Comprehensive Panel of Real-Time TaqMan™ Polymerase Chain Reaction Assays for Detection and Absolute Quantification of Filoviruses, Arenaviruses, and New World Hantaviruses

Abstract

Viral hemorrhagic fever is caused by a diverse group of single-stranded, negative-sense or positive-sense RNA viruses belonging to the families Filoviridae (Ebola and Marburg), Arenaviridae (Lassa, Junin, Machupo, Sabia, and Guanarito), and Bunyaviridae (hantavirus). Disease characteristics in these families mark each with the potential to be used as a biological threat agent. Because other diseases have similar clinical symptoms, specific laboratory diagnostic tests are necessary to provide the differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. We designed 48 TaqMan™-based polymerase chain reaction (PCR) assays for specific and absolute quantitative detection of multiple hemorrhagic fever viruses. Forty-six assays were determined to be virus-specific, and two were designated as pan assays for Marburg virus. The limit of detection for the assays ranged from 10 to 0.001 plaque-forming units (PFU)/PCR. Although these real-time hemorrhagic fever virus assays are qualitative (presence of target), they are also quantitative (measure a single DNA/RNA target sequence in an unknown sample and express the final results as an absolute value (e.g., viral load, PFUs, or copies/mL) on the basis of concentration of standard samples and can be used in viral load, vaccine, and antiviral drug studies.

Introducing EbolaCheck: potential for point-of-need infectious disease diagnosis

“…The need for a true point-of-need NAT was acute, yet no in vitro diagnostic test had received regulatory clearance. 'Homebrew' assays were based on Trombley et al (the 'Trombley' assays; US Army Medical Research Institute for Infectious Disease; USAMRIID) [9] or Panning et al [10]. These eventually received US FDA emergency use authorization (EUA; EZ1 assay) or were made commercially available under the selfcertification CE marking principles (Altona RealStar Ò Filovirus Screen) [11] Molecular diagnostics for infectious diseases at the point of need Following 9/11 and the subsequent airborne viral disease pandemics, efforts were made to develop decentralized, point-ofcare NATs [12].…”

“…The Trombley assay sets for Ebolavirus Zaire GP and NP [9] were migrated to EbolaCheck (Trombley+) to minimize delays, avoid complex licensing negotiations, and on account of emerging field performance evaluation data. Multiplexed use of the Trombley+ assay sets also discriminate vaccinated from infected patients; NP is not found in the two most advanced EVD clinical vaccine candidates [19,20], a problem in on-going vaccination clinical trials pursued by other R2HC-funded programs (Gilbert S, personal communication).…”

Genome sequence analysis of Ebola virus in clinical samples from three British healthcare workers, August 2014 to March 2015

“…In our study presented here, sequence analysis of the NP gene, the target for widely used diagnostic detection assays [1], identified no SNPs within the regions where diagnostic primers bind. The GP gene product is the viral receptor, and the target of neutralising antibodies.…”

“…The GP gene product is the viral receptor, and the target of neutralising antibodies. Synonymous SNPs are present in locations where primers and probe bind for real-time detection methodologies based on the GP gene [1] ( Table 2). …”

“…Confirmation of EVD diagnosis in all three patients was performed using PCR assays targeting the NP gene [1]. Samples for sequencing were treated with DNase I (Life Technologies) and purified using an RNA Clean and Concentrator kit (Zymo).…”