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Rapid Detection of Pathogenic Bacteria in Fishery Products by IMS Combined with Luciferase-NADH: FMN Oxidoreductase System

Author: WangJing
Tutor: LinHong
School: Ocean University of China
Course: Aquatic Products Processing and Storage Engineering
Keywords: fishery products pathogenic bacteria enzyme system IMS rapid detection
CLC: TS254.7
Type: Master's thesis
Year: 2009
Downloads: 195
Quote: 1
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Abstract


Pathogenic bacteria is the primary problem of food safety, which can cause a variety of food-borne diseases and do great harm to people’s health. The critical measures to ensure food safety is to develop new, rapid, sensitive and specific methods for the detection of pathogenic bacteria.Based on the bioluminescence principles and immunomagnetic bead separation technology, a luciferase-NADH:FMN oxidoreductase enzyme system which can detetermine the pathogenic bacteria in fishery products was established. The two-enzyme system was used to detect the pathogenic bacteria via detecting bacteria intracellular NADH. Then the system was applied to the rapid detection of Listeria monocytogenes and Salmonella typhimurium in fishery products. The results were as follows:1. In order to suit for requirements of NADH, the bacterial luciferase-NADH:FMN oxidoreductase system was established and optimized. The detection limit of NADH was 1×10-10 mol/L, which was better than the methods of ultraviolet spectrophotometry and fluorescence method (1×10-5 mol/L, 1×10-8 mol/L, respectively).2. The bacterial luciferase-NADH:FMN oxidoreductase system is unable to identify the intracellular NADH of different bacteria strains. To improve specificity of detection system, IMS was used as the pre-treatment of sample. The efficiency of IMS by commercial IMBs was evaluated. The detection limit of L. monocytogenes was 4×102 cfu/mL and the detection limit of S. typhimurium was 5 cfu/mL. The efficiency of bacteria binding of concentration of bacteria was improved by changing the concentration of beads from 20μL to 60μL. Anti-Vibrio cholerae immunomagnetic bead was prepared, and some research was done on the performance analysis. The research provides the good technical support for more species of bacteria in fishery products.3. The hot Tris-HCl method was selected as the extraction method of intracellular NADH. The contents of intracellular NADH per bacteria were determined by using two-enzyme system. The content of L.monocytogenes is 2.38×10-17 mol/cell, and S. typhimurium’s is 2.78×10-16 mol/cell. The result of contents of intracellular NADH per bacteria provides the technical support for quantitative biosensor.4. The process to determine the L. monocytogenes and S. typhimuriumus in pure culture and in fishery products by IMS combined with luciferase-NADH:FMN oxidoreductase system was investigated. The system could directly detect （without pre-enrichment） L. monocytogenes and S. typhimuriumus （108 cfu/mL（g）） from pure culture and fishery products in an hour. Under experiment condition, there was a fine linear correlation between the bacteria count and luminescence intensity. But the sensitivity of detection system should be futher studied.5. To improve the sensitivity of detection system, a pre-enrichment technology was studied. The EB broth and SC broth was chosen for selective enrichment broth. Some research was done on pre-enrichment of L. monocytogenes、S. typhimurium and other competitive bacteria. The result showed that EB broth had a strong inhibitory effect to competitive bacteria. When the samples with 1 cfu/g of L. monocytogenes were enriched 32 h in the EB broth, and the samples with 1 cfu/g of S. typhimurium were enriched 12 h in the SC broth, it could be fit the two-enzyme system’s sensitivity and specificity. Adding per-enrichment, 1 cfu/g of L. monocytogenes could be detected in 34 h, and 1 cfu/g of S. typhimuriumus could be detected in 14 h.In summary, IMS combined with the bacterial luciferase-NADH:FMN oxidoreductase system is a specific, sensitive, rapid and simple method for the detection of pathogenic bacteria in fishery products.

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CLC: > Industrial Technology > Light industry,handicrafts > Food Industry > Aquatic products processing industry > Standards and inspection of aquatic products
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