Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR

Farkas, Kata; Hillary, Luke S.; Thorpe, Jamie; Walker, Duard L.; Lowther, James; McDonald, James E.; Malham, Shelagh K.; Jones, Davey L.

doi:10.3390/mps4010017

Cited by 54 publications

(49 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This knowledge was exploited for the design of novel in-house methods that combining random hexamers-based reverse transcription and nested PCR/real-time PCR amplification against four highly stable regions of SARS-CoV-2 RNA. The evaluation of our novel assays highlighted the improved LOD (up to two copies/PCR reaction) as compared with one-step RT-PCR methods [19,20], and the significantly improved sensitivity as compared with in-house CDC/2019-nCoV_N1-based assay. Interestingly, more than half of the positive samples were detected by using only one assay, highlighting the anticipated on-going degradation of SARS-CoV-2 RNA in wastewater and clearly demonstrates that SARS-CoV-2 RNA detection in wastewater is genomic region dependent.…”

Section: Discussionmentioning

confidence: 97%

Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater

Avgeris

Adamopoulos

Galani

et al. 2021

IJMS

View full text Add to dashboard Cite

Considering the lack of effective treatments against COVID-19, wastewater-based epidemiology (WBE) is emerging as a cost-effective approach for real-time population-wide SARS-CoV-2 monitoring. Here, we report novel molecular assays for sensitive detection and mutational/variant analysis of SARS-CoV-2 in wastewater. Highly stable regions of SARS-CoV-2 RNA were identified by RNA stability analysis and targeted for the development of novel nested PCR assays. Targeted DNA sequencing (DNA-seq) was applied for the analysis and quantification of SARS-CoV-2 mutations/variants, following hexamers-based reverse transcription and nested PCR-based amplification of targeted regions. Three-dimensional (3D) structure models were generated to examine the predicted structural modification caused by genomic variants. WBE of SARS-CoV-2 revealed to be assay dependent, and significantly improved sensitivity achieved by assay combination (94%) vs. single-assay screening (30%–60%). Targeted DNA-seq allowed the quantification of SARS-CoV-2 mutations/variants in wastewater, which agreed with COVID-19 patients’ sequencing data. A mutational analysis indicated the prevalence of D614G (S) and P323L (RdRP) variants, as well as of the Β.1.1.7/alpha variant of concern, in agreement with the frequency of Β.1.1.7/alpha variant in clinical samples of the same period of the third pandemic wave at the national level. Our assays provide an innovative cost-effective platform for real-time monitoring and early-identification of SARS-CoV-2 variants at community/population levels.

show abstract

Section: Discussionmentioning

confidence: 97%

Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater

Avgeris

Adamopoulos

Galani

et al. 2021

IJMS

View full text Add to dashboard Cite

show abstract

“…Peer-reviewed studies have compared PEG precipitation with other concentration methods for the recovery of a range of viruses and relevant surrogates (36)(37)(38)(39)(40). PEG precipitation (10%) has been preferred by a number of studies for providing the highest recovery of viruses and surrogates, and for its simple approach (i.e., low-cost equipment, accessible, no pre-treatment steps) (41)(42)(43)(44)(45)(46). Many of these recent findings are aligned with the results of this study, and Lu et al (2020) found that PEG methods were among the most common due to reliability, broad applicability, and accessibility (45) with that of non-enveloped MS2 by RT-qPCR and found that MS2 showed differences in recovery and was not a suitable indicator to validate the extraction and recovery of enveloped viruses (42).…”

Section: Discussionmentioning

confidence: 99%

Polyethylene glycol (PEG) methods are superior to acidification for secondary concentration of Adenovirus and MS2 in water

McLellan

Weir

Lee

et al. 2021

Preprint

View full text Add to dashboard Cite

Enteric viruses are a leading cause of waterborne illness worldwide and surveillance studies lack standardization in method selection. The most common and cost-effective approach to concentrating viruses from water samples involves virus adsorption and elution (VIRADEL) procedures, followed by secondary concentration. There is a lack of consistency in how secondary concentration methods are practiced and some methods may have better recovery for particular groups of viruses. Secondary concentration methods typically involve precipitation and the most common methods employ organic flocculation (OF) by acidification at a pH of 3.5, or precipitation by polyethylene glycol (PEG) in combination with the addition of NaCl. In this study, the recovery of coliphage MS2 using the plaque assay and human adenovirus strain 41 (HAdV41) using cell-culture and qPCR assays were evaluated by OF and PEG secondary concentration of spiked samples of wastewater, surface water, and groundwater. The recovery of MS2 and HAdV41 by PEG precipitation was significantly higher than that by OF (p<0.0001) when viruses were detected by culture based methods and marginally better when HAdV41 was enumerated by qPCR (p<0.019). The recovery of HAdV41 by qPCR ranged from 75.3% to 94.4% (n=36). The mean recovery of MS2 by OF was 4.4% (0.9%-7.7%; n=14) and ranged from 57.1% to 87.9% (n=28) for the PEG methods. The poor recovery of MS2 by OF was attributed to inactivation or poor stability at acidic conditions as MS2 were not recovered in the supernatant following OF and centrifugation. The inconsistency and lack of justification for method selection in many studies calls for a systematic study to inform guidance and standardization with respect to the application of concentration methods for various water types and viral pathogens.IMPORTANCEMS2 should not be used as a process control for methods involving acidification and culture-based detection. The dense floc produced by the PEG method may have contributed to higher recoveries as the pellet was more compact and stable than the loose pellet formed by OF. Standard methods for the detection of enteric viruses and surrogates that involve acidification could be modified with PEG precipitation to uphold virus recovery and minimize inactivation.

show abstract

“…On each sampling occasion, 500-1000 ml of crude influent was collected by manual grab sampling between 08.00 and 10.00 h, reflecting peak sewage flow and aiming to capture the highest community faecal load. Suspended solids were then removed from a 150 ml aliquot of each sample by centrifugation (3000 g, 4 °C, 30 min), and viral nucleic acids collected by subsequent PEG (polyethylene glycol 8000) precipitation for >18 hours based on methods described previously [34,35] with some minor modifications [36]. PEG precipitates collected by centrifugation (10,000g, 4 °C, 30 min), and pellets, resuspended in 200 μl of phosphate buffer saline, prior to RNA extraction in the Cardiff University COVID-19 Testing Service laboratory using automated BOMB protocol [33].…”

Section: Collection and Preparation Of Wastewater Samples For Varlock Assaysmentioning

confidence: 99%

VarLOCK - sequencing independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance

Nan

Hoehn

Hardinge

et al. 2022

Preprint

View full text Add to dashboard Cite

The COVID-19 pandemic continues to pose a threat to the general population. The ongoing vaccination programs provide protection to individuals and facilitate the opening of society and a return to normality. However, emergent and existing SARS-CoV-2 variants capable of evading the immune system endanger the efficacy of the vaccination strategy. To preserve the efficacy of SARS-CoV-2 vaccination globally, aggressive and effective surveillance for known and emerging SARS-CoV-2 Variants of Concern (VOC) is required. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. In this work, we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to allow rapid and sensitive discrimination of VOCs, that can be used at point of care and/or implemented in the pipelines of small or large testing facilities, and even determine proportion of VOCs in pooled population-level wastewater samples. This technology aims to complement the ongoing sequencing efforts to allow facile and, crucially, rapid identification of individuals infected with VOCs to help break infection chains. Here, we show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants. In addition, we show the rapid adaptability of the technique for new and emerging VOCs such as Omicron.

show abstract

Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR

Cited by 54 publications

References 18 publications

Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater

Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater

Polyethylene glycol (PEG) methods are superior to acidification for secondary concentration of Adenovirus and MS2 in water

VarLOCK - sequencing independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance

Contact Info

Product

Resources

About