中国人群乙型肝炎病毒基因分型不同方法和标准的比较  

Comparison of different genotyping methods for hepatitis B virus in a Chinese population

作　　者：李可可[1] 刘莹[1] 姚品芳[2] 陈真[2] 王红[1] 李劲[1] 

机构地区：[1]中南民族大学生命科学学院,湖北省武汉市430074 [2]湖北省肿瘤医院,湖北省武汉市430079

出　　处：《世界华人消化杂志》2014年第16期2306-2316,共11页World Chinese Journal of Digestology

基　　金：武汉市科技计划基金资助项目;No.2013060501010152~~

摘　　要：目的:将来自中国不同地区不同民族乙型肝炎检测"大三阳"学生的外周血乙型肝炎病毒(hepatitis B virus,HBV)进行基因分型并探讨不同方法对HBV基因分型的效果进行比较,建立较为客观精确的HBV基因分子分型方法和标准.方法:反映HBV-S区的型特异性的聚合酶链式反应-限制性酶切片断长度多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)方法进行基因分型;依据HBV-S区的型特异性的PCR扩增片段大小直接进行分型;通过对NCBI数据库中共计220个条目已确认B、C、D型的HBV-S基因DNA序列的同源性比对,构建一个以HBV的S区DNA序列分子进化树为基础的基因分型标准.对待检样品S区的PCR产物进行DNA测序并与标准进化树的DNA序列进行比对并重新构建进化树而进行HBV基因分型.结果:在共计216例乙型肝炎检测"大三阳"(乙型肝炎两对半检查中,HBsAg、HBeAg和HBcAb均为阳性)样品中,第1种分型方法鉴定出:B型155例、C型41例、D型7例,尚有13例不能确定是C型还是D型.进一步经第2种分型方法确认其中10例未分型的均为C型,余3例不能确定是C型还是D型.经测序、比对及构建分子进化树,测序的30例中与PCRRFLP分型特别是亚型的结论多不相同.并且首次在国内发现3例HBV-G型(为广西壮族和瑶族男性).结论:3种方法比较各有优劣.PCR-RFLP法较为精细可以分到亚型,但该法的缺陷是不能区分C2型和D2型.根据PCR片段直接分型法较为快捷简便,但引物特异性不高,分型不准确.将待检HBV-S基因DNA序列导入标准进化树的分型方法较为准确客观,是HBV基因分型的"金标准".但针对HBV的普遍变异和种群异质性,需要建立HBV-S的蛋白分型标准.AIM： To genotype hepatitis B virus（HBV） in college students who tested positive HBsAg, HBeAg, and HBcAb using three different methods and compare the advantages and disadvantages of these genotyping methods. METHODS： According to the specificity of HBV-S, polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP） or direct genotyping based on the length of PCR amplified fragment were used for HBV genotyping. By means of homology alignment of 220 items of HBV-S gene sequences with HBV-B/C/D type identified in NCBI database, a molecular evolutionary tree as a genotyping standard has been constructed. The test samples were genotyped by carrying out HBV-S gene PCR amplification, sequencing, alignment and constructing an HBV-S region evolutionary tree together with the standard phylogenic tree. RESULTS： In a total of 216 HBV positive samples, the genotyping result of PCR-RFLP was：genotype B, 155; genotype C, 41; genotype D,7; Unknown, 13. Of these unknown cases, 10were identified as genotype C by the direct genotyping method based on the length of PCR amplified fragment, and 3 were still determined.The genotyping results for 30 selected samples were substantially different between the method of the sequencing-evolutionary tree and PCRRFLP. Three cases of HBV genotype G, which come from the men of Zhuang and Yao nationalities in Guangxi, were first discovered in China using the sequencing-evolutionary tree method. CONCLUSION： The three genotyping methods have different advantages and disadvantages：PCR-RFLP can distinguish between sub-genotypes, but can not distinguish C2 and D2; direct genotyping method based on PCR amplified fragment size is simple and direct, but its accuracy and specificity are not high; the sequencingevolutionary tree method is accurate and specific, representing a ＂golden＂ standard for HBV genotyping.

关 键 词：乙型肝炎病毒基因分型 PCR-RFLP 进化树 

分 类 号：R512.62[医药卫生—内科学]