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目的探讨炭疽芽孢杆菌DNA提取过程中的安全操作技术。方法分别采用常规提取法-热裂解法、热裂解+0.45μm滤膜过滤法进行炭疽芽孢杆菌和蜡样菌群DNA提取,采用实时荧光定量聚合酶链反应(Real-time PCR)方法进行炭疽芽孢杆菌鉴定和毒力基因的检测。结果常规提取法-热裂解法提取得到的炭疽芽孢杆菌DNA培养后出现了细菌菌落生长,生长比例占68.75%,热裂解+0.45μm滤膜过滤法提取得到的炭疽芽孢杆菌和蜡样菌群DNA培养后无细菌菌落生长。Real-time PCR检测中,炭疽芽孢杆菌疑似菌株均检出rpo B、pag A和cap基因。结论以热裂解+0.45μm滤膜过滤法进行炭疽芽孢杆菌DNA提取,对提取的DNA进行培养验证无菌生长后再进行下一步试验,可以有效地降低炭疽芽孢杆菌DNA提取过程中的生物安全风险。
Objective To explore the safe operation of Bacillus anthracis DNA extraction process. Methods The genomic DNAs of Bacillus anthracis and Corynebacterium sp. Were extracted by conventional methods of pyrolysis, pyrolysis and pyrolysis + 0.45μm membrane filtration, respectively. Real-time quantitative PCR (Real-time PCR) Bacillus identification and detection of virulence genes. Results Bacillus anthracis DNA extracted by the conventional pyrolysis method showed bacterial colony growth, the proportion of which was 68.75%, and that of Bacillus anthracis and waxy bacterium was extracted by pyrolysis + 0.45μm membrane filtration No bacterial colonies grow after culture. Real-time PCR detection, Bacillus anthracis suspected strains were detected rpo B, pag A and cap genes. Conclusion Bacillus anthracis DNA extraction by pyrolysis + 0.45μm membrane filtration method, the culture of the extracted DNA to verify the sterile growth before proceeding to the next step can effectively reduce the biosecurity risk of Bacillus anthracis DNA extraction process .