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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 a rapid real-time PCR detection system for TaqMan-MGB probe of Shigella to provide technical support for the rapid isolation and identification of Shigella from patients, food and environment. Methods According to the sequence of ipaH gene of Shigella, a pair of specific primers and TaqMan-MGB probe were designed. Through the optimization of PCR amplification system and reaction conditions, a real-time fluorescent PCR rapid detection method of Shigella TaqMan-MGB probe was established. Specificity of the method was verified by adding a sample of known Shigella concentration to the test method; using Shigella standard strains, isolates and pathogenic bacteria such as Escherichia coli, Salmonella, Vibrio parahaemolyticus and Staphylococcus aureus to verify the specificity . Results The Shigella sonnei was detected by real-time fluorescent PCR assay of TaqMan-MGB probe of Shigella in this study. The Ct value has a good correspondence with the logarithm of template concentration (Y = -3.93 × log (X) + 37.34, R = 0.999). The lowest detection concentration was 30 cfu / ml. The results of three strains of Shigella and 30 strains of Shigella were all positive. However, Salmonella, Vibrio parahaemolyticus and Escherichia coli , Staphylococcus aureus 91 other bacteria Ct value> 35 or amplification curve into a smooth straight line. There was no significant difference between the method of routine isolation and identification (P> 0.05, χ2 = 0.27). For pure bacteria and food samples the entire testing process only 2h and 10h. Conclusion The real-time PCR detection of Shigella TaqMan-MGB probe has the advantages of strong specificity, high sensitivity, easy operation and so on. It has good application prospect and research value.