蜡样芽孢杆菌特异性基因筛选及聚合酶链式反应检测方法的建立

Screening of the specific genes and development of a PCR assay for the detection of Bacillus cereus

该研究以蜡样芽孢杆菌ATCC 14579的基因组为模板,利用比较基因组学方法筛选并通过聚合酶链式反应(polymerase chain reaction,PCR)验证获得3个蜡样芽孢杆菌的特异性基因,用于食品中蜡样芽孢杆菌的快速检测,并对引物的特异性、灵敏度、抗干扰能力和人工污染检测限等进行评价。结果表明,筛选获得的3个基因片段特异性较好。其中以gene2626设计的引物gFA2特异性最好,检测灵敏度可达359. 5 fg/μL。抗干扰评价结果表明在牛肉背景菌群浓度5. 28×10^7CFU/m L和猪肉背景菌群浓度7. 75×10^6CFU/m L的干扰下,蜡样芽孢杆菌的最低检出限为3. 74×10^3CFU/m L。人工污染试验结果表明,蜡样芽孢杆菌经增菌培养10 h后,人工污染的牛肉和猪肉样品中蜡样芽孢杆菌的检出限分别为4. 62和8. 38 CFU/g。因此该研究筛选的特异性基因及建立的PCR检测方法在食品中蜡样芽孢杆菌的快速检测方面具有一定的应用潜力。

Using the genome of Bacillus cereus ATCC 14579 as a template,the specific target of Bacillus cereus was screened by bioinformatics and comparative genomics analysis and three specific genes of Bacillus cereus were identified by PCR protocol. Then,these three specific genes were used for molecular biological detection of Bacillus cereus in food. The primers were evaluated by its specificity,sensitivity,anti-interference capability and artificial contamination. The results showed that three screened primers had better specificity and there was no specific band in non-Bacillus cereus. The primer gFA2 based on gene2626 had the best sensitivity which reached 359. 5 fg/μL. The anti-interference results showed that in the presence of natural background flora enriched from beef( 5. 28 ×10^7 CFU/m L) and pork( 7. 75 × 10^6 CFU/m L) samples,the minimum detection limit of Bacillus cereus was 3. 74 × 10^3 CFU/m L. After 10 h enrichment cultivation of Bacillus cereus,the detection limits of three primers to artificially contaminated beef and pork samples were 4. 62 CFU/g and 8. 38 CFU/g,respectively. In conclusion,the specific gene screened in this study and the PCR assay could be widely used for rapidly detection of Bacillus cereus in food safety validation.