Rapid detection of anti-Vaccinia virus neutralizing antibodies

Kramski, Marit; Drozd, Anna; Lichtfuss, Gregor F.; Dabrowski, Piotr Wojtek; Ellerbrok, Heinz

doi:10.1186/1743-422x-8-139

Cited by 11 publications

(13 citation statements)

References 13 publications

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“…(22)(23)(24). A new real-time PCR amplicon was designed for the SIGV G gene and for the VARV HA gene.…”

Section: Methodsmentioning

confidence: 99%

“…A capC gene carrying plasmid (B. anthracis, plasmid pX02) was provided by the Robert-Koch-Institut (21). For DNA virus, the variola virus (VARV) HA gene was synthesized and ligated into pMA-RQ by Geneart, Regensburg, Germany, and the vaccinia virus (VACV) plasmid carrying the LE gene was provided by the Robert-Koch-Institut (22). For RNA viruses, quantitative Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV) NP-gene RNA standards were used as described previously (23,24).…”

Section: Methodsmentioning

confidence: 99%

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Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

et al. 2013

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Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms. Syndromic panels for infectious and emerging infectious diseases have been suggested to be of value in point-of-care (POC) diagnostics for developing countries and for biodefense (1). Since the introduction of molecular diagnostics and in particular real-time PCR, ample proof of its sensitivity and specificity has been generated. Indeed, molecular diagnostics are deemed superior to bacterial culture techniques or serological diagnostics (2-4). It has even been suggested to entirely eliminate the old methods in order to streamline centralized laboratories for molecular diagnostics (5-7).In recent years alternative isothermal amplification methods which can be categorized into (i) T7 promoter-driven amplifications (transcription-mediated amplification [TMA], nucleic acid sequence-based amplification [NASBA], and single primer isothermal amplification [SPIA]), (ii) strand displacement methods (strand displacement amplification [SDA], loop-mediated isothermal amplification [LAMP], and smart amplification [SmartAmp]), (iii) helicase-dependent amplification (HDA), (iv) recombinase polymerase amplification (RPA), and (v) rolling-circle amplification (RCA) methods (8-12) have been developed. Some were purposely designed for isothermal amplification starting from RNA (TMA, NASBA, and SPIA), whereas others initially targeted DNA (SDA, LAMP, HDA, RPA, and RCA) and were only later adapted for RNA targets. Nonspecific intercalating fluorophores or fluorescent primers have been used for real-time detection in LAMP, SDA, HDA, and RCA, and specific detection probe formats have been developed for NASBA, RCA, HDA,.In isothermal and exponential RPA, the phage recombinase UvsX and its cofactor UvsY form a nucleoprotein complex with oligonucleotide primers to scan for homologous sequences in a DNA template. Recognition of a specific homologous sequence leads to the initiation of strand invasion of the com...

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“…(22)(23)(24). A new real-time PCR amplicon was designed for the SIGV G gene and for the VARV HA gene.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

et al. 2013

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“…Few studies have used PCR as an approach to measure virus neutralization assay endpoints (23)(24)(25). As far as we are aware, this is the first report of a real-time PCR-based neutralization assay being used in the diagnosis of a flavivirus infection.…”

Section: Discussionmentioning

confidence: 99%

Neutralization Assay for Zika and Dengue Viruses by Use of Real-Time-PCR-Based Endpoint Assessment

et al. 2017

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The global spread and infective complications of Zika virus (ZKV) and dengue virus (DENV) have made them flaviviruses of public health concern. Serological diagnosis can be challenging due to antibody cross-reactivity, particularly in secondary flavivirus infections or when there is a history of flavivirus vaccination. The virus neutralization assay is considered to be the most specific assay for measurement of anti-flavivirus antibodies. This study describes an assay where the neutralization endpoint is measured by real-time PCR, providing results within 72 h. It demonstrated 100% sensitivity (24/24 ZKV and 15/15 DENV) and 100% specificity (11/11 specimens) when testing well-characterized sera. In addition, the assay was able to determine the correct DENV serotype in 91.7% of cases. The high sensitivity and specificity of the real-time PCR neutralization assay makes it suitable to use as a confirmatory test for sera that are reactive in commercial IgM/IgG enzyme immunoassays. Results are objective and the PCR-based measurement of the neutralization endpoint lends itself to automation so that throughput may be increased in times of high demand.KEYWORDS dengue, flavivirus, serology, Zika Z ika virus (ZKV) and dengue virus (DENV) are members of the family Flaviviridae, genus Flavivirus, which also includes yellow fever, Japanese encephalitis, and West Nile viruses. There are at least 4 closely related but serologically distinct DENV, designated DENV serotypes 1 through 4 (1). There is only transient and weak crossprotection among the 4 DENV serotypes, so individuals living in an area where DENV is endemic can be infected up to 4 times (2).ZKV and DENV have spread across the tropics in Africa, Asia, the Pacific, and the Americas with their vector mosquitoes Aedes aegypti and A. albopictus (3, 4). Cocirculation of ZKV and DENV types 1 through 4 has been documented in both French Polynesia and Brazil (5, 6) and likely occurs in many other tropical countries. Incursions into temperate countries have also occurred, with autochthonous transmission observed in Europe (DENV in southeastern France, Croatia, and Madeira Island) and the United States (DENV and ZKV in Florida) (7-9).Both ZKV and DENV infection can produce a wide spectrum of illnesses with many asymptomatic or subclinical infections (10, 11). ZKV's potential to cause fetal central nervous system abnormalities, growth restriction, and death (12) has necessitated ZKV testing of potentially exposed pregnant women, among whom nonspecific serological activity may occur, and asymptomatic individuals with low pretest probabilities of positive ZKV serology.Direct detection of ZKV nucleic acids has limited utility in asymptomatic patients, as the periods of viremia and shedding in urine and saliva are relatively brief, occurring

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“…consortium. Viruses in this panel have been quantified by quantitative real-time PCR using published assays [14, 15] and were investigated in a blinded manner to test the suitability of the ABICAP assay to discern highly pathogenic viruses in a bioterrorist setting.…”

Section: Methodsmentioning

confidence: 99%

Rapid and sensitive point-of-care detection of Orthopoxviruses by ABICAP immunofiltration

Stern

Olson

Smith

et al. 2016

Virol J

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BackgroundThe rapid and reliable detection of infectious agents is one of the most challenging tasks in scenarios lacking well-equipped laboratory infrastructure, like diagnostics in rural areas of developing countries. Commercially available point-of-care diagnostic tests for emerging and rare diseases are particularly scarce.ResultsIn this work we present a point-of-care test for the detection of Orthopoxviruses (OPV). The OPV ABICAP assay detects down to 1 × 104 plaque forming units/mL of OPV particles within 45 min. It can be applied to clinical material like skin crusts and detects all zoonotic OPV infecting humans, including Vaccinia, Cowpox, Monkeypox, and most importantly Variola virus.ConclusionsGiven the high sensitivity and the ease of handling, the novel assay could be highly useful for on-site diagnostics of suspected Monkeypox virus infections in areas lacking proper laboratory infrastructure as well as rapid on-site testing of suspected bioterrorism samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0665-5) contains supplementary material, which is available to authorized users.

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Rapid detection of anti-Vaccinia virus neutralizing antibodies

Cited by 11 publications

References 13 publications

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

Neutralization Assay for Zika and Dengue Viruses by Use of Real-Time-PCR-Based Endpoint Assessment

Rapid and sensitive point-of-care detection of Orthopoxviruses by ABICAP immunofiltration

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