利用EMA-qPCR建立快速检测猕猴桃溃疡病菌活菌的方法

摘要

利用叠氮溴乙锭(ethidium monoazide bromide,EMA)与实时荧光定量PCR技术相结合(EMA-qPCR),建立了一种有效快速检测猕猴桃溃疡病菌活菌的方法.以猕猴桃溃疡病菌ITS序列为检测靶标,菌体经EMA渗透处理,再进行qPCR特异性扩增.结果显示,qPCR检测灵敏度为2 cfu;当EMA的浓度为2.0 μg/mL时,能有效抑制1.0×107 cfu/mL经高温灭活的死菌的扩增,对活菌的扩增没有影响.当活菌数在1.0×101～1.o×105 efu范围内,每个qPCR反应体系中活菌数与Ct值呈线性相关(R2 =0.988).不同温度处理活菌菌悬液后用EMA-qPCR检测猕猴桃溃疡病菌的存活情况并与平板计数法进行比较,结果表明待检样品可在4℃和20℃短期保存.对疑似带病猕猴桃材料进行EMA-qPCR检测,结果表明能减少猕猴桃溃疡病菌PCR的假阳性结果.本研究建立的EMA-qPCR方法是一种有效检测猕猴桃溃疡病菌活菌的方法,能有效避免PCR检测实际样品可能造成的假阳性结果.%A method to rapidly detect only viable cells of Pseudomonas syringae pv.actinidiae (Psa) was established by EMA-qPCR.The ITS sequence was used as the target gene for qPCR detection of Psa.Samples were treated with EMA prior to DNA extraction.DNA was then amplified by qPCR to detect only viable Psa cells.The sensitivity of qPCR detection was 2 cfu.2 tμg/mL EMA could completely inhibit the PCR amplification of DNA derived from dead cells with the concentration of 1.0× 107 cfu/mL,but no inhibition to viable cells.A standard curve was generated relating the number of viable cells to the Ct values of the EMA-qPCR.A linear range of DNA amplification was observed from 1.0 × 101-1.0 × 105 cfu genomic targets per PCR.EMA-qPCR method was used to evaluate the survival rate of Psa treated with different temperatures for a short time,and compared with the method of plate counting.The results indicated that samples can be stored for a short time under 4℃ and 20℃.The data of EMA-qPCR detection on kiwifruit field samples indicated that 3 tμg/mL EMA could successfully inhibit PCR amplification of DNA from dead bacteria in filed samples.The EMA-qPCR method established in this study can effectively avoid false positive results of Psa detection.