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Sewer Là Gì

Use this guidance to implement wastewater-based disease surveillance. Wastewater-based disease surveillance is a rapidly developing science, and tech269.com will continue to update guidance and information as it becomes available.

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Testing methods overview

Multiple testing methods and laboratory workflows are used to quantify SARS-CoV-2 in wastewater across the United States. Laboratory controls can ensure that results are comparable by accounting for method performance and data quality. Based on the levels of SARS-CoV-2 in wastewater, methods can be adapted to higher or lower detection limits as needed. For example, if levels of SARS-CoV-2 RNA are sufficiently high in wastewater, small volumes of wastewater (e.g., 1 ml) may be tested without additional concentration processes. Testing methods include sample processing steps, use of laboratory controls, and implementation of biosafety measures to ensure that data can be interpreted for public health use.


Overview of wastewater sample processing and testing for SARS-CoV-2

After sample collection, the first step in SARS-CoV-2 wastewater testing is sample preparation. A matrix recovery control should be spiked into the sample during this step. The second step is sample concentration. The third step is RNA extraction from the concentrated wastewater sample. The final step is RNA measurement. Along with measurement of SARS-CoV-2 RNA in this step, several laboratory controls should also be measured, including matrix recovery controls, human fecal normalization, quantitative measurement controls, and controls to assess molecular method inhibition.

Sample processing

Sample processing for measuring SARS-CoV-2 RNA in wastewater involves sample preparation, sample concentration, RNA extraction, and RNA measurement methods. Methods selected at each step must be tailored for use with wastewater, which is a chemically and biologically complex and variable mixture. Evaluate the performance of these wastewater sample processing procedures using appropriate laboratory controls. Proper biosafetyprotocols for processing wastewater samples that may contain SARS-CoV-2 should be followed and are described later on this webpage.

Sample preparation

Properly storing and preparing wastewater samples helps ensure that SARS-CoV-2 RNA wastewater measurements are accurate.

Storage: Refrigerate samples at 4°C immediately after collection and, if possible, process them within 24 hours to reduce SARS-CoV-2 RNA degradation and increase surveillance utility. If you cannot process samples within 24 hours after collection, you should spike a matrix recovery control into the sample prior to refrigerating it at 4°C or freezing it at -20°C or -70°C.Homogenization: Both liquid wastewater and primary sludge samples should be well-mixed prior to removing portions of collected wastewater for downstream processing. Mix by inverting samples several times (for liquid samples) or by mechanical mixing. Homogenizing samples can also include procedures to break up wastewater solids and disaggregate virus particles, such as by sonication.Sample clarification: Clarifying liquid wastewater samples by removing large solids can aid subsequent filtration-based concentration stepsif they areused for sample concentration. However, removing solids will also remove SARS-CoV-2 RNA adhered to those solids. You can clarify samples using large pore size filters (5 µm or larger) or centrifugation.

Sample concentration

Concentrating wastewater samples can improve detection of SARS-CoV-2 RNA. Concentration may be more important for untreated wastewater samples than primary sludge samples. See What to Sampleunder ‘Developing a Wastewater Sampling Strategy’ for more information on selecting a sample type.

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Concentration approaches evaluated to date that yield adequate recovery for SARS-CoV-2 detection in wastewater include:

UltrafiltrationFiltration through an electronegative membrane with sample pre-treatment by addition of MgCl2 or acidificationPolyethylene glycol (PEG) precipitationSkim milk flocculationUltracentrifugation

Consider the following factors when selecting a virus concentration method:

Sample type: For untreated wastewater samples, several filtration and precipitation methods, listed above, are available. For primary sludge samples, centrifugation is the most effective way to concentrate solids.Sample volume: Large untreated wastewater sample volumes may require dividing the sample prior to membrane filtration (due to slow filtration rate) or PEG precipitation (due to centrifuge volume constraints). Sample volumes greater than 5 L may require pre-concentration by methods designed to concentrate large volume, such as large cartridge ultrafiltration.Potential supply chain issues: Methods that require commercial filtration products, such as membrane filters or ultrafiltration cartridges, may be more sensitive to supply chain issues than other methods.Sample processing time: Concentration method selection will be constrained by method processing time and availability of laboratory personnel. Membrane filtration of turbid wastewater samples may take several hours.Availability of laboratory equipment: Centrifuge volumes and force capacity, as well as availability of membrane filtration units, will also constrain method selection.

RNA extraction

Nucleic acid extraction and purification is an essential step in isolating SARS-CoV-2 RNA from the sewage mixture. Sewage is a complex mixture with materials known to interfere with molecular viral quantification methods, so consider the following when selecting an extraction method:

Select an extraction protocol designedto produce highly purified nucleic acid extracts from environmental samples. Commercial kits are available for environmental sample extraction.Use an extraction kit or a protocol designed specifically to purify RNA and that includes RNase denaturants prior to lysis.Avoid degradation of extracted RNA due to multiple freeze-thaw cycles by aliquoting extracts into separate tubes and storing them at -70°C or below.

RNA measurement

Detection methods: Quantify SARS-CoV-2 RNA in wastewater using either RT-qPCR (reverse transcription-quantitative polymerase chain reaction) or RT-ddPCR (RT-droplet digital PCR; other forms of digital PCR are also possible but less common). Each method can be performed as either a 1-step reaction, in which RT and PCR occur in the same reaction mixture, or a 2-step reaction, in which RT and PCR are performed in separate, sequential reactions. A 1-step RT-ddPCR protocol is advantageous for wastewater because RT is performed in individual droplets, which can reduce RT inhibition compared to RT in bulk solution, as in a 2-step process and in RT-qPCR.

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Genetic targets: Primers and probes targeting regions of the SARS-COV-2 N (N1 and N2, published by tech269.com) and E genes (E_sarbeco, Corman et al., 2020 EuroSurveillance) have been reported to be sensitive and specific for quantifying SARS-CoV-2 RNA in wastewater. When possible, compare wastewater measurements using the same target genes.

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