JP6153515B2 - Method for detecting HLA-A * 24: 02 and detection kit - Google Patents

Description

  The present invention relates to a method for detecting HLA-A * 24: 02 and a detection kit.

  In recent years, new tumor-specific antigens have been identified one after another, and development of new immunotherapy is progressing accordingly. Among these, cancer immunotherapy is mainly vaccine therapy in which CD8-positive T cells specific for tumor antigens are induced in vivo. Various clinical applications have been made regarding cancer immunotherapy by vaccine therapy. By recognizing a peptide / HLA complex in which about 10 amino acid residues, which are part of a tumor-specific antigen, are bound to an HLA molecule, tumor-specific CD8-positive T cells are induced. Utilizing this fact, peptide vaccine therapy using a peptide that binds to HLA as an antigen has been developed.

  Currently, in Japan, the development of peptide vaccine therapy using peptides that bind to HLA-A * 24: 02, which many Japanese possess, is ahead. The effect of this peptide administration is expected only in patients holding HLA-A * 24: 02 allele. For this reason, it is necessary to confirm beforehand that the subject patient holds HLA-A * 24: 02 before administration.

  DNA typing is currently the most common testing method for HLA, and typical methods include PCR-SBT (Sequencing-Based Typing), PCR-SSO (Sequence Specific Oligonucleotide) and PCR-SSP (Sequence Specific Primers). ) Etc. are known.

  A method based on PCR-SSP has been reported as a method for detecting HLA-A * 24 before administration of a peptide vaccine (Non-patent Document 1).

Nakatsugawa M et al., "Comparison of speedy PCR-ssp method and serological typing of HLA-A24 for Japanese cancer patients.", "Journal of Immunoassay and Immunochemistry", April 2011, 32 (2), pp. 93-102

  Among the conventional methods, PCR-SBT has the problems that it is necessary to decode the DNA sequence of the HLA gene with a sequencer, which is complicated and requires dedicated software for analysis. Further, the conventional PCR-SSP has a problem that the number of primer sets has to be increased in order to improve accuracy. Furthermore, the conventional PCR-SSP has a risk of contamination because the PCR product must be confirmed by electrophoresis. The risk of contamination is a serious loss of reliability, especially in the field of genetic testing.

  If PCR-SBT is used, it is possible to specifically detect only the HLA-A * 24: 02 allele, although it is not easy due to the above-mentioned problems. However, in the first place, conventional PCR-SSO and PCR-SSP could not specifically detect only HLA-A * 24: 02 allele.

  Therefore, an object of the present invention is to provide a method for specifically detecting HLA-A * 24: 02 allyl while more easily and further reducing the risk of contamination as compared with conventional methods. .

  The present inventors indicate that the base sequences of A * 24: 02: 01: 01 and A * 24: 20 are the 211st base of HLA, and C in A * 24: 02: 01: 01. On the other hand, we noticed that A * 24: 20 differs only in that it is A. The present inventors use this 211th base as a target base and perform a real-time PCR by combining a forward PCR primer that forms a mismatch at the target base with a predetermined reverse PCR primer and a sequence-specific binding probe. Thus, it was found that HLA-A * 24: 02 allyl can be specifically detected.

The present invention has been completed by further research based on the above new findings. The present invention includes the following embodiments.
Item 1
(A1) A primer set comprising a forward PCR primer comprising a DNA sequence from the 3 ′ end to at least the 16th position in the DNA sequence represented by SEQ ID NO: 1.
Item 2
A primer set comprising a reverse PCR primer consisting of a DNA sequence from the 3 ′ end to at least the 20th of the primer (A1) and (A2) SEQ ID NO: 2 according to item 1.
Item 3
Item 3. The primer set according to Item 1 or 2, and (B) a partial sequence of the DNA sequence represented by SEQ ID NO: 3, wherein any one of the 429th, 437, 440, 442, 443, 447 and 449th bases A kit for real-time PCR, comprising a sequence-specific binding probe labeled with an oligonucleotide consisting of a continuous DNA sequence of 15 to 25 bases including a base; or an complementary sequence thereof.
Item 4
(1) A step of performing real-time PCR using a human DNA sample as a template using the kit according to Item 3; and (2) The human DNA sample from which a signal is detected in step (1) is HLA-A: 24: 02 A method for determining whether or not a human DNA sample is derived from a human having HLA-A: 24: 02, which comprises a step of determining that the human DNA has a human origin.
Item 5
(1) a step of performing real-time PCR using a human DNA sample as a template using the kit according to item 3; and (2) a human from which the human DNA sample from which a signal was detected in step (1) was collected, A method for screening an HLA-A: 24: 02-restricted peptide vaccine administration target group, comprising a step of selecting as a subject to be administered a HLA-A: 24: 02-restricted peptide vaccine.

  According to the present invention, it is possible to provide a method for specifically detecting HLA-A * 24: 02 allyl with high accuracy and simplicity, while further reducing the risk of contamination.

Real-time PCR on genomic DNA of HLA-A * 24: 02/02: 06 using TaqMan (registered trademark) Gene Expression Master Mix, primer set (A) and A * 24: 02 detection probe (i) It is a graph which shows the result of having performed. Real-time PCR on genomic DNA of HLA-A * 24: 20/02: 06 using TaqMan (registered trademark) Gene Expression Master Mix, primer set (A) and A * 24: 02 detection probe (i) It is a graph which shows the result of having performed. Real-time PCR on genomic DNA of HLA-A * 02: 01/02: 06 using TaqMan (registered trademark) Gene Expression Master Mix, primer set (A) and A * 24: 02 detection probe (i) It is a graph which shows the result of having performed. Real-time PCR on genomic DNA of HLA-A * 11: 01/11: 01 using TaqMan (registered trademark) Gene Expression Master Mix, primer set (A) and A * 24: 02 detection probe (i) It is a graph which shows the result of having performed. Real-time PCR on genomic DNA of HLA-A * 23: 01/29: 01 using TaqMan (registered trademark) Gene Expression Master Mix, primer set (A) and A * 24: 02 detection probe (i) It is a graph which shows the result of having performed. A graph showing the results of real-time PCR on TE buffer (negative control) using TaqMan (registered trademark) Gene Expression Master Mix, primer set (A) and A * 24: 02 detection probe (i) is there. Real-time PCR on HLA-A * 24: 02/02: 06 genomic DNA using TaqMan (registered trademark) Gene Expression Master Mix, primer set (B) and A * 24: 02 detection probe (i) It is a graph which shows the result of having performed. Using TaqMan (registered trademark) Gene Expression Master Mix, primer set (B) and A * 24: 02 detection probe (i), HLA-A * 24: 20/02: 06, HLA-A * 02: 01 / 02: 06, HLA-A * 11: 01/11: 01 and HLA-A * 23: 01/29: 01 Genomic DNA and results of real-time PCR on TE buffer (negative control) It is a graph to show. Results of real-time PCR of HLA-A * 24: 02/02: 06 genomic DNA using QuantiTect Multiplex PCR Kit, primer set (A) and A * 24: 02 detection probe (i) It is a graph to show. Using the QuantiTect Multiplex PCR Kit, primer set (A), and A * 24: 02 detection probe (i), the results of real-time PCR on HLA-A * 24: 20/02: 06 genomic DNA It is a graph to show. Using the QuantiTect Multiplex PCR Kit, primer set (A), and A * 24: 02 detection probe (i), the results of real-time PCR on HLA-A * 02: 01/02: 06 genomic DNA It is a graph to show. Results of real-time PCR on HLA-A * 11: 01/11: 01 genomic DNA using QuantiTect Multiplex PCR Kit, primer set (A) and A * 24: 02 detection probe (i) It is a graph to show. Results of real-time PCR on HLA-A * 23: 01/29: 01 genomic DNA using QuantiTect Multiplex PCR Kit, primer set (A) and A * 24: 02 detection probe (i) It is a graph to show. It is a graph which shows the result of having performed real-time PCR with respect to TE buffer (negative control) using QuantiTect Multiplex PCR Kit, primer set (A), and A * 24: 02 detection probe (i). Results of real-time PCR on HLA-A * 24: 02/02: 06 genomic DNA using the QuantiTect Multiplex PCR Kit, primer set (B), and A * 24: 02 detection probe (i) It is a graph to show. Using QuantiTect Multiplex PCR Kit, primer set (B) and A * 24: 02 detection probe (i), HLA-A * 24: 20/02: 06, HLA-A * 02: 01/02: 06, It is a graph which shows the result of having performed real-time PCR with respect to each genomic DNA of HLA-A * 11: 01/11: 01 and HLA-A * 23: 01/29: 01, and TE buffer (negative control). Real-time PCR on HLA-A * 24: 02/02: 06 genomic DNA using TaqMan (registered trademark) Gene Expression Master Mix, primer set (B) and A * 24: 02 detection probe (i) It is a graph which shows the result of having performed. Using TaqMan (registered trademark) Gene Expression Master Mix, primer set (B) and A * 24: 02 detection probe (i), HLA-A * 24: 20/02: 06, HLA-A * 02: 01 Results of real-time PCR on / 02: 06, HLA-A * 11: 01/11: 01 and HLA-A * 23: 01/29: 01 genomic DNA and TE buffer (negative control) It is a graph to show. Using TaqMan (registered trademark) Gene Expression Master Mix, primer set (B) and A * 24: 02 detection probe (i), and IPC primer set and IPC probe (i), HLA-A * 24: It is a graph which shows the result of having performed real-time PCR with respect to the genomic DNA of 02/02: 06. Using TaqMan (registered trademark) Gene Expression Master Mix, primer set (B) and A * 24: 02 detection probe (i), and IPC primer set and IPC probe (i), HLA-A * 24: It is a graph which shows the result of having performed real-time PCR with respect to genomic DNA of 20/02: 06. Using TaqMan (registered trademark) Gene Expression Master Mix, primer set (B) and A * 24: 02 detection probe (i), and IPC primer set and IPC probe (i), HLA-A * 02: It is a graph which shows the result of having performed real-time PCR with respect to the genomic DNA of 01/02: 06. Using TaqMan (registered trademark) Gene Expression Master Mix, primer set (B) and A * 24: 02 detection probe (i), IPC primer set and IPC probe (i), HLA-A * 11: 01 It is a graph which shows the result of having performed real-time PCR with respect to genomic DNA of / 11: 01. Using TaqMan (registered trademark) Gene Expression Master Mix, primer set (B) and A * 24: 02 detection probe (i), and IPC primer set and IPC probe (i), HLA-A * 23: It is a graph which shows the result of having performed real-time PCR with respect to the genomic DNA of 01/29: 01. TE buffer (negative control) using TaqMan (registered trademark) Gene Expression Master Mix, primer set (B) and A * 24: 02 detection probe (i), and IPC primer set and IPC probe (i) It is a graph which shows the result of having performed real-time PCR with respect to. Using TaqMan (registered trademark) Gene Expression Master Mix, primer set (B) and A * 24: 02 detection probe (i), and IPC primer set and IPC probe (i), HLA-A * 24: 02/02: 06, HLA-A * 24: 20/02: 06, HLA-A * 02: 01/02: 06, HLA-A * 11: 01/11: 01 and HLA-A * 23: 01 / 29:01 A photograph replacing a drawing showing the result of subjecting each PCR product to agarose electrophoresis after performing real-time PCR on each genomic DNA and TE buffer (negative control). TaqMan (registered trademark) Gene Expression Master Mix, forward PCR primer comprising the DNA sequence represented by SEQ ID NO: 12, reverse PCR primer comprising the DNA sequence represented by SEQ ID NO: 23, and probe for detecting A * 24: 02 (i) or (ii); and IPC primer set and IPC probe (i) or (ii), HLA-A * 24: 02/02: 06, HLA-A * 24: 20/02: 06, HLA-A * 02: 01/02: 06, HLA-A * 11: 01/11: 01 and HLA-A * 23: 01/29: 01 genomic DNA, and TE buffer (negative control) It is a graph which shows the result of having performed real-time PCR with respect to each. TaqMan (registered trademark) Gene Expression Master Mix, forward PCR primer comprising the DNA sequence represented by SEQ ID NO: 12, reverse PCR primer comprising the DNA sequence represented by SEQ ID NO: 23, and probe for detecting A * 24: 02 (ii); and the IPC primer set and the IPC probe (ii), while changing the concentrations of the primer and the probe, respectively, HLA-A * 24: 02/02: 06, HLA-A * 24: 20 / 02:06, HLA-A * 02: 01/02: 06, HLA-A * 11: 01/11: 01 and HLA-A * 23: 01/29: 01 genomic DNA and TE buffer (negative control) It is a graph which shows the result of having performed real-time PCR with respect to each of. Forward PCR primer comprising the DNA sequence represented by SEQ ID NO: 12, reverse PCR primer comprising the DNA sequence represented by SEQ ID NO: 23, and A * 24: 02 detection probe (ii); and IPC primer set and IPC It is a graph which shows the result of having performed real-time PCR with respect to 100 samples of Japanese-derived cell line genomes using the probe (ii). Forward PCR primer comprising the DNA sequence represented by SEQ ID NO: 12, reverse PCR primer comprising the DNA sequence represented by SEQ ID NO: 23, and A * 24: 02 detection probe (ii); and IPC primer set and IPC It is a graph which shows the result of having performed real-time PCR with respect to 100 samples of Japanese-derived cell line genomes using the probe (ii). Forward PCR primer comprising the DNA sequence represented by SEQ ID NO: 12, reverse PCR primer comprising the DNA sequence represented by SEQ ID NO: 23, and A * 24: 02 detection probe (ii); and IPC primer set and IPC It is a graph which shows the result of having performed real-time PCR with respect to 100 samples of Japanese-derived cell line genomes using the probe (ii). Forward PCR primer comprising the DNA sequence represented by SEQ ID NO: 12, reverse PCR primer comprising the DNA sequence represented by SEQ ID NO: 23, and A * 24: 02 detection probe (ii); and IPC primer set and IPC It is a graph which shows the result of having performed real-time PCR with respect to 100 samples of Japanese-derived cell line genomes using the probe (ii). Forward PCR primer comprising the DNA sequence represented by SEQ ID NO: 12, reverse PCR primer comprising the DNA sequence represented by SEQ ID NO: 23, and A * 24: 02 detection probe (ii); and IPC primer set and IPC It is a graph which shows the result of having performed real-time PCR with respect to 100 samples of Japanese-derived cell line genomes using the probe (ii). Forward PCR primer comprising the DNA sequence represented by SEQ ID NO: 12, reverse PCR primer comprising the DNA sequence represented by SEQ ID NO: 23, and A * 24: 02 detection probe (ii); and IPC primer set and IPC It is a graph which shows the result of having performed real-time PCR with respect to 100 samples of Japanese-derived cell line genomes using the probe (ii).

1. Primer set 1.1 Forward primer (A1)
The primer set of the present invention is
(A1) A primer set comprising a forward PCR primer consisting of a DNA sequence from the 3 ′ end to at least the 16th position in the DNA sequence represented by SEQ ID NO: 1.

  SEQ ID NO: 1 corresponds to the 190th to 211th partial sequence of the DNA sequence of HLA.

  The forward PCR primer (A1) is an oligonucleotide comprising a DNA sequence preferably from the 16th to 21st, more preferably from the 19th, from the 3 'end of the DNA sequence represented by SEQ ID NO: 1.

  In the forward PCR primer (A1), the base located at the 3 'end, that is, the base corresponding to the 211st base of the HLA DNA sequence is C. The HLA base sequences of A * 24: 02: 01: 01 and A * 24: 20 are respectively the 211st base of HLA, while A * 24: 02: 01: 01 is C, whereas A * 24:20 is different only in that it is A. Therefore, when the forward PCR primer (A1) is hybridized with the HLA base sequences of A * 24: 02: 01: 01 and A * 24: 20, respectively, only the HLA base sequence of A * 24: 20 is the 211st position. A single base mismatch occurs at the position. However, it is generally known that the function as a PCR primer is not impaired by a single-base mismatch. Therefore, in order to achieve PCR inhibition due to mismatch, it is usual to provide another mismatch on the 1 or 2 base 5 'base side from the mismatch site. Moreover, the mismatch that occurs between the forward PCR primer (A1) and the A * 24: 20 HLA base sequence is a so-called C-T mispair, which is generally considered to be difficult to inhibit the PCR extension reaction. Therefore, when the forward PCR primer (A1) is used, it is unlikely that the PCR extension reaction will not occur due to the mismatch even if the HLA base sequence of A * 24: 20 is used as a sample. However, contrary to expectation, the present inventors, when using the forward PCR primer (A1), do not cause an extension reaction due to mismatch when using the HLA base sequence of A * 24: 20 as a sample. I found out. Therefore, when the forward PCR primer (A1) is used, A * 24: 02: 01: 01 and A * 24: 20 can be distinguished by further combining with a predetermined reverse primer.

  Furthermore, when the forward PCR primer (A1) is used, the present inventors further combine not only A * 24: 02: 01: 01 but also A * 24: 02 in general by combining with a predetermined reverse primer. I found out that it can be distinguished from A * 24: 20.

1.2 Reverse primer (A2)
The primer set of the present invention further comprises:
(A2) Of the DNA sequence represented by SEQ ID NO: 2, a reverse PCR primer comprising a DNA sequence from the 3 ′ end to at least the 20th position may be contained.

  SEQ ID NO: 2 corresponds to the complementary sequence of the 882 to 910th partial sequence of the DNA sequence of HLA.

  The reverse PCR primer (A2) is an oligonucleotide consisting of the DNA sequence represented by SEQ ID NO: 2, preferably from the 3 'end to at least the 21st, more preferably the 23rd.

  In the reverse PCR primer (A2), two bases located from the 3 'end to the second base, that is, bases complementary to the 909th and 910th bases of the HLA DNA sequence are T and G. The HLA base sequences of A * 23: 01: 01 and A * 24: 04 and A * 24: 20 are the 909th and 910th bases of HLA, respectively, A * 24: 02: 01: 01, A * 24:04 and A * 24: 20 are C and A, whereas A * 23: 01: 01 are T and T. Therefore, when the reverse PCR primer (A2) is hybridized with the HLA base sequences of A * 23: 01: 01, A * 24: 02: 01: 01, and A * 24: 04 and A * 24: 20, respectively. A double base mismatch occurs at positions 909 and 910 only with the HLA base sequence of A * 23: 01: 01. This mismatch inhibits the PCR extension reaction. Therefore, when the reverse PCR primer (A2) is used, a PCR extension reaction does not occur due to a mismatch of two bases when the HLA base sequence of A * 23: 01: 01 is used as a sample. Therefore, when reverse PCR primer (A2) is used, A * 23: 01: 01 can be combined with A * 23: 01: 01, A * 24: 02: 01: 01, A * 24: 04 and A * 24 : Can be distinguished from 20.

  Furthermore, when the reverse PCR primer (A2) is used, the present inventors further combine A * 23: 01: 01 not only with A * 24: 02: 01: 01 but A * 24: 02 in general, A * 24: 04 and A * 24: 20.

2. Real-time PCR kit The real-time PCR kit of the present invention comprises the above-mentioned 1. 15 and (B) a partial sequence of the DNA sequence represented by SEQ ID NO: 3, comprising any one of the bases 429, 437, 440, 442, 443, 447 and 449 It contains a sequence-specific binding probe labeled with an oligonucleotide consisting of a continuous DNA sequence of ˜25 bases or its complementary sequence.

Above 1. Preferably, the primer set described in
(A1) A forward PCR primer comprising a DNA sequence from the 3rd end to the 21st to 21st DNA sequences in the DNA sequence represented by SEQ ID NO: 1, and (A2) 3 of the DNA sequences represented by SEQ ID NO: 2. 'Contains a reverse PCR primer consisting of at least the 21st DNA sequence from the end.

Above 1. More preferably, the primer set described in
(A1) a forward PCR primer comprising the DNA sequence from the 3 ′ end to the 19th DNA sequence in the DNA sequence represented by SEQ ID NO: 1; and (A2) the 3 ′ end of the DNA sequence represented by SEQ ID NO: 2. Contains reverse PCR primers consisting of DNA sequences from to 23.

2.1 Sequence-specific binding probe (B)
The sequence-specific binding probe (B) used in the present invention is a partial sequence of the DNA sequence represented by SEQ ID NO: 3, and any one of bases 429, 437, 440, 442, 443, 447 and 449 A sequence-specific binding probe labeled with an oligonucleotide consisting of a continuous DNA sequence of 15 to 25 bases or a complementary sequence thereof.

The DNA sequence represented by SEQ ID NO: 3 corresponds to the 1st to 1000th partial sequence of HLA-A * 24: 02: 01: 01.
A * 24: 02 and A * 24: 04 differ in DNA sequence at 7 bases contained in the 429th to 449th DNA sequences. Specifically, A * 24: 02 indicates that the 429, 437, 440, 442, 443, 447, and 449th bases are A, C, T, G, C, T, and C, respectively, in turn. * 24: 04 is different in DNA sequence in that C, G, C, C, T, G and G are in order.

  Specifically, for example, a sequence-specific binding probe labeled with an oligonucleotide consisting of the 427-451, 429-449 or 433-451st DNA sequence of the DNA sequence represented by SEQ ID NO: 3 can be used. .

  By using the sequence-specific binding probe (B) together with the above primer set, the 211st base is C, the 909th and 910th bases are C and A, respectively, and 429, 437, 440, 442, DNA of HLA (for example, HLA of A * 24: 02: 01: 01) in which any of the 443th, 447th and 449th bases matches the base sequence of SEQ ID NO: 3 can be detected by real-time PCR.

  Furthermore, the present inventors use not only A * 24: 02: 01: 01 but also A * 24: 02 general HLA DNA only by using the sequence-specific binding probe (B) together with the above primer set. It was found that it can be detected by real-time PCR.

  Specifically, the accuracy of the detection is an accuracy with which A * 24: 20 and A * 24: 04, which are currently reported, can be determined without being positive when targeting Japanese people. The accuracy of the above detection is the accuracy with which A * 24: 02 can be specifically detected even for a group containing an unreported allele for Japanese. Specifically, the accuracy of the detection is that the allele of the antigen type is different from A * 24, as well as A * 24: 08, A * 24: 13, A * 24: 19, A * 24: 24, A * 24: 29, A * 24: 44, A * 24: 89, A * 24: 94, A * 24: 89, A * 24: 109, and A * 24: 129 are all accurate without being positive.

  A Plus / Minus Assay can be performed by performing real-time PCR using the sequence-specific binding probe (B) together with the above primer set. In addition, this method can be detected in a single reaction system and the internal positive control reaction proceeds simultaneously, thus eliminating the possibility of false negatives due to PCR inhibition.

  The sequence-specific binding probe (B) has a label for detection by real-time PCR. The label is, for example, a fluorescent label. The labeling method is selected according to the difference in detection method in real-time PCR.

  Although it does not specifically limit as a detection system in real-time PCR, For example, what is shown below is mentioned.

  For example, there is a method in which detection is performed by using fluorescence by being hydrolyzed by the 5 'exonuclease activity of DNA polymerase (the probe used in this method is referred to as "hydrolysis probe"). A typical example of the hydrolysis probe is a commercially available TaqMan (registered trademark) Probe.

  In addition, probes labeled with different fluorescent dyes are used, and single-stranded target DNA hybridizes adjacent to each other and emits fluorescence. A method of performing detection using the fact that none of the probes hybridize and emit fluorescence (the probe used in this method is referred to as “hybridization probe”).

  Furthermore, the system which detects by performing a melting curve analysis is mentioned. Melting curve analysis refers to tracking nucleic acid annealing and thermal denaturation in real time. The Tm of the probe / target hybrid is significantly reduced when there is a mismatch. In the melting curve analysis, this fact is used to detect the presence or absence of mismatch.

  Melting curve analysis can also be performed after PCR.

  For this method, a probe labeled with one kind of substance can be used in addition to the aforementioned hybridization probe.

  Melting curve analysis can identify even single base mismatches by monitoring the melting of the hybrid of the sequence specific binding probe and the target DNA.

  In the case of using a hybridization probe, the melting curve analysis is the following analysis in detail. One oligonucleotide hybridizes to a portion where no mismatch occurs in the target DNA. This probe functions as an anchor probe. The other oligonucleotide (detection probe) binds to a moiety that can cause a mismatch. When a mismatch occurs, the Tm of this detection probe is about 5 ° C. lower than the Tm of the anchor probe. Immediately after DNA amplification is complete, perform a melting curve analysis using the appropriate device to identify the different genotypes. During this analysis, the fluorescence is continuously measured while gradually raising the temperature (for example, 0.1 to 0.2 ° C. per second). By this operation, the melting kinetics of the probe bound to the target DNA can be monitored. As the temperature increases, the short mutant probe melts. Due to the melting, the distance between the two fluorescent dyes increases and the fluorescence signal decreases. The Tm of the probe-target hybrid depends not only on the probe length and GC content, but also on the degree of homology of the hybrid. Therefore, a more fully bound probe will have a higher melting Tm, and a probe bound to DNA containing mismatches that lack stability will have a lower Tm.

As a detection method in real-time PCR, a method using Molecular Beacon, Eclipse Probes (structured Probe), HyProbe (adjacent hybridization Probes), Q Probe, and the like, a cycling probe method using a chimeric probe composed of RNA and DNA, and Invader ( Examples include the Invader Plus (registered trademark) method using a registered trademark Probe.
2.2 Other Components The PCR kit of the present invention may further contain other components (components).

  Such components are not particularly limited, and include those usually contained in PCR kits.

The components normally contained in a PCR kit are not particularly limited, and examples thereof include dNTP (deoxynucleotide triphosphate), heat-resistant DNA polymerase, Mg ²⁺ , PCR buffer and instructions.

  dNTP may contain dUTP instead of dTTP. When dNTP contains dUTP instead of dTTP, it is possible to eliminate false positives by carrying over PCR amplification products previously performed by using UNG (Uracil-N-glycosylase), an enzyme that degrades DNA containing uracil. Can be prevented. In other words, even if a PCR amplification product performed before is mixed into the reaction system, since the PCR amplification product incorporates dUTP, such an action is caused by the action of UNG, an enzyme that degrades DNA containing uracil. False positives due to carry over can be prevented. Therefore, the PCR kit of the present invention may further contain UNG.

2.2.1 Heat-resistant DNA polymerase The heat-resistant DNA polymerase is not particularly limited, but can be appropriately selected from commercially available products according to the type of probe. It should be noted that the present invention aims to determine whether or not a mismatch is formed at the 3 ′ end of the forward PCR primer (A1) hybridized with the template DNA as an index. A thermostable DNA polymerase having no 5 ′ exonuclease activity is preferred.

  When a hydrolysis probe is used as the sequence-specific binding probe (B), a thermostable DNA polymerase having 5 '→ 3' exonuclease activity is preferable. The thermostable DNA polymerase having 5 '→ 3' exonuclease activity is not particularly limited, and preferred examples include Taq DNA polymerase and Tth DNA Polymerase. Taq DNA polymerase includes not only normal Taq DNA polymerase but also chemically modified AmpliTaq Gold (registered trademark) DNA Polymerase, HotStarTaq (registered trademark) DNA Polymerase and FastStart Taq DNA Polymerase, and hot start enzymes using aptamers. A certain AptaTaq DNA Polymerase or a Hot Start PCR system (TaKaRa Taq ™ Hot Start Version etc.) using an anti-Taq antibody can also be used.

When adopting the method using Molecular Beacon Probe or Hyprobe, cycling probe method, Invader Plus method, etc., Proof-reading activity is deleted, and 5 '→ 3' exonuclease activity and 3 '→ 5 exo An enzyme having no nuclease activity is used.
Examples of such enzymes include, but are not limited to, Taq Δ exo DNA polymerase, KOD (exo-) DNA polymerase, Exo-Pfu DNA polymerase, and Vent (exo-) DNA polymerase.

2.2.2 Mg ²⁺
Mg ²⁺ is not particularly limited, and for example, MgCl ₂ and MgSO ₄ are contained in the PCR kit. Although not particularly limited, usually 1.5 to 2.5 mM Mg ²⁺ is required.

2.2.3 Buffer The buffer is usually selected according to the type of enzyme used. Although it does not specifically limit as a buffer, The same thing as the buffer contained in the commercially available PCR buffer or the commercially available PCR kit can be used. These commercial products often do not disclose the buffer composition. The standard buffer composition when using Taq polymerase is listed below.
50 mM KCl
10 mM Tris-HCl (pH 8.4 to 9.0, 25 ° C)
1.5 mM MgCl ₂
0.01% gelatin or 0.01% Triton X-100
The PCR kit of the present invention may contain a buffer having a concentration about 10 times higher than when the buffer is used. In this case, dilute the buffer before use.

3. Determination method The method of determining whether or not the human DNA specimen of the present invention is derived from a human having HLA-A: 24: 02,
(1) The above 2. A step of performing real-time PCR using the kit described in 1; and (2) a step of determining that the human DNA specimen from which the signal is detected in step (1) is derived from a human having HLA-A: 24: 02 Is a method for determining whether or not a human DNA specimen is derived from a human having HLA-A: 24: 02.

  The human DNA specimen is not particularly limited as long as it contains human genomic DNA, and examples thereof include blood, oral mucosa, hair, nails and blood stains (those obtained by dropping blood onto a filter paper solution).

  As the human DNA sample, a blood sample can be preferably used. As a blood sample, one prepared by blood collection can be used directly, or a blood sample obtained by subjecting a blood sample to a predetermined treatment can be used. The predetermined treatment is not particularly limited, and examples thereof include heating and freezing.

  For the real-time PCR, a commercially available apparatus that is usually used can be used. Although it does not specifically limit as a commercially available apparatus, For example, ABI 7500 Fast, ABI 7500 Fast Dx (Life Technologies), etc. are mentioned.

  Real-time PCR can be performed under normal conditions.

Concentrations and reaction volumes for each primer and probe are recommended, for example, when using a commercially available premix solution (eg, a mixture of Taq DNA Polymerase, dNTPs, MgCl ₂ and buffer). Can be decided according to. Although it does not specifically limit as a commercially available premix solution, For example, TaqMan (trademark) Gene Expression Master Mix (Life Technologies), QuantiTect (trademark) Multiplex PCR Kit (QIAGEN) etc. are mentioned. According to the recommendation of TaqMan (registered trademark) Gene Expression Master Mix, primer 900 nM, probe 250 nM, and reaction system can be 20 μL. Further, according to the recommendation of QuantiTect (registered trademark) Multiplex PCR Kit, the primer can be 400 nM, the probe 200 nM, and the reaction system can be 25 μL.

  Select a primer with a suitable Tm value according to the annealing temperature and extension temperature.

PCR conditions can be set as appropriate. Although it does not specifically limit, For example, the following conditions are mentioned.
(1) 50 ° C for 2 minutes (2) 95 ° C for 10 to 15 minutes (3) 95 ° C for 15 seconds (4) 68 ° C for 1 minute (5) Repeat (so that the total is 40 cycles). However, when antibody type or aptamer hot start is used, the time for activation is not required, and thus (2) may be 0 to 15 minutes.

  According to the present invention, detection of HLA-A: 24: 02 can be performed easily and with high accuracy for a group having an allyl frequency such as Japanese.

Furthermore, for example, in Southeast Asia, there are countries where A * 24: 07 allele, which is one of the A * 24 subtypes, is frequently reported. In Japan, the frequency of A * 24: 07 allele is low. The sequence difference between A * 24: 02 and A * 24: 07 is only the 412th single base. By focusing on this single base difference, a primer capable of specifically detecting A * 24: 07 can be designed. Such a primer is not particularly limited, and examples thereof include a forward primer composed of a DNA sequence represented by SEQ ID NO: 4. If the Tm value is set at around 68 ° C., a sample in which DNA amplification is observed can be determined to be A * 24: 07 allele-negative by performing PCR using this forward primer. Therefore, for the group containing A * 24: 07 allele at high frequency, based on the determination method of the present invention, by additionally using a primer capable of specifically detecting A * 24: 07, HLA-A : 24: 02 can be detected easily and with high accuracy. Moreover, HLA-A: 24: 02 can be detected easily and with high accuracy by applying the same improvement based on the present invention to other groups having different allele frequencies from Japanese.

4). Screening method of the present invention, the screening method for the HLA-A: 24: 02 restricted peptide vaccine administration group,
(1) The above 2. A step of performing real-time PCR using the kit described in 1); and (2) a human DNA sample from which a signal was detected in step (1) is treated with an HLA-A: 24: 02 restricted peptide vaccine. It is a method comprising a step of selecting as a subject to be administered.

  Currently, in Japan, the development of peptide vaccine therapy using peptides that bind to HLA-A * 24: 02 (HLA-A: 24: 02-restricted peptide vaccine), which many Japanese possess, is ahead. Yes. Examples of such HLA-A: 24: 02 restricted peptide vaccines include OTS102, OCV-101, OCV-105, WT4869, S288310, S488410 and OTSGC-A24.

  The therapeutic effect of HLA-A: 24: 02 restricted peptide vaccine is expected only in patients who have HLA-A * 24: 02 allele. Therefore, if it can be confirmed in advance that the subject patient holds HLA-A * 24: 02 before administration, subjects to be administered with the HLA-A: 24: 02 restricted peptide vaccine can be screened.

  Regarding human DNA specimens, real-time PCR conditions and target groups, see 3. above. This is the same as described in the determination method.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

1. Materials and Methods 1-1. Primer and probe design
Regarding Primer design, A * 24 without detecting A * 23: 01: 01 by setting the 909th to 910th positions to the 3 'end of Reverse Primer based on the sequence of A * 24: 02: 01: 01 : 02: 01: 01 can be detected (Bunce M, O'Neill CM et al., “Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 Primer mixes utilizing sequence-specific Primers (PCR-SSP). ”,“ Tissue Antigens ”, November 1995, 46 (5), pp. 355-67).

  The forward primer complementary to A * 24: 02: 01: 01 differs from the other alleles only in the 3 'terminal single nucleotide sequence. Specifically, the 3 ′ terminal base of the Forward Primer is C, for example, the complementary strand of A * 24: 20 is T, and in this case, a CT mispair that is most likely to cause an extension reaction is generated (Kwok S et al., "Effects of Primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.", "Nucleic Acids Res.", February 1990, 18 (4), pp. 999-1005; Huang MM et al., "Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR.", "Nucleic Acids Res.", September 1992, 20 (17), pp. 4567-73). To solve this problem, a method using LNA (Locked Nucleic Acid) for each base of Primer (Latorra D et al., "Enhanced allele-specific PCR discrimination in SNP genotyping using 3 'locked nucleic acid (LNA) Primers." “Hum Mutat.”, July 2003, 22 (1), pp. 79-85) and allele specific competitive blocker (Orou A et al., “Allele-specific competitive blocker PCR: a one-step method with applicability to pool screening” "," Hum Mutat. ", 1995, 6 (2), pp. 163-9).

A * 24: 02: 01: 01 Detection Primer and Probe sequences are shown below. Regarding the Primer, a 16-26mer Primer was designed from the 3 ′ end to the 5 ′ end.
The most widely used TaqMan (registered trademark) Probes was used as an example of the present invention.
For Probe, for example, the 429th to 449th sequence (21mer) and the 433th to 451th sequence (19mer) of A * 24: 02: 01: 01 were designed. FAM was used as the fluorescent dye. For the quencher, TAMRA and the dark quencher ATTO540Q were used. Regarding the design of Taqman Probe, if there is a single base sequence difference, it can be detected with a probe containing LNA or MGB (Ugozzoli LA et al., “Real-time genotyping with oligonucleotide probes containing locked nucleic acids.”, “Anal Biochem. ”January 2004, 324 (1), pp. 143-52; de Kok JB et al.,“ Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB Probes). ”,“ Hum Mutat. ” Since May 2002, 19 (5), pp. 554-9), any sequence difference from 429th to 449th of A * 24: 02: 01: 01 may be targeted.

In addition, the primer and probe sequences of the RNaseP gene were designed as internal positive controls to prevent sample dispensing errors and false negatives due to PCR inhibition.
Primer and Probe were purchased from Sigma Genosis or Japan Genetic Research Institute.

<A * 24: 02: 01: 01 Detection Primer, Probe>
Forward Primer (5'-3 ')
26mer GCCACTCCTCGTCCCCAGGCTCCCAC (SEQ ID NO: 5)
25mer CCACTCCTCGTCCCCAGGCTCCCAC (SEQ ID NO: 6)
24mer CACTCCTCGTCCCCAGGCTCCCAC (SEQ ID NO: 7)
23mer ACTCCTCGTCCCCAGGCTCCCAC (SEQ ID NO: 8)
22mer CTCCTCGTCCCCAGGCTCCCAC (SEQ ID NO: 9)
21mer TCCTCGTCCCCAGGCTCCCAC (SEQ ID NO: 10)
20mer CCTCGTCCCCAGGCTCCCAC (SEQ ID NO: 11)
19mer CTCGTCCCCAGGCTCCCAC (SEQ ID NO: 12)
18mer TCGTCCCCAGGCTCCCAC (SEQ ID NO: 13)
17merCGTCCCCAGGCTCCCAC (SEQ ID NO: 14)
16mer GTCCCCAGGCTCCCAC (SEQ ID NO: 15)
15mer TCCCCAGGCTCCCAC (SEQ ID NO: 16)
Reverse Primer (5'-3 ')
29mer ACGCACGTGCCCTCCAGGTAGGCTCTCTG (SEQ ID NO: 17)
28mer CGCACGTGCCCTCCAGGTAGGCTCTCTG (SEQ ID NO: 18)
27mer GCACGTGCCCTCCAGGTAGGCTCTCTG (SEQ ID NO: 19)
26mer CACGTGCCCTCCAGGTAGGCTCTCTG (SEQ ID NO: 20)
25mer ACGTGCCCTCCAGGTAGGCTCTCTG (SEQ ID NO: 21)
24mer CGTGCCCTCCAGGTAGGCTCTCTG (SEQ ID NO: 22)
23mer GTGCCCTCCAGGTAGGCTCTCTG (SEQ ID NO: 23)
22mer TGCCCTCCAGGTAGGCTCTCTG (SEQ ID NO: 24)
21mer GCCCTCCAGGTAGGCTCTCTG (SEQ ID NO: 25)
20mer CCCTCCAGGTAGGCTCTCTG (SEQ ID NO: 26)
19mer CCTCCAGGTAGGCTCTCTG (SEQ ID NO: 27)
18mer CTCCAGGTAGGCTCTCTG (SEQ ID NO: 28)
17mer TCCAGGTAGGCTCTCTG (SEQ ID NO: 29)
16mer CCAGGTAGGCTCTCTG (SEQ ID NO: 30)
Probe (5'-3 ')
(i) (FAM-) AGAACCTGCGGATCGCGCTCC (-TAMRA) (SEQ ID NO: 31)
(ii) (FAM-) CCTGCGGATCGCGCTCCGC (-ATTO540Q) (SEQ ID NO: 32)

<Internal positive control detection Primer, Probe>
Forward Primer (5'-3 ')
TGCAGATTTGGACCTGCGAG (SEQ ID NO: 33)
Reverse Primer (5'-3 ')
TGAGCGGCTGTCTCCACAAG (SEQ ID NO: 34)
Probe (5'-3 ')
(i) (HEX-) TTCTGACCTGAAGGCTCTGCGCG (-TAMRA) (SEQ ID NO: 35)
(ii) (HEX-) TTCTGACCTGAAGGCTCTGCGCG (-ATTO540Q) (SEQ ID NO: 35)

1-2. PCR master mix
Taq Δexo DNA polymerase was used as the DNA amplification enzyme.
TaqMan (registered trademark) Gene Expression Master Mix (Life Technologies) and QuantiTect Multiplex PCR Kit (QIAGEN), which are general real-time PCR master mix reagents, were used. QuantiTect Multiplex PCR Kit (QIAGEN) was performed by adding 0.25 units to 25 μl reaction of Uracil-DNA Glycosylase and heat-labile (Roche).

1-3. Human genomic DNA
HLA typed PBMC was purchased from CTL, USA, and genomic DNA was purified with QIAamp DNA Blood Mini Kit (QIAGEN). In addition, human cell-derived cell genomic DNA was purchased from the Human Science Foundation. AlleleSEQR HLA-A (Abbott Laboratories), Assign software (Conexio Genomics), UniTray (Life Technologies), Micro SSP Allele Specific HLA Class HLA-A typing was performed using I DNA Typing Tray (One Lambda Inc).

Genomic DNA was subjected to real-time PCR reaction by adding 2 μL of 100 ng / μL and 1 ng / μL.
1-4. Real-time PCR
For real-time PCR, ABI 7500 Fast and ABI 7500 Fast Dx (Life Technologies) were used.
The conditions are as follows.
TaqMan (registered trademark) Gene Expression Master Mix
After 50 ° C. for 2 min and 95 ° C. for 10 min, 40 cycles of 95 ° C. for 15 sec and 68 ° C. for 1 min were performed.
QuantiTect Probe PCR + UNG Kit, QuantiTect Multiplex PCR Kit
After 50 ° C for 2 min and 95 ° C for 15 min, 40 cycles of 95 ° C for 15 sec and 68 ° C for 1 min were performed.
2. result

2-1. Study of Primer (Tm value)
26merPrimer set (A), which is generally considered to be the longest in PCR, and the nearest neighbor pair parameter (Nearest Neighbor Parametes, Naoki Sugimoto, “Nucleic acid stability prediction using nearest neighbor pair parameter and its function Using “Relationship”, “Biophysics” (Yoshioka Shoten), vol. 33, pp. 61-67) (B), the Primer set with a Tm value of around 68 ° C was selected, and the length of the Primer was optimized. The Tm value is set to around 68 ° C when the annealing and extension temperature is set to 68 ° C. For example, if the annealing and extension temperature is 60 ° C, a primer with a Tm value of around 60 ° C is selected. Probes were selected to contain all sequences from 429th to 449th, which are different from A * 24: 04 and A * 24: 02: 01: 01 alleles. Mix was performed at the recommended concentration, and Maser Mix is TaqMan® Gene Expression Master Mix, Qu AntiTect Multiplex PCR Kit was used: TaqMan (registered trademark) Gene Expression Master Mix, Primer 900 nM, Probe 250 nM, reaction system 20 μL, QuantiTect Multiplex PCR Kit Primer 400 nM, Probe 200 nM, reaction system 25 μL. ABI7500 Fast was used for PCR.Alleles of genomic DNA were (i) A * 24: 02 / A * 02: 06, (ii) A * 24: 20 / A * 02: 06, (iii) A * 02 : 01 / A * 02: 06, (iv) A * 11: 01 / A * 11: 01 and (v) A * 23: 01 / A * 29: 01.

(A)
Forward Primer (5'-3 ')
26mer GCCACTCCTCGTCCCCAGGCTCCCAC (SEQ ID NO: 5)
Reverse Primer (5'-3 ')
26mer CACGTGCCCTCCAGGTAGGCTCTCTG (SEQ ID NO: 17)

(B)
Forward Primer (5'-3 ')
16mer TCCCCAGGCTCCCAC (SEQ ID NO: 16)
Reverse Primer (5'-3 ')
16mer TCCAGGTAGGCTCTCTG (SEQ ID NO: 29)
A * 24: 02 detection probe (i) (Common to (A) and (B))
(FAM-) AGAACCTGCGGATCGCGCTCC (-TAMRA) (SEQ ID NO: 31)

  Both master mixes are 26 mer and long Primer set (A) detects signals in alleles other than A * 24: 02, especially including A * 24: 20, (ii) A * 24: 20 / A * 02: 06 Markedly amplified the signal. However, amplification of A * 24: 02 negative genome was not observed in the Primer set (B) with Tm optimized around 68 ° C. The results of TaqMan (registered trademark) Gene Expression Master Mix (FIGS. 1 to 8) and QuantiTect Multiplex PCR Kit (FIGS. 9 to 16) are shown.

2-2. Study of Primer (IPC-Internal Positive Control)
Measurement was performed by adding the following Primer and Probe for IPC-Internal Positive Control to the Primer set reaction solution of (B). By adding an internal positive control, it is possible to prevent the PCR reaction from being inhibited for some reason or false negatives due to a sampling error. If an IPC signal is detected and no HLA-A * 24: 02 detection signal is detected, it can be determined that the signal is negative. For the master mix, TaqMan (registered trademark) Gene Expression Master Mix and QuantiTect Multiplex PCR Kit were used. Select the IPC Primer from the RNaseP gene region, set the amplification region as short as possible (69 bp) and add the Primer concentration at a low concentration (0.1 μM) so as not to affect the HLA-A * 24: 02 detection system. It was. The probe concentration of IPC was the recommended concentration of each master mix (TaqMan (registered trademark) Gene Expression Master Mix-250 nM, QuantiTect Multiplex PCR Kit-200 nM). HLA-A * 24: 02 detection Primer and Probe concentrations were the recommended concentrations for each master mix. For TaqMan (registered trademark) Gene Expression Master Mix, Primer 900 nM and Probe 250 nM, the reaction system was 20 μL, QuantiTect Multiplex PCR Kit was Primer 400 nM and Probe 200 nM, and the reaction system was 25 μL. ABI7500 Fast was used as the real-time PCR apparatus.

The results of TaqMan (registered trademark) Gene Expression Master Mix are shown (FIGS. 17 to 24).
The results of HLA-A * 24: 02 were the same as when IPC Primer and Probe were not added, and IPC signals were detected in all genomes. As a result of agarose gel electrophoresis, HLA-A * 24: 02 detection target bands (around 800 bp) were found in A * 24: 02/02: 06 and A * 11: 01/11: 01 genomes. (FIG. 25). Although A * 11: 01 allele has a difference in the sequence set in Probe, the Primer region is almost the same sequence as the A * 24: 02 sequence, and amplification was confirmed. It is confirmed that the signal is not detected by real-time PCR because of the difference in Probe sequence. IPC bands were confirmed with all genomic DNA added, reflecting the results of real-time PCR. QuantiTect Multiplex PCR Kit gave similar results.

<Internal positive control detection Primer, Probe>
Forward Primer (5'-3 ')
TGCAGATTTGGACCTGCGAG (SEQ ID NO: 33)
Reverse Primer (5'-3 ')
TGAGCGGCTGTCTCCACAAG (SEQ ID NO: 34)
IPC probe (i) (5'-3 ')
(HEX-) TTCTGACCTGAAGGCTCTGCGCG (-TAMRA) (SEQ ID NO: 35)

2-3. Optimization of Primer Real-time PCR was performed by combining the prepared Primer, and Primer was optimized. TaqMan (registered trademark) Gene Expression Master Mix (Life Technologies), which is a master mix reagent, was used. Primer and probe concentrations were 900 nM, probe concentration was 250 nM, and the reaction system was 20 μL. Genomic DNA is (i) A * 24: 02 / A * 02: 06 is 100 ng / μL, 1 ng / μL, (ii) A * 24: 20 / A * 02: 06, (iii) A * 02: 01 / A * 02: 06, (iv) A * 11: 01 / A * 11: 01, (v) A * 23: 01 / A * 29: 01 is adjusted to 100 ng / μL with TE buffer, and 2 μL to the reaction system added. ABI 7500 Fast was used as a measuring instrument. Table 1 shows the results.

  Judgment of the results was made by analyzing Baseline from 3 to 15, and when the signal was (i) 100 ng / μL, the result of 1 ng / μL was 0.05 or more, and the signals of (ii) to (v) were 0.05 or less, did. When the A * 24: 02 positive genome in (i) was 0.05 or less, or the signal was increased in the negative alleles in (ii) to (v) and the signal was 0.05 or more, it was marked as x.

2-4. Optimization of Probe A * 24: 02 detection probe used in the above study (i) (FAM-) AGAACCTGCGGATCGCGCTCC (-TAMRA) (SEQ ID NO: 31) has G (guanine) base second from the 5 'end To do. Since the G base has a fluorescence quenching action, it is desirable that it is not at the 5 ′ end. In addition, the TAMRA quencher has its own fluorescence, thus generating a background in the entire spectrum. The sequence of the probe for A * 24: 02 detection may be targeted to the 429th to 449th sequence mismatch, which is different from A * 24: 04 and A * 24: 02: 01: 01 alleles. The 433th to 451st sequences (19mer) were designed targeting the 437th to 449th sequences. Also changed from TAMRA quencher to dark quencher ATTO540Q quencher. This is A * 24: 02 detection probe (ii).

A * 24: 02 detection probe (ii)
(FAM-) CCTGCGGATCGCGCTCCGC (-ATTO540Q) (SEQ ID NO: 32)
The IPC probe was also changed from the TAMRA quencher (IPC probe (i)) to the ATTO540Q quencher (IPC probe (ii)).

Probe for IPC
(i) (HEX-) TTCTGACCTGAAGGCTCTGCGCG (-TAMRA) (SEQ ID NO: 35)
(ii) (HEX-) TTCTGACCTGAAGGCTCTGCGCG (-ATTO540Q) (SEQ ID NO: 35)
Real-time PCR was performed using the above TAMRA quencher probe (IPC probe (i)) and ATTO540Q quencher probe (IPC probe (ii)). TaqMan (registered trademark) Gene Expression Master Mix (Life Technologies), which is a master mix reagent, was used.

For Primer, Forward Primer-19mer (SEQ ID NO: 12) and Reverse Primer-23mer (SEQ ID NO: 23) were selected from the above table. Primer and probe concentrations were 900 nM, probe concentration was 250 nM, and the reaction system was 20 μL. Genomic DNA is (i) A * 24: 02 / A * 02: 06 is 100 ng / μL, 1 ng / μL, (ii) A * 24: 20 / A * 02: 06, (iii) A * 02: 01 / A * 02: 06, (iv) A * 11: 01 / A * 11: 01, (v) A * 23: 01 / A * 29: 01 is adjusted to 100 ng / μL with TE buffer, and 2 μL to the reaction system added. ABI 7500 Fast was used as a measuring instrument. The results are shown in FIG.
The improved FAM-ATTO540Q showed an increase in signal. The IPC was equivalent.

2-5. Primer, Probe concentration optimization
Primer and probe concentrations were optimized. Excess Primer generally leads to irrelevant DNA amplification, which can ultimately lead to non-homogeneous PCR products, and excess Probe can cause background to increase. The concentration was examined using the following Primer and Probe. The reaction system was 20 μL using TaqMan (registered trademark) Gene Expression Master Mix (Life Technologies) as a master mix reagent. Genomic DNA is (i) A * 24: 02 / A * 02: 06 is 100 ng / μL, 1 ng / μL, (ii) A * 24: 20 / A * 02: 06, (iii) A * 02: 01 / A * 02: 06, (iv) A * 11: 01 / A * 11: 01, (v) A * 23: 01 / A * 29: 01 is adjusted to 100 ng / μL with TE buffer, and 2 μL to the reaction system added. ABI 7500 Fast was used as a measuring instrument.

  The results are shown in FIG. Real-time PCR was performed at concentrations of A * 24: 02 detection Primer of 100 nM, 300 nM, and 900 nM. All combinations showed an increase in signal only in the A * 24: 02 positive genome. 100nM clearly had a low signal value, but 300nM and 900nM were equivalent. Next, as a result of fixing the Primer concentration to 300 nM and setting the Probe concentration to 100, 200, 400 nM, the signal increased as the concentration of both FAMProbe and HEXProbe increased. All combinations showed an increase in signal only in the A * 24: 02 positive genome. This system was shown to be independent of Primer and Primer concentrations.

<For A * 24: 02 detection>
Forward Primer-19mer (100,300,900nM)
Reverse Primer-23mer (100,300,900nM)
A * 24: 02 detection probe (ii) (FAM-) CCTGCGGATCGCGCTCCGC (-ATTO540Q) (100,200,400nM) (SEQ ID NO: 32)

<For IPC>
Forward Primer-TGCAGATTTGGACCTGCGAG (fixed at 100nM) (SEQ ID NO: 33)
Reverse Primer-TGAGCGGCTGTCTCCACAAG (fixed at 100 nM) (SEQ ID NO: 34)
Probe for IPC (ii) (HEX-) TTCTGACCTGAAGGCTCTGCGCG (-ATTO540Q) (100,200,400nM) (SEQ ID NO: 35)

2-6. Measurement of Japanese-derived cell line genome HLA-A typing was performed using 100 samples of Japanese-derived cell line (PSC cell line) genome purchased from Human Science Foundation. For typing, first use AlleleSEQR HLA-A (Abbott Laboratories) and Assign software (Conexio Genomics). For possible A * 24: 02, UniTray (Life Technologies), Micro SSP ^TM Allele Further typing was performed using Specific HLA Class I DNA Typing Tray (One Lambda Inc), and it was confirmed that A * 24: 02. Of 100 samples, A * 24: 02 positive was 68 samples and negative was 32 samples.

  The genomes of 100 samples were measured with an HLA-A * 24: 02 detection system using sequence-specific Primer and Probe (FIGS. 28 to 33). Primer and Probe were performed under the following conditions. The genome concentration was adjusted to 100 ng / μL and 1 ng / μL, and 2 μL was added. The reaction volume was 20 μL and the measuring instrument was ABI 7500 Fast Dx. The analysis method was set to Baseline 3 to 15, and a signal of 0.05 or more in 40 cycles was regarded as positive.

  Table 2 shows the results. All 68 positive genome samples gave a signal of 0.05 or higher, and 32 negative genome samples showed no increase in signal. The IPC signal showed a signal of 0.05 or more in all 100 samples.

<A * 24: 02>
Forward Primer-19mer (300nM)
Reverse Primer-23mer (300nM)
(ii) (FAM-) CCTGCGGATCGCGCTCCGC (-ATTO540Q) (200nM) (SEQ ID NO: 32)

<IPC>
Forward Primer-TGCAGATTTGGACCTGCGAG (100nM) (SEQ ID NO: 33)
Reverse Primer-TGAGCGGCTGTCTCCACAAG (100nM) (SEQ ID NO: 34)
(ii) (HEX-) TTCTGACCTGAAGGCTCTGCGCG (-ATTO540Q) (200nM) (SEQ ID NO: 35)

3. Consideration
Primer and Probe were optimized and a simple and accurate A * 24: 02 detection system was constructed. In particular, A * 24: 20 allele has a single nucleotide sequence difference from A * 24: 02, and in order to design a Primer that does not make A * 24: 20 positive in the Forward Primer, C (Primer) -T ( template) had to be designed not to extend mismatches. In this method, the length of the Primer is designed to be the shortest, and the mismatch rate is increased to prevent erroneous CT mismatch extension. In this study, the solution can be solved by using a 22mer to 16mer Forward Primer, and the measurement system is completed in a single reaction system.

  Designed with reference to the Japanese allele frequency and measured 100 samples of Japanese-derived genomic DNA, we were able to measure it with an accuracy of 100% sensitivity and specificity.

  This measurement system can also be applied outside of Japan. In Japan, A * 24: 20 allele is frequently reported among A * 24 subtypes, but A * 24: 07 allele, which is one of A * 24 subtypes, is reported frequently in Southeast Asia. Some countries have been. The sequence difference between A * 24: 02 and A * 24: 07 is only the 412th single base (A * 24: 02 (C) -A * 24: 07 (G)), and the forward primer 3 If the end is set so that the sequence is different and the Tm value is set at around 68 ° C., a system in which A * 24: 07 can be judged negative as in (GACAGGGAAAGTGAAGGCCCAC) (SEQ ID NO: 4). Thus, the accuracy can be increased by increasing the number of reaction systems.

[SEQ ID NO: 1] Forward primer [SEQ ID NO: 2] Reverse primer [SEQ ID NO: 3] Homo sapiens [SEQ ID NO: 4] Forward primer [SEQ ID NO: 5] Forward primer [SEQ ID NO: 6] Forward Primer [SEQ ID NO: 7] Forward primer [SEQ ID NO: 8] Forward primer [SEQ ID NO: 9] Forward primer [SEQ ID NO: 10] Forward primer [SEQ ID NO: 11] Forward primer [SEQ ID NO: 12] Forward primer [SEQ ID NO: 13] Forward primer [SEQ ID NO: 14] Forward primer [SEQ ID NO: 15] Forward primer [SEQ ID NO: 16] Forward primer [SEQ ID NO: 17] Reverse primer [SEQ ID NO: 18] River Primer [SEQ ID NO: 19] reverse primer [SEQ ID NO: 20] reverse primer [SEQ ID NO: 21] reverse primer [SEQ ID NO: 22] reverse primer [SEQ ID NO: 23] reverse primer [SEQ ID NO: 24] reverse primer Primer [SEQ ID NO: 25] Reverse primer [SEQ ID NO: 26] Reverse primer [SEQ ID NO: 27] Reverse primer [SEQ ID NO: 28] Reverse primer [SEQ ID NO: 29] Reverse primer [SEQ ID NO: 30] Reverse primer [ SEQ ID NO: 31] Probe [SEQ ID NO: 32] Probe [SEQ ID NO: 33] Forward primer [SEQ ID NO: 34] Reverse primer [SEQ ID NO: 35] probe

Claims (7)

(A1) a forward PCR primer consisting of a DNA sequence from the 3 ′ end to at least the 16th among the DNA sequence represented by SEQ ID NO: 1 , and
(A2) A primer set for real-time PCR , comprising a reverse PCR primer consisting of a DNA sequence from the 3 ′ end to at least the 20th of the DNA sequence represented by SEQ ID NO: 2 . The set of forward PCR primer (A1) and reverse PCR primer (A2) according to claim 1 is at least one selected from the group consisting of the following (a), (b) and (c): Real-time PCR use primer set:
(A) a forward PCR primer (A1) comprising at least one selected from the DNA sequences represented by SEQ ID NOs: 10 to 15 and a reverse PCR primer comprising at least one selected from the DNA sequences represented by SEQ ID NOs: 17 to 25 Primer set containing (A2):
(B) A primer set comprising a forward PCR primer (A1) comprising the DNA sequence represented by SEQ ID NO: 9 and a reverse PCR primer (A2) comprising at least one selected from the DNA sequences represented by SEQ ID NOs: 22 to 25:
(C) A primer set for real-time PCR comprising a forward PCR primer (A1) comprising the DNA sequence represented by SEQ ID NO: 14 and a reverse PCR primer (A2) comprising the DNA sequence represented by SEQ ID NO: 26. A partial sequence of the DNA sequence represented by real-time PCR for flops Rye-mer sets and (B) SEQ ID NO: 3 according to claim 2, of 429,437,440,442,443,447 and 449 th base A kit for real-time PCR, comprising a sequence-specific binding probe labeled with an oligonucleotide consisting of a continuous DNA sequence of 15 to 25 bases containing any one base; or a complementary sequence thereof. The sequence-specific binding probe is a sequence-specific binding probe labeled with an oligonucleotide consisting of the DNA sequences 427-451, 429-449, and 433-451 of the DNA sequence represented by SEQ ID NO: 3. Item 6. A kit for real-time PCR according to item 3. The kit for real-time PCR according to claim 3 or 4, wherein the sequence-specific binding probe is a sequence-specific binding probe labeled with an oligonucleotide having a DNA sequence represented by SEQ ID NO: 31 or 32. (1) a step of performing real-time PCR using a human DNA sample as a template using the kit according to any one of claims 3 to 5 ; and (2) a human DNA sample in which a signal is detected in step (1) is HLA. A method for determining whether or not a human DNA specimen is derived from a human having HLA-A: 24: 02, which comprises a step of determining that the human DNA having A: 24: 02 is derived. (1) a step of performing real-time PCR using a human DNA sample as a template using the kit according to any one of claims 3 to 5 ; and (2) collection of a human DNA sample from which a signal is detected in step (1) A method for screening an HLA-A: 24: 02-restricted peptide vaccine administration target group, comprising a step of selecting an original human as a subject to be administered with an HLA-A: 24: 02-restricted peptide vaccine.

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