同时检测小鼠微小病毒(MVM)和小鼠细小病毒(MPV)的荧光定量PCR的建立及应用

目的 建立同时检测小鼠微小病毒(MVM)和小鼠细小病毒(MPV)的荧光定量PCR方法，并进行初步应用。方法 比对NCBI上发表的MVM和MPV基因组序列，设计1对引物和探针，可同时检测MVM和MPV。考察MVM-MPV引物探针的特异性和灵敏度，并对178份清洁级小鼠粪便DNA样本进行检测。结果 MVM-MPV荧光定量PCR方法最佳线性范围为109～104拷贝/μL，标准曲线的线性关系良好，R2值可达0.99，灵敏度为101拷贝/μL，特异性强。应用MVM-MPV探针对178份小鼠粪便DNA检测，结果为2份阳性样本，经MVM、MPV特异性探针鉴定，2份阳性样本均为MPV感染。阳性样本经全基因组测序后与NCBI网站上MPV(NC_001630.1)序列比对，一致率为96%。结论 建立的MVM-MPV荧光定量PCR方法，能够有效快速地同时检出小鼠细小病毒和小鼠微小病毒。

Establishment and Application of the Real-time PCR for Detecting Minute Virus of Mouse (MVM) and Mouse Parvovirus (MPV) Simultaneously in Laboratory Mice

Objective To establish and apply the real-time PCR for detecting minute virus of mouse (MVM) and mouse parvovirus (MPV) simultaneously in laboratory mice. Method According to the genome sequences of MVM and MPV from NCBI, primers and probes were designed. The linearity, specificity, and sensitivity of the method have been investigated. 178 mouse faeces DNA were detected by the real-time PCR. Result The real-time PCR showed a linear range of 1×109-1×104copies/μL, high specificity, which showed no cross-reaction with other parvovirus. The sensitivity of real-time PCR was 101copies/μL. The result of 178 clean grade mouse faeces by PCR screening were that two out of them were MPV positive. After the whole genome sequencing and blasting with NCBI (NC_001630.1) sequence, the agreement rate was 96%. Conclusion The real-time PCR method detecting MVM and MPV simultaneously were successfully established, which could be applied to routine testing.