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

Publisher: BioMed Central Country of Publication: England NLM ID: 101202210 Publication Model: Electronic Cited Medium: Internet ISSN: 1479-7364 (Electronic) Linking ISSN: 14739542 NLM ISO Abbreviation: Hum Genomics Subsets: MEDLINE

Publication: 2012- : London : BioMed Central
Original Publication: London : Henry Stewart Publications, c2003-

Wastewater*/virology ; Influenza A virus*/genetics ; Influenza A virus*/isolation & purification ; Influenza B virus*/genetics ; Influenza B virus*/isolation & purification ; Multiplex Polymerase Chain Reaction*/methods; Humans ; Sensitivity and Specificity ; Respiratory Syncytial Viruses/genetics ; Respiratory Syncytial Viruses/isolation & purification ; Reproducibility of Results ; Influenza, Human/diagnosis ; Influenza, Human/virology ; Influenza, Human/genetics ; Respiratory Syncytial Virus, Human/genetics ; Respiratory Syncytial Virus, Human/isolation & purification ; Real-Time Polymerase Chain Reaction/methods ; SARS-CoV-2/genetics ; SARS-CoV-2/isolation & purification

Background: After the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M).
Results: The assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen's correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance.
Conclusions: Overall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.
(© 2024. The Author(s).)

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Keywords: Influenza A; Influenza B; RT-ddPCR; Respiratory Syncytial Virus; Wastewater

Date Created: 20240520 Date Completed: 20240520 Latest Revision: 20240522

20240522

PMC11103825

10.1186/s40246-024-00614-8

38769549