Matrix extension to bovine feces and evaluation of semi-automated DNA extraction methods for the detection of Campylobacter jejuni in bovine and canine fecal samples using gyrA PCR

We evaluated a semi-automated method for DNA extraction, expanded to herbivore feces a gyrA PCR method for the detection of Campylobacter jejuni in canine feces, added an internal positive control (IPC) DNA to specimens before DNA extraction, and multiplexed the gyrA PCR assay with the IPC assay. The addition of IPC DNA and multiplexing of the IPC assay did not affect the performance of the gyrA PCR assay. The method was repeatable with no significant difference in results across 6 d (F = 0.715; p = 0.614 with R 2 = 0.818). In a single-laboratory randomized method test evaluating detection in bovine feces, sensitivity was 96.1%, specificity was 100%, and accuracy was 94.1%. In a multi-laboratory randomized method test (M-RMT) in bovine feces, agreement was 90% between laboratories on test results with κ = 0.80 (95% CI [0.64, 0.96]). Comparison of manual DNA extraction kits with a semi-automated extraction method in canine feces found 88.9% agreement, with κ = 0.78 (95% CI [0.64, 0.92]). In a second M-RMT in canine feces, accuracy was 78% with 90% agreement between the laboratories and κ = 0.80 (95% CI [0.64, 0.96]), even though inoculation of the intended fractional level was lower than intended at 45 cfu/g feces. Overall, the gyrA PCR assay was extended to an additional matrix, the inclusion of an IPC DNA and assay was successful, and the gyrA PCR assay for the detection of Campylobacter jejuni provided repeatable results in multiple laboratories.