay results Pathogen
Source
DLAMP result
Mycoplasma bovis (MB)
7 isolate strains from Guangxi, GVRI
+
Bovine herpes virus 1 (BHV-1)
2 reference strains (Barta Nu, BK125), CVCC; 3 isolate strains
+
from Guangxi, GVRI 1 isolate strain from Yunnan, YNCIQ
Bovine virus diarrhoea virus (BVDV)
14 isolate strains from Guangxi, GVRI; 3 reference strains
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(NADL, Oregon CV24, BA), CVCC
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Bovine respiratory syncytial virus (SRSV)
-
Bovine parainfluenza type 3 (BPI3)
1 isolate strain from Guangxi, GVRI
-
Mycoplasma agalactiae
2 reference strains (PG2, SI), CVCC; 2 isolate strains from
-
Guangxi, GVRI
1 isolate strain from Guangxi, GVRI
Mycobacterium bovis
1 isolate strain from Guangxi, GVRI
Porcine Mycoplasma hyopneumoniae
1 reference strain (354, PV11), CVCC
A
N
U
Mycoplasma conjunctivae
Mycoplasma mycoides subsp. capri
Pasteurella multocida
ED
Pasteurella haemolytica
PT
Foot-and-mouth disease virus (FMDV) Vesicular stomatitis virus (VSV)
-
3 reference strains (Y-goat, C88021, PGI), CVCC
-
4 reference strains (C87011, PG3, C87-13, C87001), CVCC
-
1 reference strain (C52-1), CVCC
-
3 reference strains (P19, P-2225, C467), CVCC
-
3 serotypes (A, O, Asia I) of inactivated viruses, YNCIQ
-
2 serotypes (New Jersey, Indiana) of inactivated viruses,
-
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Mycoplasma mycoides subsp. mycoides
-
YNCIQ 1 reference strain (AV1711), CVCC
-
Bovine rotavirus (BRV)
8 isolate strains from Guangxi, GVRI; 2 reference strains
-
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Rinderpest virus (RPV)
5 serotypes (4, 8, 9, 17, 18) of inactivated viruses, YNCIQ
-
Peste des petits ruminants virus (PPRV)
1 inactivated virus, YNCIQ
-
Escherichia coli (ETEC)
3 reference strains (C83919, C83920, C83924), CVCC
-
A
Bluetongue virus (BTV)
(NCDV, C486), CVCC
GVRI = Guangxi Veterinary Research Institute. YNCIQ = Yunnan Entry-Exit Inspection and Quarantine Bureau. CVCC = Chinese Veterinary Culture Collection Center. 22
Table 2 Sequences of primers Primers Outer MB
Inner
uvrC
MB-F3 MB-B3 MB-FIP MB-BIP
Outer Inner
BHV-1
BHV-1-F3 BHV-1-B3 BHV-1-FIP
gB BHV-1-BIP
Sequence (5’-3’)
TM (°C)
CCTGTCGGAGTTGCAATTGTT CGGTCAACTTCAACTTGAATTTG CY5TACCGCCATCAGCTATAACTAAGTCATGAGC GCAGTGCTGATGTTG TCCCTGTTATTGGATTAGTAAAAAACATATCT AGGTCAATTAAGGCTTTGG GGACGATGTGTACACGGC CTCGATCTGCTGGAAGCG FAMTCGTACGGGTACACCGAGCGTACCGCACGG GCACCT TACATGTCGCCCTTTTACGGGCCCGGCGAGT AGCTGGT
61 60 60/63* 61/59*
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Target gene
56 59 60/65* 56/68*
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Target pathogen
N
U
* The inner primers are FIP = F2 + F1c, BIP = B2 + B1c
A
Table 3 Evaluation of 125 swab samples for MB and BHV-1 by using DLAMP,
Pathogen
DLAMP result
M
real-time PCR and PCR
Detection rate
Real-time PCR
PCR
ED
(%)
MB
BHV-1
MB
BHV-1
56
44.8%
58
0
56
0
BHV-1
17
13.6%
0
18
0
17
2
1.6%
2
2
2
2
75
60%
60
20
58
19
PT
MB
CC E
MB + BHV-1
A
Total
23
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-1
24
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-2
25
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-3
26
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figr-4
27