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赵婷婷,王会娟,王蕾,谭毅,王胜,张道华,赖国旗.2016.检测小鼠肝炎病毒的SNaPshot分型技术及其应用.动物学杂志,51(6):1084-1091.
检测小鼠肝炎病毒的SNaPshot分型技术及其应用
Typing Mouse Hepatitis Viruses Using SNaPshot Assay
投稿时间:2015-06-16  修订日期:2016-08-23
DOI:DOI: 10.13859/j.cjz.201606016
中文关键词:  小鼠肝炎病毒  SNaPshot检测  酶联免疫吸附试验
英文关键词:Murine Hepatitis Virus  SNaPshot assay  Enzyme linked immunosorbent assay
基金项目:重庆市科委应用开发项目(No. CSTC2014YYKFA110033)
作者单位E-mail
赵婷婷 湖北医药学院 zttmtt@sina.cn 
王会娟 重庆医科大学  
王蕾 重庆医科大学  
谭毅 重庆医科大学  
王胜 重庆医科大学  
张道华 重庆医科大学  
赖国旗 重庆医科大学 a68895078@21cn.com 
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中文摘要:
      建立一种能对MHV1、MHV3、JHM、A59 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,SNaPhot分型检测MHV cDNA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MHV1、MHV3、JHM、A59进行分型检测,并且具有灵敏、特异、准确的优点。
英文摘要:
      SNaPshot assay is a single base extension assay which, by extending a base after four primers, displays color of the base through capillary gel electrophoresis to judge the genotype of samples. We aimed to establish a SNaPshot assay to type mouse hepatitis viruses (MHV), including MHV1, MHV3, JHM, and A59. The SNaPshot assay was developed after polymerase chain reaction (PCR) using cDNA from MHV strains two pairs of universal PCR primers and four single-base primers that were designed according to the conserved sequence of the four strains. The PCR products were analyzed by capillary gel electrophoresis. The sensitivity and specificity of the SNaPshot assay were analyzed using gradient dilution method and compared with human Hepatitis B virus (HBV). The accuracy of the SNaPshot assay was analyzed using 41 murine (Mus musculus) serum samples by comparing with ELISA and sequencing the samples containing MHVs. The optimal conditions of SNaPshot assay were that T1﹣T4 primers were 0, 3, 10, and 15 Ts in modification, 4︰6︰5︰10 in concentration, and 16 bp, 19 bp, 26 bp and 31 bp in length. The sensitivity of the SNaPShot assay was 1.25 mg/L (MHV cDNA) (Fig. 3), the specificity was 100% (Fig. 4), and the accuracy was 100% (41/41, Table 3), which was confirmed by ELISA, and the sequencing data. The SNaPshot assay is a sensitive, specific, and accurate method for typing MHV1, MHV3, JHM, and A59.
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