重组酶聚合酶扩增方法快速检测志贺菌

Rapid detection of Shigella by recombinant-enzyme polymerase amplification

目的建立一种重组酶聚合酶扩增(RPA)方法检测志贺菌。方法依据志贺菌属侵袭性质粒抗原H(ipaH)基因的保守序列设计特异性引物,建立RPA检测方法;通过检测梯度稀释的志贺菌标准菌株DNA,评价RPA的灵敏度;通过检测志贺菌、出血性大肠埃希菌O157:H7、沙门菌、副溶血弧菌、粪肠球菌评价RPA的特异性。结果建立的RPA方法在35℃反应20min,即可检测到志贺菌ipaH基因,检测灵敏度为10ng/μl志贺菌基因组DNA,比PCR高100倍;且与其他肠道病原菌无交叉反应。结论本研究建立的志贺菌RPA检测方法敏感性、特异性强,操作简单快速,为志贺菌的现场快速检测提供一种新思路。

Objective To develop a method for the detection of Shigella based on recombinant enzyme polymerase amplification(RPA).Methods The specific primers were designed based on the conserved sequence of Shigella ipa H gene,to establish RPA detection method.The sensitivity of the RPA method was evaluated by detecting genomic DNA of standard Shigella in gradient dilution.The specificity of RPA was assessed by detecting standard strain of Shigella,Escherichia coli O157:H7,Salmonella,Vibrio parahemolyticus and Enterococcus faecalis.Results The established RPA method could amplify the target ipa H gene fragment incubated at 35℃for 20 min.The detection limit was 10-5ng/μl genome DNA of Shigella,which was 100 times lower than PCR,and the method has no cross-reaction with other intestinal pathogens.Conclusion The RPA method established in this study was sensitive,specific,simple and rapid operation,and provides a new idea for the rapid detection of Shigella in field.