Bovis ISSN 2398-2993
Mycobacterium avium subsp. paratuberculosis (MAP) assay
Synonym(s): Johne's assay, Johne's testing, fecal sampling
Contributor(s): Carsten Schroeder
, James Breen
The technique described is that used by INDICAL BIOSCIENCE, formerly QIAGEN. Other laboratories will offer alternative PCR protocols to detect MAP.
- Although culture from fecal sample has been regarded as the benchmark for Mycobacterium avium subsp. paratuberculosis MAP detection, it is labor intensive and can take several weeks.
- Real-time polymerase chain reaction (PCR) is becoming more widely used and enables test results within hours.
- The technique described below, for detection of MAP in bovine fecal samples, combines optimized F-MAP sample pretreatment (reducing the number of steps and amount of time required), reliable DNA extraction and sensitive real-time PCR.
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- DNA is extracted from bovine fecal samples using F-MAP pre-treatment in combination with MagAttract 96 cador Pathogen Kit and INDICAL’s pre-treatment in combination with the QIAamp cador Pathogen Mini Kit (INDICAL BIOSCIENCE) or cador Pathogen 96 QIAcube HT Kit (INDICAL BIOSCIENCE).
- Challenges remain in extracting MAP DNA from fecal samples, these include:
- MAP clusters in the sample.
- Thick mycobacterial cell walls.
- PCR inhibitors.
- Sample pre-treatment is therefore essential in obtaining accurate PCR results and innovation in sample preparation will increase precision and reliability in PCR results.
- The system described above combines optimized lysis buffer and mechanical disruption with special beads.
- Other laboratories may not utilise this technology and may instead undertake the more traditional heating step, which although effective, increases the time taken to obtain results.
MAP DNA isolation:
- After pre-treatment, MAP DNA extraction/isolation is performed using either spin-column (QIAamp cador Pathogen Kit, cador Pathogen 96 QIAcube HT Kit) or magnetic bead (MagAttract 96 cador Pathogen Kit) technology.
- It is strongly recommended to add the Internal Control DNA to the sample lysis buffer to monitor the extraction.
Sensitive real-time PCR:
- The bactotype MAP PCR Kit is used to amplify the DNA extracted. This features:
- A ready-to-use master mix.
- A heterologous extraction and amplification control.
- TaqMan-based chemistry that can be used on real-time PCR cyclers commonly used in veterinary laboratories, with a total amplification time of about 1.40 hours (based on the QIAGEN Rotor-Gene Q).
- The test described is available from INDICAL BIOSCIENCE www.indical.com.
- Alternative MAP PCRs are available from other laboratories.
- The bactotype MAP kit detected 5 MAP DNA copies per sample with a correlation coefficient ≥0.998 and with high efficiency on all instruments tested.
- High analytical and diagnostic specificity.
- Testing a panel of non-Mycobacteria and non-MAP strains no unspecific results were detected.
- All 240 MAP-negative samples tested scored correctly negative, resulting in a diagnostic specificity of 100%.
Technique (intrinsic) limitations
- MAP infection usually occurs during the neonatal period.
- It can take weeks, months, or even years for fecal shedding to commence.
- Therefore, all fecal PCRs can only detect MAP once the organism is being shed in the feces.
- Research is ongoing to find tests which will detect this organism earlier in the animal’s life.
- PCR cannot distinguish between active and passive infection.
- It can be difficult to interpret whether being PCR+ for MAP in feces reflects a transitory infection or true shedding.
- Often fecal PCR is used to confirm diagnosis in cows who have been identified as positive via milk PCR. A positive result to both of these tests is generally accepted as confirming a positive infection status.
- The confirmatory role that fecal PCR provides is important as many milk buyers will not allow milk from MAP ELISA positive cows to go into the bulk tank.
- Recent references from PubMed and VetMedResource.
- Prendergast D M, Pearce R A, Yearsley D, Ramovic E, Egan J (2018) Evaluation of three commercial PCR kits for the direct detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine faeces. Vet J 241, 52-57 PubMed.
Other sources of information
- Gaunitz Ch, Engemann C, Labitzke M, Hennart S, Schroeder C (2014): bactotype MAP real time PCR - combining optimized sample extraction with sensitive detection, 12th International Colloquium on Paratuberculosis, Parma, Italy, Proceedings
- The detailed F-MAP pretreatment protocol, for optimized pretreatment of fecal samples in the detection of MAP, is available from firstname.lastname@example.org.
- Swift et al. Presentation to the European Association of Veterinary Laboratory Diagnosticians Congress (2018). Detection of active infection of new-born calves by MAP in first days of life. http://www.pbdbio.com/site/assets/files/1115/pbd_biotech_and_biosellal_johnes_trial_-_detection_of_infection_of_newborn_calves_by_map.pdf