The detection sensitivity and the pre-enrichment efficacy of “[TA10] Pathogenic Bacterial Multiplex PCR Detection System” which detects Salmonella spp., Listeria monocytogenes, and Escherichia coli O157 : H7 simultaneously with high sensitivity was evaluated. When this method was applied for the detection of each of the pathogenic bacteria from 16 kinds of the spiked fruits and vegetables, 1∼9 cell(s) per 25 g of inoculated sample could be detectable after enrichment in TA10 broth for 22 hr by the kit. To evaluate the pre-enrichment efficacy of the kit for PCR detection, the correlation between the pH decrease by enrichment conditions and the growth capability of three pathogens were examined in TA10, TSB, and BHI broth. The growth inhibition was apparent in the high acidity samples (ex., grapefruit, pineapple, and lemon), and L. monocytogenes especially exhibited slower growth compared with that of the other pathogens, possibly due to a pH decrease in all cultured broth except TA10 broth. The pH neutralization of high acidity samples before pre-enrichment did not improve growth in all broth. The results suggest that the buffering capacity of an enrichment broth preventing a significant pH decrease due to bacterial growth would be more important than neutralizing the sample before enrichment to improve detection sensitivity by PCR or conventional culture especially in high acidic food samples.
We have developed a duplex reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a fluorescent quenching-based system for the simultaneous and rapid detection of norovirus (NoV) genogroup I (GI) and genogroup II (GII) genomes in a single tube. After amplifying, NoV genomes could be identified visually in a tube by adding only quencher-labeled oligonucleotides, which have complementary sequences to the fluorescence-labeled primers. This assay requires no further tests such as electrophoresis or hybridization. In the evaluation tests with 70 NoV RT-PCR-positive fecal specimens, the green and red colors for GI and GII, respectively, and sometimes with yellow color for NoV GI and GII mixture, could be identified easily by the naked eye under UV light. The detection limits for NoV GI and GII genomes by duplex RT-LAMP were estimated to be between 103 and 104 copies/tube. Our duplex RT-LAMP assay allows for rapid, simple and simultaneous detection and supports genogroup-screening test of NoV GI and GII genomes from fecal specimens in most laboratories. Furthermore, this fluorescent quenching-based system is potentially useful for all gene amplification assays in a wide range of gene-related fields due to its simple principle.
To clarify the route and source of Vibrio vulnificus infection, we analyzed contamination in commercially available crustaceans with V. vulnificus and conducted serotyping and drug sensitivity tests with various antimicrobial agents. We isolated V. vulnificus from 328 of 4,769 samples (6.9%) of crustaceans. V. vulnificus were most frequently isolated from 272 of 1954 mantis crabs (13.9%), followed by 22 of 434 (5.1%) Japanese mud shrimp and 34 of 2,381 (1.4%) shrimp. Regarding the isolation rates of V. vulnificus according to region, V. vulnificus was isolated in 10 of 12 prefectures (83.3%) surveyed and was most frequently isolated in Miyagi (40.0%), followed by Kagoshima (20.8%), but was not found in any samples from Kagawa or Fukui, indicating a regional difference. As a result of serotyping, 208 of 328 (63.4%) examined strains were differentiated into 14 serotypes : serotype O19 was the most frequently observed (13.7%), followed by O16 (10.3%), O14 (7.3%) and O23 (7.3%). The results of MIC90 in drug susceptibility tests were compared. Ciprofloxacin was the lowest, followed by Meropenem, Minocycline, Tetracyclin, Doxycycline, Chloramphenicol and Nalidixic acid and all strains were susceptible to all of these 7 drugs. However, Lincomycin, Cefmetazole, Kanamycin, Amikacin, Ampicillin, Piperacillin, Cefaloridine, Cefalotin, Cefozopran, Gentamicin, Erythromycin, Cefotaxime and Latamoxef showed high MIC90, and many strains were resistant to those 13 drugs.