Abstract: Objective　To establish a duplex real-time fluorescent RT-qPCR assay that can simultaneously detect mouse norovirus and rotavirus, and validate its method ology.Methods　By comparing the genome sequences of MNV and MRV published by NCBI, conservative sequences of MNV VP1 gene and MRV NSP5 gene were selected to design primer probes. By optimizing the concentration of primers and probes, a Taqman real-time fluorescence RT-qPCR assay was established to simultaneously detect mouse norovirus and rotavirus in one reaction. To verify the quantitative range and minimum detection limit of the double and single method using RNA standards of MNV and MRV; the RNA standards for MNV and MRV were diluted at 101-105 copies/μL each, and equal volume mixed together to form a concentration matrix to verify whether different concentrations of MNV and MRV interfere with quantitative result. To verify the reproducibility of the assay by detecting RNA standards of 101,103,105copies/μL inter and intra batch. To apply the assay by testing 544 mouse fecal samples. At the same time, 89 identical mouse fecal samples were tested using conventional RT-PCR, and the differences between the two method were compared.Results　The established duplex RT-qPCR assay can achieve effective quantification of MNV and MRV in the range of 101-108copies/μL. All parameters of the standard curve were within the normal range: R2 value is 0.999, slope (MNV-3.332, MRV-3.250), amplification efficiency (MNV 99.6%, MRV 103.1%). The standard curve of the simplex assay was similar to that of the duplex assay, and the differences in parameter values were not significant; the minimum detection limits were as follows: both MNV simplex and duplex assays had the same detection limit of 3.125 copies/μL, while MRV simplex and duplex assays had a detection limit of 6.25 copies/μL and 3.125 copies/μL, respectively. The coefficient of variation of Ct values for each concentration within the batch was 0.63%-1.24%, and the coefficient of variation between batches was 3.60%-5.57%. The interference test showed that the detection of low concentration standard samples (MNV101-103copies/μL,MRV 102及103copies/μL) was susceptible to interference from high concentration templates,resulting in higher quantitative values. To detect clinical samples of mouse feces, the positive rate of MNV was 1.8% (10/544), and no positive samples were detected for MRV. 10 of 90 identical mouse fecal samples were tested positive for MNV by duplex real-time fluorescent RT-qPCR and 8 samples were positive by conventional RT-PCR. One sample was tested positive for MRV by both assays and there was no statistical difference in the result between the two assays.Conclusion　The duplex real-time fluorescence RT-qPCR asssy can be used for rapid and efficient routine health monitoring of MNV and MRV in experimental mice. 

Key words: murine norovirus, mouse rotavirus(epidemic diarrhea virus of infant mice), duplex real-time fluorescent RT-qPCR

摘要： 目的　建立可同时检测小鼠诺如病毒和轮状病毒的双重实时荧光RT-qPCR检测方法并对其进行方法学验 证。方法　通过比对NCBI公布的小鼠诺如病毒(MNV)及小鼠轮状病毒(MRV)基因组序列,分别选取MNV VP1 基因及MRV NSP5基因保守序列设计引物探针,通过优化引物及探针浓度,建立可在一次反应中同时检测小鼠诺 如病毒和轮状病毒的Taqman实时荧光RT-qPCR检测方法。使用MNV及MRV的RNA标准品验证双重及单重方 法的定量范围及最低检测限;MNV及MRV RNA标准品各设10¹~10⁵ copies/μL 等5个浓度,等体积混合形成浓度 矩阵,单因素方差分析比较不同浓度的MNV及MRV之间是否会对定量结果产生干扰;对10¹,10³,10⁵ copies/μL 3 个浓度的RNA标准品进行批间及批内检测,验证方法的重复性;通过检测544份小鼠粪便样品验证方法实际应用 性能,同时用普通RT-PCR法检测相同的90份小鼠粪便样品,卡方检验比较两种方法结果的差异。结果　建立的 双重RT-qPCR方法可在10¹~10⁸ copies/μL范围实现对MNV及MRV的有效定量,标准曲线各参数均在正常范围: R2值均为0.999,斜率(MNV -3.332,MRV -3.250)、扩增效率(MNV 99.6%,MRV 103.1%)、单重法标准曲线与双 重法相似,各参数值差别不大;最低检测限分别为:MNV单重及双重法均为3.125 copies/μL,MRV单重法为 6.25 copies/μL,双重法为3.125 copies/μL;批内各浓度Ct值变异系数为0.63%~1.24%,批间变异系数为3.60%~ 5.57%;干扰实验表明低浓度标准品的检测(MNV10¹~10³ copies/μL,MRV 10²及10³ copies/μL )易受到高浓度对 应模板的干扰,导致所测定量值偏高;用所建立的双重RT-qPCR检测临床样品,结果MNV阳性率为1.8%(10/ 544),MRV未检出阳性样品。检测相同的90份小鼠粪便样品,双重实时荧光RT-qPCR检出10份MNV阳性样品, 普通RT-PCR可检出8份;两种方法均可检出1份MRV阳性样品,经卡方检验两者结果无统计学差异。结论　本 研究所建立的双重实时荧光RT-qPCR方法可用于实验小鼠中MNV及MRV的快速高效的常规健康监测。

关键词: 小鼠诺如病毒, 小鼠轮状病毒(乳鼠流行性腹泻病毒), 双重实时荧光定量反转录聚合酶链式反应