Method for detecting RNA viruses

Abstract

Provided is a method for detecting an RNA virus in a biological sample, the method comprising: (1) a step for preparing a sample solution containing a biological sample, a protease, a nucleic acid that does not become a target of nucleic acid amplification, and at least one additive selected from the group consisting of chaotropic reagents and surfactants; (2) a step for preparing a nucleic acid amplification reaction solution containing the sample solution prepared in step (1) and containing a polypeptide having reverse transcriptase activity and DNA polymerase activity or a polypeptide having reverse transcription activity and a polypeptide having DNA polymerase activity; and (3) a step for amplifying a nucleic acid of the RNA virus in the reaction solution prepared in step (2).

Description

Technical Field

[0001] The present invention relates to a method for detecting RNA viruses contained in a biological sample, a composition for the method, and a kit.

Background Art

[0002] Detecting viruses contained in a biological sample is important in clinical diagnosis and the like, and a method has been carried out in which nucleic acids in a biological sample are amplified by PCR or the like, the amplified nucleic acids are detected, and the virus is identified. However, since various biological substances other than nucleic acids are contained in a biological sample, if nucleic acid amplification is carried out without purifying nucleic acids from the biological sample, amplification inhibition or detection inhibition occurs. Therefore, when detecting a target virus in a biological sample, for example, a method is adopted in which nucleic acids are first purified from the biological sample using a column or the like and then subjected to a nucleic acid amplification reaction.

[0003] In addition, methods using a treatment method for a nucleic acid-containing sample or an additive to a reaction solution for suppressing amplification inhibition by a biological substance have been reported in a nucleic acid amplification reaction solution, but it cannot be said that the influence on the nucleic acid amplification reaction has been completely eliminated. For example, a method has been reported in which a protease is allowed to act on an RNA virus and then RT-Nested PCR is performed for detection (see Patent Document 1). In addition, in nucleic acid extraction from a virus, a method has been reported in which a reducing agent or a polysaccharide-degrading enzyme is allowed to act on a biological sample and then RT-PCR is performed for detection (see Patent Document 2).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the method of first purifying nucleic acids from a biological sample using a nucleic acid binding carrier, etc., and then subjecting it to a nucleic acid amplification reaction, there is a risk that the operator will be infected with the virus until the virus is inactivated, and therefore the sample must be handled at a containment level suitable for the virus. In other words, there was a problem of high cost and risk. The object of the present invention is to provide a highly sensitive method for detecting RNA viruses in a biological sample that reduces the risk of infection to the operator, is not expensive, and is not complicated to operate. [Means for solving the problem]

[0006] In view of the above problems, the present inventors conducted diligent studies and found that by preparing a sample solution containing a biological sample and at least one additive selected from the group consisting of a proteolytic enzyme, nucleic acids that are not targets for nucleic acid amplification, a chaotropic reagent, and a surfactant, it is possible to efficiently amplify nucleic acids derived from target RNA viruses in a nucleic acid amplification reaction containing polypeptides having reverse transcription activity and DNA polymerase activity, and to detect them with high sensitivity, thus completing the present invention.

[0007] The present invention [1] A method for detecting RNA viruses in a biological sample, comprising the following steps: (1) A step of preparing a sample solution comprising a biological sample and at least one additive selected from the group consisting of a proteolytic enzyme, nucleic acids that are not targets for nucleic acid amplification, chaotropic reagents, and surfactants. (2) A step of preparing a nucleic acid amplification reaction solution comprising the sample solution prepared in step (1) above, a polypeptide having reverse transcription activity and a polypeptide having DNA polymerase activity, or a polypeptide having reverse transcriptase activity and DNA polymerase activity, and (3) A step of amplifying the nucleic acid of the RNA virus in the reaction solution prepared in step (2), Methods that include, [2] The method according to [1], wherein the proteolytic enzyme is proteinase K, thermolysin, or pronase. [3] The method according to [1] or [2], wherein the chaotropic reagent is guanidine or a salt thereof, urea, iodine or a salt thereof, or a combination thereof. [4] The method according to any one of [1] to [3], wherein the biological sample is at least one selected from the group consisting of oral swabs, pharyngeal swabs, nasal swabs, nasopharyngeal swabs, nasal aspirates, sputum, bronchial lavage fluid, bronchoalveolar lavage fluid, rectal swabs, saliva, blood, urine, and fecal suspension. [5] The method according to any one of [1] to [4], wherein the RNA virus is at least one selected from the group consisting of influenza virus, RSV, metapneumovirus, parainfluenza virus, SARS coronavirus, MERS coronavirus, measles virus, norovirus, rotavirus, sapovirus and HIV. [6] A method for preparing a sample solution for nucleic acid amplification, comprising the step of preparing a mixture containing a biological sample, a protease, a nucleic acid that is not a target for nucleic acid amplification, a chaotropic reagent, and at least one additive selected from the group consisting of a surfactant. A composition for a method of detecting RNA viruses in a biological sample as described in any one of [7][1] to [5], comprising: (1) a sample solution comprising a biological sample and at least one additive selected from the group consisting of proteases, nucleic acids that are not targets for nucleic acid amplification, chaotropic reagents and surfactants; and (2) a polypeptide having reverse transcriptase activity and a polypeptide having DNA polymerase activity, or a polypeptide having reverse transcriptase activity and DNA polymerase activity. A composition containing, A kit for a method of detecting RNA viruses in a biological sample as described in any one of [8][1] to [5], (1) At least one additive selected from the group consisting of proteolytic enzymes, nucleic acids that are not targets for nucleic acid amplification, chaotropic reagents, and surfactants, and (2) polypeptides having reverse transcription activity and polypeptides having DNA polymerase activity, or polypeptides having reverse transcriptase activity and DNA polymerase activity, kits including [9] A sample solution for nucleic acid amplification comprising a biological sample and at least one additive selected from the group consisting of a protease, a nucleic acid that is not a target for nucleic acid amplification, a chaotropic reagent, and a surfactant. Regarding etc. [Effects of the Invention]

[0008] By eliminating the need for specialized equipment, reagents such as organic solvents, and time required for nucleic acid extraction and purification, a simple pretreatment of biological samples can be performed, enabling the preparation of a nucleic acid amplification sample solution suitable for nucleic acid amplification without risk to the operator. The target nucleic acid can be detected by nucleic acid amplification using this sample solution. Therefore, the present invention provides a method for detecting RNA viruses in biological samples that is safe, simple, and enables highly sensitive detection. [Modes for carrying out the invention]

[0009] In this specification, "heat resistance" refers to resistance to heat treatment. For example, if the enzymatically optimal temperature is 40°C, then improved "heat resistance" can be said to exist if activity can be maintained at 50°C or above, 60°C or above, or even 70°C or above. For example, in the case of reverse transcriptase derived from Moloney's mouse leukemia virus, the optimal temperature of the wild-type enzyme is 37-42°C, so a reverse transcriptase mutant that maintains enzyme activity at, for example, 43°C or above, preferably 45°C or above, and even more preferably 50°C or above, can be said to be "heat-resistant" or "have improved heat resistance."

[0010] The details are explained below.

[0011] 1. Method for detecting RNA viruses in biological samples according to the present invention The present invention provides a method for detecting RNA viruses in a biological sample, comprising the steps of: (1) preparing a sample solution containing a biological sample and at least one additive selected from the group consisting of a protease, a nucleic acid that is not a target for nucleic acid amplification, a chaotropic reagent, and a surfactant; (2) A step of preparing a nucleic acid amplification reaction solution comprising the sample solution prepared in step (1) above, a polypeptide having reverse transcription activity and a polypeptide having DNA polymerase activity, or a polypeptide having reverse transcription activity and DNA polymerase activity, and (3) A step of amplifying the nucleic acid of the RNA virus in the reaction solution prepared in step (2), It is characterized by encompassing.

[0012] [RNA virus to be detected] The RNA viruses detected by the method of the present invention are not limited to those with RNA as their genome. Examples include double-stranded RNA viruses (dsRNA), single-stranded positive-sense RNA viruses (+-type), and single-stranded negative-sense RNA viruses (--type). While not particularly limited, examples include influenza virus, respiratory syncytial virus (RSV), metapneumovirus, parainfluenza virus, SARS coronavirus (including SARS-CoV-2, which is also referred to as 2019-nCoV in this specification), MERS coronavirus, measles virus, norovirus, rotavirus, sapovirus, and HIV.

[0013] [Biological sample] The biological sample used in the present invention is a sample suspected of containing an RNA virus, and includes not only the sample itself collected from a living body, but also its derivatives, such as suspensions, lysates, and diluents. The biological sample is not particularly limited, and examples include oral swabs, throat swabs, nasal swabs, nasopharyngeal swabs, nasal aspirates, sputum, bronchial lavage fluids, alveolar lavage fluids, rectal swabs, saliva, blood, urine, or fecal suspensions. In addition to the above samples, environmental samples suspected of containing RNA viruses include environmental water (seawater, river water, lake water, sewage, domestic wastewater, industrial wastewater, etc.), and suspensions of samples obtained by wiping operations such as swabs. The objects of such wiping include manufacturing equipment for foods, etc., experimental equipment, medical equipment, floors, walls, workbenches, sinks, various instruments and equipment in kitchens and toilets, etc. Each of the above biological samples can be used in the present invention, or a plurality of biological samples can be mixed and then used in the present invention.

[0014] [Additive] The additive used in the present invention is used for the pretreatment of biological samples, and is not particularly limited. Examples include proteolytic enzymes, nucleic acids that are not targets for nucleic acid amplification, chaotropic reagents, and surfactants.

[0015] The proteolytic enzyme is not particularly limited as long as it can act on the envelope, capsid, etc. of RNA viruses or nucleases derived from biological samples and release the RNA genome present in the capsid without degrading it. For example, serine protease, acidic protease (aspartic protease, glutamic protease), alkaline protease, semi-alkaline protease, metalloprotease, cysteine protease, N-terminal threonine protease, etc. may be used alone or in combination. The proteolytic enzyme may also be an endopeptidase. As an example of an endopeptidase, proteinase K is a representative of serine protease, but thermolysin, pronase or their mutants can also be preferably used. In the present invention, the use concentration of the proteolytic enzyme during the pretreatment of the biological sample may be determined appropriately. For example, the final concentration range of proteinase K or its mutant is preferably in the range of 3 U / ml to 150 U / ml, more preferably in the range of 3 U / ml to 120 U / ml, and particularly preferably in the range of 30 U / ml to 100 U / ml. When combined with a chaotropic reagent or a surfactant, it is in the range of 1 U / ml to 120 U / ml, preferably in the range of 2 U / ml to 100 U / ml. In this specification, the activity of proteinase K is defined as 1 U, which is the amount of enzyme that releases Folin-positive amino acids corresponding to 1.0 μmol of tyrosine per minute at 37 °C and pH 7.5 using denatured bovine hemoglobin as a substrate.

[0016] In addition, the nucleic acid that is not the target of nucleic acid amplification refers to DNA and / or RNA having a base sequence different from that of the RNA derived from the RNA virus to be detected in the present invention, or nucleic acid analogs, etc. Various nucleic acids derived from viruses and organisms other than the RNA virus to be detected can be used as the nucleic acid that is not the target of nucleic acid amplification, and there is no particular limitation, but it may be nucleic acid derived from Escherichia coli, nucleic acid derived from Bacillus subtilis, or nucleic acid derived from yeast. Preferably, ribosomal RNA derived from Escherichia coli or yeast is used.

[0017] Furthermore, a chaotropic reagent is a substance that reduces the interaction between water molecules, thereby destabilizing the structure of molecules present in the solution. Examples of chaotropic reagents, though not particularly limited, include guanidine and its salts, urea, iodine and its salts, and combinations thereof. For example, examples of guanidine salts include guanidine hydrochloride, guanidine nitrate, guanidine carbonate, and guanidine thiocyanate. Examples of iodides include lithium iodide, sodium iodide, potassium iodide, magnesium iodide, and calcium iodide. In the present invention, the final concentration of the chaotropic reagent is not limited, but can be used in the range of 0.5 w / V% to 5 w / V%, preferably 1 w / V% to 4 w / V%, and particularly preferably 2 w / V% to 4 w / V%. Furthermore, while not limited, the final molar concentration of chaotropic ions can be used in the range of, for example, 40 μmol / ml to 450 μmol / ml, preferably 80 μmol / ml to 340 μmol / ml, and particularly preferably 160 μmol / ml to 340 μmol / ml.

[0018] The surfactants that can be used in the present invention are not particularly limited, but examples include anionic surfactants (sodium dodecyl sulfate, sodium N-lauroyl sarcosinate, sodium deoxycholate, etc.), nonionic surfactants (Triton® X-100, Tween® 20, Nonidet® P-40, Brij® 35, etc.), and cationic surfactants. For example, when using sodium dodecyl sulfate, the final concentration can be used in the range of 0.001 to 1% by mass percentage (w / v %), preferably in the range of 0.005 to 1%, and more preferably in the range of 0.005 to 0.5%. It goes without saying that this concentration should be appropriately adjusted in combination with the proteolytic enzyme and RT-qPCR reagent used.

[0019] [Polypeptides used for nucleic acid amplification] In the method of the present invention, a polypeptide having reverse transcription activity for synthesizing cDNA from the genomic RNA of an RNA virus and a polypeptide having DNA-dependent DNA polymerase activity (referred to herein as DNA polymerase activity) for amplifying the DNA fragment derived from the cDNA can be used in the nucleic acid amplification reaction. The polypeptide having reverse transcription activity and the polypeptide having DNA polymerase activity may be separate polypeptides, or they may be a single polypeptide possessing both activities. Examples of polypeptides having reverse transcription activity include HIV reverse transcriptase, AMV reverse transcriptase, M-MLV reverse transcriptase, C therm. polymerase, and Tth polymerase, as well as PrimeScript® RTase, SuperScript® RTase, RevERTra Ace® RTase, SMARTScribe® RTase, Quantiscript RTase, ProtoScript RTase, and the like.

[0020] Unless otherwise specified, the polypeptides having DNA polymerase activity described herein are DNA-dependent DNA polymerases. Suitable polymerases include DNA polymerases belonging to Family A (Pol I type), DNA polymerases belonging to Family B (α type), and mixtures of the two types of polymerases. Preferably, a heat-resistant DNA polymerase is used. The DNA polymerase is not particularly limited, but examples include Taq DNA polymerase and Tth DNA polymerase derived from thermophilic bacteria, and KOD DNA polymerase and Pfu DNA polymerase derived from thermophilic archaea. In certain embodiments, it is preferable to use an α-type DNA polymerase (or a DNA polymerase belonging to Family B) that offers excellent accuracy. While there are no particular limitations on the α-type DNA polymerase, commercially available polymerases such as PrimeSTAR DNA polymerase (Takara Bio Inc.), Tks Gflex DNA polymerase (Takara Bio Inc.), Pfu DNA polymerase, or KOD DNA polymerase (Toyobo Inc.) can be used. Furthermore, a single polypeptide possessing both reverse transcription activity and DNA polymerase activity may also be used, for example, Tth polymerase.

[0021] The polypeptide having the reverse transcription activity and the polypeptide having DNA polymerase activity, or the polypeptide having both activities, may be in its native form or a mutant, as long as it is compatible with the method of the present invention.

[0022] In the detection method of the present invention, the polypeptide having reverse transcription activity and the polypeptide having DNA polymerase activity, or the polypeptide having both activities, may be a hot-start enzyme that combines with an antibody of the polypeptide to prevent nonspecific amplification before the reaction. Furthermore, it may be combined with elongation factors such as PCNA and various substances known to improve the efficiency of nucleic acid amplification reactions, such as surfactants, bovine serum albumin, acidic polymers, and amphoteric amino acids.

[0023] [Nucleic acid amplification reaction solution] The nucleic acid amplification reaction solution used in the detection method of the present invention comprises a sample solution containing a biological sample and additives, and a polypeptide having reverse transcription activity and a polypeptide having DNA polymerase activity, or a polypeptide having both reverse transcription activity and DNA polymerase activity, wherein the polypeptide is composed in such a way that it can exert its activity. When nucleic acid amplification is performed by RT-PCR, the reaction solution contains a buffer component to maintain an optimal pH, a divalent metal salt (magnesium salt, manganese salt, etc.), and dNTPs, but may also contain neutral salts, surfactants, and other components. This reaction solution may contain a primer pair for nucleic acid amplification of a specific region on the genomic RNA of the RNA virus to be detected, or a nucleic acid probe for detection (e.g., a quenching probe (Q probe), a Taqman® probe), etc. Alternatively, instead of a nucleic acid probe for detection, it may contain an intercalator dye (e.g., SYBR® Green, TB Green®), etc. Furthermore, it may contain other reagents used in the nucleic acid amplification reaction. While not particularly limited, when detecting 2019-nCoV, the primer pairs and nucleic acid probes described in the National Institute of Infectious Diseases manual "Pathogen Detection Manual 2019-nCoV Ver.2.9.1", the primer pairs and nucleic acid probes described in the Centers for Disease Control and Prevention (CDC) "2019-Novel Coronavirus (2019-nCoV) Real-time rRT-PCR Panel Primers and Probes", etc., can be suitably used. Furthermore, the nucleic acid amplification reaction may be a multiplex detection method that combines an internal standard RNA having a different sequence from the nucleic acid derived from the RNA virus to be detected, and a primer pair and nucleic acid probe for detecting the internal standard RNA, for purposes such as confirming reaction inhibition. For example, when detecting 2019-nCoV, the N-set primers, N-set No. 2 primers / probes described in the aforementioned National Institute of Infectious Diseases manual, or any of the N1 primers / probes, N2 primers / probes, or N3 primers / probes described in the aforementioned CDC manual can be suitably used.

[0024] Furthermore, in the detection method of the present invention, multiple different gene regions on the genomic RNA of the virus to be detected can be amplified, and these different gene regions can be detected with multiple probes. In this case, the multiple probes may be labeled with the same label. That is, detection of two regions at one wavelength is possible. The labeled probes may be a combination of fluorescent labels based on the FRET method, a non-FRET method, or a combination of fluorescent labels and quenching labels. Examples of the fluorescent labels include FAM, Cy5, ROX, or HEX. As the quenching substances, labels such as Eclipse or BHQ®, known as dark quenchers, can be suitably used.

[0025] While not particularly limiting the present invention, when detecting 2019-nCoV, for example, two or more primers / probes selected from the N1, N2, and N3 primers / probes published by the CDC in "2019-Novel Coronavirus (2019-nCoV) Real-time rRT-PCR Panel Primers and Probes" can be used simultaneously to prepare a nucleic acid amplification reaction solution that simultaneously detects multiple regions. In this case, by labeling the multiple probes contained in the reaction solution with the same fluorescent dye, the virus can be detected with high sensitivity.

[0026] The nucleic acid amplification reaction solution is incubated under appropriate temperature conditions so that the reactions of cDNA synthesis from the viral genome RNA target region and cDNA amplification occur sequentially. These conditions can be those well known to those skilled in the art as one-step RT-PCR. In this case, the temperature, incubation time, and cycle count can be appropriately adjusted according to the target sequence and the chain length of the primers and probes.

[0027] In the detection method of the present invention, (1) a sample solution is prepared containing a biological sample and at least one additive selected from the group consisting of a proteolytic enzyme, a nucleic acid that is not a target of nucleic acid amplification, a chaotropic reagent, and a surfactant, and this sample solution is added to the nucleic acid amplification reaction solution. Prior to adding this sample solution to the nucleic acid amplification reaction solution, it may be kept warm or heated as described below.

[0028] In the detection method of the present invention, RNA viruses in a biological sample can be subjected to reverse transcription and nucleic acid amplification reactions without purification of the nucleic acids. Therefore, the amount of template introduced into the reverse transcription and nucleic acid amplification reactions can be increased. For example, for a reaction volume of 50 μl, the amount of template solution can be 30 μl or less, allowing the introduction of biological sample processing solution (the sample solution) equivalent to 5 μl to 10 μl.

[0029] 2. Method for preparing a sample solution for nucleic acid amplification according to the present invention The nucleic acid amplification sample solution of the present invention is a mixture containing a biological sample as described in 1. above, and at least one additive selected from the group consisting of a proteolytic enzyme, a nucleic acid that is not a target for nucleic acid amplification, a chaotropic reagent, and a surfactant. Therefore, the method for preparing the nucleic acid amplification sample solution of the present invention includes the step of preparing a mixture containing a biological sample and an additive. The sample solution of the present invention may be prepared by mixing the biological sample and the additive, or by directly suspending the biological sample in a pre-prepared solution containing the additive. The sample solution may contain multiple additives. In this case, the amount of each additive added should be appropriately set based on indicators such as the detection sensitivity of RNA viruses.

[0030] The sample solution described above may be subjected to a nucleic acid amplification reaction after being held for a certain period of time, although this does not particularly limit the present invention. The holding time is not limited, but examples include 1 second to 30 minutes, preferably 10 seconds to 20 minutes, more preferably 20 seconds to 10 minutes, and even more preferably 30 seconds to 5 minutes. Furthermore, the sample solution may be held at multiple different temperatures. A sample solution containing a biological sample and at least one additive selected from the group consisting of a proteolytic enzyme, a nucleic acid that is not a target for nucleic acid amplification, a chaotropic reagent, and a surfactant may be subjected to a warming and / or heating treatment before being added to the nucleic acid amplification reaction solution. For example, it can be kept warm and / or heated at room temperature or in the range of 1°C to 99°C. In the case of a sample solution containing a proteolytic enzyme, from the viewpoint of promoting the action of the enzyme, it may be kept warm at, for example, 20°C to 90°C, preferably 30°C to 80°C, more preferably 40°C to 70°C, and even more preferably 50°C to 60°C. Examples of the incubation time include, for example, 1 second to 30 minutes, preferably 10 seconds to 20 minutes, more preferably 20 seconds to 10 minutes, and even more preferably 30 seconds to 5 minutes. For example, although not limited to these, the sample solution may be incubated at 55°C for 5 to 10 minutes. Furthermore, the sample solution may be subjected to high-temperature treatment to inactivate the proteolytic enzyme. Examples of high-temperature treatment temperatures include, for example, 91°C to 99°C, preferably 93°C to 97°C. Examples of high-temperature treatment times include, for example, 1 second to 10 minutes, preferably 30 seconds to 5 minutes. The sample solution may be subjected to high-temperature treatment after the incubation treatment described above, or it may be subjected to high-temperature treatment only without incubation treatment. Therefore, the method for preparing a nucleic acid amplification sample solution of the present invention may further include a step of holding the mixed solution containing the biological sample and additives for a certain period of time. In another embodiment, the method for preparing a nucleic acid amplification sample solution of the present invention may further include a step of incubation and / or heating the mixed solution containing the biological sample and additives.

[0031] 3. Composition for detecting RNA viruses in biological samples according to the present invention The composition of the present invention is a composition used as a nucleic acid amplification reaction solution for use in the method for detecting the RNA virus of the present invention, and is characterized by comprising a sample solution containing a biological sample and at least one additive selected from the group consisting of a protease, a nucleic acid that is not a target for nucleic acid amplification, a chaotropic reagent, and a surfactant, as described in 1. above, and a polypeptide having reverse transcriptase activity and a polypeptide having DNA polymerase activity, or a polypeptide having both reverse transcriptase activity and DNA polymerase activity. If the sample solution contains a protease, the protease is preferably an inactivated protease, which is deactivated after the preparation of the sample solution as described in 2. above, so as not to degrade the polypeptide having DNA polymerase activity in the composition of the present invention. The composition of the present invention may further contain various components necessary for the polypeptide having reverse transcriptase activity and the polypeptide having DNA polymerase activity, or the polypeptide having both reverse transcriptase activity and DNA polymerase activity, to exert their activity. Furthermore, the composition may contain a primer pair for nucleic acid amplification of a specific region on the genomic RNA of the target RNA virus, a nucleic acid probe for detection, etc. Methods for detecting nucleic acids using the nucleic acid probe include, for example, using TaqMan® probes, Q probes, MolecularBeacons, etc. In the above method, target nucleic acids can be detected by, for example, monitoring the degradation of the nucleic acid probe or by hybridizing the nucleic acid probe with nucleic acid and performing melting curve analysis. Other conditions in the detection process should be set appropriately considering the type and sequence of the nucleic acid to be detected, the type of probe, etc. Alternatively, an intercalator dye (e.g., SYBR® Green, TB Green®) may be included instead of the detection nucleic acid probe. Although not particularly limited, when detecting the 2019-nCoV coronavirus, primer pairs and detection nucleic acid probes described in the National Institute of Infectious Diseases manual, primer pairs and detection nucleic acid probes described in the US CDC manual, etc., can be suitably used. In addition, a primer pair and detection nucleic acid probe for internal standard amplification may be included to confirm reaction inhibition.

[0032] 4. Kit for detecting RNA viruses in biological samples according to the present invention The kit of the present invention is as described in 1. above. (1) At least one additive selected from the group consisting of proteolytic enzymes, nucleic acids that are not targets for nucleic acid amplification, chaotropic reagents, and surfactants, and (2) polypeptides having reverse transcriptase activity and polypeptides having DNA polymerase activity, or polypeptides having reverse transcriptase activity and DNA polymerase activity, It is characterized by including.

[0033] The kit of the present invention may contain each of the above elements in a form suitable for carrying out the "method for detecting RNA viruses in a biological sample of the present invention" and for preparing the "composition for detecting RNA viruses in a biological sample of the present invention." For this purpose, the kit of the present invention may further include a diluent for preparing a sample solution for nucleic acid amplification, a buffer for preparing a nucleic acid amplification reaction solution for RNA virus detection, etc. Herein, the kit of the present invention may also include a reagent for detecting RNA viruses by the method of the present invention. Examples of the reagent include, but are not limited to, buffer components, divalent metal salts, dNTPs, neutral salts, etc. In one aspect of the present invention, a kit is provided that includes a polypeptide having reverse transcriptase activity and a polypeptide having DNA polymerase activity, or a polypeptide having both reverse transcriptase activity and DNA polymerase activity, a premix reaction solution containing components necessary for both polypeptides to exhibit activity in appropriate concentrations, and the above-mentioned additives.

[0034] The kit of the present invention may further include a primer pair for nucleic acid amplification of a specific region on the genomic RNA of the target RNA virus, a detection nucleic acid probe, etc. Alternatively, an intercalator dye, etc., may be included instead of a detection nucleic acid probe. Although not particularly limited, when detecting 2019-nCoV coronavirus, the primer pair and detection nucleic acid probe described in the National Institute of Infectious Diseases manual, the primer pair and detection nucleic acid probe described in the US CDC manual, etc., can be suitably used. Furthermore, the kit of the present invention may include a primer pair and detection nucleic acid probe for internal standard amplification to confirm reaction inhibition.

[0035] The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to these examples. [Examples]

[0036] Consideration of additives 1 The detection method of the present invention was investigated. First, an RNA-positive control called the N2 set, which has the nucleotide sequence described in Sequence ID No. 4 of the sequence listing and is used in the sensitivity test of the primer pair and nucleic acid probe for detection described in the National Institute of Infectious Diseases manual "Pathogen Detection Manual 2019-nCoV Ver.2.9.1", was prepared by a conventional method. Next, a commercially available sputum sample (manufactured by NOVA Biologics) was diluted 3-fold with physiological saline to use as the stock solution, and this was further diluted 20-fold with physiological saline and mixed with the above RNA-positive control to obtain a biological sample suspension. A suspension without a sputum sample was also prepared. Next, the additives mixed with the biological sample were Proteinase K (Takara Bio, approximately 400 U / ml) as a proteolytic enzyme, guanidine thiocyanate (CAS registry number 593-84-0) (Tokyo Chemical Industries, Ltd.) as a chaotropic reagent (also known as guanidine thiocyanate or guanidine thiocyanate), and sodium dodecyl sulfate (Tokyo Chemical Industries, Ltd.) as a surfactant. A control was also established in which no additives were added to the biological sample suspension.

[0037] To the above biological sample suspension, Proteinase K at a final concentration of 3.5 U / ml (175 μg / ml), 7 U / ml (350 μg / ml), 35 U / ml (1750 μg / ml), or 70 U / ml (3500 μg / ml), along with guanidine thiocyanate at a final concentration of 209 μmol / ml and sodium dodecyl sulfate at a final concentration of 0.01% (w / v), was added. The mixture was incubated at 55°C for 5-10 minutes, or heated at 95°C for 5 minutes without incubation. RNA-positive control was added to this mixture at a rate of 2.5 × 10^5 copies / reaction, and the mixture was subjected to RT-PCR. The RT-PCR reagent used was One Step PrimeScript™ III RT-qPCR Mix (Takara Bio Inc.). Specifically, 5 μl of the aforementioned mixture was mixed with 25 μl of 2×RT-qPCR Mix, the NIID_2019-nCoV_N_F primer (final concentration 0.5 μM) and NIID_2019-nCoV_N_R primer (final concentration 0.7 μM) described in Sequence ID No. 1 and No. 2 of the sequence listing, the NIID_2019-nCoV_N_P probe (final concentration 0.2 μM, 5'FAM labeled, 3'BHQ1 labeled) having the nucleotide sequence described in Sequence ID No. 3 of the sequence listing, and RNase-free H2O to a final volume of 50 μl.

[0038] A Thermal Cycler Dice® Real Time System III (Cy5) with PC (manufactured by Takara Bio Inc.) was used. The PCR conditions consisted of 45 cycles, each cycle being 52°C for 5 minutes, 95°C for 10 seconds, followed by 95°C for 5 seconds and 60°C for 30 seconds. The results are shown in Table 1.

[0039] [Table 1]

[0040] As shown in Table 1, when high concentrations of Proteinase K (35-70 U / ml, 175 μg / ml-3500 μg / ml, labeled "ProK" in the table) were added, the Ct values ​​were almost equivalent to those without a biological sample suspension, eliminating the influence of the biological sample suspension. Furthermore, by combining 3.5 U / ml of Proteinase K with guanidine thiocyanate (labeled "GT" in the table) or 3.5 U / ml of Proteinase K with 0.01% sodium dodecyl sulfate (labeled "SDS" in the table), the Ct values ​​were almost equivalent to those without a biological sample suspension, eliminating the influence of the biological sample suspension. From the above, it was confirmed that high concentrations of Proteinase K, or combinations of Proteinase K with guanidine thiocyanate and / or sodium dodecyl sulfate, are promising as additives for the present invention. In addition, it was possible to detect RNA viruses in the sample even when the 55°C treatment of the sample solution was omitted. [Examples]

[0041] Consideration of additives 2 The detection method of the present invention was investigated using an inactivated virus. In this example, NATtrol® Influenza A / B Positive Control (manufactured by ZeptoMetrix) was used as the inactivated RNA virus. The undiluted solution or a 10- to 20-fold dilution with physiological saline was mixed with the sputum sample prepared in Example 1 to obtain a biological sample suspension. A suspension without a sputum sample was also prepared.

[0042] The additives used were Proteinase K as a proteolytic enzyme, guanidine thiocyanate as a chaotropic reagent, and sodium dodecyl sulfate as a surfactant.

[0043] The experiment was conducted as follows: Proteinase K at a final concentration of 3.5 U / ml or 70 U / ml, guanidine thiocyanate at a final concentration of 2.5% (w / v), or sodium dodecyl sulfate at a final concentration of 0.01% (w / v) were added to the above-mentioned biological sample suspension. The mixture was then held at room temperature or 55°C for 5 minutes, followed by heating at 95°C for 5 minutes, and then evaluated by RT-qPCR. One Step PrimeScript (trademark) III RT-qPCR Mix (manufactured by Takara Bio Inc.) was used as the RT-qPCR reagent. 5 μl of biological sample suspension containing the aforementioned additive was mixed with 25 μl of 2×RT-qPCR Mix, IAmg-F01 and IAmg-R01 primers (final concentration 0.2 μM) having the nucleotide sequences described in Sequence ID No. 5 and No. 6 of the sequence listing, IAmg-PR01 probe for influenza A detection (final concentration 0.2 μM, 5'FAM labeled, 3'BHQ1 labeled) having the nucleotide sequence described in Sequence ID No. 7 of the sequence listing, and RNase-free H2O to a final volume of 50 μl.

[0044] A Thermal Cycler Dice® Real Time System III (Cy5) with PC (manufactured by Takara Bio Inc.) was used. The PCR conditions consisted of 45 cycles, each cycle being 52°C for 5 minutes, 95°C for 10 seconds, followed by 95°C for 5 seconds and 60°C for 30 seconds. Amplification results were evaluated by comparing the Ct value with a control without additives. The results are shown in Table 2.

[0045] [Table 2]

[0046] As shown in Table 2, when a high concentration of 70 U / ml of Proteinase K (indicated as "ProK" in the table) was added to a 20-fold dilution of the undiluted biological sample suspension, the Ct value was equivalent to that of the case without the biological sample suspension, indicating that the effect of the biological sample suspension was eliminated. Furthermore, it was confirmed that combining 3.5 U / ml of Proteinase K with guanidine thiocyanate (indicated as "GT" in the table) resulted in a Ct value equivalent to that of the case with the addition of 70 U / ml of Proteinase K. In addition, when combining 3.5 U / ml of Proteinase K with 0.01% sodium dodecyl sulfate (indicated as "SDS" in the table), it was confirmed that diluting the biological sample suspension 10 to 20 times resulted in a Ct value equivalent to that of the case with the addition of 70 U / ml of Proteinase K. Based on the above, we have confirmed that a combination of high concentrations of Proteinase K, guanidine thiocyanate, and / or sodium dodecyl sulfate is promising as an additive for detecting RNA from a sample in which an inactivated virus has been spiked into a biological sample that closely resembles a real specimen. [Examples]

[0047] Examination of biological samples The detection method of the present invention was investigated using an inactivated virus. As the inactivated virus, NATtrol® SARS_CORONA Positive Control (manufactured by ZeptoMetrix), a commercially available inactivated RNA virus, was used and diluted 1.2-fold and 12-fold with physiological saline. In addition, the biological sample used was either a commercially available saliva (manufactured by LEE BIOSOLUTIONS) in its original form or diluted with a virus transport solution (product name: Virus Transport Solution (VTM): manufactured by Sugiyama Gen Co., Ltd.). A biological sample suspension was prepared by mixing this biological sample with the above inactivated virus solution. Proteinase K was used as the additive mixed with the biological sample suspension as a proteolytic enzyme.

[0048] The experiment was conducted as follows: Proteinase K at a final concentration of 70 U / ml or 17.5 U / ml was added to the above biological sample suspension, left at room temperature for 5 minutes, then maintained at 55°C for 5 minutes, and subsequently heated at 95°C for 5 minutes, after which it was evaluated by RT-qPCR. The same RT-qPCR reagent used in Example 1 was used. 10 μl of biological sample solution containing the aforementioned additive was mixed with 2×RT-qPCR Mix, PCR Forward Primer N1_SARS COV_F and R primers (final concentration 0.2 μM) having the nucleotide sequences described in Sequence ID No. 8 and 9 of the sequence listing, PCR Forward Primer N2_SARS COV_F and R primers (final concentration 0.2 μM) having the nucleotide sequences described in Sequence ID No. 11 and 12 of the sequence listing, N1_SARS COV_P probe for SARS_CORONA detection (final concentration 0.2 μM, 5'Cy5 labeled, 3'BHQ3 labeled) having the nucleotide sequence described in Sequence ID No. 10 of the sequence listing, N2_SARS COV_P probe for SARS_CORONA detection (final concentration 0.2 μM, 5'Cy5 labeled, 3'BHQ3 labeled) having the nucleotide sequence described in Sequence ID No. 13 of the sequence listing, and RNase Free H2O to a final volume of 50 μl.

[0049] The thermal cycler and PCR conditions were the same as in Example 1. The amplification results were evaluated by comparing the Ct value with that of a control without additives. The results are shown in Table 3.

[0050] [Table 3]

[0051] As shown in Table 3, nucleic acid amplification was confirmed in all samples, including saliva stock solution and saliva-VTM mixture to which inactivated virus was added. Furthermore, the Ct values ​​were similar regardless of whether 70 U / ml or 17.5 U / ml of Proteinase K (indicated as "ProK" in the table) was added. From the above, it was confirmed that the present invention is effective even when detecting RNA from samples in which inactivated virus has been spiked into biological samples such as saliva. [Examples]

[0052] Investigation of uracil-N-glucosidase (UNG) treatment For the detection method of the present invention, the same biological sample suspension and additives as in Example 3 were used.

[0053] In this example, the processing method for the biological sample suspension up to RT-PCR was the same as in Example 3. Furthermore, the evaluation of the RT-qPCR method was carried out under the same conditions as in Example 3, except that the RT-PCR reagent was replaced from One Step PrimeScript™ III RT-qPCR Mix (Takara Bio Inc.) to one Step PrimeScript™ III RT-qPCR Mix, with UNG (Takara Bio Inc.). The thermal cycler and PCR conditions were the same as in Example 1. The amplification results were evaluated by comparing the Ct value with a control without additives. The results are shown in Table 4.

[0054] [Table 4]

[0055] As shown in Table 4, nucleic acid amplification was confirmed in all cases where inactivated virus was added to either the saliva stock solution or the saliva-VTM mixture. Furthermore, regarding the additive, equivalent Ct values ​​were obtained when either 70 U / ml or 17.5 U / ml of Proteinase K (indicated as "ProK" in the table) was added. From the above, it was confirmed that the present invention is also effective in RT-qPCR including uracil-N-glucosidase and dUTP to prevent contamination by the amplified product. [Industrial applicability]

[0056] By using the detection method of the present invention, nucleic acids derived from RNA viruses contained in a sample can be detected without requiring the isolation of nucleic acids, thus making a significant contribution to the field of clinical diagnosis. [Sequence Listing Free Text]

[0057] SEQ ID NO: 1:PCR Forward Primer NIID_2019-nCOV_N_F2 SEQ ID NO: 2:PCR Reverse Primer NIID_2019-nCOV_N_R2 SEQ ID NO: 3:Probe NIID_2019-nCOV_N_P2. 5’-end is labeled FAM and 3’-end is labeled BHQ1 SEQ ID NO: 4:RNA positive control sequence for 2019-nCoV N2 set SEQ ID NO: 5:PCR Forward Primer IAmg-F01 SEQ ID NO: 6:PCR Reverse Primer IAmg-R01 SEQ ID NO: 7:Probe IAmg-PB1. 5’-end is labeled FAM and 3’-end is labeled BHQ1 SEQ ID NO: 8 PCR Forward Primer N1_SARS COV_F SEQ ID NO: 9 PCR Reverse Primer N1_SARS COV_R SEQ ID NO: 10 N1_SARS COV_P. 5’-end is labeled Cy5 and 3’-end is labeled BHQ3 SEQ ID NO: 11 PCR Forward Primer N2_SARS COV_F SEQ ID NO: 12 PCR Reverse Primer N2_SARS COV_R SEQ ID NO: 13 N2_SARS COV_P. 5’-end is labeled Cy5 and 3’-end is labeled BHQ3

Claims

1. A method for detecting RNA viruses in a biological sample, comprising the following steps: (1) A sample solution containing a biological sample selected from the group consisting of saliva, sputum, oral swabs, pharyngeal swabs, nasal swabs, nasopharyngeal swabs, nasal aspirates, and bronchial lavage fluid, and 35 U / ml to 70 U / ml of proteinase K, is kept at room temperature for 5 minutes, and then treated at 91 to 99°C for 5 minutes. (2) A step of preparing a nucleic acid amplification reaction solution comprising the sample solution treated in step (1) above, a polypeptide having reverse transcription activity and a polypeptide having DNA polymerase activity, or a polypeptide having reverse transcription activity and DNA polymerase activity, and (3) A step of amplifying the nucleic acid of the RNA virus in the reaction solution prepared in step (2), A method that includes

2. The method according to claim 1, wherein the sample solution of (1) further comprises guanidine or a salt thereof.

3. The method according to claim 1 or 2, wherein the RNA virus is at least one selected from the group consisting of influenza virus, RSV, metapneumovirus, parainfluenza virus, SARS coronavirus, MERS coronavirus, measles virus, norovirus, rotavirus, sapovirus, and HIV.

4. A mixture containing a biological sample selected from the group consisting of saliva, sputum, oral swabs, pharyngeal swabs, nasal swabs, nasopharyngeal swabs, nasal aspirates, and bronchial lavage fluid, and 35 U / ml to 70 U / ml of proteinase K, is kept at room temperature for 5 minutes, and then treated at 91 to 99°C for 5 minutes. A method for preparing a sample solution for nucleic acid amplification, which includes the following.

5. A composition for a method of detecting RNA viruses in a biological sample according to any one of claims 1 to 3, (1) A sample solution comprising proteinase K in a concentration of 35 U / ml to 70 U / ml and a biological sample selected from the group consisting of saliva, sputum, oral swabs, pharyngeal swabs, nasopharyngeal swabs, nasal aspirates, and bronchial lavage fluid, wherein the sample solution is kept at room temperature for 5 minutes and then treated at 91 to 99°C for 5 minutes, and (2) polypeptides having reverse transcriptase activity and polypeptides having DNA polymerase activity, or polypeptides having reverse transcriptase activity and DNA polymerase activity, A composition containing the following:

6. A kit for a method of detecting RNA viruses in a biological sample according to any one of claims 1 to 3, (1) Proteinase K, and (2) polypeptides having reverse transcriptase activity and polypeptides having DNA polymerase activity, or polypeptides having reverse transcriptase activity and DNA polymerase activity A kit that includes this.

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