Journal Article

Development of a non-infectious control for viral hemorrhagic fever PCR assays

Bromhead, Collette, Knox, Matthew A., Hayman, David TS

2024

Overview

Assay validation is an essential component of disease surveillance testing, but can be problematic in settings where access to positive control material is limited and a safety risk for handlers. Here we describe a single non-infectious synthetic control that can help develop and validate the PCR based detection of the viral causes of Crimean-Congo hemorrhagic fever, Ebola virus disease, Lassa fever, Marburg virus disease and Rift Valley fever. We designed non-infectious synthetic DNA oligonucleotide sequences incorporating primer binding sites suitable for five assays, and a T7 promotor site which was used to transcribe the sequence. Transcribed RNA was used as template in a dilution series, extracted and amplified with RT-PCR and RT-qPCR to demonstrate successful recovery and determine limits of detection in a range of laboratory settings. Our results show this approach is adaptable to any diagnostic assay requiring validation of nucleic acid extraction and/or amplification, particularly where sourcing reliable, safe material for positive controls is infeasible.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Access control / Acids / Amplification / Assaying / Binding sites / Biological assay / Biology and life sciences / Coccidioidomycosis / Crimean hemorrhagic fever / Diagnosis / Dilution / DNA Primers - genetics / Ebola virus / Evaluation / Feces / Fever / Gene sequencing / Haemorrhage / Handlers / Hemorrhagic fever / Hemorrhagic Fevers, Viral - diagnosis / Hemorrhagic Fevers, Viral - virology / Humans / Infectious diseases / Lassa fever / Marburg disease / Medical research / Medicine and health sciences / Medicine, Experimental / Monkeypox / Nucleic acids / Nucleotide sequence / Oligonucleotides / Pandemics / Pathogens / PCR / Polymerase chain reaction / Public health / Real-Time Polymerase Chain Reaction - methods / Research and Analysis Methods / Rift Valley fever / RNA, Viral - genetics / Sensitivity and Specificity / Sentinel health events / Temperature / Testing / Vector-borne diseases / Viral diseases / Viruses / Zoonoses