巢氏PCR-RFLP和多重PCR检测HBV基因型、基因亚型方法的比较  

Comparison of nested PCR-restriction fragment length polymorphism with mutiple-PCR by type-specific primers for HBV genotyping and subgenotyping

作　　者：张烜榕[1] 李威[1] 申元英[1] 任来峰[2] 李强[1] 沈茹[1] 赵海平[1] 

机构地区：[1]大理学院基础医学院微生物学与免疫学教研室,云南大理671000 [2]山西大学汾阳医学院微生物学与免疫学教研室,山西汾阳032200

出　　处：《军医进修学院学报》2008年第4期280-282,共3页Academic Journal of Pla Postgraduate Medical School

基　　金：国家自然科学基金资助项目(30560136);云南省中青年学术技术带头人后备人才培养基金(2006py01-57)

摘　　要：目的:建立HBV基因型和亚型的分型方法,并分析相关的两种方法特异性和敏感性。方法:采用型特异性引物的巢氏PCR-RFLP和六种主要的HBV型特异性引物和亚型特异性引物的多重PCR方法,分别检测了100例患者标本。结果:两法不一致率为50%(27/54)。型特异性引物巢氏PCR-RFLP法检测出B基因型41例(41%),C基因型25例(25%),B+C基因型34例(34%),B j亚型3例(7.3%),Ba亚型为38例(92.7%),Cs亚型为21例(84%),Ce亚型为3例(12%),1例C型未分出亚型(4%)。多重PCR法检出B型18例(33.3%),C型7例(13%),B+C混和型5例(9.3%),B2亚型2例(11%),C1亚型2例(28.5%)。巢氏PCR-RFLP法型检出率100%亚型检出率98.5%,多重PCR法型检出率55.6%,亚型检出率仅为16%,前者高于后者(P<0.05)。结论:巢氏PCR-RFLP鉴定HBV基因型、基因亚型较多重PCR敏感性高,重复性好,但耗时长,费用高。Objective： To compare the efficiency between nested PCR with type-specific primers and PCR-RFLP method and improved muhiplex-PCR method in HBV genotyping and subgenotyping. Methods： The nested PCR with PCR-RFLP method and the improved muhiplex-PCR method were used to identify HBV genotypes and subgenotypes of 100 sera of patients with HBV infection from three hospitals in Dali. Results： The discrepancy between the nested PCR with PCR-RFLP method and the im- proved multiplex-PCR method was 50% （27/54）. Of 100 sera from patients with HBV infection determined by the nested PCR with PCR-RFLP, the proportion of genotype B, C, and B ＋ C were 41% （41/100） , 25% （25/100）and 34% （34/100） ; subgeno- type Bj, Ba, Cs and Ce were 7.3 % （3/41 ）, 92.7% （ 38/41 ）, 84% （21/25 ） and 12% （ 3/25 ） respectively. Only one of genotype C was unidentified 4% （1/25）. By the improved muhiplex-PCR, genotype B,C,and B ＋ C were 33.3% （ 18/54）, 13% （7/54） and 9.3% （5/54） ; subgenotypeBa and Cs were 11% （2/18） and 28.5% （2/7）. The detections rate of PCR-RFLP were 100% in genotype and 98.5% in subgenotype, which were significantly higher than that of multiplex-PCR 55.6% and 16% （P 〈 0. 05）. Condutaon： The nested PCR with PCR-RFLP is more reliable and simple to identify HBV genotypes and subgenotypes.

关 键 词：聚合酶链反应 多态性 限制性片段长度 肝炎病毒 乙型 基因型 

分 类 号：R373.21[医药卫生—病原生物学]