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

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Overview

  • 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. 
The technique described is that used by INDICAL BIOSCIENCE, formerly QIAGEN.  Other laboratories will offer alternative PCR protocols to detect MAP.

Sampling

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Tests

Methodologies

  • 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).
Pretreatment:
  • 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).

Availability

  • The test described is available from INDICAL BIOSCIENCE www.indical.com.
  • Alternative MAP PCRs are available from other laboratories.

Validity

Sensitivity

  • 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.

Specificity

  • 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.

Further Reading

Publications

Refereed Papers

  • 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

Organisation(s)


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