Establishment And Application Of The Multiplex PCR For Three Main Canine Respiratory Tract Viruses

Canine respiratory disease, which occurs less in summer as compared with other seasons of the year, is a common disease in dogs. In this paper, we established a multiplex PCR method for diagnosing three viruses, namely canine distemper virus(CDV), canine parainfluenza virus(CPIV) and canine adenovirus type-2(CAV-2), which all can cause canine respiratory disease.Based on the complete genome of CDV CPIV and CAV-2published on GeneBank, and then using the NCBI blast to get each conserved sequence by conservative analysis:470-1989bp of CDV F gene sequence,0-1658bp of CPIV N gene sequence and the25635-26130bp of the CAV-2complete genome. Extract RNA and DNA of virus by Trizol method from the sample of U.S.Pfizer vaccine, and then reverse the transcripts into cDNA. Then the cDNA and DNA extracted formerly were generated to multiplex PCR. According to which optimal primer combinations were designed. Eventally a multiplex PCR method was established to amplify the CDV(500bp),CPIV(293bp) and CAV-2(655bp) by optimizing the PCR reaction system and process. Taking the diseased nucleic acid of the positive sample of PCR testing to do the experiments proved that the multiplex PCR was highly specific and sensitive.Collect randomly47samples form39cases of dogs with respiratory samples. And detecting them with the method mentioned above. The results are as follows:the percentage of CDV positive cases was20.5%, CPIV and CAV-2positive cases both were5.13%. The results were compared with the dogs cases who suspected got CDV with the test version showing positive. PCR method is validated angle from the nucleic acid of the virus. Which is emerging in recent years and is a molecular biology techniques with several superiorities such as high clinical detection efficiency and according rate with normal detection methods, as well as sampling conveniently. The result of CDV detection with PCR and colloidal gold test strip for statistical analysis with T test and Fisher exact test. The PCR detection rate of CDV and CDV colloidal gold detection rate were analyzed, obtained they were significant difference.The method of conventional test version is validated form the nucleocapsid antigen.The two detection methods applications can greatly improve the diagnosis rate which is operated more fast and much easily of three Combination of these two detection methods applied in clinical can greatly improve the diagnosis rate of dogs infected by these three viruses.