Target sequence detection method and target sequence detection device

Abstract

To provide nucleic acid examination methods for quick and simple detection of mutations in genes and devices for use in the examination.SOLUTION: Provided herein is a method for detecting a target sequence comprising the steps of: obtaining a template nucleic acid comprising the target sequence; obtaining an amplification product to which a dye that binds to double stranded DNA is binding, where the amplification product is produced by conducting nucleic acid amplification using a primer set to amplify a certain region including the target sequence; obtaining a melting curve of the amplification product; and detecting the target sequence on the basis of the melting curve. Also provided is a device for use in the method.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present specification relates to a method for detecting a target sequence and an apparatus for detecting a target sequence. [Background technology]

[0002] Viruses survive by repeatedly mutating to improve their adaptability to their environment. Viral mutations cause changes in resistance to the host's immune system and interactions with host cells, which in turn affect the target organism, cell type, and infectivity, severity, and vaccine effectiveness. Therefore, quickly identifying viral mutations and understanding their genetic diversity and the spread of mutant strains is crucial for infectious disease control. Furthermore, detecting genetic mutations in organisms, not just viruses, is useful in various industries and medical fields.

[0003] Methods for detecting such mutations include genome sequencing by the Sanger method, PCR-RFLP, the TaqMan probe method, and high resolution melting analysis (HRM). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2021-90404 Summary of the Invention [Problem to be solved by the invention]

[0005] However, genome sequencing and PCR-RFLP pose cost issues when it comes to surveillance of infectious diseases, etc. Furthermore, although the TaqMan probe method can be performed using the commonly used PCR method, the probes are expensive, and probe design is not easy, making it difficult to respond to mutations quickly.

[0006] In addition, the HRM method detects mutations by melting PCR products at high resolution and detecting differences in the melting curve profile. However, it has been difficult to design appropriate primers for regions where mutations are concentrated in viruses.

[0007] Therefore, until now, even if a specific mutation in a virus has been identified, detecting that mutation using PCR has been time-consuming and costly.

[0008] The present specification provides a nucleic acid testing method that can quickly and easily detect mutations in genes, and an apparatus for such testing. [Means for solving the problem]

[0009] The present inventors focused on melting curves and found that similar mutations can be easily, quickly, and accurately distinguished using melting curves. Furthermore, they found that multiple mutations can also be rapidly detected using melting curves. Based on these findings, the present specification provides the following means.

[0010] [1] A method for detecting a target sequence, comprising: Obtaining a template nucleic acid containing a target sequence; a step of performing a nucleic acid amplification reaction on a predetermined region of the template nucleic acid containing the target sequence using a primer set to obtain an amplification product to which a dye that binds to double-stranded DNA is bound; obtaining a melting curve of the amplification product; detecting the presence or absence of the target sequence based on the melting curve; A method comprising: [2] The method according to [1], wherein the template nucleic acid is obtained as an amplification product by performing a nucleic acid amplification reaction on a nucleic acid in a biological sample. [3] Furthermore, obtaining a control template nucleic acid that is identical to the template nucleic acid except that it contains a control target sequence that is a control of the target sequence; the step of obtaining an amplification product is a step of performing a nucleic acid amplification reaction on a mixture containing one or more of the control template nucleic acids in a ratio relative to the template nucleic acid in the presence of the primer set and the binding dye, to obtain a mixture containing the amplification product and an amplification product derived from the control template nucleic acid; the melting curve acquisition step is a step of acquiring a melting curve for the mixture, The method according to [1] or [2], wherein the detection step is a step of detecting the target sequence based on a melting curve of the mixture. [4] The method according to any one of [1] to [3], wherein the target sequence is a mutation in a viral nucleic acid. [5] The target sequence is a mutation in a variant of the novel coronavirus (SARS-CoV-2), The method according to [4], wherein a primer set specified by at least one selected from the group consisting of the following base sequence pairs (a) to (g) is used to obtain the amplification product: (a) SEQ ID NO: 2 and SEQ ID NO: 3 (b) SEQ ID NO: 4 and SEQ ID NO: 5 (c) SEQ ID NO: 6 and SEQ ID NO: 7 (d) SEQ ID NO: 8 and SEQ ID NO: 9 (e) SEQ ID NO: 10 and SEQ ID NO: 11 (f) SEQ ID NO: 12 and SEQ ID NO: 13 (g) SEQ ID NO: 14 and SEQ ID NO: 15 [6] The method according to [5], wherein the template nucleic acid is obtained by a nucleic acid amplification reaction using a primer set specified by at least one selected from the group consisting of the following base sequence pairs (h) to (n) for each of the selected primer sets specified by at least one selected from the group consisting of the base sequence pairs (a) to (h): (h) SEQ ID NO: 16 and SEQ ID NO: 17 (i) SEQ ID NO: 18 and SEQ ID NO: 19 (j) SEQ ID NO: 20 and SEQ ID NO: 21 (k) SEQ ID NO: 22 and SEQ ID NO: 23 (l) SEQ ID NO: 24 and SEQ ID NO: 25 (m) SEQ ID NO: 26 and SEQ ID NO: 27 (n) SEQ ID NO: 28 and SEQ ID NO: 29 [7] A method for identifying mutations in virus mutants, comprising: obtaining two or more types of template nucleic acids containing two or more types of mutations of the virus mutant; performing a nucleic acid amplification reaction on the two or more types of template nucleic acids under first reaction conditions in the presence of two or more types of first primer sets and a dye that binds to double-stranded DNA, to obtain two or more types of amplification products that contain the two or more types of mutations and are bound to the binding dye; heating the two or more amplification products from a first temperature to a second temperature to obtain two or more melting curves; detecting the two or more mutations based on the two or more melting curves; Equipped with the nucleic acid amplification reaction is carried out under the first reaction conditions in two or more reaction fields specific to each of the two or more template nucleic acids to obtain the two or more amplification products, and the two or more amplification products in the two or more reaction fields are heated from the first temperature to a second temperature to obtain the two or more melting curves. [8] The mutation is a mutation in a mutant strain of the novel coronavirus (SARS-CoV-2), The identification method according to [7], wherein the first primer set is a primer set specified by at least one selected from the group consisting of the following base sequence pairs (a) to (g): (a) SEQ ID NO: 2 and SEQ ID NO: 3 (b) SEQ ID NO: 4 and SEQ ID NO: 5 (c) SEQ ID NO: 6 and SEQ ID NO: 7 (d) SEQ ID NO: 8 and SEQ ID NO: 9 (e) SEQ ID NO: 10 and SEQ ID NO: 11 (f) SEQ ID NO: 12 and SEQ ID NO: 13 (g) SEQ ID NO: 14 and SEQ ID NO: 15 [9] The color-classifying method according to [8], wherein the first primer set is a primer set specified by at least the base sequence pairs (b) to (e).

[10] The method of identifying nucleic acids in a biological sample according to [8] or [9], wherein a nucleic acid amplification reaction is carried out under second reaction conditions in the presence of two or more second primer sets to obtain one or more template nucleic acids.

[11] The mutation is a mutation in a mutant strain of the novel coronavirus (SARS-CoV-2), The first primer set is a primer set specified by at least one selected from the group consisting of the following base sequence pairs (a) to (g): The identification method according to

[10] , wherein the second primer set is a primer set specified by the following base sequence pairs (h) to (n), respectively, with respect to the first primer set specified by the base sequences (a) to (g): (h) SEQ ID NO: 16 and SEQ ID NO: 17 (i) SEQ ID NO: 18 and SEQ ID NO: 19 (j) SEQ ID NO: 20 and SEQ ID NO: 21 (k) SEQ ID NO: 22 and SEQ ID NO: 23 (l) SEQ ID NO: 24 and SEQ ID NO: 25 (m) SEQ ID NO: 26 and SEQ ID NO: 27 (n) SEQ ID NO: 28 and SEQ ID NO: 29

[12] A kit for identifying mutations in a virus mutant strain, comprising: The virus mutant is a mutant strain of the novel coronavirus (SARS-CoV-2), A kit comprising a primer set identified by at least one selected from the group consisting of the following base sequence pairs (a) to (g): (a) SEQ ID NO: 2 and SEQ ID NO: 3 (b) SEQ ID NO: 4 and SEQ ID NO: 5 (c) SEQ ID NO: 6 and SEQ ID NO: 7 (d) SEQ ID NO: 8 and SEQ ID NO: 9 (e) SEQ ID NO: 10 and SEQ ID NO: 11 (f) SEQ ID NO: 12 and SEQ ID NO: 13 (g) SEQ ID NO: 14 and SEQ ID NO: 15

[13] A device for detecting a target sequence, a sample mounting module capable of preparing multiple reaction fields; a liquid supply / drainage module that supplies one or more types of liquid to the plurality of reaction fields on the sample mounting module and / or discharges liquids from the plurality of reaction fields; First and second temperature control modules capable of controlling the temperature of the liquid in the plurality of reaction fields; a light source module capable of irradiating light into the plurality of reaction fields; a fluorescence detection module that detects fluorescence within the plurality of reaction fields; At least one control unit; Equipped with The at least one control unit an apparatus for performing a process of: activating the liquid supply / drainage module to prepare a reaction solution for nucleic acid amplification by adding, to the plurality of reaction fields, at least a first primer set that amplifies a predetermined region of a template nucleic acid including the target sequence; activating the first temperature control module to perform a nucleic acid amplification reaction under first reaction conditions in the plurality of reaction fields to obtain a plurality of amplification products to which a dye that binds to double-stranded DNA is bound; activating the second temperature control module, the light source module, and the fluorescence detection module to obtain a plurality of melting curves for the plurality of amplification products in the plurality of sample wells; and detecting the target sequence based on the plurality of melting curves.

[14] The at least one control unit is moreover, The apparatus according to

[13] , wherein the liquid supply / drainage module is operated to carry out a process of diluting nucleic acids in the biological samples in the plurality of reaction fields to a predetermined concentration in the nucleic acid amplification reaction solution.

[15] The at least one control unit is The apparatus according to

[13] or

[14] further comprises activating the liquid supply / drainage module to add a primer set for obtaining at least the template nucleic acid by a nucleic acid amplification reaction to the plurality of reaction fields on the sample-mounted module to prepare a reaction solution for template nucleic acid amplification, and activating the first temperature control module to carry out a nucleic acid amplification reaction in the plurality of reaction fields to obtain the template nucleic acid as an amplification product.

[16] A system for detecting a target sequence, comprising: a sample mounting module capable of preparing multiple reaction fields; a liquid supply / drainage module that supplies one or more types of liquid to the plurality of reaction fields on the sample mounting module and / or discharges liquid from the plurality of reaction fields; First and second temperature control modules capable of simultaneously controlling the temperatures of the plurality of reaction fields; a light source module capable of irradiating light into the plurality of reaction fields; a fluorescence detection module that detects fluorescence within the plurality of reaction fields; At least one control unit; Equipped with The at least one control unit a process of operating the liquid supply / drainage module to prepare a reaction solution for nucleic acid amplification by adding to the plurality of reaction fields at least a primer set that amplifies a predetermined region of a template nucleic acid containing the target sequence; operating the first temperature control module to carry out a nucleic acid amplification reaction in the plurality of reaction fields to obtain a plurality of amplification products to which a dye that binds to double-stranded DNA is bound; operating the second temperature control module, the light source module, and the fluorescence detection module to obtain a plurality of melting curves for the plurality of amplification products in the plurality of reaction fields; and detecting the target sequence based on the plurality of melting curves. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a diagram showing an example of the flow of a method for detecting a target sequence. [Figure 2A] FIG. 1 shows the cDNA of the spike protein of SARS-CoV-2 and the mutation sites. [Figure 2B] Another diagram showing the cDNA and mutation sites of the spike protein of SARS-CoV-2. [Figure 2C] Another diagram showing the cDNA and mutation sites of the spike protein of SARS-CoV-2. [Figure 3]FIG. 1 shows an example of a block diagram of a target sequence detection device. [Figure 4] FIG. 1 is a diagram showing an example of a flow of a process for detecting a target sequence using a detection device. [Figure 5] FIG. 1 shows melting curves obtained by HRM analysis in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0012] This specification relates to a method for detecting a target sequence, a method and kit for identifying a viral mutant strain, a target sequence detection device, a target sequence detection system, and the like. According to the target sequence detection method disclosed herein (hereinafter also referred to as the present method), a predetermined region of a template nucleic acid containing the target sequence is amplified using a primer set to obtain an amplification product to which a dye that binds to double-stranded DNA is bound, a melting curve of this amplification product is obtained, and the target sequence is detected based on the melting curve. This allows for easy detection of the presence or absence of a target sequence through quicker and simpler steps: a nucleic acid amplification reaction using primers and a dye that binds to the template nucleic acid, followed by acquisition of a melting curve. Furthermore, this method does not require the use of a probe for the target sequence, thereby eliminating the cost and time required for probe design and prototype testing. This enables rapid mass detection of mutations in viral infections and the like. Furthermore, this method allows for detection of such mutations even in the early stages of infection spread or shortly after mutations have occurred.

[0013] In this detection method, by designing a primer set so that the length of the amplified product is, for example, 60 bp or more and 150 bp or less, it is possible to detect the target sequence quickly and accurately.

[0014] Furthermore, for example, a template nucleic acid of 130 bp to 300 bp in length is obtained by a nucleic acid amplification reaction, and then a predetermined region of this template nucleic acid is amplified with a primer set to obtain an amplified product, thereby performing a two-step nucleic acid amplification reaction (nested PCR) to enhance the detection sensitivity of the target sequence.

[0015] This detection method is useful for detecting mutations of causative viruses of viral infections that require detection with a short lead time and short testing time.

[0016] The method and kit for identifying a virus mutant disclosed herein also enable rapid and easy identification of mutant strains with high accuracy. The target sequence detection device and system disclosed herein also enable rapid and easy detection of target sequences, similar to the detection method, and enable highly accurate detection of target sequences.

[0017] The techniques disclosed in this specification are useful in technical fields related to genes and living organisms, such as the medical, sanitation, environmental, agricultural, industrial, and research fields.

[0018] The method for detecting a target sequence disclosed in the present specification will be described in detail below.

[0019] In this specification, the term "test sample" is not particularly limited. The test sample may be any sample that may contain the target sequence to be detected. For example, the test sample may be a sample collected directly from nature or a living organism, a sample obtained by roughly purifying or purifying such a sample, a sample obtained by extracting DNA or RNA from such a sample, or a sample obtained by increasing the content of DNA or RNA in such a sample or by reverse transcribing the DNA or RNA by known nucleic acid amplification methods or the like.

[0020] Test samples include, for example, various biologically derived liquid samples such as blood, urine, and saliva, which are artificially collected or naturally isolated from various animals, including humans and livestock, as well as cell and tissue samples, and processed samples from these samples, such as those that have undergone nucleic acid extraction, amplification, and reverse transcription. Test samples also include various parts or forms of samples artificially collected or naturally isolated from plants or other organisms, including viruses. Test samples also include foods, beverages, processed products, and other items that may contain target nucleic acids.

[0021] Furthermore, in this specification, nucleic acids may refer to natural nucleic acids such as DNA and RNA possessed by organisms (including viruses; the same applies hereinafter), as well as artificially synthesized nucleic acids. Nucleic acids have bases in at least a portion thereof that form hydrogen bonds with the primers disclosed in this specification. DNA is not particularly limited and may be naturally occurring DNA that can be possessed by organisms, as well as DNA artificially introduced into organisms. For example, it includes genomic DNA as well as DNA on vectors such as plasmids. RNA is also not particularly limited and may be naturally occurring RNA that can be possessed by organisms, as well as RNA introduced into organisms from outside. Examples of RNA include mRNA, tRNA, rRNA, ncRNA such as siRNA and miRNA, ribozymes, and double-stranded RNA.

[0022] Examples of target sequences include sequences related to specific genes in the organism, mutations and other sequences related to genes that characterize the individual organism, sequences that characterize diseases, pathogens, or mutant strains in the animal or plant, mutations in the sequence, and sequences with other characteristics. More specifically, examples include single-nucleotide mutations (e.g., substitution mutations) such as single nucleotide polymorphisms (SNPs), deletions, and insertion mutations. The term "target sequence" refers to only a base sequence that differs from the base sequence of a control to be compared. Therefore, for example, when a target sequence consists of only one single-base substitution, the target sequence can be a single base. Also, for example, when a target sequence contains two or more single-base substitutions, the target sequence can be a continuous base sequence (approximately 2mers to 30mers) whose ends are defined by single-base substitutions.

[0023] Examples of organisms to be tested include pathogens of infectious diseases and other diseases, and pathogens of infectious diseases and other diseases for which genetic mutations need to be determined. Examples of such pathogens include influenza virus, coronavirus, adenovirus, enterohemorrhagic Escherichia coli, Shigella, Salmonella, Campylobacter jejuni and coli, Vibrio parahaemolyticus, human parvovirus, hepatitis B and C viruses, and human immunodeficiency virus.

[0024] (Method for detecting target sequence) The detection method disclosed herein includes the steps of obtaining a template nucleic acid containing a target sequence, performing a nucleic acid amplification reaction using a primer set that amplifies a predetermined region of the template nucleic acid containing the target sequence to obtain an amplification product to which a dye that binds to double-stranded DNA is bound, obtaining a melting curve of the amplification product, and detecting the target sequence based on the melting curve. An example of a flow chart corresponding to this detection method is shown in Figure 1.

[0025] <Step of Obtaining Template Nucleic Acid Containing a Target Sequence> As shown in Figure 1, this detection method includes a step of obtaining a template nucleic acid. This template nucleic acid is a nucleic acid that serves as a template for an amplification product for obtaining a melting curve in a subsequent step. The template nucleic acid may be, for example, DNA or RNA contained in a sample isolated or collected from a living organism. Alternatively, for example, the template nucleic acid may be obtained as a nucleic acid present in a sample isolated or collected from a living organism.

[0026] For example, when the sample is saliva and the target sequence is a portion of the RNA of an RNA virus, the sample can be used as the template nucleic acid as is. It is preferable to remove RNase and the like by conventional methods. When nucleic acid in a biological sample is used as the template nucleic acid as is, it is preferable to inactivate or remove DNAase, viruses, and the like in advance, as appropriate, by conventional methods. Furthermore, if necessary, a step of removing contaminants such as proteins that may interfere with the subsequent nucleic acid amplification reaction may be performed by conventional methods.

[0027] In some cases, the amount of nucleic acid that can serve as template nucleic acid in a biological sample is small. For example, if the amount of template nucleic acid is 10 7This is the case when the number of copies is less than 100. In such cases, even if a nucleic acid amplification reaction is performed using such a nucleic acid as a template nucleic acid, a sufficient amount of amplification product may not be obtained in a certain cycle, which may reduce the accuracy of melting curve analysis. Furthermore, fluctuations in the amount of amplification product may reduce the accuracy of melting curve analysis. In such cases, the nucleic acid in the test sample can be amplified by a nucleic acid amplification reaction, and the amplification product can be obtained as a template nucleic acid. In this case, the nucleic acid amplification reaction is preferably performed by conventional PCR to obtain double-stranded DNA when the target sequence is DNA, or by reverse transcription (RT) PCR to obtain double-stranded DNA as a template nucleic acid when the target sequence is RNA. The RT-PCR may be two-step RT-PCR, which involves performing reverse transcription and conventional PCR in two steps, or one-step RT-PCR, which involves performing both in one step. When amplifying nucleic acids in a sample by a nucleic acid amplification reaction, even if the nucleic acid in the biological sample is single-stranded RNA, it ultimately becomes double-stranded DNA.

[0028] The length of the template nucleic acid is not particularly limited, but can be, for example, a DNA double-stranded chain having a length of 130 bp to 300 bp, or, for example, a DNA double-stranded chain having a length of 140 bp to 300 bp. This length ensures PCR efficiency and reliable production of true amplification products when amplification products are produced. The region to be used as the template nucleic acid can be determined by first determining a suitable region of 60 bp to 150 bp as the range of the amplification product described below, and then designing a specific primer set that includes this region and is 130 bp to 300 bp long.

[0029] The procedures and conditions for obtaining a template nucleic acid by a nucleic acid amplification reaction are well known to those skilled in the art, and can be appropriately carried out by those skilled in the art according to the protocols of commercially available PCR reagents.

[0030] <Step of Obtaining Amplification Products Bound to the Binding Dye> The present detection method may next comprise a step of obtaining an amplification product obtained by a nucleic acid amplification reaction using a primer set that amplifies a predetermined region of the template nucleic acid containing the target sequence, the amplification product having a dye that binds to double-stranded DNA bound thereto. In this step, an amplification product for obtaining a melting curve (hereinafter also referred to as a test amplification product) is obtained from the template nucleic acid of the test sample.

[0031] This step can be carried out, for example, after obtaining a biological sample such as saliva as a template nucleic acid in the previous step, and inactivating or removing biological enzymes that affect nucleic acid amplification reactions, such as DNAase and RNAase, or inactivating pathogens.

[0032] The step of obtaining an amplification product can be carried out by PCR, which can be carried out by a conventionally known PCR method.

[0033] (primer set) The primer set is designed to detect the presence or absence of a target sequence from the melting curve of the obtained test amplification product (hereinafter also referred to as the test melting curve). Detecting the presence or absence of a target sequence means, for example, when the target sequence is a mutation in a virus mutant strain, distinguishing the mutation in the target sequence from the corresponding sequence of the mutation in the wild type or other mutant strains (the homologous sequence without the mutation). The target sequence can be detected by distinguishing the melting curve of an amplification product with a mutation from the melting curve of an amplification product with a homologous sequence without the mutation. Melting curves and melting curve analysis will be explained later.

[0034] The primer set is designed so that the amplified product is 60 bp or more and 150 bp or less. The length of the amplified product is, for example, 70 bp or more and 150 bp or less. The length of each primer in the primer set is not particularly limited, but is, for example, 10 mer or more and 30 mer or less, or, for example, 14 mer or more and 28 mer or less, or, for example, 16 mer or more and 28 mer or less.

[0035] Furthermore, depending on the length of the intended amplification product, when the amplification product is, for example, 80 mer or longer, depending on the length of the primer, the target sequence may be, for example, 1 to 30 nucleotides from the 3' end of the forward primer to the 3' end, or may be, for example, 1 to 25 nucleotides or within, for example, 1 to 20 nucleotides. Furthermore, the target sequence may be, for example, 1 to 30 nucleotides from the 5' end of the reverse primer to the 5' end, or may be, for example, 1 to 25 nucleotides or within, for example, 1 to 20 nucleotides. The presence of a target sequence on the 5' or 3' end of the amplification product may result in greater differences in melting curves.

[0036] As will be explained later, the primer set can be designed so that the melting temperature (Tm) of the amplification product is set to, for example, 70°C or higher and 80°C or lower.

[0037] (binding dye) The binding dye may be any compound that intercalates between base pairs in a DNA double strand and emits fluorescence upon irradiation with excitation light. Binding dyes used in this manner are well known in real-time PCR. For example, SYBR Green (registered trademark), EVA Green (registered trademark), and SYTO 9 (registered trademark) can be used.

[0038] For example, to identify mutations in a mutant strain of SARS-CoV-2, known as the novel coronavirus, a specific primer set corresponding to the target site can be used to obtain an amplification product. Below are shown the mutations (shown as amino acid mutations) as the target sequence in this detection method and the primer sets for obtaining amplification products to detect the mutations.

[0039] Target sequences include, for example, mutations in the following target sites of the spike protein of SARS-CoV-2, such as amino acid deletion mutations at H69 / V70, amino acid substitution mutations at K417 such as K417N and K417T, amino acid substitution mutations such as L452R at L452, amino acid substitution mutations such as T478K at T478 and E484Q and E484K at E484, amino acid substitution mutations such as N501Y at N501, amino acid substitution mutations such as D614G at D614, and amino acid substitution mutations such as P681H at P681.

[0040] The wild-type and mutant base sequences for these mutations are shown in the table below. Note that the amino acid positions in the table below are positions in the amino acid sequence of the SARS-CoV-2 spike protein (NCBI Reference Sequence YP_009724390.1), and the base positions are positions in the genomic sequence of the SARS-CoV-2 spike protein (NCBI Reference Sequence: NC_045512.2, 21563-25384).

[0041] In addition, Figure 2 shows the mutation site in the base sequence (SEQ ID NO: 1) of the DNA encoding the spike protein of SARS-CoV-2 (NC_045512.2), along with the forward and reverse primers.

[0042] [Table 1]

[0043] The primer sets for obtaining amplification products for detecting these target sequences are shown below, and are also clearly shown in Figure 2.

[0044] [Table 2]

[0045] In addition, when detecting mutations at each of the above mutation sites in SARS-CoV-2, the following primer sets can be preferably used for PCR amplification after reverse transcription to obtain template nucleic acids from SARS-CoV-2 RNA.

[0046] [Table 3]

[0047] The procedures and conditions for obtaining an amplification product to which a binding dye is bound by a nucleic acid amplification reaction are well known to those skilled in the art. For example, those skilled in the art can appropriately perform the process according to the protocols of commercially available PCR reagents or real-time PCR reagents. These reagents are commercially available as reagents for melting analysis, and this process can also be performed as part of a series of processes including a subsequent melting analysis to obtain a melting curve. Alternatively, an amplification product to which a binding dye is bound may be obtained by performing PCR in the absence of the binding dye to obtain amplified nucleic acid, followed by adding the binding dye and performing heat treatment to incorporate the amplified nucleic acid into double-stranded DNA.

[0048] (Reference template nucleic acid and control template nucleic acid) When detecting a target sequence, a template nucleic acid containing the target sequence can be obtained as a reference template nucleic acid. The reference template nucleic acid is a so-called positive control. Such a reference template nucleic acid is usually prepared when designing a primer set for obtaining an amplification product. A melting curve for the reference template nucleic acid can be obtained under the same conditions as for the template nucleic acid, and used to determine the presence or absence of the target sequence.

[0049] Furthermore, when detecting a target sequence, a control template nucleic acid can be obtained that is identical to the template nucleic acid except for containing a control sequence that is a negative control and does not contain the target sequence. The control template nucleic acid is a so-called negative control. For example, when the target sequence is a mutation of a virus mutant, the control sequence is an unmutated wild-type or conventional base sequence corresponding to the target sequence. By obtaining a control template nucleic acid containing such a control sequence and obtaining a melting curve for the control template nucleic acid under the same conditions as for the template nucleic acid, the accuracy of determining the presence or absence of the target sequence can be improved.

[0050] When detecting a target sequence, depending on the target sequence, the difference in melting curves between the amplification products derived from the reference template nucleic acid and the control template nucleic acid may be small. In this case, the accuracy of determining whether the test template nucleic acid contains the target sequence may be reduced. In such cases, a template nucleic acid mixture containing one or more control template nucleic acids in a ratio relative to the test template nucleic acid is used instead of the test template nucleic acid, and a predetermined primer set is used with this mixture to finally obtain a mixture of amplification products to which the binding dye is bound. This mixture contains amplification products based on the test template nucleic acid and amplification products based on the control template nucleic acid.

[0051] A solution curve is then obtained for this mixture of amplification products, which facilitates determining whether the test template nucleic acid contains the target sequence, i.e., whether it matches the reference template nucleic acid, or whether it matches the control template nucleic acid.

[0052] This is based on the finding that even if the melting curves of an amplification product derived from a reference template nucleic acid and an amplification product derived from a control template nucleic acid appear similar at first glance, the melting curve of an amplification product of a mixture in which a control template is added to the reference template nucleic acid at a predetermined ratio can exhibit a large difference from the melting curve of an amplification product derived from the reference template nucleic acid alone.

[0053] Therefore, when the melting curves derived from the reference template nucleic acid and the control template nucleic acid are similar, it is preferable to use a mixture prepared by adding one or more control template nucleic acids to the test template nucleic acid in a ratio of the test template nucleic acid to the control template nucleic acid, as described above. In this case, it is preferable to use a mixture prepared by adding the control template nucleic acid to the reference template nucleic acid in the same ratio, as the reference template nucleic acid. It is also preferable to obtain in advance the melting curve of the amplification product of such a substituted reference template nucleic acid.

[0054] The amount of control template nucleic acid added relative to the test template nucleic acid or reference template nucleic acid is not particularly limited, and can be, for example, in the range of 10% by mass to 60% by mass, where the test template nucleic acid or reference template nucleic acid is taken as 100% by mass. The ratio may be one specific type or two or more different types. Usually, when added at a fixed ratio, a melting curve that is clearly different from the melting curve derived from the control template nucleic acid is obtained.

[0055] The acquisition of such a reference template nucleic acid and a control template nucleic acid and the acquisition of a melting curve may be performed each time a target sequence detection method is performed on a test sample, or may be stored in the memory of a target sequence detection device described below, or may be stored as a database in a data server provided via a network.

[0056] <Step of obtaining a melting curve of the amplification product> In this step, a melting curve is obtained for the test amplification product obtained in the previous step. Here, melting curve or melting curve analysis refers to a technique that applies a temperature gradient in the temperature range required to melt a DNA double strand, and measures the melting temperature (Tm) of the DNA double strand from the change in signal intensity that accompanies melting of the DNA double strand, or analyzes SNPs, heterozygotes and homozygotes, methylation patterns, etc. from changes in the shape and peaks (inflection points of signal intensity) of the melting curve. Such techniques are well known to those skilled in the art in the technical fields disclosed herein.

[0057] Melting curve analysis can be performed at any (temperature) resolution. For example, the temperature can be continuously increased at a rate within the range of about 0.1°C / sec to about 1°C / sec. Alternatively, the temperature can be increased at a rate within the range of about 0.01°C / sec to about 0.1°C / sec, or at a rate within the range of about 1°C / sec to about 3°C / sec. Melting curve analysis can be performed by increasing the temperature within a range of, for example, 75°C to 95°C, 70°C to 90°C, or 72°C to 82°C, or 70°C to 80°C, generally within a range of, for example, 5°C to 20°C, or 10°C to 15°C. Melting analysis can be performed in a chamber or as part of a continuous flow, for example, within a channel in a microdevice.

[0058] Data for obtaining a melting curve are generated, for example, by measuring the characteristics detected when the temperature of a nucleic acid is increased for a selected period of time. A binding dye, a common DNA double-strand intercalator, exhibits fluorescence upon irradiation with excitation light when intercalated into a DNA double strand, and the signal intensity decreases as the DNA double strand denatures. Therefore, in this detection method, a decrease in signal, which is an indicator of the denaturation (melting) of the DNA double strand, is measured over a certain period of time accompanied by a temperature change. A melting curve can be obtained from data obtained by melting analysis using techniques well known in the art. For example, the time resolution for acquiring signal intensity can be, for example, 10 to 30 times per second, or 100 to 300 times, or 1000 to 3000 times per second. A melting curve obtained by detecting signal intensity with such a time resolution is generally referred to as a high-resolution melting curve. To obtain a high-resolution melting curve, for example, a binding dye (e.g., a fluorescent dye such as the SYTO9 system) that binds to every single base can be preferably used. In this specification, the melting curve may be presented in the form of a melting curve normalized by the signal intensity at the beginning of the temperature rise, or in the form of a so-called temperature change curve, such as a change melting peak based on the temperature change and the amount of change in signal intensity, which are suitable forms for presentation on a medium such as a display. However, the melting curve in this specification is not limited to these, and refers to a collection of time-series data of temperature change and signal change obtained by melting curve analysis.

[0059] Those skilled in the art can appropriately perform melting curve analysis, for example, using a commercially available device capable of performing such analysis, following the protocol provided by the manufacturer, etc. Typically, the amplification product to which the binding dye is bound, obtained in the previous step, is preheated to a temperature lower than the temperature range for melting analysis in the tube or well in which the amplification product was obtained, and then the temperature is controlled as planned, and excitation light is irradiated to measure fluorescence.

[0060] When a melting curve is obtained for the above-described reference template nucleic acid and / or control template nucleic acid, the melting curve is obtained under the same conditions as those for the test sample.

[0061] <Step of detecting the target sequence based on the melting curve> In this step, the target sequence is detected based on the melting curve obtained for the test amplification product (hereinafter also referred to as the test melting curve). The presence of the target sequence can be confirmed, for example, when the melting curve obtained for the sample matches a predetermined reference melting curve for the target sequence. Furthermore, for example, the target sequence can also be detected based on a control melting curve obtained for a control template nucleic acid. As described above, single-base differences can be identified from differences in melting curve shape, etc. Therefore, for example, when the test melting curve for the sample does not match the control melting curve but matches the predetermined reference melting curve, the presence of the target sequence in the sample can be confirmed. Furthermore, for example, when the test melting curve for the test sample matches the control melting curve, the absence of the target sequence can be inferred. Furthermore, for example, when the test melting curve does not match either the reference melting curve or the control melting curve, the presence of other mutations in addition to the target sequence, or the absence of the target sequence or the presence of other mutations, can be inferred.

[0062] The detection of the target sequence, i.e., the match and mismatch of the melting curves, may be performed by a computer or the like equipped with at least one control unit. In this case, the melting curve of the sample as well as the reference melting curve and the control melting curve are all stored in a memory or the like and compared in the target sequence detection process by the control unit to determine the presence or absence of the target sequence.

[0063] As described above, this method enables rapid and low-cost detection of target sequences and is useful for mutation surveillance in viral infections and the like. This method can also be implemented in various embodiments. It can also be implemented as a method for identifying SARS-CoV-2 mutant strains. In this case, a primer set specified by one or more base pairs selected from the group consisting of the following base pairs (a) to (g) can be preferably used. Furthermore, a primer set specified by one or more base pairs selected from the group consisting of the base pairs (b) to (e) can be particularly preferably used. By using one or more primer sets selected from these primer sets, mutant strains can be efficiently screened. In addition, by using a primer set specified by one or more base pairs selected from the group consisting of the base pairs (a), (f), and (g), a wide range of mutant strains can be screened. In this identification method, one or more primer sets selected from (h) to (n) can be used to obtain template nucleic acids corresponding to the mutations to be detected.

[0064] (a) SEQ ID NO: 2 and SEQ ID NO: 3 (b) SEQ ID NO: 4 and SEQ ID NO: 5 (c) SEQ ID NO: 6 and SEQ ID NO: 7 (d) SEQ ID NO: 8 and SEQ ID NO: 9 (e) SEQ ID NO: 10 and SEQ ID NO: 11 (f) SEQ ID NO: 12 and SEQ ID NO: 13 (g) SEQ ID NO: 14 and SEQ ID NO: 15

[0065] (h) SEQ ID NO: 16 and SEQ ID NO: 17 (i) SEQ ID NO: 18 and SEQ ID NO: 19 (j) SEQ ID NO: 20 and SEQ ID NO: 21 (k) SEQ ID NO: 22 and SEQ ID NO: 23 (l) SEQ ID NO: 24 and SEQ ID NO: 25 (m) SEQ ID NO: 26 and SEQ ID NO: 27 (n) SEQ ID NO: 28 and SEQ ID NO: 29

[0066] Furthermore, a kit for identifying a mutant strain of SARS-CoV-2 can suitably use, for example, one or more primer sets selected from (a) to (n) corresponding to the mutation to be detected. For example, a primer set specified by one or more base pairs selected from the group consisting of base pairs (b) to (e) can be provided, or a primer set specified by one or more base pairs selected from the group consisting of base pairs (a), (f), and (g). Furthermore, for example, a primer set specified by one or more base pairs selected from the group consisting of base pairs (h) to (n) for obtaining template nucleic acid corresponding to the mutation to be detected can be provided.

[0067] <Methods for identifying mutations in virus mutant strains> The method for detecting a target sequence can also be implemented as a method for identifying mutations in virus mutants (hereinafter also referred to as the present identification method) as described below. In the present identification method described below, various aspects of the present detection method can be applied to the parts that are common to the present detection method described above.

[0068] This identification method can comprise the steps of obtaining a template nucleic acid containing a mutation of a virus mutant strain, performing a nucleic acid amplification reaction on the template nucleic acid under first reaction conditions in the presence of a first primer set and a double-stranded DNA-binding dye to obtain two or more amplification products containing the mutation and having the binding dye bound thereto, heating the amplification products from a first temperature to a second temperature to obtain solution curves, and detecting the mutation based on the melting curves. This identification method further comprises the steps of performing the nucleic acid amplification reaction under the first reaction conditions in a reaction field specific to the template nucleic acid to obtain the amplification products, and heating the amplification products in the reaction field from the first temperature to the second temperature to obtain the melting curves.

[0069] In this identification method, two or more template nucleic acids for two or more mutations may be obtained, two or more amplification products may be obtained using two or more first primer sets, and melting curves may be obtained for each of these amplification products in a specific reaction field. The number of mutations may be, for example, three or more, four or more, five or more, or six or more.

[0070] According to this identification method, mutations in virus mutant strains can be detected all at once by performing a nucleic acid amplification reaction for each template nucleic acid under first reaction conditions, and then obtaining a melting curve by heating the resulting amplification product from a first temperature to a second temperature.As a result, screening and monitoring of virus mutations can be performed efficiently and quickly, enabling appropriate measures to combat viral infections.

[0071] In order to carry out this discrimination method, it is useful to design a specific primer set that can obtain an amplification product containing the mutation by a nucleic acid amplification reaction under first reaction conditions for each template nucleic acid containing the mutation, and that can exhibit a melting curve that is distinguishable from the amplification product of a control template nucleic acid within a temperature range from a first temperature to a second temperature.

[0072] When designing such a primer set, for example, mutations in the nucleotide sequence of the virus mutant are first identified, and a primer set is designed to include the mutations, for example, as described above, taking into account melting curve analysis, so that the total length of the amplification product is 150 bp or less and the primer sequence does not contain any mutations. Furthermore, the primer set is designed to be amplifiable under the first reaction conditions. Note that the reaction conditions for nucleic acid amplification reactions are generally determined in advance based on the enzymes used, etc. While not particularly limited, typical reaction conditions include, for example, 30 to 40 cycles of thermal denaturation at approximately 90°C, priming at approximately 60°C, and DNA extension reaction at approximately 70°C. The reaction time for the nucleic acid amplification reaction is also not particularly limited, but is, for example, approximately 1 to 1.5 hours in total. Primer sets can be designed using common PCR primer design sites (e.g., Primer3) or software.

[0073] Furthermore, when designing a primer set, it is important to consider whether the amplification product of the control template nucleic acid and the amplification product of the mutant type can be distinguished by melting curve analysis within the range from the first temperature to the second temperature. The first temperature to the second temperature can be set within a range that includes two or more melting temperatures of two or more amplification products and that allows for the generation of unique melting curves. Typically, the starting temperature of the heating range described above is the first temperature, and the ending temperature is the second temperature. The heating rate is also as described above. The time required for melting curve analysis is not particularly limited, but is, for example, approximately 10 to 20 minutes overall, including processing.

[0074] Whether or not the primers are distinguishable by melting curve analysis can be confirmed, for example, using the Melting Curve Predictions Software available at uMELT (www.dna-utah.org). If it is determined by uMELT that the melting curves of the amplification products are distinguishable, the nucleotide sequences of the primer sets can be determined.

[0075] For mutant strains of SARS-CoV-2, the above-mentioned primer sets (a) to (g) can be used as the first primer set. In particular, primer sets (b) to (e) can be used.

[0076] It is known that A to T (T to A) substitutions in base sequences are difficult to distinguish by melting curve analysis, and overlapping melting curves may be obtained even with uMELT. In such cases, melting curve analysis using the spike method described above should be applied.

[0077] In this identification method, for example, nucleic acid amplification reactions performed by supplying two or more first primer sets for each of two or more template nucleic acids can be performed in separate reaction fields for each template nucleic acid and the specific first primer set used for that template nucleic acid, or, for example, simultaneously. That is, individual template nucleic acids, the first primer sets specific to the template nucleic acids, and the binding dye are mixed in a single reaction field, such as a single well or tube, and nucleic acid amplification reactions for all template nucleic acids are performed simultaneously under the first reaction conditions. This avoids the problem of reduced specificity of the amplification reaction due to interference between primer sets when two or more primer sets are used simultaneously. Such nucleic acid amplification reactions can be performed using a commercially available PCR device. Note that the reaction field referred to here corresponds to the individual wells or tubes on the sample stage of the target sequence detection device described below.

[0078] Similar to the detection method described above, to obtain two or more types of template nucleic acids, a nucleic acid amplification reaction may be performed on nucleic acids derived from a biological sample using a second primer set. For such nucleic acid amplification reactions, the second primer set can be designed according to the mutation to be identified. The second primer set is designed to obtain a larger amplification product than the amplification product obtained with the first primer set. For example, for mutant strains of SARS-CoV-2, the primer sets (h) to (n) described above can be used in conjunction with the first primer set.

[0079] The nucleic acid amplification reaction for obtaining such template nucleic acids can also be carried out in individual reaction fields depending on the intended mutation. The two or more template nucleic acids thus obtained are each supplied to a new reaction field, and a nucleic acid amplification reaction is carried out using the first primer set.

[0080] Furthermore, in this identification method, a melting curve is obtained for each amplification product obtained in each reaction field, for example, by performing melting curve analysis in which the amplification products are collectively heated from a first temperature to a second temperature. The melting curve thus obtained is compared with a reference melting curve, a control melting curve, or the like, to identify mutations. The melting curves obtained for the amplification products obtained in each reaction field can be obtained using a commercially available real-time PCR device or the like. The melting curve comparison can also be performed using this type of processing software using a computer.

[0081] As described above, in this identification method, multiple reaction fields, each producing a single type of amplification product, are prepared, a different type of amplification product is obtained in each reaction field, and a melting curve is individually obtained for each of the multiple amplification products obtained in the multiple reaction fields. This allows for efficient and rapid identification of two or more mutations. The nucleic acid amplification reaction and melting curve analysis generally take approximately 1 to 1.5 hours and 15 to 30 minutes, respectively, making it possible to identify multiple mutations in a total of approximately 1 to 2 hours. Even when performing a nucleic acid amplification reaction to obtain template nucleic acid, it generally takes approximately 2 to 3 hours to identify multiple mutations. When, for example, 24, 48, or 96 individual reaction fields are prepared, two or more, three or more, four or more, five or more, or six or more mutations can be efficiently and rapidly identified for nucleic acids in biological samples derived from multiple specimens (patients). Furthermore, this identification method also allows for the identification of mutant strains identified by a mutation profile based on the presence or absence of two or more mutations.

[0082] In the above description of the present identification method, one type of template nucleic acid has been described as containing one type of mutation. However, one type of template nucleic acid may contain two or three types of mutations, and even in such a template nucleic acid, it may be possible to identify the presence or absence of two or three types of mutations by appropriately selecting a primer set.

[0083] According to the present specification, the first primer set (a) to (g) and the second primer set (h) to (n) used in this identification method are provided individually and in combination as a kit for identifying mutations in mutant strains of SARS-CoV-2.

[0084] <Target sequence detection device> The target sequence detection device disclosed herein (hereinafter also referred to as the present device) can include a sample-mounted module capable of preparing multiple reaction fields; a liquid supply / drainage module that supplies one or more types of liquid to the multiple reaction fields on the sample-mounted module and / or drains liquids from the multiple reaction fields; first and second temperature control modules that can adjust the temperature of the liquids in the multiple reaction fields; a light source module that can irradiate the multiple reaction fields with light; a fluorescence detection module that detects fluorescence in the multiple reaction fields; and at least one control unit. This device can detect target sequences quickly and efficiently. The first temperature control module is configured to control the temperature in a nucleic acid amplification reaction, and the second temperature control module is configured to obtain a melting curve (melting curve analysis) of double-stranded DNA. This device can efficiently and accurately detect target sequences, such as viral mutations, by obtaining the amplification products of the nucleic acid amplification reaction and performing melting curve analysis of the amplification products.

[0085] This device is configured as an apparatus equipped with at least one control unit, and the target sequence detection process executed by multiple modules is carried out based on a series of processes by the control unit. An example of a block diagram of this device is shown in Figure 3.

[0086] (Control unit) The control unit provided in this device includes a processor such as a CPU, and is further equipped with a separate memory and input / output interface, and is configured as a so-called computer. The control unit is configured to be able to input and output signals to and from each module. The control unit can also be connected to an external device as appropriate via a known network, allowing input and output to and from the external device. Furthermore, the control unit is configured to be able to input and output data as appropriate from an input unit provided in this device. Furthermore, this device is equipped with a display or other display device as necessary, which can display the results of the control unit's determination of the presence or absence of the target sequence and the melting curve for each sample.

[0087] The control unit is capable of loading from memory and executing a program for executing the process of detecting the target sequence executed by the module of the device. The process of determining the presence or absence of the target sequence performed by the control unit involves comparing a melting curve obtained from the sample with a control melting curve or a reference melting curve pre-stored in memory, determining the presence or absence of the target sequence based on the agreement between the melting curve obtained from the sample and the reference melting curve, and determining the presence or absence of the control sequence based on the agreement between the melting curve obtained from the sample and the control melting curve.

[0088] Furthermore, when multiple template nucleic acids each containing multiple target sequences are prepared for the same sample, multiple amplification products are prepared, and multiple melting curves are obtained, the control unit can individually determine the presence or absence of each of these multiple target sequences.

[0089] The control unit can store in memory a combination of sample identification information, a melting curve, and determination information regarding the presence or absence of one or more target sequences, and output the information appropriately to a display device or external device, etc. In addition to the presence or absence of the target sequence, if appropriate determination conditions can be set and the presence of new mutations or other mutations can be estimated, such estimated information can also be stored in memory and output to a display device, etc.

[0090] (Sample mounting module) The sample mounting module is a module capable of preparing multiple reaction fields. For example, it can mount a rectangular plate (so-called well plate) with multiple reaction fields, which is commonly used in this type of device, or a sample stage with multiple reaction fields of various shapes. It also allows the sample stage to be moved or transported to other modules, such as a position suitable for supplying and discharging liquid using a liquid supply and discharge module or a position suitable for temperature control using a temperature control module. It may also be configured to mount multiple sample stages and move and hold them in appropriate positions in any order. The multiple reaction fields may be, for example, 24, 48, or 96, and the reaction fields are spaced apart from one another. The reaction fields are not particularly limited, but may be, for example, shallow recesses, tubular recesses, or recesses capable of accommodating tubes, tubes, or tubes, or tubes and openings capable of accommodating tubes.

[0091] (liquid supply / drainage module) A liquid supply / drainage module is a module that supplies one or more types of liquid to multiple reaction fields and / or drains liquid from the multiple reaction fields. The liquid supply / drainage module is not particularly limited, but may include, for example, a group of tapered supply / drain tips for supplying liquid into the reaction fields and / or draining liquid from the reaction fields, piping connected to these, a liquid storage section for supplying the necessary liquid to the piping, and a drainage storage section for storing liquid drained from the reaction fields. The group of supply / drain tips and the group of piping may each be provided uniquely for each liquid storage section. Furthermore, this module includes a gas pressure adjustment mechanism in part of the piping for supplying and draining liquid, as well as cleaning. The gas pressure adjustment mechanism is also configured to adjust the amount of liquid supplied to or drained from the reaction fields in response to a signal from the control unit. Two or more types of liquid supply / drainage modules may be provided as needed.

[0092] The liquid reservoirs can be filled with reagents, diluents, washing solutions, etc. that are necessary for detecting the target sequence. The liquid reservoirs may be provided outside the device.

[0093] The liquid supply / drainage module is also configured to allow one or more test samples to be set so that the liquid test sample can be supplied into the reaction field. The test sample is configured to be set up in the liquid supply / drainage module in a state filled, for example, in a sample supply kit or the like that includes reaction fields such as multiple tubes and wells for the test sample.

[0094] (Temperature control module) The first and second temperature control modules are configured to be capable of simultaneously controlling the temperatures of multiple reaction spaces (raising, lowering, maintaining a constant temperature, etc.) and to be able to control the temperature of the liquid in the reaction spaces. The first temperature control module is configured as a module capable of carrying out, for example, a nucleic acid amplification reaction. A plurality of first temperature control modules may be provided in order to carry out nucleic acid amplification reactions in parallel.

[0095] The second temperature control module has a temperature control capability sufficient to enable melting analysis and high-resolution melting, and the temperature profile is set by a signal from the control unit. For example, the second temperature control module can continuously increase the temperature at a rate within the range of about 0.1°C / sec to about 1°C / sec. Alternatively, the temperature may be increased at a rate within the range of about 0.01°C / sec to about 0.1°C / sec, or at a rate within the range of about 1°C / sec to about 10°C / sec. (From the paragraph regarding temperature resolution in the method)

[0096] Such temperature control modules are well known in the art. Furthermore, multiple second temperature control modules can be provided to perform multiple melting curve acquisition processes in parallel. On the other hand, providing a single second temperature control module for multiple first temperature control modules enables melting curve analysis with good reproducibility. For example, based on the relationship between the time required for the nucleic acid amplification reaction performed by the first temperature control module and the time required for the melting curve acquisition performed by the second temperature control module, amplification products performed by, for example, four or more, five or more, six or more, seven or more, or eight first temperature control modules can be processed by a single second temperature control module.

[0097] (light source module) The light source module is a module that irradiates light into multiple reaction spaces. The light source may be any light source capable of irradiating light of a wavelength sufficient to excite the binding dye used for detecting the target sequence at the required illuminance, and such modules are well known in the art.

[0098] (Fluorescence detection module) The fluorescence detection module is a module that detects fluorescence in multiple reaction fields. It is designed to detect the fluorescence intensity from each well using a sensor and output it as a signal to a control unit. The control unit processes the input signal as needed and stores it in memory or the like as fluorescence intensity. Such modules, such as two-dimensional photodiode arrays (image sensors), are well known in the art. In this device, particularly when considering application to melting curve analysis, the time resolution of detecting fluorescence intensity from the reaction fields can be set to, for example, 10 to 30 times per second, or, for example, 100 to 300 times, or 1,000 to 3,000 times per second.

[0099] The light source module and the fluorescence detection module can be provided independently, or the light source module and the fluorescence detection module can be provided in association with the first temperature control module that performs the nucleic acid amplification reaction. In this way, the amount of amplification product obtained in the nucleic acid amplification reaction using the first temperature control module can be monitored by detecting the intensity of fluorescence generated by the binding dye.

[0100] Furthermore, the light source module and the fluorescence detection module (especially having a detection time resolution suitable for melting curve analysis) may be provided in association with the second temperature control module, thereby enabling melting curve analysis using the second temperature control module to be performed with high accuracy.

[0101] In this device, the control unit is configured to appropriately operate the necessary modules to carry out the individual steps of the above-described method for detecting a target sequence.

[0102] The process of preparing a template nucleic acid containing a target sequence involves, for example, filling multiple sample wells of the device with the template nucleic acid. When nucleic acids from biological samples are used as test samples, these samples are supplied to a sample stage equipped with a well plate having multiple reaction fields, outside of the device. The test samples may be supplied to the well plate via the device's liquid supply / drainage module. If these biological samples require heating to inactivate enzymes such as DNAase or viruses, the operator may load the sample stage onto the sample loading module, and the control unit may then output a signal to the temperature control module to perform an appropriate heating or dilution process, thereby carrying out the heating process. If the biological test sample requires dilution, the control unit may output a signal to the liquid supply / drainage module to dilute the sample at a preset dilution ratio.

[0103] For example, when the control unit executes a process for obtaining a template nucleic acid in a test sample as an amplification product by a nucleic acid amplification reaction, the control unit operates the liquid supply / discharge module to add at least a primer set for obtaining the template nucleic acid by a nucleic acid amplification reaction to multiple reaction fields on the sample-mounted module to prepare a reaction solution for template nucleic acid amplification, and operates the temperature control module to perform the nucleic acid amplification reaction in the multiple reaction fields to obtain the template nucleic acid as an amplification product. Note that the sample-mounted module may be moved as appropriate to execute these processes for multiple sets of sample stages.

[0104] Furthermore, for example, a signal can be output to the liquid supply / drainage module to perform a process of diluting a template nucleic acid-containing liquid containing a template nucleic acid, such as a biological sample, to an appropriate predetermined concentration in a nucleic acid amplification reaction solution to obtain an amplification product bound with a binding dye.

[0105] For example, when the control unit executes a process for obtaining an amplification product bound with a binding dye, the control unit operates the liquid supply / discharge module to prepare a reaction solution for nucleic acid amplification by adding at least a primer set that amplifies a predetermined region of a template nucleic acid, and if necessary, a binding dye for double-stranded DNA to multiple reaction fields, and operates the temperature control module to carry out a nucleic acid amplification reaction in the multiple reaction fields and obtain multiple amplification products bound with the binding dye.

[0106] Next, an example of the process executed by the control unit to detect a target sequence based on multiple melting curves is described with reference to Figure 4. Below, we will explain the previously described identification method, i.e., the mutant screening process in which the presence or absence of six mutations (N501Y, E484K, K417N / T, L452R, D614G, P681H / R) is determined as a target sequence in saliva collected from 16 patients infected with SARS-CoV-2, and mutant strains are identified from the mutation profile. Also, below, we will explain the case where the device has a control melting curve and a reference melting curve for the target sequence to be detected stored in the memory of the computer that serves as the control unit.

[0107] First, an experimenter heats saliva samples collected from 16 individuals at 95°C for 5 minutes and roughly purifies the RNA using a known column or other suitable method to prepare test samples. These 16 test samples are loaded into a sample supply kit, which is then set up in a designated location on the sample loading module. The experimenter then inputs instructions into the system to perform a nucleic acid amplification reaction using six second primer sets to obtain six template nucleic acids corresponding to the six mutations previously prepared in the liquid supply / drainage module, a nucleic acid amplification reaction using a first primer set to obtain amplification products for melting curves, and a process to identify the six mutations for each sample from the 16 individuals through melting curve analysis.

[0108] For each test sample, the liquid supply / discharge module injects a predetermined amount (e.g., 2 μl) into a total of six wells in a 2 x 3 grid of a well plate (an example of a sample stage) with a total of 96 wells arranged in an 8 x 12 grid on the sample mounting module, thereby injecting test samples from 16 people into all 96 wells.

[0109] Furthermore, the liquid supply / discharge module supplies, to six wells per sample, one set each of reverse transcriptase and DNA polymerase for the reverse transcription reaction to amplify DNA from RNA and the nucleic acid amplification reaction, and six types of second primer sets for obtaining template nucleic acids each containing six types of mutations, as well as various substrates (dNTPs) for the nucleic acid amplification reaction, buffer solution and water required for the nucleic acid amplification reaction, etc., to a predetermined amount (e.g., 20 μl).

[0110] Next, the first temperature control module performs the heat treatment required for the reverse transcription reaction and nucleic acid amplification reaction in each of these wells. As a result of the heat treatment, template nucleic acids containing one mutation are generated in each of the six wells per sample, resulting in a total of six types of template nucleic acids. The duration of this heat treatment is not particularly limited, but is generally about one hour.

[0111] After cooling for a certain period of time following completion of the amplification reaction, the liquid supply / discharge module aspirates a predetermined amount of the reaction solution containing one type of template nucleic acid from each well, performs two-stage dilution using another two sets of well plates, and supplies the resulting reaction solution diluted to a predetermined dilution factor, for example, 1000-fold, to a new well plate in the same position as the original. Note that when two-stage dilution of the nucleic acid amplification reaction solution and supplying the diluted solution to the new well plate, the sample loading module appropriately loads the well plate or transports it to a predetermined position.

[0112] Next, the liquid supply / discharge module supplies six types of first primer sets corresponding to six types of mutations to the template nucleic acid in the six wells per sample, and adds a binding dye that binds to the DNA double strand, a heat-resistant DNA polymerase, dNTPs, a buffer solution, water, etc. to a predetermined volume (e.g., 20 μl).

[0113] Next, the first temperature control module performs the heat treatment required for the nucleic acid amplification reaction in each of these wells. Through the heat treatment, a DNA double-stranded test product derived from the test sample is generated in each well. The DNA double-stranded test product is the same size as or smaller than the template nucleic acid containing six types of mutations, and the mutations can be identified by subsequent melting curve analysis. The duration of this heat treatment is not particularly limited, but is generally about 1 to 1.5 hours. During this heat treatment, the light source module irradiates each well with an excitation light source of a wavelength suitable for the binding dye as needed, and the fluorescence detection module detects the fluorescence emitted from each well and processes it as a signal. As the amount of the DNA double-stranded amplification product increases with the nucleic acid amplification reaction, the increase in the amount of fluorescence due to the binding dye can be monitored.

[0114] Thereafter, the well plate in which the test amplification products have been produced in the wells is transferred to the second temperature control module by the sample loading module.

[0115] The second temperature control module heats the reaction solution containing the test amplification product in the well plate, for example, to 95°C to strongly thermally denature the test amplification product, then cools it, for example, to 40°C, and after preheating it to 65°C, increases the temperature from 75°C to 95°C at a heating rate of 1°C / sec to perform melting curve analysis to obtain a melting curve.

[0116] The light source module irradiates each well with excitation light corresponding to the bound dye, and the fluorescence detection module detects the predetermined fluorescence at a resolution of, for example, 25 times per second and outputs the signal to the control unit.

[0117] The control unit uses known melting curve analysis software to determine the presence or absence of six mutations for each of 16 test samples obtained from 16 patients. For each sample, the six test melting curves for the six mutations are compared with the reference melting curves corresponding to each mutation, and the presence or absence of each mutation in the sample is determined based on the agreement between the test melting curve and the reference melting curve (the melting curve of a positive control containing the mutation). If necessary, a more accurate determination can be made by referring to the control melting curve (the melting curve of a negative control containing no mutation) corresponding to each mutation.

[0118] Furthermore, the control unit can obtain mutation profile information, which indicates the profile of each of the six mutations for each of the 16 samples, from the results of determining the presence or absence of each of the six mutations for each of the 16 samples.The control unit can also compare the obtained mutation profile information with already known mutation profiles for various mutant strains to identify which of the known mutant strains the mutant strains detected from the 16 samples correspond to.

[0119] According to the above embodiment of the present device, test samples derived from specimens are supplied to wells arranged in a 2 x 3 grid in a 96-well (8 x 12) plate, which reduces the probability that specimens derived from different patients will be placed in adjacent grids, thereby reducing the risk of specimens being contaminated with each other.

[0120] In the above embodiment, a single well plate is used, but if the sample mounting module can mount multiple well plates, the mutation detection process can be performed for additional samples. When multiple well plates are used, a different plate can be used for each patient, and a different plate can be used for each target sequence.

[0121] Furthermore, in the above embodiment, the template nucleic acid is also obtained using the second primer set, thereby improving the accuracy of melting curve analysis of the amplified products using the first primer set. That is, the generation of amplified products due to mispriming by the first primer set is suppressed or avoided, and a sufficient amount of the intended amplified product is generated by any first primer set, thereby improving the accuracy of the melting curve analysis.

[0122] Furthermore, in the above embodiment, the device is described as having a sample mounting module, a liquid supply / drainage module, first and second temperature control modules, a light source module, and a fluorescence detection module, each of which is provided individually. However, the device may be configured to have multiple modules of one or more types, or two or more sets of all of these modules. Depending on the time each module is dedicated to a process, the content of the mutation identification process, and the number of mutations to be detected, multiple modules or sets may be provided as appropriate. Such a configuration enables efficient target sequence detection and mutation identification for a large number of samples.

[0123] In the above embodiment, the device is described as a device for detecting a target sequence, but it can also be implemented as a device for identifying mutations in virus mutants. Furthermore, the module and at least one control unit in the device are described as constituting a single device, but these do not necessarily need to constitute a single device and may be connected via a known network. Therefore, instead of the device, the device can also be implemented as a system for detecting a target sequence and a system for identifying mutations in virus mutants.

[0124] This specification also discloses the following aspects of the device and its use. In the following aspects, the detection of possible mutations in a virus mutant strain is exemplified, and a well plate is used as an example of a sample stage equipped with multiple reaction fields.

[0125] <Aspect 1: Device for detecting multiple mutations (target sequences) on a single-well plate and its use> The device of this embodiment may include a liquid supply / drainage module, a sample loading module, one or more first temperature control modules, a light source module and a fluorescence detection module attached to each of the first temperature control modules, a single second temperature control module, a light source module and a fluorescence detection module attached to the second temperature control module, a data comparison unit, a judgment unit, a control unit equipped with a memory for storing a reference test curve and mutant strain information, etc., and a display.

[0126] When using the device of embodiment 1, the test sample is subjected to virus inactivation, for example, by heat treatment, and then a predetermined amount is supplied by the liquid supply / discharge module into the wells of the reaction field prepared in the sample-mounted module. The test sample containing the template nucleic acid is supplied to each well, for example, according to the number of mutations to be detected. The test sample is diluted as necessary, and reagents necessary for the nucleic acid amplification reaction are prepared in advance at predetermined concentrations and supplied to each well. Furthermore, a first primer set corresponding to the mutation is also supplied to the well in which the test sample is prepared. A binding dye is also added at a predetermined concentration.

[0127] The sample loading module then transfers the well plate, prepared for nucleic acid amplification reaction, to the first temperature control module, which then carries out the nucleic acid amplification reaction according to a predetermined temperature control schedule.

[0128] When multiple test samples are processed simultaneously, the above process is performed for each test sample. Multiple test samples can be processed on a single well plate. When nucleic acid amplification reactions are performed on different test samples on a single well plate, mutual contamination can be suppressed or avoided by adopting the above-mentioned arrangement.

[0129] In addition, when multiple test samples are processed simultaneously, multiple well plates are used as necessary. When multiple well plates are used, for example, multiple first temperature control modules are used to perform nucleic acid amplification reactions sequentially or in parallel so that the reactions are completed within a fixed time interval. Here, the fixed time interval is based on the processing time for melting curve analysis in the second temperature control module to be subsequently performed.

[0130] The sample-carrying module then transfers the well plate, which has completed the nucleic acid amplification reaction in the first temperature-regulating module, to the second temperature-regulating module, which, for example, preheats the well plate to 65°C for 1 second under strict temperature control, then heats it to 95°C at a ramp rate of 1°C / second. The excitation light emitted from the excitation light source in the light source module causes the bound dye to emit fluorescence, which passes through the color filter of the fluorescence detection module. The fluorescence intensity is then detected by an optical sensor in the fluorescence detection module. The detection signal is stored in memory, and a test melting curve corresponding to the number of mutations to be detected is obtained.

[0131] The sample loading module ejects the well plate for which melting curve acquisition has been completed from the second temperature control module. Furthermore, if there is a well plate for which nucleic acid amplification reaction has been completed, the sample loading module supplies a new well plate to the second temperature control module and similarly acquires another melting curve to be tested.

[0132] In this device, a melting curve obtained from an amplification product of a test sample to detect a specific mutation is compared with a reference melting curve for the specific mutation stored in memory in advance to determine whether the test sample contains the mutation. If the specific mutation to be detected is present in the test sample, the test melting curve obtained specifically for the mutation and the reference melting curve prepared in advance for the mutation will match. If the mutation is not present in the test sample, the melting curves will not match. The determination unit included in the control unit can have built-in algorithms and databases regarding matches and mismatches between the test melting curve and the reference melting curve. The determination unit can determine whether a mismatch in the melting curves is due to test variation or a mutation, and stores the obtained determination results in memory. The determination unit performs the above-mentioned mutation detection process for the mutation to be detected and stores each determination result in memory.

[0133] The determination unit can further compare the mutation profile (information on the presence or absence of the mutation to be detected) of the nucleic acid in the test sample obtained in this manner with the mutation profiles of multiple known mutant strains pre-stored in memory, and determine which of the known mutant strains the virus from which the nucleic acid in the test sample is derived corresponds to.

[0134] In the device of this embodiment, even when multiple different well plates are used, a single second temperature control module is used to sequentially process the multiple well plates to obtain melting curves, thereby avoiding variations that may arise from multiple second temperature modules and enabling highly accurate mutation detection. The device of this embodiment is capable of both processing a large number of test samples and detecting mutations with high accuracy.

[0135] The device of this embodiment and its use are useful when detecting only a limited number of mutations in a large amount of test sample, such as multiple mutations that characterize a specific mutant strain.

[0136] In the device and use of this embodiment, the nucleic acid amplification reaction, which requires a long processing time, and the acquisition of melting curves, for which accurate temperature control is important, are performed simultaneously and in parallel for multiple primer sets, and judgment is made possible, thereby enabling identification of mutant strains specified by a combination of multiple mutations with high accuracy and in a short time.

[0137] The device of this embodiment and its use are useful, for example, in detecting multiple mutations in a test sample extracted from a population of test samples derived from virus-positive patients to identify a mutation profile, such as multiple mutations that a specific mutant strain may possess.

[0138] Furthermore, when the apparatus of this embodiment is equipped with multiple first temperature control modules, the following efficient use becomes possible. For example, if the nucleic acid amplification reaction takes 1 hour and the melting curve acquisition using the second temperature control module takes 15 minutes, the total time required for these processes for the test sample is roughly 1 hour and 15 minutes (135 minutes). To process multiple well plates using a single second temperature control module in continuous operation, five first temperature control modules are provided, based on the approximate value of 5 obtained by dividing the total required time by the time required to acquire the melting curve. This allows the nucleic acid amplification reaction to be started sequentially every 15 minutes for each of the five well plates in parallel, allowing the melting curve acquisition using the second temperature control module to be efficiently performed continuously for each of the five well plates, dramatically shortening the overall required time.

[0139] A suitable number of the first temperature joules is appropriately set based on various conditions such as the time required for the nucleic acid amplification reaction, the conditions for obtaining a melting curve, and the number of mutations (target sequences) to be detected and identified.

[0140] <Aspect 2: Device for detecting multiple mutations (target sequences) on a single-well plate and its use> The device of this embodiment has the same configuration as the device of Embodiment 1. The device of this embodiment is used in the same way as in Embodiment 1, except that a plurality of template nucleic acids is obtained from the test sample prior to obtaining a plurality of amplification products for detecting a plurality of mutations in the test sample.

[0141] In this embodiment, prior to carrying out a nucleic acid amplification reaction using the first primer set, various reagents for the nucleic acid amplification reaction and a second primer set in place of the first primer set corresponding to the mutation are first supplied to the wells of a well plate prepared in the sample-mounted module by the liquid supply / discharge module, and then a test sample is supplied to these wells.The well plate is then transferred by the sample-mounted module to the first temperature control module, and a nucleic acid amplification reaction is carried out by the first temperature control module to obtain template nucleic acids for each mutation.

[0142] Thereafter, the nucleic acid amplification reaction solution in the wells is diluted, for example, 1000-fold, as necessary, by the liquid supply / discharge module, and supplied to a new well plate prepared with a first primer set and various amplification reaction reagents corresponding to the mutation. Thereafter, as in embodiment 1, a nucleic acid amplification reaction is carried out to obtain multiple template nucleic acids for each mutation in the wells, and multiple melting curves are obtained by the second temperature control module to determine the presence or absence of mutations.

[0143] In the device of this embodiment, the nucleic acid amplification reaction is performed in two stages, which increases the total time required to detect mutations in a test sample. Therefore, when processing a large amount of test samples, such as using multiple well plates, it is more advantageous to provide the device of this embodiment with multiple first temperature control modules. The number of first temperature control modules is appropriately set based on various conditions, such as the time required for the nucleic acid amplification reaction, the conditions for obtaining a melting curve, and the number of mutations (target sequences) to be detected and identified, as described in embodiment 1.

[0144] <Aspect 3: Device for detecting a single mutation (target sequence) on a single-well plate and its use> The device of this embodiment is specified as the device of Embodiment 1, but including a plurality of first temperature regulation modules. The use of the device of this embodiment is the same as that of Embodiment 1, except that well plates corresponding to a plurality of mutations to be detected are prepared, a well plate is prepared for each mutation, i.e., for each first primer set, a nucleic acid amplification reaction is performed for each well plate using the first temperature regulation module, and a test melting curve is obtained for each of the obtained plurality of well plates using a single second temperature regulation module, a light source module, and a fluorescence detection module.

[0145] More specifically, for example, when multiple test samples are present, one mutation in the multiple test samples is detected in one well plate, and another mutation is detected in another well plate.

[0146] Because the apparatus of this embodiment includes multiple second temperature control modules, nucleic acid amplification reactions using multiple well plates can be performed sequentially or in parallel, as needed, so that the nucleic acid amplification reactions are completed at regular time intervals that take into account the acquisition of melting curves in the subsequent stage. This allows for efficient nucleic acid amplification reactions with long processing times. Furthermore, like the apparatus of embodiment 1, the apparatus of this embodiment uses a single second temperature control module, thereby reducing or avoiding variability due to the second temperature module, enabling highly accurate mutation detection. This allows for both the processing of multiple test samples and highly accurate mutation detection. Furthermore, the apparatus of this embodiment can also set different nucleic acid amplification reaction conditions for each different mutation, i.e., for each different first primer set. This allows the nucleic acid amplification reaction to be optimized for each primer set, suppressing nonspecific amplification and ensuring stable amplification product acquisition.

[0147] The device of this embodiment and its use are useful, for example, when detecting only a limited number of mutations in a large number of test samples, including multiple mutations that characterize a specific mutant strain.

[0148] The above describes methods and devices for detecting target sequences, such as mutations in viruses, but these are merely examples and do not limit the scope of the claims. The claimed technology includes various modifications and variations of the specific examples exemplified above. The technical elements described in this specification or drawings exhibit technical utility alone or in various combinations, and are not limited to the combinations described in the claims at the time of filing. Furthermore, the technology exemplified in this specification or drawings simultaneously achieves multiple objectives, and achieving any one of these objectives is itself technically useful. [Example]

[0149] Examples will be described below as specific examples to more specifically explain the disclosure of this specification. The following examples are intended to illustrate the disclosure of this specification, but are not intended to limit the scope thereof. [Example]

[0150] (Detection of nine mutations of the novel coronavirus (SARS-CoV-2) - Evaluation using an RNA positive control) In this example, melting curve analysis was performed using a primer set designed to target mutations in the novel coronavirus nucleotide sequence, using RNA prepared as a positive control. The following shows the nine mutations at the seven targeted mutation sites and the seven primer sets for the seven mutation sites.

[0151] [Table 4]

[0152] [Table 5]

[0153] In this example, to obtain positive control RNA, DNA fragments with the same sequence were first prepared by artificial gene synthesis. Both wild-type DNA fragments without mutations and mutant DNA fragments with mutations were prepared. Each DNA fragment was 400 to 500 bp in length. The base sequence was obtained from NCBI reference resquence: NC_045512.2 and GISAID (www.gisid.org). The single-base substitution mutations corresponding to the mutations shown in the table above are shown below. The base positions are based on reference resquence: NC_045512.2. To obtain RNA, the DNA fragments contained a T7 promoter sequence (5'-TAATACGACTCACTATAGG-3') upstream.

[0154] From the synthesized DNA fragments, various positive control RNAs corresponding to the control template nucleic acid and the reference template nucleic acid were prepared by in vitro T7 transcription.

[0155] In some cases, it may be difficult to distinguish between wild-type and mutant base sequences, such as when a base sequence is substituted with, for example, A to T (T to A). In such cases, a spike method is performed in which wild-type RNA or DNA is added. As an example, the mutant positive control RNA shown in Table 4, which contains a mutation at N501 and a mutation at E484, was externally added with the wild-type positive control at a ratio of 50% by mass relative to 100% by mass of the mutant positive control, to create mutant positive controls (N501Y, E484K, E484Q).

[0156] For each of these RNAs, RT-PCR was performed under the following conditions using the primer sets listed in Table 5. Melting curve analysis was performed using the LightCycler 96 System (Roche Diagnostics) as the HRM analyzer and the accompanying Gene Scanning Software (Roche Diagnostics). The analysis results are shown in Figure 5.

[0157] [PCR reaction mixture] RNA solution (RNA concentration 10 6 copies / mL) 2 μL HRM master mix 10 μL (MeltDoctor HRM Master Mix,Thermo Fisher Scientific) Reverse transcriptase 0.2 μL (SuperScript IV One-Step RTPCR, Thermo Fisher Scientific) Forward primer (10 μM) 0.8 μL Reverse primer (10 μM) 0.8 μL Appropriate amount of water Total 20 μL

[0158] [PCR and HRM conditions] Reverse transcription: 10 minutes at 52°C Heat denaturation: 98°C for 10 min PCR (40 cycles): 95°C for 10 seconds, 60°C for 30 seconds, 72°C for 30 seconds Heat denaturation: 95℃ for 60 seconds Cooling: 40°C for 60 seconds Preheat: 65°C for 1 second HRM: 75℃~95℃, temperature rise at 1℃ / sec with 25 acquisitions Then cooling

[0159] Figure 5 shows the melting peak curves obtained by melting curve analysis. As shown in Figure 5, the DNA double strands, which were nucleic acid amplification products obtained from the wild-type (conventional) and mutant-type RNA positive controls for each mutation, were all distinguishable from each other. Furthermore, for K417 and E484, two mutation forms were contained in a single template RNA, but they were also clearly distinguishable.

[0160] Furthermore, N501Y and E484Q each exhibited melting curves that were extremely difficult to distinguish from the wild type (conventional type). However, by adding 50% wild-type RNA positive control exogenously, it was found that they exhibited melting curves that were significantly different from the wild-type positive control, demonstrating that these mutations can be easily distinguished.

[0161] From the above, it was found that the primer set used in this example can identify a total of nine types of mutations in the new coronavirus. [Example]

[0162] (Detection of nine mutations of the novel coronavirus (SARS-CoV-2) in saliva from infected individuals) Samples were collected from patients in accordance with the "Guidelines for Novel Coronavirus Infection (COVID-19) Pathogen Testing," and the collected saliva was heat-treated at 95°C for 5 minutes. The amount of virus contained in COVID-19-positive samples varies depending on the patient. Since some samples with low viral loads are difficult to distinguish in Example 1, we confirmed whether mutations could be reliably detected by amplifying the viral RNA contained in the samples using reverse transcription-PCR.

[0163] The primer set used in the nucleic acid amplification reaction to obtain template nucleic acid (double-stranded DNA) from nucleic acid (RNA) in the sample is shown below. The reaction solution and PCR conditions were as follows:

[0164] [Table 6]

[0165] [Reaction solution for template nucleic acid] Sample 2 μL PCR enzyme 0.4 μL (KOD Fx neo, manufactured by Toyobo Co., Ltd.) PCR Buffer 10 μL (Buffer (2x Buffer for KOD FX Neo, manufactured by Toyobo Co., Ltd.) 2 mM dNTPs 4 μL 0.3 μL of each primer (10 μM) Reverse transcriptase 0.2 μL (SuperScript IV One-Step RTPCR, Thermo Fisher Scientific) Appropriate amount of water Total 20 μL

[0166] [PCR conditions for template nucleic acid] Reverse transcription: 10 minutes at 52°C, 4 minutes at 94°C PCR (30 cycles): 98°C for 10 seconds, 60°C for 30 seconds, 68°C for 30 seconds, cooling

[0167] The reaction solution containing the template nucleic acid was diluted 1000-fold with water, and the following PCR reaction and HRM analysis were carried out. The same primer set as in Example 1 was used. As a control, a sample collected from a patient was used as is, and the two types of RNA fragment solutions prepared in Example 1 were used as positive controls.

[0168] In order to detect mutations at N501 and E484 in the specimen, template nucleic acid PCR was performed on the wild-type positive control in the same manner as for the specimen to obtain wild-type template DNA. The wild-type template DNA was diluted 1000-fold with water to obtain a solution, which was then externally added at 50% by mass relative to 100% by mass of the template nucleic acid from the specimen to form a diluted reaction solution, which was then subjected to the following PCR and HRM.

[0169] [PCR reaction mixture] Diluted reaction solution 2 μL HRM master mix 10 μL (SuperScript IV One-Step RTPCR, Thermo Fisher Scientific) Forward primer (10 μM) 0.8 μL Reverse primer (10 μM) 0.8 μL Appropriate amount of water Total 20 μL

[0170] [PCR and HRM conditions] Heat denaturation: 98℃ for 10 minutes PCR (40 cycles): 95°C for 10 seconds, 60°C for 30 seconds, 72°C for 30 seconds Heat denaturation: 95℃ for 60 seconds Cooling: 40°C for 60 seconds Preheat: 65°C for 1 second HRM: 75℃~95℃, temperature rise at 1℃ / sec with 25 acquisitions Then cooling

[0171] Melting curve analysis was performed using the LightCycler 96 System (Roche Diagnostics) as the HRM analyzer and the accompanying Gene Scanning Software (Roche Diagnostics). For samples using template nucleic acid amplified from the specimen, melting peak curves were obtained that clearly distinguished the presence or absence of nine types of mutations. In contrast, no clear melting peak curves were obtained for the control sample, in which the specimen was used as is. These results demonstrate that by increasing the amount of template nucleic acid to a certain level, mutations can be reliably and accurately identified in a larger number of specimens. [Sequence List Free Text]

[0172] SEQ ID NOs: 2 to 29: Primers

Claims

1. A method for detecting a target sequence, comprising: obtaining a template nucleic acid containing the target sequence; a step of performing a nucleic acid amplification reaction on a predetermined region of the template nucleic acid containing the target sequence using a primer set to obtain an amplification product of 60 bp to 150 bp bound to a dye that binds to double-stranded DNA; obtaining a melting curve of the amplification product; detecting the presence or absence of the target sequence based on the melting curve; Equipped with the step of obtaining a template nucleic acid is a step of performing a nucleic acid amplification reaction on a nucleic acid in a biological sample to obtain an amplification product of 130 bp or more and 300 bp or less as the template nucleic acid; moreover, obtaining a control template nucleic acid that is identical to the template nucleic acid except that it contains a control target sequence that is a control of the target sequence, in the same step as obtaining the template nucleic acid; the step of obtaining an amplification product is a step of performing a nucleic acid amplification reaction on a mixture containing one or more of the control template nucleic acids in a ratio relative to the template nucleic acid in the presence of the primer set and the binding dye, to obtain a mixture containing the amplification product and an amplification product derived from the control template nucleic acid; the melting curve acquisition step is a step of acquiring a melting curve for the mixture, A detection method, wherein the detecting step is a step of detecting the target sequence based on a melting curve of the mixture.

2. 10. The method of claim 1, wherein the target sequence is a mutation in a viral nucleic acid.

3. the target sequence is a mutation in a variant of the novel coronavirus (SARS-CoV-2), The method according to claim 2, wherein a primer set specified by at least one selected from the group consisting of the following base sequence pairs (a) to (g) is used to obtain the amplification product: (a) SEQ ID NO: 2 and SEQ ID NO: 3 (b) SEQ ID NO: 4 and SEQ ID NO: 5 (c) SEQ ID NO: 6 and SEQ ID NO: 7 (d) SEQ ID NO: 8 and SEQ ID NO: 9 (e) SEQ ID NO: 10 and SEQ ID NO: 11 (f) SEQ ID NO: 12 and SEQ ID NO: 13 (g) SEQ ID NO: 14 and SEQ ID NO: 15

4. The method described in claim 3, wherein the template nucleic acid is obtained by a nucleic acid amplification reaction using a primer set identified by at least one selected from the group consisting of the following base sequence pairs (h) to (n), which correspond to the selected base sequence pairs (a) to (g), respectively: (h) SEQ ID NO: 16 and SEQ ID NO: 17 (i) SEQ ID NO: 18 and SEQ ID NO: 19 (j) SEQ ID NO: 20 and SEQ ID NO: 21 (k) SEQ ID NO: 22 and SEQ ID NO: 23 (l) SEQ ID NO: 24 and SEQ ID NO: 25 (m) SEQ ID NO: 26 and SEQ ID NO: 27 (n) SEQ ID NO: 28 and SEQ ID NO: 29

5. A method for identifying mutations in virus mutants, comprising: obtaining two or more types of template nucleic acids each containing at least one type of mutation among the two or more types of mutations of the virus mutant; carrying out a nucleic acid amplification reaction under first reaction conditions in the presence of a first primer set for each of the two or more types of template nucleic acids and a dye that binds to double-stranded DNA, to obtain two or more types of amplification products of 60 bp to 150 bp, each of which is bound to the binding dye and contains at least one mutation from the two or more types of mutations; heating the two or more amplification products from a first temperature to a second temperature to obtain two or more melting curves; detecting the two or more mutations based on the two or more melting curves; Equipped with the step of obtaining two or more types of template nucleic acids is a step of supplying two or more types of second primer sets to nucleic acids in a biological sample, respectively, and performing a nucleic acid amplification reaction under second reaction conditions to obtain two or more types of amplification products having a length of 130 bp or more and 300 bp or less from the biological sample as the two or more types of template nucleic acids; the nucleic acid amplification reaction is carried out under the first reaction conditions in two or more reaction fields specific to each of the two or more types of template nucleic acids to obtain the two or more types of amplification products, and the two or more types of amplification products in the two or more reaction fields are heated from the first temperature to the second temperature to obtain the two or more types of melting curves.

6. The mutation is a mutation in a mutant strain of the novel coronavirus (SARS-CoV-2), The first primer set is a primer set specified by at least one selected from the group consisting of the following base sequence pairs (a) to (g): The method according to claim 5, wherein the second primer set is a primer set specified by at least one selected from the group consisting of the following base sequence pairs (h) to (n), which correspond to the selected base sequence pairs (a) to (g), respectively: (a) SEQ ID NO: 2 and SEQ ID NO: 3 (b) SEQ ID NO: 4 and SEQ ID NO: 5 (c) SEQ ID NO: 6 and SEQ ID NO: 7 (d) SEQ ID NO: 8 and SEQ ID NO: 9 (e) SEQ ID NO: 10 and SEQ ID NO: 11 (f) SEQ ID NO: 12 and SEQ ID NO: 13 (g) SEQ ID NO: 14 and SEQ ID NO: 15 (h) SEQ ID NO: 16 and SEQ ID NO: 17 (i) SEQ ID NO: 18 and SEQ ID NO: 19 (j) SEQ ID NO: 20 and SEQ ID NO: 21 (k) SEQ ID NO: 22 and SEQ ID NO: 23 (l) SEQ ID NO: 24 and SEQ ID NO: 25 (m) SEQ ID NO: 26 and SEQ ID NO: 27 (n) SEQ ID NO: 28 and SEQ ID NO: 29

7. A device for detecting a target sequence, comprising: a sample mounting module capable of preparing multiple reaction fields; a liquid supply / drainage module that supplies one or more types of liquid to the plurality of reaction fields on the sample mounting module and / or discharges liquids from the plurality of reaction fields; first and second temperature control modules capable of controlling the temperature of the liquid in the plurality of reaction fields; a light source module capable of irradiating light into the plurality of reaction fields; a fluorescence detection module that detects fluorescence within the plurality of reaction fields; At least one control unit; Equipped with The at least one control unit an apparatus for performing a process of: activating the liquid supply / drainage module to prepare a reaction solution for nucleic acid amplification by adding, to the plurality of reaction fields, at least a first primer set that amplifies a predetermined region of a template nucleic acid including the target sequence; activating the first temperature control module to perform a nucleic acid amplification reaction under first reaction conditions in the plurality of reaction fields to obtain a plurality of amplification products, the amplification products having a dye that binds to double-stranded DNA bound thereto; activating the second temperature control module, the light source module, and the fluorescence detection module to obtain a plurality of melting curves for the plurality of amplification products in the plurality of reaction fields; and detecting the target sequence based on the plurality of melting curves.

8. The at least one control unit moreover, The apparatus according to claim 7 , wherein the liquid supply / drainage module is operated to carry out a process of diluting nucleic acids in the biological sample in the plurality of reaction fields so that the nucleic acid in the nucleic acid amplification reaction solution has a predetermined concentration.

9. The at least one control unit 9. The apparatus according to claim 7 or 8, further comprising: an apparatus for performing a process of preparing a reaction solution for template nucleic acid amplification by operating the liquid supply / drainage module to add a primer set for obtaining at least the template nucleic acid by a nucleic acid amplification reaction to the plurality of reaction fields on the sample-mounting module; and an apparatus for performing a process of performing a nucleic acid amplification reaction in the plurality of reaction fields to obtain the template nucleic acid as an amplification product by operating the first temperature control module.

10. 1. A system for detecting a target sequence, comprising: a sample mounting module capable of preparing multiple reaction fields; a liquid supply / drainage module that supplies one or more types of liquid to the plurality of reaction fields on the sample mounting module and / or discharges liquids from the plurality of reaction fields; First and second temperature control modules capable of simultaneously controlling the temperatures of the plurality of reaction sites; a light source module capable of irradiating light into the plurality of reaction fields; a fluorescence detection module that detects fluorescence within the plurality of reaction fields; At least one control unit; Equipped with The at least one control unit a process of operating the liquid supply / drainage module to prepare a reaction solution for nucleic acid amplification by adding to the plurality of reaction fields a primer set that amplifies at least a predetermined region of a template nucleic acid including the target sequence; operating the first temperature control module to perform a nucleic acid amplification reaction in the plurality of reaction fields to obtain a plurality of amplification products to which a dye that binds to double-stranded DNA is bound; operating the second temperature control module, the light source module, and the fluorescence detection module to obtain a plurality of melting curves for the plurality of amplification products in the plurality of reaction fields; and detecting the target sequence based on the plurality of melting curves.

11. The system described in claim 10, used to detect mutations in viral mutant strains.

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