HCV RNA基因分型多色荧光PCR筛查和确认方法的建立

Multiplex real-time reverse transcription-PCR screening and conforming assay for determination of hepatitis C virus genotypes

目的建立多色荧光丙型肝炎基因分型检测方法。方法针对HCV5'NCR区特异性基因设计一对通用引物和Ⅰ/Ⅱ/Ⅲ/Ⅳ型特异性探针,Ⅰ型和Ⅲ型探针标记FAM荧光染料,Ⅱ/Ⅳ型标记VIC荧光染料,分别建立Ⅰ/Ⅱ型和Ⅲ/Ⅳ型两管双色荧光PCR扩增系统。分别采用测序和本研究方法对97份丙型肝炎阳性血清进行分型,比较两种方法分型结果的一致性,并对双色荧光法检测的2889例HCV分型结果进行分析。结果在97份丙型肝炎阳性血清中,双色荧光法分出Ⅰ型65例(67.0%),Ⅱ型25例(25.8%),Ⅲ型2例(2.1%),Ⅰ/Ⅱ混合型3例(3.1%),有2例HCVRNA定量为(1～3)×103IU/ml弱阳性标本未分出型;测序法分出Ⅰ型61例(62.9%),Ⅱ型24例(24.7%),Ⅲ型2例(2.1%),Ⅰ/Ⅱ混合型2例(2.1%),有8例HCVRNA定量结果为(1～5)×103IU/ml的阳性标本测序法未分出型,有1例Ⅰ/Ⅱ混合型标本测序无法判断,而荧光法分型明确。我院采用本研究建立的方法检测2889例临床丙型肝炎标本,2268份分出基因型,其中Ⅰ型1545例(68.1%),Ⅱ型702例(31.0%),Ⅰ/Ⅱ混合型18例(0.8%),Ⅲ型3例(0.1%),高HCV载量血清全部涵盖在Ⅰ/Ⅱ/Ⅲ型中,未发现Ⅳ型和其他型的病例。结论本研究建立的双色荧光HCV基因分型的方法具有良好的敏感性、特异性、可重复性且省时省力,由于近99%的HCV阳性血清为Ⅰ/Ⅱ型,所以临床可选择Ⅰ/Ⅱ型试剂进行常规检测,对高病毒载量而非Ⅰ/Ⅱ型的标本可采用Ⅲ/Ⅳ型试剂进一步分型明确。

Objective To develop and evaluate a new,simple multiplex real-time reverse transcription-PCR（MRRTP） method for genotyping of HCV fitting for clinical laboratory routine detection.Methods Multiplex real-time polymerase chain reaction（PCR） and Taqman probes（Ⅰ/Ⅲmodified with FAM,Ⅱ/Ⅳmodified with VIC respectively） targeting the 5＇ non-coding region had been used.The method was compared with sequencing assay on 97 serum samples representing genotypesⅠ/Ⅱ/Ⅲ,and was applied on a further 2889 clinical samples.Results MRRTP typing of the 97 samples showed genotypeⅠin 65（67.0%）,genotypeⅡin 25（25.8%）,genotypeⅠand genotypeⅡmixed in 3（3.1%）,genotypeⅢin 2（2.1%）,and no genotypeⅣwere detected,while 2 samples of HCV RNA quantitation less than 3×103 IU/ml were non-reactive.In controlling method of sequence,genotypeⅠin 61（62.9%）,genotypeⅡin 24（24.7%）,genotypeⅠand genotypeⅡmixed in 2（2.1%）,genotypeⅢin 2（2.1%）,and no genotypeⅣwere detected also.There was a complete concordance with sequence with the exception of 1 sample,which were of genotypeⅠandⅡby MRRTP,but genotypeⅡby sequence.There were 8 samples of HCV RNA quantitation less than 5×103 IU/ml were not detected in sequence method,only 2 samples were not detected in MRRTP method.Out of 2889 consecutive clinical samples between 2008 and 2009,2268 could be typed by the MRRTP assay;68.1% were genotypeⅠ,30.9% were genotypeⅡ,genotypeⅠmixed genotypeⅡwere only 0.79% and genotypeⅢ were 0.1%,none of genotypeⅣand others were detected.Conclusions The method is overall accurate and provides an attractive alternative for genotyping because processing time and costs are significantly reduced.MRRTP ofⅠ/Ⅱprobe screening method is fitting for clinical amplification in China（almost 99%）.Inclusion of probes targeting genotypes Ⅲ less than 0.1 percent is required for the method to be useful in areas where these genotypes are present.