LAMP和PCR法检测痢疾志贺菌的特异性和灵敏性比较

Specificity and sensitivity of LAMP method detecting Shigella dysenteriae and its comparison with PCR

目的建立环介导等温扩增技术(LAMP)快速检测痢疾志贺菌,并对其特异性、灵敏度与传统PCR进行比较。方法以痢疾志贺菌侵袭性质粒抗原H基因(ipaH)为靶序列,设计6条特异性引物(内引物、外引物和环引物各2条),优化LAMP反应体系及反应条件,检测LAMP反应的灵敏度、特异性及模拟样品,同步与PCR进行比较。结果 LAMP法和PCR均能特异性地扩增出志贺菌靶DNA,而其他非志贺菌均未扩增出特有条带。检测细菌纯培养物和模拟食品的灵敏度LAMP法分别为5.3×101cfu/ml、6.8×101cfu/ml,PCR法分别为5.3×102cfu/ml和6.8×102cfu/ml。结论利用LAMP技术,可快速、灵敏、简便地检测痢疾志贺菌,与PCR方法比较,特异性强、操作简便、检测成本低、耗时短,有望发展成为快速检测痢疾志贺菌的有效手段。

Objective The aim was to detect Shigella dysenteriae rapidly by using the loop-mediated isothermal amplification（LAMP） technology,and to compare the specificity and sensitivity of the LAMP to that of conventional PCR.Methods The sequence of ipaH of Shigella was used as target sequences to design the six primers.The reaction conditions of LAMP were established and optimized.Results The LAMP assay detection limit was detecting 5.3×101 cfu/ml of pure Shigella dysenteriae and 6.8×101 cfu/ml in raw meat.The traditional PCR assay detection limit was 5.3×102 cfu/ml and 6.8×102 cfu/ml,respectively.Conclusion LAMP technology for detection Shigella was rapid and specificity.It was a more sensitive method for Shigella detection than the PCR method.We have evaluated this simple tool for the detection of Shigella dysenteriae in food and build a technology platform.