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目的:基于TaqMan-MGB探针的荧光定量PCR技术,研发出一种定量检测食品污染物中空肠弯曲菌的方法,为食品污染物调查提供快速、可靠的监测工具。方法:根据TaqMan-MGB探针原理,在空肠弯曲菌基因组保守区设计引物和探针,探针的5′端用FAM荧光标记,3′端标记MGB,建立基于TaqMan-MGB探针的实时荧光定量PCR方法。并对其特异性、灵敏度、重复性进行评价,然后对收集的市场食品污染物调查样本进行检测,与常规方法做对照。结果:所建立的实时荧光定量PCR方法可准确、特异的检测空肠弯曲菌,最低检测限为10 cfu/ml;对50例市场污染物调查样本进行评价,结果显示6份食品污染物调查样本中空肠弯曲菌为阳性,其余为阴性,结果与常规培养检测结果一致。结论:空肠弯曲菌TaqMan-MGB探针荧光定量PCR方法具有结果可靠、操作简便等优势,比目前常规检测方法更灵敏,该方法可用于食物污染物调查、临床检验等多个方面。
OBJECTIVE: To develop a method for the quantitative detection of Campylobacter jejuni in food contaminants based on the TaqMan-MGB probe-based fluorescent quantitative PCR technique, and to provide a fast and reliable monitoring tool for the investigation of food contaminants. Methods: According to the principle of TaqMan-MGB probe, primers and probes were designed in the conserved region of Campylobacter jejuni. The probes were labeled with FAM fluorescence at the 5 ’end and MGB at the 3’ end to establish real-time fluorescence based on TaqMan-MGB probe Quantitative PCR method. And its specificity, sensitivity and repeatability were evaluated, and then the collected samples of food contaminants from the market were tested and compared with the conventional methods. Results: The established real-time fluorescence quantitative PCR method could accurately and specifically detect Campylobacter jejuni with the minimum detectable limit of 10 cfu / ml; 50 samples of market pollutants were evaluated and the results showed that in 6 samples of food contaminants survey Campylobacter jejuni positive, the rest is negative, the results and routine culture test results. Conclusion: The TaqMan-MGB probe fluorescence quantitative PCR method of Campylobacter jejuni has many advantages, such as reliable results, simple operation and so on. It is more sensitive than the conventional detection methods. This method can be used in many aspects such as food contamination investigation and clinical examination.