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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 establish and use the HCV genotyping technique PCR-reverse dot blot (PCR-RDH) to investigate the distribution of hepatitis C virus (HCV) genotypes and subtypes in Guangdong Province. Methods The bioinformatics software was used to design a specific capture probe and biotin-labeled primer for the 5’UTR and the core protein region (C region) of HCV. A PCR-reverse dot blot of HCV genotype was established technology. HCV genotypes and subtypes were detected in 115 serum samples of patients with chronic hepatitis C by using this technique. HCV in 38 samples of them was amplified by RT-PCR, sequenced and phylogenetic tree analysis was used to determine the HCV genotype and subtype Type, in order to evaluate the accuracy of reverse hybridization and clinical value. Results Among 115 serum samples, the detection rate of HCV genotype and subtype was 96.5% (111/115) by reverse hybridization, and all 15 negative samples were negative. Among the 111 genotypes, 63 were type 1b (56.8%), 9 (8.1%) were type 2a, 4 (3a) were type 3 (3.6%), 6 were type 3b 25.2%), 1b / 2a mixed type in 1 case (0.9%). After sequencing, the accuracy of this method was 100% and the specificity was 100%. Conclusion The HCV genotype reverse spot hybridization assay is accurate, reliable, simple and economical, efficient and suitable for clinical testing. The genotypes of HCV genotypes in Guangdong are predominantly type 1b, showing a decrease in the proportion of type 1b and an increase in the proportion of type 3a and type 6a.