Abstract

This work aimed to detect Campylobacter species from naturally contaminated turbid pond water by PCR. A total of 16 water samples were collected from a turbid village pond. Four methods of DNA extraction were applied to centrifuge pellets from eight 100 ml pond water samples prior to attempted detection of Campylobacter by PCR without an enrichment step. These methods were (1) Tris-HCl and sodium dodecyl sulfate followed by phenol:chloroform:isoamylalcohol extraction followed by treatment with DNA clean up kit, (2) proteinase K, (3) Chelex® 100, and (4) boiling. The other eight pond water samples (10 ml and 100 ml) were filtered and filters were incubated overnight in Preston enrichment broth. The centrifuge pellets obtained from enrichment cultures were treated by proteinase K for DNA extraction. Primers CF03 and CF04 for the flagellin genes (fla A and fla B) of Campylobacter jejuni and Campylobacter coli were used for amplifying the extracted DNA. The DNA extracted from eight-100 ml pond water samples that were not subject to selective enrichment was never amplified with primers CF03 and CF04, hence Campylobacter was not detected. In contrast, the DNA that was from samples that were subjected to a selective enrichment step in Preston broth prior to PCR assay always gave amplified bands of 340-380 bp, therefore the presence of Campylobacter was confirmed. Detection of campylobacters
from naturally contaminated, turbid, environmental water may not be feasible by direct PCR assay because of low numbers and the presence of high concentration of humic matter and other PCR inhibitors. The enrichment of water samples in selective broth, however, facilitated PCR detection of Campylobacter probably by increasing cell number and by diluting PCR inhibitors.