Development of an IMS-PCR assay for the detection of Mycobacterium avium ssp. paratuberculosis in water.
OBJECTIVE
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.


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.


CONCLUSIONS
A sensitive method for the detection of Map in water involving addition of 0.4% Tween 80, centrifugation and IMS-PCR was developed.
Whan
L
L
Food Microbiology, The Queen's University of Belfast, Belfast, UK.
Ball
H J
HJ
Grant
I R
IR
Rowe
M T
MT
D001431:Bacteriological Techniques; D002498:Centrifugation; D004269:DNA, Bacterial / Q000032:analysis; D005374:Filtration; D018189:Immunomagnetic Separation; D016927:Mycobacterium avium subsp. paratuberculosis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014871:Water Microbiology
D004269:DNA, Bacterial