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, where at least one primer of the primer pair 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; a base sequence C is a partial sequence of the base sequence A having three 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, there are situations where it is necessary to specifically detect target nucleic acids, such as Mycoplasma genitalium, a sexually transmitted disease, and Mycoplasma pneumoniae, a respiratory infection. 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. In addition, the sexually transmitted diseases Neisseria gonorrhoeae and Neisseria meningitidis are also difficult to distinguish for the same reason, and there is a possibility of erroneous judgment.

[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 unexpectedly found that when a nucleic acid amplification reaction is carried out using a primer, it is possible to specifically detect only the target nucleic acid by adding a specific mismatch base to the primer. 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 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 the target nucleic acid using at least one pair of primer sets, characterized in that at least one primer of the at least one pair of primer sets comprises a base sequence D complementary to a partial base sequence C of the target nucleic acid comprising three 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 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. [Item 3] The method according to item 1 or 2, 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%. [Item 4] The method according to any one of Items 1 to 3, wherein the length of at least one of the primers is 15 bases or more. [Item 5] The method according to any one of Items 1 to 4, wherein the length of at least one of the primers is 15 to 35 bases. [Item 6] The method according to any one of Items 1 to 5, wherein the base sequence C of the target nucleic acid contains 3 to 7 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 7] The method according to any one of Items 1 to 6, wherein at least one mismatched base in the at least one primer is located closer to the 3' end than to the center of the primer. [Item 8] The method according to any one of Items 1 to 7, wherein the number of mismatched bases in the at least one primer is 1 to 3. [Item 9] The method according to any one of Items 1 to 8, further comprising a step of detecting a target nucleic acid using a probe that specifically binds to the target nucleic acid amplified in the target nucleic acid amplification step. [Item 10] The method according to Item 9, wherein the probe is labeled with a fluorescent quenching dye that is quenched upon interaction with guanine. [Item 11] The method according to item 9 or 10, wherein a melting curve analysis is performed in the target nucleic acid detection step. [Item 12] The method according to any one of Items 1 to 11, 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 13] The method according to any one of Items 1 to 12, 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 14] A primer set for specifically detecting a target nucleic acid, wherein at least one primer of the primer set includes a base sequence D complementary to a partial base sequence C of a target nucleic acid including three 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 common between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid. [Item 15] A kit for specifically detecting a target nucleic acid, comprising the primer set according to Item 14. [Item 16] The kit described in Item 14 or 15, further comprising a probe that specifically binds to a target nucleic acid amplified by the primer set. 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: 13) and non-target nucleic acid (SEQ ID NO: 14) and the primers or probes (SEQ ID NOs: 2 to 5) used in Test 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 Test Example 1. The results show a comparison of detection using the primers of the present invention (SEQ ID NOs: 3 and 4) containing specific mismatched bases with detection using a primer (SEQ ID NO: 2) containing no mismatched base, with Mycoplasma genitalium as the target nucleic acid and Mycoplasma pneumoniae as the non-target nucleic acid. [Diagram 3] 1 shows the results of Test Example 2. The figures show the results of comparing the detection sensitivity for a target nucleic acid between primers with a specific mismatch base (SEQ ID NO: 3 and 4) and those without (SEQ ID NO: 2). [Figure 4] 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: 6 to 12) used in Test 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. [Diagram 5]1 shows the results of Test Example 3. The results show a comparison of detection using primers of the present invention containing specific mismatched bases (SEQ ID NOs: 7, 10, and 11) and primers not containing mismatched bases (SEQ ID NOs: 6, 8, and 9) with Chlamydia trachomatis as the target nucleic acid and Chlamydia muridarum as the non-target nucleic acid. [Figure 6] 1 shows the results of Test Example 4. The figures show the results of comparing the detection sensitivity for the target nucleic acid between primers with specific mismatched bases (SEQ ID NOs: 7, 10, and 11) and those without (SEQ ID NOs: 6, 8, and 9). 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 at least one primer of at least one pair of primer sets is designed to include a specific mismatch base in a specific region of the target nucleic acid, thereby enabling specific amplification and detection of only the target nucleic acid. Usually, when performing specific detection of a target nucleic acid, a primer that is completely complementary to the base sequence of the target nucleic acid is often used to amplify the nucleic acid. One of the features of the present invention is that, completely unexpectedly, a specific mismatch base is deliberately placed in the primer, thereby suppressing the amplification of non-target nucleic acid and enabling specific amplification and detection of only the target nucleic acid.

[0018] In one embodiment, the method of the present invention comprises at least the following steps: A process for amplifying a target nucleic acid using at least one pair of primer sets, characterized in that (i) at least one primer of the primer set is based on a base sequence D that is complementary to a partial base sequence C of the target nucleic acid that contains three 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 primer set including a primer having both the above characteristics (i) and (ii). By amplifying nucleic acid using primers having these two characteristics, it becomes possible to specifically detect only the target nucleic acid even when the base sequences of the target nucleic acid and non-target nucleic acid show high sequence identity (for example, when they show 60% or more base sequence identity). 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, a 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] (At least one pair of primer sets) At least one primer of at least one pair of primer sets used in the present invention is designed based on (i) a base sequence D complementary to a partial base sequence C of a target nucleic acid that contains three or more bases that differ between the base sequence A of the target nucleic acid and the base sequence B of a non-target nucleic acid. "At least one primer of at least one pair of primer sets" means, for example, in the case of one primer set P1, one primer P11, the other primer P12, or both primers P11 and P12 contained in the primer set P1. In the case of two primer sets P1 and P2, it means at least one primer selected from the primers P11 and P12 contained in the primer set P1, and the primers P21 and P22 contained in the primer set P2. The same applies to the case of three or more primer sets.

[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 the combination of the target nucleic acid and the non-target nucleic acid include a combination of the 23S rRNA gene sequence of Mycoplasma genitalium (e.g., SEQ ID NO: 13) as the target nucleic acid and the 23S rRNA gene sequence of Mycoplasma pneumoniae (e.g., SEQ ID NO: 14) as the non-target nucleic acid, which belongs to the same Mycoplasma genus; a combination of the ompA gene of Chlamydia trachomatis (e.g., SEQ ID NO: 15) as the target nucleic acid and the ompA gene of Chlamydia suis and / or the ompA gene of Chlamydia muridarum (e.g., SEQ ID NO: 16, SEQ ID NO: 17) as the non-target nucleic acid, which belongs to the same Chlamydia genus; gonorrhoeae opA gene sequence (e.g., SEQ ID NO: 18) as the target nucleic acid and Neisseria meningitidis opA gene sequence (e.g., SEQ ID NO: 19) as the non-target nucleic acid, which belongs to the same genus Neisseria; Bordetella pertussis IS1002 gene (e.g., SEQ ID NO: 20) as the target nucleic acid and Bordetella bronchiseptica IS1002 gene (e.g., SEQ ID NO: 21) as the non-target nucleic acid, which belongs to the same genus Bordetella, and the like are included, but are not limited thereto. 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 primer is designed based on a base sequence D complementary to a partial base sequence C of the target nucleic acid containing three 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 three different bases between the base sequences of the target nucleic acid and the non-target nucleic acid is selected, and a primer 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 "target nucleic acid region to which the primer binds", 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 3 or more, but is preferably, for example, 4 or more, or 5 or more. The upper limit of the number of different bases is also not particularly limited, but may be, for example, 7 or less, or 6 or less. The number of different bases may be, for example, within the range of 3 to 7.

[0024] At least one primer of at least one pair of primer sets used in the present invention is further characterized in that, in the target nucleic acid region (base sequence D) selected in (i) above, (ii) a mismatch base is present at a position 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 position corresponding to the base common between the base sequences A and B a mismatch base, 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 base is not particularly limited as long as it is a base other than a base complementary to a base common when the target nucleic acid and the non-target nucleic acid are aligned, and may be any base. As an example, the mismatch base may be an adenine base, a cytosine base, a guanine base, a thymine base, a universal base (e.g., a hypoxanthine base, a nebularine base, a 5-nitroindole base), or 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 a 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 adenine base that is complementary to the thymine base (such as a thymine base, a cytosine base, a guanine base, or a universal base) can be used as a mismatch base. When the common base is another base, a mismatch base can be appropriately selected and used in the same manner.

[0025] The number of mismatched bases in the at least one primer is not particularly limited as long as it is within the range of 1 to 5, but from the viewpoint of enabling specific detection between target nucleic acids and non-target nucleic acids and enabling detection with sufficient sensitivity even in the case of low copy numbers of the target nucleic acid, the number is preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2.

[0026] The position of the mismatch base in the at least one primer is not particularly limited, and may be present at the 3' end side, 5' end side, or near the center of the primer, 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 primer can be designed so that one mismatch base is present at the 3' end side of the center of the primer, and another mismatch base is present at the 5' end side of the center of the primer. From the viewpoint of more reliably enabling specific detection of the target nucleic acid and the non-target nucleic acid, it is preferable to design the primer so that at least one mismatch base is present at the 3' end side of the center of the primer. In this specification, the center of the primer refers to the position (n+1) / 2 when n is an odd number, and the position n / 2 when n is an even number.

[0027] The length of the at least one primer is not particularly limited, and may be any length that is generally available for nucleic acid amplification reactions such as PCR. For example, the length of the primer may be 15 bases or more, preferably 17 bases or more, more preferably 18 bases or more, even more preferably 19 bases or more, and even more preferably 20 bases or more. The upper limit of the length of the primer is also not particularly limited, and may be, for example, 35 bases or less, or even 30 bases or less, or 25 bases or less. The length of the primer may be, for example, 15 to 35 bases.

[0028] In one embodiment, at least one primer designed as described above may have an identity of 80% or more, preferably 85% or more, more preferably 90% or more to the base sequence D complementary to the base sequence C of the target nucleic acid. The at least one primer may have an identity of, for example, 85% or less to the base sequence F complementary to the base sequence E of the non-target nucleic acid corresponding to the base sequence C. A primer consisting of a base sequence showing 60% or more identity to the base sequence F, particularly a primer consisting of a base sequence showing 70% or more identity, tends to easily cause the amplification of the non-target nucleic acid. According to the present invention, even in such a case where the non-target nucleic acid is easily amplified, the amplification of the non-target nucleic acid can be suppressed by introducing a specific mismatch base, and the target nucleic acid can be amplified with high specificity.

[0029] In the present invention, nucleic acid amplification is performed using a primer set containing at least one of the above-mentioned primers. In this primer set, the primer containing the above-mentioned specific mismatch base may be only one, or two or more may be used in combination. For example, when one primer containing a specific mismatch base is used, it may be used in combination with a primer consisting of a base sequence that does not contain a mismatch base (completely matches the base sequence C of the target nucleic acid). Even when two or more primers containing a specific mismatch base are used, a primer consisting of a base sequence that completely matches the base sequence C of the target nucleic acid may be used in combination.

[0030] [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 at least one pair of primer sets including the above-mentioned primers. 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 the nucleic acid probe described below, the 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.

[0031] 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 that may be contained in the specimen sample of step (1) to a nucleic acid amplification reaction using at least one pair of primer sets as described above as a template; and (3) detecting the one or more nucleic acid amplification products of step (2) using one or more nucleic acid probes described below. 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.

[0032] [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, for example, when it is intended to specifically detect the target nucleic acid derived from Mycoplasma genitalium, include biological samples such as animal and plant tissues, body fluids, excrement, and cells; environmental samples such as wipes of facility walls, floors, equipment and fixtures, toilets, etc., or cleaning fluids used to clean them. 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 fluid, pharyngeal swabs, nasal swabs, sputum, saliva, oral scrapings, tissue slices, skin, vomit, feces, isolated culture colonies, catheter washings, etc., collected from subjects (such as subjects suspected of being infected with Mycoplasma genitalium). In a specific embodiment targeting the main causative bacteria of sexually transmitted diseases such as Mycoplasma genitalium, the specimen sample is preferably, for example, tissue slice, 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] In one preferred embodiment, the specimen may contain more non-target nucleic acids than the target nucleic acid, or may contain at least non-target nucleic acids at a high concentration (e.g., both target and non-target nucleic acids at a high concentration). In particular, when a high copy of non-target nucleic acid is contained, nucleic acid amplification of non-target nucleic acids that are not desired to be amplified is likely to occur. According to the present invention, even in such a case where the amplification of non-target nucleic acids is likely to occur, the amplification of the non-target nucleic acids 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, or 4000 copies or more of non-target nucleic acids, and it is possible to specifically detect the target nucleic acid even when the reaction solution contains, for example, 10000 copies or more, further 20000 copies or more, 30000 copies or more, or 40000 copies or more of non-target nucleic acids. 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, in a reaction solution subjected to a nucleic acid amplification process containing 10,000 or more copies, or even 20,000 or more copies, 30,000 or more copies, or even 40,000 or more copies of the target nucleic acid and non-target nucleic acid, respectively.

[0037] [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).

[0038] (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.

[0039] 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.

[0040] (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.

[0041] 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.

[0042] 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.

[0043] PCR enzymes using these DNA polymerases are commercially available, and examples of such enzymes include Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), and Pwo (Roche), any of which can be used in the present invention.

[0044] (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 variants (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.

[0045] (Primer set) As the primer set that can be used in the nucleic acid amplification reaction in step (2), at least one pair of primer sets containing a primer containing the specific mismatch base described above is used. By using such a primer set, for example, even when detecting a single base mutation in a specific region present in the target nucleic acid (i.e., when the base sequence identity between the target nucleic acid and the non-target nucleic acid is relatively high), the amplification of the non-target nucleic acid can be suppressed and the target nucleic acid can be specifically detected. An example of detecting such a single base mutation is, for example, detecting the mutation A2059G, A2058G, A2058T, or A2058C at position 2058 or 2059 of the 23S rRNA gene sequence of Mycoplasma genitalium, and in this case, it is preferable to use a primer set that can amplify the target nucleic acid region including position 2058 or 2059. Even when detecting a single base mutation in other infectious microorganisms, a primer set may be appropriately selected and designed so as to amplify the target nucleic acid region including the position of the single base mutation in the same manner.

[0046] [Process (3)] The embodiment of step (3) is not particularly limited, and can be carried out by any method known in the art. For example, target nucleic acids such as Mycoplasma genitalium can 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 the Mycoplasma genitalium 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) can be reduced. In particular, if the number of unintended mismatches between the target nucleic acid and the probe increases in real-time PCR, 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, and such a situation may induce a false negative. In the case of melting curve analysis, since step (3) is performed after PCR is completed, even if an unintended mismatch occurs in the binding region of the primer or probe, it is possible to detect it as long as the nucleic acid amplification product is finally obtained, so that the effect of unintended mismatches can be suppressed more than in real-time PCR. Therefore, in step (3), it is particularly preferable to detect the nucleic acid amplification product by melting curve analysis. 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).

[0047] 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) In order to obtain a specific and highly sensitive determination result in the detection of the target nucleic acid, it is preferable to use a nucleic acid probe described below that can specifically react with a nucleic acid amplification product that can be generated in the nucleic acid amplification reaction in step (2) to form a complex.

[0048] 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 nucleic acid 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, amplification of non-specific nucleic acids can be suppressed and the target nucleic acid can be specifically detected.

[0049] [Nucleic acid probe that specifically detects target nucleic acid] In one embodiment, the method for detecting a target nucleic acid of the present invention preferably uses a nucleic acid probe that specifically binds to a target nucleic acid amplified by at least one pair of primer sets described above. By using such a nucleic acid probe, it becomes possible to specifically detect a target nucleic acid, for example, in a PCR-melting curve analysis method. For example, such a nucleic acid probe preferably has the following characteristics: (A) a base sequence complementary to the base sequence of the target nucleic acid amplified by the at least one pair of primer sets, or a base sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence; and (B) Only either the 5' or 3' end is labeled.

[0050] The nucleic acid probe that can be used in the present invention is not particularly limited as long as it has the base sequence of (A). However, in the case of a nucleic acid probe labeled with a fluorescent quenching dye that is quenched by interaction with guanine, as described below, it is preferable that at least one terminal base labeled with the dye is cytosine.

[0051] In a specific embodiment, the nucleic acid probe of the present invention may be a base sequence represented by (A) in which 1 to 3 bases have been substituted, deleted, inserted or added. The number of bases that may be substituted, deleted, inserted or added may preferably be 1 or 2. When a base is substituted, deleted, inserted or added in this way, the probe is said to contain a mismatched base, and such a probe containing a mismatched base can also be preferably used in the present invention.

[0052] The nucleic acid probe that can be used in the present invention contains a mismatch base, which means that it contains a base that is not complementary to the base sequence of the target nucleic acid (when the target nucleic acid becomes double-stranded after the nucleic acid amplification reaction, the base sequence of any single-stranded nucleic acid that is dissociated from the double strand). For example, when a cytosine base is present in the base sequence of the target nucleic acid, the base at the position corresponding to the cytosine base in the probe is a base other than guanine (e.g., adenine base, cytosine base, thymine base, and universal base). For example, when detecting a region of the target nucleic acid that is easily mutated, a mismatch base (e.g., a universal base) can be selected at the position of the probe that corresponds to the easily mutated base.

[0053] The nucleic acid probe is preferably one that is labeled at only one of the 5'-end or 3'-end. In one embodiment, the nucleic acid probe of the present invention is preferably labeled to generate quenching or fluorescence when bound to a nucleic acid containing a base sequence that shows 90% or more, preferably 93% or more, more preferably 95% or more identity with a base sequence complementary to the base sequence of the nucleic acid probe, and more preferably labeled to generate quenching. The labeling substance is not particularly limited, but is more preferably a fluorescent dye.

[0054] 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).

[0055] 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.

[0056] 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.

[0057] In a particular preferred embodiment, the nucleic acid probe of the present invention is used in step (3). In this way, the nucleic acid probe of the present invention can be used to specifically detect the target nucleic acid. The method of the present invention may use one type of probe of the present invention, or may use two or more types in combination. In a particular preferred embodiment, the method of the present invention may use one type of labeled probe to specifically detect the target nucleic acid.

[0058] In one embodiment, the step (3) may include at least one of the following steps (3-a), (3-b), and (3-c): (3-a) Simultaneously with step (2), 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 and measuring the fluorescence intensity of the reaction solution, thereby monitoring the progress of the reaction (nucleic acid amplification reaction) of step (2) in real time. (3-b) After completion of step (2), 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 and measuring the fluorescence intensity of the reaction solution, thereby monitoring the progress of the reaction of step (2) (nucleic acid amplification reaction) at an endpoint. (3-c) after completion of the step (2), 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 and measuring the temperature dependence of the fluorescence intensity of the reaction solution. By the step (3-a), (3-b), or (3-c), the formation of a complex formed between a nucleic acid amplification product and a nucleic acid probe can be detected simply, quickly, and with high sensitivity. The steps (3-a), (3-b), and (3-c) may be performed in combination, for example, both steps (3-a) and (3-b), or both steps (3-a) and (3-c) can be performed. In one embodiment, from the viewpoint of more rapid detection of a nucleic acid amplification product, the step (3-b) or (3-c) is preferred. In particular, the step (3-c), i.e., the melting curve analysis method, is preferred.

[0059] (Step (3-a)) Step (3-a) is a method for monitoring the progress of a nucleic acid amplification reaction in real time (so-called real-time PCR method), and quantitative analysis is possible by comparing with a control substance of known concentration.

[0060] (Step (3-b)) In step (3-b), the progress of the nucleic acid amplification reaction is monitored at the end point, thereby enabling rapid detection of the target nucleic acid contained in the specimen. Furthermore, the approximate amount of the target nucleic acid can be estimated by comparing the fluorescence intensity at the end point. For example, the progress of the nucleic acid amplification reaction is monitored at an end point by measuring the fluorescence intensity of a reaction solution containing a nucleic acid probe labeled with a fluorescent quenching dye. After the nucleic acid amplification reaction is completed, the fluorescence intensity of the reaction solution is measured and the fluorescence intensity of the reaction solution after the reaction is compared with the fluorescence intensity of the reaction solution before the reaction, thereby confirming the presence or absence of amplification of the target nucleic acid. Alternatively, the presence or absence of the target nucleic acid in the specimen sample can also be confirmed by comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of a control reaction solution. The control reaction solution is a reaction solution to which a sample known to be negative or positive is added instead of the specimen sample to be measured. The progress of the nucleic acid amplification reaction generally needs to be monitored in real time, but for the purpose of more rapid and convenient detection, it is preferable to measure it at an end point.

[0061] (Step (3-c)) In step (3-c), measuring the temperature dependency of the fluorescence intensity may 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.

[0062] 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.

[0063] In the present invention, it is preferable to perform melting curve analysis in step (3-c) and 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 obtained 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.

[0064] 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.

[0065] [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 Mycoplasma genitalium, Chlamydia trachomatis, etc.). The reagent preferably contains at least one pair of primer sets including a primer containing a specific mismatch base as described above, and a nucleic acid probe as necessary, as well as 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 nucleic acid amplification primer set and the detection nucleic acid probe can contain multiple sets to amplify and detect multiple regions of the target nucleic acid (e.g., multiple regions of Mycoplasma genitalium). In this case, it is sufficient that at least one of the primers contained in the multiple nucleic acid amplification primer sets is designed to contain the specific mismatch base. The concentration of each component can be adjusted appropriately, but for example, the concentration of the nucleic acid probe is preferably 0.01 to 1 μM, more preferably 0.02 to 0.5 μM. When used as a nucleic acid probe set, 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.

[0066] 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.

[0067] [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 nucleic acid to be detected by Mycoplasma genitalium, Chlamydia trachomatis, 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., Mycoplasma genitalium, Chlamydia trachomatis) by including at least one pair of primer sets including a primer containing a specific mismatched base as described above, and a reagent including a nucleic acid probe 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 at least one pair of primer sets, and if necessary, a nucleic acid probe, 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 the method of use of the kit. The kit of the present invention may also include a positive control solution and / or a negative control solution. EXAMPLES

[0068] The present invention will be specifically described below by showing examples, but the present invention is not limited to these examples.

[0069] [Test Example 1: Specific detection evaluation of Mycoplasma genitalium] (1) Sample preparation To evaluate the method for specific detection of Mycoplasma genitalium, crudely extracted genomic DNA consisting of the full-length base sequence of Mycoplasma genitalium and genomic DNA consisting of the full-length base sequence of Mycoplasma pneumoniae were used as templates and diluted with sterile water to prepare 40,000 copies / test. (2) Nucleic acid amplification, melting curve analysis, and determination The above samples were added to the following reagents, and Mycoplasma genitalium and Mycoplasma pneumoniae 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.

[0070] (reagent) The positional relationship between the base sequences of Mycoplasma genitalium (target nucleic acid) and Mycoplasma pneumoniae (non-target nucleic acid) and the primers or probes used is shown in Figure 1 (in Figure 1, the base sequences of SEQ ID NOs: 2 to 4 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 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:1 3.0 μM primers shown in SEQ ID NOs: 2 to 4 0.5 μM of the probe shown by SEQ ID NO:5 (labeled at the 3' end with BODIPY-FL)

[0071] (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.

[0072] (3) Results Figure 2 shows the detection graphs obtained when Mycoplasma genitalium DNA and Mycoplasma pneumoniae DNA were detected by nucleic acid amplification and melting curve analysis using primers shown in SEQ ID NO: 2 to 4. The primer shown in SEQ ID NO: 2 has five different bases from non-target nucleic acids when converted to a forward strand. Therefore, it was thought that the primer of SEQ ID NO: 2 cannot amplify non-target nucleic acids and specific amplification of target nucleic acids would be possible. However, unexpectedly, it was shown that the DNA of different species of Mycoplasma pneumoniae was also amplified and a detection peak was generated. This is probably because the copy number used in the reaction was very high at 40,000, which made non-specific reactions very likely to occur, and it is assumed that even primers such as SEQ ID NO: 2 promoted the reaction of non-target nucleic acids. It is also assumed that the annealing and extension temperatures in the PCR thermal cycle were set low in order to perform sufficient nucleic acid amplification reactions, which promoted such non-specific reactions. On the other hand, as shown in Figure 2, even in cases where such nonspecific reactions are likely to occur, it was surprisingly demonstrated that the target nucleic acid Mycoplasma genitalium can be specifically amplified and detected by using the primer of the present invention, which has a specific mismatch base added to the primer.

[0073] The measurement results using the primers used in this test example are summarized in Table 1. This study demonstrated that by selecting the primers shown in the base sequences of SEQ ID NOs: 3 and 4, which contain mismatched bases, it became possible to detect Mycoplasma genitalium with higher specificity.

[0074] [Table 1]

[0075] [Test Example 2: Evaluation of sensitivity for detection of Mycoplasma genitalium] (1) Sample preparation To evaluate the detection sensitivity of Mycoplasma genitalium when the primer contains a mismatched base, a crudely extracted genomic DNA consisting of the full-length base sequence of Mycoplasma genitalium was used as a template and serially diluted with sterile water to prepare 50 copies / test and 10 copies / test, and used as the sample. (2) Nucleic acid amplification, melting curve analysis, and determination The above samples were added to the following reagents, and Mycoplasma genitalium was 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 if a peak was obtained in the melting curve analysis.

[0076] (reagent) 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 the nucleic acid amplification was performed normally. 0.5 μM primer shown in SEQ ID NO:1 3.0 μM primers shown in SEQ ID NOs: 2 to 4 0.5 μM of the probe shown in SEQ ID NO:5 (labeled at the 3' end with BODIPY-FL)

[0077] (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.

[0078] (3) Results Figure 3 shows a detection graph obtained when Mycoplasma genitalium DNA was detected by nucleic acid amplification and melting curve analysis using the primers shown in SEQ ID NOs: 2 to 4. As shown in Figure 3, clear detection peaks were detected even in low copy numbers such as 50 copies / test and 10 copies / test, and it was confirmed that the sensitivity was almost the same when a mismatch base was added to the primer as when a primer without a mismatch base was used.

[0079] The measurement results using the primers used in this test example are summarized in Table 2. This study demonstrated that even when primers shown in the base sequences of SEQ ID NO: 3 and 4, which contain specific mismatched bases, were used, the target nucleic acid Mycoplasma genitalium could be detected with almost the same sensitivity as when a primer shown in the base sequence of SEQ ID NO: 2, which does not contain a mismatched base, was used.

[0080] [Table 2]

[0081] [Test Example 3: 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 genomic DNA consisting of the full-length base sequence of Chlamydia muridarum were used as templates, diluted with sterile water to prepare 40,000 copies / test, and used as samples. (2) Nucleic acid amplification, melting curve analysis, and determination The above samples were added to the following reagents, and Chlamydia trachomatis and Chlamydia muridarum 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.

[0082] (reagent) The positional relationship between the base sequences of Chlamydia trachomatis (target nucleic acid) and Chlamydia muridarum (non-target nucleic acid) and the primers or probes used is shown in Figure 4 (in Figure 4, the base sequences of SEQ ID NOs: 8 to 12 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 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. 3.0 μM primers shown in SEQ ID NOs: 6 to 7 0.5 μM primers shown in SEQ ID NOs: 8 to 10 0.25 μM of the probe shown in SEQ ID NO: 12 (labeled at the 5' end with BODIPY-FL)

[0083] (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.

[0084] (3) Results FIG. 5 shows a detection graph obtained when the primers shown in SEQ ID NO: 6 to 11 were used in combination to detect Chlamydia trachomatis DNA and Chlamydia muridarum DNA by nucleic acid amplification and melting curve analysis. The primer shown in SEQ ID NO: 6 has three different bases relative to the base sequence of the non-target nucleic acid, and the primers shown in SEQ ID NO: 8 and 9 have five different bases when converted to forward strands. Therefore, it was thought that these primers would not be able to amplify the non-target nucleic acid and would be able to specifically amplify the target nucleic acid, but unexpectedly, it was shown that the DNA of different species of Chlamydia muridarum was also amplified and a detection peak was generated. This result is probably similar to the above Test Example 1, and it is assumed that the number of copies used in the reaction is as high as 40,000, which makes it very easy for non-specific reactions to occur, and that even primers such as SEQ ID NO: 8 and 9 may have promoted the reaction of non-target nucleic acids. It is also assumed that the annealing and extension temperatures in the PCR thermal cycle were set low in order to perform a sufficient nucleic acid amplification reaction, which promoted such non-specific reactions. On the other hand, as shown in Figure 5, even in cases where such non-specific reactions are likely to occur, it has been shown that the use of the primer of the present invention, which has a specific mismatch base added to the primer, enables the target nucleic acid Chlamydia trachomatis to be specifically amplified and detected.

[0085] The measurement results using the primers used in this test example are summarized in Table 3. This study demonstrated that more highly specific detection of Chlamydia trachomatis was possible by selecting the primers shown in the base sequences of SEQ ID NOs: 7, 10, and 11, which contain mismatched bases.

[0086] [Table 3]

[0087] [Test Example 4: Evaluation of sensitivity for detection of Chlamydia trachomatis] (1) Sample preparation To evaluate the detection sensitivity of Chlamydia trachomatis when the primers contain mismatches, crudely extracted genomic DNA consisting of the full-length base sequence of Chlamydia trachomatis was used as a template and serially diluted with sterile water to prepare 50 copies / test and 10 copies / test, and these were used as samples. (2) Nucleic acid amplification, melting curve analysis, and determination The above samples were added to the following reagents, and Chlamydia trachomatis was 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.

[0088] (reagent) 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 the nucleic acid amplification was performed normally. 3.0 μM primers shown in SEQ ID NOs: 6 to 7 0.5 μM primers shown in SEQ ID NOs: 8 to 10 0.25 μM of the probe shown in SEQ ID NO: 12 (labeled at the 5' end with BODIPY-FL)

[0089] (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.

[0090] (3) Results Figure 6 shows a detection graph obtained when Chlamydia trachomatis DNA was detected by nucleic acid amplification and melting curve analysis using primers shown in SEQ ID NOs: 6 to 11. As shown in Figure 6, clear detection peaks were detected even in low copy numbers such as 50 copies / test and 10 copies / test, and no significant decrease in sensitivity was observed even when mismatch bases were added to the primers, confirming that the sensitivity was almost the same as when primers without mismatch bases were used.

[0091] The measurement results using the primers used in this test example are summarized in Table 4. This study demonstrated that even when primers containing specific mismatched bases, such as those shown in the base sequences of SEQ ID NOs: 7, 10, and 11, were used, the target nucleic acid Chlamydia trachomatis could be detected with almost the same sensitivity as when primers containing no mismatched bases, such as those shown in the base sequences of SEQ ID NOs: 6, 8, and 9, were used.

[0092] [Table 4] [Industrial Applicability]

[0093] According to the present invention, for example, in a melting curve analysis method, a target nucleic acid that may be contained in a specimen sample can be detected with high specificity using a simple method. The present invention enables the detection of a target nucleic acid with high reliability, and is expected to greatly contribute to clinical diagnosis and the like.

Claims

1. A method for detecting a target nucleic acid, comprising a step of amplifying the target nucleic acid using at least one pair of primer sets, characterized in that at least one primer of the at least one pair of primer sets comprises a base sequence D complementary to a partial base sequence C of the target nucleic acid comprising three 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 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.

3. 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%.

4. The method of claim 1 , wherein the at least one primer is 15 bases or more in length.

5. The method of claim 1, wherein the at least one primer is 15 to 35 bases in length.

6. The method of claim 1, wherein the base sequence C of the target nucleic acid contains 3 to 7 bases that differ between the base sequence A of the target nucleic acid and the base sequence B of the non-target nucleic acid.

7. The method of claim 1 , wherein at least one mismatched base in the at least one primer is located toward the 3′ end of the primer rather than in the center.

8. The method of claim 1, wherein the number of mismatched bases in the at least one primer is 1 to 3.

9. The method according to claim 1 , further comprising a step of detecting a target nucleic acid using a probe that specifically binds to the target nucleic acid amplified in the target nucleic acid amplification step.

10. The method of claim 9, wherein the probe is labeled with a fluorescent quenching dye that is quenched upon interaction with guanine.

11. The method according to claim 9 , wherein the target nucleic acid detection step comprises performing a melting curve analysis.

12. 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.

13. 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.

14. A primer set for specifically detecting a target nucleic acid, wherein at least one primer of the primer set comprises a base sequence D complementary to a partial base sequence C of a target nucleic acid comprising 3 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, 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.

15. A kit for specifically detecting a target nucleic acid, comprising the primer set according to claim 14.

16. The kit of claim 15, further comprising a probe that specifically binds to a target nucleic acid amplified by the primer set.

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