ArticleApplied and environmental microbiology2004
Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk.
Article in Applied and environmental microbiology, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 55 citations in OpenAlex.
- Pyrazinamide Resistance and pncA Mutation Profiles in Multidrug Resistant Mycobacterium Tuberculosis.Infection and drug resistance · 2022Article
- Evaluation of a Commercial Real-Time PCR Kit for the Detection of Mycobacterium avium subspecies paratuberculosis in Milk.Current microbiology · 2016Article
- A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells.Scientific reports · 2016Article
- Article
- Rapid and sensitive method to identify Mycobacterium avium subsp. paratuberculosis in cow's milk by DNA methylase genotyping.Applied and environmental microbiology · 2013Article
- Effects of the probiotic Lactobacillus animalis in murine Mycobacterium avium subspecies paratuberculosis infection.BMC microbiology · 2013Article
- Rapid DNA extraction for specific detection and quantitation of Mycobacterium tuberculosis DNA in sputum specimens using Taqman assays.Tuberculosis (Edinburgh, Scotland) · 2011Article
- A novel real-time PCR assay for specific detection and quantification of Mycobacterium avium subsp. paratuberculosis in milk with the inherent possibility of differentiation between viable and dead cells.BMC research notes · 2010Article
- Isolation and detection of Mycobacterium avium subsp. paratuberculosis (MAP) from cattle in Ireland using both traditional culture and molecular based methods.Gut pathogens · 2010Article
- Comparison of milk culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis.Canadian journal of veterinary research = Revue canadienne de recherche veterinaire · 2009Article
- Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment.Extremophiles : life under extreme conditions · 2008Article
- New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis.BMC microbiology · 2006Article
- Peptide aMptD-mediated capture PCR for detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples.Applied and environmental microbiology · 2006Article
- Development and application of real-time PCR assays for quantification of genes encoding tetracycline resistance.Applied and environmental microbiology · 2005Article
- Development of an F57 sequence-based real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk.Applied and environmental microbiology · 2005Article
- Detection of Mycobacterium avium subsp. paratuberculosis in bovine milk from the state of Pernambuco, Brazil.Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]Article
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg(2+) concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 x 10(6) to 3 x 10(1) copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples.
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