Development and Application of Real-Time Recombinase Polymerase Amplification Assay for Detection of Bacillus cereus in Food(食品中蜡样芽胞杆菌实时荧光RPA检测方法的建立与应用)
摘要
The study established a simple and rapid method for the determination of Bacillus cereus. According to the 16S RNA gene sequences of Bacillus cereus available in Genbank, specific primers and exo probe were designed for establishing real-time recombinase polymerase amplification(real-time RPA).The RPA reaction was performed successfully at 39℃ and the results were obtained within 20min. This method could specifically detect Bacillus cereus, but could not detect other bacteria. The study showed that the detection limit of real-time RPA was 1.0×10-3ng/μL with genomic DNA of Bacillus cereus, which was the same as the real-time PCR method. Bacillus cereus in artificially contaminated rice samples with a bacterial concentration of 1.5×10<sup>4</sup>CFU/g could be detected after 6-13min by real-time RPA; when the bacterial concentration was 1.5×10<sup>5</sup>CFU/g, the Bacillus cereus could be detected at least 30 min by real-time PCR (the Ct value was between 20 and 31).(为实现简便快速地检测蜡样芽胞杆菌(Bacillus cereus),根据GenBank中蜡样芽胞杆菌16S RNA序列设计特异性引物和exo探针,建立了一种实时荧光重组酶聚合酶扩增方法,在39℃恒温下仅需20min即可完成检测。该方法特异性扩增蜡样芽胞杆菌16S RNA基因片段,对其他芽胞杆菌和非芽胞杆菌无扩增；以蜡样芽胞杆菌基因组DNA作为模板,该方法的检测灵敏度为1.0×10-3ng/μL,同已发表的real-time PCR方法一致。人工污染实验表明,当大米饭中蜡样芽胞杆菌污染量≥1.5×10<sup>4</sup>CFU/g时,即可通过real-time RPA方法检出,所需时间仅为6～13min；而当污染量≥1.5×10<sup>5</sup>CFU/g时,才能通过real-time PCR方法检出,所需时间至少为30min(Ct值为20～31)。)