结合内标的小鼠诺如病毒荧光定量RT-PCR检测方法的建立及应用

Establishment and application of a fluorescence real-time RT-PCR assay with an internal control for detection of murine norovirus

目的建立一种快速、特异、敏感的荧光定量RT-PCR检测方法,用于小鼠诺如病毒(murine norovirus,MNV)的检测。方法根据MNV ORF1-ORF2结合区域中保守序列设计一对特异性引物和Taqman探针,同时设计和制备了内标用于监控假阴性,建立含有内标的荧光定量RT-PCR检测体系,通过优化,得到最佳反应体系和反应条件;构建质粒标准品并以之为模板绘制标准曲线;进行特异性、敏感性和重复性试验,最后用建立的方法检测344份小鼠临床样本,验证在临床应用中的效果。结果该方法特异性强,与小鼠肝炎病毒、小鼠脑脊髓炎病毒、仙台病毒、小鼠肺炎病毒、呼肠孤病毒Ⅲ型、出血热病毒和淋巴细胞性脉络丛脑膜炎病毒不发生交叉反应。构建的荧光定量标准曲线Ct值与模板浓度呈良好的线性关系相关系数r2=0.9986,可以检测到10 copies/μL的质粒标准品,对MNV活病毒检测可检测到1.78×10-2TCID50/m L的病毒,检测灵敏度比常规RT-PCR高10倍,比病毒分离高100倍。对5份样品进行5次批内和批间重复检测,检测结果变异系数均小于2%。通过对344份临床样品检测,检测到阳性样品103份,阳性率29.94%。有5份样本结果为假阴性。结论建立的MNV荧光定量RTPCR检测方法特异性强、敏感性高、重复性好,由于加入了内标,能有效地监控假阴性的出现,适合用于MNV日常监测、临床诊断和流行病学调查。

Objective To develop a rapid, specific and sensitive fluorescence real-time RT-PCR assay for detec- tion of murine norovirus （ MNV）. Methods A pair of primers and a Taqman probe were designed targeting highly con- served sequences among MNV strains, which are located at the open reading frames 1 （ ORF1 ） -ORF2 junction region. The internal control（IC） was also designed and constructed to determine false-negative results. A real-time RT-PCR assay with the IC was established for MNV detection by optimizing reaction components and conditions. The standard curve was plotted based on the linear relationship between the amount of plasmid DNA and cycle threshold（Ct） values. In addition,the speci- ficity, sensitivity and reproducibility of the assay were evaluated. 344 clinical samples were used to determine the efficacy of this real-time RT-PCR method. Results The specificity test showed that this real-time RT-PCR assay could specifically detect MNV and had no cross reactions with mouse hepatitis virus, Theiler＇ s murine encephalomyelitis virus, Sendal virus, pneumonia virus of mice, reovirus III, Hantaan virus and lymphocytic choriomeningitis virus. The standard curve showed fine linear relationship between the amount of plasmid DNA and Ct values （ correlation coefficient r2 = 0. 9986）. The devel- oped assay was sensitive enough to detect as low as 10 copies/p,L. The lower quantification limit of MNV was estimated as 1.78 ~ 10-2 TCiDs0/mL,which was 10 times more sensitive than conventional RT-PCR method, and 100 times more sensi- tive than virus isolation method. Both the intra-batch and inter-batch coefficients of variation were less than 2% based on 5 repeated intra-batch and inter-batch test of 5 samples. 344 clinical samples were tested by this developed real-time RT-PCR assay and showed a positive ratio of 29.94% （103/344）. Conclusions This real-time RT-PCR assay is a specific, sensi- tive and reproducible assay. Moreover,the internal control in the real-time RT-PCR system can be used to determine false negative results. This assay could be exploited for routine detection, clinical diagnosis and epidemiological survey of mouse norovirus.