title: 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.
abstract: 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 (ELF1α) 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 (ELF1α) gene as an endogenous control and is applicable for diagnostic purposes in samples obtained from the three salmonid species reared in Chile.
authors.affiliation: Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile. smarshal@ucv.cl.
authors.forename: Dagoberto
authors.initials: D
authors.lastname: Sepúlveda
authors.forename: Harry
authors.initials: H
authors.lastname: Bohle
authors.forename: Alvaro
authors.initials: A
authors.lastname: Labra
authors.forename: Horst
authors.initials: H
authors.lastname: Grothusen
authors.forename: Sergio H
authors.initials: SH
authors.lastname: Marshall
mesh_terms: D000818:Animals; D001483:Base Sequence; D005393:Fish Diseases / Q000175:diagnosis*; D014644:Genetic Variation; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism; D011786:Quality Control; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012487:Salmonidae / Q000145:classification*; D016415:Sequence Alignment; D013045:Species Specificity
chemical_list: D020648:Peptide Elongation Factor 1
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