Location: Infectious Bacterial Diseases ResearchTitle: Failure to detect M. avium subspecies paratuberculosis by fluorescent in situ hybridization (FISH) in Johne’s or Crohn’s disease using a proprietary assay Author
|Greenstein, R - James J Peters Vamc|
|Su, L - James J Peters Vamc|
|Fam, P - James J Peters Vamc|
|Brown, S - James J Peters Vamc|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/5/2018
Publication Date: 6/4/2018
Citation: Greenstein, R.J., Su, L., Fam, P., Stabel, J.R., Brown, S.T. 2018. Failure to detect M. avium subspecies paratuberculosis by fluorescent in situ hybridization (FISH) in Johne’s or Crohn’s disease using a proprietary assay. Meeting Abstract. 1.2/16.
Technical Abstract: Background: M. avium subspecies paratuberculosis (MAP) causes Johne’s disease. MAP may be zoonotic, responsible for, at a minimum, Crohn’s disease. Thus, the presence or absence of MAP needs to be reliably diagnosed. The “gold standard” for detection of MAP in cattle is culture, followed by DNA sequencing. Culture of MAP in Crohn’s has been achieved in very few laboratories. The purpose of this study was to evaluate a proprietary (Affymetrix™ RNA view®) FISH assay for MAP RNA in humans with Crohn’s, using Johne’s tissue as the positive control. Methods: Intestine from steer with Johne’s disease and humans with Crohn’s were assayed according to Affymetrix’s instructions. Published genomes were used to custom design probes. IS900 for MAP and bovine and human ß-actin (as species specific eukaryotic housekeeping controls). We attempted to prevent false positive signal in the “no- probe” control, by modifying wash solutions, using recommended and derivative hydrochloric acid titration and different fluorescent filters (TritC for Texas Red and “Hope” for Cy-5.) Principal Findings: Repetitively, false positive signal was observed in our “no-probe” negative control. Attempts to correct this according to the manufacturers suggestions, and with multiple derivative techniques have been unsuccessful. Conclusions: We performed the Affymetrix™ RNA view® according to the manufacture’s instruction and used multiple variations on the manufacture’s recommended suggestions to correct for false positive signal. Repetitively, “no-probe” positive controls indicate that this assay cannot be used to reliably detect MAP in pre-frozen intestine of cattle with Johne’s disease. Nor can it be used in the possible identification of MAP in frozen tissue from humans with Crohn’s disease.