乙型肝炎病毒环介导等温扩增检测法的建立  

Establishment of a loop-mediated isothermal amplification method for detecting hepatitis B virus

作　　者：邵靓婧[1] 张琪[2] 徐睿瑶 唐慧娴[2] 韩一芳[2] 张锦海[2] 王长军[1] 

机构地区：[1]南京医科大学公共卫生学院,南京医学硕士研究生211166 [2]南京军区军事医学研究所疾病预防控制所,南京210002

出　　处：《医学研究生学报》2016年第12期1309-1314,共6页Journal of Medical Postgraduates

基　　金：国家自然科学基金(31500151);江苏省自然科学基金(BK20150094)

摘　　要：目的环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术是近年来发展起来的一种新型核酸扩增技术,文中旨在建立一种LAMP方法用于乙型肝炎病毒(hepatitis B virus,HBV)的快速检测。方法利用在线引物设计软件,针对B型和C型HBV基因序列的相对保守区域,设计LAMP引物、实时荧光定量PCR(quantitative real-time PCR,q PCR)引物和探针序列。采用羟基萘酚蓝作为显色剂,优化反应条件,分别建立HBV的可视化LAMP反应体系和q PCR反应体系。反应体系中酶量为1.5μL、反应温度为65℃为LAMP扩增的最佳条件。用2种方法同时检测临床样本(96例乙型肝炎患者、5例丙型肝炎患者、5例获得性免疫缺陷综合症患者及50例健康对照样本的血清核酸),以q PCR检测结果为金标准,评价可视化LAMP方法诊断HBV的敏感性和特异性。结果建立的LAMP方法和q PCR方法最低可检测到10 copies/μL。96例乙型肝炎患者中,q PCR法共55例扩增阳性,其中LAMP方法共42例阳性,敏感性为76.4%(42/55);对病毒滴度高于102copies/μL和低于102copies/μL样本的检测敏感性分别为96.8%(30/31)和50%(12/24)。LAMP方法可成功检出B型和C型HBV,且不会和其他经血传播的病毒发生交叉反应,特异性为100%。结论建立的LAMP方法操作简便快捷、结果判读方便,能在1 h内敏感、特异地检测出血清中的HBV,为基层单位筛查HBV提供新的方法。Objective Loop-mediated isothermal amplification （LAMP） is a novel nucleic acid amplification technology de-veloped in recent years. This study aimed to establish an LAMP method for rapid detection of hepatitis B virus （HBV）. Methods Using the Primer Explorer software 4.0, we designed LAMP primers and the primers and probe sequences of quantitative real-time PCR （qPCR） targeting the relative conservative regions of HBV B and HBV C genes. We established an LAMP reaction system and a qPCR reaction system with hydroxynaphthol blue （HNB） as a chromogenic agent and by optimizing the reaction conditions. Employing the above two methods, we examined the clinical samples collected from 96 patients with hepatitis B, 5 with hepatitis C, 5 with AIDS, and 50 healthy controls） and, with the qPCR results as the gold standard, e-valuated the sensitivity and specificity of visual LAMP in the diagnosis of HBV. Results The amount of enzyme in the reaction system was 1.5 μL and the reaction temperature was 65 as the optimized LAMP amplification conditions. The minimum limit of the LAMP method for detecting the dilutions of plasmids was 10 copies/ μL, similar to the detection sensitivity of qPCR. Of the 96 cases of hepatitis B, 55 were found positive by qPCR and 42 positive by LAMP, with a sensitivity of 76.4% （42/55）. The detection sensitivities for the samples with vims titer higher and lower than 102 copies/jjlL were 96.8% （30/31） and 50% （ 12/24） , respectively. HBV B and C were accurately detected by visual LAMP, with a specificity of 100% and no cross reaction with other blood-borne viruses. Conclusion The LAMP method established, simple and convenient, can be used to detect HBV in the serum within an hour with high sensitivity and specificity.

关 键 词：环介导等温扩增 乙型肝炎病毒 特异性 灵敏度 

分 类 号：R512.62[医药卫生—内科学]