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目的建立志贺菌环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法。方法针对志贺菌侵袭性质粒抗原H基因(ipaH)特征性保守序列设计1套4条引物,应用LAMP技术对21株志贺菌和非志贺菌进行特异性检测以确定其特异性;使用LAMP和聚合酶链式反应(polymerase chain reaction,PCR)对ipaH基因重组质粒倍比稀释液进行检测以确定其灵敏度;使用重组质粒计算其初始拷贝数与反应时间之间的线性关系以评估定量检测的可能性。结果荧光定量LAMP扩增曲线图显示,LAMP技术可以在1 h内获得结果;有较好的特异性,与其他17种食源性致病菌无交叉反应;LAMP检测技术的灵敏度高出经典PCR技术10倍以上,检测限达到200拷贝/μL;平均变异系数<5%;反应时间与模板初始浓度有良好的线性关系(R2=0.985 5)。结论志贺菌环介导等温扩增检测体系是一种快速、灵敏、特异的核酸筛查方法,为食源性疾病的分子流行病学调查提供新的检测手段。
Objective To establish a loop-mediated isothermal amplification (LAMP) assay for Shigella. Methods A set of 4 primers was designed according to the conserved sequence of ipaH antigen of Shigella spp. The specificities of 21 strains of Shigella spp. And Shigella spp. Were determined by LAMP technique to determine its specificity. LAMP and polymerase chain reaction (PCR) were used to detect the ipaH recombinant plasmid dilutions to determine its sensitivity. The recombinant plasmid was used to calculate the linear relationship between the initial copy number and the reaction time to assess the quantitative detection The possibility of. Results Fluorescence quantitative LAMP amplification showed that the LAMP technique could obtain the result within 1 h, the specificity was good and there was no cross-reaction with other 17 foodborne pathogens. The sensitivity of LAMP detection technology was higher than that of classical PCR The detection limit was 200 copies / μL. The average coefficient of variation was <5%. The reaction time was linear with the initial concentration of the template (R2 = 0.985 5). Conclusion The Shigella ring-mediated isothermal amplification assay is a rapid, sensitive and specific method for screening nucleic acid and provides a new means of molecular epidemiological investigation of food-borne diseases.