PMA-ddPCR法检测活的非可培养状态高产乙醇肺炎克雷伯菌  

PMA-ddPCR method for detecting high ethanol-producing klebsiella pneumoniae in viable but non-culturable state

作　　者：赵硕 窦晨镤 张建 袁静[1] Zhao Shuo;Dou Chenpu;Zhang Jian;Yuan Jing(Department of Bacteriology,Capital Institute of Pediatrics,Beijing 100020,China;Department of Neurosurgery,Children′s Hospital Capital Institute of Pediatrics,Beijing 100020,China)

机构地区：[1]首都儿科研究所细菌学研究室,北京100020 [2]首都儿科研究所附属儿童医院神经外科,北京100020

出　　处：《中华预防医学杂志》2024年第7期998-1003,共6页Chinese Journal of Preventive Medicine

基　　金：中国博士后基金(2023M742428);2024年北京市博士后工作经费科研活动资助;北京市朝阳区2023年度博士后科研基金;国家自然科学基金(82130065, 82104243)。

摘　　要：目的建立活的非可培养(VBNC)状态高产乙醇肺炎克雷伯菌的绝对定量方法。方法诱导高产乙醇肺炎克雷伯菌进入VBNC状态,评价乙醇产量。建立PMA-ddPCR方法通过单拷贝基因来计数高产乙醇肺炎克雷伯菌VBNC状态的活细胞基因拷贝数。进一步在VBNC状态粪便模拟中,评价ddPCR对低浓度样品检测的灵敏度和适应性。结果对高产乙醇肺炎克雷伯菌梯度稀释液进行定量时ddPCR的检测下限是qPCR的10倍。在低温、低营养状态,高产乙醇肺炎克雷伯菌第45天进入VBNC状态,通过PMA-ddPCR对VBNC状态细胞进行定量结果为(5.46±0.05)log10 DNA 拷贝数/ml。VBNC状态的乙醇产量为<2.2 mmol/L,复苏后恢复产乙醇能力。VBNC状态粪便模拟样品中ddPCR最低检测下限为3.2 log10 DNA拷贝数/ml。结论建立VBNC状态高产乙醇肺炎克雷伯菌的ddPCR检测方法灵敏度和适应性良好,可用于临床样品中VBNC状态细胞的检测。Objective To establish an absolute quantitative method for high ethanol-producing klebsiella pneumoniae in a viable non-culturable(VBNC)state.Methods High ethanol-producing Klebsiella pneumonia was induced to enter the VBNC state and then the ethanol production was evaluated.A PMA-ddPCR method was established to count the copies of live cell genes in the VBNC state of high ethanol-producing Klebsiella pneumoniae using single-copy genes.Further,the sensitivity and adaptability of ddPCR for detecting low-concentration samples were evaluated in VBNC fecal simulation.Results The lower detection limit of ddPCR for quantitative analysis of high ethanol-producing Klebsiella pneumoniae gradient diluent was 10 times that of qPCR.At low temperature and low nutritional state,high ethanol-producing Klebsiella pneumoniae entered the VBNC state on the 45th day.The quantitative results of PMA-ddPCR on VBNC state cells were(5.46±0.05)log10 DNA copies/ml.The ethanol production in the VBNC state was<2.2 mmol/L and the ability to produce ethanol was restored after recovery.The minimum detection limit for ddPCR in fecal simulated samples with VBNC state was 3.2 log10 DNA copies/ml.Conclusion The ddPCR detection method for high ethanol-producing Klebsiella pneumoniae with VBNC state has good sensitivity and adaptability,and can be used for the detection of VBNC state cells in clinical samples.

关 键 词：叠氮溴化丙锭-微液滴数字聚合酶链反应 活的非可培养状态 高产乙醇肺炎克雷伯菌 

分 类 号：R446.5[医药卫生—诊断学]