LAMP与FQ—PCR技术检测HBVDNA的特异性和灵敏度比较

目的 采用环介导等温扩增反应（Loop-mediated isothermal amplification,LAMP）技术与实时荧光定量 PCR（real-time fluorescent quantitative PCR，FQ-PCR）技术检测 HBV DNA，并对 LAMP 方法的反应条件进行优化，比较两种方法检测 HBV DNA 的灵敏度及特异性。方法 选取经医院确诊的乙肝患者的血清，提取血清中的 HBV DNA 作为扩增模板，同时，对 LAMP 方法的各种条件进行优化，并分别采用 LAMP 和 FQ-PCR 两种方法对同一 HBV DNA 模板做 10 倍梯度稀释的标本进行 HBV DNA 的检测，比较其灵敏度；且分别用两种方法对乳头瘤病毒（HPV）、EB 病毒、大肠埃希氏菌、金黄色葡萄球菌、鲍曼不动杆菌、铜绿假单胞菌以及正常人血清基因组 DNA 在相同条件下进行实验，观察两种方法在检测中是否出现假阳性结果，以确定两种方法的特异性。结果 对同一 HBV DNA 模板进行 10 倍梯度稀释后，FQ-PCR 技术在待测血清标本（2.38×107copy/ml）被稀释到 10-5，就难以进行准确检测了，而 LAMP 方法在待测血清标本被稀释到 10-7时，仍然能够获得较好的扩增结果；对 HPV、EB 病毒、大肠埃希氏菌、金黄色葡萄球菌、鲍曼不动杆菌、铜绿假单胞菌以及正常人血清基因组 DNA 进行 LAMP 和 FQ-PCR 检测，结果均为阴性。结论 采用 LAMP方法可以成功、快速、高效、特异的扩增 HBV DNA，与 FQ-PCR 方法比较，都能特异地检测 HBV DNA，但 LAMP 方法具有更高的灵敏性，操作更为简单、方便，而且不需要昂贵的仪器，更适合基层检测机构的推广使用。

Comparison of Specificity and Sensitivity for HBV DNA Detection Between LAMP and FQ-PCR Method

Objective The LAMP （Loop-mediated isothermal amplification） and FQ-PCR （real-time fluorescent quantitative PCR） were used to detect HBV DNA. To optimize LAMP reaction,and then compare the sensitivity and specificity of the two techniques. Methods HBV DNA was extracted from serum of patients in our hospital and LAMP reaction condition was optimized. Sensitivity of LAMP and FQPCR was compared in detection of HBV DNA with ten-times dilution for the same specimen. To determine the specificity of these two methods,they were applied to detect genomie DNA of papilloma virus （HPV）, Epstein-Barr （EB） virus, Escherichia coli, Staphylococcus aureus,Acinetobacter baumanii,Pseudomonas aeruginosa and normal serum to experiment under the same conditions,and observe whether there were false positive results. Results FQ-PCR could not accurately detect HBV DNA concentration when the serum （2. 38 X 107 copy/mL） was diluted to 10 a. While for LAMP, it could achieve good results when serum was diluted to 10 7. The results were all negative when LAMP and FQ-PCR were used to detect DNA of HPV, EB virus, Escherichia coli, Staphylococcus aureus, Acinetobacter baumanii, Pseudomonas aeruginosa and normal serum. Conclusion The LAMP method is successful, rapid, efficient and specific to amplify HBV DNA. LAMP and FQ-PCR are of high specificity in HBV DNA detection, while LAMP shows higher sensitivity. Compared to FQ PCR the operation of LAMP is more simple and convenient, it can be performed without expensive instruments,and it is more suitable for primary medical institutions.