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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 and sensitive method for the detection of Neisseria meningitidis by loop-mediated isothermal amplification (LAMP). Methods Four LAMP primers (two inner primers and two outer primers) were designed for six regions of Neisseria meningitidis (ctrA) gene sequence. Two loop primers were designed and the reaction conditions and reaction system were optimized . The specificity and sensitivity of this method were validated separately and compared with that of normal PCR. Results The specificity and sensitivity of primers designed for appropriate reaction conditions for the amplification of Neisseria meningitidis were good. Compared with the normal PCR method, LAMP sensitivity was 10 times higher than that of ordinary PCR. Conclusion The LAMP detection speed is faster than PCR, and the amplification reaction can be completed in 60 minutes. The experimental LAMP method can detect Neisseria meningitidis rapidly, sensitively and specifically, and is suitable for grassroots inspection departments, small laboratories and on-site monitoring.