Study On The Enzymic Bacterial Bioluminescence System And Its Application In Detection Of Bacterial Count

Total viable count (TVC) is one of the most important indicators in food qualitycontrol, and it has a vital significance in food processing, environmental monitoring,food hygiene, as well as disease prevention. As a result, the development of anaccurate, rapid and sensitive method of bacteria detection is a hot spot in research offood safety.Based on detection of NADH, bacterial luciferase bioluminescence enumeratesbacterial numbers by establishing the relationship between bacterial count andluminescence intensity. Our laboratory has used this method to detect E.Coli,Klebsiella and Vibrio Parahaemolyticus for the first time and the detection limit wasapproximately107cfu/mL. Because of low levels of cellular NADH, the previousmethod cannot meet the requirements of many industrial applications. Thus, an urgentproblem is to improve the sensitivity of bacterial determination, and it can be solvedby adding appropriate luminescence substrates such as the ubiquitous cellular pyridinenucleotides (NAD+, NADP+, NADH and NADPH) and ATP. The luminescencesignals were enhanced by amplifying reaction systems in which the oxidized forms ofpyridine nucleotides (NAD+or NADP+) were converted to the reduced forms (NADHor NADPH) by enzymatic reactions. The amplified luminescence signals have a goodrelationship with bacterial count and then the quantitative detection of bacteria wasestablished. Besides, low ATP was grown at an exponentialrate by amplificationreactions and then ATP was converted to NADH to accomplish the qualitativedetection of bacteria by bacterial bioluminescence assay.1. Based on the principle of cofactor regeneration, systems of alcoholdehydrogenase, lactic dehydrogenase, glucose-6-phosphate dehydrogenase,glutathione reductase were established through the optimization of enzymaticreaction conditions. Four systems can realize the maximum conversion betweenthe oxidized pyridine nucleotides and reduced forms, which laid a good foundation for the application of enzymatic bioluminescence system in bacterialdetection.2. To increase the sensitivity of bacterial detection, bacterial bioluminescence andNAD(P)+reduction reaction were combined. Alcohol dehydrogenase systemand glucose-6-phosphate dehydrogenase system were used to convert NAD+,NADP+to NADH and NADPH, respectively. Based on the contents of pyridinenucleotides, the luminescence intensity had a good correlation with the bacterialcount (R2=0.98) and the detection limit was1.05×105cfu/mL. The improvedmethod improves the sensitivity nearly100times compared with the previousbioluminescence methods.3. ATP amplification for ultrasensitive bioluminescence assay was used to detectbacterial count. The logarithmic growth of intracellular ATP could be realizedby means of amplification reactions. And the increased ATP was converted toNADH by reactions catalyzed by NAD synthetase and alcohol dehydrogenase.This new method allows us to detect as low as101cfu of E.Coli within120min,which sharply increase the sensitivity of bacterial luminescence method.