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

Autor: Greenstein RJ; Department of Surgery, James J. Peters Veterans Affairs Medical Center, Bronx, NY, USA. BGAxis@aol.com.; Laboratory of Molecular Surgical Research, James J. Peters Veterans Affairs Medical Center, Bronx, NY, USA. BGAxis@aol.com., Su L; Laboratory of Molecular Surgical Research, James J. Peters Veterans Affairs Medical Center, Bronx, NY, USA., Fam PS; Mental Illness Research Education and Clinical Center (MIRECC), James J. Peters Veterans Affairs Medical Center, Bronx, NY, USA., Stabel JR; Johne's Disease Research Project, USDA-ARS, National Animal Disease Center, Ames, IA, 50010, USA., Brown ST; Infectious Disease Section, James J. Peters Veterans Affairs Medical Center, Bronx, NY, USA.; Department of Medicine, Icahn School of Medicine at Mt. Sinai, New York, NY, USA.
Jazyk: angličtina
Zdroj: BMC research notes [BMC Res Notes] 2018 Jul 21; Vol. 11 (1), pp. 498. Date of Electronic Publication: 2018 Jul 21.
DOI: 10.1186/s13104-018-3601-5
Abstrakt: Objectives: 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).
Results: 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 Affymetrix™ RNA view ® cannot be used to detect MAP in pre-frozen intestine of cattle with Johne's disease.
Databáze: MEDLINE
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