| In China, transmissible gastroenteritis virus, pig epidemic diarrhea virus and porcine rotavirus are the main virus that cause diarrhea of pigs. TGEV mainly infects piglets within 2 weeks, resulting in severe watery diarrhea, vomiting, dehydration, weight loss, coarse hair disorder and so on. It can also result in high mortality which is up to 100%. Diagnosis of TGEV is particularly important. The disease can be judged by the initial clinical symptoms and pathological changes and diagnosed through isolation identification and electron microscopy of the virus, which taking a long time and great efforts. Immunological diagnosis methods and molecular biology diagnositic laboratory methods are now commonly use for detection of porcine viral diarrhea. In Comparason with the RT-PCR we commonly used, real-time RT-PCR not only has better specificity, higher sensitivity and better stability, but also accurate, convenient, high-throughput, quantitative and able to save time and reduce pollution during the electrophoresis, which gains wide attention and becomes an important molecular biology diagnostic tool.In this study, a real-time RT-PCR assay was set up for the detection of TGEV, based on SYBR Green-I fluorescent dye and three pairs of primers of TGEV N gene. The amplicied fragments were 236bp,186bp and 236bp respectively. The desired gene was transformed into DH5 a competent cells after extracted RNA from TGEV activated vaccine and connected into a cloning vector pMD-19 (Simple). Three kinds of positive recombinant plasmid (pMD-N1、pMD-N2 and pMD-N3) were extracted and measured OD280 and OD26O value which was conversed to copy number, using for PCR and sequencing. The pMD-N2 was choosen for fluorescence quantitative PCR through comparison and the stock DNA contained 6.37 ×109 genome equivalent copies of the plasmid. Serial 10-fold dilutions of DNA were made using sterilization ultrapure water. The optimization of the annealing temperature was generated and the best procedure was determined. The standard curve was constructed at concentrations from 6.37 X 107 to 6.37 X 102, indicating that there is a favourable linear correlativity. The amplification efficient was 101.8%, based on the slope value of-3.279, and the correlation coefficient, R2, for the standard curve was 0.996. The reaction was carried out with the following virus, PEDV, PRV, JEV, CSFV and PRRSV. All gave negative results except TGEV. The coefficients of variability were no more than 5%, presenting a good repeatability. The real-time fluorescence quantitative PCR detected down to 6.37 ×101copies/μL from TGEV per reaction, while the conventional RT-PCR could only detect 6.37 × 103 copies of TGEV per reaction. The result demonstrated that the assay we constructed were 100 times sensitivity than the conventional RT-PCR. These consequences reveal that the real-time fluorescence quantitative PCR assay is a highly sensitive diagnostic means and it can detect pig intestinal samples’TGEV fleetly.The following applications were built using this method:1. the detection of the 80 clinical samples from Sichuan Province, Chongqing and Shandong was performed using both one-step RT-PCR that our laboratory set up and the method we constructed. 8 samples were positive by the conventional PCR while 15 samples were positive by the fluorescence quantitative PCR, and all the 8 positive samples by the conventional PCR were also positive by the fluorescence quantitative PCR.2. The virus content testing for kinds of TGEV vaccines.TGEV N gene SYBR Green-I real-time quantative PCR method was successfully estabilished in this study. Employing the real-time RT-PCR assay developed in this study should permit diagnostic laboratories to provide the results of TGE suspect cases in clinic. Moreover, the real-time RT-PCR can also be applied for rapid quantification of TGEV in fecal, vaccine or other samples is needed and have important implications for the development of diagnositic kits. |
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