基于多酶恒温快速扩增技术检测高毒力肺炎克雷伯菌新方法的建立与应用  

A new method for detection of hypervirulent Klebsiella pneumoniae based on multienzyme isothermal rapid amplification technology

作　　者：李健 李进 向姝 LI Jian;LI Jin;XIANG Shu(Armed Police Corps Hospital,Chongqing 400060,China;不详)

机构地区：[1]武警重庆总队医院卫生防疫科,重庆400060 [2]陆军特色医学中心检验科,重庆400042

出　　处：《中华医院感染学杂志》2025年第1期13-18,共6页Chinese Journal of Nosocomiology

基　　金：重庆市科卫联合医学科研项目(2022QNXM034)。

摘　　要：目的本研究旨在建立一种基于多酶恒温快速扩增技术(MIRA)检测血流感染中高毒力肺炎克雷伯菌(含有iroB基因和p-rmpA基因)的新方法。方法根据高毒力肺炎克雷伯菌主要毒力基因iroB和p-rmpA序列设计MIRA引物组,并通过实时MIRA(Real-timeMIRA)筛选最佳的引物对,建立双重Real-timeMIRA检测高毒力肺炎克雷伯菌的最佳反应体系,进行交叉反应、最低检测限与临床诊断效能评价等试验。结果在优化条件后,建立的双重Real-timeMIRA方法在与其他菌株(不含iroB基因和p-rmpA基因)的交叉反应中表现良好,对高毒力的肺炎克雷伯菌(含iroB基因和p-rmpA基因)具有良好的特异性。该方法对高毒力肺炎克雷伯菌(含iroB基因和p-rmpA基因)的最低检测限为8×10^(2)CFU。以实时聚合酶链反应(Real-time PCR)检测结果为金标准,评价诊断效能结果显示,该方法检测临床模拟血流感染标本的敏感度和特异性均为100.00%。结论本研究建立了基于MIRA检测高毒力肺炎克雷伯菌的新方法,并在临床模拟血流感染标本中验证了其诊断效能。OBJECTIVE To establish a new method for detection of hypervirulent Klebsiella pneumoniae(harboring iroB gene and p-rmpA gene)based on multienzyme isothermal rapid amplification(MIRA)technology.METHODS MIRA primers were designed based on the sequences of major virulence genes iroB and p-rmpA in the hypervirulent K.pneumoniae.The optimal primer pairs were screened out by real-time MIRA,and the optimal reaction system based on duplex real-time MIRA was established to detect the hypervirulent K.pneumoniae.Cross reactions,minima limit of detection and evaluation of clinical diagnostic efficiency were carried out.RESULTS The established duplex real-time MIRA showed good performance in cross reaction with other strains(without harboring iroB gene and p-rmpA gene)after optimization and presented high specificity for the hypervirulent K.pneumoniae(harboring iroB gene and prmpA gene).The minimal limit of detection of the method was 8X102 CFU for the hypervirulent K.pneumoniae(harboring iroB gene and p-rmpA gene).Set the real-time polymerase chain reaction(Real-time PCR)as the gold standard,the result of evaluation of the diagnostic efficiency showed that both the sensitivity and specificity of the method were 100.00%for detection of clinical specimens of simulated bloodstream infections.CONCLUSION The new method established based on MIRA is applied for detection of hypervirulent K.pneumoniae strains,and the diagnostic efficiency of the new method is verified in the clinical specimens of simulated bloodstream infections.

关 键 词：多酶恒温快速扩增技术 高毒力肺炎克雷伯菌 引物设计 检测方法 

分 类 号：R378[医药卫生—病原生物学]