食品中志贺活菌叠氮溴乙锭实时荧光聚合酶链反应技术研究

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ipaH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）=32.10~3.19log（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。

Alive foodborne pathogen Shigella detection by EMA real-time fluorescence PCR

Objective A detection assay for alive Shigella in food was developed by using of ethidium monoazide bromide (EMA) real-time fluorescence PCR technology, which is convenient, rapid, specific and sensitive. Methods The conserved ipaH gene was chose as the specific primer and probe to detect Shigella. Samples were pretreated with EMA at different concentrations and irradiating times. The detection sensitivity, specificity and stability were evaluated by using 31 Shigella strains, 26 Listeria monocytogenes strains, 24 Salmonella strains, 25 Vibrio parahemolyticus strains, 11 Cronobacter sakazakii strains, 10 Enteropathogenic Escherichia coli strains, and 1 E. coli strain, and the comparisons of the specificity and stability in detection of 30 simulated samples were made between newly developed EMA real-time fluorescence PCR assay and routine culture. Results The Ct value of EMA real-time fluorescence PCR for alive Shigella was 32.10-3.19log (bacteria load).The lowest limit of detection was 2.20 CFU per reaction. More than 99.97% of PCR reaction for dead strains was inhibited. The Ct values of 31 Shigella strains were between 15.04 and 26.54, while 97 non-Shigella strains had Ct values35 or had no Ct values (one horizontal line appeared). The variation coefficient of Ct was less than 3 when same samples were tested every other day for five times. The results of the detections of 30 simulated samples were consistent by either using newly developed EMA real-time fluorescence PCR or routine culture, but the test time was shortened from 3-5 days (routine culture)to less than 7.5 h. Conclusion The newly developed EMA real-time fluorescence PCR for alive Shigella is rapid, convenient, specific and sensitive, it is recommended to use it in food inspection.