JP4399025B1 - Nucleic acid primer set, kit for detecting genotype of N-acetyltransferase 2 (NAT2), and detection method using the primer set - Google Patents

Abstract

An object of the present invention is to provide a nucleic acid primer and a detection probe used for the detection in order to provide a simple and inexpensive method for detecting a single nucleotide polymorphism of a NAT2 gene in a short time.
A nucleic acid primer set for amplification of LAMP for detecting a genotype in a single nucleotide polymorphism T341C of a human NAT2 gene, the FIP primer selected from the primer sets 1 to 10 described in Table 5, and When the BIP primer, the FIP primer and the BIP primer are selected from the primer sets 1 to 5 and 10 in Table 5, the sequence consists of the sequence of SEQ ID NO: 39, and the FIP primer and the BIP primer are from the primer sets 6 to 9 in Table 5. When selected, a nucleic acid primer set comprising an F3 primer consisting of the sequence of SEQ ID NO: 41 and a B3 primer consisting of the sequence of SEQ ID NO: 40 is provided.
[Selection] Figure 1

Description

  The present invention relates to a nucleic acid primer set and a detection probe used for detecting a genotype in a single nucleotide polymorphism of the N-acetyltransferase 2 (NAT2) gene.

  N-acetyltransferase 2 (NAT2) is involved in the metabolism of clinically important drugs such as the tuberculosis drug isoniazid (INH) and salazosulfapyridine. Salazosulfapyridine is a therapeutic agent for ulcerative colitis and rheumatoid arthritis.

  It is known that there is a genetic polymorphism in the gene encoding NAT2. The phenotype with high NAT2 activity is called Rapid Acetylator (RA), and the phenotype with low activity is called Slow Acetylator (SA). In Japanese, it is said that there are four types of polymorphisms (NAT2 * 4, NAT2 * 5, NAT * 6, NAT2 * 7) including wild type NAT2 * 4. Mutant alleles NAT2 * 5, NAT * 6, and NAT2 * 7 can be distinguished by examining the genotypes of single nucleotide polymorphisms T341C, G590A, and G857A, respectively. A person who has a homozygote or a complex heterozygote of this mutant allele has a high possibility of SA and has a high probability of occurrence of side effects.

  Thus, testing for mutations in the NAT2 gene is useful to avoid drug side effects. In addition, by clarifying the genotype of the NAT2 gene, drug administration and treatment suitable for each individual can be selected.

  In general, single nucleotide polymorphisms are detected by amplifying a target nucleic acid by a PCR (Polymerase chain reaction) method and detecting each of wild-type and mutant-type amplification products with a specific probe (Non-patent Document). 1). However, PCR requires complicated pretreatments such as nucleic acid extraction. In addition, complicated temperature control like a thermal cycler is essential, and further, there are inconveniences such as a reaction time of 2 hours or more. In addition, since the amplification product by the PCR method is double-stranded, the complementary strand becomes a competitor to the probe during detection, resulting in a problem that the detection sensitivity is lowered. Therefore, in order to make the amplification product into a single strand, a method of decomposing or separating the complementary strand using an enzyme or a magnetic bead has been employed, but in both cases, the operation is complicated and expensive. is there.

Jain K.K., Application of Amplicip CYP450, Mol Diagn 9,119-27 (2005)

  In view of the above problems, an object of the present invention is to provide a nucleic acid primer and a detection probe used for the detection in order to provide a simple and inexpensive method for detecting a single nucleotide polymorphism of the NAT2 gene in a short time. And

  In order to achieve the above object, according to the present invention, a nucleic acid primer set for LAMP amplification for detecting the genotype of the single nucleotide polymorphism T341C of the human NAT2 gene, the primer set 1 described in Table 5 is provided. FIP primer and BIP primer selected from -10, and when the FIP primer and BIP primer are selected from the primer sets 1 to 5 and 10 of Table 5, the sequence consists of SEQ ID NO: 39, and the FIP primer and BIP primer are A nucleic acid primer set comprising an F3 primer consisting of the sequence of SEQ ID NO: 41 and a B3 primer consisting of the sequence of SEQ ID NO: 40 when selected from the primer sets 6-9 of Table 5 is provided.

Here, primer sets 1-10 listed in Table 5 consist of the following primers:
Primer set 1: FIP primer of SEQ ID NO: 33 and BIP primer of SEQ ID NO: 30
Primer set 2: FIP primer of SEQ ID NO: 33 and BIP primer of SEQ ID NO: 31
Primer set 3: FIP primer of SEQ ID NO: 33 and BIP primer of SEQ ID NO: 32,
Primer set 4: FIP primer of SEQ ID NO: 34 and BIP primer of SEQ ID NO: 30
Primer set 5: FIP primer of SEQ ID NO: 34 and BIP primer of SEQ ID NO: 31
Primer set 6: FIP primer of SEQ ID NO: 34 and BIP primer of SEQ ID NO: 32,
Primer set 7: FIP primer of SEQ ID NO: 35 and BIP primer of SEQ ID NO: 32,
Primer set 8: FIP primer of SEQ ID NO: 36 and BIP primer of SEQ ID NO: 32,
Primer set 9: FIP primer of SEQ ID NO: 37 and BIP primer of SEQ ID NO: 32,
Primer set 10: FIP primer of SEQ ID NO: 38 and BIP primer of SEQ ID NO: 32.

  In another embodiment, a kit comprising the above nucleic acid primer set for detecting a genotype in the single nucleotide polymorphism T341C of the human NAT2 gene is provided. The kit may further include a wild type nucleic acid probe and a mutant type nucleic acid probe for detecting an amplification product obtained by amplifying the target nucleic acid using the nucleic acid primer set.

  In another embodiment, the step of amplifying the target nucleic acid using the above nucleic acid primer set and the amount of each of the wild-type amplification product and the mutant-type amplification product contained in the obtained amplification product are measured and compared. A method for detecting a genotype in a single nucleotide polymorphism T341C of the human NAT2 gene comprising the steps of:

  According to the present invention, there are provided a nucleic acid primer set for detecting a genotype in the single nucleotide polymorphism T341C of the NAT2 gene, a kit including the primer set and optionally a probe for detection, and a method for detecting the single nucleotide polymorphism. Is done. According to the present invention, the genotype of the single nucleotide polymorphism T341C in the NAT2 gene can be easily detected at low cost.

Schematic diagram of LAMP method. The schematic diagram which shows the annealing position of the intermediate product of LAMP method, and an inner primer (FIP, BIP). The schematic diagram which shows arrangement | positioning of a loop primer. The schematic diagram which shows the annealing position of the intermediate product of LAMP method and loop primer (LFc, LBc). The schematic diagram which shows the detection position of an amplification product. The plane schematic diagram of one Embodiment of a probe fixed base. The plane schematic diagram of one Embodiment of a probe fixed base. The schematic diagram which shows arrangement | positioning of a FIP primer. Sequence of NAT1 and NAT2 genes. The sequence of the NAT1 and NAT2 genes following FIG. 9A. The electropherogram of amplification by the primer set of this invention. The electropherogram of amplification by the primer set of this invention. Electrophoresis diagram showing non-specific amplification. Test result 1 for G590A detection probe. Test result 2 for G590A detection probe. Test result 3 for G590A detection probe (wild type). Test result 3 for G590A detection probe (mutant type). Test result 3 for G590A detection probe (hetero type). Test result 1 (primer set 1) for the G857A detection probe. Test result 1 for G857A detection probe 1 (primer set 5). Test result 2 for G857A detection probe. Test result 1 for T341C detection probe (primer set 6). Test result 1 for T341C detection probe (primer set 10). Results of simultaneous amplification of G590A and G857A. Results of simultaneous detection of G590A, G857A and T341A (T341C primer set 6). Results of simultaneous detection of G590A, G857A, and T341A (Primer set 10 for T341C).

  The PCR method is often used to detect single nucleotide polymorphisms, but the PCR method has the disadvantages described above. Therefore, in the present invention, a single nucleotide polymorphism is detected using a LAMP (Loop-mediated isothermal amplification) method instead of the PCR method. The LAMP method is a technique for amplifying a nucleic acid at 60 to 65 ° C. under isothermal conditions. The LAMP method has an advantage that a large amount of amplification product can be obtained in a short time compared with the PCR method. It has also been reported that it is less susceptible to impurities in the sample. By using the LAMP method, it is possible to easily amplify the target nucleic acid.

  In the detection method of the present invention, the target nucleic acid is amplified by the LAMP method, and the amounts of the wild-type amplification product and the mutant-type amplification product in the obtained amplification products are measured. When there are many wild-type amplification products, it can be determined that the genotype of the target nucleic acid subjected to the test is a wild type. Conversely, when there are many mutant amplification products, it can be determined that the target nucleic acid is mutant. In addition, when the wild-type and mutant-type amplification products are approximately equal, it can be determined that the gene is a heterogeneous gene.

  The amount of the amplification product is not limited to this, but can be measured using, for example, a nucleic acid probe. As the nucleic acid probe, a nucleic acid probe complementary to the wild type amplification product and a nucleic acid probe complementary to the mutant type amplification product are used. The amplification product and each nucleic acid probe are hybridized, and the amount of the amplification product bound to each nucleic acid probe is measured. The genotype of the target nucleic acid can be determined by comparing the amount of the amplification product bound to the wild-type nucleic acid probe with the amount of the amplification product bound to the mutant nucleic acid probe.

<Outline of LAMP method>
The outline of the LAMP method is described below. In the present specification, nucleic acids (including genomic DNA and the like) subjected to single nucleotide polymorphism detection are referred to as sample nucleic acids. A region in the NAT2 gene amplified by the LAMP method is referred to as a target nucleic acid. A product obtained by the LAMP method is referred to as an amplification product. A solution containing human genomic DNA is referred to as a sample solution.

  In the LAMP method, an F3 region, an F2 region, and an F1 region are set in order from the 5 'end side of the target nucleic acid, and a B3c region, a B2c region, and a B1c region are set in order from the 3' end side. Then, the target nucleic acid is amplified using four kinds of primers as shown in FIG. The F1c, F2c, F3c, B1, B2, and B3 regions indicate regions in the complementary strands of the F1, F2, F3, B1c, B2c, and B3c regions, respectively.

  The four types of primers used to amplify nucleic acids in the LAMP method are (1) having the same sequence as the F2 region at the 3 ′ end and complementary to the F1 region at the 5 ′ end. A FIP primer having a sequence; (2) an F3 primer comprising the same sequence as the F3 region; (3) a sequence complementary to the B2c region on the 3 ′ end side and the B1c region on the 5 ′ end side; A BIP primer having the same sequence; and (4) a B3 primer comprising a sequence complementary to the B3c region. In general, the FIP primer and the BIP primer are called inner primers, and the F3 primer and the B3 primer are called outer primers.

  When LAMP amplification is performed using the above four types of primers, an intermediate product having a dumbbell structure as shown in FIG. 2 is generated. FIP and BIP primers bind to the F2c and B2c regions in the single-stranded loop, and the extension reaction proceeds from the 3 'end of the primer and the 3' end of the intermediate product itself. For details, see Japanese Patent No. 3313358.

  The LAMP method can further shorten the amplification time by optionally using an additional primer called a loop primer. In this case, as shown in FIG. 3, the LF region is set in the portion from the F2 region to the F1 region, and the LBc region is set in the portion from the B2c region to the B1c region. These are referred to as loop primer regions. In addition to the above four types of primers, a loop primer LFc composed of a sequence complementary to the LF region and a loop primer LBc composed of the same sequence as the LBc region are used. For details, refer to WO2002 / 0249028. These loop primers LFc and LBc may be used simultaneously, or only one of them may be used. The loop primer anneals to a different loop than the loop that the FIP and BIP primers anneal, as shown in FIG. 4, to facilitate amplification by providing an additional origin of synthesis.

<Detection of LAMP amplification product; Nucleic acid probe>
When detecting a single nucleotide polymorphism, the detected polymorphic site is located in the FP region or BPc region shown in FIG. Alternatively, different polymorphisms may be located in each of the FP region and the BPc region. As described in FIG. 5, the portion from the F2 region to the F1 region is a portion that becomes a single strand in the amplification product. Similarly, the part from the B2c region to the B1c region is also a single-stranded part in the amplification product. By locating a polymorphic site to be detected in a single-stranded part, detection with a nucleic acid probe can be simplified.

  The nucleic acid probe is designed to bind to the FP region or BPc region containing the polymorphic site. That is, the nucleic acid probe has a sequence complementary to the sequence of the region containing the polymorphic site in the FP region or BPc region.

  In the amplification product, there are also an FPc region and a BP region complementary to the FP region and the BPc region, respectively. Therefore, these FPc region and BP region can be used for detection.

  In the present specification, a nucleic acid probe including a sequence complementary to a wild-type amplification product is referred to as a wild-type nucleic acid probe, and a nucleic acid probe including a sequence complementary to a mutant-type amplification product is referred to as a mutant-type nucleic acid probe.

  The nucleic acid probe is not particularly limited, but may be composed of DNA, RNA, PNA, LNA, methylphosphonate skeleton nucleic acid, or other artificial nucleic acid chain. In order to immobilize the substrate, the terminal may be modified with a reactive functional group such as an amino group, a carboxyl group, a hydrosyl group, a thiol group, or a sulfone group. It is also possible to introduce a spacer between the functional group and the nucleotide. As the spacer, for example, an alkane skeleton, an ethylene glycol skeleton or the like can be used.

<Nucleic acid probe immobilization substrate>
The nucleic acid probe is not limited to this, but can be used by being immobilized on a substrate. For the nucleic acid probe-immobilized substrate, a known device called a DNA chip or DNA microarray may be used.

  A schematic diagram of one embodiment of the probe-immobilized substrate is shown in FIG. The probe is immobilized on the immobilization region 2 on the substrate 1. The substrate 1 can be manufactured from, for example, a silicon substrate, but is not limited thereto. The probe may be immobilized by a known means. One type or a plurality of types of probes may be immobilized on one substrate 1, and the arrangement and number of probes can be appropriately changed by those skilled in the art as needed. As will be described later, when the probe is detected by fluorescence, a probe-immobilized substrate as in this embodiment can be used.

  A schematic diagram of another embodiment of the probe-immobilized substrate is shown in FIG. In the present embodiment, the substrate 11 is provided with the electrode 12. The probe is fixed to the electrode 12. The electrode 12 is connected to a pad 13 for extracting electrical information. The substrate 11 can be manufactured from, for example, a silicon substrate, but is not limited thereto. The manufacture of the electrode and the immobilization of the probe may be performed by known means. The electrode is not particularly limited, but gold, gold alloy, silver, platinum, mercury, nickel, palladium, silicon, germanium, gallium and tungsten, etc., and their alloys, or graphite, glassy carbon As well as these oxides or compounds.

  The immobilization substrate of FIG. 7 includes 10 electrodes, but is not limited thereto, and the number of electrodes arranged on one substrate can be arbitrarily changed. Also, the arrangement pattern of the electrodes is not limited to that shown in the figure, and those skilled in the art can appropriately change the design as necessary. The base 1 may be provided with a reference electrode and a counter electrode as necessary. As will be described later, when the probe is detected electrochemically, a probe-immobilized substrate as in this embodiment can be used.

<Hybridization of nucleic acid probe and amplification product>
Hybridization between the nucleic acid probe and the amplification product is performed under appropriate conditions. Appropriate conditions vary depending on the type and structure of the amplification product, the type of base contained in the detection sequence, and the type of nucleic acid probe. For example, it is carried out in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In the reaction solution, dextran sulfate which is a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, a surfactant and the like may be added. The reaction temperature is, for example, in the range of 10 ° C. to 90 ° C., and the reaction efficiency may be increased by stirring or shaking. For washing after the reaction, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 may be used.

<Detection method>
When the probe immobilized on the substrate and the amplification product are hybridized, a double-stranded nucleic acid is generated. This double-stranded nucleic acid can be detected electrochemically or by fluorescence.

(a) Current detection method A method for electrochemically detecting a double-stranded nucleic acid will be described. In this method, a double-stranded recognizer that specifically recognizes double-stranded nucleic acid is used. Examples of double strand recognizers include, for example, Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalators, bisintercalators such as bisacridine, trisintercalators, polyintercalators, etc. Not. Furthermore, these substances can be modified with an electrochemically active metal complex such as ferrocene or viologen.

  The concentration of the double-stranded recognizer varies depending on the type, but is generally used in the range of 1 ng / mL to 1 mg / mL. In this case, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 may be used.

  During or after the hybridization reaction, a double-stranded recognizer is added to the reaction solution. When a double-stranded nucleic acid is generated by hybridization, a double-stranded recognizer binds to this. Therefore, for example, a reaction current value derived from the double-stranded recognizer can be measured by applying a potential higher than the potential at which the double-stranded recognizer reacts electrochemically. At this time, the potential may be applied at a constant speed, or a constant potential may be applied by applying a pulse. During measurement, the current and voltage may be controlled using a device such as a potentiostat, a digital multimeter, and a function generator. For example, known electrochemical detection means described in JP-A-10-146183 is preferably used.

(b) Fluorescence detection method A method for detecting double-stranded nucleic acid by fluorescence will be described. In advance, the primer is labeled with a fluorescently active substance. Alternatively, detection is performed using a secondary probe labeled with a fluorescently active substance. Alternatively, multiple labels may be used. Fluorologically active materials include, but are not limited to, fluorescent dyes such as FITC, Cy3, Cy5, or rhodamine. The fluorescent substance is detected using, for example, a fluorescence detector. Using a detection device appropriate for the type of label, the labeled detection sequence or secondary probe is detected.

<Selection of nucleic acid primer>
FIG. 8 shows a schematic diagram when the single nucleotide polymorphism G590A is located between F2 and F1, that is, in the FP region. The FIP primer is a primer having the same sequence as the F2 region and a sequence complementary to the F1 region. Various types of primers can be designed as long as the F2 region and the F1 region are located across G590A.

  However, the inventors' study has revealed that the amplification efficiency of the LAMP method varies depending on the type of primer. For example, FIG. 8 illustrates four types of primers. The other three primers (BIP, F3, and B3 primers) are amplified using the common ones. As a result, Primer 1 does not amplify over a sufficient amplification time, for example, 2 hours. Primer 2 is amplified in about 1 hour, but non-specific amplification occurs between the primers. Primer 3 has no non-specific amplification between primers, but amplification takes 1.5-2 hours. Primer 4 does not show any non-specific amplification between primers, and amplification is completed within 1 hour. In such cases, a good primer for amplification is 3 or 4, and the best primer is 4. That is, an excellent primer is a primer that has high amplification efficiency and can be amplified in a short time, and does not show non-specific amplification.

  Furthermore, when an amplification product is detected using a nucleic acid probe, it is desirable that hybridization between the amplification product and the nucleic acid probe occurs with high efficiency. Therefore, whether or not the amplification product is excellent in hybridization efficiency is also considered when evaluating the primer.

  Furthermore, the human NAT2 gene shows very high homology with human NAT1. 9A and B show the sequences of the NAT2 gene and NAT1 gene. Since the NAT2 gene has high homology with the NAT1 gene, it is necessary to design primers in regions where the sequences of NAT2 and NAT1 differ in order to specifically amplify NAT2.

  Therefore, either primer is designed in a region where the sequences differ between NAT2 and NAT1. Preferably, at least one of the F1 region, F2 region, B1 region, and B2 region is designed as a region having a different sequence between NAT2 and NAT1. In addition, as shown in FIG. 9, no primer is designed at a location where a mutation is reported. In some cases, when it is necessary to design a primer at the mutation site, a base of mix or a universal base such as deoxyinosine (dI) is introduced. The other inner primer that does not sandwich a single nucleotide polymorphism is preferably designed in a region where the length from F2 to B2 is 450 bp or less, more preferably 350 bp or less. In addition, it is preferable to design both inner primers so that the length of the single-stranded loop is 100 bp or less, more preferably 70 bp or less.

  Non-specific amplification between primers is a phenomenon often seen in the LAMP reaction. Since the FIP primer contains an F1c region and an F2 region, it becomes a long-chain nucleic acid. Similarly, since the BIP primer also contains a B1c region and a B2 region, it becomes a long-chain nucleic acid. Therefore, there is a high probability that FIP primers, BIP primers, or FIP primer and BIP primer are entangled with each other and amplified using the primer as a template. In addition, since the LAMP reaction includes an F3 primer, a B3 primer, and in some cases, an LFc primer and an LBc primer in the reaction solution, the probability of a nonspecific reaction is further increased as compared with the PCR reaction. When such a nonspecific reaction occurs, the amount of the desired LAMP product using the sample nucleic acid as a template decreases.

  In addition, when a non-specific reaction occurs in a negative control reaction solution to which no sample nucleic acid has been added, white precipitates of pyrophosphate and Mg that are released along with the amplification are caused by contamination, possibly due to non-specific amplification. It is not possible to distinguish whether it is a product. Therefore, it is important to eliminate primers that cause non-specific amplification.

  Therefore, the present inventors conducted a test for selecting an optimal nucleic acid primer for amplifying the single nucleotide polymorphism part of the NAT2 gene single nucleotide polymorphism G590A, and further G857A and T341C.

[Test 1-1: Primer for G590A; FP region]
An amplification reaction was performed at 63 ° C. for 1 hour and 2 hours using six types of nucleic acid primer sets in which the polymorphic site of NAT2 G590A was designed in the FP region. The reaction was carried out with the composition described in Table 1. A human genome was used as the template DNA. In order to confirm the presence or absence of contamination and the presence or absence of non-specific amplification, negative controls were prepared by adding sterilized ultrapure water instead of the human genome in all sets. After the amplification reaction, the amplification product was confirmed by 3% agarose electrophoresis.

The nucleic acid primer sets used are shown in Table 2.

  SEQ ID NO: 8 was used for the F3 primer, and SEQ ID NO: 9 was used for the B3 primer, which were commonly used for all sets.

[Test 1-1: Results]
The amplification products obtained using the six types of primer sets shown in Table 2 were subjected to electrophoresis. The result is shown in FIG. When amplified for 1 hour, sufficient amplification products were obtained with primer sets 3 and 4 (lanes 5 and 6). When amplified for 2 hours, a sufficient amount of amplification product was obtained with primer sets 1, 2, 3, and 4 (lanes 1, 2, 5, and 6). In primer sets 18 and 19 (lanes 3 and 4), there were few amplification products or amplification was not confirmed. Moreover, non-specific amplification was not seen in all sets. From the above, it was found that good primer sets are 1, 2, 3, and 4, and the best primer sets are 3, 4. The results are summarized in Table 2.

[Test 1-2: Primer for G590A; BP region]
An amplification reaction was performed at 63 ° C. for 1 hour and 2 hours using 16 types of primer sets in which the polymorphic site of NAT2 G590A was designed in the BP region. The reaction was carried out with the composition described in Table 1. A human genome was used as the template DNA. In order to confirm the presence or absence of contamination and the presence or absence of non-specific amplification, negative controls were prepared by adding sterilized ultrapure water instead of the human genome in all sets. After the amplification reaction, the amplification product was confirmed by 3% agarose electrophoresis.

The nucleic acid primer sets used are shown in Table 3.

  SEQ ID NO: 18 was used for the F3 primer, and SEQ ID NO: 19 was used for the B3 primer, which were used in common for all sets.

[Test 1-2: Results]
The amplification products obtained using the 16 types of primer sets shown in Table 3 were subjected to electrophoresis. The result is shown in FIG. When amplified for 1 hour, a sufficient amount of amplification product was obtained with primer set 9 (lane 7). When amplified for 2 hours, a sufficient amount of amplification product was obtained with primer sets 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21. No amplification was observed with primer sets 22 and 23. In the primer sets 20 and 21, non-specific amplification as shown in FIG. 12 was observed. From the above, it was revealed that good primer sets were 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16, and the best primer set was 9. The results are summarized in Table 3.

[Test 2: Primer for G857A; BP region]
An amplification reaction was performed at 63 ° C. for 1 hour and 2 hours using six types of primer sets in which the polymorphic site of NAT2 G857A was designed in the BP region. The reaction was carried out with the composition described in Table 1. A human genome was used as the template DNA. In order to confirm the presence or absence of contamination and the presence or absence of non-specific amplification, negative controls were prepared by adding sterilized ultrapure water instead of the human genome in all sets. After the amplification reaction, the amplification product was confirmed by 3% agarose electrophoresis.

The nucleic acid primer sets used are shown in Table 4.

  SEQ ID NO: 27 was used for the F3 primer, and SEQ ID NO: 28 was used for the B3 primer, which were commonly used for all sets.

[Test 2: Results]
The amplification products obtained using the six types of primer sets shown in Table 4 were subjected to electrophoresis. When amplified for 1 hour, a sufficient amount of amplification product was obtained with primer sets 1, 2, 3, 4 and 5. When amplification was performed for 2 hours, a sufficient amount of amplification product was obtained with primer sets 1, 2, 3, 4, and 5 in the same manner. In primer set 6, amplification was not observed even when amplified for 2 hours. Moreover, non-specific amplification was not seen in all sets. From the above, it was revealed that the best primer sets were 1, 2, 3, 4, and 5. The above results are summarized in Table 4.

[Test 3: Primer for T341C; BP region]
The amplification reaction was performed at 63 ° C. for 1 hour and 2 hours using 13 types of nucleic acid primer sets in which the polymorphic site of NAT2 T341C was designed in the BP region. The reaction was carried out with the composition described in Table 1. A human genome was used as the template DNA. In order to confirm the presence or absence of contamination and the presence or absence of non-specific amplification, negative controls were prepared by adding sterilized ultrapure water instead of the human genome in all sets. After the amplification reaction, the amplification product was confirmed by 3% agarose electrophoresis.

The nucleic acid primer sets used are shown in Table 5.

As F3 primer, SEQ ID NO: 39 was used for primer sets 1, 2, 3, 4, 5, 10 , 11 , 12, and 13 . For primer sets 6, 7, 8, and 9, SEQ ID NO: 41 was used. The B3 primer used SEQ ID NO: 40 and was commonly used for all sets.

[Test 3: Results]
The amplification products obtained using the 13 types of primer sets shown in Table 5 were subjected to electrophoresis. When amplified for 1 hour, a sufficient amount of amplification product was obtained with primer sets 3, 6, 9, and 10. When amplified for 2 hours, a sufficient amount of amplification product was obtained with primer sets 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11. In primer sets 12 and 13, amplification was not observed even when amplified for 2 hours. In the primer set set 11, non-specific amplification was observed. In the primer sets 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, no non-specific amplification was observed. This reveals that good primer sets are 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and the best primer sets are 3, 6, 9, and 10. It was. The results are summarized in Table 5.

<Selection of nucleic acid primers for simultaneous amplification of polymorphic sites of G590A and G857A>
Polymorphic sites can also be placed in each of the FP region and the BP region. Thereby, two or more single nucleotide polymorphisms can be measured with one amplification product. For example, in the case of G590A and G857A, G590A can be designed in the FP region, and G857A can be designed in the FPc region. Since G590A and G857A are located slightly apart, the target length (length from F2 to B2) is about 350 bp, and although amplification efficiency is slightly reduced, amplification can be performed within 1 hour by introducing loop primers, etc. it can. As a method for simultaneously amplifying G590A and G857A, there is a method in which each product is multi-amplified in one reaction tube, but in multi-amplification, the condition setting must be strictly controlled. Further, even when strictly controlled, only one product may be preferentially amplified and the other product may not be amplified. Therefore, it is a great advantage that two or more single nucleotide polymorphisms can be measured with one amplification product by the above-described method.

[Test 4: Primer for G590A (FP region), Primer for G857A (BP region)]
Using 11 types of nucleic acid primer sets that designed the NAT2 G590A polymorphic site in the FP region and the NAT2 G857A polymorphic site in the BPc region, amplification reaction was performed at 63 ° C for 1 hour, 1.5 hours, and 2 hours. . The reaction was carried out with the composition described in Table 1. A human genome was used as the template DNA. In order to confirm the presence or absence of contamination and the presence or absence of non-specific amplification, negative controls were prepared by adding sterilized ultrapure water instead of the human genome in all sets. After the amplification reaction, the amplification product was confirmed by 3% agarose electrophoresis.

The nucleic acid primer sets used are shown in Table 6.

  The F3 primer was used in common for all sets, using SEQ ID NO: 49 and the B3 primer using SEQ ID NO: 50.

[Test 4: Results]
The amplification products obtained using the 11 types of primer sets shown in Table 6 were subjected to electrophoresis. When amplified for 1 hour, no amplification was observed in any of the primer sets. When amplified for 1.5 hours, a sufficient amount of amplification product was obtained with primer sets 1, 6, 7, and 8. When amplified for 2 hours, a sufficient amount of amplification product was obtained with primer sets 1, 2, 3, 4, 5, 6, 7, 8, and 10. With primer sets 9 and 11, no amplification was observed even when amplified for 2 hours. In the primer set set 10, nonspecific amplification was observed. In 1, 2, 3, 4, 5, 6, 7, and 8, no non-specific amplification was observed. This revealed that the good primer sets were 1, 2, 3, 4, 5, 6, 7, and 8, and the best primer sets were 1, 6, 7, and 8. The results are summarized in Table 6.

  From the above tests, the preferred inner primer provided in the present invention was determined. As will be apparent to those skilled in the art, the most important primer in the LAMP reaction is the inner primer. An inner primer is essential for the amplification reaction, but an outer primer and a loop primer are not essential. Amplification efficiency is increased by putting the outer primer and loop primer in the amplification reaction solution, but the change in amplification efficiency depending on the position is less than that of the inner primer. Therefore, any sequence may be used as the outer primer (F3 and B3 primer). For example, it is preferably designed to bind to a region within 60 bases from the 5 'end of the F2 region and a region within 60 bases from the 3' end of the B2c region. That is, the F3 region that is the same sequence as the F3 primer is located within 60 bases from the 5 'end of the F2 region, and the B3c region that is complementary to the B3 primer is within 60 bases from the 3' end of the B2c region. It is preferable to be located at. In addition, the loop primer may be designed so as to bind to a loop different from the loop to which the inner primer is bound, and may be designed in a region that does not overlap with the inner primer region.

<Selection of nucleic acid probe>
It is not preferable that the length of the nucleic acid probe is too short or too long. Although there is a difference in binding strength depending on the type of base, in general, the longer the chain length, the higher the binding strength. When the chain length of the nucleic acid probe is too short, the hybridization efficiency between the nucleic acid probe and the amplification product is lowered. On the other hand, when the chain length of the nucleic acid probe is too long, the difference of one base between the wild-type nucleic acid probe and the mutant nucleic acid probe becomes small. Therefore, non-specific binding between the wild-type amplification product and the mutant nucleic acid probe, and non-specific binding between the mutant amplification product and the wild-type nucleic acid probe are increased. Therefore, in order to detect a single nucleotide polymorphism, it is preferable to use a nucleic acid probe having an appropriate chain length such as 10 to 35 bases.

  The binding force can be represented by a temperature Tm value at which the double-stranded nucleic acid dissociates. As a technique for calculating the Tm value, for example, there are a nearest neighbor pairing method, a wallance method, a GC% method, and the like. In the present invention, the closest base pair method (Breslauer et. Al. 1986, Freier et. Al. 1986, Schildkraut et.al. 1965) is adopted. In the present invention, the calculation was performed under the conditions of Na + concentration: 50 mM and nucleic acid probe (oligonucleotide) concentration: 0.5 μM.

  Hereinafter, a test for selecting a nucleic acid probe suitably used for detection of an amplification product obtained according to the present invention was conducted.

[Test 5-1: Probe for G590A detection]
LAMP amplification was performed at 63 ° C. for 1 hour using primer set 9 for G590A detection using a human genome that was found to be heterozygous by PCR-RFLP (Polymerase chain reaction-restriction fragment length polymorphism) analysis as a template. G590A detection primer set 9 is a set that was found to be the best primer as a result of test 1-2 above. The obtained amplification product was detected with a current detection type DNA chip.

Nucleic acid probe:
Table 7 shows the nucleotide sequences of the nucleic acid probes tested. The nucleic acid probe used was a plus strand. The nucleic acid probe was thiol-modified at the 3 ′ end for immobilization on the electrode. A negative control probe having a sequence completely unrelated to the NAT2 gene sequence was used.

Nucleic acid probe immobilization substrate:
A gold electrode was prepared on a DNA chip, and a nucleic acid probe was immobilized thereon. Immobilization was performed using the strong bond between thiol and gold. A probe solution containing a nucleic acid probe whose end was thiol-modified was spotted on a gold electrode, allowed to stand for 1 hour, immersed in a 1 mM mercaptohexanol solution, and then washed with a 0.2 × SSC solution. The same probe spotted two electrodes. After washing, the substrate was washed with ultrapure water and air-dried to obtain a nucleic acid probe-immobilized substrate.

Nucleic acid probes were assigned to each electrode as follows:
1-2 electrode Negative probe (SEQ ID NO: 51)
3-4 electrode Wild type nucleic acid probe 17mer (SEQ ID NO: 52)
5-6 electrode Wild type nucleic acid probe 21mer (SEQ ID NO: 53)
7-8 electrode Wild type nucleic acid probe 26mer (SEQ ID NO: 54)
9-10 electrode Wild type nucleic acid probe 29mer (SEQ ID NO: 55)
11-12 electrodes Mutant nucleic acid probe 19mer (SEQ ID NO: 56)
13-14 electrodes Mutant nucleic acid probe 21mer (SEQ ID NO: 57)
15-16 electrode Mutant nucleic acid probe 23mer (SEQ ID NO: 58)
17-18 electrode Mutant nucleic acid probe 27mer (SEQ ID NO: 59)
19-20 electrode Mutant nucleic acid probe 30mer (SEQ ID NO: 60)
Hybridization and detection of amplification products and nucleic acid probes:
A salt having a final concentration of 2 × SSC was added to the amplification product obtained by the above amplification, and a hybridization reaction was carried out with the nucleic acid probe immobilized on the electrode. The reaction temperature was 35 ° C., 45 ° C., 50 ° C., 55 ° C., 60 ° C., and the reaction was performed for 60 minutes. Thereafter, it was gently washed with ultrapure water. The electrode was immersed in a phosphate buffer containing 50 μM Hoechst 33258 as an intercalating agent for 10 minutes, washed, and the oxidation current response of Hoechst 33258 molecules was measured.

[Test 5-1: Results]
The results are shown in FIG. As the reaction temperature increased, the resulting signal increased. Therefore, it was shown that the higher the reaction temperature, the higher the hybridization efficiency. The reaction temperature was 55 ° C and 60 ° C.

[Test 5-2: Probe for G590A detection]
Next, the reaction temperature was 55 ° C., and the reaction time was 10 minutes, 20 minutes, 40 minutes, 60 minutes, and 120 minutes.

[Test 5-2: Results]
The results are shown in FIG. The results obtained with the reaction time of 10 minutes and 120 minutes were almost the same. This revealed that the hybridization reaction reached saturation in a reaction time of 10 minutes.

  The wild-type nucleic acid probe (17G: SEQ ID NO: 52) and the mutant nucleic acid probe (19A: SEQ ID NO: 56, 21A: SEQ ID NO: 57, 23A: SEQ ID NO: 58) having a relatively short chain length are relatively It was low. The wild-type nucleic acid probe (21G: SEQ ID NO: 53, 26G: SEQ ID NO: 54, 29G: SEQ ID NO: 55) and the mutant nucleic acid probe (27A: SEQ ID NO: 59, 30A: SEQ ID NO: 60) give almost the same high signal. It was.

[Test 5-3: Probe for G590A detection]
Next, hybridization was performed in the same manner as in Test 5-1, with a reaction temperature of 55 ° C. and a reaction time of 20 minutes. Subsequently, the nucleic acid probe-immobilized substrate was washed by immersing in 0.2 × SSC washing buffer at 40 ° C., 45 ° C., and 50 ° C. for 20 minutes. The sample nucleic acids used for amplification in this test were three types of human genomes that were found to be wild homozygous, mutant homozygous, and heterozygous by PCR-RFLP analysis.

[Test 5-3: Results]
The results are shown in FIGS. Without washing (ultra-pure and only lightly washed), wild-type amplification products are detected by the mutant nucleic acid probe, and similarly mutant-type amplification products are detected from the wild-type nucleic acid probe and are non-specific. Hybridization was confirmed. The result at a washing temperature of 40 ° C. was almost the same as that without washing.

  At a washing temperature of 45 ° C., in the wild-type amplification product, a high signal value was detected by the wild-type nucleic acid probe (21G, 26G), and almost no signal value was detected by the mutant nucleic acid probe (27A). Similarly, in the mutant amplification product, a high signal value was detected by the mutant nucleic acid probe (27A), and almost no signal value was detected by the wild type nucleic acid probes (21G, 26G). Therefore, it was shown that the binding due to nonspecific hybridization was released by washing at 45 ° C. In the hetero amplification product, a high signal value was detected by both the wild type nucleic acid probe (21G, 26G) and the mutant type nucleic acid probe (27A).

  At the washing temperature of 50 ° C., the detected current value was low, and it was confirmed that the amplification product was detached from the nucleic acid probe by washing. At a washing temperature of 50 ° C., the wild-type nucleic acid probe (29G) has a signal value of the mutant-type amplification product remaining despite the decrease of the signal value of the wild-type amplification product, and still has non-specific binding. It was shown that. Similarly, the signal value of the wild-type amplification product remained in the mutant nucleic acid probe (30A) even though the signal value of the mutant-type amplification product decreased.

  As a result, it was found that an ideal detection pattern can be obtained by the wild type nucleic acid probe (21G, 26G) and the mutant type nucleic acid probe (27A). Thus, the optimal nucleic acid probes used in accordance with the present invention are wild type nucleic acid probes (21G: SEQ ID NO: 53, 26G: SEQ ID NO: 54) and mutant nucleic acid probes (27A: SEQ ID NO: 59).

  Table 7 also shows the Tm values of the nucleic acid probes used in the test. As can be seen from Table 7, the nucleic acid probe suitably used in the present invention is a wild-type nucleic acid probe having a Tm value of 62 to 70 ° C, preferably 62 to 66 ° C, and a Tm value of 61 to 69 ° C, preferably Is a mutant nucleic acid probe at 66-67 ° C.

[Test 6-1: Probe for G857A detection]
Using a human genome that was found to be heterozygous by PCR-RFLP analysis as a template, LAMP amplification was performed at 63 ° C. for 1 hour using G857A detection primer sets 1 and 5. G857A detection primer sets 1 and 5 are the sets that were found to be the best primers as a result of Test 2 above. The obtained amplification product was detected with a current detection type DNA chip.

Nucleic acid probe:
Table 8 shows the nucleotide sequences of the nucleic acid probes tested. The nucleic acid probe used was a plus strand. The nucleic acid probe was thiol-modified at the 3 ′ end for immobilization on the electrode. A negative control probe having a sequence completely unrelated to the NAT2 gene sequence was used.

Nucleic acid probe immobilization substrate:
The nucleic acid probe-immobilized substrate was prepared in the same manner as in Test 5-1.

Nucleic acid probes were assigned to each electrode as follows:
1-2 electrode Negative probe (SEQ ID NO: 51)
3-4 electrode Wild type nucleic acid probe 20mer (SEQ ID NO: 61)
5-6 electrode Wild type nucleic acid probe 23mer (SEQ ID NO: 62)
7-8 electrode Wild type nucleic acid probe 24mer (SEQ ID NO: 63)
9-10 electrode Wild type nucleic acid probe 25mer (SEQ ID NO: 64)
11-12 electrode Wild type nucleic acid probe 30mer (SEQ ID NO: 65)
13-14 electrode Mutant nucleic acid probe 24mer (SEQ ID NO: 66)
15-16 electrode Mutant nucleic acid probe 26mer (SEQ ID NO: 67)
17-18 electrode Mutant nucleic acid probe 28mer (SEQ ID NO: 68)
19-20 electrode Mutant nucleic acid probe 29mer (SEQ ID NO: 69)
21-22 electrode Mutant nucleic acid probe 31mer (SEQ ID NO: 70)
Hybridization and detection of amplification products and nucleic acid probes:
A salt having a final concentration of 2 × SSC was added to the amplification product obtained by the above amplification, and a hybridization reaction was carried out with the nucleic acid probe immobilized on the electrode. The reaction temperature was 35 ° C., 45 ° C., 50 ° C., 55 ° C., 60 ° C., and the reaction was performed for 20 minutes. Thereafter, it was gently washed with ultrapure water. The electrode was immersed in a phosphate buffer containing 50 μM Hoechst 33258 as an intercalating agent for 10 minutes, washed, and the oxidation current response of Hoechst 33258 molecules was measured.

[Test 6-1: Results]
The results are shown in FIG. FIG. 16A shows the result of primer set 1, and FIG. 16B shows the result of primer set 5. As the reaction temperature increased, the resulting signal increased. Therefore, it was shown that the higher the reaction temperature, the higher the hybridization efficiency. The reaction temperature was 55 ° C and 60 ° C.
As a result of detecting the amplified LAMP product using primer sets 1 and 5 for G857A detection, the signal increase accompanying hybridization was higher in 5 compared to primer set 1 for G857A detection. The reason for the difference in signal increase between primer sets 1 and 5 is unknown. As one of the causes, it is considered that the efficiency of hybridization changes depending on the length of the single-stranded portion in the amplification product and the position where it binds to the nucleic acid probe. Alternatively, the amount of Hoechst 33258 inserted into the amplification product may be slightly different.

[Test 6-2: G857A detection probe]
Next, the product amplified using the primer set 5 was hybridized in the same manner as in Test 5-3 at a reaction temperature of 55 ° C. and a reaction time of 20 minutes. Thereafter, the nucleic acid probe-immobilized substrate was washed by immersion in a 0.2 × SSC washing buffer at 45 ° C. for 20 minutes. The sample nucleic acids used for amplification in this test were three types of human genomes that were found to be wild homozygous, mutant homozygous, and heterozygous by PCR-RFLP analysis.

[Test 6-2: Results]
The results are shown in FIG. In the wild-type amplification product, a high signal value was detected by the wild-type nucleic acid probe (23G, 24G, 25G), and almost no signal value was detected by the mutant-type nucleic acid probe (26A, 28A, 29A). Similarly, in the mutant amplification product, a high signal value was detected by the mutant nucleic acid probe (26A, 28A, 29A), and almost no signal value was detected by the wild type nucleic acid probe (23G, 24G, 25G). In the hetero amplification product, high signal values were detected by both the wild type nucleic acid probe (23G, 24G, 25G) and the mutant type nucleic acid probe (26A, 28A, 29A).

  As a result, it was found that an ideal detection pattern can be obtained with the wild type nucleic acid probe (23G, 24G, 25G) and the mutant type nucleic acid probe (26A, 28A, 29A). Therefore, the optimal nucleic acid probe used in accordance with the present invention is a wild type nucleic acid probe (23G: SEQ ID NO: 62, 24G: SEQ ID NO: 63, 25G: SEQ ID NO: 64) and a mutant nucleic acid probe (26A: SEQ ID NO: 67, 28A: SEQ ID NO: 68, 29A: SEQ ID NO: 69).

  Table 8 also shows the Tm values of the nucleic acid probes used in the test. As can be seen from Table 8, the nucleic acid probe suitably used in the present invention is a wild-type nucleic acid probe having a Tm value of 59 to 68 ° C, preferably 64 to 65 ° C, and a Tm value of 58 to 68 ° C, preferably Is a mutant nucleic acid probe at 64 to 66 ° C.

[Test 7-1: Probe for detecting T341C]
LAMP amplification was performed at 63 ° C. for 1 hour using the T341C detection primer sets 6 and 10 using the human genomes that were found to be wild homozygous, mutant homozygous and heterozygous by sequencing analysis as templates. T341C detection primer sets 6 and 10 are the sets that were found to be the best primers as a result of Test 3 above. The obtained amplification product was detected with a current detection type DNA chip.

Nucleic acid probe:
Table 9 shows the nucleotide sequences of the nucleic acid probes tested. The nucleic acid probe used was a plus strand. The nucleic acid probe was thiol-modified at the 3 ′ end for immobilization on the electrode. A negative control probe having a sequence completely unrelated to the NAT2 gene sequence was used.

Nucleic acid probe immobilization substrate:
The nucleic acid probe-immobilized substrate was prepared in the same manner as in Test 5-1.

Nucleic acid probes were assigned to each electrode as follows:
1-2 electrode Negative probe (SEQ ID NO: 51)
3-4 electrode Wild type nucleic acid probe 16mer (SEQ ID NO: 71)
5-6 electrode Wild type nucleic acid probe 18mer (SEQ ID NO: 72)
7-8 electrode Wild type nucleic acid probe 19mer (SEQ ID NO: 73)
9-10 electrode Wild type nucleic acid probe 20mer (SEQ ID NO: 74)
11-12 electrode Mutant nucleic acid probe 16mer (SEQ ID NO: 75)
13-14 electrode Mutant nucleic acid probe 17mer (SEQ ID NO: 76)
15-16 electrode Mutant nucleic acid probe 18mer (SEQ ID NO: 77)
17-18 electrode Mutant nucleic acid probe 19mer (SEQ ID NO: 78)
19-20 electrode Mutant nucleic acid probe 20mer (SEQ ID NO: 79)
Hybridization and detection of amplification products and nucleic acid probes:
A salt having a final concentration of 2 × SSC was added to the amplification product obtained by the above amplification, and a hybridization reaction was carried out with the nucleic acid probe immobilized on the electrode. The reaction temperature was 55 ° C. for 20 minutes. Thereafter, it was washed at 45 ° C. for 20 minutes. This electrode was immersed in a phosphate buffer containing 50 μM Hoechst 33258 as an intercalating agent for 10 minutes, washed, and the oxidation current response of Hoechst 33258 molecules was measured.

[Test 7-1: Results]
The results are shown in FIG. FIG. 18A shows the result of primer set 6, and FIG. 18B shows the result of primer set 10. The wild type detection nucleic acid probe (18T, 19T) and the mutant type detection nucleic acid probe (17C, 18C, 19C) showed ideal detection patterns. In the wild-type amplification product, a high signal increase was detected from the wild-type detection nucleic acid probe (18T, 19T), and the non-specific hybrid was stripped by the mutation-type detection nucleic acid probe (17C, 18C, 19C), and the signal Little increase was seen. Similarly, in the mutant amplification product, a high signal increase was detected from the nucleic acid probe for mutation detection (17C, 18C, 19C), and non-specific hybrids were detached with the nucleic acid probe for wild type detection (18T, 19T). There was little increase in signal. In the hetero amplification product, a high signal increase was detected from the wild type detection nucleic acid probe (18T, 19T) and the mutant type detection nucleic acid probe (17C, 18C, 19C). In addition, amplification products obtained using T341C detection primer sets 6 and 10 showed almost the same detection pattern.

  As a result, the optimal nucleic acid probe used in accordance with the present invention is the wild type detection nucleic acid probe (18T: SEQ ID NO: 72, 19T: SEQ ID NO: 73) and the mutant type detection nucleic acid probe (17C: SEQ ID NO: 76, 18C: SEQ ID NO: 77, 19C: SEQ ID NO: 78).

  Table 9 also shows the Tm values of the nucleic acid probes used in the test. As can be seen from Table 9, the nucleic acid probe suitably used in the present invention is a wild-type nucleic acid probe having a Tm value of 61 to 69 ° C, preferably 66 to 68 ° C, and a Tm value of 58 to 70 ° C, preferably Is a mutant nucleic acid probe at 63-69 ° C.

[Test 8-1: G590A and G857A detection probe]
LAMP amplification was performed at 63 ° C. using the primer set 6 for detection of G590A and G857A, using the human genome that was found to be wild homozygous, mutant homozygous, and heterozygous by PCR-RFLP analysis as a template. G590A and G857A detection primer set 6 is a set that was found to be the best primer as a result of Test 4 above. The obtained amplification product was detected with a current detection type DNA chip.

Shorten amplification time with loop primers:
The G590A and G857A detection primer sets require nearly 1.5 hours for amplification. Therefore, a loop primer was introduced to verify the shortening of the amplification time. As a loop primer, the LB primer (SEQ ID NO: 80) shown in Table 6 was used, and 40 pmol was added in a 25 μl reaction solution. As a result of amplification, it became clear that amplification was performed within 1 hour by introduction of the loop primer.

DNA chip detection results:
Detection of amplification products of G590A and G857A detection primer set 6 amplified by introducing loop primers using the following nucleic acid probes that have been found to be optimal nucleic acid probes as a result of studies in tests 5 and 6 did.

Nucleic acid probe:
For G590A, the wild type detection probe (SEQ ID NO: 54) and mutant detection probe (SEQ ID NO: 59) shown in Table 7 were used, and for G857A, the wild type detection probe (SEQ ID NO: 62) shown in Table 8 was used. A mutant detection probe (SEQ ID NO: 67) was used. The G590A detection nucleic acid probe was a minus strand, and the G857A detection nucleic acid probe was a plus strand. The nucleic acid probe was thiol-modified at the 3 ′ end for immobilization on the electrode.

Nucleic acid probe immobilization substrate:
The nucleic acid probe-immobilized substrate was prepared in the same manner as in Test 5-1.

Nucleic acid probes were assigned to each electrode as follows:
1-2 electrode Negative probe (SEQ ID NO: 51)
3-4 electrode Wild type nucleic acid probe 26mer (SEQ ID NO: 54)
5-6 electrode Mutant nucleic acid probe 27mer (SEQ ID NO: 59)
7-8 electrode Wild type nucleic acid probe 23mer (SEQ ID NO: 62)
9-10 electrode Mutant nucleic acid probe 26mer (SEQ ID NO: 67)
Hybridization and detection of amplification products and nucleic acid probes:
The same method as in Test 7 was performed.

[Test 8-1: Results]
The results are shown in FIG. By using the G590A wild type detection probe (SEQ ID NO: 54), the mutant type detection probe (SEQ ID NO: 59), the G857A wild type detection probe (SEQ ID NO: 62), and the mutation type detection probe (SEQ ID NO: 67), An ideal detection result was obtained.

<Sample sample>
The specimen targeted by the present invention is not particularly limited, and for example, blood, serum, leukocytes, hair roots, oral mucosa and the like collected from humans can be used. Nucleic acid components are extracted from these specimen samples to prepare a sample solution for use in a target nucleic acid detection test. Although the extraction method is not particularly limited, for example, a commercially available nucleic acid extraction method QIAamp (manufactured by QIAGEN), Sumitest (manufactured by Sumitomo Metals) or the like can also be used.

<Kit>
From another aspect of the present invention, there is provided a kit comprising the above primer set for the LAMP method for use in the detection method of the present invention. The kit can optionally include a strand displacement type DNA synthase, a synthetic substrate, a buffer solution, and the like.

From another aspect, a nucleic acid probe for detecting an amplification product amplified by the primer set for the LAMP method is provided. Furthermore, a nucleic acid probe-immobilized substrate on which the nucleic acid probe is immobilized is provided. The probe-immobilized substrate is preferably provided as a DNA chip or a DNA microarray.
Also provided is the above kit further comprising the nucleic acid probe or nucleic acid probe-immobilized substrate.

  In another embodiment of the present invention, a method for simultaneously detecting NAT2 single nucleotide polymorphisms G590A, G857A and T341C is provided. In this embodiment, as described above, G590A, G857A and T341C are separately subjected to an amplification step, and then the amplification products are mixed to prepare a mixed solution. This mixed solution is subjected to the same detection method as described above. According to this embodiment, the genotypes for G590A, G857A and T341C can be detected simultaneously.

[Simultaneous detection of G590A, G857A, T341C]
A test was performed to detect G590A, G857A, and T341C simultaneously. Using the G590A detection nucleic acid primer set 9 (Test 1-2), G857A detection nucleic acid primer set 5 (Test 2), or T341C detection nucleic acid primer set 6 and 10 (Test 3), the human genome was 63 ° C. For 1 hour. As the human genome, three types of human genomes that were found to be wild homotype, mutant homotype, and heterotype by PCR-RFLP analysis or sequence analysis were used. After the amplification reaction, the three amplification products were mixed to prepare a mixed reaction solution.

Nucleic acid probe:
This mixed reaction solution was subjected to detection using a nucleic acid probe for G590A, a nucleic acid probe for G857A, and a nucleic acid probe for T341C. The nucleic acid probes used are as follows:
G590A wild-type nucleic acid probe (SEQ ID NO: 54), mutant nucleic acid probe (SEQ ID NO: 59),
G857A wild-type nucleic acid probe (SEQ ID NO: 62), mutant nucleic acid probe (SEQ ID NO: 67)
T341C wild-type nucleic acid probe (SEQ ID NO: 72), mutant nucleic acid probe (SEQ ID NO: 76)
All nucleic acid probes were positive strands, and the 3 ′ end was thiol-modified.

Preparation of probe nucleic acid immobilized electrode:
A probe nucleic acid-immobilized electrode was prepared in the same manner as described in Test 5-1 above.

Nucleic acid probes were assigned to each electrode as follows:
1-2 electrode Negative probe (SEQ ID NO: 51)
3-4 electrode G590A wild type nucleic acid probe 26mer (SEQ ID NO: 54)
5-6 electrode G590A mutant nucleic acid probe 27mer (SEQ ID NO: 59)
7-8 electrode G857A wild type nucleic acid probe 23mer (SEQ ID NO: 62)
9-10 electrode G857A mutant nucleic acid probe 26mer (SEQ ID NO: 67)
11-12 electrode T341C wild type nucleic acid probe 18mer (SEQ ID NO: 72)
13-14 electrode T341C mutant nucleic acid probe 17mer (SEQ ID NO: 76)
Hybridization and detection of amplification products and nucleic acid probes:
It carried out similarly to the said test 7.

[result]
The results are shown in FIG. FIG. 20A shows the results using primer sets 9, 5, and 6, and FIG. 20B shows the results using primer sets 9, 5, and 10. An ideal detection pattern was shown for each type of G590A, G857A, and T341C. This indicates that G590A, G857A, and T341C can be detected simultaneously using a DNA chip.

  DESCRIPTION OF SYMBOLS 1 ... Base | substrate, 2 ... Immobilization area | region, 11 ... Base | substrate, 12 ... Electrode, 13 ... Pad

Claims (13)

A nucleic acid primer set for LAMP amplification for detecting the genotype of the single nucleotide polymorphism T341C of the human NAT2 gene,
F3 region, F2 region and F1 region in order from the 5 ′ end side of the target nucleic acid, and B3c region, B2c region and B1c region in order from the 3 ′ end side,
A FIP primer having a nucleic acid primer having the same sequence as the F2 region on the 3 ′ end side and a sequence complementary to the F1 region on the 5 ′ end side, an F3 primer comprising the same sequence as the F3 region, and a 3 ′ end In the case of a BIP primer having a sequence complementary to the B2c region on the side and a BIP primer having the same sequence as the B1c region on the 5 ′ end side, and a B3 primer consisting of a sequence complementary to the B3c region,
FIP primers and BIP primers selected from the primer sets 1 to 10 listed in Table 5,
When the FIP primer and BIP primer are selected from the primer sets 1 to 5 and 10 of Table 5, the sequence consists of the sequence of SEQ ID NO: 39, and the FIP primer and BIP primer are selected from the primer sets 6 to 9 of Table 5 Optionally an F3 primer consisting of the sequence SEQ ID NO: 41, and
B3 primer consisting of the sequence of SEQ ID NO: 40,
A nucleic acid primer set comprising:   The nucleic acid primer set according to claim 1, wherein the FIP primer and BIP primer are selected from primer sets 3, 6, 9 and 10 listed in Table 5.   The nucleic acid primer set according to claim 1, wherein the FIP primer and the BIP primer are primers of primer set 6 or 10 described in Table 5.   The kit which comprises the nucleic acid primer set as described in any one of Claims 1-3 for detecting the genotype in single nucleotide polymorphism T341C of human NAT2 gene. A wild type nucleic acid probe and a mutant type nucleic acid probe for detecting an amplification product obtained by amplifying a target nucleic acid using the nucleic acid primer set;
The wild-type nucleic acid probe is complementary to a wild-type amplification product, has a Tm value of 61-69 ° C., and the single nucleotide polymorphism T341C site is located at least 3 bases from the end. Features
The mutant nucleic acid probe is complementary to the mutant amplification product, has a Tm value of 58 to 70 ° C., and the single nucleotide polymorphism T341C site is located 3 or more bases from the end. The kit according to claim 4, wherein   The wild-type nucleic acid probe consists of the sequence of SEQ ID NO: 72 or 73 or a complementary sequence thereof, and the mutant nucleic acid probe consists of the sequence of SEQ ID NO: 76, 77 or 78 or a complementary sequence thereof. kit.   The kit according to claim 5 or 6, further comprising a nucleic acid probe-immobilized substrate on which the wild-type and mutant nucleic acid probes are immobilized. Amplifying a target nucleic acid using the nucleic acid primer set according to any one of claims 1 to 3,
Genotype detection in the single nucleotide polymorphism T341C of the human NAT2 gene comprising the steps of measuring and comparing the amount of each of the wild-type amplification product and the mutant-type amplification product contained in the obtained amplification product Method. The measurement of the amplification product is performed by hybridizing the nucleic acid probe immobilized on the substrate and the amplification product, and measuring the amount of the amplification product bound to the nucleic acid probe,
9. The nucleic acid probe according to claim 8, wherein the nucleic acid probe is a wild-type nucleic acid probe complementary to the wild-type amplification product and a mutant nucleic acid probe complementary to the mutant-type amplification product. Detection method.   The wild-type nucleic acid probe has a Tm value of 61-69 ° C., the mutant nucleic acid probe has a Tm value of 58-70 ° C., and the single nucleotide polymorphism T341C site is at the end of each nucleic acid probe The detection method according to claim 9, wherein the detection method is located 3 bases or more inside.   The detection method according to claim 10, wherein the wild-type nucleic acid probe has a Tm value of 66 to 68 ° C, and the mutant nucleic acid probe has a Tm value of 63 to 69 ° C.   The wild-type nucleic acid probe consists of the sequence of SEQ ID NO: 72 or 73 or a complementary sequence thereof, and the mutant nucleic acid probe consists of the sequence of SEQ ID NO: 76, 77 or 78 or a complementary sequence thereof. Detection method.   The detection method according to claim 12, wherein the wild-type nucleic acid probe consists of the sequence of SEQ ID NO: 72 or a complementary sequence thereof, and the mutant nucleic acid probe consists of the sequence of SEQ ID NO: 76 or a complementary sequence thereof.

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