Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples

Summary

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.

Abstract

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.

Introduction

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 unfeasible^2,3. Current surveillance systems based on individual clinical diagnostics rely heavily on sy....

Protocol

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 detection^33,34,35. Described here is the first reported method for the detection of SARS-CoV-2 in wastewater³⁶.

1. Collection and concentration of wastewater ....

Results

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.......

Discussion

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.......

Materials

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