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目的 AP631炭疽噬菌体裂解蜡样芽胞杆菌的发现和鉴定。方法通过革兰氏染色、菌落形态特征、噬菌体裂解试验、青霉素敏感试验、溶血试验、生化试验和蛋白质毒素结晶试验对炭疽疫点现场分离的5株菌进行蜡样芽胞杆菌的鉴定,通过PCR扩增、小白鼠毒力测定试验和MLST基因检测等方法对该5株菌和本实验室保存的3株被AP631炭疽噬菌体裂解的蜡样芽胞杆菌进行毒力和基因测定。结果确定了8株可被AP631炭疽噬菌体不完全裂解的蜡样芽胞杆菌。从疫点采集分离的5株菌青霉素敏感试验阴性,炭疽毒力基因PCR扩增阴性,排除了炭疽芽胞杆菌;蛋白质毒素结晶试验染色试验为阴性,排除了苏云金芽胞杆菌;根据溶血试验和生化反应等一系列鉴别实验鉴定为蜡样芽胞杆菌。8株菌的MLST基因检测发现5株菌有新的等位基因位点或ST型。结论本研究首次证明我国使用的炭疽芽胞杆菌诊断AP631噬菌体可以不完全裂解部分蜡样芽胞杆菌,存在非特异性裂解现象,提示在今后使用噬菌体鉴定炭疽杆菌时,对于外环境分离到的可疑芽胞杆菌,特别要注意关于噬菌体不完全裂解的问题,这将为炭疽诊断标准的正确制订提供新的认识并具有重要的指导意义。
Discovery and identification of Bacillus anthracis lysed by Bacillus anthracis in AP631. Methods Bacillus cereus were identified on five strains isolated on-site by Gram stain, colony morphology, phage lysis test, penicillin sensitivity test, hemolysis test, biochemical test and protein toxin crystallization test. Virulence assay and MLST gene assay were used to test the virulence and genetics of the five strains and three strains of B. cereus lysed by the AP631 anthrax phage stored in this laboratory. As a result, eight strains of Bacillus cereus that were incompletely lysed by AP631 anthrax phage were identified. Five strains of penicillin-sensitive isolates collected from the epidemic spots were negative, the PCR amplification of anthrax virulence genes was negative, and Bacillus anthracis was excluded. The protein toxin crystallization test was negative and Bacillus thuringiensis was excluded. According to hemolysis test and biochemical reaction A series of identification experiments were identified as Bacillus cereus. Five strains of MLST gene test found that five strains have a new allele locus or ST type. Conclusions This study shows for the first time that Bacillus anthracis in our country can not completely cleave some Bacillus cereus of Bacillus anthracis by using Bacillus anthracis to diagnose Bacillus subtilis. There is nonspecific cleavage of Bacillus anthracis, which indicates that Bacillus subtilis, Special attention should be paid to the problem of incomplete phage lysis, which will provide a new understanding of the diagnostic criteria for anthrax and provide important guiding significance.