WO2022147868A1

WO2022147868A1 - Sewage surveillance for sars-cov-2

Info

Publication number: WO2022147868A1
Application number: PCT/CN2021/074675
Authority: WO
Inventors: Tong Zhang; Yu Deng; Xiawan ZHENG; Xiaoqing Xu; Leo Poon; Hein Min TUN
Original assignee: The University Of Hong Kong
Priority date: 2021-01-08
Filing date: 2021-02-01
Publication date: 2022-07-14
Also published as: CN114755376A

Abstract

Systems for sensitive and accurate detection ofinfectious agents in samples from sewage and wastewater have been established. The systems can rapidly and effectively detect and record the presence ofSARS-CoV-2 and other viruses within wastewater and sewage from specific locations. The systems employ Quantitative Polymerase Chain Reaction (RT-qPCR) to identify and quantify nucleic acids indicative ofSARS-CoV-2 and other viruses within samples obtained from sewagesheds. The systems reproducibly and accurately identify and quantitate viral RNAs for enable rapid and early monitoring ofmunicipal water systems in areas housing or related to patients infected with SARS-CoV-2 and/or other viruses, and provide databases to monitor the presence ofinfectious agents within sewagesheds on a community-wide level.

Description

SEWAGE SURVEILLANCE FOR SARS-COV-2

FIELD OF THE INVENTION

The invention is generally directed to the identification and quantification of coronavirus RNA within a sewage sample, and for surveillance of sewage for SARS-CoV-2.

BACKGROUND OF THE INVENTION

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a highly-transmissible and pathogenic coronavirus that causes acute respiratory disease in humans known as Coronavirus disease 2019 (COVID-19) . SARS-CoV-2 spread rapidly across the world from its discovery in late 2019 to early 2020, prompting The World Health Organization (WHO) to declare a global pandemic in mid-March 2020 (Hu, B., Guo, H., Zhou, P. et al. Nat Rev Microbiol (2020) . doi. org/10.1038/s41579-020-00459-7) . A little more than one year after the first case of SARS-CoV-2 was reported, over 85.6 million infections had been confirmed worldwide, and SARS-CoV-2 had been associated with more than 1.8 million deaths. The United States alone reported more than 21 million cases of COVID-19, and over 360,000 deaths associated with SARS-CoV-2 by early January 2021 (WHO Coronavirus disease (COVID-19) Weekly Epidemiological Update and Weekly Operational Update; website who. int/emergencies/diseases/novel-coronavirus-2019/situation-reports/) .

The SARS-CoV-2 virus is shed in the stool of infected individuals. An infected person can shed virus in their feces even if they do not have any symptoms, and shedding can continue for several weeks after they are no longer infectious. Thus, there is a need for sensitive sewage testing systems which can help track infections in the community and provide early warning of an increase in infection amongst a community. Sewage surveillance of SARS-CoV-2 can provide valuable information in addition to other clinical indicators of COVID-19 spread to inform and support public health actions in response to SARS-CoV-2 spread.

Therefore, it is an object of the invention to provide compositions and methods for the rapid and reproducible detection and quantitation of SARS-CoV-2 in sewage samples.

It is another object to provide methods for community-wide surveillance of SARS-CoV-2 within sewage and waste water systems, especially in communities having one or more residents identified as being infected with SARS-CoV-2 virus.

SUMMARY OF THE INVENTION

Compositions and methods for the rapid and reproducible detection and quantitation of SARS-CoV-2 in sewage samples have been developed.

Methods of sampling a sewageshed for effective detection and assessment of infectious disease agent presence in an area served by the sewageshed are provided. The methods include a step of (i) collecting a first plurality of sewage samples at a first specified sewage system location of the sewageshed. Typically, the first plurality of sewage samples are collected at approximately equal time intervals during a first collection period. The methods typically include pooling the first plurality of sewage samples to form a first composite sewage sample.

In some forms, the area served by the sewageshed is a single building, a single building complex, a single campus, a single city block, a single neighborhood, a single community, a single city, or a single district. Typically, the first specified sewage system location is one or more of a building drain pipe, a building complex drain pipe, a street sewer pipe, a pumping station, or a wastewater treatment plant.

In some forms, the first collection period is approximately proportional to the average distance of the first specified sewage system location from buildings served by the first specified sewage system location. An exemplary first collection period is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10, hours, 11 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours. An exemplary time interval at which the first plurality of sewage samples are collected is 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, or 60 minutes. In some forms, the first plurality of sewage samples comprises at least 2 sewage samples, 3 sewage samples, 4 sewage samples, 5 sewage samples, 6 sewage samples, 7 sewage samples, 8 sewage samples, 9 sewage samples, 10 sewage samples, 11 sewage samples, 12 sewages samples, 14 sewage samples, 16 sewage samples, 18 sewage samples, 20 sewage samples, 22 sewage samples, 24 sewage samples, 25 sewage samples, 30 sewage samples, 35 sewage samples, 40 sewage samples, 45 sewage samples, 50 sewage samples, 55 sewage samples, 60 sewage samples, 65 sewage samples, 70 sewage samples, 75 sewage samples, 100 sewage samples, or 125 sewage samples.

The methods optionally include a step of (ii) collecting a second plurality of sewage samples at a second specified sewage system location of the sewageshed, wherein the second plurality of sewage samples are collected at approximately equal time intervals during a second collection period. The methods typically include pooling the second plurality of sewage samples to form a second composite sewage sample. In some forms, the second specified sewage system location is different from the first specified sewage system location. In some forms, the second collection period is approximately proportional to the average distance of the second specified sewage system location from buildings served by the second specified sewage system location.

The methods optionally include a step of (iii) collecting a third plurality of sewage samples at a third specified sewage system location of the sewageshed, wherein the third plurality of sewage samples are collected at approximately equal time intervals during a third collection period. The methods typically include pooling the second plurality of sewage samples to form a second composite sewage sample. In some forms, the third specified sewage system location is different from the first specified sewage system location and the second specified sewage system location. In some forms, the third collection period is approximately proportional to the average distance of the third specified sewage system location from buildings served by the third specified sewage system location.

Methods of detecting the presence of an infectious disease agent in an area served by a sewageshed are also provided. The methods include a step of (a) concentrating a sewage sample collected from the area served by the sewageshed to provide a concentrated sewage sample. Typically, the sewage sample is concentrated by (1) centrifuging the sewage sample; (2) collecting the resultant supernatant; (3) ultra-centrifuging the supernatant; and (4) re-suspending the resultant pellet, thereby producing a concentrated sewage sample.

In some forms, the methods include steps of (b) extracting nucleic acids from the concentrated sewage sample; and (c) detecting in the extracted nucleic acids one or more nucleic acid sequences indicative of the infectious disease agent, thereby detecting the presence of the infectious disease in the area served by the sewageshed.

In some forms, following step (2) and prior to step (3) , the methods include centrifuging the supernatant of step (2) . In some forms, the methods include extracting nucleic acids from the concentrated sewage sample by (1) lysing concentrated sewage sample; (2) phenol extracting the lysed concentrated sewage sample; (3) precipitating nucleic acids from the aqueous phase of the phenol extraction; and (4) cleaning the nucleic acids in a spin column. Typically, the nucleic acid sequences indicative of the infectious disease are detected by quantitative polymerase chain reaction (qPCR) of the extracted nucleic acids. In some forms, the nucleic acid sequences indicative of the infectious disease are detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) of the extracted nucleic acids.

In some forms, the infectious disease agent is a virus, such as an RNA virus.

A preferred RNA virus that is detected by the methods is a coronavirus virus, such as a SARS-CoV-2 virus.

In some forms, one or more of the method steps are performed together with one or more controls samples. An exemplary control sample is a matrix control, for example, a sewage sample spiked with a known amount of a known infectious disease agent. In some forms, one or more of the method steps are performed on a reagent blank. An exemplary reagent blank is a no sewage control sample.

In some forms, the sewage sample is a composite sewage sample formed according the described systems and methods for sampling a sewageshed for effective detection and assessment of infectious disease agent presence.

In some forms, the qPCR is performed with primers to two or more target sequences in the infectious disease agent in separate reactions. An exemplary qPCR for use with the described methods is run for 45 cycles. In some forms, a cycle threshold (Ct) of less than 45 indicates a positive result for the primer set of the reaction. In other forms, a cycle threshold (Ct) less than 45 for none of the primer sets indicates a negative for the presence of the infectious disease agent in the sewage sample. In some forms, a cycle threshold (Ct) less than 45 for only one of the primer sets indicates the suspected presence of the infectious disease agent in the sewage sample. In other forms, a cycle threshold (Ct) of less than 45 for two or more of the primer sets indicates a positive for the presence of the infectious disease agent in the sewage sample.

A preferred infectious disease agent that is detected according to the described methods is SARS-CoV-2. In some forms, one primer set is to the N1 gene of SARS-CoV-2 and another primer set is to the E gene of SARA-CoV-2.

In some forms, the methods include one or more optional steps, including performing qPCR on a positive control, wherein the positive control has a plasmid comprising the N1 gene of SARS-CoV-2, and/or performing qPCR on a negative control, wherein the negative control has no template, and/or sequencing the amplified nucleic acid to confirm the identity of the amplified nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a workflow of surveying SARS-CoV-2 virus in sewage samples.

Figure 2 is a cartoon diagram showing the workflow and practices for quantification of SARS-CoV-2 in sewage samples.

Figures 3A-3B are graphs showing Detected Ct value over expected TCID50 per mL for each of PBS and sewage for samples processed using the QIAamp Viral RNA Mini kit (Qiagen) (Figure 3A) and TRIzol ^TM Plus RNA Purification Kit (Thermofisher) (Figure 3B) , respectively.

Figures 4A-4B are bar graphs showing Log (copies per ml sample) over expected TCID50 per mL for each of PBS and sewage for samples processed using the QIAamp Viral RNA Mini kit (Qiagen) (Figure 4A) and TRIzol ^TM Plus RNA Purification Kit (Thermofisher) (Figure 4B) , respectively.

Figure 5 is a diagram showing the single-letter code nucleic acid sequence alignment of the Query RT-qPCR product from Lok Hop House sewage and Reference SARS-CoV-2 (NCBI Accession Number MT929054.1) .

Figure 6 is a graph of daily local cases in Hong Kong on the date of sample collection.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The terms “SARS-CoV-2” and “Severe Acute Respiratory Syndrome Coronavirus 2” refer to the pathogenic coronavirus strains of the subgenus Sarbecovirus which are directly descended from the coronavirus of zoonotic origin which emerged in Asia in late 2019, and which are the causative agents of pandemic Coronavirus disease 2019 (COVID-19) in humans.

The term “N gene” refers to the gene which encodes the nucleocapsid protein, located at the 3’ region of the SARS-CoV-2 coronavirus RNA genome encoding a polyprotein. A representative N gene from the SARS-CoV-2 coronavirus is deposited in GenBank as accession No: MN908947.3, which has the nucleic acid sequence of SEQ ID NO: 1.

The term “E gene” refers to the gene which encodes the envelope protein of SARS-CoV-2 coronavirus. A representative E gene from the SARS-CoV-2 coronavirus is deposited in GenBank as accession No: MN908947.3, which has the nucleic acid sequence of SEQ ID NO: 2.

As used herein, the term "nucleic acid molecule" is used broadly to mean any polymer of two or more nucleotides, which are linked by a covalent bond such as a phosphodiester bond, a thioester bond, or any of various other bonds known in the art as useful and effective for linking nucleotides. Such nucleic acid molecules can be linear, circular or supercoiled, and can be single stranded or double stranded, e.g. single stranded or double stranded DNA, RNA or DNA/RNA hybrid. In some forms, nucleic acid molecules are or include nucleic acid analogs that are less susceptible to degradation by nucleases than are DNA and/or RNA.

The terms “targeted gene” or "target nucleic acid" or "target sequence" or "target segment" as used herein refer to a nucleic acid sequence of interest to be detected and/or quantified in the sample to be analyzed. Target nucleic acid may be composed of segments of a genome, a complete gene with or without intergenic sequence, segments or portions of a gene with or without intergenic sequence, or sequence of nucleic acids to which probes or primers are designed to hybridize. Target nucleic acids may include a wild-type sequence (s) , a mutation, deletion, insertion or duplication, tandem repeat elements, a gene of interest, a region of a gene of interest or any upstream or downstream region thereof. Target nucleic acids may represent alternative sequences or alleles of a particular gene. Target nucleic acids may be derived from genomic DNA, cDNA, or RNA. In preferred forms, the target sequence refers to a gene or genomic component within a coronavirus, which is targeted by one or more primers designed to selectively bind and amplify the gene during RT-qPCR.

As used herein, the term "primer" refers to an oligonucleotide, which is capable of acting as a point of initiation of nucleic acid sequence synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a target nucleic acid strand is induced, i.e., in the presence of different nucleotide triphosphates and a polymerase in an appropriate buffer ( "buffer" includes pH, ionic strength, cofactors etc. ) and at a suitable temperature. One or more of the nucleotides of the primer can be modified for instance by addition of a methyl group, a biotin moiety, a fluorescent tag or by using radioactive nucleotides. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. The term primer as used herein includes all forms of primers that may be synthesized including peptide nucleic acid primers, labeled primers, and the like. The term "forward primer" as used herein means a primer that anneals to the anti-sense strand of double-stranded DNA (dsDNA) . A "reverse primer" anneals to the sense-strand of dsDNA. Primers are typically at least 10, 15, 18, or 30 nucleotides in length or up to about 100, 110, 125, or 200 nucleotides in length. In some forms, primers are preferably between about 15 to about 60 nucleotides in length, and most preferably between about 25 to about 40 nucleotides in length. In some forms, primers are 15 to 35 nucleotides in length. There is no standard length for optimal hybridization or polymerase chain reaction amplification. An optimal length for a particular primer application may be readily determined in the manner described in H. Erlich, PCR Technology, Principles and Application for DNA Amplification, (1989) .

The term "amplification" as used herein refers to increasing the number of copies of a nucleic acid molecule, such as a gene or fragment of a gene, for example at least a portion of the SARS-CoV-2 RNA. The products of an amplification reaction are called amplification products. An example of in vitro amplification is RT-PCR amplification.

The term "conditions sufficient for" as used herein in connection with the disclosed methods, refers to any environment that permits the desired activity, for example, that permits specific binding or hybridization between two nucleic acid molecules or that permits reverse transcription and/or amplification of a nucleic acid. Such an environment may include, but is not limited to, particular incubation conditions (such as time and/or temperature) or presence and/or concentration of particular factors, for example in a solution (such as buffer (s) , salt (s) , metal ion (s) , detergent (s) , nucleotide (s) , enzyme (s) , etc) .

The term "contact" as used herein in connection with the disclosed methods refers to placement in direct physical association; for example in solid and/or liquid form. For example, contacting can occur in vitro with one or more primers and/or probes and a biological sample (such as a sample including nucleic acids) in solution.

As used herein, the term "sample" refers to in vitro as well as test samples obtained from a sewageshed or other municipal or environmental water source, such as a sample of water, ice, soil, sludge, or other matter obtained from a sewageshed, watershed, lake, sea, river, stream, municipal tap water, freshwater treatment site, landfill site, purified drinking water or commercially available bottled water or other beverage, or specific sewer system, for example a sewer system of an individual building, multiple buildings and large housing estates, as well as entrances of conventional sewage treatment facilities (sewage pumping stations and sewage treatment works) .

The terms “individual, ” “subject, ” and “patient” are used interchangeably, and refer to a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets.

The terms “detect, ” and “identify, ” in the context of an assay are used interchangeably and refer to the positive identification of a target, such as genetic component of a coronavirus. The identification or detection can be interpreted or assessed according to the mechanism of an assay, and identification or detection can be compared to a control or to a standard level. For example, in a RT-qPCR assay, the extent of detection of a gene or expressed gene product may be quantified as complete (i.e., 100%) or partial (i.e., 1-99.9%) of the expected or calculated level of that in a control. Quantitation can be measured as a %value, e.g., from 1%up to 100%, such as 5%, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%. For example, the relative amount of a target gene, or the activity or quantity of one or more expressed gene products can be assessed relative to a control, or relative to another experimental sample. In some forms, the detection or quantitation are compared according to the level of RNAs, or proteins corresponding to the targeted genetic element within a control cell.

As used herein, the term “sensitivity” refers to the ability of a test to correctly identify true positives, i.e., sewage samples infected with SARS-CoV-2. For example, sensitivity can be expressed as a percentage, the proportion of actual positives which are correctly identified as such (e.g., the percentage of test samples having SARS-CoV-2 correctly identified by the test as having SARS-CoV-2) . A test with high sensitivity has a low rate of false negatives, i.e., the cases of SARS-CoV-2 not identified as such. Generally, the disclosed assays and methods have a sensitivity of at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

As used herein, the term “specificity” refers to the ability of a test to correctly identify true negatives, i.e., the sewage samples that have no SARS-CoV-2 infection. For example, specificity can be expressed as a percentage, the proportion of actual negatives which are correctly identified as such (e.g., the percentage of test samples not having SARS-CoV-2 correctly identified by the test as not having SARS-CoV-2) . A test with high specificity has a low rate of false positives, i.e., the cases of sewage samples not having SARS-CoV-2 but suggested by the test as having SARS-CoV-2. Generally, the disclosed methods have a specificity of at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

As used herein, the term “accurate” refers to the ability of a test to provide a results with high sensitivity and high specificity, such as with sensitivity over about 80%and specificity over about 80%, with sensitivity over about 85%and specificity over about 85%, or with sensitivity over about 90%and specificity over about 90%.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

Use of the term "about" is intended to describe values either above or below the stated value in a range of approx. +/-10%; in other aspects the values may range in value either above or below the stated value in a range of approx. +/-5%; in other aspects the values may range in value either above or below the stated value in a range of approx. +/- 2%; in other aspects the values may range in value either above or below the stated value in a range of approx. +/- 1%.

II. Compositions

Systems and compositions that can be used to rapidly and reliably identify the presence of SARS-CoV-2 viruses within a sewage or waste water sample have been established. The systems employ RT-qPCR with primers designed to recognize two distinct genes having conserved sequences amongst SARS-CoV-2 viruses associated with the COVI-19 pandemic. The systems include one or more sets of nucleic acid primer probes for annealing to viral RNA within a test sample.

Compositions for a RT-qPCR-based molecular assay system for detection of SARS-CoV-2 viruses within a sewage sample are provided. The methods and compositions are particularly effective for the rapid and sensitive detection and quantitation of SARS-CoV-2 viruses within water samples, such as sewage samples. The systems and compositions identify SARS-CoV-2 viruses within the sample if they possess an N gene and/or E gene of a specific sequence. The RT-qPCR-based systems employ a pair of target-specific primers labelled with a detectable probe to monitor the reverse-transcription polymerase chain reaction within a mixture including an experimental sample. The RT-qPCR assay is dependent on a highly-sequence specific alignment of the primer probes with template RNA or DNA within the sample, to achieve sequence-specific detection and quantitation in real-time.

A. Viral Targets

The systems and compositions identify viruses, particularly the SARS-CoV-2 viruses, which are coronaviruses of the subgenus Sarbecovirus.

1. Coronaviruses

The coronaviruses (order Nidovirales, family Coronaviridae, genus Coronavirus) are a diverse group of large, enveloped, positive-stranded RNA viruses that cause respiratory and enteric diseases in humans and other animals (Rota, et al., Science, May 2003, Page 1/10.1126/1085952) .

Coronaviruses typically have narrow host and can cause severe disease in many animals, and several viruses, including infectious bronchitis virus, feline infectious peritonitis virus, and transmissible gastroenteritis virus, are significant veterinary pathogens. Human coronaviruses (HCoVs) are found in both group 1 (HCoV-229E) and group 2 (HCoV-OC43) and are historically responsible for～30%of mild upper respiratory tract illnesses.

At～30,000 nucleotides, their genome is the largest found in any of the RNA viruses. There are three groups of coronaviruses;

groups

1 and 2 contain mammalian viruses, while group 3 contains only avian viruses. Within each group, coronaviruses are classified into distinct species by host range, antigenic relationships, and genomic organization. The genomic organization is typical of coronaviruses, with the characteristic gene order (5’-replicase [rep] , spike [S] , envelope [E] , membrane [M] , nucleocapsid [N] -3’) and short untranslated regions at both termini. The SARS-CoV rep gene, which comprises approximately two-thirds of the genome, encodes two polyproteins (encoded by ORF1a and ORF1b) that undergo co-translational proteolytic processing. There are four open reading frames (ORFs) downstream of rep that are predicted to encode the structural proteins, S, E, M, and N, which are common to all known coronaviruses.

a. SARS-CoV-2

The systems and compositions identify the SARS-CoV-2 betacoronavirus of the subgenus Sarbecovirus. SARS-CoV-2 viruses share approximately 79%genome sequence identity with the SARS-CoV virus identified in 2003. The genome organization of SARS-CoV-2 viruses is shared with other betacoronaviruses; six functional open reading frames (ORFs) are arranged in order from 5’ to 3’: replicase (ORF1a/ORF1b) , spike (S) , envelope (E) , membrane (M) and nucleocapsid (N) . In addition, seven putative ORFs encoding accessory proteins are interspersed between the structural genes.

An exemplary nucleic acid sequence for the SARS-CoV-2 N gene is set forth in GenBank accession number MN908947.3 (SEQ ID NO: 1) :

An exemplary nucleic acid sequence for the SARS-CoV-2 E gene is set forth in GenBank accession number MN908947.3 (SEQ ID NO: 2) :

B. Samples

The described systems and compositions detect and/or quantify SARS-CoV-2 virus RNA present within a swage or wastewater sample that is a liquid. In some forms, an input sample is diluted, concentrated or otherwise obtained from a liquid, gel, emulsion, or a solid.

The described systems and compositions produce an “output” sample, including the products of the RT-qPCR, which include an amplified product labelled with the probe, based on the presence of the SARS-CoV-2 virus present within a sewage or wastewater sample.

1. Input Samples for RT-qPCR

The described systems include an input sample containing nucleic acids extracted and purified from a sample of sewage or wastewater. In some forms, the input sample is the product of a process to extract and isolate viral RNA from an environmental sample. In some forms, an input sample includes an isolated and/or purified nucleic acid, such as a viral RNA, isolated from a sewage or wastewater sample. Extracted and/or purified viral RNA for use as an input sample according to the described systems can be obtained from a sewage or wastewater sample by methods known in the art for purification of RNA.

In some forms, the input sample includes an isolated and/or purified viral nucleic acid, such as an RNA or DNA. RNA or DNA may be present within the sample in the form of intact viral genomic RNA, or fragments of viral genomic RNA. In some forms, the sample includes an isolated and/or purified nucleic acid, such as an RNA or DNA plasmid. In some forms, the input sample is in the form of a cell-free, clarified, aqueous solution.

Typically, input samples for use in the described assays are in a volume between about 0.1 μL to about 1000 μL, inclusive, preferably in a volume of about 3-5 μL, most preferably a volume of about 4 μL.

2. Origin of Input Samples

Input samples for use in the described assays can originate from any water source, and can be in any form, including liquids, frozen liquids or powders, such as freeze-dried or lyophilized samples. Preferably, the input sample is from a sewageshed or other municipal or environmental water source, such as a sample of water, ice, soil, sludge, or other matter obtained from a sewageshed, watershed, lake, sea, river, stream, municipal tap water, freshwater treatment site, landfill site, purified drinking water or commercially available bottled water or other beverage, or specific sewer system, for example a sewer system of an individual building, multiple buildings and large housing estates, as well as entrances of conventional sewage treatment facilities (sewage pumping stations and sewage treatment works) . Typically, the sample is collected and processed prior to detection of infectious agents.

A sample can be identified according to the location and timing of collection, for example, as a first, second, third, fourth or further specified sewage system location. Exemplary locations include one or more of a building drain pipe, a building complex drain pipe, a street sewer pipe, a pumping station, or a wastewater treatment plant. In some forms, the sewage sample is contained within in a container, or together with one or more devices used to obtain the sample.

3. Control Samples

In some forms, the assay includes one or more control samples which act as a control for the specificity, detection and quantification of the SARS-CoV-2 virus within a sample. Typically, negative control samples include purified RNA or DNA derived from viruses that share little or no genetic relatedness with the SARS-CoV-2 virus. Exemplary negative control viruses include RNA extracted from human coronaviruses 229E, OC43, HKU1, NL63, and OC43, MERS, camel coronavirus HKU23, human influenza A viruses (H1N1, H3N2, H5N1, and H7N9 subtypes) , avian influenza (H1, H4, H6, and H9 subtypes) , human influenza B viruses (Yamagata and Victoria lineages) , and adenovirus, enterovirus, human parainfluenza viruses (PIV1, 2, 3 and 4) , respiratory syncytial virus, human metapneumovirus, rhinovirus and human bocavirus. In some forms, negative controls can include RNA extracted from retrospective human respiratory specimens previously tested positive for any of these viruses. In some forms, the negative controls are recombinantly-produced nucleic acid vectors which lack one or more of the nucleic acid sequences required for the activity of the designed primer and probe sets that are to be used.

In some forms, positive controls to confirm the specificity and efficacy of the assay for detecting and quantifying the SARS-CoV-2 virus include viral RNA extracted from SARS-CoV-2-infected cells, as well as the RT-qPCR products of SARS coronavirus generated by E and N gene assays, cloned into plasmids.

In some forms, RNA or DNA control samples are serially diluted, to evaluate the performance of the assays.

4. Diluents, Fillers and Preservatives

In some forms, the input sample includes a diluent, filler, excipient or preservative. In some forms, the sample includes one or more reagents which function to preserve or maintain the quantity of SARS-CoV-2 virus within the sewage or wastewater sample, to create a representative input sample. Therefore, in some forms, the input sample includes one or more reagents that prevent or reduce the activity of RNAase enzymes.

C. Designed Nucleic Acid Oligonucleotide Primers and Probes

The described systems include matched sets of 5’ ( “forward” ) and 3’ ( “reverse” ) nucleic acid oligonucleotide primers configured to selectively amplify specific fragments of an infectious agent genome ( “amplicons” ) , as well as target-specific nucleic acid oligonucleotide probes configured to selectively detect/label the resulting amplicons. A preferred infectious agent is a SARS-CoV-2 virus.

To detect the presence of a SARS-CoV-2 virus within an input sample for RT-qPCR, derived from a sewageshed or wastewater sample, each matched set of primers includes a 5’ ( “forward” ) and 3’ ( “reverse” ) primer, designed to target and amplify a pre-determined fragment of one or more components of the SARS-CoV-2 virus genome. In some forms, a matched set of primers is designed to amplify a specific fragment of a single target gene of the SARS-CoV-2 virus. Exemplary genes that can be targeted include the viral replicase (ORF1a/ORF1b) gene, the viral spike (S) gene, the viral envelope (E) gene, the viral membrane (M) gene, and the viral nucleocapsid (N) gene. In other forms, a set of matched primers is designed to amplify a specific fragment of a region of the viral genome coding for a non-structural gene, or a fragment of the viral genome spanning two viral genes. Typically, a matched set of primers is designed based on the nucleic acid sequence of the genome of a currently circulating viral strain, for example, Genbank Accession number: MN908947. In some forms, primers for detection of SARS-CoV-2 viruses include primers designed to amplify a region of the SARS-CoV-2 virus N gene. In some forms, primers for detection of SARS-CoV-2 viruses include primers designed to amplify a region of the SARS-CoV-2 virus E gene. Exemplary target genes include the nucleic acid sequences of the N gene (SEQ ID NO: 1) and the E gene (SEQ ID NO: 2) of the SARS-CoV-2 virus (Genbank Accession number: MN908947) .

Nucleic acid oligonucleotide probes with a sequence which selectively binds to the fragments of the SARS-CoV-2 virus E and N genes amplified by oligonucleotide primers, respectively, are also described. Typically, each primer is present within an RT-qPCR reaction in a concentration of between about 0.1 μmol/L to about 1.0 μmol/L, preferably about 0.1 μmol/L. Typically, each probe is present within an RT-qPCR reaction in a concentration of between about 0.05 μmol/L to about 1.0 μmol/L, preferably about 0.25 μmol/L.

D. Compositions for RT-qPCR

In some forms, the RT-qPCR encompasses a two-step method, typically comprising two enzymes; the first step uses a RNA-dependent DNA polymerase, also known as a reverse transcriptase, to copy RNA into DNA (cDNA) , the second step then switches to the use of DNA polymerase such as Taq polymerase, which amplifies the cDNA as in a standard PCR test.

In preferred forms, the reverse transcriptaion (RT) and the PCR reactions are carried out in a single test tube using fluorescence-based quantitative RT-PCR.

The described assay requires reagents and apparatus for conducting RT-qPCR procedures. Typically, test agents include buffer, RNA-dependent DNA polymerase, Taq polymerase, target-specific DNA primers, and a target-specific DNA probe that is labelled at one end with a fluorescent label and at the other with a quencher. In some forms, the target-specific DNA probe further comprises an internal quencher. Exemplary fluorescent label on the target-specific DNA probe include FAM dyes, and exemplary quenchers on the target-specific DNA probe include internal

Quencher, Iowa Black FQ quenchers (IBFQ) . In further preferred forms, the probes are double-quenched probes such as 5’FAM/ZEN/3’IBFQ.

In some forms, a typical reaction volume is from about 0.1μL to about 1,000 μL, preferably about 20 μL. An exemplary monoplex RT-PCR reaction mixture includes 5 μL of 4X master reaction mixture (available from multipe commercial sources, such as TaqMan Fast Virus 1-Step Master Mix, from ThermoFisher) , 0.5 μmol/L of forward primer, 0.5 μmol/L of reverse primer, 0.25 μmol/L of probe, and 4 μl of input sample.

Typically, the assay is carried out within a thermal cycler or other apparatus suitable for conducting and monitoring necessary for conducting an RT-qPCR procedure. Suitable apparatus for conducting an RT-qPCR procedure are well known in the art and are available from multiple commercial sources, including the ViiA7 Real-Time PCR system from ThermoFisher.

III. Methods for Detecting and Quantifying Infectious Agents in Sewage

Methods for detecting and quantifying infectious agents in sewage samples using a RT-qPCR system have been developed.

Early detection is crucial for effectively controlling and monitoring the spread of Infectious Agents, such as SARS-CoV-2 viruses within sewage and waste water systems. Therefore, methods for the molecular detection of SARS-CoV-2 viral RNA are provided. The detection time ranges from several minutes to hours. The methods can detect SARS-CoV-2 in sewage samples obtained from a variety of wastewater sites including community sewer systems of individual buildings and large housing estates, as well as entrances of conventional sewage treatment facilities (sewage pumping stations and sewage treatment works) . Typically, the methods include steps for sample collection, sample preparation, and detection of infectious agents. In some forms, the methods include steps for recording and assessing the extent and spread of an infectious agent within a sewageshed or wastewater system. The data obtained from the methods can inform important public health decisions, and assist epidemiological studies.

A. Sample Collection

The methods include one or more steps for collecting one or more samples from a sewageshed. Methods of sampling a sewageshed for effective detection and assessment of infectious disease agent presence in an area served by the sewageshed include a step of: (i) collecting a first plurality of sewage samples at a first specified sewage system location of the sewageshed. Typically, the first plurality of sewage samples are collected at approximately equal time intervals during a first collection period. The methods typically include pooling the first plurality of sewage samples to form a first composite sewage sample.

The methods optionally include a step of:

(ii) collecting a second plurality of sewage samples at a second specified sewage system location of the sewageshed, wherein the second plurality of sewage samples are collected at approximately equal time intervals during a second collection period. The methods typically include pooling the second plurality of sewage samples to form a second composite sewage sample. In some forms, the second specified sewage system location is different from the first specified sewage system location. In some forms, the second collection period is approximately proportional to the average distance of the second specified sewage system location from buildings served by the second specified sewage system location.

The methods optionally include a step of:

(iii) collecting a third plurality of sewage samples at a third specified sewage system location of the sewageshed, wherein the third plurality of sewage samples are collected at approximately equal time intervals during a third collection period. The methods typically include pooling the second plurality of sewage samples to form a second composite sewage sample. In some forms, the third specified sewage system location is different from the first specified sewage system location and the second specified sewage system location. In some forms, the third collection period is approximately proportional to the average distance of the third specified sewage system location from buildings served by the third specified sewage system location.

The methods optionally include a fourth or further step of collecting a fourth or further plurality of sewage samples at a fourth or further specified sewage system location of the sewageshed. The methods typically include pooling the fourth or further plurality of sewage samples to form a fourth or further composite sewage sample. In some forms, the fourth or further specified sewage system location is different from the other specified sewage system locations. In some forms, the fourth or further collection period is approximately proportional to the average distance of the fourth or further specified sewage system location from buildings served by the fourth or further specified sewage system location.

An exemplary collection procedure carried out is as follows: Composite samples (15 min interval, 3 hours in the morning peak hours for the manhole, 12 hours for the pumping station and 24 hours for influent of WWTPs) are taken by DSD of Hong Kong and delivered to the lab in 1L plastic bottles in secondary containers. During the sample delivery, samples will be kept cool with ice in secondary containers to minimize the degradation of RNA. Samples will be stored in 4℃ refrigerator and processed within 24h.

In some forms, the sample specimen is inactivated after sample collection prior to subsequent processing, for example, virus in the samples is inactivated by pasteurizing at 60℃ for 30 mins.

B. Sample Preparation

The methods include steps to purify and concentrate the infectious agents present within the samples, to enhance the efficacy and accuracy of detection and quantitation of the detection. Therefore, the methods include one or more steps for preparing each sample, or composite sample, prior to molecular probing by RT-qPCR.

In some forms, the methods employ a purification process for the processing of a sample or composite sample (s) of sewage or waste water. The process includes:

(i) removal of large particulate matter from the liquid component of the sample to provide a primary supernatant;

(ii) ultracentrifugation of the primary supernatant to remove residual insoluble material and provide a secondary supernatant; and

(iii) extraction of RNA from the secondary supernatant to provide an input sample of purified RNA suitable for RT-qPCR.

In some forms, the methods include a step of

(a) concentrating a sewage sample collected from the area served by the sewageshed to provide a concentrated sewage sample. Typically, the sewage sample is concentrated by:

(1) centrifuging the sewage sample;

(2) collecting the resultant supernatant;

(3) ultracentrifuging the supernatant; and

(4) re-suspending the resultant pellet, thereby producing a concentrated sewage sample.

In some forms, the methods include steps of (b) extracting nucleic acids from the concentrated sewage sample, prior to detecting in the extracted nucleic acids one or more nucleic acid sequences indicative of the infectious disease agent, thereby detecting the presence of the infectious disease in the area served by the sewageshed.

In some forms, following step (2) and prior to step (3) , the methods include centrifuging the supernatant of step (2) . In some forms, the methods include extracting nucleic acids from the concentrated sewage sample by:

(1) lysing the concentrated sewage sample;

(2) phenol extracting the lysed concentrated sewage sample;

(3) precipitating nucleic acids from the aqueous phase of the phenol extraction; and

(4) cleaning the nucleic acids in a spin column.

Typically, the nucleic acid sequences indicative of the infectious disease are detected by quantitative polymerase chain reaction (qPCR) of the extracted nucleic acids. In some forms, the nucleic acid sequences indicative of the infectious disease are detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) of the extracted nucleic acids.

C. Detection of Infectious Agents within Sewage

Methods of detecting the presence of an infectious disease agent in an area served by a sewageshed are provided. A preferred RNA virus that is detected by the methods is a coronavirus virus, such as a SARS-CoV-2 virus. Typically, the detection includes one or more steps of reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) .

In some forms, the RT-qPCR is performed with primers to two or more target sequences in the infectious disease agent in separate reactions. An exemplary qPCR for use with the described methods is run for 45 cycles. In some forms, a cycle threshold (Ct) of less than 45 indicates a positive result for the primer set of the reaction. In other forms, a cycle threshold (Ct) less than 45 for none of the primer sets indicates a negative for the presence of the infectious disease agent in the sewage sample. In some forms, a cycle threshold (Ct) less than 45 for only one of the primer sets indicates the suspected presence of the infectious disease agent in the sewage sample. In other forms, a cycle threshold (Ct) of less than 45 for two or more of the primer sets indicates a positive for the presence of the infectious disease agent in the sewage sample.

In some forms, one primer set is configured to selectively combine with the N1 gene of SARS-CoV-2 viruses and another primer set is configured to selectively combine with the E gene of SARS-CoV-2.

In some forms, the methods include one or more optional steps, including performing RT-qPCR on a positive control, wherein the positive control has a plasmid comprising the N1 gene of SARS-CoV-2, and/or performing RT-qPCR on a negative control, wherein the negative control has no template, and/or sequencing the amplified nucleic acid to confirm the identity of the amplified nucleic acid.

1. Quantitative PCR

Methods for detecting the presence of SARS-CoV-2 within a sample typically include a step of contacting RNA extracted from a concentrated sewage sample with a composition including

(i) a set of primers configured to amplify one or more fragments of the SARS-CoV-2 virus;

(ii) a probe configured to bind to the amplified nucleic acid fragment; and

(iii) a RT-qPCR reaction mixture including reagents necessary for amplifying the one or more fragments of the SARS-CoV-2 virus.

The methods incubate the composition under conditions sufficient for an RT-qPCR reaction to amplify the one or more fragments of the SARS-CoV-2 virus to create an output sample.

The methods detect the one or more fragments of the SARS-CoV-2 virus and probe within the output sample,

wherein the presence of the one or more fragments of the SARS-CoV-2 virus and probe within the output sample identifies the input sample as containing SARS-CoV-2.

Typically, the contacting in step occurs within a thermal cycler or other apparatus suitable for conducting and monitoring necessary for conducting an RT-qPCR procedure.

In some forms, the Detecting step includes steps for quantifying and/or recording the number of copies of viral target RNAs within the sample.

In some forms, the methods include Recording the number of copies of viral target RNAs detected within the input sample in step. In some forms, the recording includes combining together with one or more additional pieces of datum relating to the input sample, or the environment from which the input sample is derived. For example, in some forms, the recording includes annotating the result of the assay for a sample, together with one or more time points, such as the collection time or interval time. In some forms, the recording combines the data from two or more assays to form one or more databases. For example, in some forms, the recording annotates the number of copies of viral target RNAs within each of two or more samples, together with one or more time points.

In some forms, the methods detect an amount of SARS-CoV-2 virus within sample derived from a sewage site associated with hospital isolation unit housing COVID-19 patient, whereby the sample is obtained within one, two, three, four, five, six, seven, eight, nine, or ten days or weeks or months following the initial sample collection from the sewage site.

In some forms, the methods include one or more additional steps of determining the sequence of one or more of the genes of a SARS-CoV-2 virus within a sample identified as containing SARS-CoV-2. In some forms, the methods include one or more steps for recording the sequence data from one or more genes of one or more SARS-CoV-2 viruses within one or more databases, optionally together with one or more pieces of data relating the same or different samples.

In some forms, the methods include screening one or more positive and/or negative controls. Exemplary positive controls include one or more RNA sequences encoding one or more of the target viral RNAs. Exemplary positive control RNA sequences are include plasmids, or as cells expressing SARS-CoV-2 viruses, or DNA plasmids containing the target sequences. Exemplary negative controls include one or more RNA sequences specific for one or more distinct human respiratory pathogen.

In some forms, one or more of the method steps are performed together with one or more control samples. An exemplary control sample is a matrix control, for example, a sewage sample spiked with a known amount of a known infectious disease agent. In some forms, one or more of the method steps are performed on a reagent blank. An exemplary reagent blank is a no sewage control sample.

D. Use as a Sewage Surveillance Tool

A testing method for surveying SARS-CoV-2 virus in sewage collected from sampling sites with various sites characteristics has been developed. The described methods are useful for the surveillance of infectious agents within sewage sheds and waste water systems. Exemplary sites include community sewer systems of individual buildings and large housing estates as well as entrances of conventional sewage treatment facilities (sewage pumping stations and sewage treatment works) serving up to one million residents.

In some forms, the sewage sample is a composite sewage sample formed according the described systems and methods for sampling a sewageshed for effective detection and assessment of infectious disease agent presence. In some forms, the methods include steps for recording and assessing the extent and spread of an infectious agent within a sewageshed or wastewater system. The data obtained from the methods can inform important public health decisions, and assist epidemiological studies.

As described in the Example, data collected during the 3rd and 4th wave of COVID-19 outbreak in Hong Kong and analyzing the correlations between the detected viral levels and the number of infected individuals living in the sewage shed, provided a classification scheme that generates actionable information for public health actions. Thus, in some forms, the methods provide sewage surveillance data to generate actionable information for the local council or government. In preferred forms, legal enforcement of compulsory testing is issued based on sewage surveillance obtained using the described testing method in combination with the classification scheme, for successful controlling of the spread of SARS-CoV-2 virus.

IV. Kits

Kits are also disclosed. The kits can include, for example, devices for acquisition of sewage or wastewater samples, and/or for the extraction and purification of viral RNA from the sample. In some forms, the kit includes apparatus for obtaining samples from a sewageshed, such as collection vials, syphons, syringes and/or pipettes. In some forms, kits include a set of oligonucleotide primers configured to amplify a fragment of the SARS-CoV-2 viral RNA, a nucleic acid probe configured to selectively bind and detect the fragment of the SARS-CoV-2 viral RNA amplified by the primers, and a RT-qPCR reaction mixture, including reagents and enzymes in an amount and concentration suitable for conducting at RT-qPCR. In some forms, the kit includes printed instructions for use of the reagents according to the methods described above. In some forms, the kit includes two or more of the components, packaged separately or together in the same admixture. Each of the reagents can be supplied alone (e.g., lyophilized) , or in a mixture composition. In some forms, the kit includes a supply of buffers and reagents required for multiple RT-qPCR reactions. In some forms, the kit includes one or more positive and/or negative controls for the RT-qPCR amplification of SARS-CoV-2 viral RNA.

The present invention is further understood by reference to the following non-limiting examples.

EXAMPLES

Example 1: Sewage Testing Method Evaluation

Since the shedding of SARS-CoV-2 (the causative virus of COIVD-19) in stool was reported, a large number of studies have detected the genetic signal of SARS-CoV-2 in sewage across various regions and countries. The presence of SARS-CoV-2 genetic material in sewage is reported to be ubiquitous. Viral shedding in fecal can happen in symptomatic, asymptomatic or pre-symptomatic carriers1, opening possibility for using sewage surveillance of SARS-CoV-2 as early warning for the re-emergence of COIVD-19. Infectiousness of COVID-19 prior to symptoms onset was estimated to be accounted for around 44%of the transmission, and early-warning signals for identification of mid and asymptomatic cases in communities would likely the key pre-requisite for effective strategies which could help to prevent such pre-symptomatic transmission.

A few studies have shown high-quality correlations between strength of the SARS-CoV-2 signal in sewage and the COVID-19 incidence rates in corresponding sewershed, suggesting the potential of sewage testing for assessing the prevalence or the development trend of COVID-19 in a community. For the use case of estimating community prevalence, multiple sources of uncertainty remain challenging for evaluation and computational modelling, such as viral load in stool (from 10 ^2.7 to 10 ^7.6 copies per mL in confirmed COIVD-19 cases in Hong Kong) , the degradation and distribution of virus in sewer system, and interference by matrix component in sewage on method sensitivity. As it stands, the collection and interpretation of sewage testing data is an emerging filed. The challenges of informative sewage surveillance, like method validation using proof-of-concept sites and interpretation of positive results alongside sample site characteristics, should be addressed to make it a robust supplement method to clinical surveillance.

Early detection and surveillance of SARS-CoV-2 is a key pre-requisite for effective control of COVID-19. Sewage testing has been increasingly employed as an alternative surveillance tool. Sample site characteristics can impact testing results and require further study in the early stage of use. The current study aimed to compare the implementations of sewage testing for SARS-CoV-2 across sampling sites with different sewage system characteristics.

A unique testing method for quantifying SARS-CoV-2 in sewage using heat-inactivated SARS-CoV-2 virus and sample collected from a local hospital treating COVID-19 patients. 107 sewage samples were collected covering the 3 ^rd wave of COVID-19 infection in Hong Kong (from June 8 to September 29, 2020) for testing. Surveyed sites include sewage systems associated with the proof-of-concept hospital, community sewer systems of public housing estates serving a few thousands of residents, and conventional sewage treatment systems with pumping stations and downstream treatment works. The classification scheme set forth in this study is new, not used anywhere else in the world.

A typical testing method includes three steps: 1) sewage concentration, 2) viral RNA extraction, and 3) virus detection via RT-qPCR (reverse transcription-quantitative polymerase chain reaction) . Based on general principles for sewage surveillance of SARS-CoV-2, it has been established the testing method in the current study by individually optimized the three steps of testing methods for sewage surveillance of COVID-19.

The procedures for testing the SARS-CoV-2 in sewage are outlined in Figure 1. The method for testing SARS-CoV-2 in sewage samples includes the following major experimental steps: inactivation, sample concentration, viral genetic material extraction, and quantification (Figure 1) . Ultracentrifugation is used for concentrating the SARS-CoV-2 from sewage samples. Unlike the reported practices in which swage sample is subjected to direct ultracentrifugation, a two-step separation method is used in the current study. A first step to first separate the supernatant and pellet from sewage, and the second step use the supernatant for concentrating the virus via ultracentrifugation. This two-step separation method improves the recovery for SARS-CoV-2 by minimizing the effects imposed by the complex matrix in sewage. This method has been validated by spiking experiments.

The combination of quantification results using two primer and probe sets targeting the N and E region of SARS-CoV-2 for interpreting the testing result has been shown with high sensitivity and specificity. The new classification criteria for “negative” , “suspected” and “positive” according to RT-qPCR results of the two primers are provided.

The whole testing method is simple enough to be used in any laboratories provided that necessary equipment like ultracentrifuge and RT-qPCR machine are available. Such simple protocols enable the delivery of the testing results in a rapid way.

Materials and Methods

Pretreatment 1000 mL inactivated samples are further centrifuged at 4750 g for 30 mins on an Allegra X-15R Centrifuge (Beckman Coulter; Indianapolis, IN) to divide into two subsamples, i.e. pellet and supernatant. Two concentration methods are used in our protocols to concentrate different volume of supernatant, i.e. 30 mL (small volume, Method 1) and 1000 mL (large volume, Method 2) .

For

Method

1, 30 mL supernatant is ultracentrifugated at 150000 g for 1 h on a Centrifuge Model Allegra X-15R (Beckman Coulter) . Supernatant is removed carefully without disturbing the pellet. The pellet is further resuspended with 100 μL PBS and transport into a new 1.5 mL microcentrifugal tube for RNA extraction.

For Method 2, 1000 mL supernatant is concentrated by centrifugation at 20000 g for 30 min on a Sorvall LYNX 4000 Superspeed Centrifuge (Thermo Scientific) and further ultracentrifugation at 150000 g for 1 h on a Centrifuge Model Allegra X-15R (Beckman Coulter) . Concentrated samples (～400 μL) are collected from the resuspension of a pellet via 300 μL PBS with pipette and used for RNA extraction directly.

Based on our current methods evaluation on sewage samples, the positive rates and viral concentration of Method 1 are both higher than Method 2. In other words, Method 1 is more sensitive and workable than Method 2 for sewage samples and the spiked samples.

RNA extraction

RNA from concentrated samples are extracted using TRIzol ^TM Plus RNA Purification Kit (Thermofisher) and used for SARS-CoV-2 detection. A reagent blank (200 μL RNase-free water in the extraction kit) is used as the negative control for the RNA extraction and quantification steps. The details of the protocol are shown below (slightly modified from the original protocol of Thermofisher. webpage thermofisher. com/order/catalog/product/12183555#/12183555)

1. Divide the concentrated samples (～400 μL) into two subsamples. Add 1 mL of TRIzol ^TM Reagent into two subsamples.

2. Pipet the lysate up and down several times to homogenize.

3. Incubate for 5 minutes to permit complete dissociation of the nucleoproteins complex.

4. Add 0.2 mL of chloroform or 50 μL of 4-bromoanisole per 1 mL of TRIzol ^TM Reagent used for lysis, then securely cap the tube.

5. Incubate for 2–3 minutes.

6. Centrifuge the sample for 15 minutes at 12,000×g at 4℃. The mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upper aqueous phase.

7. Transfer～800 μL of the colorless, upper aqueous phase containing the RNA to a new tube.

8. Add an equal volume of 70%ethanol, then mix well by vortexing.

9. Invert the tube to disperse any visible precipitate that may form after adding ethanol.

10. Transfer up to 700 μL of the two subsamples to the same spin cartridge (with collection tube)

11. Centrifuge at 12,000×g for 1 minute.

12. Discard the flow-through, then reinsert the spin cartridge into the same collection tube.

13. Repeat steps 11 and 12, until the entire two subsamples have been processed.

14. Add 700 μL of Wash Buffer I to the spin cartridge.

15. Centrifuge at 12,000×g for 1 minute.

16. Discard the flow-through, then reinsert the spin cartridge into the same collection tube.

17. Add 500 μL of Wash Buffer II to the spin cartridge.

18. Centrifuge at 12,000×g for 1 minute.

19. Discard the flow-through, then reinsert the spin cartridge into the same collection tube.

20. Repeat steps 18 and 19 once.

21. Centrifuge at 12,000×g for 1 minute to dry the membrane.

22. Discard the collection tube, then insert the spin cartridge into a recovery tube.

23. Add 40 μL of RNase-free water to the center of the spin cartridge.

24. Incubate for 1 minute.

25. Centrifuge at>12,000×g for 2 minutes.

26. Discard the spin cartridge.

27. The recovery tube contains the purified total RNA.

28. Store the purified RNA on ice if used within a few hours. For long-term storage, store the purified RNA at–80℃.

Viral kit (QIAamp Viral RNA Mini Kit (Qiagen) ) also could be used for the small volume sample, but performance is not as good as TRIzol.

Analysis of SARS-CoV-2

The 1-step RT-qPCR was carried out for 45 cycles in 20 μl reaction mixture using TagMan Fast Virus 1-step Master Mix (Thermo Fisher, USA) . We will use the probes and primers of N1 and E genes for detection of SARS-CoV-2. The one-step RT-qPCR reaction solution was prepared as follows: 4×TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher) 5 μl, forward primer 500 nm, reverse primer 500 nm, probe 250 nm, RNA template 4 μl, and DEPC-treated water to 20 μl.

The conditions used for RT-qPCR were as follows: 50℃ for 5 minutes, 95℃ for 20 seconds, 45 cycles of 95℃ for 5 seconds and 55℃ for 30 seconds. If the Ct value of a wastewater sample was≤45, the sample was considered to have SARS-CoV-2 RNA signal.

To quantify the copy number of virus, the standard curves for the target regions were generated by using serial dilutions of a plasmid carrying the target gene with concentrations ranging from 10 to 107 copies per reaction.

The quantification limit for the control plasmid was 10 copies per reaction. For quality assurance and quality control (QA/QC) , we used the reagents in RNA extraction kit as the negative control (called as the “reagent blank” ) for RNA extraction and quantification steps. The no template control (NTC) was included as the negative control for RT-qPCR.

Detection

1) "Negative" : no Ct≤45 for any primer set.

2) "Suspected" : only one primer has Ct≤45.

3) "Positive" : Ct≤45 for at least two primer sets.

Quantification (concentration calculation) for those Ct≤45

1) Calculated based on the standard curves of different primer sets.

2) If the calculated copy number per reaction is lower than the theoretical limit (1 copy/reaction) , will report as “<10 copy/L sewage” which is the quantification limit of the current method.

If there is quantification result for more than one primer sets, report the highest concentration.

Results

Quality Assurance And Quality Control (QA/QC)

The method involves a two-step concentration procedure for SARS-CoV-2, followed by RNA extraction, and quantification of SARS-CoV-2 genetic signal via a N-gene-specific RT-qPCR (Figure 2) . To ensure the reliability of the results, a quality assurance and quality control (QA/QC) checklist was implemented as shown in Figure 2. Different concentration protocols were compared and evaluated. The detection and quantification assays were then validated for the sewage matrix. Quality indicator were included at individual steps: the reagents in RNA extraction kit was used as the negative control (called as the “reagent blank” ) for RNA extraction and quantification steps. No template control (NTC) and N2-carrying plasmid were used as the negative and positive control for RT-qPCR, respectively. Sample metadata together with experimental methods and results were thoroughly explored as a part of the QA/QC (data not shown) .

Concertation Method

The methods for concentrating the SARS-CoV-2 genetic signal were first assessed using the same sewage sample collected from a hospital treating COVID-19 patients. Commonly applied methods including 0.45 μm membrane filtration, PEG precipitation, AlCl3 precipitation, ultracentrifuge, and ultrafiltration were evaluated. By comparing the type of concentration methods and the processed volume of sewage, it was observed that the centrifugal ultrafiltration applied to a small sample volume (90 mL) has comparable performance for virus concentration when compared with membrane filtration or precipitation applied to large sample volume up to 1 L. But regarding the sensitivity of the testing method, a larger sample volume allows for an elevated concentration factor of sample, which is expected to lower the detection limit for quantification assays. The data agreed with this hypothesis. As shown in the Table 1, for the assay No. 3 using a small sample volume of 90 mL and the assay No. 6 using a large sample volume of 840 mL, despite the comparative final concentration of virus in sewage, the copy number per RT-qPCR reaction of the former was nearly 10-fold lower that latter. This result suggested that the large sample volume can increase the detection of virus in sewage by enhancing sensitivity. However, since the method using large volumes can make samples intractable to process in laboratories, the method for small sample volume which has comparative performance in the final testing result was used in this study.

RT-qPCR Determinations of Viral Genetic Signal in Sewage Matrix

The primer-probe set in the N-gene-specific RT-qPCR has been reported with high efficiency and analytical sensitivity (Pan, Y. et al., The Lancet Infectious Diseases 2020, 20, (4) , 411-412; Chu, DK et al., Clinical chemistry 2020, 66, (4) , 549-555; Vogels, CB et al., Nature microbiology 2020, 5, (10) , 1299-1305) , enabling the detection of SARS-CoV-2 viral RNA ranging from 10 to 10 ⁷copies per reaction. Since the sewage matrix is very different from clinical specimens, studies were carried out to examine to what extents the genetic extraction and quantification of genetic signal can be impacted by the complexity of the sewage matrix. By spiking heat-inactivated SARS-CoV-2 into a small volume of sewage sample and the control sample (Phosphate Buffered Saline (PBS) solution) , the influence of the sewage matrix was assessed by comparing the concentrations of viral RNA extracts obtained by two commonly used RNA extraction kits (QIAamp Viral RNA mini kit and TRIzol ^TM Plus RNA purification kit) . The matrix effect of sewage was negligible with respect to the detected Ct value (Figures 3A-3B) , indicating that the applied quantification approach has considerable utility for sewage samples. Regarding the viral concentration quantified in copy number of virus per mL sample, the sewage matrix did not yield difference over 0.6 log-unit per mL (Figures 4A-4B) . For RNA extracts obtained by QIAamp Viral RNA mini kit and TRIzol ^TM Plus RNA purification kit, the matrix effects of sewage ranged 0.1-0.6 and 0.1-0.2 log viral copy number per mL of sample, respectively.

Detection rate and dilution effect in the trail of a hospital treating COVID-19 patients

As shown in Table 2, among 107 sewage samples tested covering the 3rd wave of COVID-10 infections in Hong Kong (from June 8, 2020 to September 29, 2020) , the signal of SARS-CoV-2 was detected in 20 out of 107 (19%) sewage samples. These included 7 (out of 12, 58%) samples collected from sites associated with a hospital treating COVID-19 patients, 4 (out of 8, 50%) samples collected from individual buildings and combined manhole of public housing estates, and 10 (out of 87, 11%) samples collected from sewage pumping stations and sewage treatment works which serve population ranging from around 40 thousands to more than 1 million. Figure 6 shows the daily local cases in Hong Kong on the date of sample collection.

Overall, the established sewage testing method for SARS-CoV-2 is technically feasible for the sewershed scaling from manholes of individual buildings to entrances of large sewage treatment facilities. The average detection rates of 58%, 50%, and 10%were observed for samples collected from the hospital trail, community sewer system, and sewage treatment facilities, respectively. The comparable detection rates for the hospital trail and community sewer system suggested the effectiveness of the current method in providing presence/absence information of SARS-CoV-2 for the community sewage.

Since the sites associated with hospital trail served as positive control for sites being tested, the incidence of detection of positive signal (i.e., the detection rate) in hospital trail sites may reflect the effects of randomness in providing the presence/absence information. A detection rate of 66.7% (10 out of 15) was reported for hospital sewage collected at a downstream pumping station.

The highest level of SARS-CoV-2 (1975 copy/mL sewage) was observed in sewage directly collected from the manhole (PMH-1) of the isolation ward of PMH. Such virus concentration was higher than the reported 255 copy/L and 633 copy/L for samples collected at the adjusting tanks of a hospital. Along this hospital trail, the samples taken from a manhole (PMH-2) at the downstream had a range of 0.4～16 copy/mL, while the further downstream samples at PMH-3 (Waterboat Dock SPS) had 46 copy/mL (July 23) and 0.7 copy/mL (August 18) . These results show the dilution of the signal along the sewage pipeline from upstream to downstream.

Sensitivity of the method for testing sewage treatment facilities

In general, negative or suspected positive results were randomly observed for 87 samples from sewage treatment facilities due to the dilution of the signal along the pipeline from upstream to downstream. The randomness of positive results for quantifying SARS-CoV-2 in the samples from sewage treatment facilities is prone to dilution of genetic signal in the sewer system which may reach the marginal level of the detection method. To use sewage surveillance of SARS-CoV-2 at sewage treatment facilities as an indicator for the COVID-19 outbreak in a large district, efforts should be concentrated to address challenge of the low concentration or diluted signal.

Summary

This is the first study that reports the sewage testing for SARS-CoV-2 in Hong Kong. A sewage testing method has been validated using heat-inactivated SARS-Cov-2 and the applicability of the method has been confirmed in determining the genetic signal of SARS-CoV-2 in manholes outside buildings, pipes connecting community sewage, and entrances to downstream pumping stations and conventional treatment works. Especially, performance of the sewage testing method by analyzing “positive” samples from a hospital trial was evaluated. It has revealed 7 out of 12 samples (58%) in the hospital trail were positive and observed the dilution of virus signal along the pipeline of the hospital trail. Missing of signal was also observed, which can be probably due to the indicated the effects of sampling randomness at upstream hospital manhole or the dilution at downstream sewage treatment facilities. This is the first study that implemented sewage testing for a hospital trial and compared results across sewer system characteristics to provide more context for the interpretation of positive sewage samples.

The study indicated that interpretation of sewage surveillance results alongside sampling site characteristics is more informative to generate assessment principles and actionable recommendations for decision makers. Use of sewage surveillance of SARS-CoV-2 to obtain early warning signals for potential community outbreak has been mainly reported in retrospective studies (3, 13, 22-24) , while only two studies have successfully demonstrated this use case for large sewage treatment works covering 28,000～101,000 population (12, 16) . In the current study, sewage testing has been used for providing information on the presence of infected individuals shedding SARS-CoV-2 to single-building sewer system serving about one thousand residents before the first cases of COVID-19 were identified. Despite the promising results for public estates, monitoring at individual building level for a big area is very resource-demanding. Since monitoring virus concentration at downstream sewage treatment facilities can be used to infer the infection trends (25) , testing for sewage collected from sewage treatment facilities is more applicable for longitudinal sewage surveillance. But this strategy is also fraught with challenges when the concentration of SARS-CoV-2 virus in sewage is low. This is a ratio of infected cases to population in the catchment (assuming the viral load per individual is the same) . To overcome dilution effects at downstream sewage treatment facilities, method with increased sensitivity is desired. Improved sensitivity of current testing methods is possible by lowering the quantification limits of the whole method, including optimizations of sampling strategy to peak hour of shedding fecal matter, evaluations of virus concentration and extraction protocols for larger volume of sewage sample, as well as assessments of primer-probe sets and kits in the RT-qPCR. For the quantification of SARS-CoV-2 using RT-qPCR, a comparison for different primer-probe sets targeting various genetic loci of the virus assists to discern their performance for sewage samples. The utilization of a larger reaction volume for one-step RT-qPCR with increased sample template was considered, aiming to lower the detection limit.

For the interpretation of positive samples, the analytical limitations should be specified. Implementation of suitable quality indicators for process is considered as essential for testing performance and also a way to improve reproducibility and reliability of the testing results. Sequence-based identification of virus is a useful quality indicator for the testing method. Besides, a clear separation in the handling and process of samples is needed to lower the risk of cross-contamination of samples. However, negative samples do not imply the absence of the virus. The randomness in sampling is one of the sources of uncertainty. Such uncertainty has also been observed in this study. Sample taken from Lok Yan House on 29 July was tested negative for SARS-CoV-2 but there was a reported infection case (#2881) in that building before the sampling time. This implies that the applied testing method can miss the positive case in a building or a small catchment area, as known from the basic principle. On the other hand, if detected, the signal can be very strong.

Assessment of the correlation between sewage surveillance data and clinic testing data is critical to generate actionable information in advance of the next surge of COVID-19. Longitudinal sewage analysis covering the majority of the COVID-19 outbreak will assist to avoid potential misinterpretation of the sewage testing results. For example, a study tested 116 sewage samples from local sewage treatment works during the entire outbreak period from January to May 2020 (first cases were reported on March 3, 2020) and explored the correlations to the infection rate in the community. This study demonstrated the high-quality correlations between the strength of genetic signal of SARS-CoV-2 in sewage and the number of infected cases (27) . And it was concluded that the dynamics of SARS-CoV-2 in sewage treatment works can be used to foreshadow the trend of COVID-19 transmission, with 4-10 days in advance of clinical cases reporting. It should be noted that in principle, the time lag of 4-10 day is a reflection of the transmission rate in the community, so this number is perhaps not applicable for the situation in other regions like Hong Kong. In comparison, in the current study, such correlation analysis is limited by the low concentrations of SARS-CoV-2 in the tested sewage samples (general a few copies per mL of sewage compared with up to 1500 copies per in the reported study) , as well as the relatively low prevalence of infection cases in local communities. On the top of serving early waning and treading analysis, pinpointing the number of infected individuals in a sewershed as well as the integration of current data source to set actionable assessment criteria can be the ways forward for this emerging method.

References

1. Xu, Y.; Li, X.; Zhu, B.; Liang, H.; Fang, C.; Gong, Y.; Guo, Q.; Sun, X.; Zhao, D.; Shen, J., Characteristics of pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral shedding. Nature medicine 2020, 26, (4) , 502-505.

2. He, X.; Lau, E.H.; Wu, P.; Deng, X.; Wang, J.; Hao, X.; Lau, Y.C.; Wong, J.Y.; Guan, Y.; Tan, X., Temporal dynamics in viral shedding and transmissibility of COVID-19. Nature medicine 2020, 26, (5) , 672-675.

3. Nemudryi, A.; Nemudraia, A.; Wiegand, T.; Surya, K.; Buyukyoruk, M.; Cicha, C.; Vanderwood, K.K.; Wilkinson, R.; Wiedenheft, B., Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater. Cell Reports Medicine 2020, 1, (6) , 100098.

4. Wu, F.; Xiao, A.; Zhang, J.; Gu, X.; Lee, W.L.; Kauffman, K.; Hanage, W.; Matus, M.; Ghaeli, N.; Endo, N., SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases. medRxiv 2020.

5. Stadler, L.B.; Ensor, K.; Clark, J.R.; Kalvapalle, P.; LaTurner, Z.W.; Mojica, L.; Terwilliger, A.L.; Zhuo, Y.; Ali, P.; Avadhanula, V., Wastewater Analysis of SARS-CoV-2 as a Predictive Metric of Positivity Rate for a Major Metropolis. medRxiv 2020.

6. Pan, Y.; Zhang, D.; Yang, P.; Poon, L.L.; Wang, Q., Viral load of SARS-CoV-2 in clinical samples. The Lancet Infectious Diseases 2020, 20, (4) , 411-412.

7. Cheung, K.S.; Hung, I.F.; Chan, P.P.; Lung, K.; Tso, E.; Liu, R.; Ng, Y.; Chu, M.Y.; Chung, T.W.; Tam, A.R., Gastrointestinal manifestations of SARS-CoV-2 infection and virus load in fecal samples from the Hong Kong cohort and systematic review and meta-analysis. Gastroenterology 2020.

8. Ahmed, W.; Angel, N.; Edson, J.; Bibby, K.; Bivins, A.; O'Brien, J.W.; Choi, P.M.; Kitajima, M.; Simpson, S.L.; Li, J., First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community. Science of The Total Environment 2020, 138764.

9. Haramoto, E.; Malla, B.; Thakali, O.; Kitajima, M., First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan. medRxiv 2020.

10. Zhang, D.; Ling, H.; Huang, X.; Li, J.; Li, W.; Yi, C.; Zhang, T.; Jiang, Y.; He, Y.; Deng, S., Potential spreading risks and disinfection challenges of medical wastewater by the presence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) viral RNA in septic tanks of Fangcang Hospital. Science of The Total Environment 2020, 741, 140445.

11. Zhang, D.; Yang, Y.; Huang, X.; Jiang, J.; Li, M.; Zhang, X.; Ling, H.; Li, J.; Liu, Y.; Li, G., SARS-CoV-2 spillover into hospital outdoor environments. medRxiv 2020.

12. Randazzo, W.; Truchado, P.; Cuevas-Ferrando, E.; Simón, P.; Allende, A.; Sánchez, G., SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area. Water Research 2020, 115942.

13. Randazzo, W.; Cuevas-Ferrando, E.; Sanjuan, R.; Domingo-Calap, P.; Sanchez, G., Metropolitan Wastewater Analysis for COVID-19 Epidemiological Surveillance. Available at SSRN 35866962020.

14. Wurtzer, S.; Marechal, V.; Mouchel, J. -M.; Maday, Y.; Teyssou, R.; Richard, E.; Almayrac, J.L.; Moulin, L., Evaluation of lockdown impact on SARS-CoV-2 dynamics through viral genome quantification in Paris wastewaters. medRxiv 2020.

15. Green, H.; Wilder, M.; Middleton, F.A.; Collins, M.; Fenty, A.; Gentile, K.; Kmush, B.; Zeng, T.; Larsen, D.A., Quantification of SARS-CoV-2 and cross-assembly phage (crAssphage) from wastewater to monitor coronavirus transmission within communities. medRxiv 2020.

16. Medema, G.; Heijnen, L.; Elsinga, G.; Italiaander, R.; Brouwer, A., Presence of SARS-Coronavirus-2 RNA in sewage and correlation with reported COVID-19 prevalence in the early stage of the epidemic in the Netherlands. Environmental Science & Technology Letters 2020.

17. Chu, D.K.; Pan, Y.; Cheng, S.M.; Hui, K.P.; Krishnan, P.; Liu, Y.; Ng, D.Y.; Wan, C.K.; Yang, P.; Wang, Q., Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia. Clinical chemistry 2020, 66, (4) , 549-555.

18. Vogels, C.B.; Brito, A.F.; Wyllie, A.L.; Fauver, J.R.; Ott, I.M.; Kalinich, C.C.; Petrone, M.E.; Casanovas-Massana, A.; Muenker, M.C.; Moore, A.J., Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT–qPCR primer–probe sets. Nature microbiology 2020, 5, (10) , 1299-1305.

19. Rocha, J.; Manaia, C.M., Cell-based internal standard for qPCR determinations of antibiotic resistance indicators in environmental water samples. Ecological Indicators 2020, 113, 106194.

20.

J.; Koritnik, T.;

V.; Trkov, M.;

M.; Berginc, N.; Prosenc, K.; Kotar, T.; Paragi, M., Detection of SARS-CoV-2 RNA in hospital wastewater from a low COVID-19 disease prevalence area. Science of The Total Environment 2020, 143226.

21. Achak, M.; Alaoui, S.B.; Chhiti, Y.; Alaoui, F.E.M. h.; Barka, N.; Boumya, W., SARS-CoV-2 in hospital wastewater during outbreak of COVID-19: A review on detection, survival and disinfection technologies. Science of The Total Environment 2020, 143192.

22. La Rosa, G.; Mancini, P.; Ferraro, G.B.; Veneri, C.; Iaconelli, M.; Bonadonna, L.; Lucentini, L.; Suffredini, E., SARS-CoV-2 has been circulating in northern Italy since December 2019: Evidence from environmental monitoring. Science of the Total Environment 2020, 750, 141711.

23. Chavarria-Miró, G.; Anfruns-Estrada, E.; Guix, S.; Paraira, M.; Galofré, B.; Sáanchez, G.; Pintó, R.; Bosch, A., Sentinel surveillance of SARS-CoV-2 in wastewater anticipates the occurrence of COVID-19 cases. MedRxiv 2020.

24. Fongaro, G.; Stoco, P.H.; Souza, D.S.M.; Grisard, E.C.; Magri, M.E.; Rogovski, P.; Schorner, M.A.; Barazzetti, F.H.; Christoff, A.P.; de Oliveira, L.F.V., SARS-CoV-2 in human sewage in Santa Catalina, Brazil, November 2019. medRxiv 2020.

25. Schmidt, C., Watcher in the wastewater. Nature biotechnology 2020, 38, (8) , 917-920.

26. O'Reilly, K.M.; Allen, D.J.; Fine, P.; Asghar, H., The challenges of informative wastewater sampling for SARS-CoV-2 must be met: lessons from polio eradication. The Lancet Microbe 2020, 1, (5) , e189-e190.

27. Wu, F.; Xiao, A.; Zhang, J.; Moniz, K.; Endo, N.; Armas, F.; Bonneau, R.; Brown, M.A.; Bushman, M.; Chai, P.R., SARS-CoV-2 titers in wastewater foreshadow dynamics and clinical presentation of new COVID-19 cases. Medrxiv 2020.

Claims

A method of sampling a sewageshed for effective detection and assessment of infectious disease agent presence in an area served by the sewageshed, the method comprising:

collecting a first plurality of sewage samples at a first specified sewage system location of the sewageshed, wherein the first plurality of sewage samples are collected at approximately equal time intervals during a first collection period; and

pooling the first plurality of sewage samples to form a first composite sewage sample.
The method of claim 1, wherein the area served by the sewageshed is a single building, a single building complex, a single campus, a single city block, a single neighborhood, a single community, a single city, or a single district.
The method of claim 1 or 2, wherein the first specified sewage system location is a building drain pipe, a building complex drain pipe, a street sewer pipe, a pumping station, or a wastewater treatment plant.
The method any one of claims 1-3, wherein the first collection period is approximately proportional to the average distance of the first specified sewage system location from buildings served by the first specified sewage system location.
The method of any one of claims 1-4, wherein the first collection period is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10, hours, 11 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours.
The method of any one of claims 1-5, wherein the time intervals at which the first plurality of sewage samples are collected is 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, or 60 minutes.
The method of any one of claims 1-6, wherein the first plurality of sewage samples comprises at least 2 sewage samples, 3 sewage samples, 4 sewage samples, 5 sewage samples, 6 sewage samples, 7 sewage samples, 8 sewage samples, 9 sewage samples, 10 sewage samples, 11 sewage samples, 12 sewages samples, 14 sewage samples, 16 sewage samples, 18 sewage samples, 20 sewage samples, 22 sewage samples, 24 sewage samples, 25 sewage samples, 30 sewage samples, 35 sewage samples, 40 sewage samples, 45 sewage samples, 50 sewage samples, 55 sewage samples, 60 sewage samples, 65 sewage samples, 70 sewage samples, 75 sewage samples, 100 sewage samples, or 125 sewage samples.
The method of any one of claims 1-7 further comprising:

collecting a second plurality of sewage samples at a second specified sewage system location of the sewageshed, wherein the second plurality of sewage samples are collected at approximately equal time intervals during a second collection period; and

pooling the second plurality of sewage samples to form a second composite sewage sample.
The method of claims 8, wherein the second specified sewage system location is different from the first specified sewage system location.
The method of claim 8 or 9, wherein the second collection period is approximately proportional to the average distance of the second specified sewage system location from buildings served by the second specified sewage system location.
The method of any one of claims 8-10 further comprising:

collecting a third plurality of sewage samples at a third specified sewage system location of the sewageshed, wherein the third plurality of sewage samples are collected at approximately equal time intervals during a third collection period; and

pooling the third plurality of sewage samples to form a third composite sewage sample.
The method of claims 11, wherein the third specified sewage system location is different from the first specified sewage system location and the second specified sewage system location.
The method of claim 8 or 9, wherein the third collection period is approximately proportional to the average distance of the third specified sewage system location from buildings served by the third specified sewage system location.
A method of detecting the presence of an infectious disease agent in an area served by a sewageshed, the method comprising:

(a) concentrating a sewage sample collected from the area served by the sewageshed, wherein the sewage sample is concentrated by:

(1) centrifuging the sewage sample;

(2) collecting the resultant supernatant;

(3) ultracentrifuging the supernatant; and

(4) resuspending the resultant pellet,

thereby producing a concentrated sewage sample;

(b) extract nucleic acids from the concentrated sewage sample; and

(c) detecting in the extracted nucleic acids one or more nucleic acid sequences indicative of the infectious disease agent, thereby detecting the presence of the infectious disease in the area served by the sewageshed.
The method of claim 14 further comprising, following step (2) and prior to step (3) , centrifuging the supernatant of step (2) .
The method of claim 14 or 15, wherein the nucleic acids are extracted from the concentrated sewage sample by:

(1) lysing concentrated sewage sample;

(2) phenol extracting the lysed concentrated sewage sample;

(3) precipitating nucleic acids from the aqueous phase of the phenol extraction; and

(4) cleaning the nucleic acids in a spin column.
The method of any one of claims 14-16, wherein the nucleic acid sequences indicative of the infectious disease are detected by quantitative polymerase chain reaction (qPCR) of the extracted nucleic acids.
The method of any one of claims 14-17, wherein the nucleic acid sequences indicative of the infectious disease are detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) of the extracted nucleic acids.
The method of any one of claims 14-18, wherein the infectious disease agent is a virus.
The method of any one of claims 14-19, wherein the infectious disease agent is an RNA virus.
The method of any one of claims 14-20, wherein the infectious disease agent is a coronavirus virus.
The method of any one of claims 14-21, wherein the infectious disease agent is SARS-CoV-2.
The method of any one of claims 14-22 further comprising performing the method steps on a matrix control, wherein the matrix control is a sewage sample spiked with the infectious disease agent.
The method of any one of claims 14-23 further comprising performing the method steps on a reagent blank, wherein the reagent blank is a no sewage control sample.
The method of any one of claims 14-23, wherein the sewage sample is a composite sewage sample formed according the method of any one of claims 1-13.
The method of any one of claims 17-25, wherein the qPCR is performed with primers to two or more target sequences in the infectious disease agent in separate reactions.
The method of claim 26, wherein the qPCR is run for 45 cycles.
The method of claim 26 or 27, wherein a cycle threshold (Ct) less than 45 indicates a positive for the primer set of the reaction.
The method of any one of claims 26-28, wherein a cycle threshold (Ct) less than 45 for none of the primer sets indicates a negative for the presence of the infectious disease agent in the sewage sample.
The method of any one of claims 26-28, wherein a cycle threshold (Ct) less than 45 for only one of the primer sets indicates a suspected for the presence of the infectious disease agent in the sewage sample.
The method of any one of claims 26-28, wherein a cycle threshold (Ct) less than 45 for two or more of the primer sets indicates a positive for the presence of the infectious disease agent in the sewage sample.
The method of any one of claims 26-31, wherein the infectious disease agent is SARS-CoV-2.
The method of claim 32, wherein one primer set is to the N1 gene of SARS-CoV-2 and another primer set is to the E gene of SARA-CoV-2.
The method of any one of claims 26-33 further comprising performing qPCR on a positive control, wherein the positive control has a plasmid comprising the N1 gene of SARS-CoV-2.
The method of any one of claims 26-34 further comprising performing qPCR on a negative control, wherein the negative control has no template.
The method of any one of claims 26-35 further comprising sequencing the amplified nucleic acid to confirm the identity of the amplified nucleic acid.