【摘要】 目的建立一种适合现场使用、灵敏、特异的脑膜炎奈瑟菌(N.men)检测方法。方法针对N.men的ctrA基因设计环介导等温扩增(LAMP)特异性引物和核酸侵入反应探针,对LAMP扩增产物进行核酸级联侵入反应和纳米金颗粒探针杂交检测。优化反应体系和反应条件后进行敏感性和特异性实验,并与Real-Time PCR检测方法进行比较。结果以核酸级联侵入反应和纳米金颗粒探针显色技术检测LAMP扩增产物和毛细管电泳法具有相同的敏感性和特异性;LAMP结合核酸级联侵入反应和纳米金探针显色技术的检测敏感性为10copies DNA分子/反应,且与其他病原体之间无交叉反应。在临床样本检测方面,LAMP结合核酸级联侵入反应和纳米金探针显色技术与Real-Time PCR检测效果相当。结论结合LAMP、核酸级联侵入反应和纳米金探针显色技术建立的N.men检测方法灵敏、特异,无需特殊仪器,适用于基层检测和现场快速检测。更多还原

【Abstract】 Objective To develop a sensitive and specific field test method for detection of Neisseria meningitidis(N.men).Methods Loop-mediated isothermal amplification(LAMP)primers and invasion reaction probes were designed based on the conserved regions of ctrA gene.After LAMP amplification,the amplicons were detected by cascade invasive reaction and nanoparticle hybridization.After optimizing the reaction system and conditions,sensitivity and specificity experiments were carried out;the results were compared with those by Real-time PCR.Results The sensitivity and specificity of cascade invasive reaction and nanoparticle hybridization for detection of LAMP products were the same as those by capillary electrophoresis.The sensitivity of LAMP-cascade invasive reaction and nanoparticle hybridization assay was 10 copies of DNA molecule/reaction.No cross-reaction with other pathogens was found.In terms of clinical sample detection,LAMP-cascade invasive reaction and nanoparticle hybridization and Real-time PCR were of good consistency.Conclusion The LAMP-cascade invasive reaction combined with nanoparticle hybridization assay developed in this study was sensitive and specific for detection of N.men,which requires no complicated instruments and is suitable for low-equipment setting laboratory and on-site use.更多还原