New Triplex Real-Time PCR Assay for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Feces

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Abstract
In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2sequences ofMycobacterium aviumsubsp.paratuberculosisfrom bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105M. aviumsubsp.paratuberculosisstrains of bovine, ovine, and human origin and 100 non-M. aviumsubsp.paratuberculosisstrains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogramM. aviumsubsp.paratuberculosisDNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection ofM. aviumsubsp.paratuberculosisDNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqManmgband locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection ofM. aviumsubsp.paratuberculosisfrom bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 102CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknownM. aviumsubsp.paratuberculosisstatus resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.