猪主要腹泻病毒多重RT-PCR检测方法的建立及初步应用

Establishment and preliminary application of multiplex RT-PCR method for detection of major porcine diarrhea viruses

为建立检测猪主要腹泻病毒的多重RT-PCR方法,本试验参考GenBank中登录的猪传染性胃肠炎病毒(transmissible gastroenteritis virus of swine,TGEV)M基因、猪萨佩罗病毒(porcine sapelovirus,PSV)VP1基因、猪δ冠状病毒(porcine deltacoronavirus,PDCoV)N基因和猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)N基因的序列,分别设计相应的引物,构建4种病毒的阳性质粒。以阳性质粒为标准品,通过优化反应条件和反应程序,建立了检测猪主要腹泻病毒的多重RT-PCR方法,并确定了该检测方法的特异性、灵敏性和重复性。结果显示,该方法具有较高的灵敏性,对PSV、TGEV、PDCoV和PEDV的最低检测量分别为1.37×10^(2),1.44×10^(2),1.51×10^(2)和1.56×10^(2)拷贝/μL。而扩增猪细小病毒(porcine parvovirus,PPV)、猪瘟病毒(swine fever virus,CSFV)、猪圆环病毒Ⅱ型(porcine circovirus-2)PCV-2、猪繁殖和呼吸障碍综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)和猪伪狂犬病病毒(porcine pseudorabies virus,PRV),结果均呈阴性,具有良好的特异性。重复性试验结果显示,在同等条件下扩增4种病毒的阳性质粒,3次均出现同样的目的条带。应用建立的方法检测了采集的48份腹泻猪的小肠组织和粪便样品,结果检测出2份PSV、7份PDCoV和11份PEDV,与单重RT-PCR检测结果的总符合率为91.66%。同时发现2份PSV和PDCoV混合感染,3份PEDV和PDCoV混合感染,2种检测方法对混合感染检测结果相一致。对2份阳性样品的PCR产物进行测序,结果与检测结果相符合。本试验建立了检测猪主要腹泻病毒的多重RT-PCR方法,为临床检测PSV、PDCoV、TGEV、PEDV和流行病学调查提供了便捷有效的方法。

In order to establish a multiplex RT-PCR method for detection of porcine major diarrhea viruses,the corresponding primers were designed according to the sequences of transmissible gastroenteritis virus(TGEV)M gene,porcine Sapero virus(PSV)VP1gene,porcine deltacoronavirus(PDCoV)N gene and porcine epidemic diarrhea virus(PEDV)N gene registered in GenBank,respectively.The plasmids harbouring the gene fragment of the four viruses were constructed,and used as the standard templates.Through the optimization of reaction conditions and reaction procedures,a multiplex RT-PCR method for the detection of major porcine diarrhea virus was established,and the specificity,sensitivity and reproducibility of the detection method were tested.The results showed that the minimum detection limits of PSV,TGEV,PDCoV and PEDV were 1.37×10^(2),1.44×10^(2),1.51×10^(2) and 1.56×10^(2) copies/μL,respectively.The amplification of porcine parvovirus(PPV),swine fever virus(CSFV),porcine circovirus type II(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),porcine pseudorabies virus(PRV)were all negative when using this multiplex RT-PCR method.The detection method showed a good repeatability for the four viruses amplified three times under the same conditions.These results indicated that the established multiplex RT-PCR method had good sensitivity,specificity and repeatability.Detection of the 48small intestine tissues or feces from pigs with diarrhea using the established method revealed two-positive PSV,seven-positive PDCoV,and eleven-positive PEDV specimens,which showed a total coincidence rate of 91.66%with the single RT-PCR method.In addition,two samples were found as mixed infection of PSV with PDCoV,and three samples as mixed infection of PEDV and PDCoV with consistent detection results for the two detection methods.Sequencing results from two positive samples further confirmed the detection results.The established multiplex RT-PCR method for detection of porcine major diarrhea viruses provided a more effective method for clinical detection and epidemiological investigation on PSV,TGEV,PDCoV and PEDV.