Performance Evaluation of TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR Multiplex Assay for the Detection of Respiratory Viruses

Abstract

Purpose

To detect and differentiate co-infection with influenza and respiratory syncytial virus during the COVID pandemic, a rapid method that can detect multiple pathogens in a single test is a significant diagnostic advance to analyze the outcomes and clinical implications of co-infection. Therefore, we validated and evaluated the performance characteristics of TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR multiplex assay for the detection of SARS-CoV-2, Flu A/B, and RSV using nasopharyngeal and saliva samples.

Materials and Methods

The method validation was performed by using culture fluids of Influenza A virus (H3N2) (A/Wisconsin/67/2005), Influenza B virus (B/Virginia/ATCC4/2009), RSV A2 cpts-248, SARS-CoV-2 (USA-WA1/2020) and quantitative RNA controls of Influenza A virus (H1N1) strain A/PR/8/34 (VR-95DQ), RSV A2 (VR-1540DQ) and SARS-CoV-2 (MN908947.3 Wuhan-Hu-1) from ATCC and Zeptometrix, NY, USA. A total of 110 nasopharyngeal specimens and 70 saliva samples were used for the SARS-CoV-2 detection, and a total of 70 nasopharyngeal specimens were used for Influenza and RSV detection. Total RNA was extracted from all the samples and multiplex PCR was performed using TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR multiplex assay. The assay was used for SARS-CoV-2 variant (B.1.1.7_601443, B.1.617.1_1662307, P.1_792683, B.1.351_678597, B.1.1.529/BA.1).

Results

Validation controls showed accurate and precise results. The correlation study found the accuracy of 96.38 to 100% (95% CI) in nasopharyngeal and 94.87 to 100% (95% CI) in saliva for SARS-CoV-2 and 91.1 to 100% (95% CI) for both Influenza A/B and RSV. The diagnostic efficiency of this assay was not affected by SARS-CoV-2 variant, including Omicron.

Conclusion

The TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR multiplex assay is a rapid method to detect and differentiate SAR-CoV-2, Flu A and B, and RSV in nasopharyngeal and saliva samples. It has a significant role in the diagnosis and management of respiratory illnesses and the clinical implications of co-infection.

Keywords:

• TaqMan
• SARS-CoV-2
• Flu A/B
• respiratory syncytial virus
• RSV
• multiplex

Acknowledgment

We are grateful to the laboratory staff and management of Patients Choice Laboratories for their immense support during the study. We acknowledge Mr Tyler McKee for checking the English in this manuscript.

Disclosure

The authors report no conflicts of interest in this work.