Detection And Identification Of Five Porcine Diarrhea Viruses In Clinical Samples By Multiplex Gel Based-PCR And Evagreen Based-real Time PCR Assays

Recently porcine diarrhea of high morbidity and morbidity spread in china has caused a great damage to pig industry. Five diarrhea association viruses, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine group A rotaviruses (GAR), porcine group C rotaviruses (GCR) and porcine circovirus 2 (PCV2), have been reported to be the major or high risk causes of diarrhea disease. Mixed infections were found to be common in pig herd, which make diagnosis of the disease more difficult. Tradition methods have disadvantages to diagnose mixed infection. In the present study gel based multiplex polymerase chain reaction (multiplex PCR) and EvaGreen based multiplex real-time PCR assays have been established to detect and identify these porcine diarrhea associated viruses respectively.Gel based multiplex PCR was first developed. By downloading sequence from GenBank and aligning them by Megalign software, each primer pairs were designed from the conservative region of each virus. The assay produced amplification products of 126 bp, 242 bp, 319 bp, 394 bp and 508 bp for GCR, GAR, TGEV, PEDV and PCV2, respectively, which could be easily differentiated on gel electrophoresis,while no amplicon was produced with non-target viruses, demonstrating high specifity of the assay. After optimization of the reaction system and reaction condition,the sensitivity was up to 5×100 copies,5×101 copies,5×100 copies,5×100 copies and 5x100 copies for GCR, GAR, TGEV, PEDV and PCV2, respectively in single PCR and 500 copies for all viruses in the multiplex PCR. These results revealed this assay was a specific, sensitive detection approach for clinical diagnosis of both single and mixed infections of porcine diarrhea associated viruses.One hundred and seventy-two clinical specimens were tested for the five viruses using by Gel based PCR assay. The positive rates detected by the multiplex PCR were 5.81% for GCR, 2.32% for GAR, and 8.72% for TGEV, 20.92% for PEDV and 46.51%for PCV2. In addition, co-infection was detected in 22.09% of the samples, of which 16.86% was positive for PEDV and PCV2. Among them, 64 samples were also tested by singleplex PCRs. Similar positive rates were obtained between the single and multiplex PCR assays. The data indicate that the developed multiplex PCR is a useful tool for clinical detection and identification of porcine diarrhea associated viruses.Primers for the EvaGreen based real-time PCR were designed with special consideration of Tm value of amplicon for each viruse which should be at least 2?difference. Results showed the Tm value of TGEV, GAR, GCR, PEDV and PCV2 amplicon is 75.07 ± 0.15?,77.77 ± 0.06?,79.63 ± 0.25?,83.60 ±0.17? and 88.63 ± 0.06?,similar with the multiplex qPCR Tm value. The variability of the intra-assay and inter-assay was tested using triplicate assays of standards. triplicate assays showed the coefficients of variation (CV) was less than 0.5% for Tm,3.5% for Ct, respectively. The Tm differences of these five viruses could be easily differentiated on melt curve. No specific melting peak was generated with nontarget viruses or negative control. The sensitivity of the EvaGreen based real-time PCR was 5 copies,5 copies,5 copies, 5 copies and 50 copies of TGEV,GAR, GCR,PEDV and PCV2 in single PCR assays, and 5 copies, 5 copies, 50 copies, 5 copies and 50 copies of above viruses in the multiplex PCR with single template. In addition, the sensitivity of the multipllex assay was 50 copies with all five viral templates present. The results reveal these assays are specific, sensitive and repeatable for detection of these five viruses.The developed EvaGreen based real-time PCR was then applied to detect 172 clinical samples for these five viruses. The positive rates were 5.23% for TGEV, 3.48%for GAR, and 7.56% for GCR, and 25.58% for PEDV, and 44.76% for PCV2. In addition, co-infections were detected in 22.66% of the samples. Among them PEDV and PCV2 co-infection rates (18.02%) were the highest. Ninety samples were also tested with singleplex real-time PCRs and similar result was obtained. These data indicat that the assay could be used for epidemiology of these porcine diarrhea associated viruses.