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

Abstract Assay validation is an essential component of disease surveillance but can be problematic in low resource settings where access to positive control material is limited and a safety risk for handlers. Here we describe techniques for validating the PCR based detection of Crimean-Congo hemorrhagic fever orthonairovirus , Ebola virus, Lassa virus, Marburg virus and Rift Valley Fever phlebovirus . We designed non-infectious synthetic DNA oligonucleotide sequences incorporating primer binding sites suitable for multiple 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 are adaptable to any assay requiring validation of nucleic acid extraction and/or amplification, particularly where sourcing reliable, safe material for positive controls is infeasible. Author Summary The majority of zoonoses originate in wildlife and tend to emerge from biodiverse regions in low to middle income countries, frequently among deprived populations of at-risk people with a lack of access to diagnostic capacity or surveillance. Diseases such as Crimean-Congo Hemorrhagic Fever, Rift Valley Fever, Ebola Virus Disease, Marburg Virus Disease and Lassa Fever are viral hemorrhagic fevers (VHFs) and among the most neglected and serious threats to global public health. This threat is partly due to the severity of disease caused by these pathogens, but also because their geographical distribution is close to human populations with often limited access to medical or diagnostic laboratory services. In our study we describe and validate techniques for PCR based detection of five VHF viruses using a synthetic, multi-target non-infectious positive control. Our work has applications in assay design and optimization, particularly where access to source material is problematic or requires high level biosafety containment, as is the case with VHF viruses. This approach can help learners train in techniques used in nucleic acid extraction, amplification, and sequencing of VHF viruses, but can be used for any targets, with potential for multiplexing from a single positive control.