基于多重PCR技术检测蜡样芽胞杆菌3种毒素基因方法的建立及评价

Establishment and evaluation of detection method of three toxin genes of Bacillus cereus based on multiplex PCR

目的:基于多重PCR技术,建立一种快速检测蜡样芽胞杆菌nhe、cytK和entFM 3种毒素基因的方法,并评价其效能。方法:煮沸法提取蜡样芽胞杆菌DNA,以蜡样芽胞杆菌nhe、cytK和entFM特异性毒素基因作为靶基因,采用NCBI primer-BLAST 5.0软件设计特异性引物,优化反应体系。建立三重PCR检测体系,并检测其特异性、灵敏度和稳定性。结果:煮沸法提取蜡样芽胞杆菌DNA,浓度为227 mg·L^(-1),纯度为1.50~1.60。采用多重PCR体系同时检测蜡样芽胞杆菌3种毒素致病基因,于退火温度56℃时,蜡样芽胞杆菌基因引物nhe499的扩增片段为499 bp,基因引物cytK191的扩增片段为191 bp,基因引物entFM363的扩增片段为363 bp。PCR体系扩增蜡样芽胞杆菌3种毒素基因后条带清晰明亮,且无非特异性条带,与金黄色葡萄球菌、大肠埃希菌和铜绿假单胞菌均无扩增,表明蜡样芽胞杆菌基因引物nhe499、cytK191和entFM363特异性强;3种引物在同一反应体系中互不干扰。灵敏度检测,多重PCR体系最低检测限可同时达到10^(-1)mg·L^(-1);稳定性检测,多重PCR体系自制备起每6个月检测稳定性一致。结论:建立的多重PCR检测体系对于蜡样芽胞杆菌nhe499、cytK191和entFM363 3种毒素基因灵敏度高、特异性强,检测结果准确稳定,检测体系具有良好稳定性,适用于快速检出蜡样芽胞杆菌的3种不同致病毒素基因。

Objective:To establish a method for rapid detection of three toxin genes of Bacillus cereus nhe,cytK and entFM based on multiplex PCR,and to evaluate its efficiency. Methods:The DNA of Bacillus cereus was extracted by boiling method. The specific toxin genes of Bacillus cereus nhe,cytK and entFM were used as target genes,and the specific primers were designed by NCBI primer-BLAST 5. 0software to optimize the reaction system. The triple PCR detection system was established and the specificity,sensitivity and stability were detected. Results:The Bacillus cereus DNA was extracted by boiling method with a concentration of 227 mg·L^(-1)and a purity of 1. 50. 60. The pathogenic genes of three toxins of Bacillus cereus were detected simultaneously by multiplex PCR system. When the annealing temperature was 56 ℃,the amplification fragments of Bacillus cereus gene primer nhe499,gene primer cytK191 and gene primer entFM363 were 499,191 and 363 bp,respectively. The bands of three toxin genes of Bacillus cereus amplified by PCR system were clear and bright,and there were no non-specific bands,and no amplification with Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa,indicating that the primers of nhe499, cytK191 and entFM363 of Bacillus cereus genes had strong specificities,the three primers did not interfere with each other in the same reaction system,and the lowest detection limit of multiplex PCR system was 10^(-1)mg·L^(-1). The stability test was consistent with the self-made preparation of multiple PCR system every 6 months. Conclusion:The established multiplex PCR detection system has high sensitivity and specificity for three toxin genes of Bacillus cereus nhe499,cytK191 and entFM363,the detection results are accurate and stable,and the detection system has good stability,which is suitable for rapid detection of three different pathogenic toxin genes of Bacillus cereus.