Detection of Mycobacterium avium subsp paratuberculosis in formalin-fixed paraffin-embedded intestinal tissue by IS900 polymerase chain reaction.
OBJECTIVE
To evaluate and compare methods for DNA extraction from formalin-fixed, paraffin-embedded tissues and methods for detection of Mycobacterium avium subsp paratuberculosis by IS900 PCR for confirmation of Johne's disease in ruminants.


METHODS
A laboratory study.


METHODS
Three methods of DNA extraction of differing complexity and two PCR protocols using different pairs of IS900 primers were compared. Sensitivity and specificity were assessed using samples from ruminants with and without histological evidence of Johne's disease.


RESULTS
The simplest method of DNA extraction, which involved two cycles of boiling and freezing followed by centrifugation, gave more consistent results than two methods that required solvent extraction of paraffin, proteinase digestion and DNA purification. The sensitivity of detection of M avium subsp paratuberculosis in paraffin blocks stored for 1 to 6 years from 34 cases of Johne's disease in sheep, cattle and goats was 88% for a 229 bp IS900 PCR assay and 71% for a 413 bp assay, using the detection of acid-fast bacilli by Ziehl Neelsen staining of histological sections from the same blocks as the gold standard test. PCR results correlated with the abundance of acid-fast organisms in the tissues. No false positive reactions were detected.


CONCLUSIONS
PCR for identification of M avium subsp paratuberculosis in formalin-fixed, paraffin-embedded intestinal tissues from ruminants is a rapid and useful method. A simple method of sample preparation is effective. Amplification of short fragments of IS900 is more effective than amplification of longer fragments.
Whittington
R J
RJ
NSW Agriculture, Elizabeth Macarthur Agricultural Institute, Camden.
Reddacliff
L
L
Marsh
I
I
Saunders
V
V
D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification*; D015511:Goat Diseases / Q000382:microbiology*; D006041:Goats; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016612:Paraffin Embedding / Q000662:veterinary; D010283:Paratuberculosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000382:microbiology*
D017931:DNA Primers; D004269:DNA, Bacterial