伪狂犬病病毒和猪圆环病毒4型二重SYBR Green Ⅰ荧光定量PCR检测方法的建立

Development of a duplex SYBR Green I-based real-time quantitative PCR assay for detection of porcine pseudorabies virus and porcine circovirus type 4

本研究设计、合成了分别靶向伪狂犬病病毒(PRV)gE基因和猪圆环病毒4型(PCV4)Cap基因保守区域的特异性引物,并在优化反应条件的基础上,建立了能够同时检测和鉴别PRV和PCV4的二重SYBR GreenⅠ荧光定量PCR方法。结果显示,该方法具有良好的线性关系,R2=0.999。在同一个反应体系中,能够特异地检测到PRV和PCV4的特定荧光信号,而其他病原体无信号;对PRV和PCV4的检测限分别为48.5 copies/μL和40.8 copies/μL。利用建立的二重荧光定量PCR方法对34份猪临床样品进行检测,结果显示,PRV检出率为44.12%(15/34),PCV4检出率52.94%(18/34),且二者混合感染的检出率为32.35%(11/34),比常规PCR方法更加灵敏。该方法是检测PRV和PCV4的一种快速、灵敏、特异的方法。

An SYBR green I-based duplex fluorescence quantitative PCR(qPCR)assay was established here to detect porcine pseudora-bies virus(PRV)and porcine circovirus type 4(PCV4)simultaneously based on the optimization of reaction system and amplification condi-tions.The specificity test showed that,in the same reaction system,specific signals were detected for PRV and PCV4,while other pathogens showed no signal.The sensitivity test showed that the detection limits for the recombinant plasmid standards of PRV and PCV4 were 48.5 copies/μL and 40.8 copies/μL,respectively.In this study,34 clinical samples with respiratory diseases and diarrhea were tested by this as-say with the positive rates of 44.12%(15/34)for PRV and 52.94%(18/34)for PCV4,respectively;and the positive rate of PRV and PCV4 co-infection was 32.35%(11/34).The above results indicated that the duplex fluorescence quantitative PCR method established in this study was sensitive,specific and reproducible,which provided technical support for the detection of PRV wild virus and PCV4.