| Budget Amount *help |
¥3,980,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥480,000)
Fiscal Year 2007: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
|
|---|
| Research Abstract |
Diarrheagenic Escherichia coli (DEC) comprise a variety of pathotypes: enteropathogenic E. coli (EPEC); shigatoxin-producing E. coil (STEC); enterotoxigenic E. coli (ETEC); enteroinvasive E. coli (EIEC); enteroaggregative E. call (EAggEC); and diffusely adhering E. coil (DAEC). However, the source and routes of infection have not been clarified because these organisms are scarcely detected among numerous coliform bacteria. To exhaustively detect DEC in food, we have developed multiplex real-time PCR assays against enterovirulent genes (eae, stx1, stx2, elt, est, virB, aggR, astA and afaB). Primers and TaqMan probes were designed to amplify and quantify the genes in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 96 DEC strains, 2 strains of Vibrio cholerae El Tor Ogawa, and Shigella dysenteriae serotype 1. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0.99. Subsequently, ground meat samples and enrichment broths were spiked with DEC. Detection limits varied from 3.6×10^2 to 6.0×10^3 CFU/ml, depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU/10g) were found to be positive by screening the enrichment broth using multiplex real-time PCR. As a preliminary test, 148 food samples were exhaustively assayed by multiplex real-time PCR; 41 samples (28%) were positive for DEC pathotypes. The present multiplex real-time PCR system allows the efficient and simultaneous determination of various DEC pathotypes in food.
|
