Abstract
The manufacturing processes of many electronic and medicinal products demand the use of high-quality water. Hence the water supply systems for these processes are required to be examined regularly for the presence of microorganisms and microbial biofilm. Several bacteria detection methods have been employed for this purpose including ATP luminescence assay, polymerase chain reactions, fluorescent in situ hybridization, and a β-glucuronidase-based assay. Among them, the ATP luminescence assay is a rapid, sensitive and easy to perform method. The first aim of this study is to investigate whether ATP regeneration from inorganic pyrophosphate (PPi), a product of the ATP luminescence assay, can stabilize the bioluminescence signals in ATP detection. ADPglc pyrophosphorylase (AGPPase), which catalyzes the synthesis of ATP from PPi in the presence of ADPglc, was selected because the system yields much lower luminescence background than the commercially available ATP sulfurylase/adenosine 5´ phosphosulfate (APS) system which was broadly used in pyrosequencing technology. The method could also be used to stabilize the luminescence signals in detection of Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus either in broth or biofilm, and a detection limitation of 103 CFU/ml can be achieved. The second aim of this study is to increase the detection sensitivity from 104 CFU/ml to 102 CFU/ml of bacteria. One possible approach is to regenerate ATP simultaneously from both AMP and PPi, two major products of ATP luminescence assay, in a one-tube reaction. Regenerating AMP into ATP involves two consecutive enzyme reactions: adenylate kinase converting AMP and UTP into ADP and UDP; followed by acetate kinase catalyzing acetyl phosphate and ADP into ATP and acetate. Unfortunately, the AGPPase-based assay resulted in a high luminescence background when coupled with adenylate kinase. Therefore, ATP sulfurylase/APS system was used in the AMP-PPi Bi-directional ATP amplification (BDAA) system to replace AGPPase-based assay. These findings indicate that the AGPPase-based and the AMP-PPi bi-pathway ATP regeneration systems could increase the detection limitation to 102 CFU which will find many practical applications such as detection of bacterial biofilm in water pipelines.