Development of improved PCR to prevent false positives and false negatives in the detection ofTetracapsula bryosalmonae, the causative agent of proliferative kidney disease

Abstract

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.

Diagnostic polymerase chain reaction assay to detect Kudoa neurophila (Myxozoa: Multivalvulida) in a marine finfish hatchery

“…Although very faint, yet consistent products were attained at an estimated 0.01 spore, human error caused by the inability to visualise such a weak signal is likely to result in a false negative outcome. Sensitivity for this diagnostic assay is similar to previous myxozoan single-round PCR based tests developed to date (Kent et al 1998, Palenzuela et al 1999, Morris et al 2002 and is more sensitive than current diagnostic methods. Accuracy in diagnosis is also improved by the ability to process whole fish, whole brains and spinal cords, whereas techniques such as microscopic examination of 5 µm sections only examines a portion of the targeted tissue introducing greater probability of error.…”

“…Fourteen adult blue throat wrasse and 20 striped trumpeter from each life stage at 23, 44, 77 and 136 dph were tested for parasites using wet squash preparations (Wet), H&E stained 5 µm sections (H&E) and PCR. Positive and negative infections are shown as + and -respectively and the percentage of positive infections are calculated at the bottom of each column for the blue throat wrasse model 1998, Palenzuela et al 1999, Morris et al 2002, Yokoyama et al 2000. The single-round PCR test was able to specifically amplify a fragment of the SSU rDNA from Kudoa neurophila and strong amplicons were always visualised from fish known to harbour the parasite infection at the terminal stage as determined by traditional histology.…”

“…The test is also designed to utilise commercially available molecular, easy to operate biology kits that have been shown here to be effective, efficient and reliable when used in the field situation. Myxozoan detection research has determined similar outcomes using the PCR approach to diagnosis and health management for both wild fishery management and aquaculture worldwide (Saulnier & de Kinkelin 1997, Andree et al 1998, Kent et al 1998, Palenzuela et al 1999, Morris et al 2002, Yokoyama et al 2000. The single-round PCR test was able to specifically amplify a fragment of the SSU rDNA from Kudoa neurophila and strong amplicons were always visualised from fish known to harbour the parasite infection at the terminal stage as determined by traditional histology.…”

“…A very sensitive and specific DNA-based diagnostic test using standard polymerase chain reaction (PCR) molecular technology is a management tool capable of detecting early developmental stages of the parasite or very light but mature infections in the fish host. The ability of PCR to perform this function is particularly useful as it can detect sub-clinical myxozoan infections much earlier than routine histology (Saulnier & de Kinkelin 1997, Andree et al 1998, Kent et al 1998, Palenzeula et al 1999, Yokoyama et al 2000, Morris et al 2002. In the case of K. neurophila, traditional techniques such as light microscope examination of stained sections and wet squash preparations are only effective in detecting the terminal spore forming stage and, in rare instances, presporogonic plasmodial stages of the disease.…”

Complex interaction between proliferative kidney disease, water temperature and concurrent nematode infection in brown trout

“…The yield was determined by spectrophotometry using the NanoDrop photometer (NanoDrop Technologies). PCR was performed according to the protocol by Morris et al (2002), with some modifications. The primer pair PKX3F (CTA AGT ACA TAC TTC GGT AGA) and PKX4R (CCG TTA CAA CCT TGT TAG GAA), described by Kent et al (1998), was used.…”