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

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          Abstract

          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.

          Supplementary Information

          The online version contains supplementary material available at 10.1186/s40246-024-00614-8.

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          Most cited references37

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          PCR inhibitors - occurrence, properties and removal.

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          The polymerase chain reaction (PCR) is increasingly used as the standard method for detection and characterization of microorganisms and genetic markers in a variety of sample types. However, the method is prone to inhibiting substances, which may be present in the analysed sample and which may affect the sensitivity of the assay or even lead to false-negative results. The PCR inhibitors represent a diverse group of substances with different properties and mechanisms of action. Some of them are predominantly found in specific types of samples thus necessitating matrix-specific protocols for preparation of nucleic acids before PCR. A variety of protocols have been developed to remove the PCR inhibitors. This review focuses on the general properties of PCR inhibitors and their occurrence in specific matrices. Strategies for their removal from the sample and for quality control by assessing their influence on the individual PCR test are presented and discussed. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.
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            The effects of the COVID-19 pandemic on community respiratory virus activity

            Eric Chow, Timothy Uyeki, Helen Chu (2022)
            The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused substantial global morbidity and deaths, leading governments to turn to non-pharmaceutical interventions to slow down the spread of infection and lessen the burden on health care systems. These policies have evolved over the course of the COVID-19 pandemic, including after the availability of COVID-19 vaccines, with regional and country-level differences in their ongoing use. The COVID-19 pandemic has been associated with changes in respiratory virus infections worldwide, which have differed between virus types. Reductions in respiratory virus infections, including by influenza virus and respiratory syncytial virus, were most notable at the onset of the COVID-19 pandemic and continued in varying degrees through subsequent waves of SARS-CoV-2 infections. The decreases in community infection burden have resulted in reduced hospitalizations and deaths associated with non-SARS-CoV-2 respiratory infections. Respiratory virus evolution relies on the maintaining of a diverse genetic pool, but evidence of genetic bottlenecking brought on by case reduction during the COVID-19 pandemic has resulted in reduced genetic diversity of some respiratory viruses, including influenza virus. By describing the differences in these changes between viral species across different geographies over the course of the COVID-19 pandemic, we may better understand the complex factors involved in community co-circulation of respiratory viruses. The COVID-19 pandemic has had a considerable impact on respiratory virus infections worldwide. In this Review, Chu and colleagues discuss the changes in community spread and consequent infections by respiratory viruses other than severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) throughout the course of the pandemic, and describe the impact on the evolution and genetic diversity of these viruses.
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              Diagnostic Accuracy of Novel and Traditional Rapid Tests for Influenza Infection Compared With Reverse Transcriptase Polymerase Chain Reaction: A Systematic Review and Meta-analysis.

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                Author and article information

                Contributors
                Athina Markou: atmarkou@chem.uoa.gr
                Journal
                Journal ID (nlm-ta): Hum Genomics
                Journal ID (iso-abbrev): Hum Genomics
                Title: Human Genomics
                Publisher: BioMed Central (London )
                ISSN (Print): 1473-9542
                ISSN (Electronic): 1479-7364
                Publication date (Electronic): 20 May 2024
                Publication date PMC-release: 20 May 2024
                Publication date Collection: 2024
                Volume: 18
                Electronic Location Identifier: 48
                Affiliations
                Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, ( https://ror.org/04gnjpq42) 15771 ZografouAthens, Greece
                Article
                Publisher ID: 614
                DOI: 10.1186/s40246-024-00614-8
                PMC ID: 11103825
                PubMed ID: 38769549
                SO-VID: fe287671-6620-4e80-ad59-7c64615c8cc9
                Copyright © © The Author(s) 2024
                License:

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                Date received : 29 December 2023
                Date accepted : 1 May 2024
                Categories
                Subject: Research
                Custom metadata
                issue-copyright-statement © BioMed Central Ltd., part of Springer Nature 2024

                ScienceOpen disciplines: Genetics
                Keywords: influenza a,influenza b,respiratory syncytial virus,wastewater,rt-ddpcr
                Data availability:
                ScienceOpen disciplines: Genetics
                Keywords: influenza a, influenza b, respiratory syncytial virus, wastewater, rt-ddpcr

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