目的 排除样品中高浓度活菌和大量辅料及死菌DNA的干扰,检测地衣芽孢杆菌活菌胶囊中金黄色葡萄球菌。方法 将叠氮溴化丙锭(PMA)、实时荧光定量PCR(qPCR)和微孔膜过滤法相结合,收集滤液后离心,富集菌体,优化PMA工作浓度、暗孵育时间和曝光时间,确定PMA最佳处理方案。采用不同浓度金黄色葡萄球菌的死菌与活菌进行PMA-qPCR方法验证,同时考察方法灵敏度和特异性。结果 样品采用5.0 μm的聚醚砜膜过滤,辅料截留率和滤液活菌计数最高。PMA工作质量浓度40 μg·mL-1,暗孵育时间10 min,曝光时间10 min为最佳处理方案,该处理方案下PMA既不影响活菌DNA的PCR扩增,又能进入死菌中结合DNA抑制其扩增。本法高效快速、实时准确,全过程筛选鉴定只需4 h,最低检测限为1×104 CFU·mL-1。结论 本法灵敏度高、特异性强,适用于地衣芽孢杆菌活菌制品中金黄色葡萄球菌的检测。
Abstract
OBJECTIVE To exclude the influences of high concentration of live bacteria and a large amount of excipients in the sample and the DNA interference of dead bacteria on the detection of Staphylococcus aureus in Bacillus licheniformis live capsules. METHODS Combining the azide propidium bromide (PMA), qPCR, with the microporous membrane filtration method, the filtrate was collected and the bacteria were enriched by centrifugal method, and the working concentration, dark incubation time and exposure time of PMA were optimized to determine the best treatment plan for PMA. Different concentrations of dead and live Staphylococcus aureus were used to verify the PMA-qPCR method, the sensitivity and specificity were also investigated. RESULTS When the sample was filtered with 5.0 μm polyethersulfone membrane, the highest excipient retention rate and viable bacteria count in the filtrate were obtained. The best treatment procedure was as follows: PMA working concentration 40 μg·mL-1, dark incubation time 10 min, exposure time 10 min. Under this procedure, PMA did not affect the PCR amplification of live bacteria DNA, and could enter dead bacteria to bind DNA and inhibit its amplification. The whole process of screening and identification took only 4 h, and the minimum detection limit was 1×104 CFU·mL-1. CONCLUSION This method is efficient, fast, real-time and accurate, with high sensitivity and specificity, which is suitable for the detection of Staphylococcus aureus in Bacillus licheniformis live product.
关键词
叠氮溴化丙锭 /
实时荧光定量PCR /
微生态活菌制品 /
地衣芽孢杆菌活菌胶囊 /
金黄色葡萄球菌
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Key words
propidium monoazide /
qPCR /
probiotics /
Bacillus licheniformis live capsule /
Staphylococcus aureu
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中图分类号：
R917
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参考文献
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脚注
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基金
浙江省基础公益研究计划项目资助(LGC19H300001)
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