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Detection of Mycobacterium avium subsp paratuberculosis in formalin‐fixed paraffin‐embedded intestinal tissue by IS 900 polymerase chain reaction
Author(s) -
WHITTINGTON RJ,
REDDACLIFF L.,
MARSH I.,
SAUNDERS V.
Publication year - 1999
Publication title -
australian veterinary journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.382
H-Index - 59
eISSN - 1751-0813
pISSN - 0005-0423
DOI - 10.1111/j.1751-0813.1999.tb10315.x
Subject(s) - paratuberculosis , proteinase k , dna extraction , polymerase chain reaction , biology , mycobacterium , microbiology and biotechnology , staining , dna , virology , bacteria , gene , biochemistry , genetics
Objectives To evaluate and compare methods for DNA extraction from formalin‐fixed, paraffin‐embedded tissues and methods for detection of Mycobacterium avium subsp paratuberculosis by IS 900 PCR for confirmation of Johne's disease in ruminants. Design Alaboratory study. Procedure Three methods of DNA extraction of differing complexity and two PCR protocols using different pairs of IS 900 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 Mavium 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 IS 900 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. Conclusion 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 IS 900 is more effective than amplification of longer fragments.