冷藏对虾中腐败希瓦氏菌活菌EMA-qPCR检测方法的建立

Establishment of EMA-qPCR Detection Method of Viable Cells of Shewanella putrefaciens in Frozen Shrimps

【目的】将叠氮溴乙锭(EMA)与实时荧光PCR技术相结合,建立快速检测腐败希瓦氏菌活菌的方法。【方法】用不同浓度的EMA和作用时间优化对腐败希瓦氏菌活菌的EMA-qPCR检测方法。研究该方法对活菌的最低检出限及能抑制死菌DNA扩增的最高浓度。研究该检测方法对不同比例死、活腐败希瓦氏菌的检测效果。用10株不同细菌验证该方法的特异性。检测101尾冷藏南美对虾中的腐败希瓦氏菌,并用平板培养法和qPCR方法对结果进行验证。【结果】EMA-qPCR检测最优方法为用终浓度20μg/mL的EMA处理细菌20 min。建立的EMA-qPCR法能够最低检测活菌的浓度为1 CFU/反应,能有效抑制1.0×10~7CFU/反应腐败希瓦氏菌死菌细胞DNA的扩增。对于死活混合菌,EMA-qPCR能够选择性扩增活菌DNA。该检测方法特异性良好。EMA-qPCR、平板培养和qPCR分别在16、12和19尾对虾中检测到腐败希瓦氏菌。【结论】EMA-qPCR检测技术能够快速、准确的区分腐败希瓦氏菌死菌和活菌,可作为水产品中腐败希瓦氏菌活菌的新检测方法。

【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 realtime 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/m L EMA was demonstrated to completely inhibit the PCR amplification from DNA derived from 1.0×10~7 CFU/reaction dead cells. The detection limit was 1 CFU/reaction and the maximum inhibitory concentration of dead bacteria was 1×10~5 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 q PCR.【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.