PMA-ddPCR method for detecting high ethanol-producing klebsiella pneumoniae in viable but non-culturable state
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.
万方数据
维普
