CN112626204A - Primers and method for detecting HLA-B1502 typing useful for guiding administration of carbamazepine - Google Patents
Primers and method for detecting HLA-B1502 typing useful for guiding administration of carbamazepine Download PDFInfo
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Abstract
The invention discloses a primer and a method for detecting HLA-B1502 typing, wherein the primer comprises a forward primer, a reverse primer, an internal reference primer and a sequencing primer for specifically amplifying HLA-B1502 typing sequences; combined with Sanger sequencing technology, the kit can rapidly and accurately detect HLA-B1502 typing and can be used for guiding the administration of carbamazepine.
Description
Technical Field
The invention belongs to the fields of life science and biotechnology, and particularly relates to a primer and a method for detecting HLA-B1502 typing which can be used for guiding carbamazepine administration, wherein a common PCR (polymerase chain reaction) and Sanger sequencing technology are adopted.
Background
Carbamazepine is a clinically common antiepileptic drug, has wider clinical application, and is also a common drug for diseases such as trigeminal neuralgia, glossopharyngeal neuralgia, manic depression and the like. Many years of research have shown that aromatic antiepileptic drugs such as Carbamazepine (CBZ) can cause drug eruptions, about 10% of which are caused by carbamazepine, including non-bullous cutaneous adverse reactions Maculopapule (MPE) and hypersensitivity syndrome (HSS), as well as bullous cutaneous adverse reactions Steven-Johnson syndrome (SJS), toxic epinecrotic lysis (TEN). Wherein SJS and TEN are serious drug eruptions which can endanger life, and the death rate is up to more than 30%.
Carbamazepine attracts attention because of causing drug eruptions and even severe drug eruptions, and the occurrence of fatal adverse reactions such as SJS and TEN has certain correlation with HLA-B1502 genes of patients, and the correlation with HLA-B1502 is firstly proved by Han population in Taiwan area of China. Analysis further showed that HLA-B1502 was significantly associated with SJS/TEN in the Han population, southeast Asia countries.
The FDA in the united states has approved a recommendation in the kamazepine package for screening HLA-B1502 alleles in han and south-east asian populations before taking carbamazepine, and individuals positive for HLA-B1502 should be cautious with carbamazepine to avoid severe skin toxicity; relevant documents of clinical examination center of health and wellness Commission of China "technical guidelines (trial) for detecting genes of drug metabolizing enzymes and drug action targets", it is also clear that "people carrying HLA-B1502 alleles must use carbamazepine and phenytoin with caution to avoid causing SJS/TEN".
The proportion of people carrying the HLA-B1502 allele, reported in Chinese population, is 4% -22%. Therefore, before a patient selects carbamazepine for treatment, the HLA-B1502 genotyping is necessary, and experimental basis can be provided for clinical safe medication.
The current typing methods for HLA are mainly of type 3: serological, cytological and molecular biology typing methods. Among them, serological and cytological typing methods are rarely used at present because of the difficulty in obtaining highly specific antibodies and specific cells and the cumbersome operation. With the development of molecular biology techniques, rapid and accurate HLA genotyping established at the DNA molecular level has gradually replaced traditional serotyping methods. The molecular biology typing method mainly comprises polymerase chain reaction-allele genome specific primers (PCR-SSP), polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSO) and polymerase reaction-direct sequencing method (PCR-SBT). The PCR-SBT can directly determine the sequence of the HLA genotype, has high resolution and can directly discover new alleles, but because of heterozygotes, accurate results of each haplotype can be obtained only after homologous chromosomes are separated, the complexity of operation is increased, and the detection cost is correspondingly higher; PCR-SSP and PCR-SSO belong to low-resolution HLA typing methods, wherein PCR-SSP (sequence specific primer PCR) is a HLA genotyping method which is applied in clinic and basic research, the typing principle is that a set of primers specific to each allele or group specificity is designed according to the nucleotide sequence of each allele of HLA, and the gene fragment is amplified through PCR specificity, thereby achieving the purpose of analyzing HLA polymorphism. Compared with other HLA genotyping methods, the PCR-SSP avoids the steps of hybridization, enzyme digestion and the like, particularly has the characteristics of simplicity, convenience, time saving, small required sample amount, low requirement on blood quality, high specificity, easy mastering of technical conditions and the like, has intuitive result and convenient analysis, and is the HLA genotyping technology which is firstly applied to clinic. In summary, the research uses a PCR-SSP combined with gene sequencing method to perform HLA-B1502 gene typing detection, and provides experimental basis for clinical safe medication.
Disclosure of Invention
The invention aims to provide a primer and a method for detecting HLA-B1502 genotyping for guiding carbamazepine administration, which can rapidly and accurately detect HLA-B1502 genotyping in a patient by adopting common PCR combined with Sanger sequencing technology and provide guidance for carbamazepine administration. The primer for detecting HLA-B1502 typing comprises the following components: the primer for specifically amplifying HLA-B1502 typing has the base sequence as follows: HLA-B1502-F: TGTAAAACGACGGCCAGTGAACACACAGATCTCCAAGACCA HLA-B1502-R: AACAGCTATGACCATGCAGCCATACATCCTCTGGATGA are provided.
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 comprises primers actin-F and actin-R for detecting the reference genes, wherein the base sequences are as follows:
actin-F：CTAACTGCGCGTGCGTTCT
actin-R：AGTCCTTAGGCCGCCAGGGG。
further, the concentration of the forward primer and the reverse primer in the system is 0.1875 uM.
Further, the primer of the reference gene is used in a concentration ratio of: actin-F: actin-R ═ 1: 1.
The invention also provides a method for detecting HLA B1502 typing of a guide carbamazepine drug, which comprises the following steps:
1. extracting genome DNA in peripheral blood or bone marrow;
2. amplifying the DNA in the step 1 by using amplification primers HLA-B1502-F/HLA-B1502-R and actin-F/actin-R, determining that the sample is negative in HLA-B1502 typing when the internal reference actin-F/actin-R amplifies a target band and the HLA-B1502-F/HLA-B1502-R amplifies a non-target band, and performing sequencing detection on the sample when the two pairs of primers amplify the target band;
3. sequencing the amplification product in the step 2 by using sequencing primers M13-F and M13-R;
4. comparing the sequencing result in the step 3 with the wild type HLA-B1502 allele sequence, and determining that the sample is positive in HLA-B1502 typing if the sequencing result is completely consistent with the HLA-B1502 allele sequence, otherwise, the sample is negative;
wherein the PCR amplification primers are:
HLA-B*1502-F：TGTAAAACGACGGCCAGTGAACACACAGATCTCCAAGACCA
HLA-B*1502-R：AACAGCTATGACCATGCAGCCATACATCCTCTGGATGA。
further, the sequencing primer base sequence is:
M13 F：TGTAAAACGACGGCCAGT
M13 R：AACAGCTATGACCATG。
further, the final concentration of the template DNA in the amplification system was 0.25ng/uL or more.
The invention also provides a kit for detecting and guiding the HLA B1502 typing of carbamazepine medication, which comprises a primer for detecting and specifically amplifying the HLA-B1502 typing, and the base sequence of the primer is as follows:
HLA-B*1502-F：TGTAAAACGACGGCCAGTGAACACACAGATCTCCAAGACCA
HLA-B*1502-R：AACAGCTATGACCATGCAGCCATACATCCTCTGGATGA。
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 comprises primers actin-F and actin-R for detecting the reference genes, wherein the base sequences are as follows:
actin-F：CTAACTGCGCGTGCGTTCT
actin-R：AGTCCTTAGGCCGCCAGGGG。
has the advantages that: firstly, the invention can carry out sequence analysis on all 15 subtypes of HLA-B1502 and other highly similar types, designs specific primers aiming at HLA-B1502 type classification, can exclude most types which are not HLA-B1502 in HLA-B through common PCR and agarose gel electrophoresis identification, has the sensitivity of 81.25 percent, can amplify target bands from all 15 subtypes of HLA-B1502, has the specificity of 100 percent, and can accurately determine the positive type of HLA-B1502 type by combining sequencing result comparison analysis.
Secondly, the invention adopts PCR technology, and can optimize the amplification efficiency and construct a stable amplification system by adjusting the reaction conditions such as primer concentration, annealing temperature and the like. The primer and the method for detecting the HLA-B1502 typing which can be used for guiding the carbamazepine medicament administration disclosed by the invention not only have the advantages of simple and mature method, high detection sensitivity and good specificity, but also reduce the detection cost and difficulty compared with other HLA typing methods.
And thirdly, the result judgment is simple, only the pair of primers, namely actin-F and actin-R, has the result by using the amplification primers and the internal reference primers, so that the template and the experimental process have no problems, and the result is effective. When the amplification of the HLA-B1502-F and the HLA-B1502-R has no result, the sample can be directly judged to be negative by the HLA-B1502 allele; when the amplification of the HLA-B1502-F and the HLA-B1502-R has a result, the patient is possible to be positive, a sequencing test is needed, and when the sequencing result completely conforms to the sequence of the HLA-B1502 allele, the HLA-B1502 gene in the sample is determined to be positive.
Drawings
FIG. 1 is a sample-verified agarose gel electrophoresis chart, wherein M is Marker DL 2000, 1-12 are unknown HLA typed blood DNA, the primer of the invention is used for amplifying a target band, the target band is clear, and the size of a product is correct.
FIG. 2 is a cross-sectional view of the PCR product sequencing alignment of samples 5 and 7.
Detailed Description
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.
Example 1
A primer for detecting HLA-B1502 typing which can be used for guiding the administration of carbamazepine is a specific amplification primer designed for typing HLA-B1502 gene and has the base sequence:
HLA-B*1502-F：TGTAAAACGACGGCCAGTGAACACACAGATCTCCAAGACCA
HLA-B*1502-R：AACAGCTATGACCATGCAGCCATACATCCTCTGGATGA；
the primer also comprises a sequencing primer, and the base sequence of the sequencing primer is as follows:
M13 F：TGTAAAACGACGGCCAGT
M13 R：AACAGCTATGACCATG
the primer also comprises a primer for detecting an internal reference gene, and the base sequence of the primer is as follows:
actin-F：CTAACTGCGCGTGCGTTCT
actin-R：AGTCCTTAGGCCGCCAGGGG。
a method for HLA B1502 typing for administration of carbamazepine comprising the steps of:
(1) extracting genome DNA in peripheral blood or bone marrow;
(2) amplifying the DNA in the step (1) by using amplification primers HLA-B1502-F/HLA-B1502-R and actin-F/actin-R, determining that the sample HLA-B1502 is negative in typing when the internal reference actin-F/actin-R amplifies a target band and the HLA-B1502-F/HLA-B1502-R amplifies a non-target band, and performing sequencing detection on the sample when the two pairs of target primers amplify the non-target bands;
(3) sequencing the amplification product in the step (2) by using sequencing primers M13-F and M13-R;
(4) comparing the sequencing result in (3) with the wild type HLA-B1502 allele sequence, and determining that the sample is positive for HLA-B1502 typing if the sequence is completely consistent with the HLA-B1502 allele sequence, otherwise, the sample is negative.
Example 2
The operation flow of the blood/cell/tissue genome DNA extraction kit (Tiangen organism):
(1) extracting tissue DNA from blood: 1) mu.l of blood was taken and added to 900. mu.l of erythrocyte lysate, mixed by inversion, left at room temperature for 5 minutes, and mixed by inversion several times in the meantime. Centrifuge at 12,000 rpm for 1min, aspirate the supernatant, leave the leukocyte pellet, add 200. mu.l of buffer GA, and shake until thoroughly mixed. 2) Add 20. mu.l proteinase K solution and mix well. 3) Add 200. mu.l buffer GB, mix well by inversion, stand at 70 ℃ for 10 minutes, clear the solution, centrifuge briefly to remove beads on the inner wall of the tube cap. 4) Add 200. mu.l of absolute ethanol, mix well with shaking for 15 seconds, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cover. 5) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is put into a collecting pipe), centrifuging at 12,000 rpm for 30 s, pouring off waste liquid, and putting the adsorption column CB3 back into the collecting pipe. 6) Add 500. mu.l buffer GD (check whether absolute ethanol has been added before use) to adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, dump the waste and place adsorption column CB3 in the collection tube. 7) To the adsorption column CB3, 700. mu.l of a rinsing solution PW (previously used, whether or not absolute ethyl alcohol has been added) was added, and the mixture was centrifuged at 12,000 rpm for 30 seconds, and the waste liquid was discarded, and the adsorption column CB3 was put into a collection tube. 8) To the adsorption column CB3, 500. mu.l of a rinsing solution PW was added, and the mixture was centrifuged at 12,000 rpm for 30 seconds, and then the waste liquid was discarded. 9) The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000 rpm for 2 minutes, 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. 10) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 100 mu l of elution buffer TE into the middle part of the adsorption membrane, standing for 2-5 minutes at room temperature, centrifuging for 2 minutes at 12,000 rpm, 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.
(3) Sample adding: adding 1 mul DNA into the PCR reaction solution of the detection system; directly adding 1 mul of positive control substance and negative control substance into the positive control substance and the negative control substance; blank control was supplemented with 1. mu.l of physiological saline or nothing.
Positive control: nucleic acid solution containing HLA-B1502 typing gene
Negative control: nucleic acid solution containing non-HLA-B1502 typing gene
(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:
the preparation method of the PCR amplification system reagent comprises the following steps:
wherein, the primer sequence is as follows:
note: f is an upstream primer, R is a downstream primer
(5) Electrophoresis: electrophoresis on 1.5% agarose gel at 120V for 30min, and observation on a gel imaging system.
As shown in FIG. 1, the electropherogram of the product obtained after the amplification of 12 cases of blood sample DNA with the primer was obtained. The electrophorogram shows that the amplification of the invention is effective and the band is clear.
(6) Sanger sequencing:
take 9. mu.l of PCR product and 2. mu.l of purification system. Purification was performed according to the following procedure:
mu.l of the purified product was mixed with the upper and lower sequencing primers, respectively, according to the following system:
sequencing reaction program:
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 respectively comparing the sequencing result with an HLA-B1502 reference sequence, and reporting the result according to the actual situation.
Example 3
50 clinical peripheral blood samples were taken, and the genome was extracted, reagents were prepared, and tested as described in example 2. Each sample was added to 2. mu.l of the detection system PCR reaction solution. At the same time, positive and negative are made, and blank control is performed respectively. The detection is carried out by a common PCR instrument for 100 minutes.
And (5) identifying the target bands of 10 samples by agarose gel electrophoresis, and determining that the 5 samples are positive for HLA-B1502 typing through sequencing comparison. FIG. 2 is a cross-sectional view of the PCR product sequencing alignment of samples 5 and 7.
According to the detection result, the primer and the method can simply, conveniently and rapidly detect the positive typing of HLA-B1502 and guide the administration of carbamazepine.
Sequence listing
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Claims (8)
1. A pair of primers for detecting HLA-B1502 typing that directs carbamazepine administration comprising:
the primer for specifically amplifying HLA-B1502 typing has the base sequence as follows:
HLA-B*1502-F：TGTAAAACGACGGCCAGTGAACACACAGATCTCCAAGACCA
HLA-B*1502-R：AACAGCTATGACCATGCAGCCATACATCCTCTGGATGA。
2. the primer of claim 1, further comprising a sequencing primer having the base sequence:
M13 F：TGTAAAACGACGGCCAGT
M13 R：AACAGCTATGACCATG。
3. the primers of claim 1, further comprising actin-F and actin-R primers for detecting reference genes, wherein the base sequences of the reference genes are:
actin-F：CTAACTGCGCGTGCGTTCT
actin-R：AGTCCTTAGGCCGCCAGGGG。
4. the primer of claim 1, wherein the concentration of the forward and reverse primers in the system is 0.1875 uM.
5. The primer according to claim 3, wherein the primer for the reference gene is used at a concentration ratio of: actin-F: actin-R ═ 1: 1.
6. A method of detecting HLA B1502 typing of a medication directed to carbamazepine comprising the steps of:
(1) extracting genome DNA in peripheral blood or bone marrow;
(2) amplifying the DNA in the step (1) by using amplification primers HLA-B1502-F/HLA-B1502-R and actin-F/actin-R, determining that the sample HLA-B1502 is negative in typing when the internal reference actin-F/actin-R amplifies a target strip and the HLA-B1502-F/HLA-B1502-R amplifies a non-target strip, and performing sequencing detection on the sample when the two pairs of target primers amplify the non-target strips;
(3) sequencing the amplification product in the step (2) by using sequencing primers M13-F and M13-R;
(4) comparing the sequencing result in the step (3) with the wild type HLA-B1502 allele sequence, and determining that the sample is positive in HLA-B1502 typing if the sequence is completely consistent with the HLA-B1502 allele sequence, otherwise, the sample is negative;
wherein the PCR amplification primers are:
HLA-B*1502-F：TGTAAAACGACGGCCAGTGAACACACAGATCTCCAAGACCA
HLA-B*1502-R：AACAGCTATGACCATGCAGCCATACATCCTCTGGATGA。
7. the method of claim 6, wherein the concentration of template DNA in the amplification system is at least 0.25 ng/uL.
8. The method of claim 6, wherein the sequencing primer base sequence is:
M13 F：TGTAAAACGACGGCCAGT
M13 R：AACAGCTATGACCATG。
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