实时荧光聚合酶链反应检测HBV基因型B～D方法的建立

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (06): 630-632.

实时荧光聚合酶链反应检测HBV基因型B～D方法的建立

沈建坤¹, 周荣², 王战会¹, 白培盛², 马世武¹, 张克¹, 侯金林¹

1. 南方医科大学南方医院感染内科, 广东, 广州, 510515;
2. 广州华银基因科技有限公司, 广东, 广州, 510150

出版日期:2005-06-20 发布日期:2005-06-20
基金资助:
收稿日期:2005-2-20。
基金项目:国家973计划子课题(G1999054106)
作者简介:沈建坤(1975- ),男,在读硕士研究生,主治医师,电话:13556195912,E-mail:shjk2003@163.com
通讯作者:侯金林,电话:020-61641941

Establishment and application of real-time fluoriescene-based metry polymerase chain reaction for hepatitis B virus genotyping

SHEN Jian-kun¹, ZHOU Rong², WANG Zhan-hui¹, BAI Pei-sheng², MA Shi-wu¹, ZHANG Ke¹, HOU Jin-lin¹

1. 南方医科大学南方医院感染内科, 广东, 广州, 510515;
2. 广州华银基因科技有限公司, 广东, 广州, 510150

Online:2005-06-20 Published:2005-06-20

摘要/Abstract

摘要： 目的建立实时荧光聚合酶链反应检测乙型肝炎病毒(HBV)基因型B~D方法并验证其准确性。方法比较GenBank中已发表的明确分型的143株HBV全序列,设计特异的组合引物探针,应用实时荧光聚合酶链反应HBV基因分型法对兰州地区128例慢性乙型肝炎患者血清标本进行基因型B、C、D检测,并随机对检测的基因型各3株标本进行S基因测序验证。结果 128例标本中B型检出率为20.3%(26/128),C型71.9%(92/128),D型7.8%(10/128);18株HBV克隆标本S基因测序结果与本分型法完全一致。结论实时荧光聚合酶链反应HBV基因分型法能够简便、灵敏、快速、准确地鉴定HBV基因型,适宜用于HBV基因型的大规模临床和流行病学调查。

Abstract: Objective To explore a new method for determining hepatitis B virus (HBV) genotypes B-D with real-time fluorescence-based PCR. Methods The PCR primers and probes were designed according to the analysis of 143 complete HBV genomes from GenBank and on the basis of a search for genotype-specific nucleotide sequences which were conserved in the 3 HBV genotypes. Real-time fluorescence-based PCR was performed for HBV genotyping of 128 samples collected from Lanzhou. Three samples of each genotype of B, C and D were randomly selected and their S gene was sequenced for confirmation of the results of PCR-based method. Results Real-time PCR identified genotype B in 26 (20.3%), genotype C in 92 (71.9%), and genotype D in 10 (7.8%) cases. The sequencing results of the S gene of 18 PCR-produced clones were in complete consistence with those of fluorescence-based PCR. Conclusion The real-time PCR method is convenient, highly sensitive, rapid and accurate, especially suitable in clinical and large-scale epidemiological studies.

中图分类号:

沈建坤¹, 周荣², 王战会¹, 白培盛², 马世武¹, 张克¹, 侯金林¹. 实时荧光聚合酶链反应检测HBV基因型B～D方法的建立[J]. 南方医科大学学报, 2005, 25(06): 630-632.

SHEN Jian-kun¹, ZHOU Rong², WANG Zhan-hui¹, BAI Pei-sheng², MA Shi-wu¹, ZHANG Ke¹, HOU Jin-lin¹. Establishment and application of real-time fluoriescene-based metry polymerase chain reaction for hepatitis B virus genotyping[J]. Journal of Southern Medical University, 2005, 25(06): 630-632.

参考文献

[1] 中华医学会传染病与寄生虫病分会、肝病学分会.病毒性肝炎防治方案[J].中华肝脏病杂志(Chin J Hepatol),2000,8(6):324-9.
[2] 夏国良,Nainan OV,贾志远,等.乙型肝炎病毒基因型和血清亚型在我国部分地区的分布及其特点[J].中华流行病学杂志,2001,22(5):348-51.Xia GL, Nainan OV, Jia ZY, et al. Distribution and characteristis of hepatitis B virus genotype and genotype in China partial region [J].Chin J Epidemiol, 2001, 22(5):348-51.
[3] Ding X, Mizokami M, Yao G, et al. Hepatitis B virus genotype distribution among chronic hepatitis B virus carriers in Shanghai,China[J]. Intervirology, 2001, 44(1):43-7.
[4] Okamoto HT, Suda F, Sadugawa H, et al. Typing hepatitis B virus by homology in nucleotide sequence:comparison of surface antigen subtypes[J]. J Gen Virol, 1988, 69(pt 10):2575-83.
[5] Ohba KI, Mizokami M, Ohno T, et al. Relationship between serotypes and genotypes of hepatitis B virus:genetic classification of HBV by use of surface genes[J]. Virus Res, 1995, 39(1):25-34.
[6] Lindh M. Genotyping of hepatitis B virus by restriction pattern analysis of a pre-S amplification [J]. J Virol Methods, 1998, 72(3):163-74.
[7] Natio H, Hayashi S, Abe K. Rapid and specific genotyping system for hepatitis B virus corresponding to six major genotypes by PCR using type-specific primers [J]. J Clin Microbiol, 2001, 39 (1):362-4.
[8] Yeh SH, Tsai CY, Kao JH, et al. Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis[J]. J Hepatol, 2004, 41(4):659-66.
[9] Usuda S, Okamoto H, Iwanari H, et al. Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product [J]. J Virol Methods, 1999, 80 (1):97-112.
[10] Norder H, Courouce AM, Magnius LO, et al. Complete genomes phylogenetic relatedness and structural proteins of six strains of the hepatitis B virus, four of which represent two new genotypes [J].Virology, 1994, 198(2):489-503.

[1]	梁建新，屈杏芬，曾文铤，朱科伦，张鹤，卫建筠 . 氧化苦参碱在治疗慢性乙型肝炎中抗肝纤维化的作用机制[J]. 南方医科大学学报, 2010, 30(08): 1871-1873.
[2]	赵金红；王健；江水清；项桂菊；. 慢性乙型肝炎患者CXC趋化因子Mig的表达[J]. 南方医科大学学报, 2006, 26(11): 1589-1592.
[3]	李小玲；陈永鹏；戴琳；朱幼芙；罗小克；侯金林；. 超声波评分系统评估病毒性肝炎相关性肝纤维化[J]. 南方医科大学学报, 2006, 26(08): 1200-.
[4]	王亦斌；于立新；邓文锋；苗云；叶俊生；. 丙型肝炎患者肾移植术后霉酚酸酯的应用[J]. 南方医科大学学报, 2006, 26(08): 1215-.
[5]	杨小云；温帆渊；吴静黎；周华坚；. 慢性乙型肝炎患者血清病毒标志和丙氨酸转氨酶与肝组织病理的相关性[J]. 南方医科大学学报, 2006, 26(06): 892-893.
[6]	于宏, 曹治宸, 耿建英, 贺占国, 王政民, 孙小云, 王中华. 干扰素α治疗慢性乙型肝炎患者肝纤维化的实验研究[J]. 南方医科大学学报, 2005, 25(11): 1409-1412.
[7]	施斌斌¹, 彭劼¹, 温帆渊², 侯金林¹. 百赛诺对慢性乙型肝炎患者细胞免疫应答的影响[J]. 南方医科大学学报, 2005, 25(06): 706-708.
[8]	樊和斌, 郭亚兵, 杨洁, 侯金林, 王战会, 朱幼芙, 秦劲峰. 慢性乙型肝炎患者HBV基因型及其临床意义[J]. 南方医科大学学报, 2005, 25(02): 229-230,233.
[9]	付绍杰, 于立新, 杜传福, 徐健, 刘小友, 王亦斌. 供肾质量和受者体质量与移植肾远期功能的关系[J]. 南方医科大学学报, 2004, 24(11): 1295-1296.
[10]	库热西江·托呼提¹, 哈木拉提·吾甫尔², 张利占¹, 怕丽达¹, 哈巴尔·肉孜¹, 哈力克·依米提¹, 程钦¹, 董宇莉¹, 古丽娜·玉山¹. 和田地区维吾尔族、汉族人群HBsAg携带率的流行病学调查分析[J]. 南方医科大学学报, 2004, 24(11): 1287-1288.
[11]	陈永鹏, 朱幼芙, 冯筱榕, 彭劼, 侯金林. 慢性乙型肝炎肝组织学状态相关性无创指标分析[J]. 南方医科大学学报, 2004, 24(10): 1181-1183.
[12]	杨小兵, 蒋建平, 张训. 肝病变程度和维生素K1对慢性肾功能衰竭病人肝活检术后出血的影响[J]. 南方医科大学学报, 2004, 24(07): 787-788.
[13]	于立新, 王志鹏, 付绍杰, 王亦斌, 杜传福, 徐健, 邓文锋. 丙型肝炎病毒感染对肾移植术后影响的临床研究[J]. 南方医科大学学报, 2004, 24(06): 682-684.
[14]	梁蔚芳, 刘志华, 何海棠, 骆抗先. 一株鸭乙型肝炎病毒DHBV全基因的克隆和序列分析[J]. 南方医科大学学报, 2004, 24(04): 404-407.
[15]	王艳¹, 马文丽¹, 毛向明¹, 吴清华¹, 李凌¹, 王洪敏¹, 肖维威¹, 郑文岭². 痘苗病毒寡核苷酸检测芯片的设计及研制[J]. 南方医科大学学报, 2004, 24(02): 180-183.