Development of an IMS-PCR Assay for the Detection of Mycobacterium Avium Ssp. Paratuberculosis in Water

Lett Appl Microbiol. 2005;40(4):269-73. doi: 10.1111/j.1472-765X.2005.01662.x.

Abstract

Aims: To develop a sensitive detection method for Mycobacterium avium ssp. paratuberculosis (Map) in water by modifying and optimizing an existing immunomagnetic separation polymerase chain reaction (IMS-PCR) technique.

Methods and results: Sterile distilled water (50 ml) spiked with 10(6) Map ml(-1) was subjected to either filtration (0.45 microm pore size) followed directly by IS900 PCR (method 1) or centrifugation (2500 g for 20 min) followed by IMS and IS900 PCR (method 2). Method 2 permitted the detection of Map, whereas method 1 did not. Method 2 was then optimized by adding different concentrations of Tween 80 (0.05, 0.1, 0.2, 0.4 and 0.6% v/v) to water samples spiked with Map (10(6)-1 CFU ml(-1)) prior to centrifugation, and assessing the impact of this action on the detection sensitivity of subsequent IMS-PCR. The optimum Tween 80 concentration was found to be 0.4%, which permitted the detection of 10 Map CFU ml(-1) in spiked water samples by IMS-PCR.

Conclusions: This method will be used to determine the incidence of Map in water destined for domestic use in future studies.

Significance and impact of the study: A sensitive method for the detection of Map in water involving addition of 0.4% Tween 80, centrifugation and IMS-PCR was developed.

MeSH terms

  • Bacteriological Techniques*
  • Centrifugation
  • DNA, Bacterial / analysis
  • Filtration
  • Immunomagnetic Separation*
  • Mycobacterium avium subsp. paratuberculosis / isolation & purification*
  • Polymerase Chain Reaction*
  • Sensitivity and Specificity
  • Water Microbiology*

Substances

  • DNA, Bacterial