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建立一种能对MHV_1、MHV_3、JHM、A_(59) 4种常见小鼠肝炎病毒(Murine Hepatitis Virus,MHV)进行分型检测的SNaPshot新方法。根据MHV 4种常见毒株基因序列比对结果,设计内外两对PCR通用引物和4个单碱基延伸引物,提取MHV 4种常见毒株RNA,逆转录后进行PCR扩增,纯化扩增产物,用SNaPshot方法进行单碱基延伸,将产物进行毛细管凝胶电泳,根据电泳结果分析毒株基因型。优化SNaPshot分析条件,进行灵敏度、特异性分析。用ELISA法和SNaPshot方法检测41例小鼠(Mus musculus)血清样本,将阳性样本进行克隆测序检测。当T1~T4引物修饰的poly T的数量分别为0、3、10和15,其浓度比为4︰6︰5︰10,引物大小分别为16 bp、19 bp、26 bp和31 bp时,SNa Phot分型检测MHV c DNA的最低浓度为1.25 mg/L,特异性为100%,与ELISA和T-克隆测序比较,其准确性为100%(41/41),阳性样本均为JHM毒株。实验结果说明,所建立的SNaPshot检测方法能对MHV_1、MHV_3、JHM、A_(59)进行分型检测,并且具有灵敏、特异、准确的优点。
To establish a new SNaPshot method to detect the MHV_1, MHV_3, JHM and A_ (59) four kinds of common Murine Hepatitis Virus (MHV). According to the results of sequence comparison of four common MHV strains, four pairs of PCR universal primers and four single-base extended primers were designed, and four common strains of MHV RNA were extracted, reverse transcribed and then amplified by PCR, and the amplified products , Using SNaPshot method single base extension, the product was subjected to capillary gel electrophoresis, according to the electrophoresis analysis of genotypes. Optimize SNaPshot analysis conditions for sensitivity and specificity analysis. The serum samples of 41 Mus musculus were detected by ELISA and SNaPshot methods, and the positive samples were cloned and sequenced. When the number of poly T modified by T1 ~ T4 primer was 0, 3, 10 and 15 respectively, the concentration ratio was 4︰6︰5︰10, and the primer size was 16 bp, 19 bp, 26 bp and 31 bp respectively. The lowest concentration of MHV c DNA detected by SNa-Phot typing was 1.25 mg / L and the specificity was 100%, which was 100% (41/41) compared with ELISA and T-cloning sequencing. The positive samples were all JHM Strain. The experimental results show that the established SNaPshot detection method can detect MHV_1, MHV_3, JHM and A_ (59) and has the advantages of sensitivity, specificity and accuracy.