Related Experiment Video
Updated: Aug 26, 2026
Identification of Mycobacterium Species by DNA Microarray Chip Method
Published on: June 24, 2025
Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp.
John Ikonomopoulos1, Maria Gazouli, Ivo Pavlik
1Deparment of Anatomy-Physiology, Faculty of Animal Science, Agricultural University of Athens, Iera Odos 75, Botanikos 11855, Greece. ikonomop@aua.gr
Abstract:
Mycobacterium avium subsp. paratuberculosis (MAP) can cause a very serious, often-fatal disease, namely paratuberculosis, in several animal species, especially ruminants. Recently, it has also been implicated in the pathogenesis of Infectious Bowel Disease of man. The aim of this study was to develop a molecular method for the routine detection and identification of MAP, from tissue samples of animal origin. The proposed assay would have to combine optimum performance and cost, with high reproducibility. To this goal, three laboratories in Greece and the Czech Republic undertook different parts of a study that involved evaluation of DNA extraction procedures, and PCR assays, for MAP detection. For DNA extraction we used one in-house, and one commercial method, and for the PCR we assessed a number of different assays, starting with the evaluation of primer specificity with an extended GenBank database search. Based on these results, we chose to assess a one-tube nested, 2 two-tube nested, and a single PCR assay, targeted to different genomic regions of the IS900 element. These four methods were applied on positive and negative control samples, consisted of pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) tissue samples collected from cattle with paratuberculosis and chickens with M. avium subsp. avium infection. Based on the criteria of reliability and cost, the procedure that performed better was the one-tube nested PCR assay combined with the in-house DNA extraction method. The agreement of the results obtained by the three collaborating laboratories indicates the reliability of the proposed assay even under different laboratory conditions.
Insights
A new molecular method was developed for detecting Mycobacterium avium subsp. paratuberculosis (MAP), the cause of paratuberculosis in animals. The one-tube nested PCR with in-house DNA extraction offers a reliable and cost-effective solution for routine diagnostics.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Bacteriology
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis, a significant disease in ruminants.
- MAP is also investigated for its role in human Infectious Bowel Disease.
- Accurate detection of MAP in animal tissues is crucial for disease control.
Purpose of the Study:
- To develop and validate a molecular method for routine detection and identification of MAP.
- The assay aims for optimal performance, cost-effectiveness, and high reproducibility.
- Establish a reliable diagnostic tool for animal tissues.
Main Methods:
- Evaluation of DNA extraction methods (in-house and commercial).
- Assessment of various PCR assays targeting the IS900 element, including single, two-tube nested, and one-tube nested PCR.
- Testing on pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) animal tissue samples.
Main Results:
- The one-tube nested PCR assay combined with the in-house DNA extraction method demonstrated superior performance.
- This method proved reliable and cost-effective compared to other assessed procedures.
- High agreement between three independent laboratories validated the assay's robustness.
Conclusions:
- The developed one-tube nested PCR assay with in-house DNA extraction is a reliable and cost-effective method for MAP detection.
- This assay is suitable for routine diagnostics in animal tissues.
- The method's reproducibility across different laboratories ensures its practical applicability.
