PEDV、TGEV、PoRV和PKV多重RT-PCR检测方法的建立及初步应用

为快速鉴别诊断猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PoRV)和猪嵴病毒(PKV),根据PEDV的M基因、TGEV的N基因、PoRV的VP6基因和PKV的3D基因序列设计4对特异性引物,通过PCR扩增目的片段并构建重组质粒,建立了一种可同时检测4种病毒的RT-PCR诊断方法,该方法可特异性扩增这4种病毒的相应片段,而对猪瘟病毒(CSFV)、猪圆环病毒2型(PCV-2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)均无扩增,最低检出量分别为1.33×10^4、1.33×10^3、1.33×10^4、1.33×10^5copies/μL。应用该方法对临床55份猪腹泻样品进行检测,结果检测出14份PEDV、1份PoRV和27份PKV,未检出TGEV,其中PEDV和PKV混合感染9份。上述结果表明,建立的多重RT-PCR检测方法快速、特异、敏感,可用于以上4种腹泻病毒的临床检测和流行病学调查。

Establishment and Application of A Multiplex RT-PCR Method for Detecting PEDV,TGEV,PoRV and PKV

In order to rapidly detect porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine rotavirus(PoRV)and porcine kobuvirus(PKV)simultaneously,a multiplex RT-PCR method was established in this study by designing four pairs of specific primers targeting the M gene of PEDV,N gene of TGEV,VP6 gene of PoRV and 3 Dgene of PKV,amplifying the target fragment and constructing recombinant plasmid.The results showed that the target fragments of these four viruses were amplified,but no amplification of classical swine fever virus(CSFV),porcine circovirus type 2(PCV-2),porcine reproductive and respiratory syndrome virus(PRRSV)and pseudorabies virus(PRV).The minimum detection concentrations of PEDV,TGEV,PoRV and PKV were 1.33×10^4,1.33×10^3,1.33×10^4,1.33×10^5 copies/μL,respectively.A total number of 55 fecal samples from piglets with diarrhea were tested by the established multiplex RT-PCR,of which 9 samples are PEDV positive,0 sample is TGEV positive,1 sample is PoRV positive,and 21 samples are PKV positive.The above results indicated that the multiplex RT-PCR detection method established in this study is rapid,specific and sensitive,and can be used for the clinical detection and epidemiological investigation of the above four diarrhea viruses.