Objectives

To evaluate three methods of DNA extraction from milk for the IS900 polymerase chain reaction (PCR) and compare analytical sensitivities of the PCR and modified double incubation radiometric mycobacterial culture (RMC) method.

Experimental design

The comparative evaluation of the three DNA extraction methods (Beadbeater, InstaGene and Qiagene) and determination of the detection limits of the RMC and PCR were carried out on triplicate samples of milk inoculated with serial ten-fold dilutions of Mycobacterium paratuberculosis.

Results

Among the three protocols of DNA extraction from milk, the Beadbeater method was the most efficient procedure for the preparation of M. paratuberculosis DNA template for the IS900 PCR. The average detection limit of the Beadbeater PCR system was about 70 viable M. paratuberculosis cells/50 ml sample. The InstaGene and Qiagene (QIAamp DNA Stool Kit) methods produced average detection limits by PCR of 600 and 700 cells/50 ml sample, respectively. The analytical sensitivity of the RMC was about 700 viable cells/50 ml sample.

Conclusions

The analytical sensitivity of the Beadbeater PCR system is sufficient for this test to be used for the detection of low levels of M. paratuberculosis contamination in milk. Further evaluation of this test on diagnostic samples is warranted.