WO2021247398A1

WO2021247398A1 - Systems and methods to detect pathogens

Info

Publication number: WO2021247398A1
Application number: PCT/US2021/034788
Authority: WO
Inventors: Bryan Walser
Original assignee: Pangolin Llc
Priority date: 2020-05-30
Filing date: 2021-05-28
Publication date: 2021-12-09

Abstract

Provided herein are systems and methods to monitor waste water, detect a pathogen, and control the outbreak and spread of diseases associated with the pathogen.

Description

TITLE

SYSTEMS AND METHODS TO DETECT PATHOGENS BACKGROUND

[001] The invention generally relates to wastewater sampling and pathogen surveillance.

[002] COVID-19 is a highly pathogenic respiratory disease, which exhibited an outbreak after its first appearance in Wuhan, China in December 2019. COVID-19 is caused by a novel coronavirus namely SARS-CoV-2, which causes respiratory illness with elevated fatality rate in patients, including patients with one or more of comorbidities such as obesity, hypertension and diabetes. Cases of COVID-19 in which the patient shows no symptoms of infection appear asymptomatic but may still infect or transmit the virus to the community, state, or country.

[003] The Centers for Disease Control and Prevention confirms that the virus has been found in the feces of patients diagnosed with COVID-19, and is thus present in wastewater systems. The surveillance of sewage or waste water for COVID-19 may provide details on the epidemiology and accelerate governments’ efforts to contain the virus outbreak and save human lives.

[004] The present invention attempts to solve these problems, as well as others.

SUMMARY OF THE INVENTION

[005] Provided herein are methods and systems to detect a pathogen.

[006] The systems and methods are set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the methods, apparatuses, and systems. The advantages of the systems and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the systems and methods as claimed. [007] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

DETATEED DESCRIPTION OF THE INVENTION

[008] The foregoing and other features and advantages of the invention are apparent from the following detailed description of exemplary embodiments, read in conjunction with the accompanying drawings. The detailed description and drawings are merely illustrative of the invention rather than limiting, the scope of the invention being defined by the appended claims and equivalents thereof.

[009] Embodiments of the invention will now be described with reference to the Figures, wherein like numerals reflect like elements throughout. The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive way, simply because it is being utilized in conjunction with detailed description of certain specific embodiments of the invention. Furthermore, embodiments of the invention may include several novel features, no single one of which is solely responsible for its desirable attributes or which is essential to practicing the invention described herein.

[010] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. It will be further understood that the terms “comprises,” “comprising,” “includes,” and/or “including,” when used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[Oil] 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. The word “about,” when accompanying a numerical value, is to be construed as indicating a deviation of up to and inclusive of 10% from the stated numerical value. The use of any and all examples, or exemplary language (“e.g ” or “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

[012] References to “one embodiment,” “an embodiment,” “example embodiment,” “various embodiments,” etc., may indicate that the embodiment(s) of the invention so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment,” or “in an exemplary embodiment,” do not necessarily refer to the same embodiment, although they may.

[013] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological, industrial, electrical, software, and mechanical arts. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[014] Definitions

[015] The term “reverse transcriptase” describes a class of polymerases characterized as RNA- dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA which can then be cloned into a vector for further amplification or manipulation.

[016] Reverse transcription polymerase chain reaction (“rt-PCR”) is a laboratory technique combining reverse transcription of RNA into complementary DNA (“cDNA”) and amplification of specific DNA targets using polymerase chain reaction (PCR). Rt-qPCR, allows for the quantification of a small quantity of RNA. RT-PCR measure the amount of a specific RNA by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR).

[017] “Real-Time Quantitative Reverse Transcription PCR (“qRT-PCR”) is a PCR technology that enables reliable detection and measurement of products generated during each cycle of PCR process. This technique is enabled by introduction of an oligonucleotide probe which is designed to hybridize within the target sequence. Cleavage of the probe during PCR because of the 5' nuclease activity of Taq polymerase can be used to detect amplification of the target-specific product by a detection method. In qPCR/RT-qPCR, the accumulation of PCR product (amplicon) is monitored continuously (i.e., in real time) by measuring the fluorescence signal of a reporter using various chemistries. The amplification reaction enables identification of the cycles during which near-logarithmic PCR product generation occurs. This allows the assay to reliably quantify the DNA or RNA content in a given sample. The increase in fluorescent signal recorded during the assay is proportional to the amount of DNA synthesized during each amplification cycle. Individual reactions are characterized by the cycle fraction at which fluorescence first rises above a defined background fluorescence, a parameter known as the threshold cycle (Ct) or crossing point (Cp). The lower the Ct, the more abundant the initial target. The technique permits accurate quantification of target molecules over a wide dynamic range.

[018] The term “primer,” as used herein, refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of four different nucleoside triphosphates and an agent for extension (for example, a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. [019] A primer is preferably a single-stranded DNA. The appropriate length of a primer depends on the intended use of the primer but typically ranges from about 6 to about 225 nucleotides, including intermediate ranges, such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25 to 250 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for the amplification of a given target sequence is well known in the art and described in the literature cited herein. [020] Primers can incorporate additional features which allow for the detection or immobilization of the primer but do not alter the basic property of the primer, that of acting as a point of initiation of DNA synthesis. For example, primers may contain an additional nucleic acid sequence at the 5' end which does not hybridize to the target nucleic acid, but which facilitates cloning or detection of the amplified product, or which enables transcription of RNA (for example, by inclusion of a promoter) or translation of protein (for example, by inclusion of a 5’-UTR, such as an Internal Ribosome Entry Site (IRES) or a 3’-UTR element, such as a poly(A)n sequence, where n is in the range from about 20 to about 200). The region of the primer that is sufficiently complementary to the template to hybridize is referred to herein as the hybridizing region.

[021] As used herein, a primer, probe, or amplicon is “specific,” for a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer, probe, or amplicon hybridizes primarily to the target nucleic acid. Typically, a primer, probe, or amplicon is specific for a target sequence if the primer-target duplex stability is greater than the stability of a duplex formed between the primer and any other sequence found in the sample. One of skill in the art will recognize that various factors, such as salt conditions as well as base composition of the primer, probe, or amplicon and the location of the mismatches, will affect the specificity of the primer, and that routine experimental confirmation of the primer, probe, or amplicon specificity will be needed in many cases. Hybridization conditions can be chosen under which the primer, probe, or amplicon can form stable duplexes only with a target sequence. Thus, the use of target- specific primer, probe, or amplicon under suitably stringent amplification conditions enables the selective amplification of those target sequences that contain the target primer, probe, or amplicon binding sites.

[022] As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus fiiriosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase, among others. “RNA polymerase” catalyzes the polymerization of ribonucleotides. The foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase, which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerase (“RNAP”) include, for example, RNA polymerases of bacteriophages (e.g. T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, Syn5 RNA polymerase), and E. coli RNA polymerase, among others. The foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase. The polymerase activity of any of the above enzymes can be determined by means well known in the art.

[023] Detection System

[024] The present invention is a detection system that applies to separate sanitary sewer systems, combined sewer systems, or standalone sewer systems. Separate sanitary sewer systems are designed to transport sewage alone in underground pipe or tunnel system from houses and commercial buildings to treatment facilities or disposal. In municipalities served by sanitary sewers, separate storm drains may convey surface runoff directly to surface waters. Sanitary sewers are part of an overall system called a sewage system or sewerage. Sanitary sewers are distinguished from combined sewers, which combine sewage with storm water runoff in one pipe. A combined sewer system is a sewage collection system of pipes and tunnels designed to simultaneously collect surface runoff and sewage water in a shared system. A standalone sewer system may be a septic tank where sewage is collected for home or small building where there is no separate or combined sewer system. “Wastewater system” herein means a separate sewer system, a combined sewer system, or a standalone sewer system that is defined by a geographical region. “Wastewater” herein means the type of wastewater that is produced by a community of people. Wastewater is characterized by volume or rate of flow, pressure, physical condition, chemical and toxic constituents, and its bacteriologic, virological, or pathogenic status (which organisms it contains and in what quantities). Wastewater consists mostly of greywater (from sinks, bathtubs, showers, dishwashers, and clothes washers), blackwater (the water used to flush toilets, combined with the human waste that it flushes away); soaps and detergents; and toilet paper. In alternative embodiments, the system may incorporated for testing finished drinking water, treated water, disinfected water, irrigation water, and water obtained from wells, rivers, lakes and recreational waters such as swimming pools. Other embodiments, the system samples and analyzes food (such as fruits, vegetables, meat and prepared food items), swabs taken from slaughter lines, and meat surfaces, as well as swabs taken from environmental surfaces from slaughter houses, and meat preparation facilities, soil and clinical and veterinary samples including stool and biopsy samples.

[025] Generally speaking, the detection system monitors sewage or untreated wastewater for pathogen levels in a city, state, or country and comprises detecting a pathogen in the wastewater system and providing feedback for the detector to adjust to varying parameters of the pathogen and to optimize detection of the pathogen due to wastewater conditions. A pathogen is a bacterium, virus, or other microorganism that can cause disease. Pathogens detectable by the system, include, but are not limited to: prions, bacterium, viruses, fungi, algae, or parasites. In one embodiment, the pathogen is the virus COVID-19.

[026] In some wastewater systems, pathogens are far more dilute in wastewater, especially in comparison to bodily fluids, such as plasma, in which they are usually measured, and the wastewater matrix is a complex environment to for the detection. In some wastewater systems, pathogens can become more concentrated in wastewater due to wastewater conditions, drought, and collection methods. As previously mentioned, wastewater contains a diverse abundance of chemical and biological targets which can give extremely detailed information about the populations generating it. A detection system detects levels of pathogens as distinct from non pathogen levels in the wastewater matrix, and the subsequent analysis of specific pathogens may provide the basis for actionable measures for a city, state, or country.

[027] In one embodiment, the detection system comprises extraction methods for the pathogen, and PCR and non-PCR methods to detect the pathogen in wastewater. The detection system comprises analytical tools and algorithms for the analysis of a wide number of pathogens in wastewater. The detection system measures the levels of a pathogen in the wastewater system in real time and normalizes to the population of the wastewater system among other variables, to provide information on public health in real time. This detection system comprises spatial and temporal comparisons across the entire wastewater system and different wastewater systems in cities, states, and countries. The detection system establishes real time levels of pathogens and spread of the pathogens through wastewater systems in discrete locations, time points, and geographies.

[028] Wastewater includes pathogenic DNA/RNA residues from bacteria, viruses and fungi. The detection in influent wastewater is from human sources and hence indicates what diseases or pathogens are circulating within a population. Viruses pose a significant threat due to their high mutation rates and ability to adapt to new hosts, e.g. humans, particularly in the case of RNA viruses, where higher nucleotide substitution rates can result in rapid adaption and spreading in new host populations. The detection system provides for viral surveillance of these high mutation rates in the wastewater system, according to one embodiment.

[029] The detection system detects rising levels of the pathogen above an infection level, threshold or rate of detection indicates that the city, state, or country may require containment measures to delay or stop the spread of the pathogen. The threshold level of pathogen considers the population above N people, N% of relevant population, and size of population. In one embodiment, the infection level or threshold of the pathogen may be above about 0.1 million, about 0.2 million, about 0.3 million, about 0.4 million, about 0.5 million, or about 0.6 million genome units fL of wastewater, according to one embodiment. In another embodiment, the infection level or threshold of the pathogen may be above about 10, about 20, about 30, about 40, or about 50 genome units per mL of wastewater. In one embodiment, for the COVID-19 pathogen, as many as about 600,000 to about 30,000,000 viral genomes of SARS-CoV-2 per mL of fecal material, assuming a fecal load of about 100-400 g feces/day/person with a density of about 1.06 g/mL. Normalization of population of the pathogen’s infection level in the wastewater ensures that a significant increase in pathogen concentration in a wastewater sample does not correspond to an increase in population in the serviced wastewater area or a decrease in the diluent as might occur during a dry season or drought, or other conditions affecting the testing environment, rather than the pathogen itself.

[030] In one embodiment, the detection system comprises a detector to detect the pathogen, which may be placed at any location along the wastewater system with access to the wastewater. Such locations may be junctions, upstream facilities, nursing home, hospitals, industrial, and residential waste water junctures. The detector may include a collection, sampling, or separation method to isolate the pathogen from wastewater. The detector may employ molecular methods to detect the pathogen, including I. PCR methods and/or II. Non-PCR methods.

[031] I PCR methods

[032] In one embodiment, the detection system comprises PCR methods to detect the pathogen, and detecting the pathogens with high sensitivity in wastewater, regardless if the pathogen is infectious or non-infectious. PCR includes primers specific for the pathogen. In one embodiment, the primer is specific for a SARS-CoV-2 gene. In some embodiments, the PCR includes a primer having a specific nucleotide sequence selected from the coding region of: (a) the region spanning NSP1, NSP2, NSP3 of the SARS-CoV-2 gene (Accession No. YP_009725297.1, Accession No. P89070, or Accession No. B5APV2); (b) the region spanning Nl, N2, and N3 loci of the SARS- CoV-2 nucleocapsid gene; (c) region spanning E gene (SARS-Cov2 Wuhan-Hu isolate sequence 124 NC_045512.); or the region spanning the SARS-CoV-2 S gene. In other embodiments, the detection system comprises an amplicon having a specific nucleotide sequence selected from the coding region of: (a) the region spanning NSP1, NSP2, NSP3 of the SARS-CoV-2 gene (Accession No. YP_009725297.1, Accession No. P89070, or Accession No. B5APV2); (b) the region spanning Nl, N2, and N3 loci of the SARS-CoV-2 nucleocapsid gene; (c) region spanning E gene (SARS-Cov2 Wuhan-Hu isolate sequence 124 NC_045512.); or the region spanning the SARS- CoV-2 S gene.

[033] In alternative embodiments, the detection system comprises digital PCR (dPCR) or real time quantitative reverse transcription polymerase chain reaction (qRT-PCR). In dPCR, the absolute quantification of target genes is calculated using Poisson distribution statistics via the partitioning of DNA/RNA samples into tens and thousands of reaction wells. Due to this partitioning effect, PCR inhibitory substances have demonstrated to have less of an effect in environmental samples, including wastewater, when analyzed via dPCR. In one embodiment, microfluidics dPCR and or high density microarray dPCR may be deployed in the detection system. Microfluidic dPCR array chip uses a microfluidics sample handling system to split one wastewater sample into hundreds of individual reaction chambers that reside on an “integrated fluidic circuit” or chip, which allows performance of about 10,000 PCRs per chip.

[034] In one embodiment, the detection system comprises detecting the pathogen by qRT-PCR. DNA probes for the qRT-PCR detection of the pathogen are selected from the group consisting of: SYBR Green, TaqMan, molecular beacons, scorpion probes, and multiplex probes. All of these probes allow the detection of PCR products by generating a fluorescent signal. While the SYBR Green dye emits its fluorescent signal simply by binding to the double-stranded DNA in solution, the TaqMan probes', molecular beacons' and scorpions' generation of fluorescence depend on Forster Resonance Energy Transfer (FRET) coupling of the dye molecule and a quencher moiety to the oligonucleotide substrates. The detection system comprises detection levels and accounts for potential loss of qRT PCR signal in the sewer from virus degradation and RNA loss.

[035] In alternative embodiments, the detection system comprises OpenArray real-time qPCR, which combines the parallelism of microarrays with the quantification capabilities, sensitivity and specificity of qPCR, can run 3000 PCR tests on one wastewater sample or as few as 64 PCR tests against 48 wastewater samples. OpenArray real-time qPCR tests for multiple pathogens and mutated pathogens, simultaneously.

[036] The detection system provides a method useful to detect a pathogen, the method comprising the step of subjecting a wastewater sample that is either extracting the RNA from said wastewater or is a cDNA equivalent to a polymerase chain reaction comprising primers adapted to produce and amplify a detectable amplicon from a gene responsible for the pathogenicity, and measuring in real time the accumulation of said amplicon during said reaction. In a preferred embodiment of the invention, to render the amplicon detectable during the reaction, the polymerase chain reaction is performed in the presence of both an enzyme having 5^' nuclease activity (a 5^' nuclease) and a probe having a detectable label released following cleavage of the probe by the action of the 5^' nuclease.

[037] In another aspect, the detection system provides a multiplexed method useful to detect at least two different pathogens in a given wastewater sample, the method comprising the step of subjecting a sample comprising RNA extracted from said wastewater, or a cDNA equivalent thereof, to a polymerase chain reaction comprising primers adapted to produce and amplify detectable amplicons that are different for each pathogen, and measuring in real time the accumulation of said amplicons during the reaction. Desirably, the multiplexed method also utilizes the 5^' nuclease susceptible probes to detect and measure accumulation of the amplicons. Greene SCPrimer software may be used to design multiplex PCR primer panels for detection and differentiation of viral pathogens

[038] For one embodiment in the detection of specific pathogen, the detection system further provides oligonucleotide primers and oligonucleotide probes useful in a polymerase chain reaction to detect the presence of a selected pathogen in the wastewater.

[039] In other embodiments, the detection system comprises a primer or probe having a nucleotide sequence selected from the coding region of: (a) the region spanning NSP1, NSP2, NSP3 of the SARS-CoV-2 gene (Accession No. YP_009725297.1, Accession No. P89070, or Accession No. B5APV2); (b) the region spanning Nl, N2, and N3 loci of the SARS-CoV-2 nucleocapsid gene; (c) region spanning E gene (SARS-Cov2 Wuhan-Hu isolate sequence 124 NC_045512.); or the region spanning the SARS-CoV-2 S gene. The probe or primer is optimized per specific pathogen and per observation site in the wastewater. Probe or primer optimization may be performed by an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize the amplicon or primer, and therefore achieve a real time comparison of the two different amplicon or primers. Amplicon or primer comparison may use algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp.

[040] In one embodiment, the primers and probes are provided in Tables 1 and 2.

[041] Table 1

[042] Primers and probes, real-time RT-PCR for 2019 novel coronavirus

[043] ^a W is A/T; R is G/A; M is A/C; S is G/C. FAM: 6-carboxyfluorescein; BBQ: blackberry quencher. ^b Optimised concentrations are given in nanomol per litre (nM) based on the final reaction mix, e.g. 1.5 pL of a 10 pM primer stock solution per 25 pL total reaction volume yields a final concentration of 600 nM as indicated in the table. [044] Table 2: Primer and probe sets used for SARS-CoV2 sequences

[045] *Total number of sequences with SNP in primer/probe region. Blank cells represent 100% homology. N/A: Not applicable. EXO and RNAseP have no homology with SARS-CoV2 sequences, FAM: 6-carboxyfluorescein, VIC: 2^'-chloro-7^'phenyl-l,4-dichloro-6-carboxy- fluorescein BHQ1: Black Hole Quencher-1, MGB: Minor Grove Binder. [046] In one embodiment, the detection system comprises PCR conditions to remove interfering substances in the wastewater include, but are not limited to: inhibition avoidance, chemical avoidance, and physical avoidance, as described below. In another embodiment, PCR inhibitors are removed before PCR is performed, as described below.

[047] In one embodiment, the PCR inhibitor methods comprise altering the primer or probe length and nucleoside optimization. A PCR probe length may be between lbp to about 250bp and target the NSP1, NSP2, or NSP3 sequence of the SARS-CoV-2 gene to amplify cDNA reverse transcribed from viral RNA. The primer length is stable against hairpin formations. Nucleosides are highly functionalized biomolecules essential for the storage of information as DNA and RNA, cellular energy transfer and as enzyme cofactors. Modified nucleosides are employed enhancing the probe and primer for SARS-CoV-2. Nucleoside optimization may be performed by an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize the nucleosides, and therefore achieve a real time comparison of the two different nucleosides. Nucleoside sequence comparison may use algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp.

[048] Post-PCR detection may be coupled with mass spectroscopy, array based hybridization, Triangulation Identification for Genetic Evaluation of Risks (TIGER), beacon-based fluorescence in situ hybridization (FISH) may also be used to rapidly identify pathogens in wastewater. TIGER employs electrospray Fourier transform ion cyclotron resonance mass spectrometer (MS) to provide base counts for each of the four nucleotide bases contained in a short (<140 nucleotides) DNA amplicon.

[049] The complexity of a wastewater matrix is challenging for biological pathogens. Composition of wastewater contains a diverse range of PCR inhibitors including fats, proteins and humic and fulvic acids, which can cause problems later during downstream processing during PCR. The detection system comprises a removal system for removing PCR inhibitors in samples in the wastewater and an extraction system for DNA/RNA for extracting DNA/RNA.

[050] A PCR Inhibitor Removal: [051] Composition of wastewater contains a diverse range of PCR inhibitors including fats, proteins and humic and fulvic acids, which can cause problems later during downstream processing during PCR. Wastewater samples inherently inhibit real-time PCR. The quality of an RNA preparation greatly affects the results obtained when analyzing it by a number of different molecular biology techniques such as qRT-PCR. Degraded RNA will produce a lower signal than in an equivalent intact RNA sample.

[052] RNA is much more susceptible to degradation than DNA. RNA is readily hydrolyzed when exposed to conditions of high pH, metal cations, high temperatures and contaminating ribonucleases. A major cause of RNA degradation is ribonuclease contamination, and the detection system protects ribonuclease contamination for the PCR and non-PCR methods, including RNA isolation, mRNA purification, RNA storage, and qRT-PCR, among others.

[053] Since ribonucleases or RNases are difficult to inactivate, in one embodiment, the detection system comprises detecting the presence of RNases in wastewater sample be tested by real-time PCR and then provide RNase inhibitors either before PCR and/or during PCR conditions. RNases may be selected from the group of: RNase A, RNase H, RNase III, RNase L, RNase P, RNase PhyM, RNase Tl, RNase E, RNase HI, RNase T2, RNase U2, RNase V, PNPase, RNase PH, RNase R, RNase BN, RNase D, RNase T, oligoribonuclease, exoribonuclease I, exoribonuclease II.

[054] RNase inhibitors may be selected from i) director inhibitors and ii) indirect inhibitors. [055] i. Direct Inhibitors

[056] Direct Inhibitors may be selected from a) allosteric inhibitors and b) competitive inhibitors. [057] a) Allosteric Inhibitors

[058] An allosteric inhibitor inhibits an RNase by binding to allosteric site alters the protein conformation in active site of enzyme which consequently changes the shape of active site of the RNase. Thus, the RNase no longer remains able to bind to its specific substrate RNA. The Rnase is unable to perform its catalytic activity i.e. RNase is now inactive.

[059] In one method of inhibiting RNases, is adding diethyl pyrocarbonate (DEPC) at a sufficient concentration. DEPC covalent modifies histidine (most strongly), lysine, cysteine, and tyrosine residues. Additional molecular biology reagents and incubating for period of time and then removing the DEPC-RNase complex from the wastewater sample. In one embodiment, DEPC reacts with the s-amino groups of lysine and the carboxylic groups of aspartate and glutamate both intra- and intermolecularly, which forms polymers of the ribonuclease.

[060] Guanidinium thiocyanate is an allosteric inhibitor of RNases at high concentration of combined with ~3-mercaptoethanol. Guanidinium is an effective inhibitor of most enzymes due to its chaotropic nature. However, if RNA is dissolved in guanidinium, then it must first be purified from the guanidinium prior to being used in an enzymatic reaction.

[061] Vanadyl-ribonucleoside complexes (VRC) may be used to inhibit RNases during pathogen detection. As VRC strongly inhibits the translation of mRNA in cell-free systems and must be removed from RNA samples by phenol extraction.

[062] b) Competitive Inhibitors

[063] Competitive inhibitors compete for the active site of RNase, but they are not a substrate for the RNase. The competitive inhibitors in the detection system include with reversible antagonists, irreversible antagonists, or substrate flooding.

[064] Commercial RNase inhibitors

[065] Takara Bio's Recombinant RNase Inhibitor acts as a noncompetitive inhibitor of RNases A, B and C and is active over a wide pH range and in at least 1 mM DTT.

[066] APExBIO’s RNase Inhibitor is a 50 kD recombinant protein isolated from E. coli containing the ribonuclease inhibitor gene originally from mouse. The RNase inhibitor has specificity for RNases A, B and C, but cannot inhibit RNase 1, RNase Tl, SI Nuclease, RNase H or RNase from Aspergillus. It can bind several kinds of RNase non-covalently in a 1:1 ratio with high affinity.

[067] Two types of protein-based RNase inhibitors are commercially available: human placental ribonuclease inhibitor and PRIME Inhibitor™. RNases of the class A family bind tightly to these protein inhibitors and form noncovalent complexes that are enzymatically inactive. The major disadvantage of these inhibitors is that they have a specificity for inhibiting RNases A, B and C, just like HPRI, and do not inhibit RNases Tl, T2, H, Ul, U2 or CL3. They do not inhibit other classes of RNases. Another disadvantage when using placental ribonuclease inhibitor is that it denatures within hours at 37°C, releasing the bound ribonuclease. Thus, the RNA sample is only protected for only a few hours at most.

[068] RNAguard is a competitive inhibitor and a recombinant placental RNase inhibitor from Promega. PRI strongly binds to the active site of the RNase molecule within the S-protein part and forms a tight 1 : 1 complex at pH 7-8 (18, 19). PRI is a competitive inhibitor with a Ki of 4 x 10-14 M. The Ki increases with increasing NaCl concentrations, suggesting the importance of ionic interactions. The half-time of dissociation of the PRI-RNase A complex is 13 hr at 25°C.

[069] An irreversible antagonist is a type of antagonist that binds permanently to RNase, either by forming a covalent bond to the active site, or alternatively just by binding so tightly that the rate of dissociation is effectively zero at relevant time scales. Irreversible antagonists may be selected from the group consisting of: natural protein inhibitors and synthetic low molecular- weight inhibitors.

[070] A reversible antagonist binds non-covalently to the RNase, therefore can be washed out. Reversible antagonists may be selected from the group consisting of: natural protein inhibitors and synthetic low molecular-weight inhibitors

[071] In one embodiment, the RNase is removed by substrate flooding comprises exposing the wastewater to large amounts of RNA, DNA, or both before the pathogen detection. (Herring sperm, yeast RNA, etc.)

[072] In one embodiment, anionic polymers as a chemical method for RNase A inhibition, such as Polyvinyl Sulfonic Acid (PVSA; average MW »2-5 kDa), a negatively charged polymer with sulfate branches, is a potent inhibitor of RNase A16. The repeating sulfate units resemble the repeating phosphate units that form the backbone of RNA and are thought to form competitive coulombic interactions with RNase A, thereby occupying its RNA-binding sites and effectively inhibiting RNase A [073] ii. Indirect Inhibitors

[074] Indirect inhibitors include other RNase’ s, RNA digestion, and other inhibitors that are not direct inhibitors. Other inhibitors that are not direct inhibitors include aggregation methods, preferential adsorption of RNases and other inhibitors, and mechanical and chemical methods of inhibition.

[075] The pathogens may be absorbed in clay, sand, and inorganic material in the wastewater, so the preferential adsorption may isolate the pathogen in the clay, sand, and inorganic material.

[076] Enzymatic or other cleanup of the wastewater comprises applying latex beads, carbohydrates, anion exchange resins, or similar substances as part of wastewater sample collection and/or the PCR processing step.

[077] Bacteriophages and other microorganisms may include RNases. The removal of bacteriophages and other microorganisms from the wastewater may be included in the detection system.

[078] Other methods

[079] Other reagents have been used to inhibit ribonucleases including SDS, EDTA, proteinase K, heparin, to hydroxylamine-oxygen-cupric ion, bentonite and ammonium sulfate. None of these reagents are strong inhibitors alone, although their inhibitory effect may be improved by using them in combination. Like many of the RNase inhibitors, although these chemicals inhibit RNase activity, they also may inhibit other enzymes such as reverse transcriptase and DNase I. Therefore, the RNA must be purified away from the inhibitory reagents before it can be subjected to other enzymatic processes.

[080] Reducing agents are frequently used as adjuvants to RNA isolation solutions in conjunction with denaturants to reduce the disulfide bonds in RNases that are rendered accessible by the denaturant. In one embodiment, reducing reagents are selected from the group consisting of (3- mercaptoethanol, 3o dithiothreitol (DTT), dithioerythritol (DTE), and glutathione). Another such reducing agent is the amino acid cysteine. (3-mercaptoethanol is often included in RNA isolation solutions combined with guanidinium thiocyanate to reduce ribonuclease activity and solubolize proteins.)

[081] DTT's reducing activity can be accurately assayed using 5, 5'-dithiobis (2-nitrobenzoic acid) or DTNB. The reduction of DTNB mediated by DTT generates a yellow color whose absorbance can be measured at 412 nm using a spectrophotometer. RNase A, RNase 1 and RNase T1 all contain disulfide bonds and, therefore, are susceptible to reduction.

[082] The removal of RNase inhibitors from wastewater may comprise using Bovine Serum Albumin (BSA) (Fraction V, SIGMA) or milk powder at a sufficient concentration to remove RNase inhibitors from wastewater before or during PCR detection. Additional inhibition removal was carried out during following filtration of wastewater, treating the filter with EDTA for a period of time. The following inhibitor removers may be added to the wastewater: Chelex® (BIO RAD) slurry, to a final concentration of 20% and PVP-360 (ICN, Aurora, Ohio). The addition of BSA to the PCR mix to remove RNase inhibitors from wastewater being tested by PCR. Alternatively, the addition of EDTA, Chelex® 100 and PVP-360 treatment during RNA extraction to the wastewater samples before the PCR method to detect the pathogen.

[083] In one embodiment, viral particles from approximately 0.25-1 liter of sewage/wastewater /effluent samples were concentrated using first centrifugation to remove sediment and large particles. Secondary concentration of the supernatant was performed using polyethylene glycol (PEG) or alum (20mg/l) precipitation, followed by additional centrifugation. The mixture was incubated at 4°C with 100-rpm agitation for about 12h, then centrifuged at 14,000 g for 45 min at 4°C to pellet the virus particles. Virus particles were then resuspended in phosphate buffered saline (PBS). The aqueous phase (containing virus particles) was collected and filtered through a 0.22pm filter. Ultra-15 centrifugal tubes with a molecular weight cutoff of 30 kDa (Amicon) were used to further concentrate the sample to a final volume of 1 ml. Samples were stored at -20/-80°C until further analysis. After primary and secondary concentrations, viral RNA was extracted from the samples using viral RNA extraction kit (RNeasy mini kit- QIAGEN and EasyMAG -bioMerieux, France) and then stored at - 80°C.

[084] Each such embodiment, for purposes of the claims described herein, is to be assessed in the context of environmental data and other information relevant to the clinical behavior of the virus or pathogen, as well as its physio-chemical properties in vitro.

[085] II. Non-PCR methods

[086] The detection system comprises non-PCR methods selected from the group of nucleic acid microarrays, primer-capture modalities, or antibody/receptor methods to detect multiple pathogens simultaneously. The detection system comprises non-PCR methods selected from the group of nucleic acid capture optimization, advanced mass spectrometry, and electrochemical detection of the pathogen. Combining these multiplexed methods with fiberoptic sensors and lab-on-a-chip technology allows the detection system to rapidly screen, identify, and quantify multiple pathogens in real time.

[087] Nucleic acid microarrays comprise a collection of microscopic DNA spots attached to a solid surface. DNA microarrays measure the pathogen levels in wastewater or to measure multiple regions of a pathogen genome. Each DNA spot contains picomoles (10-12 moles) of a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section of a gene or other DNA element that are used to hybridize a cRNA (also called anti-sense RNA) sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target.

[088] Primer capture modalties include primer extension capture (PEC) as a method to enrich DNA eluates of targeted DNA molecules and remove nontarget molecules from pools containing both. PEC uses single-stranded DNA or RNA probes that are designed to bind specifically to sequences of the pathogen. Probes containing biotin are annealed to targets during a lengthy incubation step, after which avidin-biotin binding is used to extract the biotin-labeled probes, thus enriching for the pathogen of interest.

[089] Antibody/receptor methods of detecting the pathogen include immunological assays, ELISA, and Lateral flow immunoassays.

[090] Immunological assays for pathogen detection work on the basis of antibody-antigen interaction. The quality of an immunoassay hinges on the level of affinity and specificity of the antibody for the pathogen. In the case of the detection of whole bacteria, the antibody must bind to an epitope(s) on the cell surface. The epitope must be unique to the target pathogen in order to ensure assay specificity. Monoclonal antibodies greatly increase the reliability of immunoassays for pathogen detection by reducing the chances of non-specific binding and thus false positive results.

[091] Enzyme-linked immunosorbance assays (ELISA) may be used for pathogen detection in wastewater. In a sandwich ELISA, antibodies are immobilised to the surface of the wells of a microtiter plate. The wastewater sample to be tested is then added to the wells followed by a subsequent washing. If the target pathogen is present, it will bind to the antibody and remain bound following the wash step. A secondary antibody that will bind to another epitope on the analyte, usually conjugated with an enzyme such as horseradish peroxidase, is then added and subsequently washed. If the pathogen is present, the secondary antibody will remain bound to the pathogen in the well. The substrate to the enzyme which is conjugated to the secondary antibody is then added resulting in a colorimetric change that is directly proportional to the amount of secondary antibody that is bound and is therefore an indication of the presence of the pathogen. The intensity of the color can be compared to a standard curve that relates to known concentrations of the pathogen, allowing for the inference of the concentration of the pathogen in the wastewater sample.

[092] Lateral flow immunoassays employ immunochromatographic strips through which the wastewater sample migrates. If the pathogen is present, it will first bind to an antibody conjugate such as a color labelled antibody. It then migrates to a strip of immobilized antibody, which binds to the pathogen conjugate. If the pathogen is present, a color change will occur at the strip of antibodies. A second strip of antibodies that specifically detect the color labelled antibody acts as a control, ensuring that there has been adequate migration of the sample. [093] Advanced mass spectrometry may include matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid pathogen identification. In MALDI-TOF mass spectrometry, the ion source is matrix-assisted laser desorption/ionization (MALDI), and the mass analyzer is time-of-flight (TOF) analyzer. MALDI is a soft ionization that involves a laser striking a matrix of small molecules to make the analyte molecules into the gas phase without fragmenting or decomposing them. A laser energy absorbing matrix creates ions from large molecules with minimal fragmentation. Analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated gases, and then they can be accelerated into whichever mass spectrometer is used to analyze them.

[094] The Non-PCR detection system comprises Next Generation Sequencing (NGS) to detect the pathogen in the wastewater, providing information on the complex pathogens in samples, including identification of the diverse range of pathogens and resistance genes present in the wastewater. In one embodiment, the detection system analyses the viruses and resistance genes present via metagenomics providing key information on novel pathogens as well as re-emerging infectious diseases.

[095] The detection system may include detector updates from the wastewater system, as described in commonly assigned US provisional application serial no. 63/030,017, filed May 26, 2020

[096] In one embodiment, the detection system comprises interpreting data through an algorithm or Artificial Intelligence (AI) platform. The AI platform may interpret data over time and assess foreground variables and background variables for the detection of the pathogen in wastewater. Data, AI implementation, and algorithmic interpretation methods may be used as described in commonly assigned U.S. provisional application serial no. 63/033,627, filed June 2, 2020.

[097] Pathogens

[098] A wide variety of pathogenic organisms pass through municipal waste-water treatment systems. Any type of infection within a community is likely to lead to pathogen excretion in bodily fluids/sub stances and therefore, transported into the community sewage system. Infections may be classified into symptomatic infections, and asymptomatic infections. Symptomatic infections may result in death, severe illness, moderate severity, and mild illness-all of which are clinical diseases. Asymptomatic infections may be infection without clinical illness and exposure including colonization. [099] Table 3 classifies pathogen in categories Category A pathogens require the most intensive public preparedness efforts due to the potential for mass causalities, public fear, and civil disruption. Category B pathogens are also moderately easy to spread, but have lower mortality rates. Category C pathogens do not present a high public health threat, but could emerge as future threats

[0100] Table 3: The center for disease control select agents

[0101] Agents causing enteric and respiratory infections are released in large numbers in feces and respiratory secretions. Many of the enteric viruses such as the enteroviruses and adenoviruses may replicate both in the intestinal and respiratory tract. The number of enteric viruses detected can approach peak concentrations of 10¹² organisms per gram of stool while protozoa can approach 10⁶-10⁷ per gram. Cultivatable enteric bacterial pathogens such as Salmonella may also occur in concentrations as large as 10¹¹ per gram. The concentration of respiratory viruses ranges from 10⁵ to 10⁷ per ml of respiratory secretion. Blood-borne viruses such as HIV will be found in the feces of infected persons and many viruses will occur in the urine during infection of the host, although these excreted viruses may not be infectious. The total amount of virus released by a person is, of course, also related to the amount of feces, urine, respiratory secretion, and skin that is released by the person. On average, a person excretes between 100 to 400 g of feces and 700- 2000 ml of urine per day [0102] Examples

[0103] The previous examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0104] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.

[0105] System hardware and software

[0106] As used in this application, the terms “system” may refer to a computer-related entity, either hardware, a combination of hardware and software, software, or software in execution. For example, a system can be, but is not limited to being, a process running on a processor, a processor, an object, an executable, a thread of execution, a program, and/or a computer. By way of illustration, both an application running on a server and the server can be a component. One or more components can reside within a process and/or thread of execution, and a component can be localized on one computer and/or distributed between two or more computers.

[0107] Generally, program modules include routines, programs, components, data structures, etc., that perform particular tasks or implement particular abstract data types. Moreover, those skilled in the art will appreciate that the inventive methods can be practiced with other computer system configurations, including single-processor or multiprocessor computer systems, minicomputers, mainframe computers, as well as personal computers, hand-held computing devices, microprocessor-based or programmable consumer electronics, and the like, each of which can be operatively coupled to one or more associated devices.

[0108] The illustrated aspects of the innovation may also be practiced in distributed computing environments where certain tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules can be located in both local and remote memory storage devices.

[0109] A computer typically includes a variety of computer-readable media. Computer-readable media can be any available media that can be accessed by the computer and includes both volatile and nonvolatile media, removable and non-removable media. By way of example, and not limitation, computer-readable media can comprise computer storage media and communication media. Computer storage media includes volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer- readable instructions, data structures, program modules or other data. Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disk (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by the computer.

[0110] Communication media typically embodies computer-readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism, and includes any information delivery media. The term “modulated data signal” means a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media includes wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, RF, infrared and other wireless media. Combinations of the any of the above should also be included within the scope of computer-readable media.

[0111] Software includes applications and algorithms. Software may be implemented in a smart phone, tablet, or personal computer, in the cloud, on a wearable device, or other computing or processing device. Software may include logs, journals, tables, games, recordings, communications, SMS messages, Web sites, charts, interactive tools, social networks, VOIP (Voice Over Internet Protocol), e-mails, and videos.

[0112] In some embodiments, some or all of the functions or process(es) described herein and performed by a computer program that is formed from computer readable program code and that is embodied in a computer readable medium. The phrase “computer readable program code” includes any type of computer code, including source code, object code, executable code, firmware, software, etc. The phrase “computer readable medium” includes any type of medium capable of being accessed by a computer, such as read only memory (ROM), random access memory (RAM), a hard disk drive, a compact disc (CD), a digital video disc (DVD), or any other type of memory.

[0113] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0114] While the invention has been described in connection with various embodiments, it will be understood that the invention is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention, and including such departures from the present disclosure as, within the known and customary practice within the art to which the invention pertains.

Claims

CLAIMS What is claimed is:

1. A method of detecting a pathogen comprising: a. detecting a pathogen in wastewater; b. optimizing detection of the pathogen due to wastewater conditions; c. analyzing the levels of the pathogens.

2. The method of Claim 1, wherein optimizing detection of the pathogen comprises assessing the detection of the pathogen in the context of environmental data and other information relevant to the clinical behavior of the virus or pathogen, as well as its physio-chemical properties in vitro.

3. The method of Claim 2, wherein detecting the pathogen includes detecting levels of pathogens distinct from levels of non-pathogens in the wastewater matrix.

4. The method of Claim 3, further comprising analyzing the pathogen levels in wastewater and normalizing the pathogen levels to the population of the wastewater system.

5. The method of Claim 4, wherein detecting the pathogen comprises detecting at least 10 units of the pathogen per mL of wastewater.

6. The method of Claim 5, wherein detecting the pathogen in wastewater comprises Polymerase Chain Reaction (PCR) methods or non-PCR methods.

7. The method of Claim 4, wherein the PCR method comprises a primer specific for the pathogen; and wherein the PCR method is selected from the group consisting of digital PCR (dPCR), real-time quantitative reverse transcription polymerase chain reaction (qRT- PCR), and OpenArray real-time qPCR.

8. The method of Claim 7, wherein the primer is specific for a SARS-CoV-2 gene and having a specific nucleotide sequence selected from the group consisting of: (a) the region spanning NSP1, NSP2, NSP3 of the SARS-CoV-2 gene (Accession No. YP_009725297.1, Accession No. P89070, or Accession No. B5APV2); (b) the region spanning Nl, N2, and N3 loci of the SARS-CoV-2 nucleocapsid gene; (c) region spanning E gene (SARS-Cov2 Wuhan-Hu isolate sequence 124 NC_045512.); or (d) the region spanning the SARS-CoV- 2 S gene.

9. The method of Claim 8, wherein the PCR method comprises providing an amplicon having a specific nucleotide sequence selected from group consisting of: (a) the region spanning NSP1, NSP2, NSP3 of the SARS-CoV-2 gene (Accession No. YP_009725297.1, Accession No. P89070, or Accession No. B5APV2); (b) the region spanning Nl, N2, and N3 loci of the SARS-CoV-2 nucleocapsid gene; (c) region spanning E gene (SARS-Cov2 Wuhan-Hu isolate sequence 124 NC_045512.); or (d) the region spanning the SARS-CoV- 2 S gene.

10 The method of Claim 9, wherein the PCR method further comprises removing PCR inhibitors.

11 The method of Claim 10, wherein the removing PCR inhibitors comprises providing direct RNAse inhibitors or indirect RNase inhibitors; wherein direct RNase inhibitors are selected from the group consisting of allosteric inhibitors and competitive inhibitors; and indirect RNase inhibitors are selected from the group consisting of: aggregation methods, preferential adsorption of RNases and other PCR inhibitors, and mechanical and chemical methods of inhibition.

12 The method of Claim 6, wherein the non-PCR methods are selected from the group consisting of: nucleic acid microarrays, primer-capture modalities, antibody/receptor methods, nucleic acid capture optimization, advanced mass spectrometry, and electrochemical detection of the pathogen.

13. The method of Claim 12, wherein the antibody/receptor methods of detecting the pathogen are selected from the group consisting of: immunological assays, ELISA, and Lateral flow immunoassays.

14. The method of Claim 12, wherein the advanced mass spectrometry is assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).