抄録
A PCR method using a pair of arbitrary and 16S rDNA specific primers was developed to detect various microorganisms in contaminated food. Eight arbitrary primers consisted of 15 base nucleotide sequences were designed, and PCR was performed with one of arbitrary primers and 16S rDNA specific primer (semiAP-PCR), respectively. Genomic DNA of Escherichia coli, Bacillus subtilis, or Pseudomonas putida was used as a template for semiAP-PCR respectively, and each microorganism displayed various sizes of amplified DNA fragments by random annealing of arbitrary primers. The polymorphism of amplified fragments was also observed when total RNA of each microorganism was used as a template for reverse transcription (RT)-semiAP PCR. The detection limit of E. coli in milk was 10 CFU/ml (20 CFU/tube). When two microorganisms, E. coli and B. subtilis, were added in the sample, the observed fragments by semiAP-PCR amplification were a mixture of those of each microorganism. These "DNA fingerprints" are possible to offer a rapid detection of microbial contaminants in the fermented foods, without preparing the primers specific for each microorganism or separating the amplified fragments of each microorganisms by digestion with restriction endonucleases. Various concentrations of E. coli cells were added in yogurt, total RNA was extracted, and RT-semiAP PCR was performed. Both of the amplified fragments from E. coli and the lactic acid bacteria in the yogurt were observed, and the detection limit of E. coli to the lactic acid bacteria was 1/1,000 in the CFU ratio.
