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Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

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Sequence optimized diagnostic assay for Ebola virus detection.

Jeffrey W Koehler¹, Christopher P Stefan¹, Adrienne T Hall¹

¹Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, MD, 20102, USA.

Scientific Reports

|November 2, 2023

Summary

This summary is machine-generated.

Sequence mismatches in Ebola virus (EBOV) real-time PCR assays significantly reduce sensitivity. Redesigning assays to accommodate EBOV genetic diversity improves detection of variants and enhances diagnostic accuracy.

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High-throughput Detection Method for Influenza Virus

High-throughput Detection Method for Influenza Virus

Published on: February 4, 2012

Area of Science:

Molecular biology
Virology
Infectious disease diagnostics

Background:

Rapid pathogen identification is crucial for outbreak control and patient management.
Real-time PCR offers high sensitivity and specificity for infectious disease diagnosis.
Primer/probe sequence mismatches can compromise assay performance, leading to false negatives.

Purpose of the Study:

To assess the impact of sequence mismatches on Ebola virus (EBOV) real-time PCR assay performance.
To evaluate the effectiveness of redesigned EBOV assays in detecting diverse viral variants.

Main Methods:

Analysis of sequence variants in EBOV Makona from the 2013-2016 outbreak.
In silico assessment of primer/probe mismatch impact on assay sensitivity.
Redesign and validation of EBOV real-time PCR assays.
Comparative performance testing of original and redesigned assays on human EBOV samples.

Main Results:

One or two primer/probe mismatches caused sensitivity reductions from minimal to nearly two logs.
Redesigned EBOV assays demonstrated improved detection across tested variants.
New assays showed increased sensitivity, evidenced by lower Cq values and detection of previously missed positive samples.

Conclusions:

EBOV genetic diversity can significantly impair real-time PCR diagnostic assay sensitivity.
Assay redesign to account for sequence variants is essential for accurate EBOV detection.
Improved EBOV assays enhance diagnostic capabilities for outbreak response and patient care.