实时荧光聚合酶链式反应法快速鉴定阪崎克罗诺杆菌

Rapid identification of Cronobacter sakazaki by real-time fluorescence polymerase chain reaction

中文关键词:  实时荧光PCR  阪崎克罗诺杆菌  gyrB基因

英文关键词:real-time quantitative polymerase chain reaction  Cronobacter sakazakii  gyrB gene

中文摘要:

      目的 根据DNA旋转酶B亚基(gyrB)基因设计特异性的引物探针, 建立一种能够快速准确鉴定阪崎克罗诺杆菌的实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)方法。方法 寻找并在美国国家生物技术信息中心(National Center for Biotechnology Information, NCBI)中下载目标基因序列, 使用DNAMAN进行序列比对, Primer Express软件设计引物探针。通过特异性实验、绝对灵敏度、相对灵敏性实验、抗干扰实验对所建立方法进行方法验证。选择本实验室保存的36种40株食品中常见致病菌的标准菌株进行特异性验证。结果 多维度特异性验证实验结果显示该方法能够特异性地检测出阪崎克罗诺杆菌, 对亲缘关系较近的其他克罗诺杆菌及食品中较为常见的致病菌均无非特异性扩增。DNA检测灵敏度可以达到0.0100 ng/μL, 相对灵敏度可以达到103 CFU/mL。抗干扰实验结果显示, 将干扰菌和干扰菌DNA分别与阪崎克罗诺杆菌和阪崎克罗诺杆菌DNA进行混合检测, 对检测结果无显著影响, 说明该方法抗干扰能力良好。结论 本研究所设计的引物探针在实时荧光PCR方法下对食品样品中阪崎克罗诺杆菌的检测具有特异、快速、敏感和抗干扰的特点, 可为以后食品中阪崎克罗诺杆菌的检测提供技术支撑。

英文摘要:

      Objective To establish subsequently the real-time quantitative polymerase chain reaction (PCR) method that could quickly and accurately identify Cronobacter sakazakii, and design specific primer probes based on the DNA gyrB subunit gene. Methods The target gene sequences were searched and downloaded from National Center for Biotechnology Information (NCBI), sequence comparison was performed using DNAMAN, and primer probes were designed by Primer Express software. This established real-time quantitative PCR method was validated through specificity tests, absolute sensitivity tests, relative sensitivity tests and anti-interference tests. The 40 common pathogenic bacteria standard strains were selected for specificity validation. Results The results of multi-dimensional specificity validation showed that the method was able to specifically detect Cronobacter sakazakii, and there was no non-specific amplification for other closely related Cronobacter and common pathogenic bacteria in food. DNA detection sensitivity was 0.0100 ng/μL, while relative sensitivity was 103 CFU/mL. The anti-interference experiment results showed that mixing interfering bacteria and their DNA with Cronobacter sakazakii DNA and Cronobacter sakazakii did not significantly affect the detection results, indicating that this method had good anti-interference ability. Conclusion The primer probes designed in this study are specific, rapid, sensitive and anti-interference for the detection of Cronobacter sakazakii in food samples under real-time fluorescence PCR method. It can provide technical support for detection of Cronobacter sakazakii in the future.