Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV
November 10th, 2023
We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.
The overall goal of this assay is to administrate a multiplex approach for the simultaneous detection of commonly circulating respiratory viruses such as SARS-CoV-2, MERS, influenza A, and influenza B.The prevailing technologies driving advancements in our field include CRISPR, lateral flow amino assays, paper-based biomolecular sensors, Sherlock testing in one pot, DNA aptamers, and multiplex RT-qPCR integrated with loop-mediated isothermal amplification, LAMP. However, multiplex RT-qPCR is the most sensitive, common, and is the gold standard for diagnosing various viruses. The current experimental challenge is to establish a protocol that can be used to detect RNAs copies as low as 10 copies.
We used in-house RT-qPCR kit for the assay, which is time and cost effective. To begin, prepare the 2x buffer for RT-qPCR in DNAs/RNAs-free water Store the buffer at minus 20 degrees Celsius until further use. Next, mix the primers and the probes in a 1.5 milliliter tube.
Make up the volume with elution buffer, then store the tube at minus 20 degrees Celsius. Prepare an enzyme mix with taq polymerase and MMLV-RT in a 1.5 milliliter tube on ice. Make up the volume with taq polymerase storage buffer.
Now resuspend the viral synthetic RNAs in separate tubes with 100 microliters of Tris-EDTA buffer to make stocks of 10 to the sixth RNA copies per microliter. To mix the virus stocks add five microliters Each of SARS-CoV-2, influenza A and B to 50 microliters of Tris-EDTA buffer. Similarly mix SARS-CoV-2 and MERS-CoV to obtain a second viral mix.
Dilute both mixtures tenfold to obtain a final dilution of 10 RNA copies per microliter. To each well of a 96 well plate, pipette 2x buffer, primer, enzyme, DNAs/RNAs-free water, and the corresponding RNA mixtures. Seal the plate with an adhesive plate seal.
Then centrifuge the plate for one minute to collect the liquid at the well bottom. Next program the PCR machine to accept the fast 96 well block type with the experimental type set to standard curve. Set the reagents to TaqMan reagents and select standard run properties.
Define the gene targets and the reporter dye. Then define the sample names for each reaction to be tested and assign targets and samples to the plate layout. Now input the cycle program into the instrument.
Transfer the plate to the real-time QPCR machine in the correct orientation and press run to initiate the cycle. Choose the file location to save the experimental data. Then examine the amplification plots provided by the QPCR program.
The two developed kits were able to successfully amplify all three target genes simultaneously, with as low as 10 RNA copies per reaction. The amplification efficiencies for all viral titrations were above 99%
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Overview
This study presents the development of two probe-based one-step RT-qPCR kits designed for the simultaneous detection of common respiratory viruses, including SARS-CoV-2, MERS, and various strains of Influenza. These kits enhance the diagnostic capabilities of specialized laboratories through a cost-effective and time-efficient approach.
Key Study Components
Research Area
- Respiratory virus detection
- RT-qPCR technique
- Multiplex assay development
Background
- The necessity for rapid and sensitive diagnosis of respiratory viruses.
- The limitations of current detection technologies.
- The importance of a multiplex approach for simultaneous detection.
Methods Used
- In-house developed RT-qPCR kits
- Detection of viral RNA
- Multiplex RT-qPCR combined with specific probes
Main Results
- Successfully detected SARS-CoV-2 and MERS alongside Influenza A and B.
- Achieved amplification at RNA copies as low as 10 per reaction.
- Demonstrated high amplification efficiencies above 99% for target genes.
Conclusions
- The developed RT-qPCR kits are effective for multiplex detection of respiratory viruses.
- This study contributes to better diagnostic strategies in virology research.
Frequently Asked Questions
Executive Industry Relevance
Multiplex real-time RT-qPCR assays for SARS-CoV-2, Influenza A/B, and MERS-CoV address the critical need for rapid, sensitive, and specific detection of co-circulating respiratory viruses in discovery and translational research. These assays enable high-confidence differentiation of viral targets, supporting robust target validation and reducing ambiguity in early-stage diagnostic development. Their integration enhances predictive confidence and operational efficiency across R&D portfolios focused on infectious disease threats.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables precise interrogation of viral targets for functional validation in respiratory disease models.
- Supports biological de-risking by distinguishing closely related viral pathogens in complex samples.
- Facilitates predictive confidence in target selection and triage for diagnostic and therapeutic programs.
Screening & Assay Development
- Prepares validated multiplex assay systems for downstream screening of antiviral compounds or diagnostics.
- Delivers standardized, reproducible, and quantitative outputs for assay benchmarking and optimization.
- Enables scalable, high-throughput screening readiness for rapid evaluation of candidate interventions.
Translational & Preclinical Research
- Aligns with translational biomarker strategies by enabling simultaneous detection of multiple respiratory viruses.
- Supports continuity from discovery through preclinical validation by providing robust, quantitative viral detection.
- Reduces risk in advancing candidates by ensuring accurate pathogen identification in disease-relevant systems.
Pipeline & Workflow Integration
Multiplex RT-qPCR assays fit within the discovery-to-preclinical continuum, supporting early hypothesis testing, assay development, and translational research for infectious disease portfolios.
- Discovery Biology: Provides high-sensitivity detection for hypothesis-driven viral target validation.
- Screening: Offers reproducible, quantitative outputs for assay standardization and compound evaluation.
- Analytics: Delivers robust amplification data and specificity metrics for comparative analysis.
- Translational Research: Enables biomarker alignment and continuity across research phases.
- Enterprise Reuse: Establishes a reusable, adaptable platform for multiplex viral detection in diverse R&D settings.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in viral detection.
- Operational Value: Enhances standardization, reproducibility, and scalability of diagnostic workflows.
- Strategic Value: Improves go/no-go decision-making and capital efficiency by enabling early, accurate differentiation of pathogens.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of infectious disease programs.
Implementation Considerations
- Requires expertise in molecular virology and quantitative PCR assay design.
- Needs access to real-time PCR instrumentation and analytical software infrastructure.
- Demands cross-team standardization for assay setup, data interpretation, and quality control.
- May require adaptation for different sample types or emerging viral variants.
- Performance is contingent on primer/probe specificity and RNA quality as supported by the source.
