Tue 2 Mar, 2021
Monitoring wastewater for SARS-CoV-2 – or Coronavirus as it is more commonly known – can be a valuable data resource for an effective COVID-19 management. However, there are several challenges involved in detecting biological parameters in wastewater. Fortunately, there are also several solutions available to overcome them.
Wastewater can be a carrier medium of SARS-CoV- 2 as recent studies have conclusively shown. In 2020 Analytik Jena’s real-time PCR thermal cyclers of the qTOWER³ series even supported one of the first SARS-CoV-2 wastewater screenings in Japan. Since then, the monitoring of wastewater for SARS-CoV-2 and pathogens in general gained more and more attention. Authorities and researchers evaluating its role as a tool for public health monitoring and management. The following article demonstrates how the coronavirus detection workflow in wastewater – from sampling, enrichment, and nucleic acid extraction to real-time PCR detection - can be efficiently implemented and how it can be used as an effective tool for COVID-19 management.
"Regardless of the sample matrix, low concentrations of genes could be detected."
"We have been using the qTOWER³ G for quantifying virus genes, including SARS-CoV-2, in various environmental water samples. Regardless of the sample matrix, low concentrations of genes could be detected. The reproducibility of the results is fine. Owing to its efficient ramp rate, the reaction proceeds quickly. Filters for various fluorescent dyes are available and can be installed afterwards. Such flexibility is also fine.
Dr. Akihiko Hata, Toyama Prefectural University, Japan
Wastewater-based epidemiology (WBE) is the approach to identify both biological and chemical parameters in a gathering ground. It reflects the regional load and allows a comprehensive real-time monitoring of public health. Screening municipal wastewater for pathogens such as SARS-CoV-2 has recently been shown by various scientific publications* and can be a valuable tool for targeted COVID-19 management. Similar approaches** provided valuable insights into epidemiologic aspects of other diseases, too. Municipal wastewater monitoring for SARS-CoV-2 supports the effort to assess the regional prevalence of areas with a large population. Individual testing is necessary and crucial. However, not every potential infected person is being tested. Wastewater accumulates biological material of many different people living in a certain area - in urban areas up to millions of individuals. Monitoring this wastewater for SARS-CoV-2 occurrence provides valuable data for a macro-picture of the infection and helps to identify hot spots early. Even the screening for the presence of specific variants could be addressed. Therefore, it is an essential tool for crisis and disease management.
Due to its complex composition and high dilution handling of wastewater in this context is challenging. There are several hurdles involved in detecting the coronavirus in wastewater. There are also many local or regional factors that influence the detection. However, three main challenges can be identified.
Sample collection process
Sample collection in wastewater can be a tough task. Wastewater is a diverse medium and in certain cases it is very difficult to find samples that are representative for the area. Not every sampling spot provides the same sample composition and needs to be selected carefully. Furthermore, the wastewater effluent can variate throughout the day. An adjusted sampling strategy averaged over time is the key to obtain a representative number of samples.
Elevated sample volumes and low target concentrations
Usually, sample volumes to be extracted for real-time PCR based detection workflows are in the milliliter range. With wastewater samples it is quite different. Representative samples can go up to several liters. Laboratories must establish a process to downsize the sample volume and getting rid of components potentially inhibiting downstream applications.
Elevated volumes also mean high dilution of the sample. The concentrations of SARS-CoV-2 and other pathogens are often very low which makes them difficult to detect. Enrichment processes are necessary to lower the detection limits. Powerful instruments are essential to increase sensitivity and allow reliable target detection during real-time PCR.
High number of samples
To monitor the timeline and/or cover diverse areas of interest and to get a representative picture of the SARS-CoV-2 occurrence a high number of samples must be collected. This creates another challenge: processing this high sample amount. Automation equipment is needed to cope with potentially thousands of samples a day. In addition, automatization minimizes required manual steps and ensures reproducibility of results.
In order to overcome all challenges, we provide an efficient workflow solution for the detection of SARS-CoV-2 in wastewater - starting with representative wastewater sampling, continuing with effective enrichment and nucleic acid extraction and closing with the real-time PCR detection. Real-time PCR proofed to be the gold standard for the direct and highly sensitive detection of SARS-CoV-2 in research and clinical settings. It is ideal for the detection in wastewater as well.
Fully automated sample collection
Analytik Jena and its parent company Endress+Hauser combined their expertise to provide wastewater treatment plants and laboratories with a unique, fully automatic sampling solution for wastewater. The water sampler Liquistation CSF48 by Endress+Hauser is a stationary automatic sampler highly valued in the process industry. Thanks to automation, representative samples are collected over time in large amount and under equal conditions which benefits the reproducibility of results tremendously. Furthermore, the Liquistation CSF48 is saving time and effort for its operators, is compliant to worldwide water regulations, and easy to program, setup and maintain.
Sample enrichment by filtration and nucleic acid extraction
The challenge of low concentrations can be overcome by an efficient target enrichment process. This can easily be realized by utilizing filtration (third party). Thanks to this method the detection of very low amounts of the SARS-CoV-2 pathogen via real-time PCR becomes possible. The filter membrane is homogenized with the SpeedMill PLUS offered by Analytik Jena, one of only a few instruments on the market that can provide entire and reproducible sample homogenization combined with small foot print.
The next step is the extraction of the pathogen’s DNA and/or RNA. For the reproducible extraction of SARS-CoV-2 RNA the innuPREP AniPath DNA/RNA Kit-IPC16 in combination with the automated extraction platform InnuPure C16 touchare the ideal solution. The innuPREP AniPath DNA/RNA Kit allows for the highly efficient extraction of viral/bacterial DNA and/or RNA from broad range of starting materials. The InnuPure C16 touch provides the necessary automation for the extraction phase. Up to 16 samples can be processes simultaneously.
High sensitivity real-time PCR
For the detection phase Analytik Jena offers the most technically advanced real-time thermal cyclers on the market – the qTOWER³ series. The cyclers come with a patented fiber optic for enhanced sensitivity and extremely low detection limits. Furthermore, its thermal block design provides best thermal conductivity for optimal PCR results. In combination with a specific real-time PCR Assay (by third party, e.g. Water SARS-CoV-2 RT-PCR Test by IDEXX) and the sample preparation and enrichment methods described above the qTOWER³ can reach best sensitivity and reproducibility.
- Automated and controlled sample collection for reproducible sample quality
- Efficient enrichment allows low limits of detection
- Automated nucleic acid extraction for minimal hands-on time
- From sampling to final result in only 3 hours
- Reliable results even in challenging wastewater matrix
- Detection based on real-time PCR – the gold standard for viral diagnostics
- One workflow - multiple biological parameters
All steps are designed to fit harmoniously together and provide laboratory users with a fast, reliable, and scalable workflow for the detection of SARS-CoV-2 in wastewater.
*Lancet Gastroenterol Hepatol. 2020 Jun;5(6):533-534. doi: 10.1016/S2468-1253(20)30087-X. Epub 2020 Apr 1 (https://www.thelancet.com/pdfs/journals/langas/PIIS2468-1253(20)30087-X.pdf)
Sci Total Environ. 2021 Mar 1;758:143578. doi: 10.1016/j.scitotenv.2020.143578. Epub 2020 Nov 10 (https://www.sciencedirect.com/science/article/pii/S0048969720371096)
** Proc Natl Acad Sci U S A. 2018 Nov 6;115(45):E10625-E10633. doi: 10.1073/pnas.1808798115. Epub 2018 Oct 18 (https://www.pnas.org/content/115/45/E10625)
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