Rapid Real-Time PCR Assay for Detection and Quantitation of Mycobacterium avium subsp. paratuberculosis DNA in Artificially Contaminated Milk

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 ؋ 10 6 to 3 ؋ 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.

Development and Application of Real-Time PCR Assays for Quantification of Genes Encoding Tetracycline Resistance

“…We think that the preparation of sample-derived standards is a practical way to produce real-time PCR standards for accurate and simulta- neous quantification of a group of related genes in microbiomes. When the abundance of one bacterial species is to be quantified, real-time PCR standards can be validated using either known numbers of bacterial cells (17) or a known number of target gene templates (29). In this study, we validated each of the real-time PCR assays by using its respective sample-derived standard (Fig.…”

Development of an F57 Sequence-Based Real-Time PCR Assay for Detection ofMycobacterium aviumsubsp.paratuberculosisin Milk

“…paratuberculosis real-time PCR detection assays are available, but most of them are IS900 based, making them prone to potential false-positive signals associated with some non-M. avium subsp. paratuberculosis Mycobacterium species (15,26,27,31,32). As an alternative, only one assay based on real-time nucleic acid sequence-based amplification detection of the dnaA gene has been described (36).…”

“…The analytical sensitivity obtained here also falls within the range that was reported in studies in which M. avium subsp. paratuberculosis IS900 elements were targeted (18,26,32,34). In addition, the targeting of ISMav2 as an alternative marker for M. avium subsp.…”

Peptide aMptD-Mediated Capture PCR for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bulk Milk Samples

“…The most convenient and amenable methods for such detection of M. avium subsp. paratuberculosis DNA in milk are enrichment via immunomagnetic separation (17,28,38) and peptide-mediated capture (47) followed by PCR, as these methods can be adapted to high-throughput testing using standard laboratory automation.In the study presented here, we followed this approach, using the phage display-derived peptide aMptD (46) for the capture of M. avium subsp. paratuberculosis in milk samples.…”