Simultaneous Detection of Influenza A/B, Respiratory Syncytial Virus, and SARS-CoV-2 in Nasopharyngeal Swabs by One-Tube Multiplex Reverse Transcription Polymerase Chain Reaction

Abstract

The treatment and outcome of respiratory virus infections differ. SARS-CoV-2, as well as other respiratory viruses such as influenza virus (A and B) and respiratory syncytial virus (RSV), require simultaneous, cost-effective, and rapid differential detection. We used a gold standard five-target single-step RT-PCR to detect influenza viruses, RSV, and SARS-CoV-2, and this method can be extended to detect influenza virus subtypes. As a result, this five-target single-step RT-PCR method is ideal for differentiating respiratory viruses. The 5’ nuclease activity of Taq DNA polymerase is used in the real-time reverse transcription PCR assay. The Taq man fast viral 1-step enzyme is a 4× Master mix and five-target primer probe mix that detects influenza A, influenza B, SARS-CoV-2 ORF1ab, respiratory syncytial viruses A/B and actin. When compared with TaqMan TM and Invitrogen superscript TM III Platinum and the Meril Kit for SARS-CoV-2, the assay demonstrated 100% sensitivity, specificity, and amplification efficiency of 90.1% for target genes. In conclusion, our one-tube multiplex RT-PCR assay offers a rapid and reliable method for the simultaneous detection of influenza A/B, RSV, and SARS-CoV-2 from nasopharyngeal swabs. This assay has the potential to enhance diagnostic capabilities and improve public health responses during respiratory outbreaks, enabling timely interventions and informed decision making.

An overview on viral interference during SARS-CoV-2 pandemic

“…Lastly, introducing new and complete molecular panels in the clinical practice made it possible to identify the causative agents, allowing early isolation of the affected patients and preventing the onset of epidemiological outbreaks ( 82 , 92 ). On the other hand, multiplex PCR should be reserved for patients with symptoms and histories of sick contacts since their routine use for all inpatients might lead to an inappropriate management of positive cases since the FilmArray cannot quantify microorganisms ( 93 ).…”

Editorial: High-impact respiratory RNA virus diseases

“…Diagnosing RRV diseases involves various laboratory techniques, including molecular methods like polymerase chain reaction (PCR) (40)(41)(42)(43) The challenges of prevention, diagnosis, and treatment are examined, highlighting the necessity for continuous research, surveillance, and preparedness. To mitigate the effects of RRVs, it is essential to prioritize surveillance, prevention, diagnostics, and research efforts while fostering collaboration among scientists, healthcare professionals, and policymakers to enhance global preparedness for future outbreaks.…”

“…Diagnosing RRV diseases involves various laboratory techniques, including molecular methods like polymerase chain reaction (PCR) ( 40 – 43 ) ( Machado et al ), and serological tests to detect viral genetic material or antibodies (ELISA test) ( 44 – 48 ). Overall, understanding the characteristics, transmission, and diagnostic methods of RRVs is crucial for effectively combating these infections and improving global health preparedness.…”

Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay

“…Antigen tests can rapidly detect respiratory viral antigens [ 4 – 7 ], but with varying diagnostic accuracy between tests and moderate sensitivity compared to molecular tests [ 8 ]. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) tests are widely used for the detection and quantification of pathogenic viruses in clinical [ 9 , 10 ] or highly complex samples, such as wastewater samples [ 11 – 14 ]. However, several factors can lead to inaccurate and unreliable results due to the limitations of RT-qPCR technology [ 15 , 16 ], resulting in the implementation of ddPCR as an alternative approach.…”