Establishment of a new method to detect viable cells of Ralstonia solanacearum by EMA-qPCR

Abstract:

[Objective] A novel method to differentiate viable/dead cells of Ralstonia solanacearum was established by using a DNA dye of ethidium monoazide bromide (EMA) in combination with the real-time polymerase chain reaction (EMA-qPCR). [Methods] Samples were pre-treated with EMA prior to DNA extraction. DNA from dead cells was bound by EMA, so that only DNA from viable R. solanacearum cells can be amplified by real-time PCR. [Results] A final concentration of 2.0 mg/L EMA was demonstrated to completely inhibit the PCR amplification from DNA derived from 1.0×107 CFU/mL dead cells, but no inhibition to viable and viable but non-culturable (VBNC) cells. A standard curve was generated relating the Ct values of the EMA-qPCR to the log number of genomic targets per PCR. A linear range of DNA amplification was observed from 5.0×100 to 5.0×104 genomic targets per PCR. EMA-qPCR method was used to evaluate the survival rate of R. solanacearum treated with different temperatures for a short time, compared with the method of plate count. The results indicate that samples can be stored for a short time under room temperature and 4 °C. [Conclusion] The EMA-qPCR method established in this work can effectively avoid false positive and false negative results of the R. solanacearum detection.

Key words:Ralstonia solanacearum, Ethidium monoazide bromide (EMA), Viable but non-culturable (VBNC), Real-time PCR