快速检测单核细胞增生李斯特菌LAMP方法的建立  

To develop a loop-mediated isothermal amplification assay for quick detection of Listeria monocytogenes

作　　者：姚栋[1] 张如胜[1] 欧新华[1] 宋克云[1] 

机构地区：[1]长沙市疾病预防控制中心微生物检验科,湖南长沙410001

出　　处：《江苏大学学报（医学版）》2012年第5期394-397,共4页Journal of Jiangsu University：Medicine Edition

摘　　要：目的:建立一种检测单核细胞增生李斯特菌核酸的快速、特异的环介导等温扩增(Loop-mediated isothermalamplification,LAMP)方法。方法:针对单核细胞增生李斯特菌的李斯特菌溶血素hlyA基因设计4条LAMP引物,在水浴箱内65℃保温约60 min,完成对单核细胞增生李斯特菌的扩增,扩增产物经肉眼、SYBR Green I染色和电泳鉴定。利用LAMP和PCR方法同时检测1株单核细胞增生李斯特菌和10株非单核细胞增生李斯特菌(对照组)来验证LAMP方法的特异性。用LAMP和PCR方法分别检测一系列稀释后的单核细胞增生李斯特菌菌液,比较两者的敏感性。结果:经肉眼、加染料和电泳均能观察到1株单核细胞增生李斯特菌LAMP扩增阳性反应,10株非单核细胞增生李斯特菌没有出现LAMP扩增。LAMP敏感度比PCR高,LAMP检测单核细胞增生李斯特菌的检测下限为3 cfu/ml,PCR检测下限>300 cfu/ml。LAMP检测速度比PCR更快,只需要60 min。结论:建立了一种检测单核细胞增生李斯特菌的快速、特异的LAMP方法。Objective： To establish a loop-mediated isothermal amplification （LAMP） assay for the rapid and specific detection of Listeria monocytogenes. Methods： Four primers regions on the hlyA gene of Listeria monocytogenes were designed and used for LAMP assay. Listeria monocytogenes DNA was amplified under i- sothermal conditions （65 ~C ）for 60 rain in water bath, then the amplified product was judged by naked eye, SYBR Green 1 staining, and electropboresis analysis. To evaluate the specificity of the assay, 1 strain of Listeria monocytogenes and 10 strains of none-Listeria monocytogenes were tested by LAMP and conventional PCR assay. In addition, the detection limit of LAMP was compared with that of PCR by using the Listeria monocytogenes strain,that were serially diluted and were amplified by LAMP and PCR. Results： With one strain of Listeria monocytogenes, observation with naked eyes, SYBR Green I staining and electrophoretic a- nalysis were able to detect the products in the LAMP assay, and amplification were not observed when 10 strains of non-Listeria monocytogenes were tested. The sensitivity of LAMP was higher than that of PCR as- say. The detection limit of LAMP assay for Listeria monocytogenes was 3 efu/ml and that of PCR was 〉 300 cfu/ml. LAMP method was superior to conventional PCR for its rapid detection of Listeria monocytogenes, which can complete in 60 min. Conclusion： LAMP for rapid and specific detection of Listeria monocyto- genes was established in this study.

关 键 词：环介导等温扩增 单核细胞增生李斯特菌 溶血素基因 

分 类 号：R331[医药卫生—人体生理学]