Development of a Singleplex Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and...

Area of Science:

• Virology
• Molecular Biology
• Infectious Diseases

Background:

• Dengue virus (DENV) infection poses a significant global health challenge with no specific treatments or widely available vaccines.
• Early diagnosis is crucial for effective patient management.
• Current multiplex RT-PCR methods for DENV detection can suffer from reduced sensitivity due to primer dimer formation.

Purpose of the Study:

• To develop a sensitive and specific one-tube, singleplex real-time RT-PCR assay for the detection and quantification of all four DENV serotypes.
• To evaluate the assay's performance in clinical specimens and compare it with existing methods.
• To investigate the presence of defective virus particles in DENV-infected patients.

Main Methods:

• Development of a singleplex real-time RT-PCR targeting the DENV 3'-UTR.
• Testing the assay on clinical specimens collected from hospitalized patients.
• Comparison of assay sensitivity, specificity, and quantification efficiency with established RT-PCR assays targeting coding sequences.
• Utilizing dual-region targeting RT-PCR to enhance viral genome detection.

Main Results:

• The developed singleplex RT-PCR assay demonstrated high sensitivity (96.9%) in detecting DENV in clinical specimens from the first day of hospitalization.
• The assay showed no cross-reactivity with other flaviviruses and provided equivalent PCR efficiency and RNA quantification across all DENV serotypes.
• Performance was comparable to established real-time RT-PCR assays targeting coding sequences.
• The study identified the presence of defective virus particles in patients infected with all DENV serotypes, particularly evident during the late acute phase.

Conclusions:

• A novel singleplex real-time RT-PCR assay targeting the DENV 3'-UTR provides a sensitive and specific tool for pan-DENV detection and quantification.
• This assay improves upon multiplex RT-PCR limitations, offering high diagnostic accuracy early in infection.
• The findings highlight the presence of defective DENV particles, suggesting implications for viral pathogenesis and disease monitoring.