环介导等温扩增快速检测B群链球菌的临床应用

目的建立采用环介导等温扩增（LAMP）技术从临床标本中快速检测B群链球菌的方法。方法根据美国国家生物信息中心上提交的B群链球菌的cfb基因序列（登陆号X72754）设计特异LAMP引物，以热裂解法提取标本中细菌的DNA，然后以LAMP技术扩增cfb基因来鉴定其是否为B群链球菌。在采用LAMP技术检测B群链球菌的同时，以选择性培养法和PCR技术平行检测相同标本，并将3种方法的检测结果比较。结果在176例阴道拭子和176例直肠拭子中，选择性培养法检测到的B群链球菌例数为49和61、LAMP法检测到的B群链球菌例数为49和59，PCR法检测到的B群链球菌例数为48和58。以选择性培养法为金标准，LAMP法检测B群链球菌的灵敏度和特异度分别为96．7％和100％，PCR法检测B群链球菌的灵敏度和特异度分别为95．1％和100％。LAMP法检测B群链球菌的时间为2h左右，模拟菌株的检测结果显示该方法的最低检测限为10^2CFU／mL。结论该方法灵敏度高，特异性强，操作方便简单，适合从临床标本中快速检测B群链球菌。

The clinical application of loop-mediated isothermal amplification for detecting Group B Streptococci rapidly

Objective To detect Group B Streptococci in clinical samples by loop-mediated isothermal amplification. Methods Four specific LAMP primers were designed from the published sequence of GBS （ GenBank accession number X72754）. Genomic DNA in clinical sample was extracted by thermal cracking. Then gene cfb was amplified by LAMP, the same samples were detected by selective broth method and PCR. Results 49 and 61 strains of Group B Streptococci were detected in 176 vaginal and cervical samples by selective broth method respectively, 49 and 59 strains of Group B Streptococci were detected in 176 vaginal and cervical samples by LAMP method respectively, 49 and 58 strains of Group B Streptococci were detected in 176 vaginal and cervical samples by PCR method respectively. Compared with gold standard of selective broth method, sensitivity and specificity of LAMP method were 96.7% and 100% respectively. The sensitivity and specificity of PCR method were 95.1% and 100% respectively. Additionally the LAMP method could be finished in two hours. The assay had lowest detectable limit of 102 CFU/mL in simulated samples. Conclusion This LAMP-based assay is simple, rapid, sensitive and specific, it can be used to detecte Group B Streptococci in clinical samples.