环介导等温扩增技术快速检测脑膜炎奈瑟菌属方法的建立

Establishment of LAMP-based rapid detection of Neisseria meningitides

目的应用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术建立一种快速敏感的脑膜炎奈瑟氏菌属检测方法。方法针对脑膜炎奈瑟菌属(ctrA)基因序列的6个区域设计4条LAMP引物(2条内引物、2条外引物),同时设计2条环引物,并对反应条件和反应体系进行优化。分别验证该方法的特异性及敏感性,并与普通PCR进行了比较。结果在适宜反应条件所设计引物对脑膜炎奈瑟菌的扩增的特异性及敏感性均较好,与普通PCR方法比较,LAMP敏感性比普通PCR高10倍。结论 LAMP检测速度相比PCR更快速,在60 min内即可完成扩增反应。实验建立的LAMP方法能够快速、灵敏、特异地检测脑膜炎奈瑟氏菌,适合基层检验部门及小型实验室与现场监测等使用。

Objective To establish a rapid,sensitive detection approach for Neisseria meningitiids based on the loop-mediated isothermal amplification（LAMP） technique.Methods Four LAMP primers（two inner and two outer） were designed according to the six zones of the ctrA gene of Neisseria meningitiids,in addition to two ring primers.The reaction conditions and system were optimized.The sensitivity and specificity of the detection approach were evaluated and compared to conventional PCR methods.Results Favorable sensitivity and specificity of the designed primers were achieved in the amplification of Neisseria meningitiids.The sensitivity of LAMP was 10 times greater than that of conventional PCRs.Conclusion LAMP-based detection,where amplification was completed within 60 min,was faster than PCRs.The established LAMP technique in this study enables rapid,sensitive and specific detection of Neisseria meningitiids,which is suitable for the application in grass-root and small-scale laboratories as well as field surveillance.