反向点杂交法检测广东地区丙型肝炎病毒基因型和基因亚型

A novel method for hepatitis C virus genotyping using RT-PCR reverse dot blot hybridization technique

目的建立并应用HCV基因分型技术PCR-反向点杂交法(PCR-RDH),调查广东地区丙型肝炎病毒(HCV)基因型和亚型的分布情况。方法应用生物信息学软件针对HCV5'端非编码区(5'UTR)和核心蛋白区(C区)设计特异性捕获探针及生物素标记引物,建立HCV基因分型的PCR-反向点杂交技术。应用本技术对115例慢性丙型肝炎患者血清标本进行HCV基因型和亚型检测,同时对其中38份标本中的HCV进行RT-PCR扩增、测序、系统进化树分析确定HCV基因型和亚型,以评价反向点杂交法的准确性及临床应用价值。结果 115份血清标本中,反向点杂交法HCV基因型及亚型检出率为96.5%(111/115),15份阴性对照全部为阴性。111例检出基因型的标本中1b型63例(56.8%)、2a型9例(8.1%)、3a型4例(3.6%)、3b型6例(5.4%)、6a型28例(25.2%)、1b/2a混合型1例(0.9%)。经测序分型确定此法检测准确度为100%,特异度为100%。结论 HCV基因型反向点杂交法检测准确可靠、简便经济、高效,适用于临床检测。广东地区的HCV基因型分布以1b型为主,呈现出1b型比例下降,3a、6a型比例升高的趋势。

Objective To develop a rapid and specific method for hepatitis C virus（HCV） genotyping using reverse dot blot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong.Methods The primers and the probes targeting the 5＇untranslated region（5＇UTR） and core region of HCV genotypes 1b,2a,3a,3b and 6a were designed,and the RT-PCR reverse dot blot hybridization（PCR-RDH） method forHCV genotyping was established.A total of 115 patients with hepatitis C were genotyped using this method,and 38 of them were also genotyped by sequencing and phylogenetic analysis to evaluate the accuracy and specificity of the method.Results Of the 115 patients,111 were successfully genotyped to be 1b,2a,3a,3b,6a and mix-infection of 1b/2a at frequencies of 56.8%,8.1 %,3.6%,5.4%,25.2% and 0.9%respectively,and all the 15 healthy control samples showed negative results.The accuracy and reliability of the genotyping method of PCR-RDH was confirmed in 38 cases by amplification of HCV core and NS5B regions followed by DNA sequencing and phylogenetic analysis.Conclusions This method for HCV genotyping,with high reliability and specificity,is suitable for clinical and epidemiological investigations.The prevalence of HCV genotypes 1b and 2a decreases while 1b remains the dominant genotype in Guangdong,where the prevalence of 6a significantly increases as compared with that 10 years ago.