Development and validation of a range of endogenous controls to support the implementation of practical Taqman real‐time PCR‐based surveillance for fish diseases within aquaculture

Abstract

The use of Taqman real-time PCR-based technology has recently become more frequent in the detection of pathogens in the aquaculture industry. This interest has necessitated the development of robust and reliable pathogen-detection assays. The development of a range of endogenous control assays to be run alongside these diagnostic assays works to further increase confidence in the latter. This study describes the design of a range of endogenous control assays based on the elongation factor 1-α (EF1-α) gene specific to a range of fish species including Atlantic salmon, Salmo salar; rainbow trout, Oncorhynchus mykiss; brown trout, Salmo trutta; cod, Gadus morhua; haddock, Melanogrammus aeglefinus; saithe, Pollachius virens; whiting, Merlangius merlangus; Norway pout, Trisopterus esmarkii; carp (family Cyprinidae), roach, Rutilus rutilus; European eel, Anguilla anguilla; and herring, Clupea harengus, as well as a number of fish cell lines. Evidence is provided of the validation of these assays for specific species, a range of tissue types and cell lines as well as an example of the potential uses of these assays.

Development of a Multiplex Assay to Measure the Effects of Shipping and Storage Conditions on the Quality of RNA Used in Molecular Assays for Detection of Viral Haemorrhagic Septicemia Virus

“…Hence, the use of an endogenous control would provide a rapid analysis tool for RNA quality. This is supported by a recent study conducted by Bland et al (2012) using ELF-1α mRNA within the range of 17.76-25.97 as samples with acceptable quality for testing. When considering the use of endogenous genes as a quality control for tissue samples, the cost and the turnaround time of the analysis will increase dramatically, thus limiting their use.…”

“…Consequently, several studies have shown the importance of using an endogenous control such as ELF-1α mRNA in parallel with the targeted pathogen nucleic acid as a quality control for the samples (Ingerslev et al 2006;Jorgensen et al 2006;Snow et al 2006; Downloaded by [University of Prince Edward Island] at 17:56 17 November 2014 FIGURE 1. Agarose gel electrophoresis of total RNA extracted from Atlantic Salmon HK stored at room temperature and at −80 • C for 0, 6, 12, 24, 48, and 72 h. Christiansen et al 2011;Bland et al 2012). Recently, a new StaRT-PCR approach, which incorporates several endogenous controls (actin β1, 18S rRNA, and elongation factor 1 α), was published for accurate VHSV screening (Pierce et al 2013).…”

“…Indeed, in fish health screening, several studies have shown the importance of using endogenous genes as quality controls of the samples (Snow et al 2006;Olsvik et al 2008;Christiansen et al 2011;Bland et al 2012). These endogenous controls are widely used as reference genes, assuming that the level of their expression is unaffected under suitable conditions of sample storage (Snow et al 2006).…”

“…These endogenous controls are widely used as reference genes, assuming that the level of their expression is unaffected under suitable conditions of sample storage (Snow et al 2006). Among the identified reference genes, elongation factor 1 alpha (ELF-1α) was selected as the most suitable endogenous control gene in studies with Atlantic Salmon Salmo salar (Ingerslev et al 2006;Jorgensen et al 2006;Snow et al 2006;Christiansen et al 2011;Bland et al 2012). In addition, reference genes will ensure that the RT-qPCR testing was processed properly.…”

Susceptibility of goldsinny wrasse, Ctenolabrus rupestris L. (Labridae), to viral haemorrhagic septicaemia virus (VHSV) genotype III: Experimental challenge and pathology

“…A partial fragment of elongation factor 1a (ELF-1a) was amplified according to Bland et al (2012). PCR product was purified (MinElute, Qiagen) and quantified on 2% ethidium bromide-stained agarose gel alongside a Low DNA Mass Ladder (Life Technologies).…”