[PDF] Development of a non-infectious control for viral hemorrhagic fever PCR assays-论文阅读讨论-ReadPaper

摘要

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.

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