乳制品中金黄色葡萄球菌PCR快速检测方法的建立

Development of PCR Rapid Detection Method for Staphylococcus aureus in Dairy Products

为快速检测乳制品中的金黄色葡萄球菌,本研究将纳米氢氧化钛和纳米氢氧化锆固定在滤纸片上以去除PCR反应抑制因子,并以Nuc为靶基因,建立一种无需样品前增菌的PCR快速检测方法,同时确定该方法的特异性、灵敏度和实用性,并评估该方法所用试剂在冷冻保存条件下的稳定性与实用性。结果表明,该PCR快速检测方法在检测97株目标菌及83株非目标菌后未出现假阳性或假阴性结果;该方法无需增菌处理,可在4 h内完成检测,检测限为10^(1)CFU·25 g^(-1)或10^(1)CFU·25 mL^(-1);该方法与传统检测方法在实际乳制品样品中金黄色葡萄球菌的检出率及活菌检测率均一致(符合率100%);此外,该检测方法所用试剂在-20℃冷冻条件下可稳定保存至少12个月。综上所述,本研究建立的PCR快速检测方法特异性强、灵敏度高,且实用性良好,能有效去除样品基质中的PCR反应抑制因子,为乳制品中金黄色葡萄球菌的快速检测提供了参考依据。

To rapidly detect Staphylococcus aureus(S.aureus)in dairy products,in this study,nano-titanium hydroxide and nano-cobalt hydroxide were immobilized on a filter paper to eliminate PCR inhibitors.The PCR rapid detection method targeting the nuc primers was established without sample pre-enrichment,and the specificity,sensitivity,and application of the method were determined.The stability of the reagents stored under cryopreservation conditions were evaluated.The results showed that the PCR method had no false positive or negative results after detecting 97 target bacterial strains and 83 non-target bacterial strains.The method took less than 4 h to complete without pre-enrichment,and the detection limit was 10^(1)CFU·25g^(-1)or 10^(1)CFU·25mL^(-1).The method was consistent with the traditional detection method on the detection rate and viable bacteria detection rate of S.aureus in natural dairy product samples(the coincidence rate was 100%).Further,the reagents used in the method could be stored stably for at least 12 months under freezing conditions at-20℃.In summary,the PCR method developed in the study has outstanding specificity,high sensitivity and excellent application,and the PCR reaction inhibitors from the sample matrix could be effectively removed by the PCR method,which provides a reference for the rapid detection of S.aureus in dairy products.