Development Of ATP Regeneration System Combined With Bioluminescent Method For The Detection Of Trace Microbial

Microorganism monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, cosmetics and semiconductor production. At present the plate counting method has been commonly used to detect the microbial count in food. However, some undeniable faults existed in this method, such as big workload, time-consuming (generally need2～3days) which can’t meet the requirements of HACCP system.ATP bioluminescence assay based on firefly luciferase is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately104colony-forming units, which is insufficient for many applications.This study aims to improve the assay sensitivity by conversion of PPi, product of the luciferase reaction, back to ATP to form a chain-reaction loop.Because the PPi-recycling loop was completed using ATP sulfurylase (ATPS) and adenosine5’phosphosulfate (APS), each consumed ATP continuously produces two new ATP molecules. This approach can achieve exponential amplification of ATP. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its microbe detection sensitivity.The main research results are as follows:1.PPi-recycling loop was established:the concentration of APS of37.5umol/L, ATPS of0.1618U, pH value of7.8, Temperature control range of23～25℃. When it was saved at4℃, the energy recovery of ATPS was87%.With the increment of preservation temperature, the energy loss of ATPS was speeded. The liner range of PPi is10-11～10-8mol/mL, and the R2=0.988.2. PPi-recycling loop combined with bioluminescent method was established:the instrument optimal time was set to15s by the on-line monitoring trace of ATP regeneration process. According to the selection of microbial cell ATP extraction, heating was confirmed as the best method. Optimum ATP extraction was researched:the extraction time of10min, temperature of95℃.3. The assessment of this method:the detection limit of1.75×10-19mol/mL for ATP standard solution. The detection limit of Saccharomyces boulardii was lOCFU/mL, while E.coli and Pseudomonas was100CFU/mL The coefficient of variation was less than4.5%and7.8%for intra-and inter-assay precision, respectively. It showed that ATP regeneration method established in this paper was correct and reliable.4. The method of application:the optimized method were applied to detect the bacterial count in water from tap water pipeline and water machine, and correlations were reached at0.961when compared with the plate count method. Recoveries were from88%to107%. For other samples containing ATP (meat), the correlation of R2was0.923. It showed that this method can be used in actual detection.