Method for detecting and quantifying nucleic acid from environmental samples

Abstract

The present invention provides: a method for highly sensitive quantitative detection of RNA and / or DNA derived from viruses in environmental or fecal samples; and a highly sensitive quantitative detection kit for RNA and / or DNA derived from viruses in environmental or fecal samples.

Description

Nucleic acid detection and quantification methods from environmental samples

[0001] The present invention relates to a method for the quantitative detection of nucleic acids (RNA and / or DNA) of viral or bacterial origin in an environmental or fecal sample.

[0002] With the increasing flow of people and goods in the midst of globalization, people are exposed to the threat of various viral and bacterial infectious diseases. It is important to detect the outbreak of infectious diseases early, call for vigilance, and take countermeasures as soon as possible to minimize health damage and human and economic losses.

[0003] One example of a method for early detection of infectious disease outbreaks is sewage epidemiological surveys (WBEs). Sewage epidemiological surveys obtain epidemiological information within a treatment area through sewage analysis, and their applicability to viral or bacterial infectious diseases such as COVID-19 has been proposed (Non-Patent Document 1). Infectious disease patients can be unevenly distributed within the same city due to cluster outbreaks in hospitals, welfare facilities, schools, workplaces, and other places where people live together. By implementing sewage epidemiological surveys in society and utilizing the information obtained, it is expected that the location of infected people within a city can be identified and the regional distribution of testing and medical resources can be made more efficient.

[0004] In order to establish a method for early detection of the outbreak of such infectious diseases, there is a need for a highly sensitive method for quantitatively detecting nucleic acids derived from viruses or bacteria in environmental samples and the like.

[0005] Sci. Total Environ. , 739, 139076 (2020).

[0006] It is an object of the present invention to provide a novel method for the quantitative detection of viral or bacterial nucleic acids in environmental or fecal samples.

[0007] As a result of extensive research, the present inventors have discovered a method for quantitatively detecting viral or bacterial nucleic acids (RNA and / or DNA) in environmental or fecal samples with high sensitivity (hereinafter referred to as method (I) of the present invention) (lower part of Figure 1). For example, to detect viral or bacterial nucleic acids, particularly RNA, in samples obtained from sludge, feces, etc., commercial kits such as RNeasy Power Soil Total RNA Kit, RNeasy Power Microbiome Kit (QIAGEN), Quick-DNA Fecal / Soil Microbe Kit (ZYMO RESEARCH), NucleoBond (Takara Bio Inc.), and ISOIL (Nippon Gene Co., Ltd.) are generally used. However, these methods have poor recovery efficiency for viral or bacterial RNA and PCR inhibition (especially severe in samples obtained from sludge), making quantitative detection impossible, especially when viral or bacterial RNA is present in dilute samples (Figure 1, top). Furthermore, the long turnaround time makes them unsuitable for situations requiring quantitative detection of RNA from many samples, such as monitoring infectious disease outbreaks and epidemics in cities. In other words, the present inventors have discovered that existing methods for detecting viral or bacterial RNA from environmental samples, such as sewage, do not meet the need for early detection of infectious disease outbreaks, even in situations with a low number of infected individuals. As described in Example 1, quantitative detection of viral RNA in sewage samples using method (I) of the present invention resulted in almost no PCR inhibition and no loss of quantitation, and demonstrated quantitative detection of RNA with approximately 100 to 1,000 times higher sensitivity than known representative methods. Furthermore, it has been confirmed that quantitative detection is possible even when viral RNA is present in dilute samples. Furthermore, by changing the oligonucleotide on the solid support in step (b1) or (b2) (the oligonucleotide functions as a reverse transcription primer in step (b1)), it is possible to change the RNA and / or DNA derived from viruses or bacteria to be detected.For example, as described in the Examples, the oligonucleotide may be an oligonucleotide having an oligo(dT) sequence for detecting RNA derived from viruses or bacteria such as SARS-CoV-2, or an oligonucleotide having a base sequence specific to the nucleic acid sequence of a specific virus or bacterium for detecting RNA and / or DNA derived from that virus or bacterium. Furthermore, the required time is equivalent to or shorter than that of the above-mentioned method using a commercial kit, and the method can be performed at a cost equivalent to or less than that. For practical implementation, automation is required to quantitatively detect RNA and / or DNA from a large number of samples. However, the solid support used in steps (b1) and / or (b2) can retain the RNA and / or DNA on the solid support, eliminating the need for centrifugation and making it easier to automate.

[0008] Furthermore, the present inventors have found that the use of polyaluminum chloride (hereinafter also referred to as PAC) and / or triiron tetroxide is useful when obtaining a concentrated product from an environmental sample or fecal sample to be used in step (a) of method (I) of the present invention (hereinafter referred to as method (II) of the present invention). This method has the advantage of eliminating the need for centrifugation when obtaining a concentrated product from an environmental sample or fecal sample to be used in step (a) or the like, making it easier to automate. Flocculants used in water purification, etc., neutralize the surface charge of fine particles to make interparticle attraction dominant, thereby generating flocs. Known examples of such flocculants include inorganic flocculants such as aluminum-based and iron-based flocculants. While PAC and polyglutamic acid are widely used as flocculants for water purification, they are rarely used as reagents for concentrating and recovering RNA and / or DNA from sewage samples. The present inventors attempted to recover RNA using polyglutamic acid-containing PGα21Ca (Nippon PolyGlu Co., Ltd.) and PG-M (Nippon PolyGlu Co., Ltd.), but the aggregates were so strong that quantitative detection was difficult. On the other hand, when PAC was used, flocs were formed from sewage samples, and sufficient amounts of RNA for quantitative detection could be extracted from the flocs (concentrated products) (Figure 2). The present inventors also investigated the possibility of improving the recovery efficiency of RNA from sewage samples by combining PAC with various forms of iron (element or compound). When elemental iron (Fe) was used, the iron powder did not disperse in water and was not incorporated into the flocs at all. When FG beads (registered trademark, Tamagawa Seiki Co., Ltd.), which are beads coated with poly-GMA (glycidyl methacrylate), were used, the FG beads were incorporated into the flocs, but due to their weak magnetic force, recovery using a magnet was difficult. Similarly, iron(III) oxide (Fe ２ O ３ ), iron(II) sulfate (FeSO ４ ), polyferric sulfate, iron(II) chloride (FeCl ２ ) is also incorporated into the floc, but it is judged that it is not suitable for use due to its weak magnetic properties. ３ O ４) was incorporated into the flocs, and because of its magnetic properties, the flocs could be completely recovered using a magnet. As a result, the RNA recovery rate was further increased compared to the case of PAC alone (Figure 2), suggesting that accurate quantitative detection is possible.

[0009] The present inventors have also found that when a concentrated product of an environmental sample or a fecal sample is obtained using PAC, particularly a high-basicity PAC, and the concentrated product is dissolved under neutral conditions (particularly pH 7 to 8), the quantitative detection efficiency of nucleic acids is remarkably excellent (hereinafter referred to as method (III) of the present invention).

[0010] The basicity of ordinary PACs is about 45 to 55% (in this specification, where necessary, PACs with a basicity of about 45 to 55% will be referred to as "ordinary PACs"). PACs with a higher basicity are classified as high-basicity PACs (basicity is generally often defined as about 55 to 70%) or ultra-high-basicity PACs (basicity is generally often defined as about 70 to 90%). (In this specification, where necessary, PACs with a basicity of about 55 to 90% will be referred to as "high-basicity PACs.")

[0011] It is generally known that the pH of urban sewage is 7 to 8. When using a conventional PAC as a flocculant for sewage purification, the optimum pH for conventional PAC is approximately 4.5 to 6, so the pH of the sewage sample must be adjusted using an appropriate pH adjuster. Meanwhile, the present inventors focused on high-basicity PAC, and believed that its wide range of optimum pH would enable efficient detection of nucleic acids in sewage. The following examples demonstrate that simply adding a high-basicity PAC to a sewage sample can flocculate the sample, and subsequent quantitative detection of nucleic acids can also be performed efficiently.

[0012] For example, Water Res. 2014 Jan 1;48:375-86 describes that high basicity PAC can be used as a flocculant to remove bacteriophages, which are models of pathogenic viruses, from river water, but does not describe or suggest at all that high basicity PAC can be used in methods for quantitatively detecting nucleic acids derived from viruses or bacteria from environmental samples, etc. Furthermore, no study has been conducted on conditions under which PAC can be used as a flocculant to quantitatively detect nucleic acids derived from viruses or bacteria in environmental samples or fecal samples without inhibiting enzyme reaction systems such as quantitative PCR.

[0013] Sewage sediment (concentrated product) contains large amounts of viral or bacterial proteins and proteins from contaminants in sewage, in addition to nucleic acids derived from viruses or bacteria. Therefore, a strongly basic solution is often used to dissolve these proteins (referred to herein as a "lysis buffer"). However, using a strongly basic solution to dissolve sewage sediment is likely to inhibit the PCR reaction downstream of quantitative detection, potentially reducing the efficiency of quantitative detection of nucleic acids. Therefore, the inventors investigated conditions such as the composition and pH of the lysis buffer and found conditions that enable more sensitive quantitative detection of nucleic acids in sewage samples. Specifically, they found that dissolving sewage sediment at a pH of 7 to 8 significantly improved the sensitivity of quantitative detection of nucleic acids in sewage samples.

[0014] That is, method (III) of the present invention, which uses a step of obtaining a concentrated product of an environmental sample or a fecal sample in the presence of a high-basicity PAC (step (A1-a)) and a step of dissolving the concentrated product under conditions of pH 7 to 8 (step (A2-a)), is an efficient method for detecting nucleic acids from an environmental sample or a fecal sample, which does not require a pH adjustment step.

[0015] Furthermore, the present inventors investigated the dissolution conditions in step (A2-a) and found that heating at 50 to 60°C and adding protease more efficiently dissolved sewage sediment at a pH of 7 to 8. The following examples demonstrate that the use of protease and heating during dissolution significantly improves the efficiency of quantitative detection of RNA in sewage samples.

[0016] In addition, the present inventors have found that the recovery rate of nucleic acids increases when a dissolution buffer containing 1 to 2% (w / v) sodium lauryl sulfate (SDS) and 1 to 2 mmol / L dithiothreitol (DTT) is used to dissolve the concentrated product of an environmental sample or a fecal sample in step (A2-a), step (A2-b) described below, or step (A2).

[0017] Unlike typical silica beads, paramagnetic beads, particularly carboxyl-modified paramagnetic beads, can selectively bind nucleic acids in the presence of organic solvents without the need for chaotropic salts such as guanidine. Furthermore, carboxyl-modified paramagnetic beads are less likely to inhibit enzyme reactions because a magnet can easily separate beads with bound nucleic acids from wash buffers containing dissolved enzyme inhibitors. The inventors have found from the examples below that by using paramagnetic beads with these properties as the solid support in step (A3), nucleic acids can be efficiently separated from the concentrated product dissolved in step (A2-a), the step (A2-b) described below, or step (A2), i.e., from sewage samples, and then quantitative detection of nucleic acids can be performed with high sensitivity.

[0018] Furthermore, the present inventors have found that even when the process of concentrating an environmental sample or fecal sample in the presence of a highly basic PAC and dissolving the concentrated product of the sample is carried out under basic conditions using a dissolution buffer containing a surfactant and a reducing agent, the recovery rate of nucleic acids is increased, although the efficiency is lower than that under neutral conditions (hereinafter referred to as method (IV) of the present invention).

[0019] Additionally, the present inventors have found from the following Examples that a method comprising the steps of concentrating an environmental sample or fecal sample in the presence of PAC and dissolving the concentrated product of the sample under conditions of pH 7 to 11 is a highly sensitive method for quantitatively detecting nucleic acids (hereinafter referred to as method (V) of the present invention).

[0020] Among the viruses present in sewage, it has been shown that enveloped viruses such as SARS-CoV-2 have a high affinity for sediments in sewage and tend to localize in the sediment fraction (sediment) during sewage centrifugation (Science of the Total Environment 763 (2021) 144587), making method (I) of the present invention an effective means for extracting and detecting viral RNA. Non-enveloped viruses such as PMMoV are known to tend to exist in the supernatant fraction during sewage sedimentation, and existing PEG precipitation methods (Science of the Total Environment 807 (2022) 150722) and the Manual for Detecting Novel Coronavirus Genes in Sewage (see Example 1)) are also effective for their extraction and detection. However, there is no simple, inexpensive method that can quantitatively detect both enveloped and non-enveloped viruses with high sensitivity. The present inventors have conducted extensive research and discovered methods (III) to (V) of the present invention that can efficiently recover viruses present in both the precipitate (sediment) fraction and the supernatant fraction obtained during sewage sedimentation.

[0021] Specifically, using a total of 12 samples of urban wastewater sampled at different times from two different locations in the drainage basin where SARS-CoV-2 patients are present, the quantitative detection efficiency of viral RNA in wastewater was compared between method (V) of the present invention, method (I) of the present invention, and the known methods of PEG precipitation and ultrafiltration (Water Sci Technol (2021) 84 (8): 1997-2013). Both method (V) and method (I) of the present invention achieved a very high detection rate of 100% (12 / 12) for SARS-CoV-2 when performing qPCR, while the ultrafiltration method had a detection rate of 41.67% (5 / 12) and the PEG precipitation method had a detection rate of 16.67% (2 / 12). Furthermore, among the four methods, the amount of nucleic acid detected by method (V) of the present invention was 3.5 to 136.7 times higher on average than the other methods, demonstrating that method (V) of the present invention is the highest in both the detection rate and amount of nucleic acid detected for SARS-CoV-2. Furthermore, with regard to the detection signal of the non-enveloped virus PMMoV as an internal standard in sewage, method (V) of the present invention also had the highest detection rate, with an average nucleic acid detection amount 3.8 to 67.9 times higher than the other methods, demonstrating that method (V) of the present invention is suitable for detecting both enveloped and non-enveloped viruses in sewage.

[0022] The present invention relates to the following method (I) of the present invention. (I-1) A method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or a fecal sample, comprising: (a) extracting RNA and / or DNA from the sample or a concentrated product of the sample; (b1) obtaining immobilized cDNA by reverse transcription using the RNA hybridized to oligonucleotides on a solid support as a template, and / or (b2) hybridizing the DNA to the oligonucleotides on a solid support; and (c) pre-amplifying using the cDNA and / or DNA on the solid support as a template to obtain an amplified product in liquid phase. (I-2) The method according to (I-1), wherein the environmental sample is a sewage sample, water sample, air sample, or sample obtained from a solid surface. (I-3) The method according to (I-1) or (I-2), wherein the number of pre-amplification cycles is 1 to 14. (I-4) The method according to any one of (I-1) to (I-3), wherein the oligonucleotide is an oligonucleotide having an oligo(dT) sequence or a base sequence specific to the nucleic acid of the virus or bacterium to be detected. (I-5) The method according to any one of (I-1) to (I-4), wherein the solid support is a magnetic bead. (I-6) The method according to any one of (I-1) to (I-5), wherein the virus is an RNA virus. (I-7) The method according to any one of (I-1) to (I-5), wherein the virus is a coronavirus, influenza virus, norovirus, sapovirus, Aichi virus, adenovirus, orthopneumovirus, or polyomavirus. (I-8) The method according to (I-7), wherein the coronavirus is SARS-CoV-2. (I-9) The method according to any one of (I-1) to (I-8), comprising a step of (d) quantifying the cDNA and / or DNA on the solid support by quantitative PCR using the amplification product obtained in step (c) as a template. (I-10) The method according to (I-9), wherein the Ct value of quantitative PCR using the amplification product as a template is 18 to 35. (I-11) The method according to any one of (I-1) to (I-10), comprising a step of obtaining the concentrated product of step (a) from an environmental sample or a fecal sample using polyaluminum chloride and / or triiron tetroxide.

[0023] The present invention also includes the following: (I-12) The method according to any one of (I-1) to (I-11), wherein the RNA and / or DNA is present in the sample at a dilute concentration. (I-13) The method according to (I-12), wherein the RNA and / or DNA is present at 8,570 copies / L or less. (I-14) The method according to (I-12), wherein the RNA and / or DNA is present at 1,250 copies / L or more.

[0024] The present invention also includes the following method (II) and kit of the present invention: (II-1) A method for obtaining a concentrated product of an environmental sample or a fecal sample using polyaluminum chloride and / or triiron tetroxide, and extracting RNA and / or DNA from the concentrated product. (II-2) A method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in the sample, comprising the method described in (II-1). (II-3) A kit for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or a fecal sample, comprising triiron tetroxide and / or high basicity polyaluminum chloride.

[0025] The present invention also includes the following method (III) and kit of the present invention. (III-1) A method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or a fecal sample, comprising: (A1-a) obtaining a concentrated product of the sample in the presence of high-basicity polyaluminum chloride; (A2-a) dissolving the concentrated product under conditions of pH 7 to 8; (A3) binding the RNA and / or DNA in the concentrated product to a solid support and separating it; (B) if RNA is separated in step (A3), obtaining cDNA by reverse transcription using the RNA as a template; and (C) pre-amplifying the cDNA obtained in step (B) and / or the DNA obtained in step (A3) as a template to obtain an amplified product in a liquid phase. (III-2) The method according to (III-1), wherein the environmental sample is a sewage sample, a water sample, an air sample, or a sample obtained from a solid surface. (III-3) The method according to (III-1) or (III-2), wherein the number of pre-amplification cycles is 1 to 14. (III-4) The method according to any one of (III-1) to (III-3), wherein the solid support is magnetic beads. (III-5) The method according to any one of (III-1) to (III-4), wherein the virus is an RNA virus. (III-6) The method according to any one of (III-1) to (III-5), wherein the virus is a coronavirus, influenza virus, norovirus, sapovirus, Aichi virus, adenovirus, or polyomavirus. (III-7) The method according to (III-6), wherein the coronavirus is SARS-CoV-2. (III-8) The method according to any one of (III-1) to (III-7), comprising (D) a step of quantifying the cDNA and / or DNA by quantitative PCR using the amplification product obtained in step (C) as a template. (III-9) The method according to any one of (III-1) to (III-8), wherein the step (A2-a) comprises reacting with a protease. (III-10) The method according to (III-10), wherein the protease is proteinase K. (III-11) The method according to (III-9) or (III-10), wherein the step of reacting with a protease is carried out at 50°C to 60°C.

[0026] (III-12) The method according to any one of (III-1) to (III-11), wherein the pH adjustment step in step (A2-a) is a step of mixing a dissolution buffer containing a surfactant and a reducing agent. (III-13) The method according to (III-12), wherein the surfactant is sodium lauryl sulfate or cetyltrimethylammonium bromide. (III-14) The method according to (III-12) or (III-13), wherein the concentration of the surfactant is 0.5 to 5 weight percent by volume. (III-15) The method according to any one of (III-12) to (III-14), wherein the reducing agent is dithiothreitol. (III-16) The method according to (III-15), wherein the concentration of the reducing agent is 0.5 to 150 mmol / L. (III-17) The method according to (III-16), wherein the basicity of the high-basicity polyaluminum chloride is 60 to 75%. (III-18) The method according to any one of (III-1) to (III-17), characterized in that triiron tetroxide is used in step (A1-a). (III-19) The kit according to (II-3). (III-20) The kit according to (III-19), further comprising a buffer of pH 7 to 11 for dissolving sewage sediment.

[0027] (III-21) A method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or a fecal sample, comprising: (A1-a) obtaining a concentrated product of the sample in the presence of highly basic polyaluminum chloride; (A3-a) binding and separating the RNA and / or DNA in the concentrated product to carboxyl group-modified paramagnetic beads; (B) if RNA is separated in step (A3-a), obtaining cDNA by reverse transcription using the RNA as a template; and (C) pre-amplifying using the cDNA obtained in step (B) and / or the DNA obtained in step (A3-a) as a template to obtain an amplified product in a liquid phase.

[0028] The present invention also includes the following method (IV) of the present invention: (IV-1) A method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or a fecal sample, comprising: (A1-a) obtaining a concentrated product of the sample in the presence of high basicity polyaluminum chloride; (A2-b) dissolving the concentrated product under conditions of pH 8 to 11; (A3) binding the RNA and / or DNA in the concentrated product to a solid support and separating it; (B) if RNA is separated in step (A3), obtaining cDNA by reverse transcription using the RNA as a template; and (C) pre-amplifying the cDNA obtained in step (B) and / or the DNA obtained in step (A3) as a template to obtain an amplified product in a liquid phase.

[0029] The present invention also includes the following method (V) and kit of the present invention. (V-1) A method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or a fecal sample, comprising: (A1) obtaining a concentrated product of the sample in the presence of polyaluminum chloride; (A2) dissolving the concentrated product under conditions of pH 7 to 11; (A3) binding the RNA and / or DNA in the concentrated product to a solid support and separating it; and (B) if RNA is separated in step (A3), obtaining cDNA by reverse transcription using the RNA as a template; and optionally further comprising: (C) pre-amplifying the cDNA obtained in step (B) and / or the DNA obtained in step (A3) as a template to obtain an amplification product in a liquid phase. (V-2) The method according to (V-1), wherein the environmental sample is a sewage sample, a water sample, an air sample, or a sample obtained from a solid surface. (V-3) The method according to (V-1) or (V-2), wherein the number of pre-amplification cycles is 1 to 14. (V-4) The method according to any one of (V-1) to (V-3), wherein the solid support is a magnetic bead. (V-5) The method according to any one of (V-1) to (V-4), wherein the virus is an RNA virus. (V-6) The method according to any one of (V-1) to (V-5), wherein the virus is a coronavirus, influenza virus, norovirus, sapovirus, aichi virus, adenovirus, orthopneumovirus, or polyomavirus. (V-7) The method according to (V-6), wherein the coronavirus is SARS-CoV-2. (V-8) The method according to any one of (V-1) to (V-7), comprising (D) a step of quantifying the cDNA and / or DNA by quantitative PCR using the amplification product obtained in step (C), or the cDNA obtained in step (B) and / or the DNA obtained in step (A3) as a template. (V-9) The method according to any one of (V-1) to (V-8), wherein the polyaluminum chloride has a final concentration of 0.001 to 0.01 percent by volume in step (A1). (V-10) The method according to any one of (V-1) to (V-9), wherein the basicity of the polyaluminum chloride is 50 to 70%. (V-11) The method according to any one of (V-1) to (V-10), wherein step (A2) is performed under conditions of pH 7 to 8.(V-12) The method according to any one of (V-1) to (V-11), wherein the step (A2) comprises reacting with a protease.

[0030] (V-13) The method according to (V-12), wherein the protease is proteinase K. (V-14) The method according to (V-12) or (V-13), wherein the step of reacting the protease is carried out at 50°C to 60°C. (V-15) The method according to any one of (V-1) to (V-14), wherein the pH adjustment step in step (A2) is a step of mixing a dissolution buffer containing a surfactant and a reducing agent. (V-16) The method according to (V-15), wherein the reducing agent is dithiothreitol. (V-17) The method according to (V-15) or (V-16), wherein the concentration of the reducing agent is 0.5 to 150 mmol / L. (V-18) The method according to any one of (V-15) to (V-17), wherein the surfactant is sodium lauryl sulfate or cetyltrimethylammonium bromide. (V-19) The method according to any one of (V-15) to (V-18), wherein the concentration of the surfactant is 0.5 to 5 percent by weight or volume.

[0031] (V-20) The method according to any one of (V-1) to (V-19), further comprising using triiron tetroxide in step (A1). (V-21) The kit according to (II-3).

[0032] (V-22) A method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or a fecal sample, comprising: (A1-b) obtaining a concentrated product of the sample in the presence of 0.001 to 0.01 percent by volume of polyaluminum chloride; (A2) dissolving the concentrated product under conditions of pH 7 to 11; (A3-a) binding and separating the RNA and / or DNA in the concentrated product to carboxyl group-modified paramagnetic beads; and (B) if RNA is separated in step (A3-a), obtaining cDNA by reverse transcription using the RNA as a template; and optionally further comprising: (C) pre-amplifying the cDNA obtained in step (B) and / or the DNA obtained in step (A3-a) as a template to obtain an amplified product in a liquid phase.

[0033] (V-23) A kit for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or a fecal sample, the kit comprising polyaluminum chloride and a solid support. (V-24) The kit according to (V-23), wherein the solid support is a carboxyl group-modified magnetic bead.

[0034] Methods (I) to (V) of the present invention are methods for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in environmental samples or fecal samples with high sensitivity.

[0035] Schemes of the conventional method and the method of the present invention (I). While the conventional method (top) allows only a small portion of the sample to be used for detection, the method of the present invention (I) (bottom) maximizes the amount of sample that can be used for detection. Observed concentrations of SARS-CoV-2-derived RNA in sediment, a concentrated product obtained with PAC and / or triiron tetroxide from a sample from a raw wastewater tank at a lodging facility for patients with mild COVID-19 symptoms. Observed concentrations of SARS-CoV-2 RNA in sewage samples obtained from multiple municipal sewage systems. Observed concentrations of PMMoV RNA in sewage samples obtained from multiple municipal sewage systems.

[0036] Terms used in this specification are used in the sense commonly used in the art unless otherwise specified.

[0037] "Environmental samples" refer to samples obtained from the environment, excluding samples obtained directly from living organisms. Examples include samples obtained from sewage, sludge, water, air, solid surfaces, soil, sediments, etc. Preferred are sewage samples, water samples, air samples, or samples obtained from solid surfaces. "Sewage samples" include samples derived from sewage (e.g., influent, treated water, effluent, etc., from sewage treatment plants or septic tanks), sewage sludge (e.g., sludge itself obtained from sewage treatment plants, dehydrated sludge, etc.), etc. "Water samples" include samples derived from drinking water, tap water, surface water, groundwater, wastewater, etc. "Air samples" include samples derived from air, such as water, dust, and dirt in the atmosphere. For example, samples obtained from air sampler filters that filter the air itself, as well as dust from air conditioner filters, can be used. "Samples obtained from solid surfaces" include samples derived from moisture, oil, dust, dirt, etc., attached to solid surfaces other than living organisms. For example, samples obtained from the surfaces of doorknobs and elevator buttons can be used.

[0038] "Fecal sample" refers to a sample derived from the feces of animals such as humans, dogs, cats, monkeys, rabbits, cows, horses, chickens, hamsters, rats, and mice, and preferably human feces.

[0039] The "virus" in the "RNA and / or DNA derived from viruses or bacteria" detected by the method of the present invention is not particularly limited, but is preferably an RNA virus, particularly preferably a pathogenic RNA virus that infects humans. Examples of RNA viruses include viruses with an envelope consisting of a lipid bilayer membrane, such as coronavirus, influenza virus, human immunodeficiency virus, hepatitis C virus, Japanese encephalitis virus, orthopneumovirus, and dengue virus; and non-enveloped viruses such as norovirus, rotavirus, rhinovirus, sapovirus, Aichi virus, and pepper mild mottle virus. Examples of DNA viruses include enveloped viruses such as poxvirus and herpesvirus; and non-enveloped viruses such as adenovirus, papillomavirus, polyomavirus, and parvovirus. In the method of the present invention, the "virus" is preferably coronavirus, influenza virus, norovirus, sapovirus, Aichi virus, adenovirus, orthopneumovirus, or polyomavirus, more preferably coronavirus, influenza virus, norovirus, orthopneumovirus, sapovirus, or Aichi virus. Particularly preferred coronaviruses include SARS-CoV-2, MERS-CoV, and SARS-CoV.

[0040] The "bacteria" in the "bacterial RNA and / or DNA" detected by the method of the present invention refers to eubacteria or archaea. Preferably, the bacteria are aerobic bacteria, more preferably pathogenic aerobic bacteria that infect humans. Examples of aerobic bacteria include Salmonella, Shigella, Vibrio parahaemolyticus, Vibrio cholerae, Staphylococcus aureus, enterohemorrhagic Escherichia coli, Pneumoniae, Salmonella typhi, Haemophilus influenzae, and Pseudomonas aeruginosa, which are causative agents of bacterial food poisoning.

[0041] Examples of the "solid support" include columns, beads, test tubes, microtiter dishes, solid particles, microchips, the inner wall of a microtube, and membranes. Magnetic beads are preferred. In method (III) of the present invention, carboxyl group-modified paramagnetic beads, carboxyl group-modified diamagnetic beads, and capillaries whose inner wall is carboxyl-modified are more preferred. Carboxyl group-modified paramagnetic beads refer to paramagnetic beads whose surface is coated with carboxyl groups, and examples thereof include RNAClean XP (Beckman Coulter).

[0042] "Polyaluminum chloride (PAC)" is a compound whose chemical name is basic aluminum chloride (CAS No.: 1327-41-9) and whose molecular formula is [Al ２ (OH) ｎ Cl ６－ｎ ]m (0<n<6, m≦10). The basicity is expressed as n / 6×100 (%). The active ingredient is a small amount of sulfate ions (SO ４ ２－ ) may be introduced.

[0043] "Highly basic polyaluminum chloride (highly basic PAC)" refers to a PAC having a basicity of about 55 to 90%. The highly basic PAC is preferably a PAC having a basicity of 60 to 80%, more preferably a PAC having a basicity of 60 to 75%.

[0044] The term "lysis buffer" refers to a solution (lysis buffer) for dissolving (suspending) viral or bacterial nucleic acids, viral or bacterial proteins, and contaminant proteins in sewage sediment. The lysis buffer contains one or more surfactants and one or more reducing agents, and its form is not particularly limited. It may be in a dry or liquid form. Water is typically used as the solvent, but it may be a mixed solvent with other solvents as needed. The lysis buffer includes any of the commonly used lysis buffers for nucleic acid or protein extraction, including, but not limited to, a lysis buffer containing a buffering agent, a surfactant, a salt, a chelating agent, and a reducing agent, or solution 1 of a DNA extraction kit (Cat. No. 144-200, Shimadzu Rika Co., Ltd.). Lysis buffers I to V described in the Examples are preferred. Lysis buffer I (pH 7.5 to pH 11.0) described in Example 8 is more preferred.

[0045] "Surfactant" is a general term for substances having a structure containing both hydrophilic and hydrophobic moieties within the molecule. Examples of surfactants include anionic surfactants such as alkyl sulfate, alkyl ether sulfate, docusate, sulfonate fluorosurfactant, alkyl benzene sulfonate, alkyl aryl ether phosphate, alkyl ether phosphate, alkyl carboxylate, sodium lauroyl sarcosinate, carboxylate fluorosurfactant, sodium cholate, and sodium deoxycholate; cationic surfactants such as ethyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and cetyltrimethylammonium bromide (CTAB); amphoteric surfactants such as betaine and alkylamino fatty acid salts; and nonionic surfactants such as nonylphenoxypolyethoxyethanol (NP-40), polyoxyethylene sorbitan monooleate (Tween (registered trademark) 80), and polyoxyethylene p-t-octylphenol (Triton X-100 (registered trademark)). Preferably, it is alkyl sulfate or CTAB, and more preferably, it is lithium dodecyl sulfate (LiDS), sodium lauryl sulfate (sodium dodecyl sulfate) (SDS) or CTAB.

[0046] The term "reducing agent" refers to a chemical species that donates electrons to another chemical species. Examples of reducing agents include dithiothreitol (DTT), 2-mercaptoethanol (2-Me), tris(2-carboxyethyl)phosphine (TCEP), tributylphosphine, iodoacetamide, iron(II), and N-acetyl-L-cysteine ​​(NALC). DTT is preferred. Note that reducing agents used in methods (III) to (V) of the present invention also include those that do not inherently exhibit reducing activity but exhibit reducing activity in the reagent.

[0047] The "protease" is not particularly limited as long as it has the ability to decompose proteins to the extent that viral or bacterial nucleic acids in an environmental or fecal sample are dissolved, and examples include proteinase K, trypsin, chymotrypsin, subtilisin, etc., which belong to the serine proteases; pepsin, cathepsin D, etc., which belong to the aspartic acid proteases; and papain, cathepsin, caspase, calpain, etc., which belong to the cysteine ​​proteases. One or more of these can be used, but proteinase K is preferred because it has broad protein substrate specificity and maintains its activity even in the presence of a chelating agent or denaturing agent.

[0048] Specific embodiments of the present invention will be described below, but the present invention is not limited to these.

[0049] (Invention Related to Method (I) of the Present Invention) In step (a), RNA and / or DNA derived from viruses or bacteria in an environmental sample or fecal sample is extracted from the sample or a concentrated product of the sample. The sample may be used directly in the RNA and / or DNA extraction step, or may be concentrated by methods known in the art. Sample concentration methods generally include filtration, sedimentation, ultracentrifugation, and coarse centrifugation. For example, if it is known that a high proportion of RNA and / or DNA derived from viruses or bacteria is present in the solid fraction of a sample, highly sensitive RNA and / or DNA detection can be achieved by concentrating only the solid fraction. RNA and / or DNA can be extracted by methods known in the art using commercially available kits or conventional reagents and equipment. Examples include methods using spin columns with silica membranes and phenol-chloroform extraction methods. Furthermore, RNA and / or DNA may be purified using NucleoSpin® RNA Clean-up (Takara Bio) or the like. Solid samples or concentrated products can be crushed and used in the above method after adding a non-polar solvent such as phenol or chloroform.

[0050] In step (b1) or (b2), the RNA and / or DNA extracted in step (a) is hybridized to an oligonucleotide immobilized on a solid support to increase the recovery efficiency of RNA and / or DNA. If the RNA and / or DNA derived from a virus or bacterium is double-stranded, it is dissociated into single strands before hybridization. Dissociation from double strands into single strands can be performed by methods known in the art. Step (b1) is performed when quantitatively detecting RNA, and step (b2) is performed when quantitatively detecting DNA. Steps (b1) and (b2) can also be performed sequentially or simultaneously using the same sample after step (a). The oligonucleotide may have a sequence useful for recovering RNA and / or DNA derived from a virus or bacterium, and any oligonucleotide known in the art can be used. Examples include oligonucleotides having an oligo(dT) sequence and oligonucleotides having a base sequence specific to the nucleic acid of the virus to be detected. For example, an oligo(dT) primer can be used when extracting RNA derived from a virus (e.g., coronavirus) or bacterium with a poly(A) tail. The length of the oligonucleotide is, for example, 17 to 30 bases. An "oligonucleotide having a base sequence specific to the nucleic acid of a virus or bacterium to be detected" refers to an oligonucleotide that has at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more homology with a complementary sequence of a partial sequence of the nucleic acid of the virus or bacterium to be detected, and that does not exist in the complementary sequences of RNA and / or DNA derived from other organisms, viruses, etc. that may be present in the sample. Here, homology is expressed as a score, for example, using the search program BLAST, which uses an algorithm developed by Altschul et al. (The Journal of Molecular Biology, 215, 403-410 (1990)).

[0051] When the target of quantitative detection is viral RNA or bacterial RNA, a reverse transcription reaction is carried out as described in step (b1). At this time, the oligonucleotide immobilized on the solid support functions as a reverse transcription primer. This results in cDNA immobilized on the solid support.

[0052] In step (c), a pre-amplified product is obtained in a liquid phase using the cDNA on the solid support obtained in step (b1) and / or the DNA hybridized to the oligonucleotide on the solid support in step (b2) as a template. The reverse transcription in step (b1) and the pre-amplification in step (c) may be performed separately in two steps, or may be performed simultaneously as a single step. The number of cycles of pre-amplification is, for example, 1 to 14, 6 to 14, 6 to 10, or 10 to 14.

[0053] In step (b1) or (b2), viral or bacterial RNA and / or DNA from the sample or its enriched product is hybridized to the oligonucleotides on the solid support, thereby increasing the enrichment factor. Furthermore, in step (c), a pre-amplification product is generated in the liquid phase, so only the amplified product can be carried into quantitative PCR in step (d), eliminating the need for a cDNA cleavage step from the solid support during quantitative measurement. Furthermore, the immobilized (c)DNA can be reused as a template, allowing it to be used for PCR amplification for PCR or sequence analysis of a different target (e.g., the initial quantitative PCR can target a portion of the oligonucleotide to quantify the amount of virus, and then a different portion of the oligonucleotide can be targeted to detect viral or bacterial mutants by PCR or sequence analysis). This is extremely useful for reducing costs, tasks, and time when automating the process for practical implementation.

[0054] In step (d), quantitative PCR (qPCR, real-time PCR) is performed using the pre-amplification product obtained in step (c) as a template. Quantitative PCR can be performed by methods known in the art, using commercially available kits or conventional reagents and devices. The detection cycle threshold (Ct value) of quantitative PCR using the pre-amplification product as a template is, for example, 18 to 35. One feature of the method of the present invention is that it enables quantitative detection even when RNA and / or DNA derived from viruses or bacteria is present in a dilute sample. Because the pre-amplification product is used as a template in the quantitative PCR, even when the amount of RNA and / or DNA derived from viruses or bacteria is too small to be quantitatively detected by conventional methods, the method of the present invention increases the amount of sample that can be used for detection, allowing sufficient quantitative detection. In the conventional method described in the Examples, the lower limit of detection of RNA and / or DNA was 8,570 copies / L, while in the method described below (Method 2), it was 1,250 copies / L. It is expected that the method of the present invention will enable quantitative detection even when the RNA and / or DNA in a sample is even more dilute. In other words, the method of the present invention is expected to enable quantitative detection even in samples from situations where the number of infected people is small, and is therefore useful for early detection of the outbreak of infectious diseases.

[0055] In another embodiment of the method of the present invention, other known methods can be used instead of quantitative PCR as a method for quantifying RNA and / or DNA using the pre-amplification product as a template. Specifically, the CONAN (Cas3 Operated Nucleic Acid detection) method (DOI number: 10.1101 / 2020.06.02.20119875), the SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking) method (DOI number: 10.1101 / 2020.05.04.20091231), the LAMP (Loop-mediated Isothermal Amplification) method, the RPA (Recombinase Polymerase Amplification) method, and the like can be mentioned.

[0056] Among the methods of the present invention, examples of methods using sewage samples include the following: When detecting RNA viruses (particularly enveloped viruses such as SARS-CoV-2) in sewage, it has been reported that a high proportion of these viruses are present in the solid fraction (e.g., Sci. Total Environ., 763, 144587 (2020)). Therefore, when detecting enveloped viruses, it is preferable to extract RNA and / or DNA from the solid fraction.

[0057] (Method 1) Step (a): The entire sludge or sediment of a sewage sample (40-50 mL) is disrupted using, for example, the lysis buffer and phenol-chloroform from the RNeasy PowerSoil Kit (QIAGEN), to elute RNA and / or DNA. The aqueous layer is recovered, and 500 μL is transferred to a 2 mL tube, to which an equal volume of binding buffer (e.g., 20-100 mmol / L Tris-HCl, pH 7.5, 1.0 M LiCl, 1-2 mmol / L EDTA) is added. The mixture is incubated at 65°C for 2-5 minutes to dissolve the higher-order structure of the RNA, and then immediately cooled on ice. Step (b1): Washed solid support (e.g., Dynabeads® Oligo dT) resuspended in 200 μL of Binding Buffer. ２５Add 500 μL to 1 mL of Washing Buffer I (e.g., 10-20 mmol / L Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mmol / L EDTA) and wash by pipetting two or three times. Add 500 μL to 1 mL of Washing Buffer II (e.g., 10-20 mmol / L Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mmol / L EDTA) and wash by pipetting two or three times. Add 500 μL to 1 mL of Washing Buffer II (e.g., 10-20 mmol / L Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mmol / L EDTA) and wash by pipetting two or three times. Add 500 μL to 1 mL of Washing Buffer II (e.g., 10-20 mmol / L Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mmol / L EDTA) and wash by pipetting two or three times. After leaving it to stand for 2 minutes, completely remove the supernatant (Washing Buffer II). Add 500 μL to 1 mL of Law-Salt Buffer (e.g., 10-20 mmol / L Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mmol / L EDTA) and wash by pipetting 2 or 3 times. After leaving it to stand for 2 minutes, completely remove the supernatant (Law-Salt Buffer). Then, resuspend in 10 μL of 10 mmol / L Tris-HCl (pH 7.5) or water (RNase-free water). For example, using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific), cDNA is synthesized by mixing 10 μL of the solid support suspended in 10 μL with 10 μL of reverse transcription reaction reagent mix. The oligonucleotides on the solid support function as reverse transcription primers. The reaction is carried out at 25 ° C for 10 minutes, 37 ° C for 120 minutes, and 85 ° C for 5 minutes (reverse transcription step). Step (c): For example, using KOD One (registered trademark) (Toyobo Co., Ltd.), the reaction is carried out for 1 to 14 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute (pre-amplification step). Step (d): For example, using the QuantiTect (trademark) Probe PCR Kit (QIAGEN), qPCR is carried out using the pre-amplification product as a template.

[0058] In Method 1, the entire amount of the RNA eluate can be carried over to step (b1), and in principle, quantitative detection is thought to be possible if even one copy of RNA can be captured on a solid support. Furthermore, the present inventors have noticed the usefulness of a "pre-amplification step" in Method 1, and have found that the following two methods can also be used for quantitative detection, although they have lower detection sensitivity than Method 1.

[0059] (Method 2) Step (a): Add polyethylene glycol (4 g) and sodium chloride (0.9 g) to a sewage sample (40-50 mL), let stand overnight, and then centrifuge (12,000 g, 30 minutes to 1 hour). After discarding the supernatant, RNA is extracted from a portion (140 μl) of the resuspended pellet using, for example, the QIAamp® Viral RNA Mini Kit (QIAGEN). Steps (b1) and (c): After reverse transcription (2 hours) using, for example, the High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific), pre-amplification is performed using the cDNA as a template (2 steps). In this step, reverse transcription and preamplification can also be performed in one step using, for example, iScript Explorer One-Step RT and PreAmp Kit (Bio-Rad Laboratories, Inc.). Alternatively, the reverse transcription reaction can be performed using iScript (registered trademark) Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad Laboratories, Inc.) or Reliance Select cDNA Synthesis Kit (BIORAD), and preamplification can be performed using BIOTAQ (registered trademark) HS (Meridian BIOSCIENCE). The preamplification conditions are the same as in Method 1. Step (d): qPCR is performed using the pre-amplification product as a template in the same manner as in Method 1.

[0060] (Method 3) Step (a): Centrifuge a sewage sample (40-50 mL) (2,500-5,000 g, 10-30 minutes) (coarse centrifugation). The entire sediment suspension obtained is placed in a 2 mL tube, for example, of an RNeasy Power Microbiome Kit (QIAGEN). Note that step (a) can also be automated using a QIAcube (QIAGEN), including column purification and subsequent steps. Steps (b1) and (c): Perform reverse transcription and preamplification in the same manner as in Method 2. Step (d): Perform qPCR using the preamplification product as a template in the same manner as in Method 1.

[0061] That is, the method is as follows: A method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or a fecal sample, comprising: (a) a step of extracting RNA and / or DNA from the sample or a concentrated product of the sample; (bc-1) a step of reverse transcribing and pre-amplifying the extracted RNA, and / or (bc-2) a step of pre-amplifying the extracted DNA; and (d) a step of quantifying the cDNA and / or DNA by quantitative PCR using the amplification products obtained in steps (bc-1) and / or (bc-2) as templates.

[0062] In the method of the present invention using a sewage sample, Pepper mild mottle virus (PMMoV) can be used as an internal standard. The method of the present invention can also be used to separately quantitatively detect the target virus (e.g., SARS-CoV-2) and PMMoV. Alternatively, oligonucleotides that hybridize to the target virus and oligonucleotides that hybridize to PMMoV can be immobilized on the same solid support and used in step (b1) or (b2), thereby simultaneously capturing the target virus and PMMoV from the sewage sample. Subsequently, by adding corresponding primers, etc., steps (b1) or (b2) to (c) can be performed in the same container.

[0063] (Invention Related to Method (II) of the Present Invention) The present invention encompasses a method for obtaining a concentrated product from an environmental sample or a fecal sample using PAC and / or triiron tetroxide and extracting RNA and / or DNA from the concentrated product. Furthermore, the present invention also encompasses a method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or a fecal sample, comprising the steps of obtaining a concentrated product from the sample using PAC and / or triiron tetroxide and extracting RNA and / or DNA from the concentrated product. This method is characterized by using PAC and / or triiron tetroxide in the step of obtaining a concentrated product from an environmental sample or a fecal sample, and the subsequent steps can be performed using any of the methods described above or methods known in the art. PAC is added in an amount of, for example, 1 μL to 40 μL, more preferably 2 μL to 10 μL, and even more preferably 4 μL, per 40 mL of environmental sample or fecal sample. High-basicity PAC is particularly preferred. A high-basicity PAC is preferably a PAC with a basicity of 60 to 80%, more preferably a PAC with a basicity of 60 to 75%. Examples include PAC700A (registered trademark) (Taki Chemical), PAC300A (registered trademark) (Taki Chemical), Alphaine 83 (Daimei Chemical Industry Co., Ltd.), PAC#100W, and PAC#100H (Asada Chemical Industry Co., Ltd.). Triiron tetroxide is added, for example, in an amount of 0.01 to 1.0 g, more preferably 0.01 to 0.1 g, and even more preferably 0.02 g per 40 mL of environmental or fecal sample. As described in the explanation of step (a) above, methods known in the art can be used to extract RNA and / or DNA from the concentrated product of an environmental or fecal sample. In the case of concentrated products containing PAC, RNA and / or DNA can be efficiently recovered, particularly by a method using a basic buffer or a method using the lysis buffer of the RNeasy PowerSoil Total RNA Kit (QIAGEN) with phenol / chloroform. An example of the basic buffer is a glycine buffer.

[0064] The present invention encompasses a kit for quantitatively detecting viral or bacterial RNA and / or DNA in an environmental or fecal sample, which contains triiron tetroxide. More specifically, the present invention includes a kit for quantitatively detecting viral or bacterial RNA and / or DNA in an environmental or fecal sample, which contains triiron tetroxide and is used in the above-described method (II) of the present invention.

[0065] (Invention Related to Method (III) of the Present Invention) The present invention encompasses a method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or fecal sample, comprising the steps of obtaining a concentrated product of the sample in the presence of a highly basic PAC, dissolving the concentrated product under conditions of pH 7 to 8, and binding and separating the RNA and / or DNA to a solid support.

[0066] In step (A1-a), a concentrated product of an environmental sample or fecal sample is obtained in the presence of a high-basicity PAC. This step can be carried out in the same manner as the "Method for concentrating an environmental sample or fecal sample" described in the explanation of step (a) above, except that a high-basicity PAC is used. The preferred basicity of the high-basicity PAC and the preferred high-basicity PAC concentration per environmental sample or fecal sample are the same as those for the (high-basicity) PAC described in method (II). Iron tetroxide may also be added, as in method (II) of the present invention.

[0067] In step (A2-a), the concentrated product obtained in step (A1-a) is dissolved under conditions of pH 7 to 8. The pH adjustment step in step (A2-a) may involve, for example, mixing the concentrated product with a dissolution buffer described below to adjust the pH, or dissolving the concentrated product in a basic or acidic buffer and then adjusting the pH with an appropriate pH adjuster. The mixing ratio (volume ratio) of the environmental sample or fecal sample to the dissolution buffer is not particularly limited, but may be, for example, 1:1000 to 1:10, preferably 1:100 to 1:10. The pH of the dissolution buffer is not particularly limited as long as it is a solution pH that can dissolve viral or bacterial nucleic acids, viral or bacterial proteins, and proteins of contaminants in the sewage sediment. However, the pH of the dissolution buffer is preferably 7 to 11, and particularly preferably 7.5 to 9. The pH can be adjusted using a pH adjuster or buffer.

[0068] Examples of pH adjusters and buffers include phosphate buffer, Tris-HCl buffer, Tris-acetate buffer, HEPES buffer, MOPS buffer, MES buffer, sodium acetate buffer, sodium citrate buffer, and glycine buffer. While there are no particular limitations on the buffer, as long as it promotes the elution of nucleic acids and is suitable for nucleic acid storage, Tris-HCl buffer (100 mmol / L Tris-HCl, 10 mmol / L EDTA) adjusted to a desired pH is particularly preferred. The concentration of the surfactant in the dissolution buffer is preferably 0.5 to 5 weight percent by volume (% (w / v)), more preferably 1 to 3% (w / v), and particularly preferably 1 to 2% (w / v).

[0069] Step (A2-a) may further include a step of reacting with a protease. The concentration of the protease in the suspension with the dissolution buffer is not particularly limited depending on the type of protease, but is preferably 0.14 to 150 U / mL, more preferably 1 to 45 U / mL. In particular, when the nucleic acid elution reagent contains proteinase K as the protease at the above-mentioned concentration, the above-mentioned effects are significantly achieved. When step (A2-a) includes a step of reacting with a protease, a heating treatment may be performed. The temperature is preferably 37°C to 70°C, more preferably 50°C to 60°C, and particularly preferably 56°C. The heating time is not particularly limited, but is, for example, 5 to 60 minutes, preferably 5 to 30 minutes, and more preferably 5 to 15 minutes. When step (A2-a) does not include a step of reacting with a protease, the temperature of the dissolution buffer can be 25 to 70°C. The concentration of the reducing agent in the dissolution buffer is not particularly limited, but is, for example, 0.5 to 150 mmol / L, preferably 1 to 5 mmol / L in the case of DTT, and preferably 10 to 150 mmol / L in the case of β-mercaptoethanol.

[0070] The dissolution buffer may further contain various components. It may also contain components necessary for the subsequent nucleic acid amplification reaction, such as enzymes, salts such as magnesium salts and potassium salts, sugars such as trehalose, sugar alcohols such as glycerol, nucleic acid components, organic solvents, etc. Organic solvents are particularly acceptable because they have the effect of promoting the dissolution of RNA and / or DNA derived from viruses or bacteria. Examples of organic solvents include dimethyl sulfoxide (DMSO). Furthermore, RNase inhibitors, etc., can also be added to the extent that they do not interfere with the nucleic acid amplification reaction.

[0071] In step (A3), RNA and / or DNA in the enriched product obtained in step (A1-a) is bound to a solid support capable of binding nucleic acids and then separated. Known methods can be used to separate RNA and / or DNA from the solid support. For example, a method using a magnet to immobilize the solid support is exemplified. Here, "binding" refers to attaching nucleic acids to the solid support. The washing procedure for solid supports to which RNA and / or DNA are strongly bound is easy, and has the advantage of facilitating automation using robots, etc. The washing procedure refers to the procedure of removing organic components not bound to the solid support using 70% ethanol, etc. Magnetic beads are preferred as solid supports. Magnetic beads refer to beads having magnetic particles as cores. Examples include magnetic silica beads and magnetic cellulose beads. Carboxyl-modified paramagnetic beads are preferred. Beads that have the property of reversibly binding nucleic acids to their surface, similar to carboxyl-modified paramagnetic beads, are included in the solid support used in the method of the present invention.

[0072] In step (B), when RNA is isolated in step (A3), the RNA is used as a template to obtain cDNA by reverse transcription. Note that step (B) is not necessary when quantitative detection of DNA is desired. These steps can be performed by methods known in the art.

[0073] In step (C), the cDNA obtained in step (B) and / or the DNA obtained in steps up to step (A3) without step (B) is used as a template to pre-amplify the cDNA, yielding an amplified product in a liquid phase. These steps can be performed by methods known in the art. The pre-amplification step is essential when RNA and / or DNA derived from viruses or bacteria is present in a dilute concentration in the sample. On the other hand, when RNA and / or DNA derived from viruses or bacteria is present in a high concentration, for example, when detecting viruses from sewage from a facility with a large number of viral infections, viruses in sewage can be efficiently detected without the pre-amplification step.

[0074] In step (D), quantitative PCR is carried out using the pre-amplification product obtained in step (C) as a template in the same manner as in step (d) above.

[0075] (Method 4) Step (A1-a): High-basicity PAC (1 μL) is added to a sewage sample (10-20 mL), mixed, and then stirred (50-100 rpm, 30 minutes to 1 hour). The resulting mixture is centrifuged (3,000-4,000 g, 10 minutes), and the supernatant is discarded to obtain a concentrated product of the sample. Step (A2-a): A dissolution buffer is added to the concentrated product at a pH of 7-8 to resuspend the sewage sediment. Protease is added to the suspension and heated to 50-60°C to react and completely dissolve the sewage sediment. Step (A3): Nucleic acids in the concentrated product are bound to a solid carrier and separated. Nucleic acids can be separated from the solid carrier by, for example, immobilizing the solid carrier on a magnet such as a magnetic stand, washing the supernatant with an organic solvent such as ethanol, and then dissolving it in an inorganic solvent such as water. Steps (B) and (C): Reverse transcription and pre-amplification are performed in the same manner as in steps (b1) and (c) of Method 2. Step (D): qPCR is performed using the pre-amplification product as a template in the same manner as in step (d) of Method 1.

[0076] The present invention encompasses a kit for quantitatively detecting viral or bacterial RNA and / or DNA in an environmental or fecal sample, which kit contains a PAC, particularly a high-basicity PAC. More specifically, the kit includes a high-basicity PAC kit for quantitatively detecting viral or bacterial RNA and / or DNA in an environmental or fecal sample, which kit contains a high-basicity PAC and is used in the above-described method (III) of the present invention. The kit may further contain triiron tetroxide. The kit may further contain a buffer (dissolution buffer) of pH 7 to 11 for dissolving sewage sediment.

[0077] The present invention includes a method using magnetic beads, particularly carboxyl-modified paramagnetic beads, as a solid support. (Method 5) Step (A1-a): Obtain a concentrated product using a method similar to Method 4. Step (A3-a): Add carboxyl-modified paramagnetic beads to the suspension to bind and separate the RNA and / or DNA in the suspension. Steps (B) and (C): Perform reverse transcription and pre-amplification using a method similar to steps (b1) and (c) of Method 2. Step (D): Perform qPCR using the pre-amplification product as a template using a method similar to step (d) of Method 1.

[0078] (Invention Related to Method (IV) of the Present Invention) The present invention also includes a method for concentrating an environmental sample or fecal sample in the presence of a highly basic PAC, dissolving the concentrated product of the sample under basic conditions, and isolating nucleic acids derived from viruses or bacteria. (Method 6) Step (A1-a): Obtaining a concentrated product in the same manner as step (A1-a) of Method 4. Step (A2-b): Binding RNA in the concentrated product to a solid support in the same manner as step (A2-a) of Method 4, except under conditions of pH 8 to 11. Step (A3): Separating RNA on the solid support in the same manner as (A3) of Method 4. Steps (B) and (C): Reverse transcription and pre-amplification are performed in the same manner as steps (b1) and (c) of Method 2. Step (D): Performing qPCR using the pre-amplification product as a template in the same manner as step (d) of Method 1.

[0079] (Invention Related to Method (V) of the Present Invention) The present invention encompasses a method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or a fecal sample, comprising the steps of obtaining a concentrated product of the sample in the presence of PAC, dissolving the concentrated product under conditions of pH 7 to 11, and binding and separating the RNA and / or DNA to a solid support. Method (V) of the present invention encompasses methods (III) and (IV) of the present invention.

[0080] In step (A1), a concentrated product of an environmental sample or fecal sample is obtained in the presence of PAC. The sample can be concentrated using a method similar to the method for concentrating an environmental sample or fecal sample described in step (a) above. For example, when concentrating the sample by centrifugation, methods include leaving the sample at room temperature (20-25°C) for 1-10 minutes or stirring at 4°C for 10 minutes. The preferred concentration of PAC per volume of environmental or fecal sample is 0.001-0.01% (w / v), more preferably 0.001-0.005% (w / v). PACs used in method (V) of the present invention include ordinary PACs and high-basicity PACs. Quantitative detection of nucleic acids in environmental or fecal samples is possible regardless of the basicity of the PAC. Based on Example 7, however, a basicity of 50-70%, more preferably 60%, is preferred. For example, the PAC described in method (II) can be used. Note that triiron tetroxide may be added, as in method (II) of the present invention.

[0081] In step (A2), the concentrated product obtained in step (A1) is dissolved under conditions of pH 7 to 11. The pH of the dissolution buffer is not particularly limited as long as it is a solution pH that can dissolve viral or bacterial nucleic acids, viral or bacterial proteins, and proteins of contaminants in the sewage contained in the sewage sediment. However, the pH of the dissolution buffer is preferably 7 to 11, more preferably 7.5 to 9, and particularly preferably pH 8. In fact, when viral nucleic acids in a sample are quantitatively detected using a dissolution buffer of pH 8, the detection rate is higher than when dissolution buffers of other pHs are used, and the method has been found to be more versatile and can be used for various sewage samples under different conditions. Step (A2) can be performed in the same manner as step (A2-a) of method (III) of the present invention, except that the pH is 7 to 11, and may or may not include a step of reacting with a protease.

[0082] Steps (B), (C) and (D) can be carried out in the same manner as those in the method (III) of the present invention, and step (C) may or may not be carried out.

[0083] (Method 7) Step (A1): PAC is added to a sewage sample (10-20 mL), mixed, and then stirred (50-100 rpm, 30 minutes to 1 hour). The resulting mixture is centrifuged (3,000-4,000 g, 10 minutes), and the supernatant is discarded to obtain a concentrated product of the sample. Step (A2): A dissolution buffer is added to the concentrated product at a pH of 7-11 to resuspend the sewage sediment. Protease is added to the suspension, and the mixture is heated to 50-60°C to react and completely dissolve the sewage sediment. Steps (A3), (B), (C), and (D): Performed in the same manner as Method 4.

[0084] (Method 8) Step (A1-b): PAC is added to a sewage sample (10-20 mL) to a final concentration of 0.001-0.01% (w / v), mixed, and then stirred (50-100 rpm, 30 minutes to 1 hour). The resulting mixture is centrifuged (3,000-4,000 g, 10 minutes), and the supernatant is discarded to obtain a concentrated product of the sample. Step (A2): Carried out in the same manner as step (A2) of Method 7. Step (A3-a): Carried out in the same manner as step (A3-a) of Method 5. Steps (B), (C), and (D): Carried out in the same manner as Method 4.

[0085] Furthermore, method (V) of the present invention includes a method for quantitatively detecting nucleic acids in an environmental sample or fecal sample that can be used when a relatively large amount of RNA and / or DNA derived from viruses or bacteria is present in the sample. Specific examples include the above-mentioned (Method 4) to (Method 8) but without the pre-amplification step of step (C). An example is shown below.

[0086] (Method 9) Steps (A1) and (A2): Carry out the same method as steps (A1) and (A2) of Method 7. Step (A3): Carry out the same method as (A3) of Method 4. Step (B): Carry out reverse transcription in the same method as step (b1) of Method 2. Step (D): Carry out quantitative PCR using the cDNA obtained in step (B) and / or the DNA obtained in step (A3) as a template. A QuantiTect Probe PCR Kit (QIAGEN) or the like can be used.

[0087] The present invention encompasses a kit for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or fecal sample, comprising a PAC and a solid support.Preferably, the kit includes a kit for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in an environmental sample or fecal sample, which is used in the above-mentioned methods (III) to (V) of the present invention.

[0088] The PAC may be any PAC that can be used in step (A1) of method (V) of the present invention. There is no particular limitation on the amount of PAC to be included in the kit, but the protocol or the like attached to the kit may include a description such as "PAC is added to the environmental sample or fecal sample to give a final concentration of 0.001 to 0.01% (w / v)."

[0089] The solid support may be any solid support that can be used in step (A3) of method (III) of the present invention, preferably magnetic beads, particularly preferably carboxyl group-modified paramagnetic beads.

[0090] The kit may further contain a dissolution buffer of pH 7 to 11 for dissolving sewage sediment, and the dissolution buffer may be a buffer that can be used in step (A2-a) of method (III) of the present invention. Instead of including a dissolution buffer in the kit, the kit may include a protocol or the like that describes the composition of the dissolution buffer and instructs the purchaser of the kit to prepare the buffer themselves in accordance with the protocol or the like.

[0091] The kit may further contain a protease. There is no limitation on the protease contained in the kit, and examples thereof include proteinase K.

[0092] The kit may further contain triiron tetroxide. There is no limitation on the amount of triiron tetroxide to be contained in the kit, and the protocol or the like attached to the kit may state, for example, "0.01 to 1.0 g of triiron tetroxide is added per 40 mL of environmental sample or fecal sample."

[0093] The kit may further contain a reverse transcriptase kit, a quantitative PCR kit, and / or a preamplification kit. The reverse transcriptase kit is not particularly limited, but examples that can be used include High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific), iScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad Laboratories, Inc.), and Reliance Select cDNA Synthesis Kit (BIORAD). The quantitative PCR kit is not particularly limited, and examples thereof include TaqMan Environmental Master Mix 2.0 (ThermoFisher SCIENTIFIC) and QuantiTect Probe PCR Kit (QIAGEN). The preamplification kit is not particularly limited, and examples thereof include BIOTAQ HS (Meridian BIOSCIENCE). Reverse transcription and preamplification can be performed using a single kit, and examples thereof include iScript Explorer One-Step RT and PreAmp Kit (Bio-Rad Laboratories, Inc.).

[0094] The present invention will be described in more detail below by way of examples of the present invention, but the present invention is not limited to these examples.

[0095] In this specification, room temperature refers to 15 to 25°C.

[0096] Example 1 Detection of SARS-CoV-2 from Sewage Sediments (Included in Method (I) of the Present Invention) Sediments obtained by coarse centrifugation (5,000 g, 10 minutes) of several different sewages were tested using the method described herein. This technique was also found to be effective for nucleic acids (RNA and / or DNA) from sludge. Specifically, using the method described herein, RNA was extracted from sewage and sludge collected from a sewage treatment plant and a facility-attached septic tank, and the RNA was analyzed using Oligo dT. ２５RNA containing SARS-CoV-2 RNA and a poly(A) sequence at the 3' end was captured using a solid support immobilized with RNeasy. The solid support was then subjected to reverse transcription, followed by PCR amplification (pre-amplification), to generate an amplified product in the liquid phase using the cDNA on the solid support as a template. A portion of the amplified product generated in the liquid phase was quantitatively analyzed by quantitative PCR to calculate the copy number of SARS-CoV-2 RNA in the sample. The following example uses extraction using a vortex mechanical lysis method and is included to illustrate its use. This method utilizes carbide beads included in the RNeasy PowerSoil Total RNA Kit, but it has been shown that other beads, such as glass beads, do not reduce nucleic acid yields.

[0097] (Protocol) 1. Add up to 2 grams of sludge to a tube containing beads from the RNeasy PowerSoil Total RNA Kit. 2. Add 2.5 mL of Solution PowerBeads, 250 μL of Solution SR1, and 800 μL of IRS to the sample. 3. Add 3.5 mL of phenol / chloroform / isoamyl alcohol to the sample. 4. Place the tube in a vortex adapter and vortex the tube at the highest setting for 1 minute. 5. Centrifuge the tube at 2500 g for 10 minutes at room temperature and remove 500 μL of the aqueous layer. 6. Add 500 μL of binding buffer to the 500 μL aqueous layer and heat to 65°C for 5 minutes, then quickly cool to 4°C. 7. The entire sample (1 mL) was suspended in 200 μL of binding buffer. ２５8. The sample is fixed to a magnetic stand, the supernatant is discarded, and the sample is washed with 500 μL of Wash buffer 1. 9. The sample is fixed to a magnetic stand, the supernatant is discarded, and the sample is washed with 500 μL of Wash buffer 1. 10. The sample is fixed to a magnetic stand, the supernatant is discarded, and the sample is washed with 500 μL of Low-salt buffer. 11. The entire supernatant is discarded, and the sample is suspended in 10 μL of water. Add the sample to 10 μL of a previously prepared reverse transcriptase mixed solution, and heat at 25°C for 10 minutes, 37°C for 120 minutes, and 85°C for 5 minutes. Immobilized Oligo dT ２５ functions as a primer. 12. The sample is fixed to a strong magnet, the supernatant is discarded, and the sample is suspended in 10 μL of water. 10 μL of the bead suspension is added to a PCR reaction mix containing 40 μL of KOD one (TOYOBO) for a total volume of 50 μL. The SARS-CoV-2-CDC-N1 primer set (described in step 7 of the RNA extraction protocol using novel lysis buffer I in Example 3 below) is used. 13. Preamplification is performed for 10 cycles at 98°C for 10 seconds, 55°C for 5 seconds, and 68°C for 5 seconds. 14. The preamplification product is subjected to quantitative PCR (CDC-N1) using a method similar to step 8 in Example 3 below, and the copy number of the SARS-CoV-2 RNA N region (gene copy number / L) is calculated.

[0098] The results of RNA extraction from sewage samples and SARS-CoV-2 RNA detection using the above method (referred to herein as method (I)' of the present invention) are shown in Table 1. Samples that were below the detection limit using a conventional method using PEG precipitation (see "Manual for the Detection of Novel Coronavirus Genes in Sewage" (COVID-19 Task Force of the Japan Society on Water Environment, Japan Sewage Works Innovation Organization, March 2021, [searched April 5, 2021], Internet, <URL: https: / / www.jswe.or.jp / aboutus / pdf / SARS-CoV-2_RNA_Detection_Manual_for_Wastewater.pdf>)) were analyzed using the above method, as shown in Figure 1. As a result, the equivalent amount of sample that could be used for detection increased, resulting in a lower detection limit and detection at a Ct value of approximately 33 to 35.

[0099]

[0100] The reagents used are as follows: PowerBeads, SR1, IRS: contents of RNeasy PowerSoil Total RNA Kit (QIAGEN) Binding Buffer: 100 mmol / L Tris-HCl, pH 7.5, 500 mmol / L LiCl, 0.5% lithium dodecyl sulfate (LiDS), 1 mmol / L EDTA, 5 mmol / L DTT Wash Buffer I: 20 mmol / L Tris-HCl, pH 7.5, 500 mmol / L LiCl, 0.1% LiDS, 1 mmol / L EDTA, 5 mmol / L DTT Wash Buffer II: 20 mmol / L Tris-HCl, pH 7.5, 500 mmol / L LiCl, 1 mmol / L EDTA ・Low-Salt Buffer: 20 mmol / L Tris-HCl, pH 7.5, 200 mmol / L LiCl, 1 mmol / L EDTA

[0101] Example 2: Concentration using a magnetic agglutinant, RNA elution from aggregates using a basic buffer, and detection of SARS-CoV-2. To increase the recovery rate of RNA in sewage samples so that viral or bacterial RNA can be quantitatively detected even when it is present in dilute concentrations in the sample, we investigated the process of obtaining a concentrated product from the sample. As a result, we found that viral RNA can be efficiently recovered from sewage samples using the following method (method (II)' of the present invention).

[0102] (Protocol) 1. Add 0.02 g of triiron tetroxide to 40 mL of sewage and stir well. 2. Add 4 μL of PAC 300A (Taki Chemical, basicity: 60%, Al ２ O ３Add a 10.5% (w / v) aqueous solution (concentration: 10.5% by weight) (final concentration: 0.001% (w / v)) and stir well. After 1 minute, stir at low speed to form flocs. 3. Fix the tube to a strong magnet and leave to stand for 1 minute. 4. With the tube still fixed to the strong magnet, remove 140 μL of the supernatant and discard. 5. Remove the tube from the strong magnet and suspend in 500 μL of 0.5 M glycine buffer (pH 10.5). 6. Extract RNA from 140 μL of the suspension and the obtained 140 μL of supernatant using a QIAamp Viral RNA Mini Kit (QIAGEN). 7. Add 10 μL of the RNA product to 10 μL of a previously prepared reverse transcriptase mixture and heat at 25°C for 10 minutes, 37°C for 120 minutes, and 85°C for 5 minutes. 8. The pre-reverse transcription product is subjected to quantitative PCR (CDC-N1) in the same manner as in step 8 of Example 3 described below, to measure the copy number of the SARS-CoV-2 RNA N region (gene copy number / L).

[0103] The results are shown in Figure 2. The method of the present invention using PAC and triiron tetroxide ("PAC + Fe" in the figure) ３ O ４ The "direct extraction" method involved subjecting 40 mL of sewage to steps 6 to 8 of the above protocol. The "coarse centrifugation" method involved centrifuging at 3,100 g for 10 minutes, discarding the supernatant, and then subjecting the resulting sediment to steps 5 to 8 of the above protocol. The method using only PAC involved subjecting 40 mL of sewage to step 2 of the above protocol, stirring at 60 rpm for 30 minutes at 4°C, and centrifuging at 3,000 g for 10 minutes at 4°C. The resulting sample was suspended in 500 μL of 0.5 M glycine buffer (pH 10.5) as described in step 5 of the above protocol, and then subjecting it to steps 6 to 8 of the above protocol. The use of PAC and triiron tetroxide resulted in a reduced amount of viral RNA in the supernatant compared to the coarse centrifugation method (i.e., improved flocculation efficiency), and more RNA was recovered from the sediment compared to the coarse centrifugation method. This method allows for the efficient recovery of not only viruses adsorbed to solids, but also viruses floating in the supernatant.

[0104] Example 3: Investigation of conditions for extraction and detection of SARS-CoV-2 RNA from sewage sediments using protease treatment To investigate conditions for extracting viral or bacterial RNA contained in sediments obtained from sewage collected from various locations, RNA was extracted using various lysis buffer conditions or nucleic acid extraction methods described below, and the amount of extracted RNA was measured. The lysis buffer used during lysis was the buffer included in the RNeasy PowerSoil Total RNA Kit or the new Lysis buffer I.

[0105] (Protocol for RNA extraction and detection using novel Lysis buffer I) 1. 20 mL of sewage (urban sewage from the γ-point sewage treatment plant) was roughly centrifuged (3,000 g, 10 minutes) to obtain sewage sediment. 1 x 10 RNA was extracted from the sediment using quantitative PCR. ７Add an inactivated copy of SARS-CoV-2 and let stand on ice for 30 minutes. 2. Add 570 μL of Lysis buffer I to the sewage sediment obtained in step 1 and suspend the sediment. 3. Add 14 μL of proteinase K solution (20 mg / ml, final concentration 0.5 mg / mL) to the suspension obtained in step 2 and react under the three conditions listed below. With protease treatment + condition 1: Let stand at room temperature for 20 minutes. With protease treatment + condition 2: Let stand at 56°C for 10 minutes, then let stand at room temperature for 10 minutes. With protease treatment + condition 3: Let stand at 70°C for 5 minutes, then let stand at room temperature for 15 minutes. 4. 5. 580 μL of phenol / chloroform / isoamyl alcohol (PCI solution; pH 5.2) is added to each sample obtained in step 3 or the sample obtained in step 2 (without protease treatment) and vortexed at 3000 rpm for 1 minute. The resulting solution is centrifuged at 13,000 g for 10 minutes at room temperature, and 500 μL of the aqueous layer is separated. 5. 200 μL of the aqueous layer is purified using NucleoSpin RNA Clean-up (Takara Bio) and eluted with 40 μL of water to obtain the purified RNA product. 6. 10 μL of the purified RNA product is subjected to reverse transcription using a High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) to synthesize cDNA. 7. The cDNA samples were then subjected to PCR amplification using a primer set for SARS-CoV-2-CDC-N1 (2019-nCoV_N1-F (5'-GACCCCAAATCAGCGAAAT-3' (SEQ ID NO: 1)) and 2019-nCoV_N1-R (5'-TCTGGTTACTGCCAGTTGAATCTG-3' (SEQ ID NO: 2)) (Centers for Disease Control and Prevention, URL: https: / / www.cdc.gov / coronavirus / 2019-ncov / lab / rt-pcr-panel-primer-probes.html) and KOD Using a PCR product (Toyobo Co., Ltd.), pre-amplification is performed for 10 cycles at 98°C for 10 seconds, 55°C for 5 seconds, and 68°C for 5 seconds.8. The sample obtained in step 7 was subjected to quantitative PCR using the QuantiTect Probe PCR Kit (QIAGEN). A SARS-CoV-2-CDC-N1 primer set and probe (2019-nCoV_N1-P(5'-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3' (SEQ ID NO: 3)), <URL: https: / / www.cdc.gov / coronavirus / 2019-ncov / lab / rt-pcr-panel-primer-probes.html>) was used. A plasmid DNA containing a SARS-CoV-2 positive control sequence (nCoV-N1-Control) was also used. Plasmid (catalog number: 5004-N10001, eurofins) is serially diluted and the copy number of the SARS-CoV-2 RNA N region (gene copy number / 20 ml) is calculated.

[0106] (RNA extraction protocol using the buffer included with the RNeasy PowerSoil Total RNA Kit) 1. For 20 mL of sewage (γ-point sewage), perform the same steps as step 1 of the RNA extraction protocol using the new Lysis buffer I. 2. Add 400 μL of the PowerBead Solution buffer included with the RNeasy PowerSoil Total RNA Kit to the sewage sediment and suspend. 3. Add 40 μL of the IRS buffer included with the RNeasy PowerSoil Total RNA Kit and 130 μL of IRS buffer and suspend. 4-8. Perform the same steps as steps 4-8 of the RNA extraction protocol using the new Lysis buffer I.

[0107]

[0108] As shown in Table 2, the copy number of the SARS-CoV-2 RNA N region extracted and detected using Lysis Buffer I at pH 7.5 and the buffer included in the neutral Neasy Power Soil Total RNA Kit was shown to be very high. Furthermore, the highest RNA copy number was detected when proteinase K solution was added to Lysis Buffer I and heated at 56°C (Condition 2). Example 3 and Examples 4 and 5 below demonstrate that a method for detecting viral RNA comprising suspending sewage sediment at a pH of 7 to 11 is an excellent method for quantitatively detecting nucleic acids in sewage. Furthermore, a method for detecting viral RNA comprising suspending sewage sediment at a pH of 7 to 11, using protease and heating, is an excellent method for quantitatively detecting nucleic acids in sewage.

[0109] The reagents used are as follows: Lysis Buffer I: 100 mmol / L Tris-HCl pH 7.5, 100 mmol / L NaCl, 1% SDS, 10 mmol / L EDTA, 2 mmol / L DTT. When adjusting the pH, a small amount of NaOH or HCl was used.

[0110] Example 4 Comparative study of RNA purification methods using various magnetic beads (included in methods (III) to (V) of the present invention) To improve the efficiency of SARS-CoV-2 viral RNA recovery in sewage using a flocculant and to obtain the conditions for the associated nucleic acid purification method, we used a sample of urban sewage α to which inactivated SARS-CoV-2 virus had been added to study the presence or absence of a flocculant and the following conditions A or B, which include magnetic beads or columns capable of binding viral or bacterial nucleic acids.

[0111] (Common Protocol 4-1) 1. 1 x 10 RNA amount measured by quantitative PCR ７20 mL of sewage (urban sewage from the Gamma Point Sewage Treatment Plant) containing inactivated copies of SARS-CoV-2 before centrifugation is placed in a centrifuge tube. A sample is obtained by adding PAC 300A as a flocculant and shaking (final concentration: 0.001% (w / v) or 0.005% (w / v)), or a sample without flocculant added. 2. Each sample obtained in step 1 is stirred at 60 rpm at 4°C for 30 minutes, centrifuged at 3,000 g for 10 minutes at 4°C, and the supernatant is completely discarded. 3. Add 570 μL of lysis buffer (lysis buffer I or lysis buffer II), suspend well, and transfer to a 1.5 mL centrifuge tube. 4. 4. Add 14 μL of proteinase K solution to the tube (final concentration 0.5 mg / mL) and suspend thoroughly. Heat on a heat block at 56°C for 10 minutes, then leave to stand at room temperature for 5 minutes. 5. Add 580 μL of PCI solution (pH 5.2) to the tube and suspend by vortexing. 6. Leave the tube to stand for 1 minute, then centrifuge at 13,000 g for 10 minutes at 15°C. 7. Remove 500 μL of the upper supernatant layer from the tube and use it as a crude extract containing nucleic acids.

[0112] (Condition A: Purification method using a commercially available spin column kit) A1. 500 μL of the nucleic acid-containing crude extract sample obtained by the above common protocol 4-1 is purified using NucleoSpin RNA Clean-up (Takara Bio) and eluted with 40 μL of water to obtain an RNA sample.

[0113] (Condition B: Purification method using magnetic beads (carboxy group-modified magnetic beads)) B1. Add 180 μL of RNAClean XP (Beckman Coulter) to 100 μL of nucleic acid-containing crude extract sample obtained by common protocol 4-1 above, suspend by pipetting, and let stand for 5 minutes. B2. Secure the tube to a magnetic stand and let stand for 10 minutes. B3. Discard the supernatant from the tube and wash with 70% ethanol. B4. Discard the supernatant from the tube and let stand for 15 minutes to dry. B5. Remove the tube from the magnetic stand and suspend the pellet in 30 μL of water and let stand for 3 minutes. B6. Secure the tube to a magnetic stand, collect the supernatant, and transfer it to a new 1.5 mL centrifuge tube to use as an RNA sample.

[0114] (Common Protocol 4-2) 1. The RNA sample recovered in step A1 or step B6 is subjected to a reverse transcription reaction in the same manner as in step 6 of Example 3 to synthesize cDNA. 2. Each of the cDNAs obtained in step 1 is pre-amplified in the same manner as in step 7 of Example 3. 3. Each of the pre-amplified samples obtained in step 2 is subjected to quantitative PCR in the same manner as in step 8 of Example 3 to calculate the copy number of the SARS-CoV-2 RNA N region (gene copy number / 20 ml).

[0115] The procedure (Common Protocol 4-1) included suspending the sewage sediment in Lysis buffer I in step 3, and the detected SARS-CoV-2 RNA copy numbers (gene copies / 20 ml) are shown in Table 3.

[0116]

[0117] The procedure (Common Protocol 4-1) included suspending the sewage sediment in Lysis buffer II in step 3, and the detected SARS-CoV-2 RNA copy numbers (gene copies / 20 ml) are shown in Table 4.

[0118]

[0119] The results in Tables 3 and 4 demonstrate that SARS-CoV-2 RNA can be detected using the silica column under Condition A or the carboxyl-modified paramagnetic beads (RNAClean XP) (Beckman Coulter) under Condition B, regardless of the presence or absence of PAC. Furthermore, a comparison of the concentration of the flocculant PAC and the combination of lysis buffer revealed that SARS-CoV-2 RNA could be detected at a final concentration of 0.001% PAC and lysis buffer I compared with the absence of PAC, at a level 50-fold higher than that detected under conditions without PAC. RNAClean XP (Beckman Coulter) efficiently binds viral nucleic acids in sewage, allowing for easy washing of contaminants using magnetic manipulation and ethanol-containing water, and reveals that nucleic acids can be recovered using organic solvent-free water. In other words, the use of carboxyl-modified paramagnetic beads allows for the simple and efficient recovery and detection of nucleic acids in sewage samples.

[0120] Furthermore, from the results in Table 4, it was found that even under basic pH conditions, the recovery rate of nucleic acids increased, although the efficiency was lower than under neutral conditions.

[0121] The reagents used are as follows: Lysis Buffer I (pH 7.5): 100 mmol / L Tris-HCl, 100 mmol / L NaCl, 1% SDS, 10 mmol / L EDTA, 2 mmol / L DTT; Lysis Buffer II (pH 10.6): 260 mmol / L glycine buffer, 200 mmol / L Tris-HCl, 200 mmol / L NaCl, 2% SDS, 20 mmol / L EDTA, 2 mmol / L DTT.

[0122] Example 5: Investigation of versatile conditions for RNA detection from urban sewage with multiple characteristics (included in method (III) of the present invention). To stably detect SARS-CoV-2 RNA even from sewage samples containing dilute viral or bacterial RNA and large amounts of PCR inhibitors, we investigated the composition of a lysis buffer and heating treatment in the process of suspending the sewage sediment obtained from the sample, which efficiently dissolves nucleic acids by dissolving proteins in the sediment and cleaving disulfide bonds. Furthermore, to measure the detection sensitivity for non-enveloped viruses, we also investigated the Pepper mild mottle virus (PMMoV), which is generally present in large amounts in sewage and human feces.

[0123] (Protocol) 1. Place 10 mL of municipal sewage from sewage treatment facilities at three locations (α, β, or γ) into a centrifuge tube, add 1 μL of PAC 300A, and shake (final concentration 0.001% (w / v)). 2. Stir the tube at 4°C and 60 rpm for 30 minutes. 3. Centrifuge the tube at 3,000 g for 10 minutes at 4°C, and discard the supernatant completely. 4. Add 250 μL of one of the lysis buffers listed below (Lysis Buffer I, Lysis Buffer III, Lysis Buffer IV, Lysis Buffer V), suspend well, and transfer to a 1.5 mL centrifuge tube. 5. Add 14 μL of proteinase K solution to the tube, suspend well, and heat on a heat block at 56°C for 10 minutes, then leave to stand at room temperature for 5 minutes. 6. Add 300 μL of PCI solution (pH 5.2) to the tube and suspend by vortexing. 7. Allow the tube to stand for 1 minute, then centrifuge at 19,000 g for 10 minutes at 15°C. 8. Transfer 100 μL of the supernatant from the tube to another 1.5 mL centrifuge tube, add 180 μL of RNAClean XP (Beckman Coulter), suspend well by pipetting, and allow to stand for 5 minutes. 9. Secure the tube to a magnetic stand and allow to stand for 10 minutes. 10. Discard the supernatant from the tube and wash with 500 μL of 70% ethanol. 11. Repeat the washing procedure in 10. four times. 12. Discard the supernatant from the tube and allow to stand for 15 minutes to dry. 13. Remove the tube from the magnetic stand and suspend the pellet in 30 μL of water and allow to stand for 3 minutes. 14. The tube was fixed to a magnetic stand, and the supernatant was collected and transferred to a new 1.5 mL centrifuge tube to prepare the RNA sample. 15. 1 μg of the RNA sample was subjected to reverse transcription and 10 cycles of preamplification using the iScript® Explore One-step RT and PreAmp kit (Bio-Rad) to synthesize cDNA. 16. The cDNA obtained in step 15 was subjected to quantitative PCR using the QuantiTect Probe PCR Kit (QIAGEN) and the SARS-CoV-2CDC-N1 or PMMoV primer set and probe, respectively, as in step 7 of Example 3, to calculate the copy number of SARS-CoV-2 RNA N region or PMMoV RNA.The PMMoV primer set used was PMMV-FP1-rev (5'-GAGTGGTTTGACCTTAACGTTTGA-3' (SEQ ID NO: 4)) and PMMV-RP1 (5'-TTGTCGGTTGCAATGCAAGT-3' (SEQ ID NO: 5)) (Appl Environ Microbiol. 2013 December; 79(23): 7413-7418.). The PMMoV probe used was PMMV-Probe1 (5'-FAM-CCTACCGAAGCAAATG-MGB-NFQ-3' (SEQ ID NO: 6)). In parallel, a dilution series of plasmid DNA containing a SARS-CoV-2 positive control sequence (nCoV-N1-Control Plasmid (Cat. No.: 5004-N10001, Eurofins) and a plasmid DNA containing a PMMoV positive control sequence (5'-GAGTGGTTTGACCTTAACGTTTGAGAGGCCTACCGAAGCAAATGTCGCACTTGCATTGCAACCGACAA-3' (SEQ ID NO: 7)) was measured, and the copy numbers of SARS-CoV-2 RNA N region and PMMoV RNA were calculated. SARS-CoV-2 detected when sewage sediment was suspended in each lysis buffer. The observed concentration of RNA (gene copies / L) or PMMoV RNA (gene copies / L) is shown in FIG. 3 or FIG.

[0124] 3 and 4 demonstrate that RNA can be extracted and detected universally from multiple sewage samples when sewage sediments are suspended using high-basicity PAC as a flocculant and lysis buffer I, III, IV, or V with a pH of 7.5. Example 5 demonstrates that the method using high-basicity PAC and a lysis buffer with a pH of 7 to 8 has excellent versatility for quantitative detection of nucleic acids derived from not only enveloped viruses such as SARS-CoV-2 but also non-enveloped viruses such as PMMoV in multiple municipal sewage samples.

[0125] The reagents used are as follows: Lysis Buffer I (pH 7.5): 100 mmol / L Tris-HCl, 100 mmol / L NaCl, 1% SDS, 10 mmol / L EDTA, 2 mmol / L DTT; Lysis Buffer III (pH 7.5): 200 mmol / L Tris-HCl, 200 mmol / L NaCl, 2% SDS, 20 mmol / L EDTA, 2 mmol / L DTT; Lysis Buffer IV (pH 7.5): 100 mmol / L Tris-HCl, 100 mmol / L NaCl, 1% SDS, 10 mmol / L EDTA, 2% CTAB, 2 mmol / L DTT. V (pH 7.5): 100 mmol / L Tris-HCl, 2M NaCl, 20 mmol / L EDTA, 2% CTAB, 2 mmol / L DTT

[0126] Example 6: Examination of SARS-CoV-2 RNA extraction and detection efficiencies for multiple urban sewage samples sampled over time (included in method (III) of the present invention). SARS-CoV-2 and PMMoV were detected from sewage sediments obtained from sewage samples collected at multiple locations and consecutive time points using PAC flocculant and a lysis buffer at pH 7.5. The quantitative detection efficiency was compared with the detection and quantification efficiency of viral RNA obtained using a commercial kit for detecting viral RNA derived from general sewage or feces.

[0127] (Protocol for detecting viral RNA using PAC flocculant and pH 7.5 lysis buffer) (Hereinafter referred to as method (III)' of the present invention) 10 mL of municipal sewage was collected on six dates at the sewage treatment plant at point D or point E, and SARS-CoV-2 RNA was quantitatively detected using the same method as in Example 5. In addition, Lysis Buffer I was used in the step corresponding to step 4 of Example 5, iScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad Laboratories, Inc.) and BIOTAQ HS (meridian BIOSCIENCE) were used in the step corresponding to step 15 of Example 5, and TaqMan (registered trademark) Environmental Master Mix 2.0 (ThermoFisher SCIENTIFIC) was used in the step corresponding to step 16 of Example 5.

[0128] (Protocol for detecting viral RNA using a commercial kit) 1. Place 40 mL of municipal sewage collected on six dates at two sewage treatment plants, α or β, into a centrifuge tube, centrifuge the tube at 6,800 g for 10 minutes at 4°C, and discard the supernatant. 2. Using the RNeasy Power Microbiome Kit (QIAGEN), suspend in 650 μL of the included PM1 buffer. 3. Add 100 μL of TRIzol RNA Isolation Reagents (Thermofisher Scientific) to the suspension obtained in step 2, and mix and stir at 3,000 rpm for 10 minutes. 4. Centrifuge the tube from step 3 above at 13,000 g for 1 minute at room temperature. 5. Using 450 μL of the supernatant obtained from the tube in step 4 above, RNA is eluted in a volume of 50 μL according to the protocol provided with the PowerMicrobiome Kit. 6. Using 1 μg of the RNA obtained in step 5 above, reverse transcription and pre-amplification are performed using the iScript® Explorer One-step RT and PreAmp kit in the same manner as in Example 3. 7. The pre-amplified sample is subjected to quantitative PCR using Quantitect Probe Mix. The SARS-CoV-2 RNA concentration (copies / L) is calculated in the same manner as in Example 3.

[0129] The observed concentrations of SARS-CoV-2 RNA (gene copies / L) detected by extracting RNA from sewage samples using the method of the present invention (III) or a method using a commercial kit are shown in Tables 5 and 6.

[0130]

[0131]

[0132] As shown in Tables 5 and 6, the RNA extraction method using high-basicity PAC and a lysis buffer at pH 7.5 was suggested to be capable of quantitatively detecting 37 times more SARS-CoV-2 viral RNA on average than the method using a commercial kit for detecting viral RNA from sewage or feces. Furthermore, it was suggested that the method using high-basicity PAC and a lysis buffer at pH 7-8 is capable of efficiently quantitatively detecting nucleic acids contained in sewage under different environments.

[0133] Example 7 Comparative study of RNA purification using PACs with different basicities (included in methods (III) to (V) of the present invention). It is known that the properties of PACs change depending on their basicity. To clarify whether the basicity of PACs affects the efficiency of SARS-CoV-2 RNA recovery, an experiment was conducted to detect the added virus using PAC flocculants with different basicities in a sample of municipal wastewater spiked with inactivated SARS-CoV-2 virus.

[0134] 1. 1x10 RNA amount measured by quantitative PCR ６ 10 mL of uncentrifuged municipal wastewater (δ point sewage treatment plant municipal wastewater) containing inactivated copies of SARS-CoV-2 was placed in a centrifuge tube. PAC 250A (Taki Chemical, basicity: 50%, Al) was used as a flocculant. ２ O ３ PAC 300A (Taki Chemical, basicity: 60%, Al ２ O ３ Aqueous solution with a converted concentration of 10.5% by weight), PAC 700A (Taki Chemical, basicity: 70%, Al ２ O ３Add 1 μL of a 10.5% by weight aqueous solution (concentration: 0.001% (w / v)) and shake to obtain a sample, or shake without adding any flocculant. 2. Allow each sample obtained in step 1 to stand at 20°C for 10 minutes, then centrifuge at 3,000g for 10 minutes at 4°C and discard the supernatant completely. 3. Add 250 μL of lysis buffer I (pH 7.5) and suspend well, then transfer to a 1.5 mL centrifuge tube. 4. Add 14.25 μL of proteinase K solution (final concentration: 1.14 mg / mL or more) to the tube and suspend well, heat on a heat block at 56°C for 10 minutes, then leave to stand at room temperature for 5 minutes. 5. Add 550 μL of PCI solution (pH 5.2) to the tube and suspend by vortexing. 6. Allow the tube to stand for 1 minute, then centrifuge at 19,000g for 10 minutes at 20°C. 7. Remove 200µL of the supernatant layer from the tube to use as the nucleic acid-containing crude extract sample. 8. Add 180µL of RNAClean XP (Beckman Coulter) to 100µL of nucleic acid-containing crude extract sample obtained using the above common protocol, suspend by pipetting, and allow to stand for 5 minutes. 9. Secure the tube to a magnetic stand and allow to stand for 10 minutes. 10. Discard the supernatant from the tube, wash with 70% ethanol, and repeat step 10 a total of 5 times. 11. Discard the supernatant from the tube and allow to stand for 15 minutes to dry. 12. Remove the tube from the magnetic stand and suspend the pellet in 30µL of water and allow to stand for 3 minutes. 13. Secure the tube to a magnetic stand, collect the supernatant, and transfer to a new centrifuge tube to use as the RNA sample. 14. 15. Using 1 μg of the RNA sample recovered in step 13 and 2019-nCoV_N1-R, perform a reverse transcription reaction to synthesize cDNA using the Reliance Select cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.). 16. Perform 10 cycles of pre-amplification using the cDNA obtained in step 14, a dilution series of plasmid DNA containing a SARS-CoV-2 positive control sequence (nCoV-N1-Control Plasmid (Cat. No.: 5004-N10001, Eurofins) for copy number calculation, the SARS-CoV-2 CDC-N1 primer set, and BIOTAQ HS (Meridian BIOSCIENCE).16. The pre-amplified sample obtained in step 15 is assayed using SARS-CoV-2 CDC-N1 primers and probe and TaqMan Environmental Master Mix 2.0 (ThermoFisher SCIENTIFIC), and the copy number of the SARS-CoV-2 RNA N region (gene copy number / 10 ml) is calculated.

[0135]

[0136] The calculated copy numbers of the SARS-CoV-2 RNA N region (gene copies / 10 ml) are shown in Table 7. The addition of PAC flocculant was also confirmed to improve the detection of SARS-CoV-2 viral RNA in urban wastewater δ. Furthermore, high detection levels were observed when PAC with basicity of 50%, 60%, or 70% was used.

[0137] Example 8 Comparative study of pH conditions during sewage sediment dissolution (included in methods (III) to (V) of the present invention) To clarify the optimal pH conditions for improving the quantitative detection efficiency of SARS-CoV-2 virus RNA, an experiment to detect the added virus was carried out using a sample prepared by adding inactivated SARS-CoV-2 virus to urban sewage δ. 1. 1x10 based on the amount of RNA measured by quantitative PCR ５10 mL of sewage (urban sewage from δ-point sewage treatment plant) containing inactivated copies of SARS-CoV-2 is placed in a centrifuge tube before centrifugation. 1 μL of PAC 300A is added as a flocculant and shaken to obtain a sample (final concentration 0.001% (w / v)). 2. Each sample obtained in step 1 is stirred at 60 rpm at 4°C for 10 minutes, then centrifuged at 3,000 g for 10 minutes at 4°C, and the supernatant is completely discarded. 3. 250 μL of Lysis buffer I, adjusted to pH 6, 7.5, 8, 9, 10, or 11, is added, suspended well, and transferred to a 1.5 mL centrifuge tube. 4. Add 14.25 μL of proteinase K solution to the tube (final concentration: 1.07 mg / mL or more) and suspend thoroughly. Heat on a heat block at 56°C for 10 minutes, then leave to stand at room temperature for 5 minutes. 5. Add 550 μL of PCI solution (pH 5.2) to the tube and suspend by vortexing. 6. Leave the tube to stand for 1 minute, then centrifuge at 19,000 g for 10 minutes at 20°C. 7. Remove 200 μL of the supernatant layer from the tube to use as the nucleic acid-containing crude extract sample. 8. Add 180 μL of RNAClean XP (Beckman Coulter) to 100 μL of the nucleic acid-containing crude extract sample obtained in step 7, suspend by pipetting, and leave to stand for 5 minutes. 9. Secure the tube to a magnetic stand and leave to stand for 10 minutes. 10. Discard the supernatant from the tube and wash with 70% ethanol. 11. Discard the supernatant from the tube and let it stand for 15 minutes to dry. 12. Remove the tube from the magnetic stand and suspend the pellet in 30 μL of water and let it stand for 3 minutes. 13. Fix the tube to the magnetic stand, collect the supernatant and transfer it to a new 1.5 mL centrifuge tube to use as the RNA sample. 14. Perform a reverse transcription reaction using 1 μg of the RNA sample collected in step 13 and the 2019-nCoV_N1-R and PMMoV reverse primers with the Reliance Select cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.) to synthesize cDNA.15. Obtain a 10-cycle pre-amplified sample using the cDNA obtained in step 14, a dilution series of plasmid DNA containing a SARS-CoV-2 positive control sequence or a PMMoV positive control sequence for copy number calculation, a SARS-CoV-2 CDC-N1 primer and PMMoV primer set, and BIOTAQ HS (Meridian BIOSCIENCE). At the same time, 10 cycles of pre-amplification heat treatment are carried out using only the cDNA obtained in step 14, a dilution series of plasmid DNA containing the PMMoV positive control sequence for copy number calculation, and the PMMoV reverse primer (PMMV-RP1) and BIOTAQ HS (Meridian BIOSCIENCE), to obtain a non-pre-amplified sample in which the PMMoV cDNA fragment itself has not been pre-amplified. The SARS-CoV-2 pre-amplified and PMMoV non-pre-amplified samples obtained in step 15 are measured using the SARS-CoV-2 CDC-N1 primer and PMMoV primer set and probe, and TaqMan Environmental Master Mix 2.0 (ThermoFisher SCIENTIFIC), and the copy numbers of the SARS-CoV-2 RNA N region and PMMoV RNA (gene copy number / 10 ml) are calculated.

[0138]

[0139] The calculated copy numbers of SARS-CoV-2 RNA N region and PMMoV are shown in Table 8. These results demonstrate that when sewage sediment is dissolved under conditions of pH 7.5 to pH 11.0, SARS-CoV-2 RNA and PMMoV RNA can be quantitatively detected more efficiently than under conditions of pH 6.0.

[0140] The method of the present invention is a method for quantitatively detecting RNA and / or DNA derived from viruses or bacteria in environmental or fecal samples with high sensitivity.

Claims

1. 1. A method for the quantitative detection of RNA and / or DNA of viral or bacterial origin in an environmental or fecal sample, comprising: (A1) obtaining a concentrated product of the sample in the presence of polyaluminum chloride; (A2) dissolving the concentrated product under conditions of pH 7 to 11; (A3) binding and separating the RNA and / or DNA in the enrichment product to a solid support; and (B) if the RNA is separated in step (A3), obtaining cDNA by reverse transcription using the RNA as a template; (C) The method may include a step of preamplifying the cDNA obtained in step (B) and / or the DNA obtained in step (A3) as a template to obtain an amplification product in a liquid phase.

2. 2. The method of claim 1, wherein the environmental sample is a sewage sample, a water sample, an air sample, or a sample obtained from a solid surface.

3. The method according to claim 1 or 2, wherein the number of cycles of preamplification is 1 to 14 cycles.

4. The method according to any one of claims 1 to 3, wherein the solid support is a magnetic bead.

5. The method according to any one of claims 1 to 4, wherein the virus is an RNA virus.

6. The method according to any one of claims 1 to 5, wherein the virus is a coronavirus, influenza virus, norovirus, sapovirus, Aichi virus, adenovirus, orthopneumovirus or polyomavirus.

7. The method of claim 6, wherein the coronavirus is SARS-CoV-2.

8. The method according to any one of claims 1 to 7, further comprising: (D) a step of quantifying the cDNA and / or DNA by quantitative PCR using the amplification product obtained in step (C), or the cDNA obtained in step (B) and / or the DNA obtained in step (A3) as a template.

9. The method according to any one of claims 1 to 8, wherein the polyaluminum chloride has a final concentration of 0.001 to 0.01 mass volume percent in step (A1).

10. 10. The method according to claim 1, wherein the basicity of the polyaluminum chloride is 50 to 70%.

11. The method according to any one of claims 1 to 10, wherein the step (A2) is carried out under a condition of pH 7 to 8.

12. The method according to any one of claims 1 to 11, wherein the step (A2) comprises a step of reacting with a protease.

13. A kit for quantitatively detecting RNA and / or DNA of viral or bacterial origin in an environmental or fecal sample, comprising polyaluminum chloride and a solid support.

14. The kit according to claim 13, wherein the solid support is a carboxyl group-modified paramagnetic bead.

15. The kit described in claim 13 or 14, further comprising a protocol instructing the addition of polyaluminum chloride to an environmental sample or fecal sample to a final concentration of 0.001 to 0.01% (w / v).