微波提取苹果汁中脂环酸芽孢杆菌DNA的PCR检测方法研究

Optimization of DNA Extraction by Microwave Treatment and PCR Detection of Spoilage Alicyclobacillus acidoterrestris in Apple Juice

根据Genbank公布的酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris DSM 3922T)鲨烯环化酶序列自行设计一对引物,利用微波技术直接从果汁样品中提取目标菌DNA,对引物特异性、微波法提取DNA的扩增效果及检测灵敏度进行探讨。结果表明:微波功率1000W、处理时间30s、经5000r/min离心2min即可获得酸土脂环酸芽孢杆菌基因组DNA,所得模板质量符合聚合酶链式反应检测要求,目的条带清晰,检测时间仅为2h,检测限为200CFU/mL,有望真正应用于苹果汁生产的在线检测。

A simple and rapid method for microwave-assisted DNA extraction from A. acidoterrestris in juice sample was developed in this study. A pair of primers was designed based on the gene sequence encoding squalene epoxidase from Alicyclobacillus acidoterrestris published in the Genbank database to establish a PCR assay for detecting Alicyclobacillus acidoterrestris in apple juice. The specificity and sensitivity of the method was investigated by DNA amplification from l 1 Alicyclobacillus spp. and 7 other bacteria using the designed specific primers. The detection limit was determined using serially diluted extracts. The results of gel electrophoresis indicated that the DNA bands of microwave-assisted samples were the same as those obtained from ZR Genomic DNA Kit, implying effective bacterial cell lysis in juice samples. The limit of detection of this method was 200 CFU/mL. Only 2 hours were required to detect A. acidoterrestris in juice sample by this method. In conclusion, the microwave-assisted DNA extraction coupled to PCR detection method provides an effective and stable approach for the online detection of Alicyclobacillus spp. from apple juice.