Detection of Small Numbers ofCampylobacter jejuniandCampylobacter coliCells in Environmental Water, Sewage, and Food Samples by a Seminested PCR Assay

Abstract

A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA andflaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as ≤3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection of C. jejuni or C. coli in drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms.

Campylobacter contamination in French chicken production from farm to consumers. Use of a PCR assay for detection and identification of Campylobacter jejuni and Camp. coli

“…A sensitivity of 10 2 to 10 3 UFC ml )1 of broth after a fast extraction procedure was reported by Manzano et al (1995). Waage et al (1999) reported sensitivity lower than or equal to 3 UFC g )1 of food. Straub et al (1999) did not present any sensitivity of their technique.…”

“…DNA extraction protocols from a broth for Campylobacter detection by PCR have been described in several publications. They have been adapted for faecal samples (Rasmussen et al 1996;Lawson et al 1998;Vanniasinkam et al 1999) or for food samples (Giesendorf et al 1992;Manzano et al 1995;Winters et al 1998;Straub et al 1999;Waage et al 1999). All these studies required an enrichment step before DNA extraction.…”

“…All these studies required an enrichment step before DNA extraction. Waage et al (1999) obtained more positive samples using PCR after an enrichment step which thus seems to be necessary for slightly contaminated samples.…”

“…Consequently, no PCR inhibition was encountered with the 103 food samples and the 16 neck skins analysed. Waage et al (1999) reported problems of PCR inhibition in detecting Campylobacter in food samples. These were solved by diluting the enrichment broth before DNA extraction and not by diluting the DNA extract as in our study.…”

Detection of toxigenicVibrio choleraefrom environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

“…Not only does an enrichment procedure dilute any inhibitors present, but dead bacteria are diluted as well, thus reducing the probability of detecting them by the subsequent PCR assay. By allowing exponential bacterial growth to amplify target copy number rather than using increased numbers of ampli®cation cycles to detect less target, the chance that false-positives might be generated during extended PCR cycling, as in the case of conventional nested and semi-nested PCR assays (Starnbach et al 1989;Lindqvest 1999;Waage et al 1999), is thus minimized. In this study, enrichment in CDC broth for as little as 6 h of incubation before ampli®cation enhanced the limit of detection considerably (at least 300-fold) and as few as 4 cfu of Vibrio organisms were detectable in the assay.…”