鉴别牛肠道病毒感染复合PCR方法的建立及初步应用

Establishment and preliminary application of a complex PCR method for differentiating bovine enterovirus infection

牛肠道病毒(bovine enterovirus, BEV)感染是国内近年来报道的一种临床上以消化道、呼吸道症状为特点的新发传染病,其病原体为小RNA病毒科肠道病毒属中的成员。目前,BEV分为EV-E和EV-F 2个病毒种,两者间的核苷酸序列差异很大,属于不同的血清型/基因型,常规血清学方法难以将其区别。为建立一种鉴别EV-E和EV-F病毒的方法,本研究根椐GenBank已发表的EV-E和EV-F肠道病毒的基因组序列,设计合成引物,建立了鉴别EV-E和EV-F的复合PCR方法,并确定了该方法的特异性、敏感性和重复性。同时,应用该方法检测吉林省长春地区疑似BEV感染样品。结果显示,建立的方法具有良好的特异性,只能检测出EV-E或EV-F肠道病毒,而牛细小病毒、牛病毒性腹泻病毒等病原检测均为阴性。敏感性试验结果显示,EV-E和EV-F最低检出量分别为3.67×10^(2),5.21×10^(3)拷贝/μL。检测长春地区的32份牛粪便样品的结果显示,EV-E和EV-F病毒的检出阳性率分别为28.13%和34.38%,两种病毒混合感染率为15.63%,与间接ELISA检测方法结果一致。上述结果表明,本试验建立的复合PCR方法具有特异性强,灵敏度高、快速与简便等特点,可用于EV-E和EV-F两种肠道病毒的鉴别诊断及流行病学调查。

Bovine enterovirus(BEV)infection is a new infectious disease characterized by gastrointestinal and respiratory symptoms reported in China in recent years.The pathogen belongs to member of the genus Enterovirus within the family of Picornaviridae.Currently,two enterovirus species including EV-E and EV-F can infect cattle.These two enterovirus species share relatively low homology of nucleotide sequences and are two different serotypes/genotypes,which is difficult to be differentiated by the conventional methods.In this study,specific primers were designed based on sequence alignment analyses from the EV-E and EV-F strains published in GenBank,and used to establish the complex PCR method for differentiation of EV-E and EV-F enterovirus.Specificity assay showed that the complex PCR can only detect enterovirus,while no fragments were amplified from virus such as foot-and-mouth disease virus,bovine parvovirus,and bovine viral diarrhea virus.Sensitivity assay revealed that the minimum detection amount of EV-E and EV-F was3.67×10^(2)and 5.21×10^(3)copies/μL,respectively.Moreover,EV-E and EV-F species were easily differentiated by the fragment sizes,where fragment size of EV-E and EV-F was approximately604and 288bp,respectively.Detection of 32feces samples collected from Changchun area showed that the positive rates of EV-E and EV-F enterovirus infection were 28.13%and 34.38%,respectively.Furthermore,mixed infection rate of EV-E and EV-F was 15.63%,which was consistent with the ELISA results.In summary,the complex PCR detection method established in this study was specific,sensitive,rapid,and simple,which can be used for differential diagnosis of EV-E and EV-F infection and epidemiological investigation.