乙型肝炎病毒基因型(A-F)多引物PCR分型法的初步建立

Classification of genotyping hepatitis B virus with multiplex PCR

目的 建立一种简便易行的乙型肝炎病毒基因型分型方法（只需 ＰＣＲ）。方法 对 ＧｅｎＢａｎｋ中查获的１１４例ＨＢＶ全序列进行比较分析，找出每种基因型相对于其他５种基因型的独特序列，并根据这些独特序列设计出６对分别针对Ａ－Ｆ基因型的特异引物。用这６对引物分别对标本进行ＰＣＲ，根据阳性结果判断出标本的基因型。进一步简化该方法，用多引物对ＰＣＲ法（ＰＣＲ ｗｉｔｈ ｓｅｖｅｒａｌ ｐｒｉｍｅｒ ｓｅｔｓ ｉｎ ａ ｓｉｎｇｌｅ ｔｕｂｅ，Ｍｕｌｔｉｐｌｅｘ ＰＣＲ）将Ｂ、Ｃ、Ｄ ３种基因型的特异引物混台进行ＰＣＲ，根据扩增片断的大小判断基因型。用此方祛对已鉴定的Ｂ、Ｃ、Ｄ型标本进行比较和验证。结果 单引物对ＰＣＲ与多引物对ＰＣＲ的分型结果一致，且与以前用ＰＣＲ－ＲＦＬＰ法的分型结果一致。结论 用多引物对ＰＣＲ分型法准确易行，灵敏性高，便于推广应用。

Objective To establish a simple and practicable method to identify the different genotypes of hepatitis B virus (HBV). Methods Based on the alignment of 114 complete nucleotide sequence for HBV DNA of different genotypes, the specific sequence of each genotype was found. Six primer sets were designed for each of the six genotypes according to the genotype-specific sequence, and used separately for PCR. The genotype of HBV was identified according to the positive result of PCR. Three primer sets for B, C and D genotypes were added into a single tube for PCR reaction, and HBV was genotyped according to the length of the amplified DNA. Results There was no difference in the genotyping result of PCR by single or multiplex primers. which was identical to the PCR-RFLP method. Conclusions This multiplex PCR method is simple, precise. sensitive, and easy to use.