Establishment Of Triplex Fluorescence Quantitative RT-PCR For Identification Of TGEV、PEDV And PoRV

Transmissible gastroenteritis virus (TGEV), Porcine epidemic diarrhea virus (PEDV) and Porcine rotavirus (PoRV) are major etiological reasons of diarrhea and death in piglets. These diseases have similar clinical signs, such as watery diarrhea and dehydration. So, it’s difficult to differentiate of these diseases. In the early stage of veterinary research, Several laboratory diagnosis in pigs are made by virus Isolation, IFA, and ELISA etc., but these methods have some disadvantages of costing long time, low sensitivity and complex. With the development of molecular biology, some reserchers established polymerase chain reaction (PCR) to detect these diseases, which reduced the detection time and improved the detection sensitivity, but these methods can only achieve qualitative detection for nucleic acid and it is hard to achieve accurate quantitative. Therefore, the fast and accurate dfferential detection is required for etiologic diagnosis. This study was aimed to establish a sensitive,specific and reproducible triplex real-time RT-PCR that can differentiate TGEV, PEDV and PoRV.Primers and TaqMan hydrolysis probes were designed against the sequences in M of TGEV, M of PEDV and VP2 of PoRV. Three different amplicon with size of 834 bp,499 bp and 378 bp for TGEV, PEDV and PoRV respectively. The recombinant plasmids pMD-TGEV, pMD-PEDV and pMD-PoRV were constructed respectively. The postive recombinant plasmid were identified by PCR and sequenced. After optimization of the detection conditions,established three single fluorescent RT-PCR.On the basis of the single fluorescent RT-PCR, after optimization of the detection conditions, established a dual fluorescence RT-PCR for discrimination of TGEV, PEDV and PoRV. The minimal detection limits of TGEV, PEDV and PoRV were 2.49 copies/μL,4.36 copies/μL,4.96 copies/μL. The maximum variation coefficient of TGEV within group was2.5%, the maximum variation coefficient between group was 3.7%, the maximum variation coefficient of PEDV within group was 3.8%, the maximum variation coefficient between group was 3.4%, the maximum variation coefficient of PoRV within group was 4.3%, the maximum variation coefficient between group was 3.2%, no more than 5%, showed that the method had good repeatability; The method was applied to detect several viruses, such as classical PRRSV, PCV,, PRV. There were no specific reaction, showed that the method had high specificity.40 pathological sample were tested by the established method. The fluorescence RT-PCR for detection of TGEV detected 2 positive, positive rate is 5%; The fluorescence RT-PCR for detection of PEDV detected 12 positive, positive rate is 30%; The fluorescence RT-PCR for detection of PoRV detected 5 positives, positive rate is 12.5%. Besides, The fluorescence RT-PCR for detection of TGEV and PEDV detected 1 positives, positives rate is 2.5%; The fluorescence RT-PCR for detection of PEDV and PoRV detected 2 positives,positives rate is 5%; 40 pathological sample were tested by the dual fluorescence RT-PCR method, the results are consistent with the single fluorescent RT-PCR method, showed the established method had great value in clinical application.