PMA-ddPCR method for detecting high ethanol-producing klebsiella pneumoniae in viable but non-culturable state
10.3760/cma.j.cn112150-20240401-00265
- VernacularTitle:PMA-ddPCR法检测活的非可培养状态高产乙醇肺炎克雷伯菌
- Author:
Shuo ZHAO
1
;
Chenpu DOU
;
Jian ZHANG
;
Jing YUAN
Author Information
1. 首都儿科研究所细菌学研究室，北京 100020
- Keywords:
PMA-ddPCR;
Viable but non culturable state;
HiAlc Kpn
- From:
Chinese Journal of Preventive Medicine
2024;58(7):998-1003
- CountryChina
- Language:Chinese
-
Abstract:
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 45 th day. The quantitative results of PMA-ddPCR on VBNC state cells were (5.46±0.05) log 10 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 log 10 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.
