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Introduction
Protocol
Results
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Environment

Kvantificering og helgenomkarakterisering af SARS-CoV-2 RNA i spildevands- og luftprøver

Published: June 30, 2023
doi:
10.3791/65053
, , , , , , , , ,
1Institute of Sustainable Processes,University of Valladolid, 2Department of Chemical Engineering and Environmental Technology,University of Valladolid, 3Department of Public Health Microbiology,National Laboratory of Health, Environment and Food, 4ICBAS – School of Medicine and Biomedical Sciences,Porto University, 5Epidemiology Research Unit (EPIUnit),Instituto de Saúde Pública da Universidade do Porto, 6Laboratório para a Investigação Integrativa e Translacional em Saúde Populacional (ITR)

Summary

Denne protokol har til formål at kvantificere SARS-CoV-2 RNA i spildevands- og luftprøver, der skal anvendes til spildevandsbaserede epidemiologiske undersøgelser, og at vurdere eksponeringsrisikoen for SARS-CoV-2 i indendørs og udendørs aerosoler. Denne protokol beskriver også en flisebelagt amplicon langskabelonsekventeringstilgang til SARS-CoV-2 helgenomkarakterisering.

Abstract

Spildevandsbaseret epidemiologi har vist sig som et lovende og effektivt overvågningssystem for SARS-CoV-2 og andre smitsomme sygdomme i mange nationer. Processen involverer typisk spildevandskoncentration, nukleinsyreekstraktion, amplifikation af udvalgte genomiske segmenter og påvisning og kvantificering af det amplificerede genomsegment. Denne metode kan ligeledes udnyttes til at detektere og kvantificere infektiøse agenser, såsom SARS-CoV-2, i luftprøver. Oprindeligt blev SARS-CoV-2 formodet at sprede sig primært gennem tæt personlig kontakt med dråber genereret af et inficeret individ, mens han talte, nyser, hoster, synger eller trækker vejret. Imidlertid har et stigende antal undersøgelser rapporteret tilstedeværelsen af SARS-CoV-2 RNA i luften i sundhedsfaciliteter, hvilket etablerer luftbåren transmission som en levedygtig rute for virussen. Denne undersøgelse præsenterer en sammensætning af etablerede protokoller for at lette miljødetektion, kvantificering og sekventering af vira fra både spildevands- og luftprøver.

Introduction

I december 2019 opstod en ny sygdom kaldet COVID-19 forårsaget af et tidligere ukendt coronavirus, SARS-CoV-21. Den deraf følgende globale pandemi har udgjort en betydelig udfordring for kliniske laboratorier og folkesundhedslaboratorier verden over, da et stort antal individer kræver test for nøjagtigt at vurdere virusoverførsel og prævalens i samfundet. I mange regioner er det imidlertid ikke økonomisk muligt at opnå det nødvendige testniveau rettidigt og rumligt omfattende 2,3. De nuværende overvågningssystemer baseret på individuel klinisk diagnostik er stærkt afhængige af symptomernes sværhedsgrad og individuel rapportering, samt i hvilket omfang disse symptomer overlapper eksisterende sygdomme, der cirkulerer i befolkningen 4,5,6,7,8,9,10. Derfor bidrager et stort antal asymptomatiske tilfælde til en signifikant undervurdering af sygdomsbyrden 7,11.

På grund af disse udfordringer blev spildevandsbaseret epidemiologi (WBE) til COVID-19-overvågning foreslået som en supplerende overvågningsstrategi. WBE blev først beskrevet i 200112 og blev oprindeligt brugt til at spore kokain og andre ulovlige stoffer13. Denne tilgang bygger på den antagelse, at det er muligt at beregne den oprindelige koncentration af ethvert stof, der er stabilt i spildevand og udskilles af mennesker 8,12. WBE er med succes implementeret i mange lande som et komplementært og effektivt overvågningssystem for SARS-CoV-2 3,8,14,15,16. De fleste metoder til påvisning af humane vira i vandmiljøer følger disse trin: koncentration, nukleinsyreekstraktion, amplifikation af det eller de valgte genomsegmenter og påvisning/kvantificering af det amplificerede genomsegment3.

Et andet vigtigt miljø til påvisning og kvantificering af SARS-CoV-2 er i luftprøver. Oprindeligt blev SARS-CoV-2 antaget at blive overført hovedsageligt gennem tæt personlig kontakt med åndedrætsdråber fra aerosoler genereret af en inficeret person, mens han talte, nyser, hoster, synger eller trækker vejret17. Imidlertid begyndte flere undersøgelser at rapportere tilstedeværelsen af SARS-CoV-2 RNA i luften, især i sundhedsfaciliteter og andre lukkede rum 18,19,20,21. Der er fundet bevis for SARS-CoV-2-levedygtighed i luftprøver taget indendørs på hospitaler og andre lukkede rum, når viruskoncentrationen var tilstrækkelig høj22,23,2 4. Udendørs undersøgelser har generelt ikke fundet tegn på SARS-CoV-2, undtagen i overfyldte udendørs rum 21,25,26,27,28,29. Fra nu af er luftbåren transmission af SARS-CoV-2 blevet anerkendt som en transmissionsform30,31. En nylig gennemgangsundersøgelse viser forskellene mellem udendørs, hvor risikoen for luftbåren transmission er minimal uden for overfyldte områder, og indendørs, hvor større risici kan være til stede i dårligt ventilerede miljøer, hvor stærke kilder (dvs. antal inficerede mennesker) kan være til stede. En nylig omfattende undersøgelse har fremhævet de betydelige forskelle mellem risikoen for luftbåren transmission i udendørs versus indendørs miljøer, især i overfyldte områder med dårlig ventilation. Undersøgelsen indikerer, at risikoen for luftbåren transmission er minimal i udendørs miljøer, hvor der er en større mængde luft til rådighed til fortynding og spredning af viruspartikler32. Disse resultater har vigtige konsekvenser for folkesundhedspolitikker og retningslinjer relateret til COVID-19. Ved at anerkende de betydelige forskelle i transmissionsrisici mellem indendørs og udendørs miljøer kan politikere udvikle mere effektive strategier til at afbøde spredningen af virussen og beskytte folkesundheden.

Der er en række forskellige metoder og protokoller til påvisning, kvantificering og sekventering af SARS-CoV-2 fra forskellige miljøprøver. Denne metodeartikel har til formål at præsentere en kombination af veletablerede protokoller, der gør det muligt for laboratorier med forskellige kapacitetsniveauer at udføre miljødetektion, kvantificering og sekventering af vira fra spildevands- og luftprøver.

Protocol

Alle metoder beskrevet her er publiceret andetsteds og indeholder små modifikationer fra de oprindelige metoder. 1. Opsamling og prøvebehandling af spildevand BEMÆRK: På grund af de lave koncentrationer af SARS-CoV-2 RNA i miljøprøver er implementeringen af et koncentrationstrin afgørende for en vellykket detektion33,34,35. Her beskrives den første ra…

Representative Results

Resultaterne opsummeret i tabel 3 viser eksempler på påvisning og kvantificering af SARS-CoV-2 RNA i spildevands- og luftprøver ved hjælp af metoden beskrevet i denne artikel. Spildevandsprøver blev indsamlet fra spildevandsrensningsanlæg i Spanien og Slovenien og blev betragtet som positive, hvis Ct var mindre end 40 i mindst to af de tre replikater, med kvantificering anset for gyldig, hvis Ct havde en variation på mindre end 5%. I Spanien og Portugal blev der indsamlet indendørs og udendørs l…

Discussion

Mikrobiel og viral detektion og kvantificering ved hjælp af (RT-) qPCR-metoder har opnået udbredt accept på grund af deres bemærkelsesværdige følsomhed. Disse teknikker står imidlertid over for adskillige udfordringer, når de analyserer miljøprøver. Spildevandsprøver indeholder en overflod af hæmmende stoffer, der kan skævvride målingerne og generere vildledende resultater. For at tackle disse begrænsninger og øge præcisionen blev en kompleks protokol udtænkt, designet og implementeret. Denne protokol b…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev udført med økonomisk støtte fra regionalregeringen i Castilla y León og FEDER-programmet (projekt CLU 2017-09, UIC315 og VA266P20).

Materials

Adapter+A25+A2:D19+A2:D20+A2+A2:D19 Oxford Nanopore EXP-AMII001 Sequencing
AllPrep PowerViral DNA/RNA Kit Qiagen 28000-50 RNA extraction kit
AMPure XP Beckman Coulter A63880 PCR Purification, NGS Clean-up, PCR clean-up
ARTIC SARS-CoV-2 Amplicon Panel IDT 10011442 SARS-CoV-2 genome amplification
Blunt/TA Ligase Master Mix NEB M0367S Library preparation
CENTRICON PLUS­70 10KDA. Fisher Scientific 10296062 Concentration filters
CORIOLIS COMPACT AIR SAMPLER Bertin Technologies 083-DU001 Air sampler
Duran laboratory bottles Merck Z305200-10EA Sampling Bottles
Flow Cell (R9.4.1) Oxford Nanopore FLO-MIN106D Sequencing
General labarotory consumables (tubes, qPCR plates, etc)
Ligation Sequencing Kit Oxford Nanopore SQK-LSK109 Sequencing
LunaScript RT SuperMix Kit NEB E3010  cDNA synthesis
Mengovirus extraction control Kit Biomérieux KMG Concentration control
Nalgene General Long-Term Storage Cryogenic Tubes Thermofisher 5011-0012 Sample storage
Native Barcoding Expansion 1-12 (PCR-free Oxford Nanopore EXP-NBD104 Barcoding
NEBNext Ultra II End Repair/dA-Tailing Module NEB E7595 DNA repair
NEBNext VarSkip Short SARS-CoV-2 Primer Mixes NEB E7658 SARS-CoV-2 genome amplification
NEBNext Quick Ligation Reaction Buffer NEB B6058S Sequencing 
Phosphate buffered saline Merck P4474 Collection buffer
Phosphate-buffered saline (PBS, 1X), sterile-filtered Thermofisher J61196.AP Elution of air samples
Q5 Hot Start High-Fidelity 2X Master Mix NEB M0494S hot start DNA polymerase
Qubit RNA HS Assay Kit Thermofisher Q32852 RNA quantitation
SARS-CoV-2 RUO qPCR Primer & Probe Kit IDT 10006713 Primer-Probe mix and qPCR positive control
TaqPath 1-Step RT-qPCR Master Mix Thermofisher A15299 RT-qPCR kit
DOWNLOAD MATERIALS LIST

References

  1. Naming the Coronavirus Disease (COVID-19) and the Virus that Causes it. World Health Organization Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/naming-the-coronavirus-disease (2020)
  2. Lab Workplace Safety. Centers for Disease Control and Prevention Available from: https://www.cdc.gov/coronavirus/2019-ncov/lab/lab-safety-practices.html (2020)
  3. Gonçalves, J., et al. Centralized and decentralized wastewater-based epidemiology to infer COVID-19 transmission – A brief review. One Health. 15, 100405 (2022).
  4. Dawood, F. S., et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. The Lancet Infectious Diseases. 12 (9), 687-695 (2012).
  5. Gonçalves, J., Koritnik, T., Paragi, M. Assessment of weather and atmospheric pollution as a co-factor in the spread of SARS-CoV-2. Acta Bio-Medica: Atenei Parmensis. 92 (3), e2021094 (2021).
  6. Gonçalves, J., et al. Detection of SARS-CoV-2 RNA in hospital wastewater from a low COVID-19 disease prevalence area. The Science of The Total Environment. 755, 143226 (2021).
  7. Mizumoto, K., Kagaya, K., Zarebski, A., Chowell, G. Estimating the asymptomatic proportion of coronavirus disease 2019 (COVID-19) cases on board the Diamond Princess cruise ship, Yokohama, Japan, 2020. Eurosurveillance. 25 (10), 2000180 (2020).
  8. Polo, D., et al. Making waves: Wastewater-based epidemiology for COVID-19 – approaches and challenges for surveillance and prediction. Water Research. 186, 116404 (2020).
  9. Shmueli, G., Burkom, H. Statistical challenges facing early outbreak detection in biosurveillance. Technometrics. 52 (1), 39-51 (2010).
  10. Simonsen, L., et al. Global mortality estimates for the 2009 influenza pandemic from the GLaMOR project: A modeling study. PLoS Medicine. 10 (11), e1001558 (2013).
  11. Oran, D. P., Topol, E. J. Prevalence of asymptomatic SARS-CoV-2 infection: a narrative reivew. Annals of Internal Medicine. 173, 362-367 (2020).
  12. Daughton, C., Jones-Lepp, T. Pharmaceuticals and Personal Care Products in the Environment: Scientific and Regulatory Issues. ACS Symposium Series. , (2001).
  13. Zuccato, E., et al. Cocaine in surface waters: a new evidence-based tool to monitor community drug abuse. Environmental Health. 4, 14 (2005).
  14. Aguiar-Oliveira, M. d. e. L., et al. Wastewater-based epidemiology (WBE) and viral detection in polluted surface water: A valuable tool for COVID-19 surveillance-a brief review. International Journal of Environmental Research and Public Health. 17, 9251 (2020).
  15. García-Encina, P. A. Wastewater-based epidemiology (WBE). Water and Environment Journal. 35 (4), 1162-1163 (2021).
  16. Mao, K., Zhang, H., Pan, Y., Yang, Z. Biosensors for wastewater-based epidemiology for monitoring public health. Water Research. 191, 116787 (2021).
  17. Shereen, M. A., Khan, S., Kazmi, A., Bashir, N., Siddique, R. COVID-19 infection: Origin, transmission, and characteristics of human coronaviruses. Journal of Advanced Research. 24, 91-98 (2020).
  18. Chia, P. Y., et al. Detection of air and surface contamination by SARS-CoV-2 in hospital rooms of infected patients. Nature Communications. 11 (1), 2800 (2020).
  19. Lei, H., et al. SARS-CoV-2 environmental contamination associated with persistently infected COVID-19 patients. Influenza and Other Respiratory Viruses. 14 (6), 688-699 (2020).
  20. Razzini, K., et al. SARS-CoV-2 RNA detection in the air and on surfaces in the COVID-19 ward of a hospital in Milan, Italy. The Science of The Total Environment. 742, 140540 (2020).
  21. da Silva, P. G., Gonçalves, J., Nascimento, M. S. J., Sousa, S. I. V., Mesquita, J. R. Detection of SARS-CoV-2 in the indoor and outdoor areas of urban public transport systems of three major cities of Portugal in 2021. International Journal of Environmental Research and Public Health. 19 (10), 5955 (2022).
  22. Barbieri, P., et al. Molecular detection of SARS-CoV-2 from indoor air samples in environmental monitoring needs adequate temporal coverage and infectivity assessment. Environmental Research. 198, 111200 (2021).
  23. Lednicky, J., et al. Earliest detection to date of SARS-CoV-2 in Florida: Identification together with influenza virus on the main entry door of a university building, February 2020. PLoS One. 16 (1), 0245352 (2021).
  24. Santarpia, J. L., et al. Aerosol and surface contamination of SARS-CoV-2 observed in quarantine and isolation care. Scientific Reports. 10 (1), 12732 (2020).
  25. Chirizzi, D., et al. SARS-CoV-2 concentrations and virus-laden aerosol size distributions in outdoor air in north and south of Italy. Environment International. 146, 106255 (2021).
  26. Hadei, M., et al. Presence of SARS-CoV-2 in the air of public places and transportation. Atmospheric Pollution Research. 12 (3), 302-306 (2021).
  27. Moreno, T., et al. Tracing surface and airborne SARS-CoV-2 RNA inside public buses and subway trains. Environment International. 147, 106326 (2021).
  28. Mouchtouri, V. A., et al. Environmental contamination of SARS-CoV-2 on surfaces, air-conditioner and ventilation systems. International Journal of Hygiene and Environmental Health. 230, 113599 (2020).
  29. Setti, L., et al. Airborne transmission route of COVID-19: why 2 meters/6 feet of inter-personal distance could not be enough. International Journal of Environmental Research and Public Health. 17, 2932 (2020).
  30. SARS-CoV-2 Transmission. Centers for Disease Control and Prevention (CDC) Available from: https://www.cdc.gov/coronavirus/2019-ncov/science/science-briefs/sars-cov-2-transmission.html (2021)
  31. Coronavirus Disease (COVID-19): How is it Transmitted. World Health Organization Available from: https://www.who.int/news-room/q-a-detail/coronavirus-disease-covid-19-how-is-it-transmitted (2021)
  32. Dinoi, A., et al. A review on measurements of SARS-CoV-2 genetic material in air in outdoor and indoor environments: Implication for airborne transmission. The Science of the Total Environment. 809, 151137 (2022).
  33. Bosch, A., et al. Analytical methods for virus detection in water and food. Food Analytical Methods. 4, 4-12 (2011).
  34. Gonçalves, J., et al. Surveillance of human enteric viruses in coastal waters using concentration with methacrylate monolithic supports prior to detection by RT-qPCR. Marine Pollution Bulletin. 128, 307-317 (2018).
  35. La Rosa, G., Muscillo, M. Molecular detection of viruses in water and sewage. Viruses in Food and Water. , 97-125 (2013).
  36. Medema, G., Heijnen, L., Elsinga, G., Italiaander, R., Brouwer, A. Presence of SARS-Coronavirus-2 RNA in sewage and correlation with reported COVID-19 prevalence in the early stage of the epidemic in the Netherlands. Environmental Science & Technology Letters. 7 (7), 511-516 (2020).
  37. CDC – 2019-nCoV Real-Time RT-PCR Diagnostic Panel Fact Sheet for Healthcare Providers. Centers for Disease Control and Prevention Available from: https://stacks.cdc.gov/view/cdc/85028 (2020)
  38. Conte, M. Airborne concentrations of SARS-CoV-2 in indoor community environments in Italy. Environmental Science and Pollution Research International. 29 (10), 13905-13916 (2022).
  39. nCoV-2019 sequencing protocol v3 (LoCost). protocols.io Available from: https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye (2020)
  40. Tyson, J. R. . Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore. , (2020).
  41. . ARTIC SARS-CoV-2 Workflow Available from: https://github.com/epi2me-labs/wf-artic (2022)
  42. Li, H., et al. The sequence alignment/map format and SAMtools. Bioinformatics. 25 (16), 2078-2079 (2009).
  43. . Freyja Available from: https://github.com/andersen-lab/Freyja (2022)
  44. Li, H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 27 (21), 2987-2993 (2011).
  45. Grubaugh, N. D., et al. An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biology. 20 (1), 8 (2019).
  46. Hadfield, J., et al. Nextstrain: real-time tracking of pathogen evolution. Bioinformatics. 34 (23), 4121-4123 (2018).
  47. Aksamentov, I., Roemer, C., Hodcroft, E. B., Neher, R. A. Nextclade: clade assignment, mutation calling and quality control for viral genomes. Journal of Open Source Software. 6 (67), 3773 (2021).
  48. Markt, R., et al. Detection and stability of SARS-CoV-2 fragments in wastewater: impact of storage temperature. Pathogens. 10 (9), 1215 (2021).
  49. Kocamemi, B. A., et al. First Data-Set on SARS-CoV-2 Detection for Istanbul Wastewaters in Turkey. MedRxiv. , (2020).
  50. Randazzo, W., et al. SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area. Water Research. 181, 115942 (2020).
  51. Hoorfar, J., et al. Practical considerations in design of internal amplification controls for diagnostic PCR assays. Journal of Clinical Microbiology. 42 (5), 1863-1868 (2004).
  52. Parshionikar, S. U., Cashdollar, J., Shay Fout, G. Development of homologous viral internal controls for use in RT-PCR assays of waterborne enteric viruses. Journal of Virological Methods. 121, 39-48 (2004).
  53. Nalla, A. K. Comparative performance of SARS-CoV-2 detection assays using seven different primer-probe sets and one assay kit. Journal of Clinical Microbiology. 58 (6), 00557 (2020).
  54. Hirotsu, Y., Mochizuki, H., Omata, M. Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay. Journal of Virological Methods. 284, 113926 (2020).
  55. Ahmed, W. First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community. The Science of The Total Environment. 728, 138764 (2020).
  56. Bar-Or, I., et al. Detection of SARS-CoV-2 variants by genomic analysis of wastewater samples in Israel. The Science of the Total Environment. 789, 148002 (2021).
  57. La Rosa, G., Bonadonna, L., Lucentini, L., Kenmoe, S., Suffredini, E. Coronavirus in water environments: Occurrence, persistence and concentration methods – A scoping review. Water Research. 179, 115899 (2020).
  58. Wu, F., et al. SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases. mSystems. 5, 00614 (2020).
  59. Wurtzer, S., et al. Evaluation of lockdown effect on SARS-CoV-2 dynamics through viral genome quantification in waste water, Greater Paris, France, 5 March to 23 April 2020. European Communicable Disease Bulletin. 25 (50), 2000776 (2020).
  60. . VATar COVID-19 | Caso de Exito – Ministerio para la Transición Ecologica y el Reto Demografico Available from: https://esri.es/es-es/descubre-los-gis/casos-de-exito/administracion-/vatar-covod19-miteco-cs (2022)
  61. Nemudryi, A., et al. Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater. Cell Reports. Medicine. 1 (6), 100098 (2020).
  62. Rios, G., et al. Monitoring SARS-CoV-2 variants alterations in Nice neighborhoods by wastewater nanopore sequencing. The Lancet Regional Health. Europe. 10, 100202 (2021).
  63. Gomes da Silva, P. Environmental dissemination of SARS-CoV-2 in a University Hospital during the COVID-19 5th wave Delta variant peak in Castile-León, Spain. International Journal of Environmental Research and Public Health. 20, 1574 (2023).
  64. Gonçalves, J., et al. . Exposure assessment of SARS-CoV-2 and Nov GII/GII in aerosols generated by a municipal wastewater treatment plant. , (2022).
  65. Lednicky, J. A., et al. Isolation of SARS-CoV-2 from the air in a car driven by a COVID patient with mild illness. International Journal of Infectious Diseases. 108, 212-216 (2021).

Tags

Wastewater-based EpidemiologySARS-CoV-2COVID-19Air SamplingViral RNART-qPCRMengovirusN1 GeneN2 GeneRNA ExtractionCentrifugal UltrafiltrationComposite Wastewater SampleMolecular Biology
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Gonçalves, J., Gomes da Silva, P., Koritnik, T., Bosilj, M., Torres-Franco, A., Diaz, I., Rodriguéz, E., Marcos, E., Mesquita, J. R., García-Encina, P. Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples. J. Vis. Exp. (196), e65053, doi:10.3791/65053 (2023).
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