阅读说明：本技术 用于检测hla-b*1502等位基因的引物组、试剂盒及方法 (Primer group, kit and method for detecting HLA-B1502 allele ) 是由 赵方圆 翟瑞雪 智慧芳 倪君君 于 2020-04-30 设计创作，主要内容包括：本发明提供了用于检测HLA-B*1502等位基因的引物组、试剂盒及方法,该引物组包括3个序列特异性扩增引物对和至少一个内参基因引物对,这3个序列特异性扩增引物对的上下游引物的核苷酸序列分别由SEQ ID NO.1-6所示。利用该引物组,可对HLA-B*1502等位基因的基因型进行检测。(The invention provides a primer group, a kit and a method for detecting HLA-B1502 allelic genes, wherein the primer group comprises 3 sequence specificity amplification primer pairs and at least one internal reference gene primer pair, and the nucleotide sequences of the upstream primer and the downstream primer of the 3 sequence specificity amplification primer pairs are respectively shown by SEQ ID NO. 1-6. By using the primer group, the genotype of the HLA-B1502 allele can be detected.)

1. A primer set for detecting HLA-B1502 alleles, comprising:

a first sequence-specific amplification primer pair for amplifying HLA-B1502 allele, wherein the nucleotide sequence of the upstream primer is shown by SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 2;

a second sequence-specific amplification primer pair for amplifying HLA-B1502 allele, wherein the nucleotide sequence of the upstream primer is shown by SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 4;

a third sequence-specific amplification primer pair for amplifying HLA-B1502 allele, wherein the nucleotide sequence of the upstream primer is shown by SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 6;

at least one reference gene primer pair for amplifying reference genes.

2. The primer set of claim 1, wherein the at least one reference gene primer pair comprises:

in the first internal reference gene primer pair, the nucleotide sequence of the upstream primer is shown by SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 8;

in the second internal reference gene primer pair, the nucleotide sequence of the upstream primer is shown by SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 10.

3. Use of a primer set according to claim 1 or 2 for the preparation of a kit for the detection of HLA-B1502 alleles.

4. Kit for the detection of HLA-B1502 alleles comprising PCR reaction reagents and a primer set according to claim 1 or 2.

5. The kit according to claim 4,

the final concentration of each primer in the primer set is in the range of 20-300 nM;

the PCR reagent comprises DNA polymerase, PCR buffer solution, dNTPs and ultrapure water;

wherein the amount of the DNA polymerase is in the range of 0.5-5U and the final concentration of each dNTP in the dNTPs is in the range of 50-500. mu.M.

6. The kit according to claim 4 or 5,

the PCR amplification reaction conditions suitable for the kit comprise: 1-10min at 92-96 ℃; 5-60s at 92-98 ℃, 10-60s at 56-68 ℃ and 30s-5min at 68-72 ℃ for 25-40 cycles; 0-30min at 68-72 ℃.

Method for the amplification of an HLA-B1502 allele, wherein an HLA-B1502 allele is amplified using the primer set according to claim 1 or 2, or the kit according to any one of claims 4 to 6.

8. The method of claim 7,

the final concentration of each primer in the primer set is in the range of 20-300 nM;

the PCR reaction reagent for HLA-B1502 allele amplification comprises DNA polymerase, PCR buffer solution, dNTPs and ultrapure water;

wherein the amount of the DNA polymerase is in the range of 0.5-5U and the final concentration of each dNTP in the dNTPs is in the range of 50-500. mu.M.

9. The method according to claim 7 or 8,

the PCR amplification reaction condition for the HLA-B1502 allele amplification comprises the following steps: 1-10min at 92-96 ℃; 5-60s at 92-98 ℃, 10-60s at 56-68 ℃ and 30s-5min at 68-72 ℃ for 25-40 cycles; 0-30min at 68-72 ℃.

10. A method for detecting HLA-B1502 genotype comprising:

step 1: extracting template DNA from a sample to be detected as an amplification template;

step 2: performing a PCR amplification reaction on a PCR reaction system containing the amplification template by using the method for amplifying the HLA-B1502 allele according to any one of claims 7 to 9 to obtain a PCR product;

and step 3: detecting the PCR product by electrophoresis;

and 4, step 4: determining the HLA-B1502 genotype of the sample to be detected according to the electrophoresis detection result;

the electrophoresis detection result shows that the sample to be detected is positive in HLA-B1502 genotype when each primer pair in the primer group has a corresponding amplification band, the electrophoresis detection result shows that each reference gene primer pair in the primer group has a corresponding amplification band, and the sample to be detected is negative in HLA-B1502 genotype when at least one sequence specific amplification primer pair in the primer group does not have a corresponding amplification band.

Technical Field

The invention relates to the technical field of biological detection, in particular to a primer group, a kit and a method for detecting HLA-B1502 allele.

Background

Antiepileptic drugs such as carbamazepine, oxcarbazepine and phenytoin sodium, etc. may cause adverse skin reactions such as mild Maculopapule (MPE), Stevens-Johnson syndrome (SJS), Toxic Epidermal Necrolysis (TEN), and Drug Hypersensitivity Syndrome (DHS). Among them, SJS and TEN are serious skin and mucosal reactions that can lead to permanent disability and even fatality, with TEN fatality rates as high as 40%.

HLA (human leukocyte antigen) is a gene cluster encoding the human Major Histocompatibility Complex (MHC), located on the short arm of human chromosome 6, and is classified into HLA-I, II and III genes. Classical HLA-class I genes include HLA-A, HLA-B and HLA-C, wherein HLA-B is the most polymorphic region of the human genome, comprising more than 1600 alleles. The united states Food and Drug Administration (FDA) issued a notice at 12 months 12 of 2007 warning HLA-B1502 allele positive patients that severe and potentially fatal skin reactions may occur with carbamazepine, and recommended patients of asian descent to begin blood genetic screening prior to using carbamazepine.

At present, the drug specification of antiepileptic drugs such as carbamazepine and phenytoin suggests that patients carrying HLA-B1502 alleles should be screened for HLA-B1502 alleles before receiving antiepileptic treatments such as carbamazepine and phenytoin, and patients with positive screening results should not use antiepileptic drugs such as carbamazepine and phenytoin unless the expected benefit of the drugs is significantly greater than the risk of severe skin reactions.

Currently, the human leukocyte antigen HLA-B can be genotyped by whole genome sequencing, and the HLA-B1502 allele can be identified. However, the method has long detection period and high cost.

Disclosure of Invention

The invention provides a primer group, a kit and a method for detecting HLA-B1502 allele, which can detect HLA-B1502 genotype.

In order to achieve the purpose, the invention is realized by the following technical scheme:

in a first aspect, the present invention provides a primer set for detecting HLA-B1502 alleles, comprising: a first sequence-specific amplification primer pair for amplifying HLA-B1502 allele, wherein the nucleotide sequence of the upstream primer is shown by SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 2; a second sequence-specific amplification primer pair for amplifying HLA-B1502 allele, wherein the nucleotide sequence of the upstream primer is shown by SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 4; a third sequence-specific amplification primer pair for amplifying HLA-B1502 allele, wherein the nucleotide sequence of the upstream primer is shown by SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 6; at least one reference gene primer pair for amplifying reference genes.

In detail, different sequence-specific amplification primer pairs correspond to different key sites.

In detail, the reference gene is a conserved gene to control the quality of the amplification process of the PCR system. The reference gene may be a gene other than HLA, or may be a gene other than HLA-B1502 allele. I.e. the reference gene does not comprise the HLA-B1502 allele.

In detail, sequence specific primers can be designed based on the differences of several bases in the core sequences of different types. Thus, based on the sequence-specific primers, the genotype of HLA-B1502 allele can be determined by PCR (Polymerase Chain Reaction) combined with electrophoresis.

Specifically, the primer set can be used to amplify a sample to be detected, and the PCR amplification product can be subjected to electrophoresis detection. And the electrophoresis detection result shows that when each primer pair in the primer group has a corresponding amplification band, the sample to be detected is positive in HLA-B1502 genotype. And the electrophoresis detection result shows that each internal reference gene primer pair in the primer group has a corresponding amplification band, and when at least one sequence specific amplification primer pair in the primer group does not have a corresponding amplification band, the sample to be detected is negative to HLA-B1502 genotype.

In detail, the length of the amplified fragment of the HLA-B1502 allele amplified by the first sequence-specific amplification primer pair is 677 bp; the length of an amplified fragment of the HLA-B1502 allele amplified by the second sequence-specific amplification primer pair is 124 bp; the third sequence-specific amplification primer pair has the length of 374bp for amplifying an amplified fragment of the HLA-B1502 allele.

Further, the at least one reference gene primer pair comprises: in the first internal reference gene primer pair, the nucleotide sequence of the upstream primer is shown by SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 8; in the second internal reference gene primer pair, the nucleotide sequence of the upstream primer is shown by SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 10.

In detail, the length of an amplified fragment of the first internal reference gene primer pair amplified internal reference gene is 785 bp; the length of the amplified fragment of the second reference gene primer pair amplified reference gene is 250 bp.

Based on the above, after PCR amplification is performed by using the primer set, DNA fragments with different lengths can be generated, so that the fragments with different lengths can be distinguished by subsequent electrophoresis. And determining the HLA-B1502 genotype according to the electrophoresis detection result.

The following description will be made for each of the 5 primer pairs with reference to the above description:

a first sequence-specific amplification primer pair, an upstream primer and a downstream primer for amplifying HLA-B1502 allele, wherein the length of a corresponding amplification fragment is 677bp, and the sequences are respectively as follows:

the sequence of the upstream primer is as follows: CCGGAACACACAGATCTCCAAGACCAACA

The sequence of the downstream primer is as follows: TTTCCTCCTCTTCTCGTGGGAGGCCAT are provided.

A second sequence-specific amplification primer pair, an upstream primer and a downstream primer for amplifying HLA-B1502 allele, wherein the length of a corresponding amplification fragment is 124bp, and the sequences are respectively as follows:

the sequence of the upstream primer is as follows: ATTACTCGCGAGTCCGAGGATGGC

The sequence of the downstream primer is as follows: CTATACTGCAGGTTCCGCAGGCTCT are provided.

A third sequence-specific amplification primer pair, an upstream primer and a downstream primer for amplifying HLA-B1502 allele, wherein the length of a corresponding amplification fragment is 374bp, and the sequences are respectively as follows:

the sequence of the upstream primer is as follows: TTGCCGGAGTATTGGGACCGGAAC

The sequence of the downstream primer is as follows: GTCGCAGCCATACATCCTCTGGATGA are provided.

The first internal reference gene primer pair is used for amplifying upstream and downstream primers of an internal reference gene, the length of a corresponding amplified fragment is 785bp, and the sequences are as follows:

the sequence of the upstream primer is as follows: AGACTTGCCAAGTGGAGCACCCAA

The sequence of the downstream primer is as follows: GGAGACGCATCTTGCTCTGTGCAGAT are provided.

The second internal reference gene primer pair is used for amplifying the upstream primer and the downstream primer of the internal reference gene, the length of a corresponding amplified fragment is 250bp, and the sequences are respectively as follows:

the sequence of the upstream primer is as follows: GCGTACATGATGTTGACCTTTCCAGGG

The sequence of the downstream primer is as follows: CGCGTCGTTCTGTAACTTTTCATCAGTTGC are provided.

In a second aspect, the present invention provides the use of a primer set according to any one of the first aspect above in the preparation of a kit for detecting an HLA-B1502 allele.

In a third aspect, the present invention provides a kit for detecting an HLA-B1502 allele, comprising PCR reaction reagents and a primer set according to any one of the first aspect above.

Further, the final concentration of each primer in the primer set is in the range of 20-300 nM;

the PCR reagent comprises DNA polymerase, PCR buffer solution, dNTPs and ultrapure water;

wherein the amount of the DNA polymerase is in the range of 0.5-5U and the final concentration of each dNTP (deoxyriboside triphosphate) in dNTPs is in the range of 50-500. mu.M.

For example, the final concentration of each primer may be 20nM, 50nM, 100nM, 150nM, 200nM, 250nM, or 300 nM; the amount of DNA polymerase used may be 0.5U, 1U, 2U, 3U, 4U or 5U; each dNTP final concentration can be 50 u M, 100 u M, 200 u M, 300 u M, 400 u M, 450M or 500M.

In detail, the DNA polymerase may be Taq enzyme, KOD FX enzyme, KOD Plus enzyme, LA Taq enzyme, or the like. In detail, the PCR buffer may be a concentrated buffer corresponding to the selected DNA polymerase, and the concentration may be 2X, 5X or 10X. The reaction system can be enlarged or reduced in equal proportion. In addition, when another DNA polymerase system is replaced, the amplification can be achieved by adjusting the ratio appropriately.

For example, when KOD FX is used as the DNA polymerase and 2 × concentrated buffer is used, the above PCR system may be configured such that the following components are used: 0.2-1. mu.l of DNA polymerase, 7-15. mu.l of PCR buffer, 1-10. mu.l of a mixture of various dNTPs, 0.3-2. mu.l of each of upstream and downstream primers, 5-500ng of DNA, and an appropriate amount of ultrapure water to replenish water to 20. mu.l. Of course, other volume sizes configured in the same proportions are also possible.

Further, the PCR amplification reaction conditions suitable for the kit include: 1-10min at 92-96 ℃; 5-60s at 92-98 ℃, 10-60s at 56-68 ℃ and 30s-5min at 68-72 ℃ for 25-40 cycles; 0-30min at 68-72 ℃.

For example, for 92-96 ℃, 92 ℃, 93 ℃, 94 ℃, 95 ℃ or 96 ℃; for 1-10min, it can be 1min, 2min, 3min, 6min, 9min or 10 min; for 5-60s, it can be 5s, 10s, 20s, 30s, 40s, 50s or 60 s; 56-68 deg.C, 56 deg.C, 58 deg.C, 60 deg.C, 62 deg.C, 66 deg.C or 68 deg.C; for 10-60s, it can be 10s, 20s, 30s, 40s, 50s or 60 s; 68-72 deg.C, 70 deg.C or 72 deg.C; for 30s-5min, it can be 30s, 1min, 2min, 3min, 4min or 5 min; for 25-40, there may be 25, 30, 35 or 40; for 0-30min, it can be 0min, 5min, 10min, 20min or 30 min.

In a fourth aspect, the present invention provides a method for amplifying an HLA-B1502 allele, comprising amplifying an HLA-B1502 allele using the primer set according to any one of the first aspect or the kit according to any one of the third aspect.

Further, the final concentration of each primer in the primer set is in the range of 20-300 nM;

the PCR reaction reagent for HLA-B1502 allele amplification comprises DNA polymerase, PCR buffer solution, dNTPs and ultrapure water;

wherein the amount of the DNA polymerase is in the range of 0.5-5U and the final concentration of each dNTP in the dNTPs is in the range of 50-500. mu.M.

Further, the PCR amplification reaction conditions for the amplification of HLA-B1502 allele comprise: 1-10min at 92-96 ℃; 5-60s at 92-98 ℃, 10-60s at 56-68 ℃ and 30s-5min at 68-72 ℃ for 25-40 cycles; 0-30min at 68-72 ℃.

In a fifth aspect, the present invention provides a method for detecting HLA-B1502 genotype, comprising:

step 1: extracting template DNA from a sample to be detected as an amplification template;

step 2: performing a PCR amplification reaction on a PCR reaction system containing the amplification template by using the method for amplifying the HLA-B1502 allele according to any one of claims 7 to 9 to obtain a PCR product;

and step 3: detecting the PCR product by electrophoresis;

and 4, step 4: determining the HLA-B1502 genotype of the sample to be detected according to the electrophoresis detection result;

the electrophoresis detection result shows that the sample to be detected is positive in HLA-B1502 genotype when each primer pair in the primer group has a corresponding amplification band, the electrophoresis detection result shows that each reference gene primer pair in the primer group has a corresponding amplification band, and the sample to be detected is negative in HLA-B1502 genotype when at least one sequence specific amplification primer pair in the primer group does not have a corresponding amplification band.

In detail, in the step 1, the template DNA is human genome DNA, and a manual extraction method or a commercial kit extraction method can be selected to extract a sample to be detected to obtain human genome DNA; the sample to be detected is a biological sample such as human blood, cells, tissues or buccal swab samples containing human genome DNA.

In detail, in step 3, fragments of different lengths can be resolved by agarose gel electrophoresis or polyacrylamide gel electrophoresis.

In detail, PCR-SSP (sequence specific primer) is a PCR reaction that is primed with sequence specific primers. The invention provides a sequence specific primer, which is designed according to the difference of key bases of different types of core sequences, so that the genotype of HLA-B1502 allele can be judged by a PCR-electrophoresis method.

The invention provides a primer group, a kit and a method for detecting HLA-B1502 allelic genes, wherein the primer group can amplify the HLA-B1502 allelic genes, so that the genotype of the HLA-B1502 allelic genes can be judged according to the electrophoresis detection result of amplification products. At least the following features may be provided:

(1) the detection cost is low, and the popularization is easy. The PCR-SSP can be used for detecting the genotype of the HLA-B1502 allele, the detection can be realized by using common non-labeled primers and a common PCR instrument and combining electrophoresis, and the cost is low. Without the need for expensive fluorescent probes and expensive fluorescent PCR instruments. Therefore, the primer does not need to be marked with fluorescence, and expensive and complex equipment is not needed, so that the detection cost is greatly saved, and any detection mechanism can be developed.

(2) The detection time is short, the detection efficiency is high, and convenience and rapidness are realized. The genotype of HLA-B1502 allelic gene can be directly judged by PCR-electrophoresis without sequencing, thus being simple and efficient.

(3) The amplification result is interpreted intuitively and accurately. Based on specific primer design, an amplification system and an amplification program, the sizes of amplification products of each pair of primers can be fully distinguished, and nonspecific products or dimers cannot be generated due to interaction among amplification fragments, so that the accuracy of results is ensured.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 shows the result of agarose gel electrophoresis detection according to an embodiment of the present invention.

Detailed Description

Specifically, the reagents used in the implementation of the invention are all commercial products, and the databases used in the implementation of the invention are all public online databases. The following examples are illustrative only and are not to be construed as limiting the invention.