志贺菌TaqMan-MGB探针实时荧光聚合酶链反应快速检测方法的应用研究

Rapid detection of Shigella by real-time fluorescent PCR assay using TaqMan-MGB probes

目的建立志贺菌TaqMan-MGB探针实时荧光PCR快速检测技术,为从患者、食品和环境中快速分离和鉴定志贺菌提供技术支撑。方法根据志贺菌ipaH基因序列设计一对特异性引物和TaqMan-MGB探针;通过对PCR扩增体系和反应条件的优化,建立志贺菌TaqMan-MGB探针实时荧光PCR快速检测方法;用添加已知志贺菌浓度的样本验证方法敏感性;用志贺菌标准菌株、分离株以及大肠埃希菌、沙门菌、副溶血性弧菌、金黄色葡萄球菌等致病菌验证方法特异性。结果用本研究建立的志贺菌TaqMan-MGB探针实时荧光PCR检测方法检测志贺菌,其Ct值与模板浓度的对数值具有很好的对应关系(Y=-3.93×log(X)+37.34,R=0.999),最低检测浓度为30cfu/ml,3株志贺菌标准株,30株志贺菌分离株检测结果均为阳性;而沙门菌、副溶血性弧菌、大肠埃希菌、金黄色葡萄球菌等91株其他细菌的Ct值均>35或扩增曲线成一平滑直线。与常规分离鉴定方法比较差异无统计学意义(P>0.05,χ2=0.27)。对于纯菌和食品样品整个检测过程仅需2h和10h。结论志贺菌TaqMan-MGB探针实时荧光PCR检测技术具有特异性强,敏感性高,易操作等优点,有很好的应用前景和研究价值。

Objective To establish an approach for rapid identification of Shigella isolated from patients, food and environment, taking advantage of real-time fluorescent PCR assay featuring TaqMan-MGB probes. Methods A couple of primers and a TaqMan-MGB probe were specifically designed according to the published sequence of Shigella ipaH gene. PCR amplification system and the reaction condition optimized, a TaqMan-MGB probe-based rapid detection of Shigella using real-time fluorescent PCR was developed. By adding strain samples of known concentrations, the sensibility of assay was evaluated. The specificity was validated using Shigella isolates and standard strains, Escherichia coli, Salmonella, Vibrio parahaemolyticus, and Staphylococcus aureus. Results Good corresponding relationship （Y = -3.93 × log（X） ＋ 37.34 ,R = 0. 999 ）between Ct value and the logarithm of bacterial concentration was present in the TaqMan-MGB probe-based real-time fluorescent PCR assay for Shigella detection. The minimum detectable concentration of Shigella was 30 cfu/ml. All the results of three Shigella standard strains and 30 isolates were positive, while those of 91 other bacterial strains such as Escherichia coli, Salmonella, Vibrio parahaemolyticus, and Staphylococcus aureus, were negative with the Ct value greater than 35 or the amplification curve turning into a smooth line. Compared with conventional identification methods, there was no significant difference （ P 〉 0.05 ,x^2 = 0.27 ）. However, the entire process of assay cost merely two and ten hours for pure bacteria and food samples. Conclusion The TaqMan-MGB probe-based real time fluorescent PCR assay served as a promising approach for easy, rapid and highly specific and sensitive detection of Shigella.