Detection of Mycobacterium avium subsp paratuberculosis in formalin-fixed paraffin-embedded intestinal tissue by IS900 polymerase chain reaction

Objectives To evaluate and compare methods for DNA extraction from formalin -fixed, paraffin-embedded tissues and methods for detection of Mycobacteriu m avium subsp paratuberculosis by IS900 PCR for confirmation of Johne's dis ease in ruminants. Design A laboratory study. Procedure Three methods of DNA extraction of differing complexity and two P CR protocols using different pairs of IS900 primers were compared. Sensitiv ity and specificity were assessed using samples from ruminants with and wit hout 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 resu lts than two methods that required solvent extraction of paraffin, proteina se digestion and DNA purification. The sensitivity of detection of Mycobact erium avium subsp paratuberculosis in paraffin blocks stored for 1 to 6 yea rs 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 reaction s were detected. Conclusion PCR for identification of Mycobacterium avium subsp paratubercul osis in formalin-fixed, paraffin-embedded intestinal tissues from ruminants is a rapid and useful method. A simple method of sample preparation is eff ective. Amplification of short fragments of IS900 is more effective than am plification of longer fragments.