乙型肝炎病毒B基因亚型（Ba，Bj）半巢式PCR检测方法的建立  

Establishment of a semi-nested PCR for identifying the sub-genotypes（Ba and Bj） of hepatitis B virus of genotype B

作　　者：王杰[1] 高俊薇[1] 李杰[1] 庄辉[1] 王佳[1] 李雅娟[1] 金晖[1] 

机构地区：[1]北京大学医学部病原生物学系,100083

出　　处：《中华流行病学杂志》2008年第2期177-180,共4页Chinese Journal of Epidemiology

基　　金：国家“十五”科技攻关课题资助项目（2004BA718802）

摘　　要：目的建立一种灵敏特异且简便易行的乙型肝炎病毒（HBV）B基因Ba和Bj亚型分型方法。方法用DNAStar软件比较分析GenBank中登录的B基因型HBV（HBV／B）和C基因型HBV（HBV／C）的基因序列，分别设计出HBv／B型特异性引物HB及Ba和目基因亚型特异性引物BA和BJ。第一轮PCR反应以HBV／B型特异性引物HB作为上游引物，以日本学者Sugauchi等设计的引物HBAS-4V作为下游引物（外引物）；第二轮PCR反应同样以HB作为上游引物，亚型特异性引物BA和BJ作为下游引物（内引物），在同一个反应管中进行。根据PCR产物片段的大小判定B基因亚型。随机选取实验室内经型特异性引物PCR法鉴定为HBv／B（56份）及经preS／S区序列测定证实为HBV／C（15份）的HBV慢性感染者血清标本共71份，以研究设计的引物进行半巢式PCR反应，检测Ba和Bj基因亚型，并以载有HBVBi亚型基因组的质粒作为尉亚型的阳性对照。然后随机选取15份B型样本的第一轮PCR产物直接测序，利用Blast和DNAStar软件将测序结果与GenBank中登录的Ba及Bj亚型序列进行同源性分析，验证该半巢式PCR法的准确性。结果56份HBV／B血清标本经该半巢式PCR法检测，均为Ba亚型，随机选取的15份B型PCR产物直接测序，结果均为Ba亚型，与研究中应用的半巢式PCR方法检测结果一致。15份HBV／C的血清标本检测结果均为阴性。结论建立了HBVBa和Bi亚型半巢式PCR法。该方法灵敏、特异、简便、易行，可用于临床和流行病学大样本检测。Objective To establish a sensitive, specific, simple and practicable method for identifying the two sub-genotypes （Ba and Bj） of genotype B isolates of hepatitis B virus （HBV） （HBV/ B）. Methods The entire nucleotide sequences of HBV/B and HBV/C were obtained from GenBank, compared and analyzed with DNAStar software. The specific primers for HBV/B （HB） and Ba （BA）, Bj （BJ） were designed respectively. HB as HBV/B specific primer （sense） and HBAS-4V （designed by Japanese scientists Sugauchi et al） as a universal outer primer （antisense） were used in the first-round PCR. In the second-round PCR, HB was also used as sense primer while BA and BJ as inner primers （antisense） and they were added into a single tube for PCR reaction. The two sub-genotypes of HBV/B were identified according to the length of the PCR products. A total of 71 HBV DNA-positive serum samples were selected randomly from our laboratory, including 56 HBV/B samples identified by type-specified PCR method and 15 HBV/C samples identified by direct sequencing in preS and S Region （preS/S）. All the 71 samples were detected with this semi-nested PCR method. A plasmid containing full length genomic DNA of HBV/Bj, was presented by Professor Kenji Abe as positive control of Bj. Then, the first-round PCR products of 15 HBV/B were randomly selected and sequenced directly, and their sequences were compared phylogenetically with the above known Ba and Bj sequences using Blast and DNAStar softwares to confirm the results of semi-nested PCR. Results 56 HBV/B samples were all identified as HBV/Ba by our semi-nested PCR method. 15 randomly selected PCR products were all sequenced as HBV/Ba. All of the 15 HBV/C samples were negative. Conclusion A simple, rapid, sensitive and specific method for identifying sub-genotypes Ba and Bj was established whith might be used for large-scale clinical and epidemiological studies.

关 键 词：乙型肝炎病毒 B基因型 半巢式聚合酶链反应 

分 类 号：R4[医药卫生—临床医学] R51