PMID- 24040749
OWN - NLM
STAT- MEDLINE
DCOM- 20140630
LR  - 20211021
IS  - 1746-6148 (Electronic)
IS  - 1746-6148 (Linking)
VI  - 9
DP  - 2013 Sep 16
TI  - Design and evaluation of a unique RT-qPCR assay for diagnostic quality control 
      assessment that is applicable to pathogen detection in three species of salmonid 
      fish.
PG  - 183
LID - 10.1186/1746-6148-9-183 [doi]
AB  - BACKGROUND: The detection of pathogens at early stages of infection is a key 
      point for disease control in aquaculture. Therefore, accurate diagnostic 
      procedures are a must. Real-time PCR has been a mainstay in diagnostics over the 
      years due to its speed, specificity, sensitivity, reproducibility and throughput; 
      as such, real-time PCR is a target for improvement. Nevertheless, to validate a 
      novel diagnostic tool, correct setup of the assay, including proper endogenous 
      controls to evaluate the quantity and quality of the samples and to detect 
      possible sample degradation, is compulsory. This work aims to design a unique 
      RT-qPCR assay for pathogen detection in the three salmonid species reared in 
      Chile. The assay uses elongation factor 1 alpha as the single endogenous control, 
      thus avoiding the need for multiple endogenous controls, as well as multiple 
      validations and non-comparable quality control parameters. RESULTS: The in vivo 
      and in vitro analyses of samples from Salmo salar, Oncorhynchus mykiss and 
      Oncorhynchus kisutch showed that when primers were accurately selected to target 
      conserved regions of the elongation factor 1 alpha (ELF1alpha) gene, a single novel 
      RT-qPCR assay yielding similar and reproducible Ct values between the three 
      species could be designed. The opposite occurred when an assay originally 
      designed for Salmo salar was tested in samples from the two species of the genus 
      Oncorhynchus. CONCLUSIONS: Here, we report the design and evaluation of an 
      accurate trans-species RT-qPCR assay that uses the elongation factor 1 alpha 
      (ELF1alpha) gene as an endogenous control and is applicable for diagnostic purposes 
      in samples obtained from the three salmonid species reared in Chile.
FAU - Sepulveda, Dagoberto
AU  - Sepulveda D
AD  - Laboratorio de Genetica e Inmunologia Molecular, Instituto de Biologia, Facultad 
      de Ciencias, Pontificia Universidad Catolica de Valparaiso, Valparaiso, Chile. 
      smarshal@ucv.cl.
FAU - Bohle, Harry
AU  - Bohle H
FAU - Labra, Alvaro
AU  - Labra A
FAU - Grothusen, Horst
AU  - Grothusen H
FAU - Marshall, Sergio H
AU  - Marshall SH
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20130916
PL  - England
TA  - BMC Vet Res
JT  - BMC veterinary research
JID - 101249759
RN  - 0 (Peptide Elongation Factor 1)
SB  - IM
MH  - Animals
MH  - Base Sequence
MH  - Fish Diseases/*diagnosis
MH  - Genetic Variation
MH  - Molecular Sequence Data
MH  - Peptide Elongation Factor 1/genetics/metabolism
MH  - Quality Control
MH  - Real-Time Polymerase Chain Reaction/*veterinary
MH  - Reproducibility of Results
MH  - Reverse Transcriptase Polymerase Chain Reaction/*veterinary
MH  - Salmonidae/*classification
MH  - Sequence Alignment
MH  - Species Specificity
PMC - PMC3848472
EDAT- 2013/09/18 06:00
MHDA- 2014/07/01 06:00
PMCR- 2013/09/16
CRDT- 2013/09/18 06:00
PHST- 2013/02/25 00:00 [received]
PHST- 2013/09/03 00:00 [accepted]
PHST- 2013/09/18 06:00 [entrez]
PHST- 2013/09/18 06:00 [pubmed]
PHST- 2014/07/01 06:00 [medline]
PHST- 2013/09/16 00:00 [pmc-release]
AID - 1746-6148-9-183 [pii]
AID - 10.1186/1746-6148-9-183 [doi]
PST - epublish
SO  - BMC Vet Res. 2013 Sep 16;9:183. doi: 10.1186/1746-6148-9-183.