Failure to detect M. avium subspecies paratuberculosis in Johne's disease using a proprietary fluorescent in situ hybridization assay

BMC Research Notes
Robert J GreensteinSheldon T Brown

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants. The "gold standard" of MAP detection is by culture, DNA sequencing possibly supplemented by identification of Ziehl-Neelsen positive mycobacteria. The purpose of this study was to evaluate a proprietary (Affymetrix™ RNA view®) fluorescent in situ hybridization (FISH) assay for MAP RNA. Intestine from a steer with documented Johne's disease was assayed according to the manufacturer's instructions. Probes were custom designed for MAP and bovine β-actin (as the eukaryotic housekeeping gene) from published genomes. We attempt to prevent false positive signal in the "no-probe" control, by modifying wash solutions, using recommended hydrochloric acid titration and different fluorescent filters (TritC for Texas Red and "Hope" for Cy-5). 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. It is concluded that when performed according to manufactures instruction and with multiple variations on the manufactures recommended suggestions to correct for false positive signal, that the Affy...Continue Reading

References

Jun 1, 1989·Digestive Diseases and Sciences·G GitnickM Palieschesky
Sep 1, 1986·Journal of Clinical Microbiology·R J ChiodiniJ A Coutu
Nov 1, 1984·Journal of Clinical Microbiology·R J ChiodiniJ A Coutu
Sep 3, 1996·Proceedings of the National Academy of Sciences of the United States of America·D MishinaR J Greenstein
Aug 4, 2004·Lancet·Robert J Greenstein, Michael T Collins
Aug 15, 2012·Infection and Immunity·Dayle A KeownJacqueline I Keenan
Jul 14, 2017·Frontiers in Microbiology·Matteo RicchiBarbara Bertasi

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Methods Mentioned

BETA
IS900
Assay
Fluorescence

Software Mentioned

Affymetrix
HisTech

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