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

Abstract

In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from 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 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. 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 picogram M. avium subsp. paratuberculosis DNA 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 of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqMan mgb and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from 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 10 2 CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status 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.Mycobacterium avium subsp. paratuberculosis is the causative agent of ruminant paratuberculosis (Johne's disease), which has become a worldwide problem. There is controversy regarding its zoonotic capacity and potential role in the human Crohn's disease (14). Because of these reasons, a rapid, costeffective, and automated diagnosis of this pathogen is a high priority task not only for animal breeders but also for the food production industry and for public health institutions. Culturebased detection of M. avium subsp. paratuberculosis is timeconsuming, labor-intensive, and therefore not suitable. The PCR has been shown to be a powerful tool in microbiological diagnostics (12,43). Guidelines for diagnostic quality assurance have been set by the International Organization for Standardization (7,8). Standardized PCR and real-time PCR methods should fulfill numerous criteria, including a high detection probability with regard to the investigated matrix, the sample preparation, and DNA extraction as well as high specificity, robustness, and user-friendly protocols. In this context the real-time PCR technology offers the possibility for a onestep and closed-tube reaction (13).As a molecular reference marker for the confirm...

Development and validation of a triplex real-time PCR for rapid detection and specific identification of M. avium sub sp. paratuberculosis in faecal samples

“…Regarding single IS900 PCR, many reports have highlighted the lack of specificity of IS900 for Map detection. Previously published IS900 PCR protocols could indeed also amplify IS900-like sequences (Kim et al, 2002;Rajeev et al, 2005;Tasara et al, 2005), which have prompted the use of PCR assays excluding IS900 (Schö nenbrü cher et al, 2008). Nevertheless, IS900 primers designed according to IS900-like sequences could specifically amplify Map (Kawaji et al, 2007).…”

Fecal culture and two fecal-PCR methods for the diagnosis of Mycobacterium avium subsp. paratuberculosis in a seropositive herd

“…According to Withlock et al (2000), the disadvantages of some detection test are due mainly because of the intermittent shedding of microorganisms. This means that the sensitivity of direct tests to detect symptomatic animals is high, but low for detection of infected/subclinical animals (Nielsen and Toft, 2008;Schönenbrücher et al, 2008;Whittington et al, 2011).…”

“…The nested IS900 assay can detect 0.01 pg of DNA (corresponding to 10 genomes) when extracted from a pure culture, while the F57 assay can detect 0.1 pg of DNA (corresponding to 100 genomes; Radomski et al, 2013). Vansnicka et al (2004), Tasara and Stephan (2005), and Schönenbrücher et al (2008) recommend including the F57-PCR assay to confirm the presence of MAP after a positive IS900-PCR. According to this, our results (F57-PCR negative results and some positive results by IS900-PCR), can be considered MAP-unspecific by IS900-PCR, and confirmed as negative by the F57 insertion detection.…”

“…Usually, the specificity of fecal culture is considered to be almost 100% if the isolates obtained are confirmed to be MAP by molecular methods such as polymerase chain reaction (PCR; Nielsen and Toft, 2008;Schönenbrücher et al, 2008;Whittington et al, 2011). Fecal culture has been used as an acceptable standard technique for detecting the infection status of animals -related to elimination rate-, for estimating the sensitivity of other diagnostic tests (e.g.…”

Therapeutic Potential of a Candidate relA Deletion Mutant Vaccine of Mycobacterium Avium Subsp. Paratuberculosis

“…RT-PCR targeting Map-specific single copy gene, F57, was used for a correct quantification (Schonenbrucher et al 2008). DNA extraction was done in duplicate, and the RT-PCR for each DNA sample was conducted in duplicate.…”