Foundation And Application On The Method Of Simultaneous Detection Of Porcine Circovirus Type2and Porcine Respiratory And Deproductive Syndrome Virus By One-Step Multiplex RT-PCR

Porcine circovirus type2(PCV-2) and Porcine respiratory and reproductive syndrome virus (PRRSV) Co-infection was been reported in many countries in the world. Porcine multisystem wasting syndrome (PMWS) was one of the typical disease caused by the PCV-2and PRRSV Co-infection. PCV-2was the cause for PMWS, but the simple infection by PCV-2could not cause PMWS. Other pathogenic also played an important role in the development of PMWS, e.g. PRRSV. PMWS which had high morbidity and mortality broke out fast and suddenly and spread widely. It will bring a huge loss to worldwide swine breeding, and has caused extreme attention of the experts. In clinical, it is difficult to identify the pathogeny which cause the PMWS by pathological change. Technology of molecular biology provides a specific and sensitive diagnostic method, e.g.PCR. But normal PCR only can amplify the nucleic acid of suspicious samples by the specificity primer. So it need long time and more cost for detection. As far as the complex of co-infection of PMWS is concerned, a multiplex PCR which can detect many kinds of virus in a reaction system is need to be designed.Two pairs of primers (PH1/PH2and PT1/PT2) were designed according to the conservative sequences of PCV-2ORF2and PRRSV ORF5respectively, and has established a specific single-PCR. PCV-2DNA and PRRSV RNA were extracted from the mixed sample of PCV-2and PRRSV culture by the virus genome DNA/RNA extraction kit, and amplified by the one-step multiplex RT-PCR using the two sets of primers mentioned above. The results showed that the two specific frangments of560bp PCV-2and398bp PRRSV were amplified, but negative results were obtained with other six porcine pathogens. The sensitivity assay indicated that10ng DNA of PCV-2and5 ng RNA of PRRSV could be detected using this method.30suspicious samples were detected by the one-step multiplex PCR method, and the results were in consistent with that of the normal PCR. So one-step multiplex PCR method established is specific, sensitive and stable for the rapid detection of PCV-2and PRRSV. The DNA and RNA of virus were extracted at the same time by this method, and the DNA and RNA were amplified meantime in one reaction tube. This method can save more cost and time, so it can be spread to the differential diagnosis for DNA and RNA virus Co-infection. This mode has active sense for advancing the application of multiplex PCR on veterinary.