CN112626201A - Primers and method for detecting HLA-B5701 typing - Google Patents

Primers and method for detecting HLA-B5701 typing Download PDF

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CN112626201A
CN112626201A CN202110056149.1A CN202110056149A CN112626201A CN 112626201 A CN112626201 A CN 112626201A CN 202110056149 A CN202110056149 A CN 202110056149A CN 112626201 A CN112626201 A CN 112626201A
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hla
primer
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董春燕
王淑一
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Hefei Aidikang Medical Laboratory Co ltd
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Abstract

The invention discloses a primer for detecting HLA-B5701 typing, which comprises a primer for amplifying HLA-B5701 and a sequencing primer; the polymerase reaction-direct sequencing method (PCR-SBT) can be used for quickly detecting the HLA-B5701 allele related to the liver injury caused by the flucloxacillin administration. The detection result completed by the method is accurate, and the method has important reference significance for the safety of the clinical medication of the flucloxacillin.

Description

Primers and method for detecting HLA-B5701 typing
Technical Field
The invention belongs to the fields of life science and biotechnology, and particularly relates to a primer for detecting HLA-B5701 allele, which can be used for rapidly detecting HLA-B5701 typing related to liver injury caused by flucloxacillin medication by adopting a polymerase reaction-direct sequencing method (PCR-SBT) and can be used for guiding the medication of the flucloxacillin clinically.
Background
The liver is one of the metabolic organs of the human body, and most of the drugs need to be decomposed and metabolized in the liver, so the liver also becomes one of the target organs for drug and metabolite attack. Then, during the course of using drugs, the disease caused by toxic damage caused by the drugs themselves or their metabolites, or the allergic reaction of liver to drugs and metabolites is called drug-induced liver injury (DILI), also called drug-induced liver disease (DILD), which is a serious adverse drug reaction, a complex disease. With the recent increase in the reports of liver damage caused by various drugs, DILI has become a non-negligible public health problem. According to the World Health Organization (WHO) statistics, DILI has risen to the 5 th cause of death worldwide, directly affecting patient treatment and recovery. In the united states, DILI has become the leading cause of acute liver failure. DILI is also becoming more severe in China, and according to statistics, DILI patients account for l% -5% of liver disease patients, 10% of acute hepatitis patients and 13% -30% of fulminant liver failure patients. However, in practice, because the clinical manifestations of DILI are complex, laboratory examinations are not specific, and their true incidence is often underestimated. In addition, DILI incidence is increasing with the advent of new drugs on the market.
Previous studies have shown that DILI occurs in association with factors such as pharmaceutically active metabolites, metabolic enzyme and transporter activity, cholestasis, calcium imbalance, mitochondrial dysfunction, and oxidative stress. However, as research continues, more and more studies have shown that DILI occurs in association with individual Human Leukocyte Antigen (HLA) allelic polymorphisms. Its polymorphism is the result of the presence of different morphogens, i.e.alleles (alleles), at the same position on homologous chromosomes. There are studies to find relevant information between liver damage due to specific drugs and major HLA gene sites by candidate gene association studies and whole genome association analysis (as shown in table 1). Among them, flucloxacillin has been reported to cause cholestatic hepatitis as a clinically commonly used penicillinase-resistant antibiotic, and the adverse reaction is more likely to occur in women, the elderly and patients with long-term medication. The HLA class I gene B5701 is highly related to DILI caused by flucloxacillin and is an allele with the strongest association with DILI. Although GWAS data suggest that it is highly correlated with flucloxacillin-induced DILI, since the incidence of this adverse reaction is low (8.5/10 ten thousand), the positive predictive value is only 0.0012, and an average of 13513 patients tested could predict one DILI.
HLA, also known as human MHC, is a group of closely linked genes that control cell-cell recognition and regulate immune responses. The HLA gene family is located on the short arm (6p21.31) of the No. 6 human chromosome, has the full length of 3.6Mb, accounts for 0.1 percent of the number of bases of a human genome, and is a region which is most complex, most abundant in polymorphism, most concentrated in related genes of immune function and most closely related to diseases so far. The DNA fragment can be divided into 3 regions (as shown in FIG. 1) according to its chromosomal arrangement, and the HLA class I region is located at the telomere side and has a length of about 2000kb, including A, B, C three loci, and encodes the alpha chain (i.e., heavy chain) of HLA-A, HLA-B, HLA-C molecules. HLA class I molecules are distributed on all tissue cells, but the expression level is different, and the HLA class I molecules are transplantation antigens on cell membranes and are main antigens causing rejection reaction after transplantation. The HLA class II region is located on the centromere side, has the length of about 1000kb, and comprises three regions of DR, DQ and DP, wherein each region comprises a plurality of A and B genes respectively encoding the alpha chain and the beta chain of HLA class II molecules (HLA-DR, HLA-DQ and HLA-DP). HLA class II molecules are mainly distributed on immune cells, and can be used as recognition markers among immune cells to induce immune response and regulate the interaction among the immune cells, thereby playing an important role in immune response. The class III gene region is located between HLA-I, II gene regions and is composed of some genes related to complement and some inflammatory factors. HLA genes encode a group of polymorphic proteins positioned on the cell surface, can combine and present antigen peptides for T cells to recognize, activate the T cells to start specific immune response, and is directly related to individual immune response difference.
Therefore, the research on the polymorphism of the HLA-B5701 gene related to DILI caused by flucloxacillin in the project is beneficial to the deep research on the generation mechanism of the DILI, improves the safety of the flucloxacillin clinical medication, realizes individualized treatment, is also beneficial to the early screening of the medicine which is easy to cause hepatotoxicity in the research and development of new medicines, and has important clinical application and economic value.
Disclosure of Invention
The invention aims to provide a primer for detecting HLA-B5701 allele, which can rapidly detect HLA-B5701 typing related to liver injury caused by flucloxacillin medication by adopting a PCR-SBT method.
The primer for detecting the HLA-B5701 allele comprises the following components:
the primer for amplifying the HLA-B5701 has the base sequence as follows:
HLA-B*5701-F：TGTAAAACGACGGCCAGTGAACATGAAGGCCTCCGCG
HLA-B*5701-R：AACAGCTATGGCCATACATCACCTGGATGATGTG。
further, the kit also comprises a sequencing primer, wherein the base sequence of the sequencing primer is as follows:
M13-F：TGTAAAACGACGGCCAGT
M13-R：AACAGCTATGACCATG。
further, the kit also comprises a primer for detecting the internal reference gene, and the base sequence of the primer is as follows:
actin-F：CTAACTGCGCGTGCGTTCT
actin-R：AGTCCTTAGGCCGCCAGGGG。
the invention also provides a method for detecting the HLA-B5701 allele, which comprises the following steps:
a method of detecting HLA-B5701 alleles comprising the steps of:
(1) extracting sample genome DNA;
(2) amplifying the DNA extracted in the step (1) by using a primer HLA-B5701-F/HLA-B5701-R for amplifying the HLA-B5701 allele and a primer actin-F/actin-R for amplifying the reference gene;
(3) sequencing the product amplified by the HLA-B5701-F/HLA-B5701-R in the step (2) by using a sequencing primer M13 to obtain an amplified gene sequence;
(4) comparing the gene sequence obtained in the step (3) with the HLA-B5701 sequence in a known gene library, judging a sequencing result, and determining whether the sample is an HLA-B5701 typing;
wherein the PCR amplification primers are respectively as follows:
HLA-B*5701-F：TGTAAAACGACGGCCAGTGAACATGAAGGCCTCCGCG
HLA-B*5701-R：AACAGCTATGGCCATACATCACCTGGATGATGTG
actin-F：CTAACTGCGCGTGCGTTCT
actin-R：AGTCCTTAGGCCGCCAGGGG。
the sequencing primer base sequence is:
M13-F：TGTAAAACGACGGCCAGT
M13-R：AACAGCTATGACCATG。
the invention also provides a kit for detecting HLA-B5701 allele, which comprises:
(i) blood DNA extraction reagent;
(ii) detection system PCR amplification reaction solution: comprises a primer for amplifying HLA-B5701 allele and a primer for amplifying reference gene, and the base sequences are respectively:
HLA-B*5701-F：TGTAAAACGACGGCCAGTGAACATGAAGGCCTCCGCG
HLA-B*5701-R：AACAGCTATGGCCATACATCACCTGGATGATGTG
actin-F：CTAACTGCGCGTGCGTTCT
actin-R：AGTCCTTAGGCCGCCAGGGG
(iii) sequencing system reagent: comprises a sequencing primer, and the base sequence of the sequencing primer is as follows:
M13-F：TGTAAAACGACGGCCAGT
M13-R：AACAGCTATGACCATG
(iv) a positive control and a negative control.
Has the advantages that: the invention designs a primer for amplifying HLA-B5701 allele. A stable amplification system is constructed by adopting a PCR-SBT technology. By adjusting the reaction conditions such as primer concentration and annealing temperature, the amplification efficiency can be optimized. The method for detecting whether the patient can use the flucloxacillin medicine or not by using the sequencing technology can detect the typing of HLA-B5701, has good specificity and simple and convenient operation, and reduces the detection cost and difficulty compared with an NGS method.
Drawings
FIG. 1 is an agarose gel electrophoresis of HLA-B5701 allele sample screening, wherein sample No. 17 is positive for HLA-B5701 allele.
FIG. 2 shows a sequencing partial map of a positive sample of HLA-B5701 allele number 17 confirmed by sequencing.
FIG. 3 is an agarose gel electrophoresis of a primer specificity experiment for amplification of HLA-B5701 alleles.
Detailed Description
Example 1
The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings. It should be noted that the conventional conditions and methods not described in the examples are generally employed by those skilled in the art according to the routine procedures: such as OsOb and Kingston, fourth edition, or following the manufacturer's suggested procedures and conditions.
A primer for detecting HLA-B5701 genotype, which is designed to span the intron of HLA-B gene and is designed to be specific amplification primer in the 2 nd and 3 rd exons, comprising:
the base sequences of the primer for amplifying the HLA-B5701 gene and the primer for amplifying the reference gene are respectively as follows:
HLA-B*5701-F：TGTAAAACGACGGCCAGTGAACATGAAGGCCTCCGCG
HLA-B*5701-R：AACAGCTATGGCCATACATCACCTGGATGATGTG
actin-F：CTAACTGCGCGTGCGTTCT
actin-R：AGTCCTTAGGCCGCCAGGGG
the primer also comprises a sequencing primer, and the base sequence of the sequencing primer is as follows:
M13-F：TGTAAAACGACGGCCAGT
M13-R：AACAGCTATGACCATG
a kit for detecting HLA-B5701 genotype comprises
(i) Blood DNA extraction reagent;
(ii) detecting a system PCR reaction solution;
(iii) sequencing system reagents;
(iv) a positive control and a negative control.
Wherein, the PCR amplification reaction solution of the detection system comprises: 2 times PCR Buffer; 2mM dNTPs; KOD FX DNA Polymerase (1U/. mu.L); amplifying HLA-B5701 upstream and downstream primers (10 mu M); and amplifying upstream and downstream primers (10 mu M) of the internal reference gene.
The sequencing system reagent comprises: sequencing purification solution (ExoI:0.6U, CIP:1.2U), EDTA (125mmol), absolute ethanol, 75% ethanol, HIDI (highly deionized formamide), sequencing primers: and upstream and downstream primers (3.2 μm) for detecting HLA-B5701.
Example 2
(1) The operation flow of extracting the blood genome DNA is as follows:
1) to 200. mu.L of blood was added 20. mu.L of LProteinase K solution, and the mixture was mixed well.
2) Adding 200 μ L buffer GB, mixing thoroughly, standing at 70 deg.C for 10min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover.
3) Adding 200 μ L of anhydrous ethanol, shaking thoroughly to mix for 15s, wherein flocculent precipitate may appear, and centrifuging briefly to remove water beads on the inner wall of the tube cover.
4) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (adsorption column is put into a collecting pipe), centrifuging at 12,000rpm (13,400 Xg) for 30s, pouring off waste liquid, and putting adsorption column CB3 back into the collecting pipe.
5) To the adsorption column CB, 500. mu.L of the buffer GD (to check whether or not absolute ethanol has been added before use) was added, and the mixture was centrifuged at 12,000rpm (13,400 Xg) for 30 seconds, and the waste liquid was discarded, and the adsorption column CB3 was put into a collection tube.
6) To the adsorption column CB3, 600. mu.L of a rinsing solution PW (previously used, whether or not absolute ethanol was added or not was checked), centrifuged at 12,000rpm (13,400 Xg) for 30 seconds, the waste liquid was discarded, and the adsorption column CB3 was put into a collection tube.
7) Operation 6 is repeated.
8) The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000rpm (13,400 Xg) for 2min, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material. (Note: the purpose of this step is to remove the residual rinsing solution in the adsorption column, and the residual ethanol in the rinsing solution will affect the subsequent enzyme reaction (enzyme digestion, PCR, etc.) experiment).
9) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50 μ L of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12,000rpm (13,400 Xg) for 2min, and collecting the solution into the centrifuge tube.
(2) Reagent preparation: preparing X mul of PCR reaction liquid of a detection system according to the parts of detected people, and subpackaging 18 mul of each part:
x18. mu.l reaction solution X (n specimen +1 part positive control +1 part negative control +1 part blank control)
And n is the number of detected samples.
The preparation method of the PCR amplification system reagent comprises the following steps:
Figure BDA0002900953860000061
wherein, the primer sequence is as follows:
Figure BDA0002900953860000062
note: f is an upstream primer, R is a downstream primer
(3) Sample adding: adding 2 mul DNA into the PCR reaction solution of the detection system; directly adding 2 mul of negative control substance into the negative control; blank control was supplemented with 2. mu.l of physiological saline or nothing.
(4) Amplification: the detection is carried out on a conventional PCR instrument, and available instruments include ABI veriti (Applied Biosystems, USA) and the like. The reaction conditions were as follows:
Figure BDA0002900953860000071
(5) electrophoresis: electrophoresis on 1.5% agarose gel at 110V for 35min, and observation on a gel imaging system.
(6) Sanger sequencing:
take 9PCR products and 2. mu.l purification system. Purification was performed according to the following procedure:
Figure BDA0002900953860000072
mu.l of the purified product was mixed with the upper and lower sequencing primers, respectively, according to the following system:
Figure BDA0002900953860000073
sequencing reaction program:
Figure BDA0002900953860000074
Figure BDA0002900953860000081
and (3) a precipitation link:
adding 2 mu l of 125mmol EDTA into the product after the sequencing reaction, and standing for 5 min; adding 15 mul of absolute ethyl alcohol, and mixing evenly by vortex; centrifuging at 3700rpm for 30 min; inverting, centrifuging for 15sec, adding 50ml 70% ethanol, and mixing by vortex; centrifuging at 3700rpm for 15 min; inverting and centrifuging for 15sec, and placing on a metal bath at 95 ℃; after addition of 10. mu.l CBL, denaturation was carried out for 5min and finally sequencing was carried out on a sequencer (ABI3730) at-20 ℃ for 2 min.
(7) And (5) judging a result: and (3) comparing the sequencing result with an HL-B5701 typing reference sequence, and reporting the result according to the actual mutation condition.
Example 3
20 blood samples of the physical examination were taken, genomic DNA was extracted, reagents were prepared and the samples were tested for the presence of HLA-B5701 allele as described in example 2. Each sample was added to 2. mu.l of the detection system PCR reaction solution. The amplification time is 160 minutes by using a common PCR instrument. After PCR amplification, the result shows that in 20 samples, sample No. 17 has a band, and the band is single and has the correct size, which indicates that sample No. 17 is HLA-B5701; after sequencing, the HLA-B5701 allele was confirmed by alignment with standard sequences. The results of agarose gel electrophoresis of 20 samples are shown in FIG. 1, and the alignment chart of sample No. 17 is shown in FIG. 2.
Example 4
10 clinical samples of known type (no HLA-B5701 type) were taken, genomic DNA was extracted, reagents were prepared and primer specificity was determined as described in example 2. Each sample was added to 2. mu.l of the detection system PCR reaction solution. The amplification time is 160 minutes by using a common PCR instrument. After PCR amplification, the results showed that no band was found in any of the 10 samples after PCR amplification, no HLA-B5701 allele could be amplified, and the primer specificity was good. The results of agarose gel electrophoresis of 10 clinical specimens are shown in FIG. 3.
The detection results are combined, so that the primer can amplify the HLA-B5701 allele, has good specificity, is completely correct in sequencing, has no set peak, and can be used for detecting the HLA-B5701 allele.
Sequence listing
<110> Yidekang laboratory Co., Ltd for medical examination of Hefei Aidikang
<120> primers and method for detecting HLA-B5701 typing
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aacagctatg gccatacatc acctggatga tgtg 34
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Claims (5)

1. Primer for detecting the HLA-B5701 allele, comprising:
the primer for amplifying the HLA-B5701 has the base sequence as follows:
HLA-B*5701-F：TGTAAAACGACGGCCAGTGAACATGAAGGCCTCCGCG
HLA-B*5701-R：AACAGCTATGGCCATACATCACCTGGATGATGTG。
2. the primer of claim 1, further comprising a sequencing primer having the base sequence:
M13-F：TGTAAAACGACGGCCAGT
M13-R：AACAGCTATGACCATG。
3. the primer according to claim 1, further comprising a primer for detecting an internal reference gene, which has a base sequence of:
actin-F：CTAACTGCGCGTGCGTTCT
actin-R：AGTCCTTAGGCCGCCAGGGG。
4. a method of detecting HLA-B5701 alleles comprising the steps of:
(1) extracting sample genome DNA;
(2) amplifying the DNA extracted in the step (1) by using a primer HLA-B5701-F/HLA-B5701-R for amplifying the HLA-B5701 allele and a primer actin-F/actin-R for amplifying the reference gene;
(3) sequencing the product amplified by the HLA-B5701-F/HLA-B5701-R in the step (2) by using sequencing primers M13-F and M13-R to obtain an amplified gene sequence;
(4) comparing the gene sequence obtained in the step (3) with the HLA-B5701 sequence in a known gene library, judging a sequencing result, and determining whether the sample is an HLA-B5701 typing;
wherein the PCR amplification primers are respectively as follows:
HLA-B*5701-F：TGTAAAACGACGGCCAGTGAACATGAAGGCCTCCGCG
HLA-B*5701-R：AACAGCTATGGCCATACATCACCTGGATGATGTG
actin-F：CTAACTGCGCGTGCGTTCT
actin-R：AGTCCTTAGGCCGCCAGGGG。
5. the method of claim 4, wherein the sequencing primer base sequence is:
M13-F：TGTAAAACGACGGCCAGT
M13-R：AACAGCTATGACCATG。
CN202110056149.1A 2021-01-15 2021-01-15 Primers and method for detecting HLA-B5701 typing Pending CN112626201A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140342919A1 (en) * 2013-03-10 2014-11-20 Johanna M. Seddon Markers related to age-related macular degeneration and uses therefor
CN105177152A (en) * 2015-09-29 2015-12-23 福州艾迪康医学检验所有限公司 Method and primers for detecting HLA-B*51 allelomorphic gene
CN105861694A (en) * 2016-05-11 2016-08-17 中南大学湘雅三医院 Biomarker for predicating idiosyncratic liver injuries of child patients and application of biomarker
CN106636370A (en) * 2016-11-30 2017-05-10 武汉海吉力生物科技有限公司 Nucleic acid, kit and method for detecting human HLA-B*5701 allele

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140342919A1 (en) * 2013-03-10 2014-11-20 Johanna M. Seddon Markers related to age-related macular degeneration and uses therefor
CN105177152A (en) * 2015-09-29 2015-12-23 福州艾迪康医学检验所有限公司 Method and primers for detecting HLA-B*51 allelomorphic gene
CN105861694A (en) * 2016-05-11 2016-08-17 中南大学湘雅三医院 Biomarker for predicating idiosyncratic liver injuries of child patients and application of biomarker
CN106636370A (en) * 2016-11-30 2017-05-10 武汉海吉力生物科技有限公司 Nucleic acid, kit and method for detecting human HLA-B*5701 allele

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANN K DALY ET AL.: ""HLA-B*5701 genotype is a major determinant of drug-induced liver injury due to flucloxacillin"", 《NATURE GENETICS》 *
章伟等: ""HLA-B新的等位基因B*5136的确认和分析"", 《中华医学遗传学杂志》 *

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Application publication date: 20210409