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Identification of Mycobacterium Species by DNA Microarray Chip Method
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Development of a novel DNA extraction method for identification and quantification of Mycobacterium avium subsp.

Kun Taek Park1, Andrew J Allen2, William C Davis1

  • 1Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, United States.

Journal of Microbiological Methods
|February 19, 2014
PubMed

Insights

A new pre-treatment method significantly improves Mycobacterium avium subsp. paratuberculosis (Map) DNA extraction from tissues. This enhances the accuracy of real-time PCR (RT-PCR) for detecting Map, aiding Johne's disease and Crohn's disease research.

Area of Science:

  • Veterinary Microbiology
  • Molecular Diagnostics
  • Infectious Diseases

Background:

  • Mycobacterium avium subsp. paratuberculosis (Map) causes Johne's disease in ruminants and is implicated in human Crohn's disease.
  • Accurate detection of Map in tissues is crucial but hindered by DNA extraction challenges and PCR inhibitors.
  • Current real-time PCR (RT-PCR) methods for Map detection show variable sensitivity.

Purpose of the Study:

  • To develop and validate a novel pre-treatment method for enhanced Map DNA extraction from clinical specimens.
  • To improve the reliability and sensitivity of RT-PCR-based Map detection.
  • To facilitate accurate identification and quantification of Map in affected tissues.

Main Methods:

  • A novel pre-treatment protocol was developed to optimize DNA recovery and reduce PCR inhibitors.
  • The pre-treatment method was combined with the DNeasy Blood and Tissue kit (Qiagen).
  • The efficacy of the improved extraction method was assessed using three RT-PCR assays and compared against culture methods, analyzing Kappa values and correlation coefficients.

Main Results:

  • The novel pre-treatment method significantly increased Map DNA recovery and decreased PCR inhibitors (p<0.05).
  • No PCR inhibition was observed when the method was used with the DNeasy kit across three RT-PCR assays.
  • High correlation (r=0.940) and excellent agreement (Kappa=0.849) were found with culture methods using the IS900 probe.
  • Field sample analysis revealed higher positive identification rates (5 vs 3) using the improved DNA extraction method.

Conclusions:

  • The developed pre-treatment method substantially enhances Map DNA extraction efficiency from tissue samples.
  • This improved method overcomes limitations of current RT-PCR assays, reducing inhibition and increasing sensitivity.
  • The optimized protocol represents a more powerful tool for Map identification and quantification in diverse applications.
Keywords:
Crohn's diseaseJohne's diseaseMycobacterium paratuberculosisPCR inhibitorReal-time quantitative PCRTissue DNA extraction

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