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Molecular Assay Rapidly Detects Hemorrhagic Fever Virus

By Labmedica International staff writers
Posted on 08 Nov 2017
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Image: The Applied Biosystems 7500 Real-Time PCR systems offer high performance, multicolor real-time polymerase chain reactions (Photo courtesy of Thermo Fisher Scientific).
Image: The Applied Biosystems 7500 Real-Time PCR systems offer high performance, multicolor real-time polymerase chain reactions (Photo courtesy of Thermo Fisher Scientific).
Crimean-Congo Hemorrhagic fever Virus (CCHFV) is a rapidly emerging vector-borne pathogen and the cause of a virulent hemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia.

Crimean–Congo hemorrhagic fever (CCHF) is a viral disease and symptoms may include fever, muscle pains, headache, vomiting, diarrhea, and bleeding into the skin. Onset of symptoms is less than two weeks following exposure to a tick-bite and complications may include liver failure. In those who survive, recovery generally occurs around two weeks after onset.

A collaborating team of international scientists led by those at the UK National Infection Service, (Public Health England, Porton Down, UK) developed an isothermal recombinase polymerase amplification (RPA) assay for molecular detection of CCHFV. A collection of CCHFV strains representing each of the following S-segment clades: Asia 1, Asia 2, Africa 2, Africa 3 and Europe 1 were cultured and viral RNA was extracted using a standard RNA extraction kit.

The CCHF RPA assay was performed in a 50μL volume using a TwistAmp Exo-RT kit. The products of the RPA were purified using a a PCR purification kit, then run on a 2% Agarose gel. The CCHF real-time polymerase chain reaction (RT-PCR) assay was performed in a 20μL volume using a Superscript III Platinum One-step quantitative RT-PCR kit and run on an ABI 7500 real-time PCR system.

The investigators reported that the assay showed rapid, detection of viral extracts/synthetic virus RNA of all seven S-segment clades of CCHFV, with high target specificity, in less than 10 minutes. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan.

The authors concluded that the RPA assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries. The study was published on October 13, 2017, in the journal Public Library of Science Neglected Tropical Diseases.

Related Links:
UK National Infection Service


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