三磷酸腺苷生物发光法快速测定食品中细菌的含量

Development of a rapid microplate bioluminescence assay for determination of bacteria′s adenosine-5′-triphosphate in food

建立了测定食品中细菌三磷酸腺苷（—帅）的生物发光分析方法。生物发光原理基于萤光素酶、虫萤光素、氧气、ATP和Mg^2＋的催化氧化反应。产生的光信号强度与ATP含量成一定关系。考察了各种物理化学参数对反应的影响。反应体系最优化条件：pH7．4、牛血清白蛋白（BSA）浓度为1．0g／L和室温反应。方法检出限为1．0×10^12mol／L。线性范围1．0×10^-7-1．0×10^-11mol／L，分析时间30min，批内变异和批间变异分别小于4％和5％。研究了部分食品中细菌检测的样品前处理方法，应用于糕点和饮料样品中细菌的测定，加标回收率范围为82．1％～115％。检测结果与传统培养方法检测结果相关性良好。

A bioluminescence assay （BL） method with high sensitivity, rapidity, and reproducibility was established for the determination of bacteria＇s adenosine-5＇-triphosphate （ATP） in food samples. The bioluminescence was based on a subsequent reaction of firefly luciferase, lucifefin, oxygen, ATP and Mg^2 ＋ . The BL emission intensity was direcfly proportional to the amount of ATP presented in a sample solution. The effects of several physicochemical parameters upon the BL assay were studied. Such conditions were optimized as pH value of 7.4, the concentration of BSA of 1.0 g/L and the reaction of room temperature. The proposed method exhibited advantages in a low minimum detectable concentration of 1.0×10^12mol/L and showed a wide linear range of 1.0×10^-7-1.0×10^-11mol/L, as well as less total assay time of only 30 min. The coegicient of variation was less than 4% and 5% for intra- and inter-assay precision, respectively. This method has been successfillly applied to the evaluation of ATP in cake and drink samples with recoveries from 82.1% to 115%. It showed a good correlation with the traditional method.