葡糖杆菌的SYBR Green Ι荧光定量PCR方法的建立

Establishment of Real-time Fluorescence Quantitative PCR for Gluconobacter using SYBR Green I

摘要:

基于GenBank提供的葡糖杆菌的16S rRNA保守序列设计PCR引物，利用SYBR Green Ι荧光定量PCR技术建立一种快速灵敏检测酸乳制品中葡糖杆菌的方法。用本研究建立的SYBR Green Ι荧光PCR方法对3株葡糖杆菌以及18株非葡糖杆菌进行特异性检测，并用凝胶电泳验证其可靠性，结果显示，本研究所设计的引物具有良好的特异性。对扩增结束后的PCR产物进行溶解曲线分析，证实此引物的扩增产物存在引物二聚体，但可通过溶解曲线的出峰时间排除非特异性扩增。利用SYBR Green Ι荧光定量PCR检测葡糖杆菌建立的标准曲线相关性良好，R2=0.9968，对葡糖杆菌进行灵敏度检测，最低检出限可达75 CFU/mL。利用该方法可成功检测出5份人工污染样品中的葡糖杆菌，研究表明，该方法灵敏度高、操作简便快捷，适用于酸乳制品的定量检测。

Abstract:

PCR primer was designed according to 16S rRNA conserved sequence of Gluconobacter from the GenBank, and a rapid and sensitive fluorescence quantitative PCR using SYBR Green Ι for Gluconobacter detection in fermented dairy products was established. The established method was then used for the specific detection of a panel of three Gluconobacter strains and 18 non-Gluconobacter strains, with the reliability of the results verified by agarose gel electrophoresis. The results showed that the primers were highly specific. Melting curve analysis of the PCR amplification product showed that primer dimers were present in the amplification product. However, nonspecific amplification could be excluded using the peak time of the melting curve. The standard curve based on Gluconobacter detection using the established method had good correlation (R2 = 0.9968). The minimal detection limit was 75 CFU/mL in the sensitivity test. The established method also successfully detected Gluconobacter in five artificially contaminated samples. These results indicated that the SYBR Green Ι fluorescence quantitative PCR method used herein exhibited high sensitivity and was easy to use. Therefore, this method is suitable for the qualitative detection of Gluconobacter strains in fermented dairy products.