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Multiplexed Amplification Evaluated for Viral Hemorrhagic Fever Diagnostics

By Labmedica International staff writers
Posted on 14 Dec 2017
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Image: The Mastercycler Nexus thermal cycler (Photo courtesy of Eppendorf).
Image: The Mastercycler Nexus thermal cycler (Photo courtesy of Eppendorf).
Viral hemorrhagic fever is a severe and potentially lethal disease, characterized by fever, malaise, vomiting, mucosal and gastrointestinal bleeding, and hypotension, in which multiple organ systems are affected.

Due to modern transportation and global trade, outbreaks of viral hemorrhagic fevers have the potential to spread rapidly and affect a significant number of susceptible individuals. Therefore, urgent and robust diagnostics with an identification of the causative virus is crucial.

Scientists at the Robert Koch Institute (Berlin, Germany) have developed and evaluated a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics.

The team used an ultrahigh-multiplex panel designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform.

A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. Thermo cycling was performed in an Eppendorf Mastercycler Pro. The amplicons obtained from the virus strains were subjected to the Ion Torrent Personal Genome Machine (PGM) System for NGS analysis.

The investigators used the multiplex primer panel that comprised two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours.

The authors concluded that virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs, which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring. The study was published on November 20, 2017, in the journal Public Library of Science Neglected Tropical Diseases.

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Robert Koch Institute


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