Oligonucleotides that specifically detect target nucleic acid and uses thereof

Abstract

To provide means for specifically detecting a target nucleic acid.SOLUTION: There is disclosed a method for detecting a target nucleic acid comprising a step of amplifying a target nucleic acid using at least one set of primer pair and a step of detecting the target nucleic acid by melting curve analysis using a fluorolabeled probe that specifically binds to the target nucleic acid amplified in the amplification step, where the probe comprises a base sequence D defined as follows: a base sequence A is a sequence of the target nucleic acid; a base sequence B is a base sequence of a non-target nucleic acid corresponding to the base sequence A; base sequence C is a partial sequence of the base sequence A having one or more bases different from the base sequence B; the base sequence D is complementary to the sequence C and further has a mismatch base at a position corresponding to one to five bases common between the base sequence A and the base sequence B.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oligonucleotide for specifically detecting a target nucleic acid contained in a specimen sample. The present invention further relates to a method for specifically detecting a target nucleic acid contained in a specimen sample using the oligonucleotide, and a reagent and a kit for use in the method. [Background technology]

[0002] Many infectious diseases have very similar clinical symptoms, making it difficult to distinguish them. In addition to the very similar clinical symptoms, many closely related bacteria and viruses have similar genetic information. For example, closely related species that belong to the same taxonomic genus but are different species often have genes with similar functions and sequences. These may also be present in the same specimen, making it extremely difficult to accurately distinguish between such closely related species.

[0003] However, in order to accurately distinguish between these and provide appropriate treatment, it is clinically very important to specifically detect the target nucleic acid. Therefore, various testing methods, including the PCR method, have been developed to specifically distinguish between these.

[0004] In genetic testing such as PCR, examples of situations in which it is necessary to specifically detect target nucleic acids include the sexually transmitted disease Neisseria gonorrhea and the meningitis-causing Neisseria meningitidis. These may be detected from the same specimen, such as a throat specimen, but the genetic information of the two bacteria is partially similar, making it difficult to distinguish them. Other sexually transmitted diseases such as Chlamydia trachomatis and Chlamydia suis are also difficult to distinguish for the same reason, and there is a possibility of misdiagnosis.

[0005] Misdiagnosis of these infectious diseases leads to incorrect diagnosis, which prevents prompt and appropriate treatment. As a result, not only will treatment be delayed, but there is also concern that the infection may spread to those around you. In such cases, if highly specific differentiation of infectious diseases with similar genetic information becomes possible, it will not only enable early treatment, but also prevent infection to those around you.

[0006] To date, widely used methods for specifically detecting a target infection include a method that targets genes that are specific only to the causative microorganism of the target infection, and a method that uses BNA clamp technology to prevent false positives (Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 6242336 Summary of the Invention [Problem to be solved by the invention]

[0008] However, in some cases, it is difficult to target a gene that is specific to only the causative microorganism of the target infectious disease, such as when detecting a single base mutation in a specific region present in the target nucleic acid, or when detecting a region that exists in multiple locations per genome for the purpose of highly sensitive detection.

[0009] Furthermore, when the BNA clamp technique is used as in Patent Document 1, there are problems such as the time required for preparing analytical reagents and increased costs for the reagents.

[0010] The present invention has been made in view of the above problems in the conventional technology. That is, a main object of the present invention is to provide a new method for specifically detecting a target nucleic acid. [Means for solving the problem]

[0011] As a result of intensive research, the present inventors have unexpectedly found that when a probe is used for detection by melting curve analysis, it is possible to specifically detect only the target nucleic acid by using a probe to which a specific mismatch base has been added. This makes it possible to specifically detect only the target nucleic acid, for example, even when non-target nucleic acids derived from different species of the same genus are present in the same specimen sample. The present invention was completed based on this finding and through further investigation.

[0012] Representative embodiments of the present invention are as follows. [Item 1] A method for detecting a target nucleic acid, comprising: A step of amplifying a target nucleic acid using at least one pair of primer sets; and a target nucleic acid detection step by melting curve analysis using a fluorescently labeled probe that specifically binds to the target nucleic acid amplified in the target nucleic acid amplification step; The method is characterized in that the probe includes a base sequence D complementary to a partial base sequence C of a target nucleic acid containing one or more bases that differ between a base sequence A of the target nucleic acid and a base sequence B of a non-target nucleic acid corresponding to base sequence A, in which mismatch bases exist at positions corresponding to 1 to 5 bases common between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid. [Item 2] The method according to Item 1, wherein the 5'-terminal base or the 3'-terminal base of the probe corresponds to a different base position between base sequence A of a target nucleic acid and base sequence B of a non-target nucleic acid, and the 5'-terminal base or the 3'-terminal base is fluorescently labeled. [Item 3] The method according to item 1 or 2, wherein the base sequence C of the target nucleic acid contains two or more bases that differ between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid. [Item 4] The method according to any one of Items 1 to 3, wherein the base sequence C of the target nucleic acid contains 2 to 5 bases that differ between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid. [Item 5] The method according to any one of Items 1 to 4, wherein the target nucleic acid and the non-target nucleic acid are derived from different species in the same genus, and the method is for species-specific detection of the target nucleic acid. [Item 6] The method according to any one of Items 1 to 5, wherein the base sequence identity between the base sequence C of the target nucleic acid and the base sequence E of the non-target nucleic acid corresponding to the base sequence C is 60% or more and less than 100%. [Item 7] The method according to any one of Items 1 to 6, wherein the probe has a length of 15 bases or more. [Item 8] The method according to any one of Items 1 to 7, wherein the probe has a length of 15 to 25 bases. [Item 9] The method according to any one of Items 1 to 8, wherein at least one mismatched base in the probe is present in a region within 5 bases before and after the center of the probe. [Item 10] The method according to any one of Items 1 to 9, wherein the number of mismatched bases in the probe is 1 to 3. [Item 11] The method according to any one of Items 1 to 10, wherein the probe is labeled with a fluorescence quenching dye that is quenched by interaction with guanine. [Item 12] The method according to item 11, wherein the fluorescence quenching dye is at least one fluorescence quenching dye selected from the group consisting of fluorescein and derivatives thereof, rhodamine and derivatives thereof, and BODIPY and derivatives thereof. [Item 13] The method according to item 11 or 12, wherein the fluorescence quenching dye is at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). [Item 14] The method according to any one of items 1 to 13, wherein only one of the 5' end and the 3' end of the probe is labeled with a fluorescence quenching dye. [Item 15] The method according to any one of Items 1 to 14, wherein the labeled terminal base in the probe is cytosine. [Item 16] The method according to any one of Items 1 to 15, wherein 10,000 copies or more of non-target nucleic acid are present in the reaction solution in the target nucleic acid amplification step. [Item 17] The method according to any one of Items 1 to 16, wherein 10,000 copies or more of each of the target nucleic acid and non-target nucleic acid are present in the reaction solution in the target nucleic acid amplification step. [Item 18] The method according to any one of Items 1 to 17, wherein the target nucleic acid detection step is carried out by melting curve analysis at a detection temperature of 40 to 75°C. [Item 19] A fluorescently labeled probe for specifically detecting a target nucleic acid by melting curve analysis, characterized in that the probe contains a base sequence D complementary to a partial base sequence C of a target nucleic acid containing one or more bases that differ between a base sequence A of the target nucleic acid and a base sequence B of a non-target nucleic acid corresponding to base sequence A, in which mismatch bases exist at positions corresponding to 1 to 5 bases common between base sequence A of the target nucleic acid and base sequence B of the non-target nucleic acid. [Item 20] A fluorescently labeled probe according to Item 19, wherein the 5'-terminal base or the 3'-terminal base of the probe corresponds to a different base position between base sequence A of a target nucleic acid and base sequence B of a non-target nucleic acid, and the 5'-terminal base or the 3'-terminal base is fluorescently labeled. [Item 21] A fluorescently labeled probe according to Item 19 or 20, wherein the base sequence C of the target nucleic acid contains two or more bases which differ between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid. [Item 22] The fluorescently labeled probe according to any one of Items 19 to 21, wherein the base sequence C of the target nucleic acid contains 2 to 5 bases which differ between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid. [Item 23] A kit for specifically detecting a target nucleic acid, comprising the fluorescently labeled probe according to any one of Items 19 to 22. [Item 24] The kit described in Item 23, further comprising a primer set capable of amplifying a target nucleic acid region including a region to which the fluorescently labeled probe binds. Effect of the Invention

[0013] According to the present invention, it is possible to detect a target nucleic acid with a higher degree of specificity even when it is difficult to target a gene specific to only the causative microorganism of a target infectious disease, for example, when it is difficult to specifically identify a target nucleic acid by genetic testing, particularly when detecting a single base mutation in a specific region present in the target nucleic acid, or when detecting a region present in multiple locations per genome for the purpose of highly sensitive detection. The present invention also has a great advantage in that it can specifically measure a target nucleic acid while being simple and low-cost. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows the positional relationship between the target nucleic acid (SEQ ID NO: 10) and non-target nucleic acid (SEQ ID NO: 11) and the primers or probes (SEQ ID NOs: 1 to 3) used in Example 1. The double-underlined bases are different between the target nucleic acid and the non-target nucleic acid. The bases enclosed in boxes indicate the positions where mismatch bases were added to positions corresponding to bases common between the target nucleic acid and the non-target nucleic acid. [Diagram 2] 1 shows the results of Example 1. The figure shows the results of comparing detection when Neisseria gonorrhoeae was used as a target nucleic acid and Neisseria meningitidis was used as a non-target nucleic acid, and when a probe (SEQ ID NO: 3) containing a mismatched base was used. [Diagram 3] FIG. 1 shows the positional relationship between the target nucleic acid (SEQ ID NO: 12) and non-target nucleic acid (SEQ ID NO: 13) and the primers or probes (SEQ ID NOs: 4 to 6) used in Example 2. The double-underlined bases are different between the target nucleic acid and the non-target nucleic acid. The bases enclosed in boxes indicate the positions where mismatch bases were added to positions corresponding to bases common between the target nucleic acid and the non-target nucleic acid. [Figure 4] 1 shows the results of Example 2. The results show a comparison of detection when Chlamydia trachomatis was used as a target nucleic acid and Chlamydia suis was used as a non-target nucleic acid, and a probe (SEQ ID NO: 6) containing a mismatched base was used. [Diagram 5]FIG. 1 shows the positional relationship between the target nucleic acid (SEQ ID NO: 15) and non-target nucleic acid (SEQ ID NO: 16) and the primers or probes (SEQ ID NOs: 7 to 9) used in Example 3. The double-underlined bases are different between the target nucleic acid and the non-target nucleic acid. The bases enclosed in boxes indicate the positions where mismatch bases were added to positions corresponding to bases common between the target nucleic acid and the non-target nucleic acid. [Figure 6] 1 shows the results of Example 3. The figure shows the results of comparing detection when Bordetella pertussis was used as a target nucleic acid and Bordetella bronchiseptica was used as a non-target nucleic acid, and a probe (SEQ ID NO: 9) containing a mismatched base was used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in more detail below while showing embodiments of the present invention, but the present invention is not limited thereto. All non-patent documents and patent documents described in this specification are incorporated herein by reference. In addition, "~" in this specification means "more than or equal to, less than or equal to", for example, if "X~Y" is described in the specification, it means "more than or equal to X, less than or equal to Y". In addition, "and / or" in this specification means part or all.

[0016] In addition, in this specification, a nucleic acid primer may be referred to as an oligonucleotide primer or simply as a primer, and a nucleic acid probe may be referred to as an oligonucleotide probe or simply as a probe, and these are collectively referred to as oligonucleotides.

[0017] The present invention is based on the discovery that when detection is performed by melting curve analysis (also referred to as "melting curve analysis") using a probe, adding a specific mismatch base to the probe makes it possible to specifically detect only the target nucleic acid. Usually, when specifically detecting a target nucleic acid, a probe that is completely complementary to the base sequence of the target nucleic acid is often used for detection. One of the features of the present invention is that, completely unexpectedly, by intentionally adding a specific mismatch base to the probe, it is possible to suppress the detection of non-target nucleic acids and specifically detect only the target nucleic acid.

[0018] In one embodiment, the method of the present invention comprises at least the following steps: A step of amplifying a target nucleic acid using at least one pair of primer sets; and A target nucleic acid detection process by melting curve analysis using a fluorescently labeled probe that specifically binds to the target nucleic acid amplified in the target nucleic acid amplification process, characterized in that (i) the probe is based on a base sequence D that is complementary to a partial base sequence C of the target nucleic acid that contains one or more bases that differ between a base sequence A of the target nucleic acid and a base sequence B of a non-target nucleic acid corresponding to base sequence A, and (ii) the base sequence D contains a base sequence in which mismatch bases exist at positions corresponding to 1 to 5 bases that are common between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid.

[0019] That is, the method of the present invention is characterized by using a probe having both of the above characteristics (i) and (ii) in the melting curve analysis. By performing melting curve analysis using a probe having these two characteristics, even when the base sequences of the target nucleic acid and the non-target nucleic acid show high sequence identity (for example, when they show 60% or more base sequence identity), it becomes possible to specifically detect only the target nucleic acid. Therefore, according to the method of the present invention, when it is difficult to specifically identify the target nucleic acid by genetic testing, for example, when it is required to detect a single base mutation in a specific region present in the target nucleic acid, or when detecting a region present in multiple regions per genome (especially, the region of a multicopy gene whose sequence is similar in closely related species), it is possible to easily and inexpensively identify and detect the target nucleic acid and the non-target nucleic acid.

[0020] (Specific binding fluorescently labeled probe) The specific-binding fluorescently labeled probe used in the present invention is designed based on (i) a base sequence D that is complementary to a partial base sequence C of a target nucleic acid that contains one or more bases that differ between a base sequence A of the target nucleic acid and a base sequence B of a non-target nucleic acid.

[0021] In this specification, the term "target nucleic acid" refers to a nucleic acid intended to be detected, and the term "non-target nucleic acid" refers to a nucleic acid not desired to be detected in the detection of a target nucleic acid. For example, when the target nucleic acid is a nucleic acid derived from a certain infectious microorganism S1, the non-target nucleic acid may be a nucleic acid derived from an infectious microorganism S2 whose clinical symptoms are very similar to those of the infectious microorganism S1, and whose genetic information is also similar, and which may cause a false positive in a genetic test. Such a non-target nucleic acid may be, for example, a nucleic acid derived from an infectious microorganism S3 that belongs to the same genus as the infectious microorganism S1 in terms of biological taxonomy, but is a different species. From another perspective, such a non-target nucleic acid may be, for example, a nucleic acid that shows high sequence identity with the target nucleic acid and may cause a false positive. For example, the non-target nucleic acid may be a nucleic acid in which the identity of the base sequence between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid, and / or the identity of the base sequence between the base sequence C of the target nucleic acid and the base sequence E of the non-target nucleic acid corresponding to the base sequence C is, for example, 60% or more, further 65% or more, and further 70% or more. In this case, the upper limit of the base sequence identity is not particularly limited as long as it is less than 100%, but it can be, for example, 90% or less, preferably 85% or less. According to the present invention, even in the presence of non-target nucleic acids showing such high sequence identity, it is possible to specifically detect the target nucleic acid.

[0022] In a particular preferred embodiment, specific examples of combinations of target nucleic acids and non-target nucleic acids include a combination of the opA gene sequence (e.g., SEQ ID NO: 10) of Neisseria gonorrhoeae as the target nucleic acid and the opA gene sequence (e.g., SEQ ID NO: 11) of Neisseria meningitidis, which belongs to the same genus Neisseria, as the non-target nucleic acid; a combination of the ompA gene (e.g., SEQ ID NO: 12) of Chlamydia trachomatis as the target nucleic acid and the ompA gene of Chlamydia suis and / or the ompA gene of Chlamydia muridarum, which belongs to the same genus Chlamydia, as the non-target nucleic acid; Examples of the combination include, but are not limited to, a combination of the IS1002 gene of Bordetella pertussis (e.g., SEQ ID NO: 15) as the target nucleic acid and the IS1002 gene of Bordetella bronchiseptica (e.g., SEQ ID NO: 16) as the non-target nucleic acid, which belongs to the same Bordetella genus; a combination of the 23S rRNA gene sequence of Mycoplasma genitalium (e.g., SEQ ID NO: 17) as the target nucleic acid and the 23S rRNA gene sequence of Mycoplasma pneumoniae (e.g., SEQ ID NO: 18) as the non-target nucleic acid, which belongs to the same Mycoplasma genus, and the like. For example, when the non-target nucleic acid in the above specific example is used as the target nucleic acid, the target nucleic acid may be the non-target nucleic acid. These target nucleic acids and non-target nucleic acids may exist in the same specimen sample, and the gene sequence information is similar, which may easily lead to erroneous judgment in the detection of the target nucleic acid. According to the present invention, it is possible to differentiate infectious microorganisms having similar genetic information as described above using a simple method, thereby reducing the risk of false positives due to erroneous amplification reactions, and may lead to early treatment and prevention of the spread of infection to those around you.

[0023] In the present invention, the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid are aligned, and a probe is designed based on a base sequence D complementary to a partial base sequence C of the target nucleic acid containing one or more bases different between the base sequence A and the base sequence B. The base sequence B of the non-target nucleic acid corresponding to the base sequence A of the target nucleic acid refers to the base sequence B at the corresponding position in the non-target nucleic acid when aligned to perform a primary structure comparison with the base sequence A of the target nucleic acid according to a conventional method. In the present invention, specific detection of the target nucleic acid is possible even when the base sequence identity between the target nucleic acid and the non-target nucleic acid is high, but a region having at least one different base between the base sequences of the target nucleic acid and the non-target nucleic acid is selected, and a probe is designed to bind to the region of the target nucleic acid corresponding to the selected region (in this specification, this region is referred to as the "target nucleic acid region", the "probe binding region", etc., and the base sequence of the region is referred to as the base sequence C) (i.e., to have a substantially complementary base sequence). In this target nucleic acid region (base sequence C), the number of different bases between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid is not particularly limited as long as it is 1 or more, but is preferably, for example, 2 or more, or 3 or more. The upper limit of the number of different bases is also not particularly limited, but may be, for example, 5 or less, or 4 or less. The number of different bases may be, for example, within the range of 2 to 5.

[0024] In a particular embodiment, the specifically binding fluorescently labeled probe used in the present invention may be a probe in which the 5'-end base or the 3'-end base is designed to correspond to a different base position between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid, and the 5'-end base or the 3'-end base corresponding to the different base position is fluorescently labeled (herein, this is also referred to as the "first fluorescently labeled probe"). In the first fluorescently labeled probe, the number of bases that differ between the base sequences of the target nucleic acid and the non-target nucleic acid in the probe binding region may be small, for example, 1 or 2, or may be 1. In the first fluorescently labeled probe, even if the number of bases that differ between the base sequences of the target nucleic acid and the non-target nucleic acid in the probe binding region is small (that is, even if the region with a very high base sequence identity between the base sequence C of the target nucleic acid and the base sequence E of the non-target nucleic acid is used as the probe binding region), the target sequence can be specifically detected.

[0025] In another embodiment, the specifically binding fluorescently labeled probe used in the present invention is designed so that the 5'-end base or 3'-end base corresponds to the position of a common base between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid, and the 5'-end base or 3'-end base corresponding to the position of the common base may be a probe that is fluorescently labeled (herein, this is also referred to as a "second fluorescently labeled probe"). When a base different between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid does not exist at the end of the probe binding region as in the second fluorescently labeled probe, the number of bases different between the base sequences of the target nucleic acid and the non-target nucleic acid in the probe binding region is preferably 2 or more, more preferably 2 to 5, and even more preferably 3 or 4. In the case of the second fluorescently labeled probe, it is possible to detect the target nucleic acid more specifically by designing it to a region where a plurality of different bases exist in this way.

[0026] The specifically binding fluorescently labeled probe used in the present invention is further characterized in that, in the target nucleic acid region (base sequence D) selected in (i) above, (ii) mismatch bases are present at positions corresponding to 1 to 5 bases common between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid (between the base sequence C of the target nucleic acid and the base sequence E of the non-target nucleic acid). In this way, by deliberately making the positions corresponding to the bases common between the base sequence A and the base sequence B mismatch bases, it becomes possible to highly distinguish even target nucleic acids and non-target nucleic acids derived from closely related species that show high sequence identity. The mismatch bases are not particularly limited as long as they are not bases complementary to the bases common when the target nucleic acid and the non-target nucleic acid are aligned, and can be any base. As an example, the mismatch bases can be adenine bases, cytosine bases, guanine bases, thymine bases, universal bases (e.g., hypoxanthine bases, nebularine bases, 5-nitroindole bases), and the like. For example, when the common base is an adenine base when the target nucleic acid and the non-target nucleic acid are aligned, a base other than the thymine base that is complementary to the adenine base (such as a cytosine base, a guanine base, an adenine base, or a universal base) can be used as the mismatch base, and when the common base is a thymine base when the target nucleic acid and the non-target nucleic acid are aligned, a base other than the thymine base (such as a thymine base, a cytosine base, a guanine base, or a universal base) can be used as the mismatch base. When the common conserved base is another base, a mismatch base can be appropriately selected and used in the same manner.

[0027] The number of mismatched bases in the at least specifically binding fluorescently labeled probe is not particularly limited as long as it is within the range of 1 to 5. From the viewpoint of enabling specific detection between the target nucleic acid and the non-target nucleic acid and enabling detection with sufficient sensitivity even in the case of a low copy number of the target nucleic acid, the number is preferably 1 to 4, more preferably 1 to 3, even more preferably 1 or 2, and particularly preferably 1.

[0028] The position of the mismatch base in the specifically binding fluorescently labeled probe is not particularly limited, and may be present at the 3' end side, 5' end side, or near the center of the probe from the center, but when two or more mismatch bases are added, it is preferable that the mismatch bases are not added consecutively (i.e., at least one (e.g., 2 or more, 5 or more, or 10 or more) match bases are present between the mismatch bases). For example, as one preferred embodiment, the probe can be designed so that one mismatch base is present at the 3' end side of the center of the probe and another mismatch base is present at the 5' end side of the center of the probe. From the viewpoint of more reliably enabling specific detection of the target nucleic acid and the non-target nucleic acid, it is preferable that at least one mismatch base is present near the center of the probe, and for example, it is preferable to design it so that it is present within 7 bases around the center of the probe, preferably within 6 bases around the center of the probe, and more preferably within 5 bases around the center of the probe. In this specification, the center of the probe refers to the position (n+1) / 2 when the total length of the base sequence of the probe is n, and refers to the position n / 2 when n is an odd number.

[0029] The length of the specifically binding fluorescently labeled probe is not particularly limited, and may be any length that is generally available for melting curve analysis detection such as PCR. For example, the length of the probe may be 15 bases or more, preferably 16 bases or more, more preferably 17 bases or more, even more preferably 18 bases or more, and even more preferably 19 bases or more. The upper limit of the length of the probe is not particularly limited, and may be a length that is generally available for melting curve analysis detection such as PCR, for example, 25 bases or less, or even 23 bases or less, or 20 bases or less. The length of the probe may be, for example, 15 to 25 bases.

[0030] In one embodiment, the specifically binding fluorescently labeled probe designed as described above may show 80% or more, preferably 85% or more, more preferably 90% or more identity to the base sequence D complementary to the base sequence C of the target nucleic acid. The specifically binding fluorescently labeled probe may show, for example, 90% or less, preferably 85% or less identity to the base sequence F complementary to the base sequence E of the non-target nucleic acid corresponding to the base sequence C. A probe consisting of a base sequence showing 60% or more identity to the base sequence F, particularly a probe consisting of a base sequence showing 70% or more identity, tends to easily cause erroneous detection in melting curve analysis. According to the present invention, even in such a case where the non-target nucleic acid is easily detected, the detection of the non-target nucleic acid can be suppressed by introducing a specific mismatch base, and the target nucleic acid can be detected with high specificity.

[0031] In the present invention, detection is performed by melting curve analysis using the above-mentioned specifically binding fluorescently labeled probe. In the present invention, only one fluorescently labeled probe containing the above-mentioned specific mismatched base may be used, or two or more may be used in combination, but from the viewpoint of simpler and more sensitive detection, it is preferable to use only one.

[0032] The above-mentioned fluorescently labeled probe for specific detection may be one that is labeled at both the 5'-end and the 3'-end, but it is preferable that only one of the 5'-end or the 3'-end is labeled. In one embodiment, the fluorescently labeled probe for specific detection of the present invention is preferably labeled so as to cause quenching or fluorescence when bound to a target nucleic acid containing a base sequence that shows, for example, 80% or more, preferably 85% or more, more preferably 90% or more identity to the complementary sequence of the probe, and more preferably is labeled so as to cause quenching. The labeling substance is not particularly limited, but it is more preferable that it is a fluorescent dye.

[0033] The fluorescent dye may be either a fluorescent substance that generates fluorescence or a fluorescent substance that quenches fluorescence by hybridizing with a target nucleic acid amplification product to form a complex, but is preferably a fluorescent substance that quenches fluorescence when hybridized with a target nucleic acid amplification product, and is particularly preferably a fluorescence quenching dye that is quenched by interaction with guanine when hybridized with a target nucleic acid amplification product (for example, a fluorescence quenching dye that is quenched by interaction with guanine). Specific examples of the fluorescent quenching dye include, but are not limited to, at least one fluorescent quenching dye selected from the group consisting of fluorescein and its derivatives (e.g., fluorescein isothiocyanate (FITC)), rhodamine and its derivatives (e.g., 5-carboxyrhodamine 6G (GR6G), tetramethylrhodamine (TAMRA), carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), and BODIPY and its derivatives (e.g., BODIPY-FL, BODIPY-FL / C3, BODIPY-FL / C6, BODIPY-5-FAM, BODIPY-TMR, BODIPY-TR, BODIPY-R6G, BODIPY-564, BODIPY-581, BODIPY-591, BODIPY-630, BODIPY-650, BODIPY-665).

[0034] More specifically, examples of the fluorescence quenching dye that is quenched by interaction with guanine include at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G (CR6G), TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue), and these fluorescence quenching dyes can be suitably used in the present invention.

[0035] In a particular preferred embodiment, a probe having a terminal base labeled with a fluorescence quenching dye that is cytosine is more preferred. When such a probe hybridizes to a target nucleic acid amplification product, it can form a base pair with a guanine base in the nucleic acid amplification product and interact with it to quench the light, making it very easy to measure the change in the fluorescence intensity of the reaction solution.

[0036] In a particular preferred embodiment, the fluorescently labeled probe of the present invention that specifically detects a target nucleic acid is used in the detection step of step (3) described below. In the method of the present invention, one type of fluorescently labeled probe that specifically detects a target nucleic acid may be used in step (3) described below, or two or more types may be used in combination. In a particular preferred embodiment, the method of the present invention can specifically detect a target nucleic acid by using one type of fluorescently labeled probe that specifically detects a target nucleic acid in step (3) described below.

[0037] [Method for specifically detecting target nucleic acid] In one embodiment, the method for specifically detecting a target nucleic acid that may be contained in a specimen sample is a method using the above-mentioned probe. The method may be a method for determining whether a target nucleic acid is present or absent in a specimen sample. The method may also be a method for quantifying a target nucleic acid that may be contained in a specimen sample. In a specific embodiment, by using at least one pair of nucleic acid primer sets described below, a target nucleic acid that may be contained in a specimen sample can be specifically and highly sensitively detected, for example, in a PCR-melting curve analysis method or the like.

[0038] In certain embodiments, a method for specifically detecting a target nucleic acid comprises at least the following steps (1), (2), and (3): (1) providing a specimen sample that may contain a target nucleic acid; (2) subjecting the target nucleic acid contained in the specimen sample of step (1) to a nucleic acid amplification reaction using at least one pair of primer sets described below as a template; and (3) detecting the one or more nucleic acid amplification products of step (2) using one or more of the nucleic acid probes described above; It is preferable that the method includes the steps of: (1) performing step (2) by PCR reaction (including the case of RT-PCR reaction); and (3) performing step (3) by melting curve analysis (PCR-melting curve analysis). Steps (1), (2), and (3) may be performed in the same reaction solution. In addition, two or more steps among steps (1), (2), and (3), for example, step (1) and step (2), may be performed consecutively or simultaneously.

[0039] [Process (1)] The specimen sample in step (1) is not particularly limited as long as it may contain the target nucleic acid. Examples of specimen samples include biological samples such as animal and plant tissues, body fluids, excrement, and cells, and environmental samples such as wipes of facility walls, floors, equipment, fixtures, toilets, etc., or cleaning fluids used to clean them, when the specific detection of a causative microorganism of a sexually transmitted disease, such as a target nucleic acid derived from Neisseria gonorrhea, are intended. Suitable examples of specimen samples include, but are not limited to, blood, blood culture fluid, cervical scrapings, urethral scrapings, male urethral scrapings, urine, pus, cerebrospinal fluid, pleural effusion, pharyngeal swabs, nasal swabs, sputum, saliva, oral scrapings, tissue slices, skin, vomit, feces, isolated culture colonies, catheter cleaning fluid, etc., collected from a subject (such as a subject suspected of infection with a sexually transmitted disease such as Neisseria gonorrhea). In a specific embodiment targeting a major causative bacterium of sexually transmitted diseases such as Neisseria gonorrhea, the specimen sample is preferably, for example, a tissue section, skin, vomit, urine, isolated culture colony, cervical scraping, urethral scraping, male urethral scraping, urine, or throat swab, and more preferably urine. When a biological sample is used as the specimen sample to be measured, depending on the type of biological sample, etc., pretreatment such as dilution or suspension, centrifugation, enzyme treatment, filtration, heat treatment, acid treatment, alkali treatment, organic solvent treatment, crushing treatment, grinding treatment, or nucleic acid extraction may be performed, although this is not particularly limited.

[0040] The method of collecting and preparing the specimen is not particularly limited, and known methods can be used depending on the type and purpose of the specimen. In a particular preferred embodiment, the specimen does not need to be a sample from which DNA has been isolated or purified, and for example, a sample collected from a living body may be treated with a protease (e.g., proteinase K) and / or heat treated (e.g., at 60 to 100°C for 1 second to 10 minutes) to degrade and denaturate the sample, and DNase (DNA degrading enzyme) present in the sample may be degraded and removed in advance, and the sample may be used as is.

[0041] The method of nucleic acid extraction is not particularly limited, and a known method can be used depending on the type and purpose of the specimen. For example, a kit sold by each manufacturer may be used for nucleic acid extraction. Nucleic acid extraction may also be performed using an automatic extraction and purification device.

[0042] In a particular preferred embodiment, the specimen may be one in which the nucleic acid purification step, which is usually considered essential in conventional nucleic acid amplification reactions, has been omitted. When performing nucleic acid purification, in addition to the need for a dedicated reagent, there is a problem that the operation is complicated, laborious, and time-consuming. When using a specimen sample from which the nucleic acid purification step has been omitted, the time from collection of the specimen sample to obtaining the genetic test result can be shortened, and for example, the time from collection of the specimen sample to obtaining the genetic test result can be within one day, preferably within half a day, more preferably within six hours, even more preferably within three hours, and of these, preferably within two hours (for example, within one hour). Thus, when the method of the present invention is performed using a specimen sample that has not undergone a nucleic acid purification step, the labor of nucleic acid purification can be omitted, and the target nucleic acid can be specifically detected simply and in a short time.

[0043] In one preferred embodiment, the specimen may contain more non-target nucleic acids than the target nucleic acid, or may contain at least a high concentration of the target nucleic acid (e.g., both the target nucleic acid and the non-target nucleic acid are high concentration). In particular, when the specimen contains a high copy of the non-target nucleic acid, the amplification of the non-target nucleic acid, which is not desired to be amplified, is likely to occur. According to the present invention, even in such a case where the amplification of the non-target nucleic acid is likely to occur, the amplification of the non-target nucleic acid can be suppressed and the target nucleic acid can be specifically amplified. From this viewpoint, the present invention is suitable, for example, when the reaction solution to be subjected to the nucleic acid amplification step contains 1000 copies or more, further 2000 copies or more, 3000 copies or more, 4000 copies or more, or 5000 copies or more of the non-target nucleic acid, and it is possible to specifically detect the target nucleic acid even when the reaction solution contains, for example, 10000 copies or more of the non-target nucleic acid. In certain embodiments, a specimen sample containing high copies of both the target nucleic acid and non-target nucleic acid may be used, for example, the reaction solution subjected to the nucleic acid amplification step may contain 1000 or more copies, or even 2000 or more copies, 3000 or more copies, 4000 or more copies, or 5000 or more copies of each of the target nucleic acid and non-target nucleic acid, for example, 10000 or more copies of each of the target nucleic acid and non-target nucleic acid.

[0044] [Process (2)] In one embodiment, the nucleic acid amplification reaction in step (2) is preferably carried out by a nucleic acid amplification method (carrying out a nucleic acid amplification reaction using one or more primer sets). The nucleic acid amplification method is a technique for amplifying several copies of a target nucleic acid to a level at which it can be visualized, i.e., to hundreds of millions of copies or more, and is widely used not only in the field of life science research, but also in fields such as clinical diagnosis, food hygiene inspection, and environmental inspection. Examples of such nucleic acid amplification methods include PCR, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, and TRC. These techniques have already been established in the technical field, and a method can be selected according to the purpose. The nucleic acid amplification method is preferably, but is not limited to, the PCR method (including the RT-PCR method).

[0045] (PCR reaction) PCR is a reaction catalyzed mainly by DNA polymerase. PCR usually includes three steps, namely (i) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (ii) annealing of a primer to a template single-stranded DNA, and (iii) extension of the primer using DNA polymerase, which are set as one cycle, and the cycle is repeated. Examples of DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and mutants thereof. In the present invention, it is preferable to use a DNA polymerase belonging to family B from the viewpoints of simplicity, speed, high sensitivity, and resistance to amplification inhibition by a sample. In addition, when step (3) is performed by melting curve analysis, it is preferable to use a DNA polymerase belonging to family B that does not have 5'→3' exonuclease activity from the viewpoint of using a fluorescence quenching probe.

[0046] The conditions of the PCR reaction are not particularly limited as long as the reaction proceeds. For example, the first step (i) may be performed at 80 to 100°C for about 0 to 300 seconds (for example, about 0.5 to 300 seconds), the second or subsequent (repeated) step (i) may be performed at 80 to 100°C for about 0.5 to 300 seconds, step (ii) may be performed at 35 to 80°C for about 1 to 300 seconds, and step (iii) may be performed at 35 to 85°C for about 1 to 300 seconds. It is preferable to repeat the cycle from step (i) to (iii) 30 to 70 times. The temperature and time of the cycle repeated here may be changed every 1 to 3 cycles. When the temperature of step (ii) and step (iii) is set to a low temperature, non-specific amplification is likely to occur, but in the present invention, the target nucleic acid can be specifically detected even in such a case.

[0047] (DNA polymerase) The DNA polymerase that can be used in the nucleic acid amplification reaction, particularly the PCR reaction, in step (2) is preferably, but not limited to, a DNA polymerase belonging to family B. The DNA polymerase belonging to family B is not particularly limited, but is preferably a DNA polymerase derived from Archea, more preferably a DNA polymerase derived from bacteria of the genus Pyrococcus and Thermococcus. In addition, suitable DNA polymerases also include variants thereof that do not lose DNA polymerase activity derived from Archea belonging to family B. Examples of the variants include variants in which 1 to 3 amino acids are deleted, substituted, inserted and / or added in the wild-type amino acid sequence, or variants that show 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more amino acid sequence identity with the wild-type amino acid sequence. Specifically, examples of the DNA polymerase variants include, but are not limited to, variants aimed at enhancing polymerase activity, deleting exonuclease activity, adjusting substrate specificity, and the like.

[0048] Examples of DNA polymerases derived from the genus Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus furiosus, Pyrococcus sp. GB-D, Pyrococcus woesei, Pyrococcus abyssi, and Pyrococcus horikoshii, as well as mutants derived therefrom that have not lost their DNA polymerase activity.

[0049] Examples of DNA polymerases derived from the genus Thermococcus include, but are not limited to, DNA polymerases isolated from Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9doN-7), and Thermococcus siculi, and mutants thereof that have not lost their DNA polymerase activity. Preferably, DNA polymerases derived from Thermococcus kodakaraensis and mutants thereof (e.g., KOD-derived DNA polymerase lacking 3'→5' exonuclease activity) can be particularly preferably used in the present invention from the viewpoint of excellent extensibility and thermostability.

[0050] PCR enzymes using these DNA polymerases are commercially available, and examples include Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), Pwo (Roche), and the like, any of which can be used in the present invention.

[0051] (DNA polymerase derived from KOD) In the present specification, KOD-derived DNA polymerase (also referred to as KOD DNA polymerase) refers to Thermococcus kodakaraensis-derived DNA polymerase and its mutants (for example, KOD-derived DNA polymerase in which 3'→5' exonuclease activity has been deleted by substituting, deleting, inserting and / or adding 1 to 3 amino acids in the naturally occurring amino acid sequence). In one preferred embodiment, the nucleic acid amplification reaction in step (2) uses such a KOD-derived DNA polymerase. Compared to Taq DNA polymerase, which is a DNA polymerase belonging to family A, KOD DNA polymerase is superior in accuracy, amplification efficiency, extensibility and resistance to amplification inhibition by inhibitors derived from a test sample. In the present invention, the use of such a KOD DNA polymerase is more preferable in terms of simple, rapid, highly sensitive and specific detection of a target nucleic acid, as shown in the examples described later.

[0052] (Primer set) The primer set that can be used in the nucleic acid amplification reaction in step (2) is not particularly limited as long as it can amplify the target nucleic acid to which the fluorescently labeled probe can bind. The primer set is designed so that one primer is complementary to the DNA extension product of the other primer. For example, the primer set that can be used in the nucleic acid amplification reaction may include a primer consisting of a base sequence that completely matches the base sequences of both the target nucleic acid and the non-target nucleic acid, or may include a primer consisting of a base sequence that contains, for example, 1 to 7 or 1 to 5 bases that are mismatched with either or both of the target nucleic acid and the non-target nucleic acid. However, it is preferable to use at least one pair of primer sets consisting of primers consisting of a base sequence that completely matches the base sequence of the target nucleic acid.

[0053] [Process (3)] The melting curve analysis in step (3) is not particularly limited in its embodiment, so long as it uses a fluorescently labeled probe containing the above-mentioned specific mismatch base. For example, target nucleic acids such as Neisseria gonorrhoeae may mutate in the same way as other infectious microorganisms. Here, if an unintended mismatch occurs between the base sequence of the primer or probe and the base sequence of the target nucleic acid of a Neisseria gonorrhoeae mutant strain, the binding strength of the primer or probe to the target gene or the nucleic acid amplification product derived therefrom (target nucleic acid) may decrease. In particular, if the number of unintended mismatches increases between the target nucleic acid and the probe in the real-time PCR method, the probe cannot sufficiently bind to the target nucleic acid, so that the rise of the amplification curve may be delayed or may not rise at all, which may induce a false negative. In the case of the melting curve analysis method, since step (3) is performed after the completion of PCR, even if an unintended mismatch occurs in the binding region of the primer or probe, it is possible to detect it as long as a nucleic acid amplification product is finally obtained, so that the influence of unintended mismatches can be suppressed more than in the real-time PCR method. Furthermore, by detecting the nucleic acid amplification products using melting curve analysis, it may be possible to detect them in a shorter time (for example, 45 minutes or less, preferably 40 minutes or less, and more preferably 35 minutes or less from the start of the nucleic acid amplification reaction to the completion of melting curve analysis).

[0054] In one embodiment, step (3) comprises the following steps (3-1) and (3-2): (3-1) a step of hybridizing a nucleic acid amplification product that can be produced by the nucleic acid amplification reaction of the step (2) with a nucleic acid probe to form a complex; and (3-2) detecting the complex of step (3-1) The present invention is characterized in that, in order to obtain a specific and highly sensitive determination result in the detection of a target nucleic acid, the above-mentioned specifically binding fluorescently labeled probe capable of specifically reacting with and forming a complex with a nucleic acid amplification product that can be generated in the nucleic acid amplification reaction in step (2) is used.

[0055] The hybridization in step (3-1) is preferably carried out under temperature conditions that allow sufficient hybridization of the nucleic acid amplification product and the fluorescently labeled probe. Examples of such temperature conditions include, but are not limited to, a temperature at least 5°C lower than the Tm value of the nucleic acid probe, more preferably at least 10°C lower. Hybridization at a temperature lower than the Tm value in this way tends to cause amplification of non-target nucleic acids, but according to the present invention, even when sufficient hybridization is performed at a temperature lower than the Tm value in this way, detection of non-specific nucleic acids in melting curve analysis can be suppressed, and the target nucleic acid can be specifically detected.

[0056] In one embodiment, step (3) may include the following melting curve analysis method (melting curve analysis method) of step (3'): (3') a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution of the nucleic acid amplification reaction after the step (2) is completed, and measuring the temperature dependence of the fluorescence intensity of the reaction solution.

[0057] (Process (3')) In step (3'), measuring the temperature dependency of the fluorescence intensity can specifically mean measuring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from low to high. The melting temperature (Tm value) specific to the nucleic acid probe used can be obtained by first differentiating the obtained fluorescence intensity with respect to temperature. The fluorescence intensity may also be converted into a fluorescence quenching rate or the like according to the purpose. The detection and analysis of target nucleic acids using the Tm value is called melting curve analysis. In general, the Tm value refers to the temperature at which the proportion of an oligonucleotide that forms a double strand with its complementary strand is equal to the proportion that does not form a double strand and is single-stranded. Since the Tm value is a value specific to a base sequence, melting curve analysis can be used as a method for analyzing base sequence polymorphisms of target nucleic acids. Base sequence polymorphisms referred to here include single nucleotide polymorphisms, base substitutions, base deletions, base insertions, etc.

[0058] As an example, melting curve analysis is also applied to SNP analysis. When there is a mutation in the base sequence of the target nucleic acid relative to the probe, the bases mismatch when the probe hybridizes, and the Tm value is generally low. Therefore, single nucleotide polymorphism analysis (SNP analysis) can also be performed by comparing the magnitude of the Tm value.

[0059] In the present invention, melting curve analysis is performed in step (3') to monitor in real time the intensity of the fluorescence or quenching signal generated as the probe dissociates from the target sequence in response to temperature change. When the first derivative value obtained by differentiating the change in emission or quenching signal intensity with increasing temperature by the temperature change value is plotted, the melting temperature can be displayed as a peak in the resulting melting curve (temperature-first derivative curve). In this specification, the melting temperature displayed as a peak when a melting curve analysis is performed using a specific probe is referred to as the detection temperature of the probe.

[0060] When a mutation occurs in the target nucleic acid, a double-stranded gene with a mismatch is formed by performing PCR. In this way, the double-stranded gene with a mismatch easily denatures (dissociates) the double-stranded structure, and the detection temperature may be lower than that of a wild-type gene without a mismatch during melting curve analysis. Therefore, when a mutation occurs in the target nucleic acid that causes an unintended mismatch with the probe sequence, a change in the detection temperature (lowering) can be detected.

[0061] [Reagents for specifically detecting target nucleic acids] In another embodiment, the present invention provides a reagent for specifically detecting a target nucleic acid (e.g., a target nucleic acid of Neisseria gonorrhoeae, Chlamydia trachomatis, Bordetella pertussis, etc.). The reagent preferably contains at least the fluorescently labeled probe that specifically detects the specific mismatched base described above, and, if necessary, in addition to the nucleic acid primer, at least components necessary for nucleic acid amplification reaction and detection. The necessary components can be known. For example, the reagent of the present invention preferably contains at least a primer set for nucleic acid amplification such as PCR, DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and inorganic salts such as magnesium salts. The primer set for nucleic acid amplification and the fluorescently labeled detection probe can contain multiple sets to amplify and detect multiple regions of the target nucleic acid (e.g., multiple regions of Neisseria gonorrhoeae). In this case, it is sufficient that the multiple fluorescently labeled nucleic acid detection probes are designed to contain the specific mismatched bases. The concentration of each component can be adjusted appropriately, but for example, the concentration of the fluorescently labeled probe is preferably 0.01 to 1 μM, more preferably 0.02 to 0.5 μM. When a fluorescently labeled probe set is used, each nucleic acid probe contained in the probe set is preferably within the above-mentioned concentration range. The concentration of each primer in the primer set is preferably 0.01 to 10 μM. The concentration of the DNA polymerase is preferably 0.01 to 1 U / μL, more preferably 0.02 to 0.5 U / μL. The concentration of the deoxyribonucleoside triphosphates (dNTPs) is preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM. The concentration of inorganic salts such as magnesium salts is preferably 0.1 to 10 mM, more preferably 1 to 5 mM.

[0062] Furthermore, for the purpose of suppressing non-specific amplification and promoting the reaction, the reagent of the present invention may contain additives known in the art. Examples of additives for suppressing non-specific amplification include known anti-DNA polymerase antibodies, phosphoric acid, etc. Examples of additives for promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single-stranded binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, carnitine, Triton, Tween 20, and Nonidet (registered trademark) P40. In order to facilitate the determination of false negatives, the reagent of the present invention preferably contains an internal control known in the art. In the present invention, these additives may be used alone or in combination of two or more.

[0063] [Reagent kit for specifically detecting target nucleic acid] In another embodiment, the present invention provides a kit for specifically detecting a target nucleic acid (e.g., a target nucleic acid of Neisseria gonorrhoeae, Chlamydia trachomatis, Bordetella pertsys, etc.). The kit of the present invention is not particularly limited as long as it is configured to detect (including distinguish) a target nucleic acid (e.g., Neisseria gonorrhoeae, Chlamydia trachomatis, Bordetella pertsys) by including a fluorescently labeled probe containing a specific mismatched base as described above, and by including a reagent containing a nucleic acid primer and / or other additives as necessary. For example, the kit of the present invention may optionally include a reagent capable of detecting or quantifying the presence of a target substance, and / or an instruction manual for use that explains a method of use, etc. For example, the kit of the present invention may be provided in such a manner that the fluorescently labeled probe containing a specific mismatched base, and if necessary, a nucleic acid primer, components necessary for a nucleic acid amplification reaction, and components necessary for detecting a nucleic acid amplification product are sealed in the same container or in separate containers, and packaged in, for example, a single package, and includes information on how to use the kit. The kit of the present invention may also include a positive control solution and / or a negative control solution. EXAMPLES

[0064] The present invention will be specifically described below by showing examples, but the present invention is not limited to these examples.

[0065] [Test Example 1: Evaluation of specific detection of Neisseria gonorrhoeae] (1) Sample preparation To evaluate a method for specifically detecting Neisseria gonorrhoeae, crudely extracted genomic DNA consisting of the full-length base sequence of Neisseria gonorrhoeae and genomic DNA consisting of the full-length base sequence of Neisseria meningitidis were diluted with sterile water to prepare a test concentration of 10,000 copies. (2) Nucleic acid amplification, melting curve analysis, and determination The above samples were added to the following reagents, and Neisseria gonorrhoeae (NG) and Neisseria meningitidis (NM) were detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for nucleic acid amplification and melting curve analysis. The results were judged as positive when a peak was obtained in the melting curve analysis.

[0066] (reagent) The base sequences of Neisseria gonorrhoeae (target nucleic acid) and Neisseria meningitidis (non-target nucleic acid) and the positional relationship of the primers or probes used are shown in Figure 1 (note that in Figure 1, the base sequence of SEQ ID NO: 2 was converted to a forward strand for alignment). The following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) was used after adjusting the amount used as described in the respective instruction manuals. An internal control (IC) of known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.5 μM primer shown in SEQ ID NO:1 3.0 μM primer shown in SEQ ID NO:2 0.25 μM of the probe shown in SEQ ID NO:3 (labeled at the 3' end with BODIPY-FL)

[0067] (Nucleic acid amplification and melting curve analysis) 94℃・30 seconds (One cycle) 97℃・1 second 58℃・5 seconds 68℃・5 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.4℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0068] (3) Results FIG. 2 is a detection graph obtained when the probe shown in SEQ ID NO: 3 was used to detect Neisseria gonorrhoeae DNA and Neisseria meningitidis DNA by nucleic acid amplification and melting curve analysis. The probe used in this test example was designed so that its end corresponds to the position of a different base between the base sequences of the target nucleic acid and non-target nucleic acid (the double underlined base in FIG. 1), and the end base corresponding to the position of the different base was fluorescently labeled, and a mismatch was inserted near the center of the probe (corresponding to the first fluorescently labeled probe). As shown in FIG. 2, it was shown that specific detection of Neisseria gonorrhoeae was possible when a probe designed as described above with a specific mismatch base was used. In addition, when the same test as above was performed using a fluorescently labeled probe designed in the same way as SEQ ID NO: 3 except that it did not contain a mismatch base, a rise in fluorescence was observed even in the detection of Neisseria meningitidis DNA, which is a non-target nucleic acid, and it was confirmed that a non-specific reaction occurred.

[0069] The measurement results using the probe used in this test example are summarized in Table 1. This study demonstrated that specific detection of Neisseria gonorrhoeae was possible in melting curve analysis by selecting a probe represented by the base sequence of SEQ ID NO: 3, which contains a mismatched base. In this test example, the copy number used in the reaction was very high at 10,000, and the annealing and extension temperatures in the PCR thermal cycle were set low, which are conditions that tend to cause non-specific reactions. However, it was revealed that the present invention enables specific detection of the target nucleic acid.

[0070] [Table 1]

[0071] [Test Example 2: Evaluation of specific detection of Chlamydia trachomatis] (1) Sample preparation To evaluate the method for specific detection of Chlamydia trachomatis, crudely extracted genomic DNA consisting of the full-length base sequence of Chlamydia trachomatis and Chlamydia suis were diluted with sterile water to prepare a test concentration of 10,000 copies / test. (2) Nucleic acid amplification, melting curve analysis, and determination The above samples were added to the following reagents, and Chlamydia trachomatis (CT) and Chlamydia suis (CS) were detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for nucleic acid amplification and melting curve analysis. The result was judged as positive when a peak was obtained in the melting curve analysis.

[0072] (reagent) The positional relationship between the base sequences of Chlamydia trachomatis (target nucleic acid) and Chlamydia suis (non-target nucleic acid) and the primers or probes used is shown in Figure 3 (in Figure 3, the base sequences of SEQ ID NOs: 5 and 6 were converted to forward strands for alignment). The following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) was used after adjusting the amount used as described in the respective instruction manuals. An internal control (IC) of known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 3.0 μM primer shown in SEQ ID NO:4 0.5 μM primer shown in SEQ ID NO:5 0.25 μM of the probe shown in SEQ ID NO:6 (labeled at the 3' end with BODIPY-FL)

[0073] (Nucleic acid amplification and melting curve analysis) 94℃・30 seconds (One cycle) 97℃・1 second 58℃・5 seconds 68℃・5 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.4℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0074] (3) Results FIG. 4 is a detection graph obtained when Chlamydia trachomatis DNA and Chlamydia suis DNA were detected by nucleic acid amplification and melting curve analysis using the probe shown in SEQ ID NO: 6. The probe used in this test example was designed so that its end corresponds to the position of a base common to the base sequences of the target nucleic acid and non-target nucleic acid (the double underlined bases in FIG. 3 correspond to the positions of bases that differ between the base sequences of the target nucleic acid and non-target nucleic acid), and a mismatch was introduced near the center of the probe (corresponding to the second fluorescently labeled probe). As shown in FIG. 4, it was shown that specific detection of Chlamydia trachomatis was possible when a probe with a specific mismatch base added as described above was used. In this test example, a fluorescently labeled probe designed in the same way as SEQ ID NO: 6 except that it did not contain a mismatch base was separately prepared and the same test as above was performed. It was confirmed that a rise in fluorescence was observed in the detection of Chlamydia suis DNA, which is a non-target nucleic acid, and a non-specific reaction occurred.

[0075] The measurement results using the probe used in this test example are summarized in Table 4. This study demonstrated that specific detection of Chlamydia trachomatis was possible in melting curve analysis by selecting a probe represented by the base sequence of SEQ ID NO: 6, which contains a mismatched base. In this test example, the copy number used in the reaction was very high at 10,000, and the annealing and extension temperatures in the PCR thermal cycle were set low, making the conditions prone to non-specific reactions, but it became clear that the present invention enables specific detection of the target nucleic acid.

[0076] [Table 2]

[0077] [Test Example 3: Evaluation of specific detection of Bordetella pertussis] (1) Sample preparation To evaluate the method for specific detection of Bordetella pertussis, crudely extracted genomic DNA consisting of the full-length base sequence of Bordetella pertussis and genomic DNA consisting of the full-length base sequence of Bordetella bronchiseptica were diluted with sterile water to prepare a test concentration of 10,000 copies. (2) Nucleic acid amplification, melting curve analysis, and determination The above samples were added to the following reagents, and Bordetella pertussis and Bordetella bronchiseptica were detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for nucleic acid amplification and melting curve analysis. The result was judged as positive when a peak was obtained in the melting curve analysis.

[0078] (reagent) The positional relationship between the base sequences of Bordetella pertussis (target nucleic acid) and Bordetella bronchiseptica (non-target nucleic acid) and the primers or probes used is shown in FIG. 5 (note that in FIG. 5, the base sequence of SEQ ID NO: 8 was converted to a forward strand for alignment). The probe used in this test example corresponds to a second fluorescently labeled probe whose end corresponds to the position of a base common between the base sequences of the target nucleic acid and the non-target nucleic acid. The following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) was used by adjusting the amount used as described in the respective instruction manuals. An internal control (IC) of a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.5 μM primer shown in SEQ ID NO:7 3.0 μM primer shown in SEQ ID NO:8 0.25 μM of the probe shown in SEQ ID NO:9 (labeled at the 3' end with BODIPY-FL)

[0079] (Nucleic acid amplification and melting curve analysis) 94℃・30 seconds (One cycle) 97℃・1 second 58℃・5 seconds 68℃・5 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.4℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0080] (3) Results As in Test Examples 1 and 2, it was shown that specific Bordetella pertussis can be detected in melting curve analysis by adding a specific mismatch base to the fluorescent detection probe. In this test example, a fluorescently labeled probe designed to be the same as SEQ ID NO: 9 except that it does not contain a mismatch base was separately prepared and the same test as above was performed. As a result, a rise in fluorescence was observed even in the detection of Bordetella bronchiseptica DNA, which is a non-target nucleic acid, and it was confirmed that a non-specific reaction occurred.

[0081] FIG. 6 is a detection graph obtained when Bordetella pertussis DNA and Bordetella bronchiseptica DNA were detected by nucleic acid amplification and melting curve analysis using the probe shown in SEQ ID NO: 9. The measurement results using the probe used in this test example are summarized in Table 3. This study showed that specific detection of Bordetella pertussis was possible by selecting the probe shown in the base sequence of SEQ ID NO: 9 containing a mismatched base. In this test example, the copy number used in the reaction was very high at 10,000, and the annealing and extension temperatures in the PCR thermal cycle were set low, making the conditions prone to non-specific reactions, but it was revealed that the present invention enables specific detection of the target nucleic acid.

[0082] [Table 3] [Industrial Applicability]

[0083] By utilizing the method of the present invention, a target nucleic acid that may be contained in a sample can be detected simply, highly sensitively, and specifically in a melting curve analysis method. The present invention enables highly reliable detection of a target nucleic acid, and is expected to greatly contribute to clinical diagnosis and the like.

Claims

1. 1. A method for detecting a target nucleic acid, comprising: A step of amplifying a target nucleic acid using at least one pair of primer sets; and a target nucleic acid detection step by melting curve analysis using a fluorescently labeled probe that specifically binds to the target nucleic acid amplified in the target nucleic acid amplification step; The method is characterized in that the probe includes a base sequence D that is complementary to a partial base sequence C of a target nucleic acid that includes one or more bases that differ between a base sequence A of the target nucleic acid and a base sequence B of a non-target nucleic acid corresponding to base sequence A, and in which mismatch bases exist at positions corresponding to 1 to 5 bases that are common between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid.

2. The method according to claim 1, wherein the 5'-terminal base or the 3'-terminal base of the probe corresponds to a different base position between a base sequence A of a target nucleic acid and a base sequence B of a non-target nucleic acid, and the 5'-terminal base or the 3'-terminal base is fluorescently labeled.

3. The method of claim 1 , wherein the base sequence C of the target nucleic acid contains two or more bases that differ between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid.

4. The method of claim 1, wherein the base sequence C of the target nucleic acid contains 2 to 5 bases that differ between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid.

5. The method of claim 1, wherein the target nucleic acid and the non-target nucleic acid are each derived from different species of the same genus, and the method is for species-specific detection of the target nucleic acid.

6. The method according to claim 1, wherein the base sequence identity between the base sequence C of the target nucleic acid and the base sequence E of the non-target nucleic acid corresponding to the base sequence C is 60% or more but less than 100%.

7. The method of claim 1 , wherein the probe is 15 bases or more in length.

8. The method of claim 1, wherein the probe is 15 to 25 bases in length.

9. The method according to claim 1 , wherein at least one mismatched base in the probe is present in a region within 5 bases before and after the center of the probe.

10. The method according to claim 1, wherein the number of mismatched bases in the probe is 1 to 3.

11. The method of claim 1, wherein the probe is labeled with a fluorescent quenching dye that is quenched upon interaction with guanine.

12. 12. The method of claim 11, wherein the fluorescence quenching dye is at least one fluorescence quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives.

13. 11. The method of claim 10, wherein the fluorescence quenching dye is at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).

14. The method of claim 1, wherein the probe is labeled with a fluorescent quenching dye at only one of the 5' end or the 3' end.

15. The method of claim 1 , wherein the terminal base that is labeled in the probe is a cytosine.

16. The method according to claim 1 , wherein 10,000 or more copies of non-target nucleic acid are present in the reaction solution in the target nucleic acid amplification step.

17. The method according to claim 1 , wherein the target nucleic acid and non-target nucleic acid are each present in a reaction solution at 10,000 or more copies in the target nucleic acid amplification step.

18. The method according to claim 1, wherein the step of detecting the target nucleic acid is carried out by melting curve analysis at a detection temperature of 40 to 75°C.

19. A fluorescently labeled probe for specifically detecting a target nucleic acid by melting curve analysis, characterized in that the probe contains a base sequence D complementary to a partial base sequence C of a target nucleic acid containing one or more bases that differ between a base sequence A of the target nucleic acid and a base sequence B of a non-target nucleic acid corresponding to the base sequence A, in which mismatch bases exist at positions corresponding to 1 to 5 bases that are common between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid.

20. 20. The fluorescently labeled probe according to claim 19, wherein the 5'-terminal base or the 3'-terminal base of the probe corresponds to a different base position between a base sequence A of a target nucleic acid and a base sequence B of a non-target nucleic acid, and the 5'-terminal base or the 3'-terminal base is fluorescently labeled.

21. 20. The fluorescently labeled probe according to claim 19, wherein the base sequence C of the target nucleic acid contains two or more bases which differ between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid.

22. 20. The fluorescently labeled probe according to claim 19, wherein the base sequence C of the target nucleic acid contains 2 to 5 bases which differ between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid.

23. A kit for specifically detecting a target nucleic acid, comprising the fluorescently labeled probe according to any one of claims 19 to 22.

24. The kit according to claim 23, further comprising a primer set capable of amplifying a target nucleic acid region including a region to which the fluorescently labeled probe binds.

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