CN113308532A - Specific primer probe combination for detecting human HLA-B1502 gene mutation, kit and method thereof - Google Patents
Specific primer probe combination for detecting human HLA-B1502 gene mutation, kit and method thereof Download PDFInfo
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Abstract
The invention provides a specific primer probe combination for detecting human HLA-B1502 gene mutation, which comprises the following primers and probe sequences: the primer and probe sequences for human HLA-B1502 gene are shown in SEQ ID No. 1-3, and the primer and probe sequences for reference gene RPPH gene segment are shown in SEQ ID No. 4-6. The invention also provides a kit and a method for detecting the human HLA-B1502 gene mutation. The invention solves the problems of complicated process, complex operation, long time consumption and high cost of the method and the kit for detecting the human HLA-B1502 gene mutation in the prior art, realizes simple, quick and high-accuracy detection of the human HLA-B1502 gene mutation, and effectively reduces the generation of SJS/TEN caused by taking carbamazepine and other medicaments by patients.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a specific primer probe combination for detecting human HLA-B1502 gene mutation, a kit and a method thereof.
Background
Epilepsy is a chronic brain disease caused by various causes, and is mainly characterized in that recurrent and excessive synchronous discharge of cerebral neurons leads to paroxysmal, sudden and transient brain dysfunction, and various clinical epileptic seizures, electroencephalogram epileptic waves and other abnormal changes are accompanied. Carbamazepine (CBZ), Lamotrigine (LTG), Oxcarbazepine (OXC), phenytoin sodium (PHT) and tolidine (PB) all have similar aromatic structures and are widely used antiepileptic drugs (AEDs) for the control of epilepsy, trigeminal neuralgia and the like. However, such drugs often cause adverse skin reactions (cADRs), which afflict clinicians for a long time. cADRs account for 10% -30% of all reported adverse drug reactions, including mild Maculopapular (MPE), drug hypersensitivity syndrome (HSS) to the severe, potentially fatal Stevens-Johnson syndrome (SJS) and/or Toxic Epidermal Necrolysis (TEN).
Since the lethality of SJS is around 5% and that of TEN is around 30%, induction of SJS/TEN once a patient takes carbamazepine can lead to very serious consequences and can cause irreparable losses. It has been shown that SJS/TEN can be reduced in patients taking carbamazepine by screening for HLA-B15: 02 gene mutations and intervening in the treatment regimen according to the results of the test.
At present, common methods for detecting HLA-B1502 gene mutation include a PCR-sanger sequencing method, a qPCR method, a next generation sequencing method, a PCR electrophoresis method and the like. Among them, the PCR-sanger sequencing method has the advantages of accurate result, but has the disadvantages of complicated process, complex operation, long time consumption and low flux. The second-generation sequencing method is similar to the sanger sequencing method in advantages and disadvantages, accurate in result, complex in operation, long in time consumption and high in sequencing cost. The PCR electrophoresis method and the like are complicated in operation and take a long time. The qPCR method is a commonly used method for detecting the mutation of the HLA-B1502 gene.
Currently available on the market for the detection of HLA-B15: 02 qPCR kit for gene mutation, comprising: a kit using a dye method, and a kit using a probe method. Generally, the probe method has high sensitivity and requires less template, while the dye method cannot amplify multiple targets simultaneously without using the dissolution curve analysis, and if the dissolution curve analysis is used, the time consumption is longer than that of the probe method. The qPCR kit had HLA-B15: 02 gene mutation identification, and two or three sets of identification are also adopted. In China, the difference between the detection results of one set and two or three sets is not large, and the difference between the accuracy rates is not more than 1%.
Disclosure of Invention
The invention aims to provide a specific primer probe combination for detecting human HLA-B1502 gene mutation, a kit thereof and a method, thereby solving the problems of complicated process, complex operation, long time consumption and high cost of the method and the kit for detecting human HLA-B1502 gene mutation in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to a first aspect of the present invention, there is provided a specific primer probe combination for detecting human HLA-B1502 gene mutation, comprising the following primers and probe sequences: upstream primer Fp1 for human HLA-B1502 gene: 5'-GCGAGTCCGAGGATGGCGCCC-3', respectively; downstream primer Rp1 for human HLA-B1502 gene: 5'-TTGTAGTAGCCGCGCAGGTTC-3', respectively; probe1 recognizing mutation of human H LA-B1502 gene: 5'-FAM-AACACACAGATCTCCAAGAC CAACACACAG-BHQ 1-3'; upstream primer Fp2 for reference gene human RPPH gene fragment: 5'-CCGCCTCTGGCCCTAGT-3', respectively; downstream primer Rp2 for reference gene human RPPH gene fragment: 5'-GCCACGAGCTGAGTGCGT-3', respectively; probe2 recognizing the conserved sequence specificity of the segment of the reference gene human RPPH gene: 5'-VIC-TGTCACTCCACTCCCATGTCCCTTGG-BHQ 1-3'.
According to a second aspect of the present invention, there is provided a kit for detecting mutations in the human HLA-B1502 gene, the kit comprising a specific primer probe set for detecting mutations in the human HLA-B1502 gene as described above.
The kit also comprises a reaction solution, pure water, a positive control and a negative control; wherein the reaction solution comprises the following components: tris buffer solution, MgCl2, dNTP, primers, probes, Taq enzyme, UDG enzyme, PCR enhancer and water; the positive control is a positive control containing HLA-B15: a 02 genotype partial sequence and a Control gene partial sequence, and the negative Control is a plasmid without a DNA sequence of a region to be detected.
According to a third aspect of the present invention, there is also provided a TaqMan probe real-time fluorescence PCR method for detecting mutations in human HLA-B1502 gene, for non-disease diagnostic purposes, comprising the steps of:
1) specific primer probe combination is designed according to the sequence characteristics of human HLA-B1502 gene and reference gene human RPPH gene; 2) obtaining extracted genome DNA of a sample to be detected;
3) in the same reaction system, the primer probe combination is mixed according to a certain proportion and added into the reaction solution; 4) carrying out real-time quantitative fluorescent PCR detection; 5) when the following conditions are met, the result can be judged that the human HLA-B15: 02 gene is mutated to be positive: ct value of RPPH is less than or equal to 35; b: ct value for HLA-B1502 < Ct value for RPPH.
According to a preferred embodiment of the present invention, the final concentrations of the primers and the probes are respectively as follows, wherein the final concentration is 20. mu.L: fp 1: 0.1-0.4 mu M; rp 1: 0.1-0.4 mu M; probe 1: 0.08-0.3 mu M; fp 2: 0.1-0.4 mu M; rp 2: 0.1-0.4 mu M; probe 2: 0.08 to 0.3 μ M.
According to a preferable embodiment of the present invention, the reaction system comprises 20 μ L of the reaction solution: 1 to 1.5U of DNA polymerase, 0.2 to 0.45mM of DNTP (A, G, C, U), 0.3 to 0.6U of UDG enzyme, 45 to 60mM of Tris-HCl (pH 9), 18 to 22mM (NH)4)2SO4，1.8～2.2mM Mg2+SO40.8 to 1.2% of glycerol and 0.08 to 0.12mg/ml of BSA.
According to a preferred embodiment of the present invention, the amplification procedure used in step 4) is: 30 ℃, 10min, 1 cycle; at 95 ℃, 10min, 1 cycle; 95 ℃, 30s, 65 ℃, 30s, 40 cycles.
The method is not only suitable for EDTA anticoagulated peripheral whole blood, but also suitable for oral swab samples.
According to the kit provided by the invention, genome sequences which can distinguish the 15:02 genotype from other genotypes in the HLA-B gene are selected, and PCR amplification primers and fluorescence labeled probes which can be specifically combined with a DNA template are specifically designed aiming at the sequences. In the PCR extension reaction process, the 5' exonuclease activity of Taq enzyme cuts the 5' end fluorescent group of the probe, so that the probe is dissociated and gets rid of the shielding of the 3' fluorescent quenching group in the reaction system. Therefore, when excited by laser with a specific wavelength, the fluorescence which can be detected by a detection instrument is emitted, and the DNA sample of the HLA-B15: 02 gene carrier is detected.
According to the kit provided by the invention, a set of primer probes is adopted to distinguish three HLA-B1502 mutant fragments different from other HLA-B alleles, the accuracy is up to 99%, and more than 500 clinical samples are tested, so that false negative results do not exist. The kit is simple to operate and short in time consumption, the extracted human DNA can be used for on-machine test, and the detection result can be obtained within 1 and half hours, so that medical workers are effectively helped to intervene in a treatment scheme according to the detection result, the occurrence of SJS/TEN caused by the patient taking carbamazepine is reduced, and the health of the patient is guaranteed.
In conclusion, the invention provides a specific primer probe combination, a kit and a method for detecting human HLA-B15: 02 gene mutation, which are simple and rapid and can ensure high accuracy.
Drawings
FIG. 1 shows the results of the test using the kit of the present invention in case one;
FIG. 2 shows the results of the test using the comparative kit in case one.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
Example 1 design of primers and probes
According to this preferred embodiment, the invention is directed to human HLA-B15: the primer and the probe are designed according to the different characteristic sequences of the 02 gene and other HLA-B gene mutation, and meanwhile, the primer and the probe are designed by taking the human RPPH gene segment as an internal reference gene, the primer and the probe designed according to the invention can be specifically combined with the nucleic acid of the human HLA-B15: 02 gene mutant, so that the segment is amplified, a fluorescent signal is emitted in the amplification process, and the HLA-B15: 02 mutation, the presence of signal from the RPPH gene, marked that sampling and the entire amplification process were not affected, helped to eliminate false negatives.
Specifically, the primer and probe sequences and system concentrations designed by the invention are shown in the following table 1:
TABLE 1 primer and Probe sequences and concentrations
Wherein, SEQ ID No: 1 and 2 are amplification primers aiming at an HLA-B gene fragment, and SEQ ID No: 3 is the fragment used to screen 15:02 mutant probes; SEQ ID No: 4 and 5 are amplification primers aiming at the reference gene human RPPH gene segment, and SEQ ID No: 6 is a probe specific to human conserved sequence of human RPPH gene segment.
Example 2A kit for detecting human HLA-B1502 Gene mutation
According to this preferred embodiment, there is provided a kit for detecting mutations in human HLA-B1502 gene, the kit comprising primer and probe sequences as shown in table 1 above.
1. The main components
TABLE 2 major Components
2. Storage condition and shelf life
The kit is stored at-20 +/-5 ℃ and has the validity period of 12 months.
3. Adapted for instruments
Applied BiosystemsTM7500 real-time quantitative PCR System (Life Technologies Holdings Pte Ltd.).
4. Sample requirement
1.5-2 mL of EDTA anticoagulated peripheral whole blood or buccal swab sample.
5. Inspection method
5.1 reagent preparation
1) In the reagent preparation area, the reaction solution, the positive control and the negative control of the kit are taken out from the storage area at-20 ℃ in a refrigerator and are thawed at room temperature. When the reagent is used for the first time, 900 mu L of water is added into the reaction solution, and the mixture is inverted and mixed until the reaction solution is completely dissolved.
2) The reaction, positive control and negative control are then transferred to the sample preparation area.
5.2 sample preparation
In the sample preparation area, a peripheral whole blood sample or a buccal swab sample is taken, and the sample DNA is extracted according to the nucleic acid extraction instructions. DNA extraction kit recommendation: AllPrep DNA/RNA Mini Kit, Qiagen.
If the sample is pre-extracted DNA, the DNA sample is taken out from the storage area at-20 ℃ in a refrigerator and is thawed at room temperature.
5.3 reaction System preparation
1) In the sample preparation area, the reaction system was prepared in 8-tube or 96-well plates according to the following table 3.
TABLE 3 reaction System
| Composition (I) | Volume of |
| Reaction solution | 18μL |
| Sample(s) | 2μL |
The amounts of the respective components used in the reaction system of 20. mu.L are shown in Table 4 below. It should be understood that the present reaction system may be varied in equal proportion with the change in volume.
TABLE 4 reaction solution
| Name of composition | Dosage of |
| DNA polymerase | 1～1.5U |
| DNTP(A,G,C,U) | 0.2～0.45mM |
| UDG enzymes | 0.3～0.6U |
| Tris-HCl(PH＝9) | 45～60mM |
| (NH4)2SO4 | 18～22mM |
| Mg2+SO4 | 1.8～2.2mM |
| Glycerol | 0.8～1.2％ |
| BSA | 0.08～0.12mg/ml |
| Oligonucleotide mixtures | Final concentrations refer to the final concentrations in Table 1 |
2) After completion of the reaction system preparation, the reaction mixture is transferred to a PCR amplification detection zone.
5.4 amplification assay
5.4.1 Place the reaction 8-line or 96-well plate on a centrifuge for short centrifugation, ensuring that the reaction system is at the bottom of the 8-line or 96-well plate.
5.4.2 opening the fluorescent quantitative PCR instrument, and putting the sample to be detected into a machine detection bin. The instrument control software was then turned on.
5.4.3 setting the reaction program according to the prompt
5.4.3.1, the first time they operate on the inspection machine, the following should be done.
Click on "advanced setting" and set the reaction program in the experimental attributes as per table 5 below.
TABLE 5 reaction procedure
Then in the reaction plate setup, click "define genes and samples" and then click "add new genes" button. Then, the 'gene 1' is renamed to 'HLA-B1502', a report group is selected to be 'FAM', and other settings are unchanged; the "Gene 2" was renamed as "Control", the reporter group was selected as "VIC", and the other settings were unchanged.
Then, in the reaction procedure, the reaction system was changed to "20", and the reaction amplification procedure was set up as in the following Table 6.
TABLE 6 amplification procedure
Then click the "save" button, select "save as template" and save as "HLA-b1502. edt". The template is then closed.
5.4.3.2 if the machine used has completed the operations in 5.4.3.1, please proceed as follows.
Click the "open" button to open the file HLA-b1502. edt.
After the file is opened, a sample to be tested is set at the "define sample" in the "reaction plate setting". Then click "specify gene and sample", in this panel, the position of the sample to be tested is marked clearly. All samples were then selected and in the "genes assigned" column, HLA-B1502 and Control were all checked.
Selecting "Control" among "reference genes"; in "reference Dye", the "ROX" is selected.
After all settings are completed, the "Start" button in the upper right corner is clicked to Start the test.
5.5 analysis of results
After the reaction program is operated, entering an analysis interface, clicking analysis setting at the upper right corner, modifying the CT threshold settings of HLA-B1502 and Control to be 0.1, and clicking application analysis setting.
5.5.1 Positive judgment value
When the following conditions are satisfied simultaneously, the result can be judged as positive for HLA-B15: 02.
Ct value of RPPH is less than or equal to 35.
Ct value for HLA-B1502 < Ct value for RPPH.
5.5.2 interpretation of test results
If the Ct value of RPPH is >35, there may be a problem with the sample quality and a re-test is required. If the result of the re-detection is still that the Ct value of RPPH is greater than 35, the re-sampling detection is needed.
If the Ct value of the RPPH is less than or equal to 35, the Ct value of the HLA-B1502 is more than or equal to the Ct value of the RPPH, and the sample needs to be detected again. If the result of the re-detection is that the Ct value of RPPH is less than or equal to 35 and the Ct value of HLA-B1502 is greater than or equal to the Ct value of RPPH, the sequencing is recommended to carry out result confirmation.
5.5.3 limitations of the test method
The detection result of the kit is only referred by a clinician, can be only used for judging the genotype and cannot be used as the only basis for clinical medication.
Should be operated by personnel with professional experience or trained qualification.
Improper sample collection, transport, handling, and improper experimental handling and environment can lead to false negative or false positive results.
5.5.4 product Performance index
The accuracy is as follows: detecting 5 positive reference substances, wherein the coincidence rate of the detection result is up to 100%; and (3) detecting 12 negative reference products, wherein the coincidence rate of the detection results is up to 100%. The detection results of 448 blood samples and 493 oral swab samples are compared with the sequencing result of sanger, and the positive coincidence rate reaches 100 percent and the negative coincidence rate reaches 99 percent.
Detection limit: the product can detect 10ng of human genome DNA with the A260/A280 purity of 1.80-2.00 at the lowest.
Precision: the precision reference substances of 2 cases are repeatedly detected for 10 times, the detection results are all corresponding types, and the coincidence rate is 100%.
Anti-interference performance: the final test result is not affected by the interference of 0.1g/L or less of hemoglobin in the blood sample, the final test result is not affected by the interference of 342 μmol/L or less of bilirubin, the final test result is not affected by the interference of 15mM or less of Mg2+, the final test result is not affected by the interference of 37mM or less of triglyceride, and the final test result is not affected by the interference of 7.5g/L or less of sodium EDTA. Blood content of 5% or less in the buccal swab sample does not affect the final test result, saliva content of 10% or less in the buccal swab sample does not affect the final test result, toothpaste content of 1% (w/v) or less does not affect the final test result, and cephalosporin content of 10g/L or less does not affect the final test result.
Case 1
Using the kit provided in example 2 and the comparative kit, 3 samples known to carry HLA-B15: 02 mutations and 3 samples not carrying mutations were tested, respectively.
The comparative kit employs the primers and probe sequences SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9 shown in Table 7 below, instead of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively, of the kit provided in example 2.
TABLE 7 primer and Probe sequences for comparison kits
The kit provided by embodiment 2 of the present invention and the comparison kit are used for detecting the sample, and the detection results are shown in fig. 1 and fig. 2, wherein the detection result of the kit of the present invention is completely consistent with the clone sequencing result of the sample (fig. 1), and meanwhile, the comparison kit can detect the sample carrying HLA-B15: 02, but also detects the negative sample (fig. 2), so the specificity of the comparison reagent is poor. Therefore, the test result of the kit provided by the invention is more accurate.
Case 2
We randomly selected 100 clinical samples, and tested the 100 samples using the Kit provided in example 2 of the present invention and the HLA-B15: 02 mutant gene assay Kit (shiji biology, taiwan, trade name "human leukocyte antigen B site 1502 gene assay Kit (PCR-fluorescence method) PG1502 Detection Kit" approved by the drug administration). The results are shown in Table 8 below.
TABLE 8 test results
According to the table, the kit provided by the invention is completely consistent with the detection result of the HLA-B15: 02 mutant gene detection kit approved and registered by the drug administration. However, the kit provided by the invention adopts one reaction to complete the amplification of the internal reference and HLA-B15: 02 mutant genes, while the comparative kit needs two reactions to complete the amplification, and the workload is larger.
The reason is that the comparison kit adopts a SYBR nucleic acid fluorescent dye method, and because the fluorescent wavelength of the dye is single, real-time quantitative analysis cannot be realized when two gene fragments are amplified in one hole, so that the reference gene adopted by the kit adopts one reaction, and HLA-B15: 02 mutation needs to be identified by one reaction. The kit adopts a TaqMan probe method, and can be used for labeling different genes by adopting fluorescent labels excited and emitted by a plurality of different wave bands. Therefore, the kit of the invention adopts FAM fluorescein and VIC fluorescein labels for the HLA-B gene segment and the RPPH gene segment respectively, and can complete real-time monitoring of amplification of the two gene segments in the same reaction, so that the expected effect can be achieved by only one reaction. The reaction time of the kit of the invention is about 1 hour and 20 minutes, while the reaction time of the comparative kit is about 1 hour and 45 minutes.
Therefore, the kit provided by the invention is completely consistent with the detection result of the HLA-B15: 02 mutant gene detection kit approved and registered by the medical supervision agency, realizes accurate detection of human HLA-B15: 02 gene mutation, shortens the detection time, saves the detection cost and has better application prospect.
The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.
SEQUENCE LISTING
<110> Shanghai Kangli diagnostic technology Co., Ltd
<120> a primer probe combination for detecting human HLA-B1502 gene mutation and its kit, and method
<160> 9
<170> PatentIn version 3.5
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Claims (7)
1. A specific primer probe combination for detecting human HLA-B1502 gene mutation is characterized by comprising the following primers and probe sequences:
upstream primer Fp1 for human HLA-B1502 gene: 5'-GCGAGTCCGAGGATGGCGCCC-3', respectively;
downstream primer Rp1 for human HLA-B1502 gene: 5'-TTGTAGTAGCCGCGCAGGTTC-3', respectively;
probe1 recognizing human HLA-B1502 gene mutation: 5'-FAM-AACACACAGATCTCCAAGACCAACACACAG-BHQ 1-3';
upstream primer Fp2 for reference gene human RPPH gene fragment: 5'-CCGCCTCTGGCCCTAGT-3', respectively;
downstream primer Rp2 for reference gene human RPPH gene fragment: 5'-GCCACGAGCTGAGTGCGT-3', respectively; and
probe2 recognizing the conserved sequence specificity of the segment of the reference gene human RPPH gene: 5'-VIC-TGTCACTCCACTCCCATGTCCCTTGG-BHQ 1-3'.
2. A kit for detecting human HLA-B1502 gene mutation, comprising the specific primer probe combination according to claim 1 for detecting human HLA-B1502 gene mutation.
3. The kit according to claim 2, further comprising a reaction solution, pure water, a positive control and a negative control; wherein the reaction solution comprises the following components: tris buffer, MgCl2dNTP, a primer, a probe, Taq enzyme, UDG enzyme, a PCR enhancer and water; the positive control is a positive control containing HLA-B15: a 02 genotype partial sequence and a Control gene partial sequence, and the negative Control is a plasmid without a DNA sequence of a region to be detected.
4. A TaqMan probe real-time fluorescent PCR method for detecting mutations in the human HLA-B1502 gene for non-disease diagnostic purposes, comprising the steps of:
1) designing a primer probe combination according to claim 1 according to the sequence characteristics of human HLA-B1502 gene and reference gene human RPPH gene;
2) obtaining extracted genome DNA of a sample to be detected;
3) in the same reaction system, the specific primer probe combination of claim 1 is mixed according to a certain proportion and added into a reaction solution;
4) carrying out real-time quantitative fluorescent PCR detection;
5) when the following conditions are met, the result can judge that the sample to be detected is positive through human HLA-B15: 02 gene mutation: ct value of RPPH is less than or equal to 35; b: ct value for HLA-B1502 < Ct value for RPPH.
5. The TaqMan probe real-time fluorescent PCR method according to claim 4, wherein the final concentrations of the primer and the probe are respectively as follows, wherein the final concentrations of the primer and the probe are calculated by 20 μ L: fp 1: 0.1-0.4 mu M; rp 1: 0.1-0.4 mu M; probe 1: 0.08-0.3 mu M; fp 2: 0.1-0.4 mu M; rp 2: 0.1-0.4 mu M; probe 2: 0.08 to 0.3 μ M.
6. The TaqMan probe real-time fluorescent PCR method according to claim 4, wherein the reaction system is calculated by 20 μ L, and the reaction solution comprises the following components: 1 to 1.5U of DNA polymerase, 0.2 to 0.45mM of dNTP (A, G, C, U), 0.3 to 0.6U of UDG enzyme, 45 to 60mM Tris-HCl (pH 9), 18 to 22mM (NH)4)2SO4，1.8～2.2mM Mg2+SO40.8 to 1.2% of glycerol and 0.08 to 0.12mg/ml of BSA.
7. The TaqMan probe real-time fluorescent PCR method according to claim 4, wherein the amplification procedure adopted in the step 4) is: 30 ℃, 10min, 1 cycle; at 95 ℃, 10min, 1 cycle; 95 ℃, 30s, 65 ℃, 30s, 40 cycles.
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