Abstract
The aim of this study was to evaluate a SYBR Green based real-time PCR assay using well-characterized primers to detect Campylobacter jejuni in naturally contaminated dairy farm environmental samples. Specificity of the assay was determined with 62 C. jejuni strains and 120 non-C. jejuni strains. Peak melting temperature obtained with melting curves specific for C. jejuni was 77.5 degrees C. Standard curves were constructed using mean threshold cycle (C(T)) and various concentrations of C. jejuni ranging from 10(0) to 10(8) colony forming units (CFU)/mL, which resulted in a linear relationship between C(T) and log input DNA. Correlation coefficients of standard curves based on pure culture of C. jejuni in broth and spiked cells in lagoon water were R(2) = 0.995 (slope = 3.21) and R(2) = 0.988 (slope = 3.22), respectively, and sensitivity limits were <10 and >10(3) CFU/mL, respectively. After 24-h enrichment, total C. jejuni counts of all samples spiked with 10(0) CFU/mL reached >10(5) CFU/mL, and the detection limit was improved from >10(3) CFU/mL to <10 CFU/mL of inoculum in broth. Eighty-two dairy farm environmental samples, including fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding material, were analyzed. The real-time PCR assay detected C. jejuni in 25 (30.4%) of 82 samples, with 17 (68%) of these samples being culture positive for C. jejuni. All samples that were positive by standard culture methods were also positive by the real-time PCR method. Mean C( T ) values of 48-h enriched cultures for 17 PCR-positive/culture-positive samples and eight PCR-positive/culture-negative samples were 21.4 +/- 3.6, and 34.6 +/- 1.5 (p < 0.0001), respectively. C( T ) values for negative samples were >38.0. These results indicate that the SYBR Green real-time PCR assay provides a specific, reproducible, and simple method for detecting C. jejuni in dairy farm environmental samples.
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