摘要
以非洲猪瘟病毒(ASFV)、猪瘟病毒(CSFV)和猪水泡病毒(SVDV)为研究对象,建立可以同时检测和鉴别诊断这3种病毒混合感染的多重荧光RT-PCR方法,并与单荧光RT-PCR进行比较。经荧光RT-PCR标准曲线分析,建立的所有多重和单重荧光RT-PCR扩增效率E值为90%~110%,R2>0.98,扩增效果良好。敏感性分析结果表明,与单重荧光RT-PCR检测限相比,多重荧光RT-PCR的Ct值略有升高,但结果仍可达到1~10拷贝/μL。特异性分析结果表明,所建立的单重和多重荧光RT-PCR方法特异性均为100%,引物和探针只能扩增出各自的目标病原,无交叉反应。ASFV、CSFV和SVDV单重荧光RT-PCR与多重荧光RT-PCR之间的相关系数分别为0.9988、0.9978和0.9976,与单重荧光RT-PCR相比,建立的多重荧光RT-PCR没有产生明显的干扰和敏感性降低现象。对20份CSFV阳性样品、25份ASFV阳性样品进行验证,100%符合;分别从200份血液和300份肉制品中筛查到ASFV阳性8份和10份,并与单重荧光RT-PCR检测结果相吻合。建立的多重荧光RT-PCR方法可以实现1次取样、1次分析,同时检测和鉴别3种病毒的目的,适用于临床样品混合感染的快速筛选。
One multiplex real-time RT-PCR method to detect and differentiate mix-infection of classical swine fever virus(CSFV),African swine fever virus(ASFV)and swine vesicular disease virus(SVDV)was established and compared with its corresponding single real-time RT-PCR method in this study.Analysis on real-time standard curves of RT-PCR showed that all the PCR amplification efficiencies(E)were between 90%-110%,the correlation coefficients(R2)were all over 0.98,and the curve slopes were good.Sensitivity analysis between single and multiplex real-time RT-PCR indicated that the detection Ct value of multiplex real-time RT-PCR was a little higher than that of single RT-PCR,but was still up to 1-10 copies/μL,without significant difference between them.The specificity of the established single and multiple fluorescence RT-PCR methods were 100%,and the primers and probes could only amplify the respective target pathogens.The correlation coefficients between ASFV,CSFV and SVDV were 0.9988,0.9978 and 0.9976 respectively.The established multiplex real-time RT-PCR did not produce significant interference and sensitivity reduction.Twenty positive samples of CSFV and 25 positive samples of ASFV were tested,the newly developed multiplex real-time RT-PCR achieved the anticipated result.Eight ASFV positive samples and 10 ASFV positive samples were screened out from 200 blood samples and 300 meat samples,respectively,and the results were in good agreement with those detected by single real-time RT-PCR.This method can detect and identify three viruses simultaneously on the basis of one reaction,and is suitable for screening for mix-infection of different viruses in clinical samples.
作者
史喜菊
刘艳华
马贵平
李炎鑫
刘全国
赖平安
高志强
徐立伟
SHI Xiju;LIU Yanhua;MA Guiping;LI Yanxin;LIU Quanguo;LAI Pingan;GAO Zhiqiang;XU Liwei(Beijing Customs,,Beijing 100026,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2021年第3期426-431,共6页
Chinese Journal of Veterinary Science
基金
国家重点研发计划资助项目(2016YFD0500705)。
