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This protocol aims to quantify SARS-CoV-2 RNA in wastewater and air samples to be used for wastewater-based epidemiology studies and to assess the exposure risk to SARS-CoV-2 in indoor and outdoor aerosols. This protocol also describes a tiled amplicon long-template sequencing approach for SARS-CoV-2 whole genome characterization.
Wastewater-based epidemiology has emerged as a promising and efficacious surveillance system for SARS-CoV-2 and other infectious diseases in many nations. The process typically involves wastewater concentration, nucleic acid extraction, amplification of selected genomic segments, and detection and quantification of the amplified genomic segment. This methodology can similarly be leveraged to detect and quantify infectious agents, such as SARS-CoV-2, in air samples. Initially, SARS-CoV-2 was presumed to spread primarily through close personal contact with droplets generated by an infected individual while speaking, sneezing, coughing, singing, or breathing. However, a growing number of studies have reported the presence of SARS-CoV-2 RNA in the air of healthcare facilities, establishing airborne transmission as a viable route for the virus. This study presents a composite of established protocols to facilitate environmental detection, quantification, and sequencing of viruses from both wastewater and air samples.
In December 2019, a novel disease called COVID-19 emerged, caused by a previously unknown coronavirus, SARS-CoV-21. The resulting global pandemic has presented a significant challenge to clinical and public health laboratories worldwide, as a large number of individuals require testing to accurately assess virus transmission and prevalence in the community. However, in many regions, achieving the necessary level of testing in a timely and spatially comprehensive manner is economically unfeasible2,3. Current surveillance systems based on individual clinical diagnostics rely heavily on symptom severity and individual reporting, as well as the extent to which these symptoms overlap with existing diseases circulating in the population4,5,6,7,8,9,10. Consequently, a high number of asymptomatic cases contributes to a significant underestimation of disease burden7,11.
Due to these challenges, wastewater-based epidemiology (WBE) for COVID-19 surveillance was proposed as a complementary surveillance strategy. WBE was first described in 200112, and was initially used to trace cocaine and other illegal drugs13. This approach relies on the assumption that it is possible to calculate the initial concentration of any substance that is stable in wastewater and excreted by humans8,12. WBE has been successfully implemented in many countries as a complementary and efficient surveillance system for SARS-CoV-23,8,14,15,16. The majority of methods to detect human viruses in aquatic environments follow these steps: concentration, nucleic acid extraction, amplification of the genomic segment (or segments) chosen, and detection/quantification of the amplified genomic segment3.
Another important environment for the detection and quantification of SARS-CoV-2 is in air samples. Initially, SARS-CoV-2 was thought to be transmitted mainly through close personal contact with respiratory droplets from aerosols generated by an infected person while speaking, sneezing, coughing, singing, or breathing17. However, several studies began to report the presence of SARS-CoV-2 RNA in the air, especially in healthcare facilities and other enclosed spaces18,19,20,21. Evidence of SARS-CoV-2 viability in air samples taken indoors in hospitals and other enclosed spaces has been found when the virus concentration was sufficiently high22,23,24. Outdoor studies have generally found no evidence of SARS-CoV-2, except in crowded outdoor spaces21,25,26,27,28,29. As of now, airborne transmission of SARS-CoV-2 has been recognized as a mode of transmission30,31. A recent review study shows the differences between outdoors, where risks of airborne transmission are minimal outside of crowded areas, and indoors, where larger risks could be present in poorly ventilated environments in which strong sources (i.e., number of infected people) could be present. A recent comprehensive review study has highlighted the substantial differences between the risks of airborne transmission in outdoor versus indoor environments, particularly in crowded areas with poor ventilation. The study indicates that the risk of airborne transmission is minimal in outdoor environments, where there is a larger volume of air available for the dilution and dispersion of virus particles32. These findings have important implications for public health policies and guidelines related to COVID-19. By recognizing the significant differences in transmission risks between indoor and outdoor environments, policymakers can develop more effective strategies to mitigate the spread of the virus and protect public health.
There are a variety of methods and protocols for the detection, quantification, and sequencing of SARS-CoV-2 from different environmental samples. This method article aims to present a combination of well-established protocols that allow laboratories with different capacity levels to perform environmental detection, quantification, and sequencing of viruses from wastewater and air samples.
All methods described here have been published elsewhere and contain small modifications from the original methods.
1. Wastewater collection and sample pre-processing
NOTE: Due to the low concentrations of SARS-CoV-2 RNA in environmental samples, the implementation of a concentration step is crucial for a successful detection33,34,35. Described here is the first reported method for the detection of SARS-CoV-2 in wastewater36.
2. Quantification of SARS-CoV-2 RNA by real-time-quantitative polymerase chain reaction (RT-qPCR)
NOTE: The below protocol is according to the CDC 2019-Novel Coronavirus (2019-nCoV) RT-PCR diagnostic panel37. Divide the primer/probe mix into several aliquots to avoid freezing and thawing cycles.
3. Sequencing variants in wastewater and data analysis
NOTE: The described protocol is a modified protocol created by Quick et al.39,40. It uses two sets of primers for SARS-CoV-2 genome amplification by PCR tiling methodology-ARTIC primers and VarSkip primers. A combination of primers is used to guarantee the best genome coverage and to minimize the possibility of novel mutations causing primers of one type to fail. In general, the protocol is divided into three parts: reverse transcription (RT) and amplicon generation, sequencing library preparation, and sequencing and data analysis.
The results summarized in Table 3 show examples of the detection and quantification of SARS-CoV-2 RNA in wastewater and air samples using the method described in this article. Wastewater samples were collected from wastewater treatment plants in Spain and Slovenia and were considered positive if the Ct was less than 40 in at least two of the three replicates, with quantification considered valid if the Ct had a variation of less than 5%. In Spain and Portugal, indoor and outdoor air samples were collecte...
Microbial and viral detection and quantification using (RT-)qPCR methods have garnered widespread acceptance due to their remarkable sensitivity. However, these techniques face numerous challenges when analyzing environmental samples. Wastewater samples contain an abundance of inhibitory substances that can skew measurements and generate misleading results. To tackle these limitations and enhance precision, a complex protocol was conceived, designed, and implemented. This protocol was tailored by combining protocols from...
The authors have no competing economic interests or other conflicts of interest.
This work was performed with financial support from the Regional Government of Castilla y Leon and the FEDER program (projects CLU 2017-09, UIC315 and VA266P20).
Name | Company | Catalog Number | Comments |
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 |
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