基于cgcA基因实时荧光PCR快速检测穆汀斯克罗诺杆菌的研究

Rapid Detection of Cronobacter muytjensii by Real-time PCR Based on cgcA Gene

摘要:

为实现奶粉中常见污染菌穆汀斯克罗诺杆菌的快速检测与控制，本文根据cgcA基因序列设计出特异性探针和引物，建立了运用实时荧光定量PCR法检测穆汀斯克罗诺杆菌的反应体系和反应条件，并对该法的特异性、灵敏度、稳定性进行评价，并进行人工染菌样品检测实验。特异性结果表明，对穆汀斯克罗诺杆菌的荧光PCR检测结果的特异性为100%；灵敏度结果表明，穆汀斯克罗诺杆菌检出限在440 cfu/mL；稳定性结果表明，穆汀斯克罗诺杆菌组内实验CV在0.48~0.69%之间波动，而组间实验CV在0.69~0.73%之间波动；人工染菌样品试验表明，在增菌6 h后能检出阳性。本研究所建立的实时荧光定量PCR法特异性好、灵敏度高、稳定性强，有望成为快速检测食品中穆汀斯克罗诺杆菌污染的新方法，具有很好的研究价值和应用前景。

Abstract:

To achieve the rapid detection of the common contaminating bacteria C. muytjensii in milk powder, specific probes and primers were designed based on the cgcA gene. The reaction system and reaction conditions for the detection of C. muytjensii were developed using quantitative real-time polymerase chain reaction (PCR) assay. Additionally, the specificity, sensitivity, and reproducibility of this method were evaluated, and the method was applied for the detection of artificially contaminated samples. The results showed that the specificity of real-time PCR assay was 100%; the sensitivity results revealed a detection limit of 440 cfu/mL for C. muytjensii. The reproducibility test results showed that the intra-assay coefficient of variations (CVs) ranged from 0.48% to 0.69%, while inter-assay CVs ranged from 0.69% to 0.73%. For the artificially contaminated samples, a positive result was obtained after 6 h of enrichment. The established real-time PCR assay exhibited good specificity, high sensitivity, and high reproducibility, and has promising potential to be a novel method for the rapid detection of C. muytjensii in food as well as a good value in research and application.