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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 establish a loop-mediated isothermal amplification (LAMP) technique for rapid detection of hepatitis B virus (HBV) by grassroots medical institutions. Methods According to the hepatitis B virus surface antigen gene sequence published by the National Institutes of Health (NIH) gene sequence database (Accession Number: V00867), two pairs of specific LAMP primers were designed according to the pre-S gene of virus surface antigen , DNA was extracted as an amplification template, LAMP reaction system was optimized, the reaction was carried out at 65 ℃ for 1 h, and the reaction results were judged by visual inspection or agarose gel electrophoresis. Meanwhile, a pair of specific primers was designed by polymerase chain reaction 35 cases of hepatitis B infected sera were amplified, comparing the specificity and sensitivity of the two methods in the test. Results Totally 20 cases were positive for serum LAMP in 35 cases of hepatitis B virus and 20 cases were positive for common PCR. The serial dilution of the same virus template was 10-8 for LAMP, while the detection limit of ordinary PCR was 10-6. Conclusion LAMP can detect the HBsAg gene rapidly, sensitively and specifically. The experimental apparatus is simple and does not require special testing equipment. It is suitable for the use of various testing institutions in grass roots.