基于EMA-qPCR的茄科青枯菌活体检测技术的建立

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

摘要:

【目的】利用特异性核酸染料叠氮溴乙锭(Ethidium monoazide bromide, EMA)与实时荧光定量PCR技术相结合, 建立一种能有效区分青枯菌死活细胞的检测方法。【方法】样品DNA制备前经EMA渗透预处理, 再进行实时荧光定量PCR特异扩增菌体DNA。【结果】终浓度为2.0 mg/L的EMA能有效排除1.0×107 CFU/mL灭活青枯菌细胞DNA的扩增, 对活细胞和不可培养状态(Viable but non-culturable, VBNC)活菌的DNA扩增均没有影响。当每个定量PCR反应体系中的活细胞在5.0×100?5.0×104 CFU范围内时, 扩增Ct值与定量PCR反应体系中活细胞CFU对数值呈良好的负相关性(R2=0.992 5)。比较EMA-qPCR法和平板计数法对经过不同温度短期保存的青枯菌检测结果发现, 待检样品可在24 °C与4 °C冷藏条件下短期保存。【结论】本研究建立的EMA-qPCR方法能有效检测青枯菌VBNC细胞和有效区分死活菌, 避免或减少青枯菌PCR检测的假阳性和假阴性。

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.