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易海华, 赵金伟, 房超, 王云飞, 宋阳威, 徐波, 吴萍兰, 徐政. 志贺菌环介导等温扩增检测方法建立[J]. 中国公共卫生, 2011, 27(3): 380-382. DOI: 10.11847/zgggws2011-27-03-64
引用本文: 易海华, 赵金伟, 房超, 王云飞, 宋阳威, 徐波, 吴萍兰, 徐政. 志贺菌环介导等温扩增检测方法建立[J]. 中国公共卫生, 2011, 27(3): 380-382. DOI: 10.11847/zgggws2011-27-03-64
YI Hai-hua, ZHAO Jin-wei, FANG Chao, . Development of genetic diagnostic method of Shigella based on loop-mediated isothermal amplification[J]. Chinese Journal of Public Health, 2011, 27(3): 380-382. DOI: 10.11847/zgggws2011-27-03-64
Citation: YI Hai-hua, ZHAO Jin-wei, FANG Chao, . Development of genetic diagnostic method of Shigella based on loop-mediated isothermal amplification[J]. Chinese Journal of Public Health, 2011, 27(3): 380-382. DOI: 10.11847/zgggws2011-27-03-64

志贺菌环介导等温扩增检测方法建立

Development of genetic diagnostic method of Shigella based on loop-mediated isothermal amplification

  • 摘要: 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。

     

    Abstract: Objective To develop a method of loop-mediated isothermal amplification (LAMP) for detection of Shigella.Methods According to conserved nucleotide of Shigella ipaH gene and principle of LAMP, we designed a set of LAM Preaction system containing four primers.Twenty-two strains of Shigella and non-Shigella were detected with LAMP to evaluate the specificity of the method.The dilution of the ipaH gene recom binant plasmid was detected with PCR and LAMP to evaluate the sensitivity of the method.Inaddition, we evaluated the linearity between initial template copys Log value and reaction time to explore the feasibility of quantitative determination.Results The LAMP assay could be finished with in 1 hour and was no cross-reaction with other closely related members of food pathogens.The detectability was 200 copies/reaction and was 10 times more sensitive than PCR.The coefficient of variance between tests was less than 5%.The kinetics curves showed a good linearity between initial template copys Log value and reaction time with a correlation coefficient of 0.985 5.Conclusion The genetic diagnostic method of Shigella based on LAMP is rapid, sensitiv e and specific for detection of Shigella.

     

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