关键词: 多重RT-PCR, PEDV, PEV-9, PKV, 检测

Abstract:

The aim of this study was to establish a triplex for simultaneously detecting porcine epidemic diarrhea virus (PEDV)，porcine enterovirus-9 (PEV-9)，and porcine kobuvirus (PKV).Two pairs of special primers were designed according to the published sequences of PEDV，PKV in GenBank，one primers of PEV-9 were synthesized according to reference.A mixture of 3 paris of primers was used for amplification of viral nucleic acids，yielding 3 different amplicons with sizes of 681 bp，454 bp，and 313 bp for PEDV，PKV and PEV-9 respectively by optimizingthe reaction condition.Testing of the sensitivity of multiplex RT-PCR indicated that the lowest detection limits were 5.97 pg for PEDV，0.11 pg for PEV-9，0.74 pg for PKV，respectively.For the specificity test，all of pathogens in the negative controls were found no amplicons.A total of 46 specimens from piglets with acute diarrheas collected from Sichuan province were tested by triplex RT-PCR，and the positive rate were as follows：PEDV 76.1%，PKV 39.1%，PEV-9 73.9%，and the positive accordance rate between simplex and triplex PCR of PEDV，PEV-9，PKV were 100%，91%，and 94% respectively.In addition，the fact that three pathogens were detected simultaneously in one sample indicated mixed infection in the diarrhea disease.It was also suggested that the pathogenic mechanisms of three kinds of pathogens is somehow synergetic.This study showed that the triplex RT-PCR may be a useful tool for rapid and sensitive etiological diagnosis for acute viral diarrheas in piglets and provides an effective technical support for pathogenic molecular epidemiology investigation.Besides，it established the foundation that we can further explore the relationship between the traditional diarrhea pathogens and potential new intestinal pathogens.

Key words: triplex RT-PCR, PEDV, PEV-9, PKV, detection