Detection of Mycobacterium avium subsp. paratuberculosis in bovine manure using Whatman FTA card technology and lightcycler real-time PCR

Carmela V. Jaravata, Wayne L. Smith, Gabriel J. Rensen, Juliana M. Ruzante, James S Cullor

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

A modified forensic DNA extraction and real-time fluorescent polymerase chain reaction assay has been evaluated for the detection of Mycobactenum avium subsp. paratuberculosis (MAP) in bovine fecal samples using primers and fluorescent resonance energy transfer (FRET) probes targeting the IS900 gene sequence of MAP. DNA was successfully extracted from manure samples by utilizing the Whatman FTA® card technology, which allows for simple processing and storage of samples at room temperature. The FTA® cards were washed and subjected to a Chelex-100 incubation to remove any remaining polymerase chain reaction (PCR) inhibitors and to elute the DNA from the FTA® card. This isolated DNA was then subjected to direct real time fluorescent PCR analysis. Detection of MAP DNA from bovine fecal samples spiked with known concentrations of viable MAP cells was obtained. The detection limits of the assay was consistently found to be between 102 and 104 colony forming units [CFU]/g, with some samples containing as low as 10 CFU/g, yielding positive assay results. This cost-efficient assay allows reporting of results as early as 4 h after fecal collection, which can be particularly useful in high-throughput herd screening.

Original languageEnglish (US)
Pages (from-to)212-215
Number of pages4
JournalFoodborne Pathogens and Disease
Volume3
Issue number2
DOIs
StatePublished - Jun 2006

Fingerprint

Paratuberculosis
Mycobacterium avium
Manure
Mycobacterium avium subsp. paratuberculosis
cattle manure
paratuberculosis
Real-Time Polymerase Chain Reaction
quantitative polymerase chain reaction
Technology
DNA
polymerase chain reaction
assays
sampling
Stem Cells
Gene Targeting
cattle
Energy Transfer
energy transfer
probes (equipment)
animal manures

ASJC Scopus subject areas

  • Microbiology
  • Applied Microbiology and Biotechnology
  • Infectious Diseases
  • Food Science

Cite this

Detection of Mycobacterium avium subsp. paratuberculosis in bovine manure using Whatman FTA card technology and lightcycler real-time PCR. / Jaravata, Carmela V.; Smith, Wayne L.; Rensen, Gabriel J.; Ruzante, Juliana M.; Cullor, James S.

In: Foodborne Pathogens and Disease, Vol. 3, No. 2, 06.2006, p. 212-215.

Research output: Contribution to journalArticle

Jaravata, Carmela V. ; Smith, Wayne L. ; Rensen, Gabriel J. ; Ruzante, Juliana M. ; Cullor, James S. / Detection of Mycobacterium avium subsp. paratuberculosis in bovine manure using Whatman FTA card technology and lightcycler real-time PCR. In: Foodborne Pathogens and Disease. 2006 ; Vol. 3, No. 2. pp. 212-215.
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