食品中蜡样芽胞杆菌实时荧光RPA检测方法的建立与应用

Development and Application of Real-Time Recombinase Polymerase Amplification Assay for Detection of Bacillus cereus in Food

为实现简便快速地检测蜡样芽胞杆菌(Bacillus cereus),根据Gen Bank中蜡样芽胞杆菌16 S 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20 min即可完成检测。该方法特异性扩增蜡样芽胞杆菌16 S 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≥1.5×104CFU/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≥1.5×105CFU/g时,才能通过real-time PCR方法检出,所需时间至少为30 min(Ct值为20~31)。

The study established a simple and rapid method for the determination of Bacillus cereus. According to the 16 S 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 20 min. 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^-3 ng/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 × 10^4 CFU/g could be detected after 6-13 min by real-time RPA;when the bacterial concentration was 1. 5 × 10^5 CFU/g,the Bacillus cereus could be detected at least 30 min by real-time PCR（ the Ct value was between 20 and 31）.