Abstract
A conserved nucleic acid fragment of the nucleocapsid gene of Swine Transmissible Gastroenteritis Coronavirus (TGEV) was chosen as the target, six special primers were designed successfully. Loop-mediated isothermal amplification (LAMP) was developed to detect the TGEV by incubation at 60°C for 1 h and the product specificity was confirmed by HphI digestion. Standard curves with high accuracy for TGEV quantization was constructed by adding 1 × SYBR greenI in the LAMP reaction. The assay established in this study was found to detect only the TGEV and no cross-reaction with other viruses, demonstrating its high specificity. By using serial sample dilutions as templates, the detection limit of LAMP was about 10 pg RNA, 10 times more sensitive than that of PCR and could be comparable to the nest-PCR.
Highlights
Swine Transmissible Gastroenteritis Coronavirus (TGEV), as a member of the coronaviridae, is a kind of single-stranded RNA virus, which produces villous atrophy and enteritis, leading to the serious financial loss to the whole pig industry
Samples Swine Transmissible Gastroenteritis Coronavirus (TGEV, strain H), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Pesudorabies (PRV), Porcine Parvovirus (PPV) derived from their passages in cell culture were provided by Shanghai Entry-Exit Inspection and Quarantine Bureau (SHCIQ); nucleic acids of Footand-mouth Disease Virus (FMDV) and Classical Swine Fever Virus (CSFV) were obtained from Chinese Academy of Inspection and Quarantine (CAIQ)
Detection of TGEV by Loop-mediated isothermal amplification (LAMP) TGEV deprived from the cell culture was first qualitatively analyzed by LAMP
Summary
Swine Transmissible Gastroenteritis Coronavirus (TGEV), as a member of the coronaviridae, is a kind of single-stranded RNA virus, which produces villous atrophy and enteritis, leading to the serious financial loss to the whole pig industry. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA/RNA with high specificity, sensitivity and rapidity under isothermal condition [5]. It has already found wide application in RNA virus detection, such as Foot-and-mouth Disease Virus[6], Swine Vesicular Disease Virus[7], Taura Syndrome Virus [8], Severe Acute Respiratory Syndrome Coronavirus and H5N1 Avian Influenza Virus[9,10]. LAMP method was applied in developing qualitative and quantitative detection system of TGEV, while its specificity and sensitivity were assessed
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