A Multiplex One-Step RT-qPCR Protocol to Detect SARS-CoV-2 in NP/OP Swabs and Saliva

Area of Science:

• Molecular Biology
• Virology
• Infectious Diseases

Background:

• The global spread of SARS-CoV-2 necessitates accurate and accessible diagnostic methods.
• Current molecular tests for SARS-CoV-2 RNA often have restrictive specimen collection requirements, limiting their application.
• Variations in specimen types (nasopharyngeal/oropharyngeal swabs, saliva) and collection media (VTM, saline) complicate laboratory validation and increase costs.

Purpose of the Study:

• To develop and validate a cost-effective multiplex reverse-transcription real-time PCR (RT-qPCR) assay for SARS-CoV-2 detection.
• To create a versatile assay capable of analyzing diverse specimen types and collection media.
• To streamline laboratory workflows for SARS-CoV-2 diagnostics.

Main Methods:

• Development and validation of a multiplex RT-qPCR assay for detecting SARS-CoV-2.
• Testing the assay's performance with nasopharyngeal/oropharyngeal swabs and saliva samples.
• Evaluation of different collection media, including viral transport medium (VTM), saline, and preservative fluid.
• Description of optimal laboratory setup and unidirectional workflow for RT-qPCR.

Main Results:

• The developed multiplex RT-qPCR assay demonstrated high sensitivity and specificity for SARS-CoV-2 detection.
• The assay successfully detected viral RNA in both saliva and nasopharyngeal/oropharyngeal swabs collected in various media.
• The single assay accommodates multiple specimen types and collection media, simplifying laboratory procedures.

Conclusions:

• The validated multiplex RT-qPCR assay offers a cost-effective and flexible solution for SARS-CoV-2 detection.
• This unified approach reduces the need for multiple test validations, saving costs and infrastructure.
• The assay supports diverse specimen types and collection media, enhancing diagnostic accessibility and efficiency.