基于qPCR快速检测米饭中产呕吐毒素蜡样芽孢杆菌的研究

为了建立基于QPCR的快速方法检测米饭中产呕吐毒素蜡样芽孢杆菌,首先采用煮沸法提取蜡样芽孢杆菌基因组DNA,并利用普通PCR方法验证引物特异性,然后通过在米饭样品中添加目标菌,模拟受污染的实际样本,用QPCR技术定量检测米饭中产呕吐毒素蜡样芽孢杆菌。结果表明该方法具有快速、特异性强、灵敏度高和稳定性好的优点,能对产呕吐毒素蜡样芽孢杆菌定量。不经过增菌培养,实际样品的检测限为9.8×101CFU/g;经过2 h的增菌培养,检测限能达到100CFU/g;并且米饭中添加其他杂菌后,不影响对蜡样芽孢杆菌的检测。建立的QPCR方法适用于米饭等相关淀粉类食品中产呕吐毒素蜡样芽孢杆菌的检测,从而为监测该菌所致食品污染以及早期相关食物中毒提供快速定量检测方法。

Study on rapid detection of emetic Bacillus cereus by qPCR

Quantitative PCR method was developed for rapid and accurate detection of emetic Bacillus cereus in cooked rice. Experiments were carried out as described below: DNA was isolated by boiling methods and the specificity of primers was tested with conventional PCR. Cooked rice was inoculated with emetic B. cereus for imitating contaminated sample and then was detected by qPCR. Results showed that the qPCR assay was rapid, specific, sensitive and stable, which can detect emetic B. cereus accurately. The limit of detection for emetic B. cereus was as low as 9.8×10exp1 CFU/g in cooked rice sample and the assay was suitable for detection of 10exp0 CFU/g emetic B. cereus in cooked rice after a two-hour short enrichment in LB broth. In addition, the Ct values acquired by qPCR assay were maintained even in the presence of other microorganisms which contaminated the cooked rice frequently. The established qPCR method for emetic B. cereus detection is efficient, which can be used for detection of emetic B. cereus in cooked rice and other starch-derived food. Therefore, an emergency diagnosis method was developed for monitoring emetic B. cereus caused food contamination and detecting early food poison and foodborne diseases.