多对型特异性引物巢式PCR检测乙型肝炎病毒基因型分析

Detection for genotypes of hepatitis B virus by nested PCR with multiplex pairs of primers

目的采用多对型特异性引物通过巢式PCR法检测HBeAg阳性患者血清中乙肝病毒(HBV)基因型的分布情况。方法根据从前S1基因到S基因中的保守序列设计出10条内外引物,并将其中8条内引物分成A、B两组分别扩增A、B、C和D、E、F型HBV,然后将第2轮PCR的两组产物分别用3%琼脂糖进行电泳,根据PCR产物片段大小直接判定HBV基因型。用该法检测15例慢性乙肝患者血清中HBV基因型,了解HBV基因型分布情况。结果 HBV基因分型结果为B型7例(46.7%)、C型4例(26.7%)、B+C型2例(13.3%),另有2例未能分型。结论人群HBV优势基因型以B型为主,C型次之。

Objective To determine the genotypes of hepatitis B virus （HBV） by nested PCR with multiplex pairs of genotype-specific primers.Methods Ten outer and inner primers were designed on the basis of the conserved nature of nuleotide sequences in regions of the Pre-S1 through S genes,in which 8 inner primers were devided into mix A and B to amplify HBV of genotype A,B,C and D,E,F respectively.The two different products from one sample in second-round PCR were separately electrophoresed on a 3% agarose gel.Genotypes of HBV were determined directly by the size of PCR products.This nested PCR was used in the genotyping of HBVs in 15 patients with chronic hepatitis B.Results Of the 15 patients with chronic hepatitis B,7 （46.7%） were genotype B and 4 （26.7%） were genotype C,2 （13.3%） were genotype B＋C.Conclusion With the application of this nested PCR,the predominant HBV genotypes in human are confirmed to be genotypes B and C.