Development and evaluation of a real-time RT-PCR assay for the detection of Ebola virus (Zaire) during an Ebola outbreak in Guinea in 2014–2015

Dedkov, Vladimir G.; Magassouba, N’Faly; Safonova, Marina V.; Deviatkin, Andrei A.; Dolgova, Anna; Pyankov, Oleg V.; Sergeev, A.A.; Уткин, Д. В.; Odinokov, G. N.; Сафронов, Валентин; Агафонов, А. П.; Maleev, V V; Shipulin, German A.

doi:10.1016/j.jviromet.2015.11.007

Cited by 20 publications

(17 citation statements)

References 14 publications

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“…Note that such specimens are suitable only for PCR. Also, note that a well-established assay is necessary for each particular virus diagnosis 8,23 , and unknown virus samples must be sent to assigned laboratories for further investigations 19,20,21 . Mandatory and recommended requirements to the list of laboratory equipment for respiratory virus diagnostic PCR tests must be recognized.…”

Section: Discussionmentioning

confidence: 99%

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Remote Laboratory Management: Respiratory Virus Diagnostics

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Avadhanula

Michel

et al. 2019

JoVE

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An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

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Section: Discussionmentioning

confidence: 99%

“…The ability to rapidly estimate viral loads in a specimen makes PCR an efficient tool for viral disease screening 17,18 . The implementation of novel, molecular diagnostic assays allows expansion of diagnostic capabilities for viruses such as Ebola 19,20,21 , influenza 8,22 , and tuberculosis (TB) 23 .…”

Section: Introductionmentioning

confidence: 99%

Remote Laboratory Management: Respiratory Virus Diagnostics

Петрова

Avadhanula

Michel

et al. 2019

JoVE

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“…Ebola virus RNA genome encodes seven viral proteins in the following order nucleoprotein, phosphoprotein (VP35), matrix protein (VP40), GP, replication‐transcription protein (VP30), matrix protein (VP24), and polymerase (L), with an additional soluble GP produced from an edited GP mRNA . Molecular assays based on RT‐PCR have been successfully applied in the diagnosis of Ebola virus genome, their targets for detection being exclusively on the L, nucleoprotein or GP gene . In this study, we selected the primers/probe set for EBOV GP, VP24 and VP40 gene detection and tested them in one‐step qRT‐PCR assays.…”

Section: Discussionmentioning

confidence: 99%

“…and polymerase (L), with an additional soluble GP produced from an edited GP mRNA (20,21). Molecular assays based on RT-PCR have been successfully applied in the diagnosis of Ebola virus genome, their targets for detection being exclusively on the L, nucleoprotein or GP gene (8,(10)(11)(12)(13)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). In this study, we selected the primers/probe set for EBOV GP, VP24 and VP40 gene detection and tested them in one-step qRT-PCR assays.…”

Section: Discussionmentioning

confidence: 99%

Rapid detection and quantification of Ebola Zaire virus by one‐step real‐time quantitative reverse transcription‐polymerase chain reaction

Ticer

Carrión

2017

Microbiology and Immunology

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Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection and preventing further transmission. Here, a one-step qRT-PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross-reactivity being observed among them. The limits of detection of the assays ranged from 10 to 10 copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture-propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene- and strain-specific. The RT-PCR assays detected viral RNAs in blood samples from virus-infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples.

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“…The cDNA region (126 bp) equivalent to L gene of LASV (strain Josiah) that included the primer and probe target sequences was generated using step-out amplification, as previously described (Dedkov et al, 2016). The final PCR product was purified using a MinElute Gel Extraction kit (Qiagen, Germany), ligated into the pGEM-T plasmid vector (Promega, USA), and transformed into Escherichia coli (XL1-Blue strain) (Maniatis et al, 1989).…”

Section: Generation Of Positive-control Samplesmentioning

confidence: 99%