Development And Application Of SYBR Green Ⅰ And Taq-Man Fluorescence Quantitative Real Time PCR For Detection Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea(PED) caused by porcine epidemic diarrhea virus(porcine epidemic diarrhea virus, PEDV) is a highly contagious intestinal disease, characterized with diarrhea, vomiting, dehydration and even death. All ages of pigs are susceptible and the mortality rate of the neonatal piglets affected with PED can be as high as 100%, Presently, the widely used diagnostic methods for detection of PEDV include RT-PCR, immunohistochemistry, serological tests, virus isolation and identification, electron microscopy and ELISA, However, these methods aforementioned have some disadvantages in terms of specificity and sensitivity. Fluorescence-based quantitative real time PCR is a fast and accurate assay with very high specificity and sensitivity which can accurately quantify a few nucleic acid copies of pathogens, and this method can be used for a early diagnosis and assessment of severity degree of the infectious diseases and thus to help us take timely intervention measures for the prevention and control of the disease.To establish two kinds of q PCRs for detection of PEDVs, a pair of specific primers, a probe specific to the minor groove binder(MGB) and a corresponding specific primers for amplification of MGB were designed based on the most conserved regions of PEDVs after a multialignment analysis. Via RT-PCR amplification, cloning and sequencing, one SYBR Green-based and one Taq-Man-based qPCR were established and evaluated. The results showed that the two kinds of qPCRs had good specificities and there were no cross reations with common intestinal pathogens, including PRRSV, CSFV, TGEV and, both methods had high sensitilities, The detection limit of SYBR Green I-based q PCR and Taq-Man-based qPCR were 103 copies/μl and 10 copies/μl respectively. The two qPCRs established also had good reproducibilities The statistical analysis of detection results showed that the coefficients of variation of intra and inter groups tested by SYBR Green I qPCR were 0.19%- 0.53% and 0.3%- 2.1%, resepectively.The results tested by using these two methods in a survey for PEDV infection indicated that was higher than conventional endpoint RT-PCR(76.8%) that the positive rates for PEDV determined by SYBR Green I qPCR and Taq-Man qPCR were 92.8% and 100%, respectively which was much higher among Jiangxi diarrheal piglets demonstrated.By utilizing the Taq-Man qPCR method established, one-step growth curve of PEDV CV777, a vaccine PEDV strain, on Vero-81 cells, was measured. The results indicated that inoculations with different MOI had similar growth cruve. The replication rate of the virus post infection(PI) of 0-24 hr was no significant differences at PI of 24-48 hr displayed an exponential increase, at PI of 48-72 hr showed a decreased replication rate, at PI 72-96 hr, the replication rate of the virus reached the plateau, and at PI of 96-120 hr, the replication rate started decreasing. The results provided useful information in respect of the growth pattern of vaccine strain of CV777 virus on Vero-81 cells, and laid a foundation for identification and characterization of wild-types of PEDVs which will be benefiting the development anad production of PEDV vaccines.