Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay

Area of Science:

• Virology
• Molecular Biology
• Infectious Diseases

Background:

• The 2014 United States enterovirus D68 (EV-D68) outbreak caused severe respiratory illness.
• Accurate and rapid detection of EV-D68 is crucial for public health surveillance.

Purpose of the Study:

• To develop and evaluate a specific real-time reverse transcriptase PCR (RT-PCR) assay for EV-D68 detection.
• To create a tool for identifying and studying EV-D68 outbreaks.

Main Methods:

• Sequencing of the circulating EV-D68 genome.
• Computational selection of a target region within the VP1 gene.
• Development and analytical validation of a strain-specific RT-PCR assay.

Main Results:

• The developed RT-PCR assay demonstrated high sensitivity with a limit of detection of 4 copies per reaction.
• The assay specifically detected EV-D68, including divergent strains, and did not cross-react with other enteroviruses or rhinoviruses.
• The assay was more sensitive than existing commercial assays and FDA-approved multiplex respiratory panels.

Conclusions:

• The developed real-time RT-PCR assay is a sensitive and specific tool for EV-D68 detection.
• This assay can effectively identify EV-D68 in clinical specimens, aiding in outbreak investigation.
• The assay is valuable for monitoring current and future EV-D68 activity.