Development of a widely applicable positive control strategy to support detection of infectious salmon anaemia virus (ISAV) using Taqman real‐time PCR

Snow, Michael; McKay, P; Matějusová, Iveta

doi:10.1111/j.1365-2761.2008.00972.x

Cited by 33 publications

(36 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The tag-specific probe integrated in the master mix can hybridize only with the LPC, and only then can the tagspecific fluorophore be detected during amplification. A similar approach has been described by Snow et al (30). In general these authors used the same strategy, but they cloned the tagged positive control prior to in vitro transcription.…”

Section: Discussionmentioning

confidence: 99%

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy

Hoffmann

Freuling

Wakeley³

et al. 2010

J Clin Microbiol

View full text Add to dashboard Cite

To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories.

show abstract

Section: Discussionmentioning

confidence: 99%

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy

Hoffmann

Freuling

Wakeley³

et al. 2010

J Clin Microbiol

View full text Add to dashboard Cite

show abstract

“…For instance, the design of a plasmid-based positive control that contains a unique target site for a probe is one such approach. 19 The positive control is detected by the first probe specific to the target and also by a second probe (different fluorophore) that targets a unique sequence. Both probes are added to test samples, and positive results are detected by the first probe.…”

Section: Investigation Of Artifactual Resultsmentioning

confidence: 99%

Selection of a Cutoff Value for Real-Time Polymerase Chain Reaction Results to Fit a Diagnostic Purpose: Analytical and Epidemiologic Approaches

et al. 2011

View full text Add to dashboard Cite

Abstract. Diagnostic laboratories frequently select a subjective cutoff value for real-time amplification assays, above which a threshold cycle (Ct) value is deemed false. Commonly, higher Ct values are interpreted as amplification or fluorescence artifacts, or cross contaminations. Although the implementation of Ct cutoff might be reasonable, its justification and selection should be based on evidence. The current article reviewed evidence-based strategies to select Ct cutoffs grouped in analytical and epidemiologic approaches. Analytical strategies use criteria gathered during the assay development and include fluorescence threshold, reaction endcycle, limit of detection, and artifact investigation. Variability in amplification efficacy across test runs may induce some instability in an intended Ct cutoff and requires some standardization or normalization procedures. Epidemiologic strategies use criteria based on either the probability or the cost of a false test result associated with a specified cutoff. Cutoffs, depending on the intended purpose of the test, can be selected graphically to minimize the probability of either false-positive or false-negative results by using two-graph receiver operating characteristics curves. The assay's diagnostic sensitivity and specificity may vary with the tested population, thus, the estimated two-graph receiver operating characteristics curve is population dependent and should be established for the targeted population. Although the selection of a cutoff based on misclassification cost depends on infection prevalence, the selection based on predictive values does not. To optimize the test average diagnostic performance, the Ct cutoff should be selected when diagnostic odds ratio is maximal. Epidemiologic approaches were illustrated by selecting Ct cutoffs for a real-time assay for Infectious salmon anemia virus.

show abstract

“…23 The APC plasmid DNA was used as a positive control in separate reaction wells and to generate standard curves. The APC plasmid was used to determine absolute ENV gene copy number based on a plasmid molecular weight of 1.3 × 10 6 g/mole (both assays), which equates to 4.7 × 10 11 copies/µg; copy number calculations were as described.…”

Section: Primer Probes and Artificial Positive Controls Developmentmentioning

confidence: 99%

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

et al. 2016

View full text Add to dashboard Cite

Abstract. Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

show abstract

Development of a widely applicable positive control strategy to support detection of infectious salmon anaemia virus (ISAV) using Taqman real‐time PCR

Cited by 33 publications

References 4 publications

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy

Selection of a Cutoff Value for Real-Time Polymerase Chain Reaction Results to Fit a Diagnostic Purpose: Analytical and Epidemiologic Approaches

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

Contact Info

Product

Resources

About