食品中沙门菌实时荧光PCR快速检测方法的研究

Rapid Detection of Salmonella in Food Samples by Real-time PCR

目的利用荧光定量PCR技术,建立食品中沙门菌污染的快速敏感特异的检测方法。方法以沙门菌的fimY基因作为靶序列,设计一对引物和探针,以沙门菌菌株提取核酸DNA作为模板,优化引物和探针的浓度比和Mg^(2+)浓度,以沙门菌和10种相关细菌考核检测体系的灵敏性、稳定性和特异性,并初步应用于样品的检测。结果本研究建立的反应体系在引物和探针的浓度为0.8μmol/L,1.0μmol/L,Mg^(2+)浓度为3 mmol/L时,具有良好的特异性和敏感性。在10株相关菌株的检测中,除沙门菌出现很好的阳性外,其余菌株均为阴性。在纯菌条件下,定量检测低限33 cfu/ml。稳定性分析表明:同一样品重复检测3次Ct值的变异系数均小于5%。检测样品结果显示实时PCR方法较传统方法敏感、快捷、简便。结论该方法特异性强,稳定性高,操作简便快捷,适应食品微生物检验发展需要,具有较大的推广及应用价值。

Objective To establish a rapid, sensitive and specific Salmonella detection method with real -time PCR. Methods Based on Salmonella rim Y gene, a pair of primers and a probe were designed. DNA extracted from Salmonella strain was used as template. Magnesium concentration, primer/probe concentration were optimized. Specificity, sensitivity and stability analyses were performed by Salmonella and 10 associated bacteria strains. The constructed real - time PCR method was primarily used for detection of Salmonella in food samples. Results The best Mg^2＋ concentration was 3 mmol/L. Primer concentration and probe concentration were 0. 8 μmol/L and 1.0 μmoL/L, respectively. Test showed that the probe was highly conservative and specific. The results of all 10 bacteria strains were negative except for strains of Salmonella. The detection limit of the method was 33 cfu/ml of pure cultures of Salmonella . Stability test showed that co - efficient variables were all less than 5% in 4 different samples. Primary application showed that real - time PCR method was more sensitive, easier and faster than conventional culture method for detection of Salmonella in food. Conclusion Real - time PCR method was easy to handle, rapid and of high sensitivity and specificity. It meets the need of development of food microorganism examination, and is valuble to be further extended.