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

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