A rapid, cheap and sensitive detection method of Mycobacterium avium subsp. paratuberculosis (MAP) in raw milk was needed for routine usage. We developed two duplex real time qPCR systems specific for MAP detection. These real time qPCR assays amplify the multicopy element IS900 for qualitative analysis and the single copy element F57 for quantitative analysis. Both assays incorporate an internal amplification control amplified with the same primers as the targets and the same probes are used in both assays. The specificity of the assays was confirmed by the testing of 6 different MAP isolates, 12 isolates of other mycobacteria or bacterial species and 4 different mammalian DNAs. The sensitivity of the developed assays and isolation efficiency were demonstrated through the analysis of raw milk samples artificially contaminated with MAP cells and with plasmids containing cloned fragments of the targets (IS900 and F57). The developed assays for milk analysis were applied to samples from one farm with two faecal shedding cows. Three hundred and forty five individual milk samples were tested by real time qPCR assays and by cultivation. Hundred and eleven (32.5%) individual milk samples were positive by the real time qPCR, no milk sample was culture positive. The spread of MAP in individual, tank and bulk tank milk samples was also monitored.

Veterinary Research Institute Hudcova 70 621 00 Brno Czech Republic

References provided by Crossref.org

Utilisation of Actiphage in combination with IS900 qPCR as a diagnostic tool for rapid determination of paratuberculosis infection status in small ruminant herds

First Evidence of the Presence of the Causative Agent of Caseous Lymphadenitis-Corynebacterium pseudotuberculosis in Dairy Products Produced from the Milk of Small Ruminants

Experimental Evidence of the Long-Term Survival of Infective African Swine Fever Virus Strain Ba71V in Soil under Different Conditions

A viability assay combining palladium compound treatment with quantitative PCR to detect viable Mycobacterium avium subsp. paratuberculosis cells

A Novel Approach to the Viability Determination of Mycobacterium avium subsp. paratuberculosis Using Platinum Compounds in Combination With Quantitative PCR

Recovery of Mycobacteria from Heavily Contaminated Environmental Matrices

Application of the Actiphage® Assay to Detect Viable Mycobacterium avium subsp. paratuberculosis Cells in Fresh Sheep and Goat Milk and Previously Frozen Milk and In-Line Milk Filters

Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR

Phage Amplification Assay for Detection of Mycobacterial Infection: A Review

Efficiency of DNA Isolation Methods Based on Silica Columns and Magnetic Separation Tested for the Detection of Mycobacterium avium Subsp. Paratuberculosis in Milk and Faeces

The Impact of the Antimicrobial Compounds Produced by Lactic Acid Bacteria on the Growth Performance of Mycobacterium avium subsp. paratuberculosis

Sequence Analysis of Changes in Microbial Composition in Different Milk Products During Fermentation and Storage

Magnetic Separation Methods for the Detection of Mycobacterium avium subsp. paratuberculosis in Various Types of Matrices: A Review

An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds

A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything

Avian Mycobacteriosis: Still Existing Threat to Humans

Changes in Microbial Composition of Wastewater During Treatment in a Full-Scale Plant

Spread of Mycobacterium avium subsp. paratuberculosis through soil and grass on a mouflon (Ovis aries) pasture

Microscopy, culture, and quantitative real-time PCR examination confirm internalization of mycobacteria in plants

Influence of Stress Connected with Moving to a New Farm on Potentially MAP-Infected Mouflons