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目的利用叠氮溴乙锭(ethidium monoazide bromide,EMA)实时荧光聚合酶链反应(PCR)技术,建立一种简便、快速、特异、灵敏的在食品中检测志贺活菌的方法。方法根据志贺菌ipa H基因保守序列设计特异性引物和探针。用不同EMA浓度、不同光照次数优化样品EMA前处理条件。用已知菌验证志贺活菌检测的敏感性和志贺死菌检测的抑制性。用31株志贺菌、26株单增李斯特菌、24株沙门菌、25株副溶血弧菌、11株阪崎肠杆菌、10株致病性大肠埃希菌和1株大肠埃希菌验证方法的特异性和稳定性。同时用本研究方法和常规分离培养法,对30件模拟样品进行实样验证。结果志贺活菌EMA实时荧光PCR方法的循环阈值(Ct)=32.10~3.19×log(菌量)(R2=0.994)。最低检测浓度为2.20 CFU/反应。对死菌DNA抑制效率≥99.97%。31株志贺菌Ct值最低为15.04,最高为26.54,而97株非志贺菌的Ct值均>35或无Ct值(呈一条直线)。重复试验Ct值变异系数<3。30件模拟样品采用本研究方法和常规分离培养法的检测结果一致,但采用EMA实时荧光PCR方法耗时不超过7.5 h,而常规分离培养方法则需要3~5 d。结论 EMA实时荧光PCR技术是一种快速简便、特异性强、灵敏度高、仅检测志贺活菌的方法,建议在食品检测中推广应用。
Objective To establish a simple, rapid, specific and sensitive method for the detection of Shigella viability using ethidium monoazide bromide (EMA) real-time fluorescence polymerase chain reaction (PCR). Methods Specific primers and probes were designed according to the conserved sequence of ipa H gene of Shigella. The EMA pretreatment conditions were optimized with different EMA concentrations and different illumination times. Susceptibility of the test of Shiga positive bacteria and inhibition of the test of Shiga kawachi were verified with known bacteria. Thirty-one strains of Shigella, 26 strains of Listeria monocytogenes, 24 strains of Salmonella, 25 strains of Vibrio parahaemolyticus, 11 strains of Enterobacter sakazakii, 10 strains of pathogenic Escherichia coli and 1 strain of Escherichia coli Verify the specificity and stability of the method. At the same time, 30 samples were verified by the method of this study and routine isolation and culture method. Results The cycle threshold value (Ct) of real-time fluorescent PCR of Shigella live cells was 32.10 ~ 3.19 × log (R2 = 0.994). The minimum detectable concentration was 2.20 CFU / reaction. Inhibition of dead bacteria DNA ≥ 99.97% efficiency. The lowest Ct value of 31 strains of Shigella was 15.04 and the highest was 26.54, while 97 strains of non-Shigella had Ct values> 35 or no Ct values (a straight line). The coefficient of variation (Ct) was less than 3.30 in the replicate test. The results of the proposed method were the same as those of the conventional method. However, the EMA real-time PCR method did not take more than 7.5 h, while the conventional method required 3 ~ 5 d. Conclusion EMA real-time fluorescence PCR is a rapid, simple, specific and sensitive method for detecting only live shigella. It is suggested that it should be widely applied in food testing.