蜡样芽胞杆菌DNA分子分型研究

Study on DNA molecular typing of Bacillus cereus

目的 建立蜡样芽胞杆菌分子分型方法 ,用于蜡样芽胞杆菌食物中毒的快速溯源。方法 15株蜡样芽胞杆菌进行生化分型 ,同时进行vrrA基因PCR扩增和聚丙烯酰胺凝胶电泳 银染检测PCR扩增产物 ,所有PCR扩增产物进行序列分析 ,并用ClustalW(ebi.ac .uk)分析软件对DNA序列进行同源性比较。结果 传统生化分型 :15株蜡样芽胞杆菌中 12株可分为 3个型 ,3株不能分型 ;主要生化型为 1型、2型和 4型。分子分型 :15株蜡样芽胞杆菌都可分为 7个型。结论 vrrA基因可作为蜡样芽胞杆菌分子分型的一个多态性遗传标记。蜡样芽胞杆菌分子分型方法与传统生化分型方法相比 ,将传统生化分型所需的 4 8h甚至更长时间缩短到 5h ,具有简便快速准确的优点 ,可做到快速溯源。

Objective To develop a molecular typing method of B.cereus for fast tracking of B.cereus food poisoning. Methods Fifteen B.cereus strains were typed by the classic bio-chemical assays and vrrA gene of 15 B.cereus was detected by PCR. The amplicons were then detected by silver staining of polyacrylamid gel. In order to analyze the differences of 15 vrrA DNA fragments, both strands of each amplicon were sequenced and DNA sequence files were analysed by using ClustalW (ebi.ac.uk) software. Results According to the classic bio-chemical typing protocols, of 15 B.cereus strains, 12 strains were typed into 3 subtypes and 3 strains could not be typed. The main types were type 1, type 2, & type 4. Meanwhile , 15 B.cereus were typed into 7 genotypes by molecular typing. Conclusion VrrA gene is one of the polymorphic genetic molecular marker. The molecular typing method is simple, rapid (5 hours less) and accurate in comparing classic bio-chemical typing method. So the molecular typing analysis could be used to track rapidly and analyze the difference between B.cereus strains.