Development of an automated, DNA amplification technique-based method for detection of food-poisoning bacteria

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The purpose of this research project was to develop a quick and easy system to confirm food poisoning cases by combining the DNA amplification method or polymerase chain reaction (PCR) method for a particular target gene of the causative bacterium and an automated sample-processing and DNA-detecting equipment. The most important point was to establish an approach to obtain the oligonucleotide primers and amplification conditions which are the key factors determining the specificity and sensitivity of detection. We have adapted an empirical but sure method ; we repeatedly screened many test strains to determine the two parameters for each target gene of the food-poisoning bacterium. We then obtained primer sets and amplification conditions for the following target genes : the thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) genes of Vibrio parahaemolyticus, heat-labile and -stable enterotoxin genes and Vero toxin genes of Escherichia coli, enterotoxin genes and toxic shock syndrome toxin genes of Staphylococcus aureus, and cholera toxin gene of Vibrio cholerae.We also developed a method to detect amplified DNA fragments so that the above PCR can be performed in an automated equipment. The PCR products were labeled with biotin, trapped through affinity with streptoavidin onto the wells of a 96-well microtiter plate. The trapped PCR products were detected by oligonucleotide probes labeled nonisotopically (alkaline phosphatase). The feature of our detection method was that a few copies of the trget gene could be detected in 45 minites which is faster than any other microtiter-based non-isotopic methods reported so far.