JP6460626B2 - Probe preparation method, probe set, and phenotyping gene detection method - Google Patents

Description

  The present invention relates to a probe preparation method, a probe set, and a phenotyping gene detection method.

  In an organism, a gene that determines a phenotype may have a variety of base sequences. For example, RNA viruses, particularly HIV (human immunodeficiency virus) are highly susceptible to mutation because they have reverse transcriptase with low fidelity (accuracy). Therefore, HIV acquires various phenotypes, such as acquiring resistance to anti-HIV drugs, along with gene mutation.

  In recent years, CCR5 inhibitors have been developed as anti-HIV (human immunodefectivity virus) drugs. When HIV invades CD4 positive lymphocytes, it selectively binds to the complement receptor CCR5 (C-C chemokin receptor 5) or CXCR4 (C-X-C chemokin receptor 4) on its cell membrane. Break into. A CCR5 inhibitor selectively binds to CCR5 and changes its conformation to inhibit the entry of CCR5-directed HIV into cells.

  Thus, because the CCR5 inhibitor binds selectively to CCR5 and not to CXCR4, the patient undergoes a tropism assay to confirm that the infecting virus is a CCR5-directed virus. Treatment with CCR5 inhibitors is necessary.

Examples of directivity inspection include genotype inspection and phenotype inspection (see Non-Patent Document 1).
The genotype test is a method for evaluating the directionality of an infecting virus by performing genetic analysis of the HIV env region proliferating in the body and comparing the obtained base sequence with a database or a certain algorithm.
The phenotype test consists of amplifying the env region from the infected virus, reconstructing an infectious virus having the gene in this amplified region, and measuring the HIV-derived protease activity in the presence of a CCR5 inhibitor. This is a method for evaluating the directionality of an infecting virus.
Here, the env region is a region that encodes a protein that forms a shell that covers the virus, and the protein has a role of binding to a receptor on the surface of the host cell and allowing the virus to enter the host cell.

Sing, T., Low, AJ, Beerenwinkel, N., Sander, O., Cheung, PK, Domingues, FS, Buch, J., Daumer, M., Kaiser, R., Lengauer, T., Harrigan, PR , Antivir Ther 12, 1097-1106 (2007).

  However, the above-described directivity inspection requires a time of about one week to one month, and requires a high cost for the inspection, so there is still room for improvement.

  The present invention has been made in view of the above circumstances, and according to the present invention, it is possible to provide a rapid and inexpensive probe preparation method, probe set, and phenotype-determining gene detection method.

  In order to solve the above-described problems, the present inventors have conducted extensive research and found that the problems can be solved through a predetermined probe selection process. One embodiment of the present invention provides the following (1) to (4).

(1) The probe production method in one embodiment of the present invention is a method for producing a probe for specifically detecting a specific phenotyping gene from a group of phenotyping genes having diversity of base sequences, A plurality of fragment sets including a fragment A in a specific phenotyping gene and a fragment B corresponding to the fragment A in another phenotyping gene based on known sequence information of the phenotyping gene group A fragment setting step for setting; and a probe selection step for selecting the fragment set of probe candidates from a plurality of the fragment sets using the Tm values of the fragment A and the fragment B.
(2) The probe set according to an embodiment of the present invention is obtained by using the probe preparation method described above.
(3) The probe set according to one embodiment of the present invention includes a DNA comprising the base sequence represented by SEQ ID NO: 1, a DNA comprising the base sequence represented by SEQ ID NO: 2, and a base represented by SEQ ID NO: 3. It comprises DNA comprising a sequence and DNA comprising a base sequence represented by SEQ ID NO: 4.

(4) The phenotyping gene detection method in one embodiment of the present invention is a method for specifically detecting a specific phenotyping gene from a group of phenotyping genes having diversity of base sequences, It has a detection step of detecting a double-stranded nucleic acid obtained by hybridizing a nucleic acid derived from a typing gene and at least one type of probe selected from the probe set described above.

  According to the present invention, it is possible to provide a rapid and inexpensive probe preparation method, probe set, and phenotyping gene detection method.

It is the figure which showed the one aspect | mode of the process in the probe preparation method of this invention. It is the figure which showed the one aspect | mode of the process in the probe preparation method of this invention. It is the figure which showed the one aspect | mode of the process in the probe preparation method of this invention. It is the figure which showed the one aspect | mode of the process in the probe preparation method of this invention. It is the figure which showed the one aspect | mode of the process in the probe preparation method of this invention. It is the figure which showed the one aspect | mode of the process in the probe preparation method of this invention. It is the result of having plotted the difference of the Tm value in an Example. It is a result based on the linear discriminant in an Example. It is a measurement result of the melting curve in an Example. It is a measurement result of the melting curve in an Example. It is a measurement result of the melting curve in an Example. It is a measurement result of the melting curve in an Example. It is a measurement result of Tm value in an example. It is a graph which shows the relationship between the variation | mutation number of template RNA in an Example, and (DELTA) Tm. It is a graph which shows the relationship between the variation | mutation number of template RNA in an Example, and (DELTA) Tm. It is a graph which shows the relationship between the variation | mutation number of template RNA in an Example, and (DELTA) Tm. It is a graph which shows the relationship between the variation | mutation number of template RNA in an Example, and (DELTA) Tm. It is a graph which shows the relationship between the number of hydrogen bonds and (DELTA) Tm in an Example. It is a graph which shows the relationship between the number of hydrogen bonds and (DELTA) Tm in an Example. It is a graph which shows the relationship between the number of hydrogen bonds and (DELTA) Tm in an Example. It is a graph which shows the relationship between the number of hydrogen bonds and (DELTA) Tm in an Example. It is a graph which shows the linear discrimination | determination result in an Example. It is a graph which shows the evaluation result in an Example. It is a graph which shows the result of the electrophoresis in an Example. It is a graph which shows the result of the electrophoresis in an Example. It is a graph which shows the result of the electrophoresis in an Example. It is a measurement result of the melting curve in an Example. It is a graph which shows Tm value in an Example.

≪Probe fabrication method≫
[First Embodiment]
HIV-1 (human immunogenicity virus type 1) is a CCR5-directed virus that selectively binds to CCR5 (hereinafter also referred to as R5 virus) or CXCR4 that selectively binds to CXCR4 when entering the host cell. It is roughly classified into a directional virus (hereinafter also referred to as X4 virus).
Examples of such a gene group that defines cell directivity (hereinafter also referred to as a phenotype-determining gene group) include a variable region of a coat glycoprotein, particularly a third variable region (V3 region). The V3 region is a genetic region included in the env region described above. HIV is a highly susceptible virus, and amino acid changes in the V3 region caused by genetic mutations affect phenotypes such as cell tropism of the virus. Therefore, the V3 region present in the cells of the infected person can be said to be a phenotype-determining gene group having a variety of base sequences.
In the present embodiment, the detection target is a gene that determines cell directivity of an RNA virus, particularly cell directivity of a human immunodeficiency virus.

  That is, the probe preparation method of the present embodiment is a probe preparation method for specifically detecting a R5 virus determinant gene from a gene group that defines cell directivity existing in a sample of an HIV-1 infected person.

Hereinafter, each process in the probe manufacturing method of this embodiment is demonstrated.
The probe creation method of the present embodiment includes a fragment setting step and a probe selection step.
In the fragment setting step, a fragment set including a fragment A in a specific phenotyping gene and a fragment B corresponding to the fragment A in another phenotyping gene is converted into known sequence information of the phenotyping gene group. Set more than one based on. Here, the specific phenotyping gene is a phenotyping gene that is specifically detected by the probe to be prepared. In this example, the specific phenotyping gene is the R5 virus phenotyping gene. Fragment A is a fragment of a phenotyping gene that is specifically detected by the probe to be prepared. In this example, it is a fragment of an R5 virus phenotyping gene. The other phenotyping genes are phenotyping genes other than phenotyping genes that are specifically detected by the probe to be prepared. In this example, the other phenotyping gene is the X4 virus phenotyping gene. Fragment B is a fragment of a phenotyping gene other than the phenotyping gene that is specifically detected by the probe to be prepared. In this example, it is a fragment of the X4 virus phenotyping gene. The known sequence information of the phenotype determining gene group is the base pair sequence information included in the phenotype determining gene group, and is sequence information stored in advance in a gene database or the like. In this example, the known sequence information of the phenotype-determining gene group is HIV-1 gene sequence information stored in the HIV sequence database.

  Here, a specific procedure in the fragment setting step will be described. In the fragment setting step, gene sequence information of the V3 region of R5 virus is acquired from the gene sequence information of HIV-1 stored in the HIV sequence database. Next, among the obtained gene sequence information of the R5 virus, a fragment A having a plurality of base pairs is set in order from the end of the V3 region. Here, the V3 region contains about 100 base pairs. In this fragment setting step, a plurality of consecutive base pairs are set as one fragment A out of about 100 base pairs included in the V3 region.

  Here, the number of consecutive base pairs is, for example, 10 to 30 pairs. That is, the number of bases of fragment A and fragment B is, for example, 10 to 30. A plurality of fragments A are set by setting the fragments A in order from the base pair at one end to the base pair at the other end of the V3 region.

  In the fragment setting step, the gene sequence information of the V3 region of the X4 virus is acquired from the HIV-1 gene sequence information stored in the HIV sequence database. Next, among the obtained gene sequence information of the X4 virus, a fragment B having a plurality of base pairs is set in order from the end of the V3 region. Also for this fragment B, in the same manner as the fragment A described above, among about 100 base pairs included in the V3 region, a plurality of consecutive base pairs (for example, 10 to 30 pairs) are defined as one fragment B. Set. A plurality of fragments B are set by setting the fragments B in order from the base pair at one end to the base pair at the other end of the V3 region.

In the probe selection step, a fragment set of probe candidates is selected from a plurality of fragment sets using the Tm values of fragment A and fragment B. Here, the Tm (Melting temperature) value includes the melting temperature of DNA.
A specific procedure in this probe selection step will be described. In the probe selection step, first, the difference between the Tm value (first Tm value) of a certain fragment A and the Tm value (second Tm value) of the fragment B corresponding to this fragment A is obtained. Here, the fragment B corresponding to a certain fragment A is a fragment B indicating a base pair at the same position as the base pair position of the V3 region indicated by the certain fragment A among the plurality of fragments B. As an example, when 100 base pairs are included in the V3 region, the base pair at one end of the V3 region is set as the base pair 1, and the base pair at the other end is set as the base pair 100. In this case, when a certain fragment A is a fragment indicating the region from base pair 18 to base pair 45, fragment B corresponding to this fragment A is the base pair 18 to base pair of the plurality of fragments B. Fragment B showing up to 45 regions. In the following description, the position of the base pair having a smaller number in the base pair region in the V3 region of a certain fragment is referred to as the start position of the certain fragment. As an example, when a certain fragment is a fragment showing a region from base pair 18 to base pair 45, base pair 18 is described as the start position of this fragment. Moreover, when a certain fragment is a fragment indicating a region from base pair 18 to base pair 45, this fragment is also referred to as a fragment whose starting position is base pair 18.

Next, with the difference in Tm values obtained as described above on the vertical axis and the start position of the fragment from one end of the V3 region on the horizontal axis, for each start position of fragment A and fragment B, Create a graph plotting the difference in Tm values. Here, one end of the V3 region is, for example, the 5 ′ end of the V3 region. Next, in the created graph, a fragment A and a fragment B in which the position of the base pair at which the difference in Tm value takes the maximum value and the start position coincide with each other are selected from the plurality of fragments A and the plurality of fragments B. .
That is, in the probe selection step, a step of obtaining a difference between a Tm value of a fragment A included in a fragment set and a Tm value of a fragment B among a plurality of fragment sets for each of the plurality of fragment sets, and a plurality of fragments Among the sets, when the position from the 5 ′ end of the fragment set is plotted on the first axis and the difference in Tm value for the fragment set is plotted on the second axis, the difference in Tm value Selecting a fragment set exhibiting a maximum value.

In the selection of the fragment set, the Tm values of the fragment A and the fragment B are used, and the fragment set having a relatively low correlation coefficient p-value among a plurality of fragment sets based on the linear discriminant based on the Mahalanobis generalized distance. Are selected as probe candidates. Here, the linear discriminant based on Mahalanobis's general distance is an expression (2) described later.
As an example, the p value of the correlation coefficient is obtained for each of the plurality of fragment sets selected as described above, and a predetermined number of fragment sets are selected from the one with the lower p value of the obtained correlation coefficient. The p value of this correlation coefficient is the positive discrimination rate when discriminating HIV-1 specimens into the first group (here, the group of X4 virus strains) and the second group (here, the group of R5 virus strains). The lower the p value, the higher the positive discrimination rate. Therefore, if a fragment set having a low correlation coefficient p value is used, it is possible to discriminate whether a sample belongs to the X4 virus strain group or the R5 virus strain group with a high positive discrimination rate. . In addition, the smaller the fragment set for determining the group to which the sample belongs, the faster and cheaper the determination can be made.
As an example, when there are 20 fragment sets having a maximum difference in Tm value and 4 fragment sets are selected from the 20 fragment sets, the correlation coefficient p is set for each of the 20 selected fragment sets. The value is obtained, and four fragment sets are selected from the one with the lower p value of the obtained correlation coefficient.

  In addition, the Tm values of the fragment A and the fragment B described above may be calculated in advance, or may be calculated by conversion in the Tm value conversion step shown below. In this case, the Tm value conversion step converts the Tm value for each of the plurality of fragments A and the plurality of fragments B set in the fragment setting step. In this case, the Tm value may be converted by the closest base pair method.

  By the probe production method of the present embodiment, DNA comprising the base sequence represented by SEQ ID NO: 1, DNA comprising the base sequence represented by SEQ ID NO: 2, and DNA comprising the base sequence represented by SEQ ID NO: 3 Thus, a probe set containing DNA consisting of the base sequence represented by SEQ ID NO: 4 is obtained.

[Second Embodiment]
The probe preparation method of this embodiment is a method used for type determination of viruses other than HIV described above.
The phenotype determining gene according to the present embodiment is, for example, a carcinogenic risk determining gene in HPV (Human papillomavirus). By using a probe set for detecting such a gene, the infectious virus of an HPV-infected person is a low-risk type such as HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, CP 6108 type. It can be determined whether there is a high-risk type such as HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82 type.
In addition, the phenotype determining gene according to the present embodiment is, for example, an interferon (IFN) sensitivity determining gene in HCV (hepatitis C virus). By using a probe set for detecting such a gene, the effect of IFN treatment can be predicted. Examples of HCV genotypes include 1a type, 1b type, 1c type, 2a type, 2b type, 2c type, 3a type, 3b type, 4 type, 5a type, and 6b type. It is determined that the complete remission rate ranges from 80% to 90% except for 50% and 1b.

[Third Embodiment]
The probe production method of the present embodiment utilizes ribosomal small subunit rRNA lineage analysis. The phenotyping gene according to the present embodiment is 16S rRNA in prokaryotes such as bacteria, and 18S rRNA in eukaryotes such as fungi.
The phenotype according to the present embodiment refers to an infectious disease cause such as sepsis. According to this embodiment, it is possible to produce a probe that specifically detects a bacterium having a specific infectious disease cause.

[Fourth Embodiment]
The probe manufacturing method of this embodiment is a method used for typing of HLA (Human Leukocyte Antigen).
The phenotype determining gene according to the present embodiment is, for example, a disease onset risk determining gene. By using a probe set that detects such a gene, whether the subject's HLA is likely to become diabetic (B54, DQB1 * 04: 01, DRB1 * 04: 05, etc.), or a type that is likely to develop neoplastic colitis (DRB1 * 09: 01, DR2, B52, etc.), it is possible to determine whether the type is susceptible to Behcet's disease (B51).

≪Phenotyping gene detection method≫
[First Embodiment]
The phenotyping gene detection method of this embodiment is a method for specifically detecting a specific phenotyping gene from a group of phenotyping genes having diversity of base sequences.
Hereinafter, as an example, each step in a method for specifically detecting an R5 virus-determining gene from a gene group defining cell directivity existing in a sample of an HIV-1 infected person will be described.

The method for detecting a phenotype-determining gene according to this embodiment comprises hybridizing a nucleic acid derived from an R5 virus-determining gene and at least one type of probe selected from the probe set obtained by the first embodiment of the above-described probe production method. And a detection step of detecting a double-stranded nucleic acid.
The nucleic acid derived from the R5 virus determining gene may be an RNA strand or a single-stranded DNA. First, an RNA strand synthesis method will be described.

(Synthesis method of RNA strand)
In this embodiment, first, double-stranded DNA is extracted from a sample. Examples of the sample include serum, lymphocytes, sputum, urine, cerebrospinal fluid, pleural fluid, ascites, etc. As an example, lymphocytes are preferred from the viewpoint of extracting double-stranded DNA derived from HIV-1.
For example, in the case of extracting double-stranded DNA from serum, the following method can be mentioned. A buffer solution is added to the serum sample and reacted at 50 ° C. for a whole day and night. Then, cells are lysed with SDS (Sodium dodecyl sulfate) and RNA is digested with RNase. Next, the protein is digested with proteinase K, and the protein in the extract is removed by phenol extraction. Ethanol is added to the remaining liquid, and DNA is recovered as a precipitate.
In addition, for example, in the case of extracting double-stranded DNA from lymphocytes, the following method can be mentioned. After obtaining a lymphocyte fraction by the Ficoll-paque method, DNA is extracted by the phenol / chloroform method. A specimen containing a large amount of erythrocytes is hemolyzed by adding 10 times the amount of TE buffer, and centrifuged at 10,000 rpm for 10 minutes. This operation is performed twice to extract the DNA after making a pellet sample.
In addition, for example, in the case of extracting double-stranded DNA from cocoons, the following methods can be mentioned. Add 1N NaOH to an equal amount of sample and mix at room temperature for 10 minutes, then add 1M NaH ₂ PO ₄ as it is, or centrifuge at 10000 rpm for 10 minutes. After washing twice with PBS, the resulting residue is dissolved with 40-100 μL of lysozyme solution. Thereafter, the reaction is carried out at 37 ° C. for 30 minutes to 2 hours, and proteinase K is further added at a concentration of 200 μg / ml, and the reaction is carried out at 37 ° C. overnight. Since the sample contains many cells and impurities, DNA extraction is preferably performed using the phenol / chloroform method as an example.
In addition, for example, when extracting double-stranded DNA from urine, spinal fluid, pleural fluid, or ascites, the following methods can be mentioned. The cell components may be pelleted by centrifugation and used as a sample, or may be directly used as a PCR sample without any concentration. For example, a hemolysis operation is performed on a sample containing a large amount of red blood cells. The DNA extraction method from these samples is performed using the phenol / chloroform method as an example.

  Next, a fragment containing a phenotyping gene is amplified from the double-stranded DNA. The nucleic acid amplification reaction, for example, PCR (Polymerase Chain Reaction), LAMP (Loop-Mediated Isothermal Amplification), NASBA (Nucleic Acid Sequence Based Amplification), ICAN (Isothermal and Chimerical primer-initiated Amplification of Nucleic acids), TRC (Transcription Reverse -Transcribation Concerted), SDA (Strand Displacement Amplification), TMA (Transcribion Mediated Amplif) cation), SMAP (SMart Amplification Process), RPA (Recombines polymerase amplification), HDA (Helicase-dependent amplification), and the like.

  As the reagent used for the nucleic acid amplification reaction, it is preferable to use an appropriate reagent for nucleic acid amplification reaction according to the employed nucleic acid amplification reaction. For example, when PCR is employed as the nucleic acid amplification reaction, reagents necessary for each reaction constituting the PCR, that is, dNTP and DNA polymerase are used. As the DNA polymerase, for example, a thermostable DNA polymerase such as Taq DNA polymerase, Tth DNA polymerase, Vent DNA polymerase or the like is used. ) Use functional DNA polymerase. These reagents are commercially available and are readily available.

  An aqueous component containing a template DNA and a reagent necessary for nucleic acid amplification is prepared as a solution suitable for the nucleic acid amplification reaction. For example, it is preferable to use a buffer such as Tris-hydrochloric acid to make a solution having a pH of about 7.0 to pH 9.0 so that the PCR reaction proceeds well.

  In this embodiment, a pair of primer sets that can amplify a fragment containing a phenotyping gene by, for example, a PCR reaction is used.

Next, a promoter is added to the minus strand of the fragment containing the phenotyping gene from the amplified double-stranded DNA. As an example, there may be mentioned a method of performing the nucleic acid amplification reaction using a primer obtained by adding a promoter sequence to the 5 ′ end of a sequence capable of hybridizing to the 3 ′ end of the plus strand of the fragment (see FIG. 1). . Examples of the promoter include T3 promoter, T7 promoter, SP6 promoter and the like, and T7 promoter is preferable from the viewpoint of versatility.
The step of amplifying a fragment containing a phenotype-determining gene from double-stranded DNA and the step of adding a promoter may be performed simultaneously. That is, when a fragment containing a target gene is amplified from double-stranded DNA, an amplification reaction may be performed using a primer having a promoter sequence.

  Next, an RNA chain is synthesized as a nucleic acid derived from the phenotyping gene from the fragment to which the promoter has been added (see FIG. 2). Examples of transcription systems in RNA strand synthesis include conventionally known ones that are transcribed with RNA polymerase. Examples of the RNA polymerase include T7 RNA polymerase.

(Single-strand DNA synthesis method 1)
Next, a method for synthesizing a single-stranded DNA will be described as a method for synthesizing a nucleic acid derived from an R5 virus determining gene.
In this embodiment, a DNA complementary to the RNA is synthesized from the RNA strand obtained by (RNA strand synthesis method) by reverse transcription reaction, and a complex of the DNA and the RNA is formed (FIG. 3). reference). As the reverse transcriptase used for reverse transcription, conventionally known ones are used, and examples include reverse transcriptase derived from Moloney Murine Leukemia Virus. Reverse-transcribed DNA forms a complex with RNA.

Next, the formed DNA-RNA complex is heat-treated to dissociate into an RNA strand and a DNA strand, and the RNA strand is decomposed by RNase treatment to form a single-stranded DNA. The step of synthesizing a single-stranded DNA strand as a nucleic acid derived from the phenotype-determining gene from the complex includes a step of subjecting the complex to heat treatment by adding RNase.
Examples of RNase include RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2i, and RNase V1, and RNase A is preferable from the viewpoint of versatility. Furthermore, for example, bovine pancreas-derived RNase A is preferable because it is inexpensive.

  In this step, for example, RNase is added to subject the complex to a heat treatment (see FIG. 4). From the viewpoint of dissociating DNA and RNA from the DNA-RNA complex by heat treatment and simultaneously decomposing the dissociated RNA, the heating temperature is, for example, 65 ° C to 100 ° C, 80 ° C to 100 ° C, 90 ° C to 90 ° C. 95 ° C. The processing time is, for example, 2 seconds to 1 minute, 2 seconds to 30 seconds, or 2 seconds to 6 seconds. Since RNase A is particularly excellent in heat resistance, it is preferably used in the heat treatment step.

(Single-strand DNA synthesis method 2)
The step of synthesizing the single-stranded DNA strand may include a step of treating the complex with an alkali instead of the above-described RNase treatment step.
Examples of the alkali used for the alkali treatment include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkaline earth metal hydroxides such as barium hydroxide, magnesium hydroxide and potassium hydroxide; alkalis such as sodium carbonate Examples thereof include metal carbonates, such as alkali metal hydroxides, and sodium hydroxide as an example.
The concentration of sodium hydroxide used is, for example, 1 mM to 100 mM, 5 mM to 50 mM, or 10 mM. The pH in the alkali treatment is, for example, 10-14, 12-14. The temperature in alkali treatment is 50-80 degreeC and 60-70 degreeC, for example. The reaction time in the alkali treatment is, for example, 5 to 120 minutes or 10 to 30 minutes.
Through this step, impurities such as unreacted RNA and RNA derivatives can be easily removed in the reverse transcription reaction, and a single-stranded DNA strand complementary to the RNA strand is obtained as a nucleic acid derived from a phenotyping gene. be able to.

(Single-strand DNA synthesis method 3)
As a method for synthesizing single-stranded DNA, a method utilizing an avidin-biotin bond can be mentioned. As an example, as shown in FIG. 5, PCR is performed using double-stranded DNA extracted from a sample as a template. In a pair of primer sets that can amplify a specific region of the template DNA by PCR reaction, one primer is modified with biotin. Of the double strands that are amplification products obtained by reaction using such a primer set, one strand formed by extension of the biotin-modified primer has biotin at the 5 ′ end. Therefore, only the biotin-modified single-stranded DNA can be easily recovered by using avidin-modified magnetic beads. Alternatively, one primer may be modified with avidin and biotin-modified magnetic beads may be used.
Further, the synthesis method is not limited to utilizing an avidin-biotin bond, but one primer is modified with a functional group such as an amino group, an aldehyde group, or an SH group, and the beads are subjected to an amino group, an aldehyde group, You may use what was surface-treated with the silane coupling agent which has an epoxy group etc.

(Single-strand DNA synthesis method 4)
As a method for synthesizing single-stranded DNA, a method using asymmetric PCR can be mentioned. As an example, as shown in FIG. 6, PCR is performed using double-stranded DNA extracted from a sample as a template. In a pair of primer sets (primer A and primer B) that can amplify a specific region of the template DNA by a PCR reaction, a large difference is made in the ratio of the number of primers. In the amplification product obtained by the reaction using such a primer set, a single strand obtained by extending a primer having a small number of molecules (primer B) and a primer having a large number of molecules corresponding to the single strand ( A single strand formed by extension of primer A) forms a double strand. However, the strand formed by extension of the primer A amplified beyond the number of molecules of the primer B exists as a single strand.

  The phenotyping gene detection method of the present embodiment is a double strand obtained by hybridizing the RNA or single-stranded DNA obtained as described above and at least one type of probe selected from the probe set described above. A detection step of detecting a nucleic acid;

Examples of the detection step include a step of detecting the double-stranded nucleic acid using a fluorescent dye.
Examples of the steps using a fluorescent dye include a method using a dye such as Cyber Green or SYTO9 that specifically intercalates double-stranded DNA to emit fluorescence, and a method using a probe to which a fluorescent substance is bound. It is done. Fluorescent substances include FAM (carboxyfluorescein), JOE (6-carboxy-4 ′, 5′-dichloro2 ′, 7′-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX ( 5'-hexachloro-fluorescein-CE phosphoramidite), Cy3, Cy5, Alexa568 and the like.

  Examples of the detection step using a fluorescent dye include a step of detecting the double-stranded nucleic acid using a melting curve. The melting curve refers to a curve obtained by measuring the fluorescence intensity until the double-stranded nucleic acid is denatured into a single-stranded state by gradually raising the temperature from a low temperature. The negative first derivative curve of the melting curve indicates the amount of fluorescence change with respect to the temperature change, and Tm is the place where the change amount is maximum. By using the melting curve, the affinity of the designed probe with the phenotyping gene can be quantified.

  In addition, as a detection step using a fluorescent dye, the above-mentioned phenotype-determining genes derived from each patient are spotted on a substrate, and the substrate is used as a nucleic acid microarray. And the step of measuring by.

  Since the detection process using a fluorescent dye has high sensitivity, it is excellent in that a small amount of nucleic acid sample is required for measurement and simultaneous measurement of multiple samples is easy.

Moreover, as a detection process, the process of detecting the said double stranded nucleic acid using a spectroscopic method is mentioned. Examples of the step using the spectroscopic technique include a step of detecting the double-stranded nucleic acid using the melting curve described above. Tm can be determined by increasing the temperature of the double-stranded nucleic acid and measuring the change in absorbance at 260 nm accompanying the process of dissociating into single strands.
In the detection step using the spectroscopic technique, it is not necessary to use a fluorescent dye, so that the influence of the fluorescent dye can be eliminated. Further, it is excellent in that the measurement is easy because it is not necessary to modify the probe with a fluorescent dye.

  In addition, as the Tm value calculation (conversion) step, the difference between the Tm value of the double-stranded nucleic acid calculated from the melting curve described above and the Tm value of the probe used for forming the double-stranded nucleic acid is calculated. A process is mentioned. The step of discriminating a phenotype-determining gene is based on the calculated Tm value difference and the linear discriminant based on Mahalanobis' general distance, and a specific phenotype-determining gene and other phenotype-determining genes A step of discriminating is mentioned.

  As described above, according to the present embodiment, by analyzing the interaction between the probe and the target nucleic acid, the sequence of the target nucleic acid can be analyzed without analyzing the base sequence using a DNA sequencer.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to a following example.

[Design of HIV directivity inspection probe]
Loss Alamos National Laboratory HIV Sequence Database (Los Alamos National Laboratory HIV Sequence Database) has already shown the directivity of HIV-1 subtype B from the sequence information of V3 region (R5 virus strain and 47 derived from 612 samples) (Base sequence information of V3 region of X4 virus strain derived from specimen) was obtained.
Next, in the obtained base sequence information, a fragment of 16 to 26 bases is set from the 5 ′ end of each base sequence, and the Tm value of the corresponding fragment in the virus derived from each specimen is calculated by the following formula ( 1).

In formula (1), ΔH represents the sum of nearest enthalpy changes in the hybrid [kcal / mol], ΔS represents the sum of nearest neighbor entropy changes in the hybrid [cal / mol · K], and R is gas It represents a constant (1.987 cal / deg · mol), Ct represents the total molar concentration of the nucleic acid [mol / l], and Na ⁺ represents the molar concentration of Na ⁺ [mol / l].

The difference of the Tm value calculated in the corresponding fragment (henceforth a fragment set) of R5 virus strain and X4 virus strain was plotted. The results are shown in FIG. The horizontal axis of FIG. 7 represents the number of the base at the 5 ′ end of each fragment when the 5 ′ end base of the V3 region is 1, and the vertical axis of FIG. The difference of Tm value in each site | part of V3 area | region is represented.
In the graph of FIG. 7, 20 types of fragment sets showed maximum values, and these fragment sets were used as probe candidates.

  Using the Tm value calculated based on the above formula (1) for the 20 probe candidate fragment sets, the p value is calculated based on the linear discriminant based on the Mahalanobis generalized distance expressed by the following formula (2). did.

In Expression (2), x represents a multivariable vector, ￣Xk (X Bar Kay) represents an average of the multivariable vector x, and Sk (S Kay) represents a covariance matrix of the multivariable vector x. . The equation (2), a group of X4 virus strains as the first group, when the group of R5 virus strain is a second group, when representing the sample of HIV-1 by a multi-variable vector x, D ₂ ² if ≧ _D ^{1 2,} the sample belongs to the group of the first group, i.e. X4 virus _strains, if ^D 2 2 _<D ^{1 2,} the sample belonging to groups of the second group, i.e. R5 virus strains It shows that. That is, it is possible to determine whether the specimen belongs to the group of the X4 virus strain or the group of the R5 virus strain by the sign of z in the following formula (3).

This expression (3) can also be expressed by a linear expression such as the following expression (4).

This equation (4) can be used as a discriminant for discriminating whether the specimen belongs to the group of X4 virus strains or the group of R5 virus strains. At this time, by selecting a fragment set having a relatively low p-value of the correlation coefficient from the 20 types of fragment sets, it is possible to simplify the discriminant while reducing a decrease in discrimination accuracy. Here, for each of the 20 types of fragment sets, the p values are calculated using equations (1) to (4), and among the calculated p values, the 4 types of fragment sets from the lower p value are selected as HIV directivity. Selected as inspection probes (X18, X33, X75, X80). FIG. 8 shows the discrimination results by the four selected fragment sets.
Among the calculated p values, four types of fragment sets having the lowest p value were selected as HIV directivity test probes (X18, X33, X75, X80).
X18 represents the following sequence.
CAATACAAGAAAAAGTATACATATAGGG (SEQ ID NO: 1: 27mer)
X33 represents the following sequence.
AAGTATACATATAGGACCAGGG (SEQ ID NO: 22 mer)
X75 represents the following sequence.
TGCAACAGGAGAAATAATAG (SEQ ID NO: 3: 20mer)
X80 represents the following sequence.
CAGGAGAAATAATAGAGGATATAAG (SEQ ID NO: 25 mer)

[Extraction of Genomic DNA]
Ficoll-Paque PLUS (manufactured by GE Healthcare Bioscience) was layered with blood of HIV patients (36 samples), centrifuged at room temperature and 1500 rpm for 40 minutes, and a lymphocyte layer was collected.
10 mL of PBS (phosphate buffered saline) containing 2% FCS (fetal calf serum) was added to the collected lymphocytes, centrifuged at room temperature and 1200 rpm for 10 minutes, and the supernatant was removed. The precipitate was dissolved in 5 mL of PBS containing 2% FCS, and then centrifuged at 1000 rpm for 10 minutes at room temperature, and the supernatant was removed.
Subsequently, the genomic DNA was extracted from the precipitate using QIAamp DNA Blood Mini Kit (manufactured by QIAGEN). Specifically, the precipitate was suspended in 200 μL PBS, 20 μL QIAGEN protease and 200 μL Buffer AL were added, and incubated at 56 ° C. for 10 minutes to lyse lymphocytes. To this was added 200 μL ethanol, and the entire solution was transferred to a spin column and centrifuged at 8000 rpm for 1 minute. The spin column was transferred to a new collection tube, 500 μL Buffer AW1 was added to the column, and the mixture was centrifuged at 8000 rpm for 1 minute. Further, the spin column was transferred to a new collection tube, 500 μL Buffer AW2 was added to the column, and centrifuged at 8000 rpm for 1 minute. Further, the spin column was transferred to a new collection tube and centrifuged at 14000 rpm for 1 minute. After the spin column was transferred to a 1.5 mL tube, 200 μL TE buffer was added to the column, incubated at room temperature for 1 minute, and centrifuged at 8000 rpm for 1 minute to collect a Genomic DNA lysate.

[1st PCR]
About 420 bp (7377-6955) of the C2V3 region of HIV-1 was amplified by PCR. A reaction solution was prepared so as to have the composition shown in Table 1 and held at 94 ° C. for 1 minute, followed by 5 cycles of 3-step PCR at 94 ° C. for 10 seconds, 48 ° C. for 10 seconds, and 72 ° C. for 30 seconds. 25 cycles of 3-step PCR of 5 seconds, 52 ° C. for 10 seconds, and 72 ° C. for 30 seconds were held at 72 ° C. for 1 minute, and then cooled at 4 ° C. The primer sequences used are shown below.
(1) env 6955-F (21mer, 20 μM)
[Sequence: 5′-CAGTACAATGYACACATGGGAA-3 ′] (SEQ ID NO: 5)
(2) env7377L-R (26mer, 20 μM)
[Sequence: 5′-TAGAAAAATTCCCCCTCYACAATTAAA-3 ′] (SEQ ID NO: 6)

[2nd PCR]
Using 1 μL of the reaction solution obtained by 1st PCR, about 330 bp (7337-7005) of C2V3 region of HIV-1 was amplified by PCR. A reaction solution for 2nd PCR was prepared so as to have the composition shown in Table 2, and 1 μL of the reaction solution obtained by 1st PCR was added and held at 94 ° C. for 1 minute, then 94 ° C. for 5 seconds, 48 ° C. for 10 seconds, 5 cycles of 3-step PCR at 72 ° C for 30 seconds, 30 cycles of 3-step PCR at 94 ° C for 5 seconds, 60 ° C for 10 seconds, 72 ° C for 30 seconds, held at 72 ° C for 1 minute, and then cooled at 4 ° C . The primer sequences used are shown below.
(3) env7005-F (20mer, 20μM)
[Sequence: 5′-TTAAATGGCAGTTCTAGCAGA-3 ′] (SEQ ID NO: 7)
(4) env7337B-R (20 mer, 20 μM)
[Sequence: 5′-AATTTCTGGGTCCCCTCCTG-3 ′] (SEQ ID NO: 8)
(5) env7337E-R (20mer, 20μM)
[Sequence: 5′-AATTTCTAGATCTCCCTCTG-3 ′] (SEQ ID NO: 9)

[3rdPCR]
50 μL of the reaction solution obtained by 2nd PCR was purified using QIAquick PCR Purification Kit (manufactured by QIAGEN) to obtain 50 μL of lysate. After this lysate was diluted 10 times with water, a 3rd PCR reaction solution was prepared to have the composition shown in Table 3, 10 μL of the 10-fold diluted lysate was added, and held at 94 ° C. for 1 minute. Then, 30 cycles of 3-step PCR at 94 ° C. for 5 seconds, 60 ° C. for 10 seconds, and 72 ° C. for 15 seconds were held at 72 ° C. for 1 minute, and then cooled at 4 ° C. The primer sequences used are shown below. (6) env-bridge-F (30 mer, 20 μM)
[Sequence: 5′-ATCTTGTAGAAATTAATTTGTACAAGACCCAA-3 ′] (SEQ ID NO: 10)
(7) env-T7-R (40 mer, 20 μM)
[Sequence: 5′-TAATACGACTCACTATAGGGATGTTTACAATGGCTTGTCT-3 ′] (SEQ ID NO: 11)

  By 3rd PCR, an amplification product having a T7 promoter at the 5 ′ end of the minus strand of the double-stranded DNA obtained by 2nd PCR was obtained. In this embodiment, PCR is divided into three times, but may be performed once at a time.

[Synthesis of RNA]
RNA was synthesized from the DNA obtained by 3rd PCR using RiboMAX (trade name) Large Scale RNA Production Systems (Promega).
First, a reaction solution was prepared so as to have the composition shown in Table 4, and then reacted at 37 ° C. for 2 hours.

  After the reaction, 1 μl of RNase-Free DNase (Promega) was added and incubated at 37 ° C. for 30 minutes. After completion of the incubation, purification was performed using a QIAamp MinElute Virus Spin Kit (Qiagen).

[Measurement of Melting Curve 1]
A reaction solution was prepared using the synthesized RNA so as to have the composition shown in Table 5, and then a melting curve was measured using a StepOnePlus (trade name) real-time PCR system (manufactured by Applied Biosystems). The Run method was 65 ° C. for 15 minutes / Mel curve stage / 20 ° C. for 1 minute / 95 ° C. for 15 seconds. The temperature was raised from 20 ° C. to 95 ° C. over 90 minutes.

  In Table 5, SYTO9 means SYTO9 Solution contained in the MeltDoctor (trade name) HRM master mix reagent kit. The measurement result of the melting curve at the time of using X18 is shown to FIGS. FIG. 9 shows the results when the number of mutations in RNA used as a template was zero, FIG. 10 shows the results when the number of mutations in RNA used as a template was one, and FIG. 11 shows the results when used as a template. FIG. 12 shows the results when the number of RNA mutations is two, and FIG. 12 shows the results when there are many RNA mutations used as templates. As shown in FIGS. 9 to 12, the melting curve peak (Tm value) shifted to the left as the mutation of the RNA used as the template increased, and it was confirmed that the peak disappeared when there were many mutations.

The Tm value was measured using each HIV directivity test probe (X18, X33, X75, X80) and each patient-derived RNA, and the difference from the Tm value of the wild type R5 strain was calculated. The results are shown in FIG. In FIG. 13, ID indicates the sample number of each patient. In FIG. D. Means that the Tm value was not obtained for many mutations.
Moreover, based on FIG. 13, the graph which showed the relationship between the variation | mutation number of the template RNA at the time of using each probe and the said Tm value difference ((DELTA) Tm) is shown to FIGS. As shown in FIGS. 14 to 17, the correlation coefficients in each graph are −0.87, −0.83, −0.75, and −0.77, and there is a correlation between the number of template RNA mutations and ΔTm. It was confirmed that there was. N. D. In this case, ΔTm was assumed to be −25 ° C.
Similarly, graphs showing the relationship between the number of hydrogen bonds and ΔTm in each probe are shown in FIGS. As shown in FIGS. 18 to 21, the correlation coefficients in each graph are −0.85, −0.82, −0.78, and −0.87, and there is a correlation between the number of hydrogen bonds and ΔTm. Was confirmed. N. D. In this case, ΔTm was assumed to be −25 ° C.

[Linear discrimination of Tm value by Mahalanobis generalized distance]
Using each Tm value, the Z values represented by the above formulas (3) and (4) were calculated based on the linear discriminant based on the Mahalanobis' general distance represented by the above formula (2). The linear discrimination result is shown in FIG. In FIG. 22, the horizontal axis indicates the sample number, and the vertical axis indicates the Z value. As shown in FIG. 22, it was confirmed that the R5 virus and the X5 virus were distinguished with high accuracy.
Further, FIG. 23 shows a comparison with the discrimination result by the algorithm Geno2pheno (also referred to as g2p) for identifying directivity. As shown in FIG. 23, it was confirmed that the linear discrimination based on the maharabinos pan distance showed almost the same result as the discrimination result based on Geno2pheno.

[Synthesis of DNA-RNA hybrid]
Using the RNA obtained in the above [Synthesis of RNA], a reaction solution was prepared so as to have the composition shown in Table 6, held at 65 ° C. for 5 minutes, and then placed on ice for 2 minutes. The primer sequences used are shown below.
(8) env-bridge-F (30 mer, 20 μM)
[Sequence: 5′-ATCTTGTAGAAATTAATTTGTACAAGACCCAA-3 ′] (SEQ ID NO: 10)

  Next, 4 μL of 5 × First-Strand Buffer, 1 μL of 0.1 M DTT, and 1 μL of Super Script (registered trademark) III reverse transcriptase were added to the above reaction solution, reacted at 50 ° C. for 15 minutes, and then 1 μL of 500 mM EDTA. In addition, the reaction was stopped and a DNA-RNA hybrid was synthesized.

[Synthesis of single-stranded DNA 1 (RNase treatment)]
A reaction solution was prepared using the DNA-RNA hybrid so as to have the composition shown in Table 7, heated at 94 ° C. for 5 seconds, and then maintained at 37 ° C. for 15 minutes. 10 μm of the reaction solution was subjected to electrophoresis using a 2% agarose gel to confirm that RNA in the reaction solution was decomposed. The results are shown in FIG.

In FIG. 24, RNA was migrated from the leftmost lane; DNA-RNA hybrid was electrophoresed; DNA-RNA hybrid was heated at 94 ° C. for 5 seconds, then held at 37 ° C. for 15 minutes, and electrophoresed ; RNase added to DNA-RNA hybrid, kept at 37 ° C for 15 minutes, then migrated; RNase added to DNA-RNA hybrid, heated at 94 ° C for 5 seconds, held at 37 ° C for 15 minutes, then migrated Show things.
As shown in the rightmost lane of FIG. 24, it was confirmed that RNA was degraded by adding RNase to the DNA-RNA hybrid, heating at 94 ° C. for 5 seconds, and holding at 37 ° C. for 15 minutes.

[Confirmation of single-stranded DNA (plus strand)]
In order to confirm that the single-stranded nucleic acid obtained in [Synthesis of single-stranded DNA 1 (RNase treatment)] is DNA (plus strand), the reaction solution was prepared so as to have the composition shown in Table 8. After preparation, after holding at 94 ° C. for 1 minute, one cycle of 3-step PCR of 94 ° C. for 5 seconds, 60 ° C. for 10 seconds, 72 ° C. for 15 seconds was held at 72 ° C. for 1 minute, and then cooled at 4 ° C. . 20 μL of the reaction solution was subjected to electrophoresis using a 2% agarose gel. The results are shown in FIG.
In Table 8, 2 μM Primer represents one of the following sequences.
(9) env-bridge-F (30 mer, 2 μM)
[Sequence: 5′-ATCTTGTAGAAATTAATTTGTACAAGACCCAA-3 ′] (SEQ ID NO: 10)
(10) env-bridge-R (31 mer, 2 μM)
[SEQ ID NO: 12] [Sequence: 5′-TTGCTCTACTACATGTTACAATGTGCTTGTCT-3 ′]

In FIG. 25, from the leftmost lane, an untreated sample in which only template DNA was migrated (indicated as “untreated” in FIG. 25); a reaction product in which water was added instead of 2 μM Primer (in FIG. 25, No reaction); reaction product added with primer (env-bridge-F) (shown as (+) in FIG. 25); reaction product added with primer (env-bridge-R) (Shown as (-) in FIG. 25).
As shown in the rightmost lane of FIG. 25, since a band was detected from the reaction product to which the primer (env-bridge-R) was added, it was obtained by [Synthesis of single-stranded DNA 1 (RNase treatment)]. It was confirmed that the single-stranded nucleic acid was DNA (plus strand).

[Synthesis of single-stranded DNA 2 (alkali treatment)]
A final concentration of 10 mM NaOH and a final concentration of 2 mM MgCl ₂ are added to the DNA-RNA hybrid, and the mixture is reacted at 60 ° C. for 15 minutes. The reaction solution is electrophoresed using a 2% agarose gel. The strand was confirmed to be DNA. The results are shown in FIG.

In FIG. 26, from the leftmost lane, DNA-RNA hybrids subjected to alkali treatment and migrated (untreated); DNA-RNA hybrids subjected to alkali treatment and DNase treatment and then migrated (DNase) are shown.
As shown in FIG. 26, since the band seen in the leftmost lane disappeared by DNase treatment, it was confirmed that the single-stranded nucleic acid obtained by alkali treatment was DNA.

[Measurement of melting curve 2]
Using the synthesized single-stranded DNA, prepare a reaction solution to have the composition shown in Table 9, and then measure the melting curve using StepOnePlus (trade name) real-time PCR system (Applied Biosystems). went. The Run method was 65 ° C. for 15 minutes / Mel curve stage / 20 ° C. for 1 minute / 95 ° C. for 15 seconds. The temperature was raised from 20 ° C. to 95 ° C. over 90 minutes.

  In Table 9, SYTO9 means SYTO9 Solution contained in the MeltDoctor (trade name) HRM master mix reagent kit. The measurement results of the melting curve when X18 is used are shown in FIG. FIG. 27 shows the results when the number of DNA mutations used as a template is _, and it was confirmed that the Tm value can be measured with the same accuracy as when RNA was used as a template.

[Measurement of melting curve by spectroscopic method]
Using each of the synthesized mRNA and single-stranded DNA, melting curves were measured using a spectrophotometer (manufactured by JASCO Corporation; V-650 spectrophotometer). The measurement conditions are shown below.

Starting temperature 30-40 ° C (changed according to expected Tm value)
Target temperature 60-70 ° C (change according to expected Tm value)
Gradient 2.0 ℃ / min
Data capture interval 0.3 ° C
Measurement start condition When set temperature is within ± 0.1 ℃ for 10sec. Stirrer 0rpm
Metering mode Abs
UV / Vis bandwidth 2.0 nm
Response Medium
Measurement wavelength 260 nm
Light source switching 350 nm
Light source D2 / WI

  FIG. 28 shows a graph of Tm values when X18, X33, X75, and X80 are used as HIV directivity inspection probes, respectively. As shown in FIG. 28, it was confirmed that the increase / decrease tendency of the Tm value when each probe was used was the same as the tendency when measured using SYTO 9 in [Measurement of Melting Curve 1].

Claims (18)

A method of making a probe that distinguishes between two phenotypes,
A fragment A in a phenotyping gene defining the phenotype in a virus having one phenotype, and a fragment A in a phenotyping gene corresponding to the phenotyping gene in a virus having the other phenotype A fragment setting step of setting a plurality of fragment sets including a fragment B at a position corresponding to a position based on known sequence information of the phenotyping gene group;
A probe selection step of selecting the fragment set of probe candidates from a plurality of the fragment sets using Tm values of the fragment A and the fragment B;
Including
The probe selection step for each of the plurality of fragment sets, obtaining a difference between the Tm value of fragment A and the Tm value of fragment B;
For each of the plurality of fragment sets, the position of the 5 ′ end of the fragment set from the 5 ′ end of the phenotyping gene is plotted on the first axis, and the difference in Tm value is plotted on the second axis. And a step of selecting a plurality of the fragment sets indicating the maximum value of the difference in the Tm values;
For each of the selected fragment sets, using the Tm value, a correlation coefficient p value is obtained based on a linear discriminant based on Mahalanobis's general distance, and the fragment set having a relatively low correlation coefficient p value is obtained. Selecting as the probe candidate;
A method for producing a probe, comprising:   2. The probe manufacturing method according to claim 1, further comprising a Tm value conversion step of converting Tm values of the fragment A and the fragment B. The method for producing a probe according to claim 1 or 2 , wherein the phenotype is a cell tropism of an RNA virus. 4. The probe production method according to claim 3 , wherein the RNA virus is a human immunodeficiency virus. The probe production method according to claim 4 , wherein the phenotyping gene is an env gene, and the fragment A and the fragment B are part of a V3 region. The probe preparation method according to any one of claims 1 to 5 , wherein the fragment A and the fragment B have 10 to 30 bases. Probe Preparation The method according to any one of claims 1 to 6, characterized in that the Tm value is determined by the nearest neighbor base pair method.   A probe for identifying the cell directivity of a human immunodeficiency virus, which comprises DNA comprising the base sequence represented by any of SEQ ID NOs: 1 to 4.   DNA comprising the base sequence represented by SEQ ID NO: 1, DNA comprising the base sequence represented by SEQ ID NO: 2, DNA comprising the base sequence represented by SEQ ID NO: 3, and base represented by SEQ ID NO: 4 A probe set for identifying cell directivity of a human immunodeficiency virus, comprising DNA comprising a sequence. A method for detecting cell tropism of an RNA virus, comprising:
A nucleic acid containing a phenotyping gene that defines cell directivity of the RNA virus, a probe obtained by using the probe production method according to any one of claims 1 to 7 , or a probe according to claim 8 A method for detecting a phenotype-determining gene, comprising a detection step of detecting a double-stranded nucleic acid hybridized with. The method for detecting a phenotype-determining gene according to claim 10 , wherein the double-stranded nucleic acid is detected using a fluorescent dye in the detection step. The method for detecting a phenotype-determining gene according to claim 10 , wherein the double-stranded nucleic acid is detected using a spectroscopic technique in the detection step. The phenotyping gene detection method according to any one of claims 10 to 12 , wherein the detection step detects the double-stranded nucleic acid using a melting curve. Difference in phenotype determinative genes detecting method according to claim 1 3, and Tm value of the double-stranded nucleic acid that is calculated from the melting curve, the Tm value of the probe used in the formation of the double-stranded nucleic acid Calculating
Discriminating a specific phenotype-determining gene and other phenotype-determining genes based on the calculated difference in Tm value and a linear discriminant based on Mahalanobis' generalized distance;
A phenotyping gene detection method comprising: The nucleic acid containing the phenotyping gene is
Extracting double stranded DNA from the sample;
Amplifying a fragment containing a phenotyping gene from the double-stranded DNA;
Adding a promoter to the minus strand of the fragment;
Synthesizing an RNA chain as a nucleic acid derived from the phenotyping gene from the promoter-added fragment;
The method for detecting a phenotype-determining gene according to any one of claims 10 to 14 , wherein the RNA strand is synthesized by the method. The nucleic acid containing the phenotyping gene is
Extracting double stranded DNA from the sample;
Amplifying a fragment containing a phenotyping gene from the double-stranded DNA;
Adding a promoter to the minus strand of the fragment;
Synthesizing RNA strand from the promoter-added fragment;
Synthesizing a complex of the RNA and the DNA complementary to the mRNA from the RNA strand by a reverse transcription reaction;
Synthesizing a single-stranded DNA strand as a nucleic acid derived from the phenotyping gene from the complex;
The method for detecting a phenotype-determining gene according to any one of claims 10 to 14 , which is a single-stranded DNA strand synthesized by the method. The method for detecting a phenotype-determining gene according to claim 16 , wherein the step of synthesizing the single-stranded DNA strand includes a step of heat-treating the complex and subjecting it to RNase treatment. The method for detecting a phenotype-determining gene according to claim 16 , wherein the step of synthesizing the single-stranded DNA strand includes a step of treating the complex with an alkali.