HCV反向点杂交基因分型方法的建立

HCV Genotyping by Reverse Dot Blot

目的利用反向点杂交技术建立HCV基因分型新方法。方法在HCV5'端非编码区(5'NCR)设计引物和分型探针(1.2.3.6型),采用反向点杂交分型技术对53例HCV RNA阳性(浓度均在102～107IU/mL之间)血清进行分型,并以基因序列分析作为金标准,对新方法的敏感性、特异性和重复性进行评价。结果反向点杂交基因分型新方法检出的基因型有52例:1b型32例占60.38%,2a型4例占7.55%,6a型8例占15.09%,3b型8例占15.09%;未检出的基因型有1例,用基因序列分析也未能确定型別(失败的原因应是该HCV RNA扩增产物浓度偏低2.24×102IU/mL)。本研究的方法敏感性为98.1%,特异性为100%,随机抽取20份标本再次检测的结果与前次检测的结果完全一致,重复性好。结论相对现有的型特异性PCR分型、基因芯片、核酸序列分析等各种方法,反向点杂交技术用于HCV基因分型是一种准确有效和简便经济的方法。

Objective To develop a new HCV genotyping method using the reverse dot blot technique . Methods Primers and subtype probe （1.2.3.6 type） specific for the UTR at the 5＇ （5＇NCR） regions were designed and used in this study. 53 cases of HCV RNA-positive （concentrations were between 10^2-10^7 IU/mL） samples were then analyzed using reverse dot blot and sequencing techniques. The sensitivity, specificity and reproducibility of this new method were then evaluated. Results Genotypes were positively identified in 52 samples with this new genotyping method. 32 samples were of lb-type （60.38%）, 4 samples were 2a-type （7.55%）, 8 samples were 6a-type （15.09%） and 8 samples were 3b-type （15.09%）. The genotype of one of the samples was not confirmed as the concentration of the PCR products was too low （2.24×10^2 IU/mL）. The sensitivity of this detection method was 98.1% and the specificity was 100%. Twenty samples were randomly selected and retested with this method, and the results were reproducible. Conclusion Compared with the type-specific PCR typing, gene chip and gene sequence analysis methods, reverse dot blot technique is accurate, simple and cost effective in the identification of HCV genotyping.