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Development of an IMS–PCR assay for the detection of Mycobacterium avium ssp. paratuberculosis in water
Author(s) -
Whan L.,
Ball H.J.,
Grant I.R.,
Rowe M.T.
Publication year - 2005
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2005.01662.x
Subject(s) - centrifugation , paratuberculosis , chromatography , distilled water , mycobacterium , immunomagnetic separation , biotinylation , polymerase chain reaction , chemistry , microbiology and biotechnology , biology , bacteria , biochemistry , gene , genetics
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 μ m 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.