ArticleApplied and environmental microbiology2008
New triplex real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces.
Article in Applied and environmental microbiology, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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Who cites it
29 citing papers in PubMed.
- Systematic evaluation and optimization of TaqMan qPCR assays targeting FJournal of clinical microbiology · 2026Article
- Interferon-gamma producing CD4Veterinary research · 2024Article
- Comparison of 2 PCR assays on environmental samples cultured forJournal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc · 2024Article
- Detection ofVeterinarni medicina · 2021Article
- Article
- Presence ofMicroorganisms · 2019Article
- Capacity to Elicit Cytotoxic CD8 T Cell Activity AgainstFrontiers in immunology · 2019Article
- A Mycobacterium avium subsp. paratuberculosis relA deletion mutant and a 35 kDa major membrane protein elicit development of cytotoxic T lymphocytes with ability to kill intracellular bacteria.Veterinary research · 2018Article
- Effectiveness of an inactivated paratuberculosis vaccine in Iranian sheep flocks using theIranian journal of microbiology · 2018Article
- Evaluation of different diagnostic methods for the detection of Mycobacterium avium subsp. paratuberculosis in boot swabs and liquid manure samples.BMC veterinary research · 2017Article
- An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds.Folia microbiologica · 2017Article
- Evaluation of a Commercial Real-Time PCR Kit for the Detection of Mycobacterium avium subspecies paratuberculosis in Milk.Current microbiology · 2016Article
- Improvement of sensitivity for Mycobacterium avium subsp. paratuberculosis (MAP) detection in bovine fecal samples by specific duplex F57/IC real-time and conventional IS900 PCRs after solid culture enrichment.Tropical animal health and production · 2015Article
- Mycobacterium avium Subspecies paratuberculosis: Human Exposure through Environmental and Domestic Aerosols.Pathogens (Basel, Switzerland) · 2014Article
- Development and evaluation of a novel multicopy-element-targeting triplex PCR for detection of Mycobacterium avium subsp. paratuberculosis in feces.Applied and environmental microbiology · 2014Article
- Flow cytometric detection of Mycobacterium avium subsp. paratuberculosis-specific antibodies in experimentally infected and naturally exposed calves.Clinical and vaccine immunology : CVI · 2013Article
- Composite testing for ante-mortem diagnosis of Johne's disease in farmed New Zealand deer: correlations between bacteriological culture, histopathology, serological reactivity and faecal shedding as determined by quantitative PCR.BMC veterinary research · 2013Article
- Mycobacterium avium subspecies paratuberculosis: an insidious problem for the ruminant industry.Tropical animal health and production · 2013Review
- Effects of the probiotic Lactobacillus animalis in murine Mycobacterium avium subspecies paratuberculosis infection.BMC microbiology · 2013Article
- Genotypes of Mycobacterium avium subsp. paratuberculosis from South American countries determined by two methods based on genomic repetitive sequences.Tropical animal health and production · 2012Article
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4 authors.
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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.
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