关键词: 腐败希瓦氏菌, 活菌检测, 叠氮溴化乙锭, 实时荧光定量PCR, 南美白对虾

Abstract: 【Objective】 The aim of this study was to establish a novel method to detect alive Shewanella putrefaciens by using a DNA dye of ethidium monoazide bromide (EMA) in combination with the real-time polymerase chain reaction (qPCR). 【Method】 The pretreatment conditions including EMA concentrations and irradiating times were optimized.The detection limit to viable bacteria and test results in different mix proportions of bacteria of this method were confirmed. The detection specificity was evaluated by using 10 different bacteria. 101 samples of frozen shrimp were detected by using EMA-qPCR, the results were verified by plate culture and qPCR. 【Result】 EMA-qPCR method was established to detect alive Shewanella putrefaciens. After 20 min, a final concentration of 20 μg/mL EMA was demonstrated to completely inhibit the PCR amplification from DNA derived from 1.0×10⁷ CFU/reaction dead cells. The detection limit was 1 CFU/reaction and the maximum inhibitory concentration of dead bacteria was 1×10⁵ CFU/reaction. EMA-qPCR could completely inhibit the PCR amplification from DNA derived from dead cells, but no inhibition to viable cell in different mix proportions of bacteria. The method has good specificity. Shewanella putrefaciens were detected in 16, 12 and 19 samples of shrimp by EMA-qPCR, plate culture and qPCR. 【Conclusion】 The EMA-qPCR method established in this work can rapidly and accurately distinguish between dead and living bacteria and the EMA-qPCR could be applied in aquatic products.

Key words: Shewanella putrefaciens, alive bacteria detect, ethidium monoazide bromide, real-time PCR, Litopenaeus vannamei