Submitted to: US-Japan Coop Pgm on Dev and Util of Natural Products Abstracts Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 9/4/1996
Publication Date: N/A
Technical Abstract: We have designed a multiplex PCR (mPCR) assay using two primer sets for the identification and differentiation of Campylobacter coli and Campylobacter jejuni. Primer set I targets a 450-bp fragment which is present in C. coli and C. jejuni, but is absent in other thermotolerant campylobacters. Primer set II amplifies a 160-bp sequence unique to C. jejuni. When the mPCR assay was performed on reference strains, amplification of C. coli yielded only the 450-bp fragment, whereas C. jejuni generated both the 160- and 450-bp fragments. In field trials, mPCR was used to differentiate C. jejuni and C. coli strains recovered from meat and to detect these bacteria in feces of healthy livestock. Campylobacter field strains (n=85) isolated from meat were identified by mPCR and by conventional biochemical methods. Species determination by the two methods agreed for 82 of the 85 isolates examined. By mPCR, of the 85 isolates, 23 were identified as C. coli and 62 as C. jejuni. Biochemical testing failed to speciate two isolates, which the mPCR assay identified as C. coli. We next utilized the multiplex PCR assay to determine the prevalence of C. jejuni and C. coli present in healthy livestock. Fecal suspensions were made in 0.1% peptone water and an aliquot streaked onto modified charcoal-cefazolin-sodium deoxycholate agar (mCCDA) supplemented with 0.01% amphotericin B. After incubation (48 h in 5% 02, 10% C02, 85%N2), bacterial colonies were processed directly for PCR. Of 1,057 pig fecal samples examined, 69% were positive for C. coli and 0.28% were positive for C. jejuni. Of 1,334 cattle fecal samples processed, 43% were positive for C. jejuni and 1.57% were positive for C. coli. These results indicate the application of mPCR for identifying potential human foodborne pathogens.