Method for the detection of human pathogenic viruses in water, in particular wastewater

Abstract

The present invention relates to a method for detecting viruses, in particular respiratory viruses in a water sample, in particular a wastewater sample, comprising amplifying viral nucleic acid sequences present in the sample using certain improved oligonucleotides or a set of oligonucleotides and detecting an amplification product of viral nucleic acid sequences present in the sample and thereby detecting respiratory viruses in the wastewater sample. The present invention further relates to improved oligonucleotides or a set thereof, and to a kit comprising materials for carrying out the method according to the invention. Furthermore, the invention relates to the use of the kit or an oligonucleotide or set of oligonucleotides for wastewater monitoring, in particular for the detection and quantification of human pathogenic viruses in water samples, in particular in wastewater samples, in particular as multiplex PCR.

Description

[0001] Method for the detection of human pathogenic viruses in water, in particular wastewater

[0002] The present invention relates to a method for detecting viruses, in particular respiratory viruses in a water sample, in particular a wastewater sample, comprising amplifying viral nucleic acid sequences present in the sample using certain improved oligonucleotides or a set of oligonucleotides and detecting an amplification product of viral nucleic acid sequences present in the sample and thereby detecting viruses in the wastewater sample. The present invention further relates to improved oligonucleotides or a set thereof, and to a kit comprising materials for carrying out the method according to the invention. Furthermore, the invention relates to the use of the kit or an oligonucleotide or set of oligonucleotides for wastewater monitoring, in particular for the detection and quantification of human pathogenic viruses in water samples, in particular in wastewater samples, in particular as multiplex PCR.

[0003] Background to the invention

[0004] In principle, many different pathogens can be detected in wastewater. For the poliovirus, wastewater surveillance has been an established monitoring tool in some countries for many decades. Various aspects have to be taken into account here: e.g. the relevance for public health, possible measures or the risk of reintroduction of a pathogen. In the AMELAG project, wastewater samples are currently being tested for SARS-CoV-2 and influenza A and B viruses. The benefits of wastewater surveillance for respiratory syncytial virus (RSV) and antibioticresistant bacteria are also being examined.

[0005] In wastewater monitoring, both RT-qPCR and RT-dPCR are used to quantitatively detect target pathogens and determine their concentrations. Classic probe-based qPCR is based on exponential target gene amplification and fluorescence-based quantification, which allows a broad concentration spectrum to be covered.

[0006] WO 2010 / 097490A1 describes a sensitive test for the detection of porcine adenoviruses in environmental samples, water and food. More specifically, the invention relates to a quantitative tool intended for the analysis of porcine adenoviruses as indicators of the presence of porcinecontamination. The invention further relates to a kit comprising two sequences of primers and a probe sequence capable of detecting and quantifying the aforementioned virus by PCR.

[0007] WO 2011 / 061373 A2 describes a quantitative PCR assay specifically for bovine polyomavirus that determines the levels and concentration of bovine polyomaviruses in urine and environmental samples, including municipal wastewater, slaughterhouse effluent and river water. The assay provides a quantitative method for tracing the source of contamination for the analysis of bovine polyomaviruses as indicators of the presence of bovine contamination in environmental samples.

[0008] WO 2021 / 222832A1 relates to methods and kits for detecting a virus, e.g. a respiratory virus, such as a coronavirus, in a biological sample. The invention also relates to methods and kits for detecting and / or quantifying biomarkers, e.g. antibody biomarkers against a viral antigen; biomarkers for inflammatory and / or tissue damage responses; and / or extracellular vesicles in response to a viral infection.

[0009] WO 2021 / 168478A1 describes compositions, assays, methods, diagnostic methods, kits and diagnostic kits for the specific and differential detection of SARS-CoV-2 and / or other viruses from samples, including veterinary samples, clinical samples, food samples, forensic samples, environmental samples (e.g. from soil, waste, sewage, air, water, food processing and manufacturing surfaces or the like) or biological samples obtained from a human or non-human animal.

[0010] US7563577B2 describes polynucleotides and methods for the detection and quantification of RNA viruses, such as enteroviruses and noroviruses, including in environmental samples. In one aspect, the invention provides amplification primers and labeled molecular beacons for the amplification of viral nucleic acid sequences. In another aspect, the invention provides a synthetic RNA internal control. In another aspect, the invention provides a kit for detecting the presence of enteroviruses and / or noroviruses in a sample.

[0011] Lansivaara et al. (published in: (2024). Wastewater-Based Surveillance of Respiratory Syncytial Virus Epidemic at the National Level in Finland. ACS ES and T Water, 4(6), 2403-2411. https: / / doi.org / 10.1021 / acsestwater.3c00752) describe a protocol for RSV detection inwastewater, but the test does not allow for a differentiation between genotypes A and B due to the primer binding sites.

[0012] Zhang et al. ((2006) RNA Viral Community in Human Feces: Prevalence of Plant Pathogenic Viruses. PLoS Biol 4(1): e3. https: / / doi.org / 10.1371 / journal.pbio.0040003) described a comparative metagenomic analysis of the RNA viruses found in three stool samples from two healthy human individuals.

[0013] Haramoto et al. (2013. Occurrence of Pepper Mild Mottle Virus in Drinking Water Sources in Japan. Appl Environ Microbiol 79; https: / / doi.org / 10.1128 / AEM.02354-13) described a determination of the seasonal and geographical occurrence of PMMoV in drinking water sources throughout Japan.

[0014] The GT-Digital SARS-CoV-2 Wastewater Surveillance Assay For QIAcuity™ consists of a molecular reagent kit containing all primers, probes and controls for wastewater surveillance of SARS-CoV-2 in accordance with the CDC Wastewater Surveillance Testing Method Guidance for reporting to the National Wastewater Surveillance System (NWSS). For the quantification of 1) SARS-CoV-2 (N1 and N2, published by CDC); 2) Bovine Coronavirus (BCoV), a matrix recovery control (also called a process control) that is biologically similar to SARS-CoV-2; and 3) Pepper Mild Mottle Virus (PMMoV), a stool indicator that can estimate the human stool content in a sample.

[0015] Boehm Alexandria et al. (in: "Wastewater concentrations of human influenza, metapneumovirus, parainfluenza, respiratory syncytial virus, rhinovirus, and seasonal coronavirus nucleic-acids during the COVID-19 pandemic: a surveillance study", The Lancet Microbe, vol. 4, no. 5 22 March 2023, pages E340-E348, XP093309508), disclose an assay design using an RSV-B quantification through the N-gene. This has considerable disadvantages with respect to sensitivity when comparing RS V- A with RSV-B.

[0016] Boehm Alexandria et al. (in: "More than a Tripledemic: Influenza A Virus, Respiratory Syncytial Virus, SARS-CoV-2, and Human Metapneumovirus in Wastewater during Winter 2022-2023", ENVIRONMENTAL SCIENCE & TECHNOLOGY LETTERS, vol. 10, no. 8, 20 July 2023, pages 622-627, XP093309509) disclose a multiplex PCR monitoring of wastewaterfor SARS-CoV-2, BCoV, PMMoV, Influenza A, RSV, HMPV using for each of said viruses a specific set of primer pair plus probe, i.e. a pan-RSV assay different from the present invention.

[0017] Despite considerable progress in monitoring the viral load of wastewater, there are still problems with the robustness, sensitivity and ease of use of PCR-based tests. It is therefore the object of the present invention to provide improved and at the same time commercially viable tests. Other objects and advantages will also become apparent to the skilled person after studying the description of the invention provided here.

[0018] In a first aspect, the problem of the invention is solved by providing a method for the detection of respiratory viruses in a water sample, in particular a wastewater sample, comprising the steps of i) Providing a water sample, in particular a wastewater sample, which potentially contains respiratory viruses, ii) Amplifying of viral nucleic acid sequences potentially present in the sample, comprising the use of at least one primer, in particular a primer pair comprising the sequences: ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3) and AGTGATAGATCATTGTCGCTATC (RSV P 1 Rev, SEQ ID NO: 4), and optionally GCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), and GATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and TTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6), and iii) Detecting of an amplification product of viral nucleic acid sequences present in the sample and thereby detecting of respiratory viruses in the water sample, in particular wastewater sample, preferably further comprising the use of at least one probe selected from the group of the sequences TTACTAATGCTGCTA (RSVA N I Probe, SEQ ID NO: 7), AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8), and TAGCGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9).

[0019] Preferred is a method according to the present invention, comprising the use of at least one primer and probe pair selected from the group consisting of: GCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), ATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and TTACTAATGCTGCTA (RSVA N I Probe, SEQ ID NO: 7), and ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3), AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4), and AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8), and optionallyTTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5), TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6), and TAGCGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9).

[0020] In a second aspect, the problem of the invention is solved by providing a method for the detection of viruses in a water sample, in particular a wastewater sample, comprising the steps of i) Providing a water sample, in particular a wastewater sample, which potentially contains respiratory viruses, ii) Amplifying of viral nucleic acid sequences potentially present in the sample, comprising the use of at least one primer, in particular a primer pair comprising the sequences: TTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6), and optionally GCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), and GATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3) and AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4), and iii) Detecting of an amplification product of viral nucleic acid sequences present in the sample and thereby detecting of respiratory viruses in the water sample, in particular wastewater sample, preferably further comprising the use of at least one probe selected from the group comprising the sequences TTACTAATGCTGCTA(RSVA_N_l_Probe, SEQ ID NO: 7), AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8), and TAGCGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9).

[0021] Preferred is a method according to the present invention, wherein the amplification reaction is a primer extension, a PCR, a digital PCR, a quantitative PCR, an RT-PCR, in particular an RT-dPCR, RT-ddPCR or RT-qPCR.

[0022] Further preferred is a method according to the present invention, wherein the respiratory virus is RSV-A and / or RSV-B.

[0023] In a third aspect, the problem of the invention is solved by providing a method for the detection of respiratory viruses in a water sample, in particular wastewater sample, according to the present invention, wherein the amplification further comprises the use of at least one primer selected from the group of primers comprising the sequences CAAGACCAATCYTGTCACCTCTGAC (INF A For 1, SEQ ID NO: 10),CAAGACCAATYCTGTCACCTYTGAC (INF A For 2, SEQ ID NO: 11), GCATTYTGGGACAAAVCGTCTACG (INF A Rev 1, SEQ ID NO: 12), GCATTTTGGGATAAAGCGTCTACG (INF A Rev 2 SEQ ID NO: 13), TCCTCAAYTCACTCTTCGAGCG (INF B For, SEQ ID NO: 15), CGGTGCTCTTGACCAAATTGG (INF B Rev, SEQ ID NO: 16), GACCCCAAAATCAGCGAAAT (nCOV NI For, SEQ ID NO: 18), TCTGGTTACTGCCAGTTGAATCTG (nCOV NI Rev, SEQ ID NO: 19), TTACAAACATTGGCCGCAAA (nCOV_N2_For, SEQ ID NO: 20), GCGCGACATTCCGAAGAA (nCOV_N2_Rev, SEQ ID NO: 21), where Y is selected from C or T, and optionally ACCCCGCATTACGTTTGGTGGACC (nCOV NI Probe, SEQ ID NO: 22), ACAATTTGCCCCCAGCGCTTCAG (nCOV_N2_Probe, SEQ ID NO: 23), TGCAGTCCTCGCTCACTGGGCACG (INF A-P, SEQ ID NO: 14), and CCAATTCGAGCAGCTGAAACTGCGGTG (INF B-P, SEQ ID NO: 17).

[0024] A method according to the present invention is preferred, wherein the amplification is carried out without diluting and therefore a dilution of the sample.

[0025] Wastewater samples represent pooled environmental samples derived from a population and therefore commonly contain heterogeneous mixtures of viral nucleic acids originating from multiple infected hosts. Consequently, several viral subtypes or variants may be present simultaneously, unlike many clinical samples that typically contain predominantly a single viral variant. The oligonucleotide sequences as used according to the present invention are optimized by specifically targeting regions with higher divergence between RSV A and RSV B, thereby allowing clear molecular discrimination of both subtypes in a multiplex assay. In the context of wastewater samples that are characterized by RNA degradation and other inhibitors, this allows for a robust, subtype-specific quantification from a single reaction. It was found for the assay detecting PMMoV that the present primers are multiplex -ready and compatible, and therefore no dilution is required when performing a qPCR or dPCR.

[0026] By combining the P gene targets with additional N gene sequences, the inventive approach is further optimized. The N gene was selected as target for detection of RSV-A because experimental data demonstrated that assays targeting this region provide high analytical sensitivity for RSV-A. In contrast, assays targeting the N gene for detection of RSV-B showed a substantially reduced sensitivity. It was unexpectedly found that targeting the P gene for RSV-B enabled a detection sensitivity comparable to that obtained for RS V-A using the N gene target. This balanced sensitivity between the subtype-specific assays is particularly important for wastewater-based epidemiological applications, where simultaneous circulation of multiple RSV subtypes requires comparable assay performance in order to reliably estimate the relative abundance of the viral subtypes in environmental samples.

[0027] The use of shortened probes comprising locked nucleic acid (LNA) modifications in the N and P gene regions increases probe binding affinity and improves mismatch discrimination. Importantly, the increased affinity provided by the LNA residues allows the use of substantially shorter probe sequences than would be feasible with conventional DNA probes. This shortening enables targeting of compact sequence segments that contain subtype-discriminating nucleotide differences between RS V-A and RSV-B. Without the LNA modifications, longer probes would be required to achieve sufficient binding stability, which would inevitably extend into adjacent conserved regions and thereby reduce subtype specificity. The use of LNA-modified probes therefore makes it possible to reliably discriminate RSV-A and RSV-B within the selected genomic regions and stabilizes the performance of the assay particularly in complex matrices such as wastewater.

[0028] The need to establish in-house assays is illustrated in Figure 1. The diagram clearly shows that commercially or freely available assays (such as assay 2 from the WHO Global RSV Surveillance Project report from 2019; https: / / www.who.int / publications / i / item / who-strategy-for-global-respiratory-syncytial-virus-surveillance-project-based-on-the-influenza-platform) can detect laboratory reference strains (standard reference from ATCC, 1961), but are not able to efficiently detect more recent strains from patient samples (2024), which are currently epidemiologically relevant, in particular in the wastewater context (Figure 1 A).

[0029] WO 2008 / 122598 A2 discloses RSV detection approaches including a TaqMan-based assay and an MNAzyme-based detection system. In the TaqMan assay, the forward primer shows sequence similarity to present SEQ ID No. 1, whereas the reverse primer overlaps with the region targeted by the probe used in the present invention. However, the assay has been reported to generate false positive RSV-B signals in RSV-A positive samples, indicating insufficient subtype discrimination and limiting its suitability for samples that may simultaneously contain RSV-A and RSV-B. In addition, WO 2008 / 122598 A2 describes an alternative MNAzyme-based detection approach in which one oligonucleotide component shows strong similarity tothe forward primer of the present invention and another component corresponds to the reverse primer used herein. However, this system relies on a different detection principle based on the assembly of a catalytically active MNAzyme and employs distinct partzyme sequences rather than a probe-based detection format.

[0030] An optimized variant of the RSV assay 2 from the WHO project from 2021 (Todd et al., 2021; DOI: 10.1016 / j.jviromet.2021.114171) also does not appear to have sufficient sensitivity and specificity in the wastewater context and was therefore also assessed as unsuitable (Figure IB).

[0031] A commercially available PCR assay kit (GoTaq Enviro FluA / FluB / SARS-CoV-2 / RSV, Promega), which is frequently used in the field, was successfully tested for the ability to detect RSV-A and RSV-B, but no discrimination of the two genotypes was possible with this kit (Figure 1C).

[0032] In addition, the sensitivity (1 / ct < 0.04) could not meet the necessary requirements for efficient wastewater based monitoring. A published, freely available protocol according to Lansivaara et al. 2023 (https: / / doi.org / 10.1021 / acsestwater.3c00752) was significantly better in terms of sensitivity (1 / ct > 0.04), but also did not allow discrimination between genotypes A and B (Figure ID).

[0033] Real-time PCR amplification curves for detection of RSV-A and RSV-B using different combinations of forward primers, reverse primers, and probes targeting either the N gene or the P gene are shown in Figure 6. As shown, the assay configuration employing an N gene target for RSV-A and a P gene target for RSV-B provides improved analytical sensitivity compared to the other tested combinations, as reflected by lower Ct values. This configuration also shows an enhanced signal-to-noise ratio. Such characteristics are particularly relevant for environmental samples, including wastewater, where fluorescence signals (RFU) may be reduced. The data were obtained using RNA extracted from authentic RSV-A and RSV-B viral material.

[0034] Compared to the primer-probe mix according to Zhang et al, 2006 and Haram oto et al, 2013 (https: / / doi.org / 10.1128 / AEM.02354-13, https: / / doi.org / 10.1371 / journal.pbio.0040003) as well as compared to a commercially available mix (GT-Digital SARS-CoV-2 Wastewater Surveillance, GT-Molecular), a significantly lower copy number could be detected accordingto the invention, so that the measurement can also be regarded as dPCR compatible by maintaining the linear measuring range.

[0035] This optimization enables simultaneous measurement of the surrogate parameter in the dPCR system even without prior sample dilution (FigurelF). Figure IF shows the measurement range obtained for different assays targeting the surrogate marker PMMoV. The reciprocal Ct-values between 0.025 and 0.04 correspond to the test range of digital PCR. As illustrated in the figure, only the assay developed in the present invention (referred to herein as Inhouse 2024), enables measurement of the surrogate marker without dilution in a multiplex setting within this range.

[0036] Existing RSV assays frequently target the N gene for detection of both RSV-A and RSV-B. However, the present inventors found that, while the N gene enables highly sensitive detection of RSV-A, targeting the N gene for RSV-B may result in a substantial loss of sensitivity. It was further found that targeting the P gene for RSV-B enables a sensitivity that is more comparable to that achieved for RSV-A using the N gene target, while still allowing reliable subtype-specific detection in a multiplex format.

[0037] This balanced assay performance according to the invention is particularly important for wastewater-based monitoring, where RSV-A and RSV-B may be present simultaneously and where meaningful epidemiological interpretation requires that the relative abundance of both subtypes can be assessed on the basis of assays with comparable analytical sensitivity. Accordingly, in preferred embodiments of the present invention, RSV-A is detected via the N gene and RSV-B is detected via the P gene.

[0038] By using LNAs at suitable positions, it was also possible to shorten the probe length compared to conventional probes so that particularly highly conserved regions could be selected. These sequence regions would otherwise not be available for a conventional probe design. The use of shortened, LNA-modified oligonucleotides in the N and P gene region further increases binding affinity and mismatch control and stabilizes assay performance, particularly in complex matrices and environments, such as wastewater.

[0039] A method according to the present invention is preferred, wherein the amplification reaction is a primer extension, a PCR, a digital PCR, a quantitative PCR, an RT-PCR, in particular an RT-dPCR, RT-ddPCR or RT-qPCR. In particular, the amplification is preferably a multiplex PCR.In a fourth aspect, the problem of the invention is solved by providing an oligonucleotide selected from the group consisting of oligonucleotides comprising the sequences or consisting of the sequences: ATGGCAAAAGACACCTCAG (SEQ ID NO: 3), AGTGATAGATCATTGTCGCTATC (SEQ ID NO: 4), TTGCAGCAGTTCCATATGG (SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (SEQ ID NO: 6), TTACTAATGCTGCTA (SEQ ID NO: 7), AACAAGTCACTCA (SEQ ID NO: 8), TAGCGGACCAGTATACACCATCG (SEQ ID NO: 9) and a mixture of these.

[0040] Preferred sizes of the primers according to the present invention are between 18 and 25 bases, preferably between 19 and 23 bases in length. Preferred sizes of the probes according to the present invention are between 13 and 23 bases in length, preferably between 13 and 15 bases in length. As mentioned above, using LNAs at suitable positions allows to provide shorter probes and primers, while maintaining the specificity.

[0041] Further preferred according to the present invention are primers and probes according to the present invention that consist of the sequences as indicated and extended 5’ and / or 3’ by 1 or 2 additional bases. Naturally, the binding of these extensions must not substantially interfere with the binding properties of the primers and probes according to the present invention.

[0042] Further preferred according to the present invention is a set of oligonucleotides comprising the oligonucleotides comprising the sequences or consisting of the sequences:

[0043] GCTCCAGAATATAGGCATGA (SEQ ID NO: 1), GATCTATCTCCTGCTGCTAAT (SEQ ID NO: 2), CAAGACCAATCYTGTCACCTCTGAC (SEQ ID NO: 10), CAAGACCAATYCTGTCACCTYTGAC (SEQ ID NO: H), GCATTYTGGACAAAVCGTCTACG (SEQ ID NO: 12), GCATTTTGGATAAAGCGTCTACG (SEQ ID NO: 13), TCCTCAAYTCACTCTTCGAGCG (SEQ ID NO: 15), CGGTGCTCTCTTGACCAAATTGG (SEQ ID NO: 16), GACCCCAAAATCAGCGAAAT (SEQ ID NO: 18), TCTGGTTACTGCCAGTTGAATCTG (SEQ ID NO: 19), TTACAAACATTGGCCGCAAA (SEQ ID NO: 20), GCGCGACATTCCGAAGAA (SEQ ID NO: 21), ACCCCGCATTACGTTTGGTGGACC (SEQ ID NO: 22), ACAATTTGCCCCCAGCGCTTCAG (SEQ ID NO: 23), TGCAGTCCTCGCTCACTGGGCACG (SEQ ID NO: 14), andCCAATTCGAGCAGCTGAAACTGCGGTG (SEQ ID NO: 17), wherein Y is selected from C or T.

[0044] Particularly preferred is a set of oligonucleotides comprising at least the oligonucleotides according to present invention together with at least three further oligonucleotides of the above set of oligonucleotides.

[0045] Further preferred is an oligonucleotide according to present invention or set of oligonucleotides according to the present invention, wherein the oligonucleotide(s) are labeled, for example with fluorescein amidite (FAM), FITC (fluorescein isothiocyanate), biotin, 2-[A-(2-hydroxyethyl)-4-[[2-methoxy-5-methyl-4-[(4-methyl-2-nitrophenyl)diazenyl]phenyl]diazenyl]anilino]ethanol (BHQ-1), 4'-(4-nitro-phenyldiazo)-2'-methoxy-5'-methoxy-azobenzene-4"-(N-2-4,4'-dimethoxytrityl(oxyethyl))-N-ethyl-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite (BHQ2), hexachlorofluorescein (HEX), ZEN, TAO, SUN, JOE, MAX, ATTO, ROX, Texas Red (TxR), YakYel, lABkFQ, IBRQ, Cy5.5 and / or Cy5, and / or wherein the primers and / or probes comprise DNA, RNA, PNA and / or LNA nucleotides.

[0046] In a fifth aspect, the problem of the invention is solved by providing a kit comprising materials for carrying out the method according to the present invention, for example at least one suitable oligonucleotide or set of oligonucleotides according to the present invention, buffers, reagents and / or instructions for use.

[0047] In a sixth aspect, the problem of the invention is solved by using the kit according to the present invention or an oligonucleotide or set of oligonucleotides according to the present invention for wastewater monitoring, in particular for the detection and quantification of human pathogenic viruses in water samples, in particular in wastewater samples. Preferred use is as a multiplex PCR.

[0048] As explained above, the present invention relates firstly to a method for detecting viruses, in particular respiratory viruses, in a water sample, in particular a wastewater sample

[0049] The method initially comprises providing a water sample that may potentially be contaminated with viruses or genetic material from viruses. A wastewater sample that potentially containsgenome fragments of respiratory viruses is preferred. Such samples are taken, for example, as part of wastewater surveillance from the inlets of the respective wastewater treatment plants or other accessible wastewater-carrying sewers or collecting tanks, which may also be installed in airplanes, cruise ships, trains and passenger transport vehicles.

[0050] After appropriate enrichment and extraction, in particular RNA extraction, the samples can be prepared relatively easily for PCR analysis using appropriate methods. Wastewater samples can also be stored, but this should generally not be done for longer than approx. 4 - 7 days refrigerated at 4°C for analysis.

[0051] This is followed by amplification of viral nucleic acid sequences potentially present in the sample. According to the invention, this comprises using at least one primer selected from the group of primers comprising the sequences or consisting of the sequences as a starting point: ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3) AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4), and

[0052] a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences

[0053] and optionally

[0054] GCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), and GATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and TTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6), and

[0055] a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences.

[0056] Alternatively, the above is followed by a different amplification of viral nucleic acid sequences potentially present in the sample. According to the invention, this comprises using at least one primer selected from the group of primers comprising the sequences or consisting of the sequences as a starting point:

[0057] TTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6), and

[0058] a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences

[0059] and optionallyGCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), and GATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3) and AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4), and

[0060] a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences.

[0061] With a few exceptions, the polymerase chain reaction (PCR) method is used to detect specific viral pathogens in wastewater. PCR is used to detect the genetic fingerprint, i.e. the DNA or ribonucleic acid (RNA) of a particular pathogen

[0062] In principle, amplification can be carried out by any suitable amplification reaction or amplification reaction. Preferred is the method according to the present invention, wherein the amplification reaction is a primer extension, a PCR, a digital PCR, a quantitative PCR, an RT-PCR, in particular an RT-dPCR, RT-ddPCR or RT-qPCR.

[0063] Quantitative PCR (qPCR) is particularly preferred, as it can determine the amount of virus present in the sample ("viral load") in addition to detection. For this purpose, a corresponding quantification standard consisting of viral or synthetic DNA or RNA material containing the target sequences to be amplified is usually also analyzed.

[0064] PMMoV (Pepper Mild Mottle Virus) is a plant virus with an RNA genome that is used in wastewater monitoring as a surrogate indicator for human fecal contamination. In PCR testing in wastewater, PMMoV is therefore used to normalize the concentrations of other viruses. Although PMMoV, a plant virus ingested via food, is only an indirect surrogate marker for human input and occurs in high concentrations, its RNA is often amplified in parallel with the target pathogens in expert circles. This also serves to qualitatively control sample integrity after nucleic acid extraction and to control the efficiency of reverse transcription, which can be impaired by inhibitors often found in wastewater.

[0065] The measured concentrations of the target pathogens can be normalized relative to the PMMoV concentration, depending on the context, to allow a comparable assessment of integrity and reprocessing between different samples (R H Holm, et al, Surveillance of RNase P, PMMoV, and CrAssphage in wastewater as indicators of human fecal concentration across urban sewerneighborhoods, Kentucky, FEMS Microbes, Volume 3, 2022, xtac003, https: / / doi.org / 10.1093 / femsmc / xtac003).

[0066] The final step is the detection of one or the amplification product of viral nucleic acid sequences present in the sample and thus the detection of respiratory viruses in the water sample, in particular wastewater samples.

[0067] In contrast to PCR, digital PCR (dPCR) uses a separation of the DNA molecules by limit dilution and microfluidics in a large number of separate reaction compartments. Amplification with the polymerase takes place with the separated DNA molecules. Therefore, a digital result is obtained in each reaction vessel, which can be evaluated by reading out the probe-based fluorescence. (Amplification: yes or no). Reverse transcription, which in most cases is used as a one-step approach, can be used to transcribe RNA to DNA and make it usable for dPCR,

[0068] In qPCR, a fluorescent reporter dye is used as an indirect measure of the amount of nucleic acid present during each amplification cycle. The increase in fluorescent signal mediated by a dye measurable in dsDNA-binding and bound state is directly proportional to the amount of exponentially increasing PCR product molecules (amplicons) produced during the repetitive phases of the reaction.

[0069] Alternatively, a probe (or a combination of two probes, depending on the detection chemistry) can add a level of detection specificity beyond the dsDNA-binding dye, as it binds to a specific region of the template located between the primers. The most commonly used probe format is the dual-labeled probe (DLP; also referred to as hydrolysis or TaqMan® probe). The DLP is an oligonucleotide with a 5' fluorescent label, e.g. 6-FAM™, and a 3' quenching molecule, such as one of the dark quenchers, e.g. BHQ® 1 or OQ™. The use of probes labeled with different reporter dyes enables the simultaneous detection and quantification of multiple targets in a single (multiplex) reaction. Internal quenchers can be incorporated into the probe design to further reduce background fluorescence in the non-cleaved probe, thereby further optimizing the signal-to-noise ratio. These quenchers are positioned in the center of the probe between the reporter and terminal quenchers.

[0070] The term "respiratory virus" in the context of the present invention primarily refers to the respiratory syncytial virus, also known as RS virus or RSV, an RNA virus from thePneumoviridae family. The term "respiratory virus" shall also include SARS-CoV-2 and influenza viruses. Influenza and RSV are each categorized into two clinically and epidemiologically relevant genotypes, A and B. Preferred is the method according to the present invention, wherein the respiratory virus is RSV-A and / or RSV-B.

[0071] As mentioned above, a preferred method according to the present invention further comprises using at least one probe selected from the group comprising the sequences or consisting of the sequences TTACTAATGCTGCTA(RSVA_N_l_Probe, SEQ ID NO: 7), AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8), TAGCGGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9) and a nucleic acid sequence which is at least 80%, preferably at least 90% and more preferably at least 95% identical to these sequences. Particularly preferred is the method according to the present invention, comprising the use of at least one primer and probe pair selected from the group consisting of:

[0072] GCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), ATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and TTACTAATGCTGCTA (RSVA N I Probe, SEQ ID NO: 7) or ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3), AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4), AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8), and

[0073] a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences

[0074] and optional

[0075] TTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5), TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6), TAGCGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9) and

[0076] of a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences.

[0077] Amplicons are thus amplified which can be detected with a probe during amplification in proportion to the amount of amplicon.

[0078] Alternatively, also particularly preferred is the method according to the present invention, comprising the use of at least one primer and probe pair selected from the group consisting of: TTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5),TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6), and TAGCGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9), and

[0079] a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences,

[0080] and optionally

[0081] GCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), and GATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and TTACTAATGCTGCTA (RSVA N I Probe, SEQ ID NO: 7) or ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3) and AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4), and AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8), and

[0082] a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences.

[0083] Amplicons are thus amplified which can be detected with a probe during amplification in proportion to the amount of amplicon.

[0084] According to the invention, in the process according to present invention, the primers and / or probes may be labeled, for example with fluorescein amidite (FAM), FITC (fluorescein isothiocyanate), biotin, 2-[A-(2-hydroxyethyl)-4-[[2-methoxy-5-methyl-4-[(4-methyl-2-nitrophenyl)diazenyl]phenyl]diazenyl]anilino]ethanol (BHQ-1), 4'-(4-nitro-phenyldiazo)-2'-methoxy-5'-methoxy-azobenzene-4"-(N-2-4,4'-dimethoxytrityl(oxyethyl))-N-ethyl-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite (BHQ2), hexachlorofluorescein (HEX), ZEN, TAO, SUN, JOE, MAX, ATTO, ROX, Texas Red (TxR), YakYel, lABkFQ, IBRQ, Cy5.5 and / or Cy5.

[0085] Additionally, or alternatively, the primers and / or probes in the method according to present invention may comprise DNA, RNA, PNA and / or LNA nucleotides.

[0086] Particularly preferred is a method according to the present invention wherein the probe is selected from

[0087] 5'FAM / TT+AC+TA+AT+G+CT+GC+TA / 3'BHQ-1 (RSVA N I Probe, SEQ ID NO: 7), 5'HEX / AA+C+AA+GT+C+A+CTCA / 3'BHQ1 (RSVB P I Probe, SEQ ID NO: 8),5'Cy5 / TAGCGGAC / BHQ2 / CAGTATACACCATCG / -3'BHQ2 (PMMoV_Probe2, SEQ ID NO: 9), where "+" denotes the subsequent LNA nucleotide (e.g. +A) and a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences.

[0088] As mentioned above, a method according to the present invention is particularly preferred, wherein the amplification is a multiplex PCR, in a multiplex PCR, in contrast to a standard PCR, several different primers are added to the reaction mixture.

[0089] Thus, the method according to the present invention may comprise further primers, preferably an amplification as above, further comprising the use of at least one primer selected from the group of primers comprising the sequences or consisting of the sequences CAAGACCAATCYTGTCACCTCTGAC (INF A For 1, SEQ ID NO: 10), CAAGACCAATYCTGTCACCTYTGAC (INF A For 2, SEQ ID NO: 11), GCATTYTGGGACAAAVCGTCTACG (INF A Rev 1, SEQ ID NO: 12), GCATTTTGGGATAAAGCGTCTACG (INF A Rev 2 SEQ ID NO: 13), TCCTCAAYTCACTCTTCGAGCG (INF B For, SEQ ID NO: 15), CGGTGCTCTTGACCAAATTGG (INF B Rev, SEQ ID NO: 16), GACCCCAAAATCAGCGAAAT (nCOV NI For, SEQ ID NO: 18), TCTGGTTACTGCCAGTTGAATCTG (nCOV NI Rev, SEQ ID NO: 19), TTACAAACATTGGCCGCAAA(nCOV_N2_For, SEQ ID NO: 20), GCGCGACATTCCGAAGAA (nCOV_N2_Rev, SEQ ID NO: 21),

[0090] wherein Y is selected from C or T, and a nucleic acid sequence which is at least 80%, preferably at least 90% and more preferably at least 95% identical to these sequences, and optionally ACCCCGCATTACGTTTGGGTGGACC (nCOV NI Probe, SEQ ID NO: 22), ACAATTTGCCCCCAGCGCTTCAG (nCOV_N2_Probe, SEQ ID NO: 23).

[0091] TGCAGTCCTCGCTCACTGGGCACG (INF A-P, SEQ ID NO: 14), CCAATTCGAGCAGCTGAAACTGCGGTG (INF B-P, SEQ ID NO: 17), and a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences.

[0092] One challenge in the PCR analysis of wastewater lies in the complex composition of the samples. Sediments, suspended solids and other solid components can interfere with the analysis. In addition, the sample is often diluted by industrial and domestic wastewater as wellas rainwater. Therefore, the excess liquid and solids must be separated in a multi-stage process to obtain a concentrate of the virus material. Surprisingly, it was found that the present method is particularly efficient and therefore the amplification can preferably be carried out without a special dilution of the sample.

[0093] Surprisingly, it was also found that the present method is particularly optimal when amplification is carried out under the following conditions. Primer and probe concentrations: RSV - primer, Seq. 1 0.70 pM; RSV - primer Seq. 2, 0.70 pM; RSV - primer, Seq. 3, 1.00 pM; RSV - primer, Seq. 4, 1.00 pM; PMMoV- primer, Seq. 5, 0.20 pM; PMMoV primer, Seq. 6, 0.20 pM; RSV probe, Seq. 7, 0.30 pM; RSV probe, Seq. 8, 0.50 pM; and PMMoV probe, Seq.

[0094] 9, 0.125 pM. Annealing temperature 50°C and 45 cycles.

[0095] As mentioned above, the present invention further relates to an oligonucleotide selected from the group consisting of oligonucleotides comprising the sequences or consisting of the sequences:

[0096] ATGGCAAAAGACACCTCAG (SEQ ID NO: 3), AGTGATAGATCATTGTCGCTATC (SEQ ID NO: 4), TTGCAGCAGTTCCATATGG (SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (SEQ ID NO: 6), TTACTAATGCTGCTA (SEQ ID NO: 7), AACAAGTCACTCA (SEQ ID NO: 8), TAGCGGGACCAGTATACACACCATCG (SEQ ID NO: 9) and a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences, and a mixture of these.

[0097] Particularly preferred is a set of oligonucleotides according to the present invention comprising the oligonucleotides comprising the sequences or consisting of the sequences:

[0098] CAAGACCAATCYTGTCACCTCTGAC (SEQ ID NO: 10), CAAGACCAATYCTGTCACCTYTGAC (SEQ ID NO: 11), GCATTYTGGACAAAVCGTCTACG (SEQ ID NO: 12), GCATTTTGGATAAAGCGTCTACG (SEQ ID NO: 13), TCCTCAAYTCACTCTTCGAGCG (SEQ ID NO: 15), CGGTGCTCTTGACCAAATTGG (SEQ ID NO: 16), GACCCCAAAATCAGCGAAAT (SEQ ID NO: 18), TCTGGTTACTGCCAGTTGAATCTG (SEQ ID NO: 19), TTACAAACATTGGCCGCAAA (SEQ ID NO: 20), GCGCGACATTCCGAAGAA (SEQ ID NO: 21), ACCCCGCATTACGTTTGGTGGACC (SEQ ID NO: 22), ACAATTTGCCCCCCCAGCGCTTCAG (SEQ ID NO: 23), TGCAGTCCTCGCTCACTGGGCACG (SEQ ID NO: 14), andCCAATTCGAGCAGCTGAAACTGCGGTG (SEQ ID NO: 17), wherein Y is selected from C or T, and optionally further comprising GCTCCAGAATATAGGCATGA (SEQ ID NO: 1), GATCTATCTCCTGCTGCTAAT (SEQ ID NO: 2), and a nucleic acid sequence that is at least 80%, preferably at least 90% and more preferably at least 95% identical to these sequences.

[0099] Particularly preferred is a multiplex set of oligonucleotides comprising at least the oligonucleotides according to present invention of SEQ ID NOs: 1 to 6, optionally with SEQ ID NOs: 7 to 9 together with at least three further oligonucleotides according to the present invention as above.

[0100] According to the invention, the primers and / or probes can be labeled according to present invention, for example with fluorescein amidite (FAM), FITC (fluorescein isothiocyanate), biotin, 2-[A-(2 -hydroxy ethyl)-4-[[2-methoxy-5-methyl-4-[(4-methyl-2-nitrophenyl)diazenyl]phenyl]diazenyl]anilino]ethanol (BHQ-1), 4'-(4-nitro-phenyldiazo)-2'-methoxy-5'-methoxy-azobenzene-4"-(N-2-4,4'-dimethoxytrityl(oxyethyl))-N-ethyl-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite (BHQ2), hexachlorofluorescein (HEX), ZEN, TAO, SUN, JOE, MAX, ATTO, ROX, Texas Red (TxR), YakYel, lABkFQ, IBRQ, Cy5.5 and / or Cy5.

[0101] Additionally, or alternatively, the primers and / or probes according to present invention may comprise DNA, RNA, PNA and / or LNA nucleotides.

[0102] Particularly preferred is an oligonucleotide according to the present invention selected from 5'FAM / TT+AC+TA+AT+G+CT+GC+TA / 3'BHQ-1 (RSVA N I Probe, SEQ ID NO: 7), 5'HEX / AA+C+AA+GT+C+A+CTCA / 3'BHQ1 (RSVB P I Probe, SEQ ID NO: 8), 5'Cy5 / TAGCGGAC / BHQ2 / CAGTATACACCATCG / -3'BHQ2 (PMMoV_Probe2, SEQ ID NO: 9), where "+" denotes an LNA nucleotide and a nucleic acid sequence which is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences.

[0103] Particularly preferred is an oligonucleotide according to the present invention selected from 5'FAM / TT+AC+TA+AT+G+CT+GC+TA / 3'BHQ-1 (SEQ ID NO: 7), 5'HEX / AA+C+AA+GT+C+A+CTCA / 3'BHQ1 (SEQ ID NO: 8), 5'Cy5 / TAGCGGAC / BHQ2 / CAGTATACACCATCG / -3'BHQ2 (SEQ ID NO: 9),

[0104] 5'-FAM / TGCAGTCCT / ZEN / CGCTCACTGGGCACG / 3TABkFQ (SEQ ID NO: 14),5'-YakYel / CCAATTCGA / ZEN / GCAGCTGAAACTGCGGTG / 3TABkFQ (SEQ ID NO: 17), 5'Cy5 / ACAATTTGC / ZEN / CCCCAGCGCTTCAG / 3'IABkFQ (SEQ ID NO: 23), wherein "+" denotes an LNA nucleotide and Y is selected from C or T and a nucleic acid sequence which is at least 80%, preferably at least 90% and more preferably at least 95% identical to these sequences.

[0105] As mentioned above, the present invention further relates to a kit, in particular a diagnostic kit, comprising materials for carrying out the method according to the present invention, for example at least one suitable oligonucleotide or set of oligonucleotides according to the present invention buffers, reagents and / or instructions for use. Preferably, the suitable oligonucleotide is selected from the group consisting of oligonucleotides comprising the sequences or consisting of the sequences:

[0106] ATGGCAAAAGACACCTCAG (SEQ ID NO: 3), AGTGATAGATCATTGTCGCTATC (SEQ ID NO: 4), TTGCAGCAGTTCCATATGG (SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (SEQ ID NO: 6), TTACTAATGCTGCTA (SEQ ID NO: 7), AACAAGTCACTCA (SEQ ID NO: 8), TAGCGGGACCAGTATACACACCATCG (SEQ ID NO: 9), and optionally GCTCCAGAATATAGGCATGA (SEQ ID NO: 1), GATCTATCTCCTGCTGCTAAT (SEQ ID NO: 2), and a nucleic acid sequence that is at least 80%, preferably at least 90% and further preferably at least 95% identical to these sequences, and a mixture of these.

[0107] A further aspect of the invention relates to the use of the kit according to the present invention or an oligonucleotide or set of oligonucleotides according to the present invention for wastewater monitoring, i.e. wastewater surveillance, in particular for the detection and quantification of human pathogenic viruses in water samples, in particular in wastewater samples. Preferred is the use according to the present invention, wherein the use is carried out in the context of a multiplex PCR.

[0108] The invention relates to the following items.

[0109] Item 1. Method for the detection of respiratory viruses in a water sample, in particular a wastewater sample, comprising the steps of

[0110] i) Providing a water sample, in particular a wastewater sample, which potentially contains respiratory viruses,ii) Amplifying of viral nucleic acid sequences potentially present in the sample, comprising the use of at least one primer, in particular a primer pair comprising the sequences:

[0111] ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3) and AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4),

[0112] and optionally

[0113] GCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), and GATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and TTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6), and

[0114] iii) Detecting of an amplification product of viral nucleic acid sequences present in the sample and thereby detecting of respiratory viruses in the water sample, in particular wastewater sample.

[0115] Item 2. Method according to Item 1, further comprising the use of at least one probe selected from the group comprising the sequences

[0116] TTACTAATGCTGCTA (RSVA N I Probe, SEQ ID NO: 7),

[0117] AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8), and TAGCGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9).

[0118] Item 3. Method according to Item 1 or 2,

[0119] comprising the use of at least one primer and probe pair selected from the group consisting of: GCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), ATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and TTACTAATGCTGCTA (RSVA N I Probe, SEQ ID NO: 7) or ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3), AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4), and AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8),

[0120] and optionally

[0121] TTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5), TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6), and TAGCGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9).

[0122] Item 4: Method for the detection of viruses in a water sample, in particular a wastewater sample, comprising the steps ofi) Providing a water sample, in particular a wastewater sample, which potentially contains respiratory viruses,

[0123] ii) Amplifying of viral nucleic acid sequences potentially present in the sample, comprising the use of at least one primer, in particular a primer pair comprising the sequences:

[0124] TTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6),

[0125] and optionally

[0126] GCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), and GATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3) and AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4), and

[0127] iii) Detecting of an amplification product of viral nucleic acid sequences present in the sample and thereby detecting of respiratory viruses in the water sample, in particular wastewater sample, preferably further comprising the use of at least one probe selected from the group comprising the sequences

[0128] TTACTAATGCTGCTA (RSVA N I Probe, SEQ ID NO: 7),

[0129] AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8), and TAGCGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9).

[0130] Item 5: Method according to one of Items 1 to 4, wherein the amplification reaction is a primer extension, a PCR, a digital PCR, a quantitative PCR, an RT-PCR, in particular an RT-dPCR, RT-ddPCR or RT-qPCR.

[0131] Item 6: Method according to one of Items 1 to 5, wherein the respiratory virus is RSV-A and / or RSV-B.

[0132] Item 7. A method according to any one of Items 1 to 6, wherein the primers and / or probes are labeled, for example with fluorescein amidite (FAM), FITC (fluorescein isothiocyanate), biotin, 2-[A-(2 -hydroxy ethyl)-4-[[2-methoxy-5-methyl-4-[(4-methyl-2-nitrophenyl)diazenyl]phenyl]diazenyl]anilino]ethanol (BHQ-1), 4'-(4-nitro-phenyldiazo)-2'-methoxy-5'-methoxy-azobenzene-4"-(N-2-4,4'-dimethoxytrityl(oxyethyl))-N-ethyl-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite (BHQ2), hexachlorofluorescein (HEX), ZEN, TAO, SUN, JOE, MAX, ATTO, ROX, Texas Red (TxR), YakYel, lABkFQ, IBRQ, Cy5.5 and / orCy5, and / or wherein the primers and / or probes comprise DNA, RNA, PNA and / or LNA nucleotides.

[0133] Item 8. Method according to Item 7, wherein the probes are selected from 5'FAM / TT+AC+TA+AT+G+CT+GC+TA / 3'BHQ-1 (RSVA N I Probe, SEQ ID NO: 7), 5'HEX / AA+C+AA+GT+C+A+CTCA / 3'BHQ1 (RSVB P I Probe, SEQ ID NO: 8), and 5'Cy5 / TAGCGGAC / BHQ2 / CAGTATACACCATCG / -3'BHQ2 (PMMoV_Probe2, SEQ ID NO: 9), where "+" denotes an LNA nucleotide.

[0134] Item 9: Method according to one of Items 1 to 8, wherein the amplification is a multiplex PCR.

[0135] Item 10: Method according to any one of Items 1 to 9, the amplification further comprising the use of at least one primer selected from the group of primers comprising the sequences CAAGACCAATCYTGTCACCTCTGAC (INF A For 1, SEQ ID NO: 10), CAAGACCAATYCTGTCACCTYTGAC (INF A For 2, SEQ ID NO: 11), GCATTYTGGGACAAAVCGTCTACG (INF A Rev 1, SEQ ID NO: 12), GCATTTTGGGATAAAGCGTCTACG (INF A Rev 2 SEQ ID NO: 13), TCCTCAAYTCACTCTTCGAGCG (INF B For, SEQ ID NO: 15), CGGTGCTCTTGACCAAATTGG (INF B Rev, SEQ ID NO: 16), GACCCCAAAATCAGCGAAAT (nCOV NI For, SEQ ID NO: 18), TCTGGTTACTGCCAGTTGAATCTG (nCOV NI Rev, SEQ ID NO: 19), TTACAAACATTGGCCGCAAA(nCOV_N2_For, SEQ ID NO: 20), GCGCGACATTCCGAAGAA (nCOV_N2_Rev, SEQ ID NO: 21),

[0136] where Y is selected from C or T, and optionally

[0137] ACCCCGCATTACGTTTGGGTGGACC (nCOV NI Probe, SEQ ID NO: 22), ACAATTTGCCCCCAGCGCTTCAG (nCOV_N2_Probe, SEQ ID NO: 23).

[0138] TGCAGTCCTCGCTCACTGGGCACG (INF A-P, SEQ ID NO: 14), and CCAATTCGAGCAGCTGAAACTGCGGTG (INF B-P, SEQ ID NO: 17).

[0139] Item 11: Method according to one of Items 1 to 10, wherein the amplification is carried out without a specific dilution or dilution of the sample.

[0140] Item 12: An oligonucleotide selected from the group consisting of oligonucleotides comprising the sequences or consisting of the sequences:ATGGCAAAAGACACCTCAG (SEQ ID NO: 3), AGTGATAGATCATTGTCGCTATC (SEQ ID NO: 4), TTGCAGCAGTTCCATATGG (SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (SEQ ID NO: 6), TTACTAATGCTGCTA (SEQ ID NO: 7), AACAAGTCACTCA (SEQ ID NO: 8), TAGCGGACCAGTATACACCATCG (SEQ ID NO: 9) and a mixture of these.

[0141] Item 13. A set of oligonucleotides comprising the oligonucleotides comprising the sequences or consisting of the sequences:

[0142] CAAGACCAATCYTGTCACCTCTGAC (SEQ ID NO: 10), CAAGACCAATYCTGTCACCTYTGAC (SEQ ID NO: H), GCATTYTGGACAAAVCGTCTACG (SEQ ID NO: 12), GCATTTTGGATAAAGCGTCTACG (SEQ ID NO: 13), TCCTCAAYTCACTCTTCGAGCG (SEQ ID NO: 15), CGGTGCTCTCTTGACCAAATTGG (SEQ ID NO: 16), GACCCCAAAATCAGCGAAAT (SEQ ID NO: 18), TCTGGTTACTGCCAGTTGAATCTG (SEQ ID NO: 19), TTACAAACATTGGCCGCAAA (SEQ ID NO: 20), GCGCGACATTCCGAAGAA (SEQ ID NO: 21), ACCCCGCATTACGTTTGGTGGACC (SEQ ID NO: 22), ACAATTTGCCCCCAGCGCTTCAG (SEQ ID NO: 23), TGCAGTCCTCGCTCACTGGGCACG (SEQ ID NO: 14), and CCAATTCGAGCAGCTGAAACTGCGGTG (SEQ ID NO: 17), wherein Y is selected from C or T, and optionally further including the oligonucleotides GCTCCAGAATATAGGCATGA (SEQ ID NO: 1), and GATCTATCTCCTGCTGCTAAT (SEQ ID NO: 2).

[0143] Item 14: A set of oligonucleotides comprising at least the oligonucleotides of Item 12 together with at least three oligonucleotides of Item 13.

[0144] Item 15. An oligonucleotide according to Item 12 or a set of oligonucleotides according to Item 13 or 14, wherein the oligonucleotide(s) are labeled, for example with fluorescein amidite (FAM), FITC (fluorescein isothiocyanate), biotin, 2- [A-(2 -hydroxy ethyl)-4-[ [2-methoxy-5-methyl-4-[(4-methyl-2-nitrophenyl)diazenyl]phenyl]diazenyl]anilino]ethanol (BHQ-1), 4'-(4-nitro-phenyldiazo)-2'-methoxy-5'-methoxy-azobenzene-4"-(N-2-4,4'-dimethoxytrityl(oxyethyl))-N-ethyl-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite (BHQ2), hexachlorofluorescein (HEX), ZEN, TAO, SUN, JOE, MAX, ATTO, ROX, Texas Red (TxR), YakYel, lABkFQ, IBRQ, Cy5.5 and / or Cy5, and / or wherein the primers and / or probes comprise DNA, RNA, PNA and / or LNA nucleotides.Item 16: The oligonucleotide of Item 15, wherein the oligonucleotide is selected from 5'FAM / TT+AC+TA+AT+G+CT+GC+TA / 3'BHQ-1 (SEQ ID NO: 7), 5'HEX / AA+C+AA+GT+C+A+CTCA / 3'BHQ1 (SEQ ID NO: 8), 5'Cy5 / TAGCGGAC / BHQ2 / CAGTATACACCATCG / -3'BHQ2 (SEQ ID NO: 9),

[0145] 5'-FAM / TGCAGTCCT / ZEN / CGCTCACTGGGCACG / 3TABkFQ (SEQ ID NO: 14),

[0146] 5'-YakYel / CCAATTCGA / ZEN / GCAGCTGAAACTGCGGTG / 3TABkFQ (SEQ ID NO: 17), and

[0147] 5'Cy5 / ACAATTTGC / ZEN / CCCCAGCGCTTCAG / 3TABkFQ (SEQ ID NO: 23), wherein "+" denotes an LNA nucleotide, and Y is selected from C or T.

[0148] Item 17: A kit comprising materials for carrying out the method according to any one of Items 1 to 11, for example at least one suitable oligonucleotide or set of oligonucleotides according to any one of Items 12 to 16, buffers, reagents and / or instructions for use.

[0149] Item 18: Use of the kit according to Item 16 or of an oligonucleotide or set of oligonucleotides according to one of Items 12 to 16 for wastewater monitoring, in particular for the detection and quantification of human pathogenic viruses in water samples, in particular in wastewater samples.

[0150] Item 19 Use according to Item 18, wherein the use is as multiplex PCR.

[0151] The present invention will now be further explained below with reference to, but not limited to, the examples, the figures and the attached sequence listing. The cited references are hereby incorporated by reference in their entirety.

[0152] Figure 1: Development and validation steps in the development of the multiplex assay for the detection of respiratory viruses in wastewater. Several published (A, B, D) and commercial (C) assays were compared with the assay (E) developed by the inventors within WB Eready using RT-qPCR. (F) Detection of PMMoV in wastewater (dark gray) in the context of the linear measurement range of digital RT-PCR (light gray). 1 / ct describes the reciprocal Ct value indicating the respective fluorescence channel tested.Figure 2: Compatibility of WBEready-respiratory panel 1 and 2 with RT-qPCR (A) and RT-dPCR (B).

[0153] Figure 3: Comparison of WBEready-respiratory panel 1 and 2 with representative currently commercially available respiratory multiplex PCR assays.

[0154] Figure 4: Comparison of wastewater epidemiological data collected with the WBEready respiratory panel 1 and 2 compared to freely available epidemiological data from individual testing.

[0155] Figure 5: Identification of conserved but genotype-specific regions within the genomes of RSV-A and RSV-B. The selected gene regions on the RSV N and P genes are shown. The genotypespecific assay components according to the invention (primers and probes) for RSV A and RSV B are shown in dark gray. The pan -genotypic RSV assay according to Lansivaara et al. in light gray. The first part of Figure 5 shows the 5’ regions, and the second part the 3’ regions. See also SEQ ID NOs: 26 to 47.

[0156] Figure 6: Real-time PCR amplification curves for detection of RSV-A (A) and RSV-B (B) using different combinations of forward primers, reverse primers, and probes targeting either the N gene or the P gene.

[0157] SEQ ID NO: 1 to 47 show the sequences of primer oligomers and probes as well as parts of the RSV genes as used in the invention, or according to the invention.

[0158] Table 1 : Nucleic acid sequences

[0159] SEQ Sequence Notes

[0160] ID NO:

[0161] 1 GCTCCAGAATATAGGCATGA RSV-A - primer 2 GATCTATCTCCTGCTGCTAAT RSV-A - primer 3 ATGGCAAAAGACACCTCAG RSV-B - primer 4 AGTGATAGATCATTGTCGCTATC RSV-B - primer 5 TTGCAGCAGTTCCATATGG PMMoV primer 6 TGTCTAAATAGTTCTTGCATTGTTG PMMoV primer

[0162]

[0163] 7 TTACTAATGCTGCTA RSV-A - Probe 8 AACAAGTCACTCA RSV-B - Probe 9 TAGCGGACCAGTATACACCATCG PMMoV probe 10 CAAGACCAATCYTGTCACCTCTGAC INF -A - primer 11 CAAGACCAATYCTGTCACCTYTGAC INF -A - primer 12 GCATTYTGGACAAAVCGTCTACG INF -A - primer 13 GCATTTTGGATAAAGCGTCTACG INF -A - primer 14 TGCAGTCCTCGCTCACTGGGCACG INF -A - Probe 15 TCCTCAAYTCACTCTTCGAGCG INF-B - primer 16 CGGTGCTCTTGACCAAATTGG INF-B - primer 17 CCAATTCGAGCAGCTGAAACTGCGGTG INF-B - Probe 18 GACCCCAAAATCAGCGAAAT SARS Cov - primer 19 TCTGGTTACTGCCAGTTGAATCTG SARS Cov - primer 20 TTACAAACATTGGCCGCAAA SARS Cov - primer 21 GCGCGACATTCCGAAGAA SARS Cov - primer 22 ACCCCGCATTACGTTTGGTGGACC SARS Cov - Probe 23 ACAATTTGCCCCCAGCGCTTCAG SARS Cov - Probe 24 taaaagaaatgggagaggtagctccagaatacaggcatgactctcctgattgtggga RSV-A: Protein N tgataatattatgtatagcagcattagtaataactaaattagcaggggacagatctggtc ttacagccgtgattaggagagctaataatgt

[0164] 25 tggcaagactcaggaatgaggaaagtgaaaagatggcaaaagacacatcagatga RSV-A: Protein P agtgtctctctcaatccaacatcagagaaattgaacaacctattggaagggaatgata

[0165] gtgacaatgatctatcacttgaagatttctgattagctac

[0166]

[0167] Examples

[0168] In the context of the present invention, a preferred multiplex assay was generated for the viruses Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), influenza viruses of types A (IAV) and B (IBV) and the respiratory syncytial virus of the two genotypes A and B (RSV-A / RSV-B). In parallel, the detection test for the human fecal surrogate indicator Pepper mild mottle virus (PMMoV) was also optimized so that it is compatible with all methods, workflows and devices currently used in wastewater monitoring (RT-dPCR, RT-ddPCR and RT-qPCR).The design, establishment and validation of RT-qPCR / RT-dPCR assays for the detection of circulating respiratory pathogens was carried out and wastewater epidemiological data on SARS-CoV-2, influenza viruses (A / B) and the respiratory syncytial virus (RSV-A / RSV-B) was generated for the winter of 2023 / 2024. For this purpose, wastewater samples were obtained and collected twice a week from November 2023 in cooperation with the consortium partner EGLV.

[0169] A comprehensive genome database of current virus sequences of respiratory viruses relevant to wastewater epidemiology was then created. This was used for the development of sequences for improved RT-qPCR and RT-dPCR assays. In addition to the acquisition of commercially available assays described in the literature, multiplex assays were developed according to the invention and primer / probe pairs were synthesized and the assay performance was compared with regard to sensitivity and specificity in the wastewater context.

[0170] The multiplex assays were evaluated with regard to sensitivity and specificity using inactivated authentic viruses (cultivated in the S2 or S3 laboratory) in wastewater samples that were negative or very low in abundance for the respective analyte.

[0171] For this purpose, existing relevant viruses (SARS-CoV-2, influenza A and B, RSV-A / B) have already been amplified in cell culture at the UKF Institute for Medical Virology by members of the Widera research group and other viruses have been obtained from external laboratories (e.g. influenza H5N1, FLI).

[0172] Including the developed assay for RSVA and RSVB as well as the optimized PMMoV assay, using already available assays for the detection of 1) SARS-CoV-2 (N1 and N2) as well as influenza A and influenza B (https: / / www.who.int / docs / default-source / coronaviruse / uscdcrt-pcr-panel-primer-probes.pdf?sfvrsn=fa29cb4b_2, https: / / www.cdc.gov / flu / php / laboratories / influenza-sars-cov-2-multiplex-assay.html) and 2) RSV-A, RSB-B and PMMoV, two different multiplex conditions were titrated out and crossvalidated against each other and the amplification conditions were successively optimized so that they showed no cross-reactions with each other and allowed efficient amplification under the same PCR conditions. The primer and probe concentrations, annealing temperature and number of cycles were experimentally adjusted to ensure simultaneous and efficient amplification of all target sequences.The genotype can provide clinically relevant information, particularly in combination with other mutation information, in addition to the purely epidemiological added value (https: / / doi.org / 10.1016 / S1473-3099(23)00062-2), so that special primers and probes were developed in the course of the project.

[0173] Material / Methods

[0174] Figure 1

[0175] Using the RSVA laboratory strain VR-1302 (ATCC reference material) isolated in 1961 and numerous patient samples from 2024 (2x RSVA and lx RSVB are shown as examples), the sensitivity and specificity of our self-developed RSVA / RSVB PCR assay was compared to the RSVA / RSVB PCR assay from the 2019 WHO Global Surveillance of RSV Project report (https: / / www.who.int / publications / i / item / who-strategy-for-global-respiratory-syncytial-virus-surveillance-project-based-on-the-influenza-platform), an optimized variant of Todd et al, 2021 (DOI: 10.1016 / j.jviromet.2021.114171), the commercially available RSV PCR assay GoTaq Enviro FluA / FluB / SARS-CoV-2 / RSV (Promega) and the published RSV PCR assay according to Lansivaara et al., 2023 (https: / / doi.org / 10.1021 / acsestwater.3c00752) were evaluated (A-E). The RNA from the virus-containing material was extracted using the QIAamp Viral RNAMini Kit (Qiagen).

[0176] The RT-qPCR and RT-dPCR compatibility (between CT-25 and CT-40, highlighted in light gray) of our self-developed PMMoV assay, a published PMMoV assay according to Zhang et al., 2006 (https: / / doi.org / 10.1371 / journal.pbio.0040003) and Haramoto et al., 2013 (https: / / doi.org / 10.1128 / AEM.02354-13) and the commercial GT-Digital SARS-CoV-2 Wastewater Surveillance Assay For QIAcuity (GT-Molecular) was tested with real wastewater samples (exemplary wastewater sample from 20.06.2021 shown). Nucleic acid extraction from wastewater to evaluate the PMMoV assay was performed using the previously described FESPiNA method (Wilhelm et al., 2022 (https: / / doi.org / 10.3390 / vl4091876, https: / / doi.Org / 10.1016 / j.scitotenv.2022.157375). The RT-qPCR measurements were performed in a total reaction volume of 20 pL and the use of 5 pL sample with the Biorad One-Step Reliance Mastermix (Fi. Biorad) according to the manual on a Biorad CFX96 Touch Real-Time PCR Detection System (Biorad).

[0177] Fi ure 2The multiplex assay was validated with inactivated authentic IAV (H1N1 and H3N2), IBV, SV2 (Omicron KPI .1.1), RSVA and RSVB. The nucleic acids from the virus-containing cell culture material were extracted using the QIAamp Viral RNA Mini Kit (Qiagen). Various virus concentrations were tested using RT-qPCR and RT-dPCR (concentrations found in wastewater are shown as examples).

[0178] 1 / ct describes the reciprocal Ct value indicating the respective fluorescence channel tested. RT-dPCR was performed with the QIAcuity OneStep Advanced Probe Kit (Qiagen) on a QIAcuity Digital PCR System (Qiagen) according to the publications of Wilhelm et al., 2022 (https: / / doi.org / 10.3390 / vl4091876, https: / / doi.Org / 10.1016 / j.scitotenv.2022.157375). For each reaction, 10 pL of sample was used with a total reaction volume of 40 pL and QIAcuity Nanoplates 26k 24-well plates (Qiagen) were used. The QIAcuity Software Suite version 2.5.0.1 was used for data analysis. The RT-dPCR limits shown (shown as horizontal line in B) were automatically determined by the QIAcuity software. The RT-qPCR measurements were performed in a total reaction volume of 20 pL with the Biorad One-Step Reliance Mastermix (Fi. Biorad) according to the manual on a Biorad CFX96 Touch Real-Time PCR Detection System (Fa. Biorad).

[0179] Figure 3

[0180] The assays were validated in the context of waste water-based epidemiology under realistic conditions and real wastewater samples from November 2023 to February 2024 were used as reference samples. The values shown for influenza A, influenza B, SARS-CoV-2, RSVA and RSVB of the WBEready-respiratory panels 1 and 2 are normalized with the values of the wastewater surrogate marker PMMoV. Sample preparation was performed using pressure filtration with electronegative membrane filters as previously described in the publications of Wilhelm et al, 2022 (https: / / doi.org / 10.3390 / vl4091876, https: / / doi.Org / 10.1016 / j.scitotenv.2022.157375). Research Kit 1 = GoTaq Enviro FluA / FluB / SARS-CoV-2 / RSV, Promega, IVD Kit 1: ViroQ SARS-FluA / B-RSV, BAG Diagnostics, IVD Kit 2: Allplex™ SARS CoV-2 FluA / FluB / RSV, Seegene. 1 / ct describes the reciprocal Ct value indicating the respective fluorescence channel tested. The RT-qPCR measurements were carried out in a total reaction volume of 20 pL with the Biorad One-Step Reliance Mastermix (Biorad) and were performed according to the manufacturer's instructions using a Biorad CFX96 Touch Real-Time PCR Detection System (Biorad).Figure 4

[0181] Over the period from October 2023 to the end of September 2024, the viral load (in copies per milliliter of wastewater, c / ml) of SARS-CoV-2, influenza A (IAV) and B (IBV) and resp. sync, virus A (RSVA) and B (RSVB) was determined once a week in 24-hour mixed wastewater samples from the inlet of the Emschermundung wastewater treatment plant (EGLV) using RT-dPCR. The wastewater epidemiological data were compared with the reported case numbers or the 7-day incidence for the corresponding viruses in North Rhine-Westphalia. The data were taken from the public databases and dashboards of the North Rhine-Westphalia Health Center (LZG NRW) and SurvStat of the Robert Koch Institute (RKI) (shown in the second Y-axis). Sample preparation was carried out using pressure filtration with electronegative membrane filters, as previously described in the publications by Wilhelm et al, 2022 (https: / / doi.org / 10.3390 / vl4091876, https: / / doi.Org / 10.1016 / j.scitotenv.2022.157375). RT-dPCR was performed using the QIAcuity OneStep Advanced Probe Kit (Qiagen) on a QIAcuity Digital PCR System (Qiagen) according to the publication by Wilhelm et al., 2022 (https: / / doi.org / 10.3390 / vl4091876, https: / / doi.Org / 10.1016 / j.scitotenv.2022.157375). For each reaction, 10 pL of sample was used with a total reaction volume of 40 pL and QIAcuity Nanoplates 26k 24-well plates (Qiagen) were used. QIAcuity Software Suite version 2.5.0.1 was used for data analysis.

[0182] Fi ure 5

[0183] The sequence matches within the RSV-A and RSV-B variants and the comparison of sequence identity between RSV-A and RSV-B were determined using selected RS Vs from an in-house RSV genome database (the narrower and lighter the stripe below the nucleotide sequence, the lower the sequence match; dark gray = 100% sequence identity, light gray = <100% sequence identity).

[0184] Primers / probes and concentrations for panel 1 according to the invention

[0185] SEQ Sequence Notes Final

[0186] ID concentration in NO: 20 pL reaction 10 CAAGACCAATCYTGTCACCTCTGAC INF -A - primer 1.11 pM

[0187] 11 CAAGACCAATYCTGTCACCTYTGAC INF -A - primer 1.11 pM

[0188] 12 GCATTYTGGACAAAVCGTCTACG INF -A - primer 1.67 pM

[0189] 13 GCATTTTGGATAAAGCGTCTACG INF -A - primer 0.55 pM

[0190]

[0191] 14 TGCAGTCCTCGCTCACTGGGCACG INF -A - Probe 0.55 pM

[0192] 15 TCCTCAAYTCACTCTTCGAGCG INF-B - primer 2.22 pM

[0193] 16 CGGTGCTCTTGACCAAATTGG INF-B - primer 2.22 pM

[0194] 17 CCAATTCGAGCAGCTGAAACTGCGGTG INF-B - Probe 0.55 pM

[0195] 18 GACCCCAAAATCAGCGAAAT SARS CoV - 0.50 pM primer

[0196] 19 TCTGGTTACTGCCAGTTGAATCTG SARS CoV - 0.50 pM primer

[0197] 20 TTACAAACATTGGCCGCAAA SARS CoV - 0.50 pM primer

[0198] 21 GCGCGACATTCCGAAGAA SARS CoV - 0.50 pM primer

[0199] 22 ACCCCGCATTACGTTTGGTGGACC SARS CoV - 0.25 pM Probe

[0200] 23 ACAATTTGCCCCCAGCGCTTCAG SARS CoV - 0.25 pM

[0201] Probe

[0202]

[0203] Primer / probe concentrations for panel 2

[0204] SEQ Sequence Notes Final

[0205] ID concentration in NO: 20 pL reaction 1 GCTCCAGAATATAGGCATGA RS V-A - primer 0.70 pM

[0206] 2 GATCTATCTCCTGCTGCTAAT RS V-A - primer 0.70 pM

[0207] 3 ATGGCAAAAGACACCTCAG RSV-B - primer 1.00 pM

[0208] 4 AGTGATAGATCATTGTCGCTATC RSV-B - primer 1.00 pM

[0209] 5 TTGCAGCAGTTCCATATGG PMMoV primer 0.20 pM

[0210] 6 TGTCTAAATAGTTCTTGCATTGTTG PMMoV primer 0.20 pM

[0211] 7 TTACTAATGCTGCTA RSV-A - Probe 0.30 pM

[0212] 8 AACAAGTCACTCA RSV-B - Probe 0.50 pM

[0213] 9 TAGCGGACCAGTATACACCATCG PMMoV probe 0.125 pM

[0214]

[0215] The RT-qPCR measurements were performed in a total reaction volume of 20 pL and the use of 5 pL sample with the Biorad One-Step Reliance Mastermix (Fi. Biorad) according to the manual on a Biorad CFX96 Touch Real-Time PCR Detection System (Fa. Biorad).Preferred temperature program for

[0216] Step Temperature Time (minutes seconds)

[0217] 1 50.0°C 10:00

[0218] 2 95.0°C 10:00

[0219] 3 95.0°C 00:10

[0220] 4 60.0°C 00:30

[0221] 5 Fluorescence measurement

[0222] 6 Back to step 3 for 44 more cycles

[0223] 7 40.0°C 00:30

[0224] 8 End

[0225]

[0226] RT-dPCR was performed using the QIAcuity OneStep Advanced Probe Kit (Qiagen) on a QIAcuity Digital PCR System (Qiagen) according to the publications of Wilhelm et al, 2022 (https: / / doi.org / 10.3390 / vl4091876, https: / / doi.Org / 10.1016 / j.scitotenv.2022.157375). For each reaction, 10 pL of sample was used with a total reaction volume of 40 pL and QIAcuity Nanoplates 26k 24-well plates (Qiagen) were used. QIAcuity Software Suite version 2.5.0.1 was used for data analysis.

[0227] Preferred temperature program for RT-dPCR

[0228] Step Temperature Time (minutes seconds)

[0229] 1 50.0°C 40:00

[0230] 2 95.0°C 02:00

[0231] 3 95.0°C 00:05

[0232] 4 60.0°C 00:30

[0233] 5 Fluorescence measurement

[0234] 8 End

[0235]

[0236] Results

[0237] Classical probe-based qPCR is based on exponential target gene amplification and fluorescence-based quantification, which allows a broad concentration spectrum to be covered. In contrast, the linear measurement spectrum of digital PCR is narrower than that of classical quantitative PCR (Figure IF), as digital PCR is based on the division of the sample into manyindividual reactions (endpoint PCRs) and the number of compartments in which the individual reactions take place is limited.

[0238] In order to provide practical methods that can be used on both systems, the PCR detection assay for PMMoV was optimized. An alternative primer-probe set was created and validated using a gene region that is measured with comparatively low amplification efficiency. The inventors created a comprehensive genome database of current virus sequences and used this to develop specific primers and probes for RT-qPCR and RT-dPCR assays.

[0239] Compared to the primer-probe mix according to Zhang et al, 2006 and Haram oto et al, 2013 (https: / / doi.org / 10.1128 / AEM.02354-13, https: / / doi.org / 10.1371 / joumal.pbio.0040003) and compared to a commercially available mix (GT-Digital SARS-CoV-2 Wastewater Surveillance, GT-Molecular), a significantly lower copy number could be detected, so that the measurement can also be considered dPCR compatible by adhering to the linear measuring range.

[0240] This optimization enables simultaneous measurement of the surrogate parameter in the dPCR system even without prior sample dilution (FigurelF).

[0241] For this purpose, the identification of conserved but genotype-specific sequences in the target genomes RSVA and RSVB was started. The relevant regions were analyzed from the databases described above and compared in silico using genomics software acquired for this purpose in order to design primers and probes specific for RSV A and RSV B, respectively. The primers and probes were optimized in several empirical test series so that they showed high specificity and efficiency in amplification without cross-reactivity between the genotypes (Figure IE).

[0242] Finally, the assay was realistically tested and validated by spiking real wastewater samples with authentic RSV A and RSV B and negative control wastewater samples to confirm the specificity and sensitivity in the wastewater context. For this purpose, strains from 2024 were compared with a laboratory reference strain from 1961.

[0243] The inventors optimized the assays for high sensitivity and specificity in the wastewater context and then validated them with inactivated authentic viruses in real wastewater samples. For RSV, they developed genotype-specific primers and probes using LNAs (Locked Nucleic Acids), which enable differentiation between RSV A and B. LNAs are modified nucleic acids that areused in PCR to improve the sensitivity and specificity of amplification due to their increased binding affinity and thermal stability.

[0244] PMMoV detection was optimized by an alternative primer-probe set with lower amplification efficiency to ensure compatibility with dPCR systems without sample dilution.

[0245] The inventors established a total of two sophisticated multiplex assays:

[0246] i) Panel 1 for IAV, IBV and SARS-CoV-2, whose primer sequences are known, and ii) The panel 2 according to the invention for RSVA, RSVB and PMMoV.

[0247] Panel 1 for the detection of influenza A, influenza B, SARS-CoV-2 (dual-target determination: N1 or N2). Y is selected from C or T.

[0248] SE Sequence Name

[0249] Q ID

[0250] NO:

[0251] 10 CAAGACCAATCYTGTCACCTCTGAC INF A For 1 11 CAAGACCAATYCTGTCACCTYTGAC INF A For 2 12 GCATTYTGGACAAAVCGTCTACG INF A Rev 1 13 GCATTTTGGATAAAGCGTCTACG INF A Rev 2 14 5'-FAM / TGCAGTCCT / ZEN / CGCTCACTGGGCACG / 3TABkFQ INF A-P

[0252] 15 TCCTCAAYTCACTCTTCGAGCG INF B For 16 CGGTGCTCTTGACCAAATTGG INF B Rev 17 5'- INF B-P YakYel / CCAATTCGA / ZEN / GCAGCTGAAACTGCGGTG / 3'IA

[0253] BkFQ

[0254] 18 GACCCCAAAATCAGCGAAAT nCOV NI For 19 TCTGGTTACTGCCAGTTGAATCTG nCOV NI Rev 20 5'TxR / ACCCCGCATTACGTTTGGTGGGACC / 3'IBRQ nCOV NI Pro be

[0255] 21 TTACAAACATTGGCCGCAAA nCOV_N2_For 22 GCGCGACATTCCGAAGAA nCOV_N2_Rev

[0256]

[0257] 23 5'Cy5 / ACAATTTGC / ZEN / CCCCAGCGCTTCAG / 3'IABkFQ nCOV_N2_Pro be

[0258]

[0259] The first set ("WBEready" respiratory panel 1) contains primers and probes that enable the detection of IAVM1 region (in FAM channel, 450-490 nm), IBVNS1 region (in HEX channel, 515-535 nm) and SARS-CoV-2 N gene (dual detection: target gene 1 and target gene 2 in Cy5 channel (see Figure 2A), or target gene land target gene 2 in Texas Red, 560-590 and Cy5 channel 620-650, respectively) (Figure 2A). Cy5 channel 620-650) (Figure 2A).

[0260] Panel 2 according to the invention for the detection of RSVA, RSVB and PMMoV (all sequences created and evaluated by the inventors) The suffix "+" stands for an LNA modification (e.g. "+A" stands for an LNA modification of base A). Y is selected from C or T. SEQ Sequence Name

[0261] ID

[0262] NO:

[0263] 1 GCTCCAGAATATAGGCATGA RSV N I For 2 GATCTATCTCCTGCTGCTAAT RSV N I Rev 7 5 FAM / TT+AC+TA+AT+G+CT+GC+TA / 3 BHQ-1 RSVA_N_l_Probe 3 ATGGCAAAAGACACCTCAG RSV P I For 4 AGTGATAGATCATTGTCGCTATC RSV P I Rev 8 5 'HEX / AA+C+ AA+GT+C+ A+CTC A / 3 'BHQ 1 RSVB P I Probe 5 TTGCAGCAGTTCCATATGG PMMoV_For2 6 TGTCTAAATAGTTCTTGCATTGTTG PMMoV_Rev2 9 5'Cy5 / TAGCGGAC / BHQ2 / CAGTATACACCATCG / -3'BHQ2 PMMo V Prob e2

[0264]

[0265] With the second primer / probe set according to the invention (WBEready-respiratory panel 2), RSVA (in the FAM channel), RSVB (in the HEX channel) and PMMoV (in the Cy5 channel) can be detected (Figure 2A).

[0266] A good signal-to-noise difference (Figure 2B) was achieved with the probes tested, which have internal quenchers.The inventors also optimized the amplification conditions and validated the assays in comparison to commercial kits. Finally, they tested the developed multiplex assay in the real laboratory over the course of a year and compared the results with data from individual testing, which showed a very good correlation.

[0267] Finally, the assay was first tested with authentic viruses and then validated with real wastewater samples spiked with authentic viruses grown in cell culture but previously heat-inactivated. For this purpose, samples with known concentrations of the target viruses were tested in several test series in order to check sensitivity, specificity and robustness. The multiplex assay developed was then compared with commercially available research and in vitro diagnostic (IVD) test kits (Figure 3).

[0268] Compared to all other kits tested in the wastewater context, only the test according to the invention was able to detect the respiratory viruses SARS-CoV-2, RSV and influenza sensitively enough and also enable discrimination between genotypes A and B in RSV (Figure 3).

[0269] All other kits tested showed deficits in the detectability of at least one target parameter; in one kit (In-Vitro-Diagnoistic kit 2: Allplex™ SARS CoV-2 FluA / FluB / RSV, Seegene) no target parameter could be detected.

[0270] These data also show that validation of the PCR test kits in the wastewater context is necessary in order to meet the requirements of the Public Health Services in terms of sensitivity and specificity.

[0271] The multiplex assay according to the invention was then tested in the real-world laboratory (Emscher-Lippe region). The individual measurements normalized with PMMoV from the Emschergenossenschaft / Lippeverband (EGLV) wastewater treatment plant Emschermundung for an entire year (Oct 2023 to Oct 2024) were presented as a trend line and compared with the data on the 7-day incidence obtained from the individual testing and the published case numbers in North Rhine-Westphalia (NRW) (Figure 4).

[0272] The curves for SARS-CoV-2, RSV-A and RSV-B are qualitatively congruent. The detection of influenza A and B in wastewater, on the other hand, showed a slight rightward shift in thewaste water-related data, but here too the trend values were able to reflect the curve of the individual testing, so that it can be assumed that the trend analysis of respiratory viruses in wastewater is essentially correlated with the individual case numbers.

[0273] To the inventors' knowledge, approx. 90% of the approx. 26 laboratories involved in wastewater monitoring currently use PMMoV as a surrogate marker. In addition, the influenza A assays used also detect avian influenza viruses, including H5N1 of clade 2.3.4.4b, which must be taken into account when specifying the specificity. In addition, the determination of the limits of detection and quantification (LoD / LoQ) may be subject to device-specific characteristics.

[0274] The inventors aimed to use identical forward and reverse primers for RSV-A and RSV-B with two genotype-specific, competitively binding LNA probes. This approach showed excellent discrimination, but sensitivity was insufficient for RSV-A or RSV-B depending on the region, presumably due to differential expression of individual genes in RSV-A and RSV-B. For the detection of RSV A, the N gene was identified as optimal for the specific and sensitive detection of RSV A in the approach chosen by the inventors. Using the inventors' genome database, it became clear that the forward primer and probe of Lansivaara et. al bind conserved regions in RSV A and RSV B, but 24% of the bases of the reverse primer (Rev) are not conserved in currently circulating RSV-A and RSV-B variants (see Figure 5).

[0275] For the genotype-specific detection of RSV-A, the primers and probes of the invention bind exclusively to regions that are highly conserved within the currently circulating RSV-A or RS V-B variants (see Figure 5). This was made possible in the probe in particular by the use of LNAs, which enable a melting temperature suitable for multiplexing due to stronger base binding despite significantly shorter probe lengths. In addition, the probe binds to a region with four bases that are specific for RSV-A. Due to the appropriate placement of RSV-A-specific LNAs, the probe advantageously does not bind to RSV-B sequences.

[0276] For the genotype-specific detection of RSV-B, the P gene enabled particularly sensitive detection. Analogous to the strategy described above for the detection of RSV-A, a suitable region within the P gene was found that fulfills all the above-mentioned requirements for the primers and the LNA probe.

Claims

Claims1. Method for the detection of respiratory viruses in a water sample, in particular a wastewater sample, comprising the steps ofi) Providing a water sample, in particular a wastewater sample, which potentially contains respiratory viruses,ii) Amplifying of viral nucleic acid sequences potentially present in the sample, comprising the use of at least one primer, in particular a primer pair comprising the sequences:ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3) and AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4),and optionallyGCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), and GATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and TTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6), andiii) Detecting of an amplification product of viral nucleic acid sequences present in the sample and thereby detecting of respiratory viruses in the water sample, in particular wastewater sample, preferably further comprising the use of at least one probe selected from the group comprising the sequencesTTACTAATGCTGCTA (RSVA N I Probe, SEQ ID NO: 7),AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8), and TAGCGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9).

2. Method according to claim 1,comprising the use of at least one primer and probe pair selected from the group consisting of: GCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), ATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and TTACTAATGCTGCTA (RSVA N I Probe, SEQ ID NO: 7), and ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3), AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4), and AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8),and optionallyTTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5),TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6), and TAGCGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9).

3. Method for the detection of viruses in a water sample, in particular a wastewater sample, comprising the steps ofi) Providing a water sample, in particular a wastewater sample, which potentially contains respiratory viruses,ii) Amplifying of viral nucleic acid sequences potentially present in the sample, comprising the use of at least one primer, in particular a primer pair comprising the sequences:TTGCAGCAGTTCCATATGG (PMMoV_For2, SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (PMMoV_Rev2, SEQ ID NO: 6),and optionallyGCTCCAGAATATAGGCATGA (RSV N I For, SEQ ID NO: 1), and GATCTATCTCCTGCTGCTAAT (RSV N I Rev, SEQ ID NO: 2), and ATGGCAAAAGACACCTCAG (RSV P I For, SEQ ID NO: 3) and AGTGATAGATCATTGTCGCTATC (RSV P I Rev, SEQ ID NO: 4), andiii) Detecting of an amplification product of viral nucleic acid sequences present in the sample and thereby detecting of respiratory viruses in the water sample, in particular wastewater sample, preferably further comprising the use of at least one probe selected from the group comprising the sequencesTTACTAATGCTGCTA (RSVA N I Probe, SEQ ID NO: 7),AACAAGTCACTCA (RSVB P I Probe, SEQ ID NO: 8), and TAGCGGACCAGTATACACCATCG (PMMoV_Probe2, SEQ ID NO: 9).

4. Method according to any one of claims 1 to 3, wherein the amplification reaction is a primer extension, a PCR, a digital PCR, a quantitative PCR, an RT-PCR, in particular an RT-dPCR, RT-ddPCR or RT-qPCR.

5. Method according to any one of claims 1 to 4, wherein the respiratory virus is RSV-A and / or RSV-B.

6. Method according to any one of claims 1 to 5, wherein the primers and / or probes are labeled, for example with fluorescein amidite (FAM), FITC (fluorescein isothiocyanate), biotin, 2-[7V-(2-hydroxyethyl)-4-[[2-methoxy-5-methyl-4-[(4-methyl-2-nitrophenyl)diazenyl]phenyl]diazenyl]anilino]ethanol (BHQ-1), 4'-(4-nitro-phenyldiazo)-2'-methoxy-5'-methoxy-azobenzene-4"-(N-2-4,4'-dimethoxytrityl(oxyethyl))-N-ethyl-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite (BHQ2), hexachlorofluorescein (HEX), ZEN, TAO, SUN, JOE, MAX, ATTO, ROX, Texas Red (TxR), YakYel, lABkFQ, IBRQ, Cy5.5 and / or Cy5, and / or wherein the primers and / or probes comprise DNA, RNA, PNA and / or LNA nucleotides, wherein preferably the probe is selected from 5'FAM / TT+AC+TA+AT+G+CT+GC+TA / 3'BHQ-1 (RSVA N I Probe, SEQ ID NO: 7), 5'HEX / AA+C+AA+GT+C+A+CTCA / 3'BHQ1 (RSVB P I Probe, SEQ ID NO: 8), and 5'Cy5 / TAGCGGAC / BHQ2 / CAGTATACACCATCG / -3'BHQ2 (PMMoV_Probe2, SEQ ID NO: 9), where "+ " denotes an LNA nucleotide.

7. Method according to any one of claims 1 to 6, wherein the amplification is a multiplex PCR.

8. Method according to any one of claims 1 to 7, wherein the amplification further comprises the use of at least one primer selected from the group of primers comprising the sequences CAAGACCAATCYTGTCACCTCTGAC (INF A For 1, SEQ ID NO: 10), CAAGACCAATYCTGTCACCTYTGAC (INF A For 2, SEQ ID NO: 11), GCATTYTGGGACAAAVCGTCTACG (INF A Rev 1, SEQ ID NO: 12), GCATTTTGGGATAAAGCGTCTACG (INF A Rev 2 SEQ ID NO: 13), TCCTCAAYTCACTCTTCGAGCG (INF B For, SEQ ID NO: 15), CGGTGCTCTTGACCAAATTGG (INF B Rev, SEQ ID NO: 16), GACCCCAAAATCAGCGAAAT (nCOV NI For, SEQ ID NO: 18), TCTGGTTACTGCCAGTTGAATCTG (nCOV NI Rev, SEQ ID NO: 19), TTACAAACATTGGCCGCAAA(nCOV_N2_For, SEQ ID NO: 20), GCGCGACATTCCGAAGAA (nCOV_N2_Rev, SEQ ID NO: 21),where Y is selected from C or T, and optionallyACCCCGCATTACGTTTGGGTGGACC (nCOV NI Probe, SEQ ID NO: 22), ACAATTTGCCCCCAGCGCTTCAG (nCOV_N2_Probe, SEQ ID NO: 23), TGCAGTCCTCGCTCACTGGGCACG (INF A-P, SEQ ID NO: 14), and CCAATTCGAGCAGCTGAAACTGCGGTG (INF B-P, SEQ ID NO: 17).

9. Method according to one of claims 1 to 8, wherein the amplification is carried out without a dilution of the sample.

10. Oligonucleotide selected from the group consisting of oligonucleotides comprising the sequences or consisting of the sequences:ATGGCAAAAGACACCTCAG (SEQ ID NO: 3), AGTGATAGATCATTGTCGCTATC (SEQ ID NO: 4), TTGCAGCAGTTCCATATGG (SEQ ID NO: 5) and TGTCTAAATAGTTCTTGCATTGTTG (SEQ ID NO: 6), TTACTAATGCTGCTA (SEQ ID NO: 7), AACAAGTCACTCA (SEQ ID NO: 8), TAGCGGACCAGTATACACCATCG (SEQ ID NO: 9) and a mixture of these.

11. A set of oligonucleotides comprising the oligonucleotides comprising the sequences or consisting of the sequences:CAAGACCAATCYTGTCACCTCTGAC (SEQ ID NO: 10), CAAGACCAATYCTGTCACCTYTGAC (SEQ ID NO: 11), GCATTYTGGACAAAVCGTCTACG (SEQ ID NO: 12), GCATTTTGGATAAAGCGTCTACG (SEQ ID NO: 13), TCCTCAAYTCACTCTTCGAGCG (SEQ ID NO: 15), CGGTGCTCTCTTGACCAAATTGG (SEQ ID NO: 16), GACCCCAAAATCAGCGAAAT (SEQ ID NO: 18), TCTGGTTACTGCCAGTTGAATCTG (SEQ ID NO: 19), TTACAAACATTGGCCGCAAA (SEQ ID NO: 20), GCGCGACATTCCGAAGAA (SEQ ID NO: 21), ACCCCGCATTACGTTTGGTGGACC (SEQ ID NO: 22), ACAATTTGCCCCCAGCGCTTCAG (SEQ ID NO: 23), TGCAGTCCTCGCTCACTGGGCACG (SEQ ID NO: 14), and CCAATTCGAGCAGCTGAAACTGCGGTG (SEQ ID NO: 17), wherein Y is selected from C or T.

12. A set of oligonucleotides comprising at least the oligonucleotides according to claim 10 together with at least three oligonucleotides according to claim 11.

13. Oligonucleotide according to claim 11 or set of oligonucleotides according to claim 11 or 12, wherein the oligonucleotide(s) are labeled, for example with fluorescein amidite (FAM), FITC (fluorescein isothiocyanate), biotin, 2-[A-(2-hydroxyethyl)-4-[[2-methoxy-5-methyl-4-[(4-methyl-2-nitrophenyl)diazenyl]phenyl]diazenyl]anilino]ethanol (BHQ-1), 4'-(4-nitro-phenyldiazo)-2'-methoxy-5'-methoxy-azobenzene-4"-(N-2-4,4'-dimethoxytrityl(oxyethyl))-N-ethyl-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite (BHQ2), hexachlorofluorescein (HEX), ZEN, TAO, SUN, JOE, MAX, ATTO, ROX, Texas Red (TxR), YakYel, lABkFQ, IBRQ,Cy5.5 and / or Cy5, and / or wherein the primers and / or probes comprise DNA, RNA, PNA and / or LNA nucleotides.

14. The oligonucleotide according to claim 13, wherein the oligonucleotide is selected from 5'FAM / TT+AC+TA+AT+G+CT+GC+TA / 3'BHQ-1 (SEQ ID NO: 7), 5'HEX / AA+C+AA+GT+C+A+CTCA / 3'BHQ1 (SEQ ID NO: 8), 5'Cy5 / TAGCGGAC / BHQ2 / CAGTATACACCATCG / -3'BHQ2 (SEQ ID NO: 9),5'-FAM / TGCAGTCCT / ZEN / CGCTCACTGGGCACG / 3TABkFQ (SEQ ID NO: 14),5'-YakYel / CCAATTCGA / ZEN / GCAGCTGAAACTGCGGTG / 3TABkFQ (SEQ ID NO: 17), and5'Cy5 / ACAATTTGC / ZEN / CCCCAGCGCTTCAG / 3TABkFQ (SEQ ID NO: 23), wherein "+" denotes an LNA nucleotide and Y is selected from C or T.

15. Kit comprising materials for carrying out the method according to any one of claims 1 to 9, for example at least one suitable oligonucleotide or set of oligonucleotides according to any one of claims 10 to 14, buffers, reagents and / or instructions for use.

16. Use of the kit according to claim 15 or of an oligonucleotide or set of oligonucleotides according to one of claims 10 to 14 for wastewater monitoring, in particular for the detection and quantification of human pathogenic viruses in water samples, in particular in wastewater samples, preferably in the form of multiplex PCR.

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