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.