快速检测淋球菌porA和16S rRNA基因的环介导等温扩增技术的建立

Development of A Loop-mediated Isothermal Amplification Method for Rapid Detection of porA and 16S rRNA Genes of Neisseria gonorrhoeae

目的建立快速检测淋球菌porA和16S rRNA基因的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)技术,探讨其用于淋球菌感染早期诊断的可行性。方法利用Primer-ExplorerV3软件分别针对淋球菌porA及16S rRNA基因设计6条引物(2条内引物FIP、BIP,2条外引物F3、B3,2条环引物LF、LB),以淋球菌标准菌株基因组DNA为模板,进行LAMP扩增,同时,以外引物F3、B3为PCR引物,进行PCR扩增,并比较2种扩增方法的灵敏度和特异性。结果LAMP法与PCR法灵敏度相同,可检测到10个拷贝的目的基因;其针对淋球菌porA和16S rRNA基因的引物对脑膜炎奈瑟菌、肺炎链球菌和大肠埃希菌的DNA均不能扩增。结论已成功建立了检测淋球菌porA和16S rRNA基因的LAMP技术,为淋球菌的快速检测提供了新的手段,有望成为淋球菌常规检测的简便方法。

Objective To develop a loop-mediated isothermal amplification（LAMP）technique for rapid detection of porA and 16S rRNA genes of Neisseria gonorrhoeae and investigate the feasibility of its application to early diagnosis of Neisseria gonorrhoeae infection. Methods By using Primer-Explorer V3 software, six specific primers for porA and 16S rRNA genes of Neisseria gonorrhoeae, including two inner primers FIP and BIP, two outer primers F3 and B3 as well as two loop primers LF and LB, were designed for LAMP using the genomic DNA of standard Neisseria gonorrhoeae strain as a template. The same genes were amplified by PCR us-ing F3 and B3 primers at the same time, and the sensitivities and specificities of the two methods for amplification were compared. Results The sensitivity of LAMP was in accordance with that of PCR, both of which were 10 copies of target genes. However, no DNA sequences were amplified from Neisseria gonorrhoeae, Streptococcus pneumoniae or E. coli by using the primers specific to porA and 16S rRNA genes of Neisseria gonorrhoeae. Conclusion The LAMP technique for detection of porA and 16S rRNA genes of Neisseria gonorrhoeae was successfully developed, which provided a novel tool for rapid detection of Neisseria gonorrhoeae and might be used as a simple method for routine detection.