蜡样芽胞杆菌快速检测及其毒力基因筛选

Screening specific genes and virulence genes of Bacillus cereus for its rapid identification and detection

目的筛选蜡样芽胞杆菌鉴定基因和快速检测毒力基因。方法选择分离自食品和土壤的代表性蜡样芽胞杆菌共329株,聚合酶链式反应(PCR)方法检测gyrB和groEL基因的种特异性,检测毒力基因在菌株中的分布特征。基于检测结果,用多重PCR检测方案快速检测蜡样芽胞杆菌鉴定基因及其毒力因子。结果在蜡样芽胞杆菌及其近缘芽胞杆菌中,除1株苏云金芽胞杆菌扩增阳性外,gyrB基因具有蜡样芽胞杆菌种特异性;而groEL基因在4种芽胞杆菌中均有扩增。6种毒力基因nheA、entFM、bceT、hblC、cytK和ces的携带率分别为84.19%、79.64%、49.24%、47.72%、47.11%和1.52%。选择nheA、hblC、entFM、ces、cytK和gyrB用于快速鉴定蜡样芽胞杆菌及其毒力基因,获得了双重PCR扩增体系(gyrB和cytK)与4重PCR扩增体系(nheA、hblC、entFM和ces)的最佳检测方案。结论筛选的蜡样芽胞杆菌鉴定基因和毒力基因能够全面、特异、简便、高效地检测蜡样芽胞杆菌,可为食品安全检测及快速诊断提供依据,在实际检验工作中具有良好的应用前景。

Objective To screen the specific genes and virulence genes of Bacillus cereus for its rapid identification and detection. Methods A total of 329 B. cereus strains isolated from food and soil were used to evaluate the specificity of gyrB and groEL genes for the identification of B. cereus and investigate the distribution of virulence genes with PCR method. The appropriate genes were selected to develop a multiplex PCR assay for rapid detection of B. cereus. Results Among the studied B. cereus and closely related Bacillus strains, gyrB gene was specific for B. cereus except for one B. thuringiensis strain, however, groEL gene was amplified in four species. The carrying rates of six virulence genes of nheA, entFM, bceT,hblC, cytK and ces were 84.19%, 79.64%, 49.24%, 47.72%, 47.11% and 1.52%, respectively. Six genes, gyrB, hblC, nheA,entFM, ces and cytK, were selected. Two multiplex PCR systems, including one duplex PCR(gyrB and cytK) and one quadruple PCR(nheA, hblC, entFM and ces), were optimized. Conclusion The specific genes and virulence genes of B. cereus screened can be used in the detection of B. cereus in a comprehensive, specific, simple and effective way and have good potential in laboratory detection practice.