Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards

Abstract

In the present study, 2 systems of real-time RT-PCR -one based on SYBR Green and the other on TaqMan -were designed to detect strains from any genotype of viral haemorrhagic septicaemia virus (VHSV), with high sensitivity and repeatability/reproducibility. In addition, the method was optimized for quantitative purposes (qRT-PCR), and standard curves with different types of reference templates were constructed and compared. Specificity was tested against 26 isolates from 4 genotypes. The sensitivity of the procedures was first tested against cell culture isolation, obtaining a limit of detection (LD) of 10 0 TCID 50 ml −1 (100-fold below the LD using cell culture), at a threshold cycle value (Ct) of 36. Sensitivity was also evaluated using RNA from crude (LD = 1 fg; 160 genome copies) and purified virus (100 ag; 16 copies), plasmid DNA (2 copies) and RNA transcript (15 copies). No differences between both chemistries were observed in sensitivity and dynamic range. To evaluate repeatability and reproducibility, all experiments were performed in triplicate and on 3 different days, by workers with different levels of experience, obtaining Ct values with coefficients of variation always < 5. This fact, together with the high efficiency and R 2 values of the standard curves, encouraged us to analyse the reliability of the method for viral quantification. The results not only demonstrated that the procedure can be used for detection, identification and quantification of this virus, but also demonstrated a clear correlation between the regression lines obtained with different standards, which will help scientists to compare sensitivity results between different studies.

Design and validation of a RT‐qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers

“…Consequently, and due to the fact that iv RNA standards provide more acceptable efficiencies, the use of this type of standard is advised. This differs from the results reported in a similar study to validate an RT‐qPCR procedure for the quantification of VHSV, in which either of both types of standard curves ( iv RNA and titrated virus) could be used because they showed parallel kinetics (Lopez‐Vazquez, Bandín & Dopazo ).…”

A sensitive one-step TaqMan amplification approach for detection of rubella virus clade I and II genotypes in clinical samples

“…Ten replicates were used per copy number step TaqMan-based RT-PCR assay for quantification of RV, which was not only validated through a DNA standard, but also by using RV-positive patient samples, as well as RNA extracted from cell culture supernatants, which is an accepted substitute for in vitro transcribed RNA. Both types of RNA produce similar results regarding sensitivity of real-time PCRs using TaqMan chemistry [17]. The results outlined in this study are consistent with this notion, as extracted Therien RNA had performance characteristics similar to the one observed for the RNA standard.…”

Improvement of a diagnostic procedure in surveillance of the listed fish diseases IHN and VHS

“…Several RT-qPCR assays are available to detect the IHNV or VHSV genome (Cutrín et al, 2009;Garver et al, 2011;Gjevre, Ørpetveit, Tavornpanich, & Lyngstad, 2014;Hope et al, 2010;Jonstrup et al, 2013;Liu et al, 2008;Lopez-Vazquez, Bandín, & Dopazo, 2015;Phelps, Patnayak, Jiang, & Goyal, 2012;Pierce et al, 2013;Purcell et al, 2013;Sandlund et al, 2014;Warg et al, 2014aWarg et al, , 2014b. However, diagnostic performance of most IHNV and VHSV RT-qPCR assays has not been fully evaluated in accordance with OIE guidelines (Bustin et al, 2009;Jonstrup et al, 2013;OIE, 2017d;Warg et al, 2014a).…”

“…Finally, the IHNV and VHSV RT‐qPCR assays were completed by including an endogenous control system to detect improper and impaired RT‐qPCRs resulting in false‐negative results (Hoferer et al, ; Jonstrup et al, ; Knüsel et al, ; Lopez‐Vazquez et al, ; Matejusova, McKay, McBeath, Collet, & Snow, ; Pierce et al, ). Including β‐actin as endogenous control has proven to be successful, as already demonstrated in previous studies (Hoferer et al, ; Julin, Johansen, & Sommer, ).…”