Sequence optimized diagnostic assay for Ebola virus detection

Author:

Koehler Jeffrey W.,Stefan Christopher P.,Hall Adrienne T.,Delp Korey L.,O’Hearn Aileen E.,Taylor-Howell Cheryl L.,Wauquier Nadia,Schoepp Randal J.,Minogue Timothy D.

Abstract

AbstractRapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013–2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.

Funder

Armed Forces Health Surveillance Branch

Defense Threat Reduction Agency

Publisher

Springer Science and Business Media LLC

Subject

Multidisciplinary

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