Analysis

We operate the UK’s only high-throughput laboratory dedicated to wastewater-based public health surveillance, with ISO 17025 accreditation. Our lab is equipped with advanced technologies, including QuantStudio RT-qPCR machines, BioRad dPCR, Illumina NextSeq and MiSeq, and Oxford Nanopore MinION and GridION. Field-collected samples are subjected to comprehensive laboratory analysis, which includes: 

• Identification and quantification of a wide range of target viruses, including SARS-CoV-2, influenza A and B, RSV, enterovirus, enterovirus D68, poliovirus, noroviruses, hepatitis A and E viruses, human adenoviruses, and sapoviruses. 

• Sequencing of SARS-CoV-2 variants to identify potential variants of concern (VOCs), as well as sequencing of enteroviruses. 

• Elution and quantification of intact and infectious viral particles, and by default the detection of non-infectious particles. 

• Enrichment of potentially harmful viruses for further analysis. 

• Chemical and physical wastewater quality analysis, including parameters such as pH, ammonium, phosphate, electrical conductivity, turbidity, dissolved carbon, metals, and BOD. 

• Quantification and analysis of antimicrobial resistance (AMR) genes and mobile genetic elements through high-throughput qPCR and metagenomics. 

• Pharmaceutical analysis in collaboration with the University of Bath. 

• Recovery and culturing of Clostridium difficile in partnership with the Anaerobe Reference Lab, Public Health Wales. 

• Cryptosporidium analysis in collaboration with the Crypto Reference Lab, Public Health Wales. 

• Parasite analysis for cercarial dermatitis (Swimmer’s Itch) in freshwater environments. 

• Avian bird flu analysis in natural water sources. 

For further details on methods, see our publications. 

Key Aims and Approaches: 

Optimisation of Viral Recovery from Environmental Samples 

We are continually refining methods to improve the recovery of viruses, fungi, protists, and bacteria from a range of environmental matrices, including wastewater (influent and effluent), shellfish, river and lake water, sediments, and estuarine and marine waters. 

While previous studies have focused on the extraction of specific viruses from certain environmental matrices, we are addressing combinations that have not yet been explored. Our approach compares the efficiency of traditional extraction methods with novel techniques. 

For sensitive quantification, quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR) are used alongside RT-LAMP and digital droplet-PCR (dPCR), targeting a representative sequence of the genomes of target viruses. We use HT-qPCR for AMR analysis combined with metagenomics on the Illumina NovaSeq. 

Sample Preparation for Metagenomics Analysis 

Metagenomics refers to the direct genetic analysis of genomes contained within an environmental sample. Viral genomes, which can be either double-stranded (ds) or single-stranded (ss) RNA or DNA, are extracted to create genomic libraries. The preparation process involves: 

• The complement for each harvested DNA strand is synthesised to produce dsDNA from ssDNA viruses, whilst doubling the numbers of dsDNA molecules. 

• Viral RNA is then converted to DNA by reverse transcription and then made into ds complimentary DNA (cDNA). 

Both DNA and RNA libraries from each environmental sample and barcoded sample sequenced in pools, enables us to generate large, high-coverage datasets — capabilities that have only recently become possible. 

Exploring Viral Infectivity in Environmental Samples 

Current viral diagnostics focus on genome detection via PCR, as most enteric viruses cannot be routinely cultured. A key challenge is determining whether detected viral genomes originate from viable, infectious viruses. 

To address this, we explore methods to assess viral integrity as a marker for infectivity, focusing on both genome and capsid integrity. In the absence of culture, we evaluate these markers alongside PCR. Additionally, we use cultivatable surrogate viruses, such as RNA bacteriophage or Phi 6, to determine viral infectivity and compare them with infectivity models for viruses like SARS-CoV-2. 

This dual approach helps improve our understanding of viral viability in environmental samples, complementing genetic detection methods.