基于ATP再生体系快速检测乳品中微生物

Development of a Bioluminescence Method Combined with ATP Amplification for Detection of Bacteria in Dairy Products

基于焦磷酸(pyrophosphoric acid,PPi)再生三磷酸腺苷(adenosine triphosphate,ATP)建立乳品中微生物快速检测方法。通过单因素试验优化PPi再生ATP反应条件,并考察方法的灵敏度、准确度、精密度和稳定性。结果表明,PPi再生ATP最佳反应条件为腺苷酰硫酸(adenosine phosphosulfate,APS)浓度10μmol/L,ATP硫酸化酶(ATP sulfurylase,ATPS)活力0.15 U/m L、反应p H 7.8。在最佳的ATP再生条件下偶联生物发光法,对ATP标准品、大肠杆菌、铜绿假单胞菌的检测限分别为10^(-17) mol/m L、10~2 CFU/m L和10~2 CFU/m L。工作曲线在10~2~10~7 CFU/m L范围内线性关系良好,对乳品基质的回收率为81.33%~97.78%,变异系数为14.24%~22.17%,与国标平板计数法对比显示两种方法检测结果相关性良好,相关系数为0.96。本方法快速、简单、灵敏、稳定,适用于乳品中微生物快速监测。

A bioluminescence method combined with adenosine triphosphate（ATP） amplification through regeneration from pyrophosphate（PPi） was developed to detect bacteria in dairy products. Experimental conditions were optimized by one-factor-at-a-time method. The sensitivity, precision and accuracy of this method were studied. The results showed that adenosine phosphosulfate（APS） concentration of 10 μmol/L, ATP sulfurylase（ATPS） activity of 0.15 U/m L and p H of 7.8 were found to be the optimum conditions. The method showed a good linearity within the range of 10^2–10^7 CFU/m L with lowest limits of detection（LODs） of 10^-17 mol/m L, 10^2 CFU/m L and 10^2 CFU/m L for ATP, Escherichia coli and Pseudomonas aeruginosa, respectively. The recoveries of E. coli in dairy at three spiked levels ranged from 81.33% to 97.78%, with coefficients of variation between 14.24% and 22.17%. Good correlation between this method and the standard plate count method was obtained using spiked dairy samples. The proposed method is fast, simple, sensitive, stable, and thus suited for fast detection of bacteria in dairy products.