荧光PCR直接检测食物中毒样品中A(B)型肉毒梭菌

Detection of Clostridium botulinum type A(B) strain in food poisoning sample by real-time fluorescent quantitative PCR

目的建立食物中毒样品中肉毒毒素基因的荧光PCR快速检测方法。方法对食物中毒臭豆腐样品进行前处理,直接从样品中提取基因组总DNA,对毒素基因保守区域设计特异性引物和探针,分别进行肉毒梭菌、A型、B型、E型和F型肉毒毒素基因的荧光PCR检测,FAM通道采集扩增荧光信号。结果食物中毒臭豆腐样品经过去蛋白和脂肪等前处理后,可直接提取获得食物基因组总DNA,以总DNA为模板同时进行肉毒梭菌及毒素基因的荧光PCR检测,检出含有A型、B型毒素基因,只有A型毒素基因表达的A(B)型肉毒梭菌。结论荧光PCR方法可快速检测食物中毒样品中的肉毒梭菌和毒素基因,对肉毒梭菌污染食品引起的食物中毒诊断具有参考价值。

Objective To establish a real-time fluorescent quantitative PCR method（ RT-PCR） for the rapid detection of botulinum toxin gene in poisoned food sample. Methods The samples of food poisoning stinky tofu were pretreated,the total genomic DNA was extracted directly from the sample,and specific primers and probes were designed for conserved regions of the toxin gene. C. botulinum,type A,type B,type E and type F botulinum toxin gene were conducted for fluorescence PCR detection,and FAM channel was used for acquisition amplification fluorescence signal. Results After the pretreatment removed of proteins and fats in the food poisoning stinky tofu samples,food genome total DNA can be directly extracted to obtain. Using total DNA as a template,clostridium botulinum and toxin genes were detected by fluorescent PCR simultaneously. C. botulinum type A（ B） containing only type A and type B toxin genes and only type A toxin gene in expression was detected. Conclusion The established RT-PCR method can detect C. botulinum and toxin genes in food poisoning sample directly,which may be valuable for the diagnosis of food poisoning event caused by C. botulinum toxin.