SPECIFIC DETECTION AND CONFIRMATION OF CAMPYLOBACTER-JEJUNI BY DNA HYBRIDIZATION AND PCR

Conventional detection and confirmation methods for Campylobacter jeju ni are lengthy and tedious, A rapid hybridization protocol in which a 1,475-bp chromogen-labelled DNA probe (pDT1720) and Campylobacter stra ins filtered and grown on 0.22-mu m-pore-size hydrophobic grid membran e filters (HGMFs) are used was developed. Among the environmental and clinical isolates of C. jejuni, Campylobacter coil, Campylobacter jeju ni subsp. doylei, Campylobacter lari, and Arcobacter nitrofigilis and a panel of 310 unrelated bacterial strains tested, only C. jejuni and C. jejuni subsp, doylei isolates hybridized with the probe under strin gent conditions. The specificity of the probe was confirmed when the p rotocol was applied to spiked skim milk and chicken rinse samples, Bas ed on the nucleotide sequence of pDT1720, a pair of oligonucleotide pr imers was designed for PCR amplification of DNA from Campylobacter spp . and other food pathogens grown overnight in selective Mueller-Hinton broth with cefoperazone and grow th supplements. All C. jejuni strain s tested, including DNase producing strains and C. jejuni subsp. doyle i, produced a specific 402-bp amplicon, as confirmed by restriction an d Southern blot analysis. The detection range of the assay was as low as 3 CFU per PCR to as high as 10(5) CFU per PCR for pure cultures. Ov ernight enrichment of chicken rinse samples spiked initially with as l ittle as similar to 10 CFU/ml produced amplicons after the PCR No ampl icon was detected with any of the other bacterial strains tested or fr om the chicken background microflora. Since C. jejuni is responsible f or 99% of Campylobacter contamination in poultry, PCR and HGMF hybridi zation were performed on naturally contaminated chicken rinse samples, and the results were compared with the results of conventional cultur al isolation on Preston agar, All samples confirmed to be culture posi tive for C. jejuni were also identified by DNA hybridization and PCR a mplification, thus confirming that these DNA-based technologies are su itable alternatives to time-consuming conventional detection methods. DNA hybridization, besides being sensitive ce, also has the potential to be used in direct enumeration of C. jejuni organisms in chicken sam ples.