Establishment And Application Of A Multiplex RT-PCR For Viral Diarrhea Of Porcine And Isolation Of Porcine Rotavirus

In winter, porcine epidemic diarrhea virus(PEDV),porcine transmissble gastroenteritis virus(TGEV) and porcine group A rotavirus(PGAR) are major agents in viral diarrhea of pigs.the three viruses induce similar clinical signs,lesions and epidemiology. To develop a multiplex reverse transcription polymerase chain reaction (mutiplex RT-PCR) for detection of porcine epidemic diarrhea virus (PEDV), porcine transmissble gastroenteritis virus(TGEV) and porcine group A rotavirus(PGAR). Three pairs of primers targeting the M gene, N gene, VP7 gene of PEDV, TGEV, PGAR were designed respectively. Using the three pairs of primes, A multiplex reverse transcription polymerase chain reaction (mutiplex RT-PCR) was developed.And we compared the mutiplex RT-PCR with routine RT-PCR in the field trail.In the laboratory test, we found that the detection limit of multiplex RT-PCR is 35 pg RNA of TGEV-PEDV-PGAR vaccine. And in the field trail, a total of 75 fecal specimens from pigs with diarrhea were collected in the central area of China. The relative sensitivity and specificity of multiplex RT-PCR were evaluated. The results suggesting that this assay is equal in quality to routine RT-PCR assys (sensitivity: 92%,100%,100% for PEDV, TGEV, PGAR respectively; specificity:100% for all 3 viruses). The results indicated that the multiplex RT-PCR with high sensitivity and specificity provided a new and alternative tool for the detection of PEDV, TGEV and PGAR.A tatal of 197 fecal samples from swine with diarrheal were collected from HuBei, HeNan, HuNan, JiangXi and GuangXi and tested by a mutiplex RT-PCR that can detect PEDV, TGEV and PGAR. Fifty-eight (29.44%) infections were caused by PEDV, one (0.51%) infection was caused by TGEV and fifteen (7.61%) infections were caused by PGAR. In the PEDV and PGAR positive samples, the suckling piglets,the weaning or growing pigs and the sow accounts for 24.14%,55.17%,20.69% and 13.33%,6.67%, 80.00%. TGEV was only detected in the suckling piglets. The results indicated that porcine epidemic diarrhea virus (PEDV) and porcine group A rotavirus(PGAR) are major agents in enteric diseases of pigs.Three strains of porcine group A rotavirus(PGAR) named as TM-a, WH-a and GX-a were isolated from fecal specimens of pigs with diarrhea by using MA 104 cell, and produced obvious cytopathic effects. And these viruses were identified by routine RT-PCR.Tow pairs of primers were designed according to VP6 and VP7 sequences of porcine group A rotavirus in GenBank, and VP6 and VP7 gene were amplified by RT-PCR and cloned into pMD18-T vectors. Gene sequences analysis showed that VP6 and VP7 gene sequences of TM-a were more closely related to human rotavirus R479 isolated from WuHan and human rotavirus RMC321 isolated from India; VP6 and VP7 gene sequences of WH-a were more closely related to human rotavirus LL3354 isolated from BeiJing and human rotavirus RMC321 isolated from India; VP6 gene sequence of GX-a was more closely related to porcine rotavirus CMP74/01 isolated from Thailand. These indicated that rotavirus was diversity and complexity.