猪伪狂犬病毒Taqman-MGB荧光定量PCR检测方法的建立及应用

Establishment and application of TaqMan-MGB Fluorescence Quantitative PCR for Detection of Pseudorabies Virus

建立可区分猪伪狂犬野毒株及基因缺失疫苗株的TaqMan-MGB荧光定量PCR检测方法。根据猪伪狂犬病病毒gE基因序列,设计一对特异引物和探针,通过优化反应条件,建立了可区分猪伪狂犬野毒株及基因缺失疫苗株的TaqMan-MGB荧光定量PCR检测方法,同时验证该方法的特异性、敏感性、重复性,并对30份疑似病料进行临床检测。本研究所建立的TaqMan-MGB荧光定量PCR检测方法灵敏度可达2.23×10拷贝/μL,比常规PCR检测方法高100倍;与猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)、猪繁殖和呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)均无交叉反应,具有高特异性;对30份疑似病料的TaqMan荧光定量PCR和普通PCR检测阳性率分别为40%和33%,两者符合率90%。该方法灵敏度高、特异性强、重复性好,可同时检测大量样品,可适合于PRV的临床诊断和流行病学调查。

The aim was to establish a Real-time TaqMan-MGB fluorescence quantitative PCR for distinguishing the wild strain and gene-deleted vaccine strain of PRV. A pairs of primers and a TaqMan-MGB probes were designed and synthesized according to the nucleotide sequence of the gE gene of pseudorabies virus （PRV） available in GenBank, and real-time TaqMan-MGB fluorescence quantitative PCR for distinguishing the wild strain and gene-deleted vaccine strain of PRV was established successfully through optimizing the reaction condition. Simultaneously, the diagnostic specificity, sensitivity and repeatability of the assays were verified and the 30 suspected material were detected by the established real-time TaqMan-MGB fluorescence quantitative PCR. It was demonstrated that the established TaqMan-MGB quantitative PCR assay could detect 2.23x 10 copies/μL of plasmid DNA. Its sensitivity was 100 times higher than that of the gel-based PCR, and had no cross reaction with classical swine fever virus （CSFV）, porcine reproductive and respiratory syndrome virus （PRRSV）, porcine cireovirus type 2 （PCV2） and porcine parvovirus （PPV）. The 30 suspected material were detected both by TaqMan-MGB fluorescence quantitative PCR, and gel-based PCR,respectively, the positive detection rate were 40% and 33%, the coincidence rate was 90%. The Real-time TaqMan-MGB fluorescence quantitative PCR assay was specific, sensitive, rapid and accurate that could be used for massive samples detection and the diagnosis of PRV infection.