| 杨光,陈姝,崔金环,司建华,谭家驹.丙型肝炎病毒逆向点杂交基因分型方法的建立及初步应用[J].中华流行病学杂志,2005,26(6):440-443 |
| 丙型肝炎病毒逆向点杂交基因分型方法的建立及初步应用 |
| Development and application of a new hepatitis C virus genotyping method with polymerase chain reaction reverse blot dot technique |
| 收稿日期:2004-09-16 出版日期:2014-09-18 |
| DOI： |
| 中文关键词: 丙型肝炎病毒 基因分型 |
| 英文关键词: Hepatitis C virus Genotyping |
| 基金项目: |
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| 中文摘要: |
| 目的利用逆向点杂交技术建立丙型肝炎病毒(HCV)基因分型新方法。方法在HCV5’端非编码区(5’UTR)设计引物与分型探针,将活性氨基标记的分型探针依次固定在尼龙膜上,制成HCV基因分型逆向点杂交检测膜条。分离纯化血清中的HCVRNA,经逆转录反应和生物素标记引物聚合酶链反应(PCR)巢式扩增后,将扩增产物与检测膜条杂交,过氧化物酶和四甲基联苯胺显色,判断基因分型结果。最后,通过与基因测序结果比较确定新方法的有效性。从佛山地区丙型肝炎患者中随机抽取60份经荧光定量PCR方法对HCVRNA进行过定量分析的血清,用新建方法进行HCV基因分型检测。结果新建HCV逆向点杂交基因分型方法可对所有60份HCVRNA拷贝数在102～106/ml之间的抽检血清进行基因分型,发现1b型50例,占83.3%;1a型2例,占3.3%;2a型2例,占3.3%;1a、1b混合型5例,占8.0%;1b、2a混合型1例,占1.7%;未发现2b、3a和3b型。新方法基因分型结果与测序分型结果一致。结论PCR逆向点杂交技术可以准确有效和简便经济地进行HCV基因分型,适用于临床检测与流行病学研究。在佛山地区人群中感染的HCV以1b型为主。 |
| 英文摘要: |
| Objective Using polymerase chain reaction reverse blot dot (PCR RDB) technique to establish a new method for hepatitis C v irus (HCV) genot yping and to study the distribution of HCV genotypes in Foshan area. Methods HCV primers and pr obes were designed in 5 untranslated reg ion(nt 1 nt 299) of HCV. HCV RNA in ser um was isolated and purified, and its cDNA was obtained by reversed transcription. Nested PCR using biotin labelled primers, was done. PCR products were hybridized with immobilized specific probes (genotype1ato3b) on Biodyne C membrane to genoty pe HCV by color development w hile adding POD and TMB. A certain judgment could be made according to the position of color reaction. The reliability of this new method was verified by sequencing. HCV RNA levels in serum were determined by real time fluor escent quantitative (FQ) PCR. 60 FQ PCR positive HCV sera from Foshan area w ere genotyped using this assay. Results All 60 sera could be successfully genotyped by PCR RBD. 50(83.3%) cases were found to be g enotype 1b,2(3.3%) as g enotype 1a and 2(3.3%) as genotype 2a while 5(8.0%) to be mix ture of genoty pe 1a and 1b, and 1(1.7%) to be mix ture of genotypes 1b and 2a. No genotypes 2b, 3a and 3b were found. T he results of PCR RDB g enotyping methods coincided with sequence analysis. Conclusion Newly established HCV genotyping system was proved to be sensitive, specific, precise and economic, thus suitable for clinical and epidemiologic studies.The results of HCV genotyping showed that genotype 1b was the predominant genotype in Foshan area. |
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