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Development of improved PCR to prevent false positives and false negatives in the detection of Tetracapsula bryosalmonae , the causative agent of proliferative kidney disease
Author(s) -
Morris D C,
Morris D J,
Adams A
Publication year - 2002
Publication title -
journal of fish diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.819
H-Index - 85
eISSN - 1365-2761
pISSN - 0140-7775
DOI - 10.1046/j.1365-2761.2002.00398.x
Subject(s) - biology , primer (cosmetics) , false positive paradox , parasite hosting , polymerase chain reaction , ribosomal dna , virology , microbiology and biotechnology , computational biology , pathology , gene , genetics , medicine , chemistry , phylogenetics , organic chemistry , machine learning , world wide web , computer science
Proliferative kidney disease (PKD) is an economically significant disease caused by the myxozoan parasite Tetracapsula bryosalmonae . Polymerase chain reaction (PCR) protocols using primers specific for the small subunit ribosomal RNA (18S rDNA) gene of the parasite enable detection, however, false positive and negative results can render detection inconclusive. In this study a decontamination protocol was developed, using hydroxylamine hydrochloride (H), to prevent false positives by blocking re‐amplification of carry‐over contaminants. A mimic molecule was also developed and used as a competitive internal standard coamplified with target DNA in PCRs, revealing both true and false negatives. The sensitivity of one new and two existing primer sets was assessed with all primers detecting DNA equivalent to at least eight parasite cells per gram of tissue. This improved PCR protocol canprovide more reliable testing for T. bryosalmonae .