通用型酵母菌实时荧光PCR检测方法的建立

Establishing a Universal Fluorescence Real-time PCR Method for the Detection of Yeast in Food

为建立一种快速检测鉴定食品中酵母污染菌实时荧光PCR法,根据酵母基因序列设计出通用型探针和引物,建立了运用实时荧光PCR法检测酵母的反应体系和反应条件,进而对该方法进行特异性验证,灵敏度分析及稳定性评价,并应用于产品样本的检测。特异性试验表明,酵母菌检测结果均为阳性,而细菌、霉菌均为阴性,荧光PCR检测结果的特异性为100%。灵敏度试验表明,酵母菌检出限在760 cfu/m L。稳定性试验表明,酵母菌组内实验CV在0.62-0.81%之间波动,而组间实验在0.43-0.77%之间波动。通过样品增菌检测发现在培养12 h后能检出阳性。本研究所建立的实时荧光PCR法特异性好、灵敏度高、稳定性好,具有快速、简便的特点,为快速检测食品中酵母污染提供新的方法和途径,具有很好的研究价值和应用前景。

The aim of this study was to establish a rapid fluorescence real-time PCR method for the detection of contamination in yeast present in food products. For this purpose, a universal probe and primers based on the yeast genome sequence were designed. The reaction system and conditions for fluorescence real-time PCR detection of yeast were established. The method was tested for its specificity, sensitivity, and reproducibility, and was also applied to the examination of food samples. The fluorescence real-time PCR assay was shown to display 100% specificity, as positive results for the specificity test were obtained only in yeast and not in bacteria or mold. The limit of detection of sensitivity was determined to be 760 cfu/mL. The sample was also tested for reproducibility, and the coefficient of variation （CV） was observed to fluctuate in the range of 0.62%-0.81% within the yeast group, and 0.43%-0.77% between groups. Moreover, a yeast enrichment test was performed, which demonstrated that the positive samples could be detected even after 12 h. In summary, the fluorescence real-time PCR assay established in this study showed excellent specificity, high sensitivity, and good reproducibility, in addition to being rapid and easy to use. This method could be used in the rapid detection of yeast contamination in food, and therefore can be used in research and related applications.