DNA环介导恒温扩增技术在乙型肝炎病毒表面抗原基因检测中的应用

Utilization of Loop-mediated Isothermal Amplification of DNA to the HBs Region of Hepatitis Virus B

目的建立一种适合基层医疗机构开展的快速检测乙肝病毒的环介导恒温扩增(loop-mediated isothermal amplification,LAMP)技术。方法根据美国国立卫生研究院(NIH)基因序列数据库(genbank)公布的乙型肝炎病毒表面抗原基因序列(登录号:V00867),针对病毒表面抗原pre-S基因设计两对特异性的LAMP内外引物,提取DNA作为扩增模板,优化LAMP反应体系,恒温65℃反应1h,反应结果采用目测或琼脂糖凝胶电泳法判断;同时设计一对特异性的引物采用聚合酶链式反应(PCR)对35例乙肝感染者血清进行扩增,比较两种方法在检测中的特异性和敏感性。结果35例乙肝病毒患者血清LAMP反应检测阳性20例,普通PCR法检测阳性20例;对同一病毒模板做系列倍比稀释,LAMP能检测出的极限为10-8,而普通PCR的检测极限为10-6。结论LAMP能快速、敏感、特异地检测乙肝病毒表面抗原基因,实验仪器简单,不需要特殊的检测设备,适合基层各种检测机构的使用。

Objective To set up a method of loop-mediated isothermal amplification （LAMP） for the rapid and sensitive detection of HBV which is suitable for basic medical institution.Methods According to the reporting of the genbank,the gene sequence of HBV surface antigen （accession number：V00867） was chosen,and LAMP forward primer and LAMP backward primer were designed.DNA was extracted as the amplification template,the reaction system of LAMP was optimized,and the amplification characters were estimated by eyeball or electrophoresis after the reaction was in thermostatic water bath at 65℃ for 60 minutes.A couple of specific primers were simultaneously designed to amplify the target gene of HBV from 35 samples through polymerase chain reaction.The specificity and sensitivity of the two methods were compared.Results Among the 35 serum samples selected from hepatitis B patients,there were 20 positive samples detected by LAMP,which was as the same as the PCR.The template was diluted by multi-proportion;the lowest limit of LAMP was 10-8,while that of the PCR was 10-6.Conclusions The LAMP assay is a quick,specific and sensitive method in detecting the HBV and is suitable for primary medical detection institutions.