CN112481374A - Detection method and detection kit for HLA-B1502 gene and application thereof - Google Patents
Detection method and detection kit for HLA-B1502 gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and provides a reagent for detecting a human leukocyte antigen B locus 1502 genotype (HLA-B1502), application of the reagent in preparing a kit for guiding medication of antiepileptic drugs such as carbamazepine, oxcarbazepine, phenytoin, lamotrigine and the like, a corresponding kit and a detection method thereof. HLA-B1502 genotyping adopts PCR-fluorescence probe method to detect 3 sets of MIX reaction solution, circularly amplifies target nucleic acid molecules through amplification reaction, and indirectly combines fluorescence generating group with amplified target nucleotide sequence; the amount of fluorescence generated by the fluorogenic group is measured to determine the presence of the target nucleotide. The detection reagent comprises: 3 sets of nucleic acid amplification systems of MIX reaction solution, each of which comprises an upstream primer 1 and a downstream primer 1 capable of binding with a target nucleotide, and an upstream primer 2 and a downstream primer 2 capable of binding with the target nucleotide; group 3 probes 1-4 of the fluorescence detection system used in the nucleic acid amplification system.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a detection reagent, a detection method and a kit for clinically detecting a human leukocyte antigen B site 1502 gene (HLA-B1502) for medication guidance of anti-epileptic therapeutic drugs such as carbamazepine, oxcarbazepine, phenytoin, lamotrigine and the like. The invention also comprises application of the detection reagent in preparing a kit for anti-epileptic treatment medication guidance of epileptics such as carbamazepine, oxcarbazepine, phenytoin, lamotrigine and the like, a corresponding kit and a using method thereof.
Background
Mental disorders have become a global high incidence of disease. According to statistics, over 17% of adults in China have troubles of mental diseases such as deep fertilization schizophrenia, epilepsy and the like (Phillips 2009). Epilepsy is defined by the World Health Organization (WHO) and the international anti-epileptic association (ILAE) as: a chronic brain disease caused by various etiological factors is mainly characterized in that recurrent and excessive synchronous discharge of cerebral neurons leads to paroxysmal, sudden and transient brain dysfunction, and is accompanied with various clinical epileptic seizures, electroencephalogram epileptic wave and other abnormal changes.
Epilepsy occurs in people of any age, region and race, but occurs at a higher rate in children and adolescents. According to WHO estimates, there are about fifty million epileptic patients worldwide. The epidemiological data in China show that the prevalence rate of epilepsy in China is between 4 per mill and 7 per mill. In recent years, the prevalence rate of active epilepsy is more concerned, the prevalence rate of active epilepsy in China is 4.6 per thousand, and the annual incidence rate is about 30/10 ten thousand. According to the estimation, about 600 ten thousand active epileptic patients exist in China, and about 40 ten thousand new epileptic patients exist every year. The death risk of epileptics is 2-3 times of that of the general population. Epilepsy has serious negative effects on individuals, families and society, and the WHO has listed epilepsy as one of the major neurological and mental diseases for prevention and treatment. The epileptic seizure causes great physical and psychological pains to patients, and the quality of life of the patients and families is seriously affected; the long-term taking of antiepileptic drugs and other diagnosis and treatment costs bring heavy economic burden to families; meanwhile, the problems of health care, education, employment, marital fertility and the like of epileptics are also the concerns of patients, relatives thereof and multiple social departments.
At present, the traditional antiepileptic drugs (AEDs) Carbamazepine (CBZ), oxcarbamazepine (oxcarbamazepine), Phenytoin (Phenytoin) and Lamotrigine (Lamotrigine) are still the first-line drugs for epilepsy treatment, and the market share occupies a high proportion. However, CBZ is a narrow therapeutic index drug with an effective concentration close to the toxic concentration; deviations from optimal dosages or concentrations can lead to treatment failure or severe toxicity; in clinical application, monitoring of therapeutic drugs based on pharmacokinetic indexes is required; with a low or medium degree of intra-individual variation. On 12 months 2007, the U.S. Food and Drug Administration (FDA) issued safety information on carbamazepine that CBZ caused dangerous and even fatal skin reactions (stevens-johnson syndrome SJS and toxic epidermal necrolysis TEN), especially in patients with the human leukocyte antigen allele HLA-B1502.
Human Leukocyte Antigens (HLA) are an important immunogenetic system of the human body and are the most complex polymorphic system of the human body. The nature or molecular structure of HLA as antigen for stimulating body to produce corresponding antibody is the main reason for rejection reaction in organ transplantation, and also the basis of serology and cytology matching. Recent studies have found that some alleles of HLA are closely associated with adverse reactions caused by some drugs. Results of a carbamazepine-drug hypersensitivity syndrome (CBZ-HSS) association study showed: HLA-B0801, HLA-DR3 and HLA-DQ2 alleles have relevance to CBZ-HSS.
The probability of CBZ causing SJS/TEN is low, and the incidence of SJS/TEN on carbamazepine in caucasian countries is only one to six TEN thousandths. However, some Asian countries have approximately 10 times higher SJS/TEN probabilities based on adverse drug event reports received by WHO and CBZ pharmacists. Studies in europe and china taiwan, hong kong, china show that an increased risk of SJS/TEN is associated with HLA-B1502. Information on SJS/TEN and information instructing high risk groups to receive HLA-B1502 allele tests has been added to the existing black box warnings and "warnings", "experimental tests" and "adverse reactions" sections of the drug instructions. The FDA advises physicians to have full knowledge of the drug specifications and information in the updated black box warning when prescribing carbamazepine (including the same class of drugs as carbamazepine). The information indicates that 10-15% or more of patients in china, thailand, malaysia, indonesia, philippines are likely to carry HLA-B1502, indicating that most asian patients of ancestry should receive HLA-B1502 gene testing before taking CBZ.
The clinical pharmacogenomics implementation alliance (CPIC) guidelines indicate that HLA-B1502 sites and CBZ are strongly correlated with SJS, and that gene testing is recommended prior to dosing, and positive HLA-B1502 sites do not recommend carbamazepine. Currently, there are many techniques available for HLA allele typing, mainly based on nucleic acid sequence identification methods, including, for example, sequence specific primer polymerase chain reaction (PCR-SSP), polymerase chain reaction-oligonucleotide probe dot hybridization (PCR-SSOP), polymerase chain reaction-direct sequencing (PCR-SBT). The PCR-SBT technology is more a 'gold standard' of the HLA typing method recommended by WHO at present, and the method has the advantages of high accuracy, strong specificity and the like. However, the method has the advantages and also has the disadvantages of complicated operation, long time consumption, high cost and the like. Particularly, the defects of complex operation, long time consumption and the like can not meet the detection requirement of clinical medicine and can not adapt to the concept of 'rapidness, simplicity and convenience' required by clinical medicine detection.
Disclosure of Invention
The invention aims to provide a rapid and simple HLA-B1502 allele detection method and a kit thereof.
It is another object of the present invention to provide the use of the above methods, reagents and kits.
Real-time fluorescent quantitative PCR (FQ-PCR) is a nucleic acid detection method developed on the basis of ordinary PCR, and the initial amount of nucleic acid in a sample to be detected is calculated by measuring the fluorescence value in an exponential amplification period by combining the accumulation of the fluorescence value with the amplification process of a PCR product. Compared with the traditional HLA allele typing detection method, the FQ-PCR has the characteristics of high sensitivity, strong specificity and the like, and also has the incomparable advantages of simple and convenient operation, short time consumption, quantifiability and the like of the traditional method, and the application of the FQ-PCR technology to the typing detection of the HLA allele is an innovation in the technical field of PCR. The TaqMan MGB probe technology is one of the more advantageous FQ-PCR technologies.
However, due to the specificity of the HLA-B1502 gene, accurate results are difficult to obtain by conventional TaqMan MGB probe technology. In order to detect the HLA-B1502 gene, the conventional idea is to detect the SNP associated with the HLA-B1502 gene and then obtain the HLA-B1502 allele typing result through a series of data analysis. The method has the advantages of multiple steps, long time consumption and improved accuracy of test results. The invention realizes the 3-tube combined detection of HLA-B1502 allelic gene, breaks through the conventional concept of applying the fluorescence PCR technology to HLA-B1502 genotyping, and completes the invention on the basis of the research.
In one aspect, the invention provides a method for detecting HLA-B1502 allelic gene, which can detect the gene typing of the HLA-B1502 locus more accurately, conveniently, with low cost and comprehensively.
The invention provides a detection method for detecting HLA-B1502 allele, which comprises the following steps:
1) providing MIX reaction liquid comprising a sample to be detected, a nucleic acid amplification system and a fluorescence detection system;
2) amplifying a target nucleotide nucleic acid in a sample to be detected through a nucleic acid amplification system;
3) binding the probe in the fluorescence detection system to the amplified target nucleotide nucleic acid sequence;
4) measuring the amount of fluorescence generated by the fluorogenic group, thereby determining the presence of the target nucleotide nucleic acid.
The nucleic acid amplification system comprises heat-resistant DNA polymerase, deoxynucleotide triphosphate, an upstream primer (F) 1 capable of binding with a first strand of a target nucleotide nucleic acid, a downstream primer (R) 1 capable of binding with a second strand of the target nucleotide nucleic acid, an upstream primer (F) 2 capable of binding with the first strand of the target nucleotide nucleic acid, and a downstream primer (R) 2 capable of binding with the second strand of the target nucleotide nucleic acid;
the fluorescence detection system is a TaqMan MGB specific oligonucleotide probe (P) which can be combined with target nucleotide nucleic acid and two ends of which respectively contain a fluorescence generating group and a fluorescence quenching group.
As a preferred technical scheme, the nucleic acid amplification system and the fluorescence detection system of the invention are 3 groups of MIX reaction liquids, wherein:
the upstream primer 1 and the downstream primer 1 for amplification of the target nucleotide nucleic acid in the MIX1 reaction solution comprise a primer 1 and a primer 2, and the oligonucleotide probe used in the fluorescence detection system of the MIX1 reaction solution comprises a probe 1;
the upstream primer 1 and the downstream primer 1 for amplifying the target nucleotide nucleic acid in the MIX2 reaction solution comprise a primer 3 and a primer 4, and the oligonucleotide probe used in the fluorescence detection system of the MIX2 reaction solution comprises a probe 2;
the upstream primer 1 and the downstream primer 1 for amplification of the target nucleotide nucleic acid in the MIX3 reaction solution comprise a primer 5 and a primer 6, and the oligonucleotide probe used in the fluorescence detection system of the MIX3 reaction solution comprises a probe 3.
The nucleotide sequences of the primers 1-6 are respectively shown as SEQ ID NO.1-SEQ ID NO.6, and are derived from the gene sequence of HLA-B locus.
The nucleotide sequences of the probes 1-3 are respectively shown as SEQ ID NO.9-SEQ ID NO.11, and are derived from the gene sequence of HLA-B locus.
The MIX1, MIX2 and MIX3 reaction solution can also contain a primer 7, a primer 8 and a probe 4; the primer 7 and the primer 8 are used for amplifying the reference gene, and the probe 4 identifies the reference gene amplified by the primer 7 and the primer 8.
The nucleotide sequences of the primer 7 and the primer 8 are respectively shown as SEQ ID NO.7 and SEQ ID NO.8, and are used for amplifying the sequences derived from human GAPDH genes. The nucleotide sequence of the probe 4 is shown as SEQ ID NO.12 and is derived from a human GAPDH gene sequence.
The detection reagent provided by the invention also has the following characteristics: wherein, equally include respectively in probe 1, probe 2 and the probe 3: FAM marker at 5 'end and MGB marker at 3' end; probes 4, 5 'end VIC label and 3' end MGB label.
The detection reagent provided by the present invention is also characterized by further comprising: a thermostable DNA polymerase; blank control samples for system error correction in genotype testing; and the positive control sample is used for controlling the quality of a PCR reaction system in the genotype detection process.
The detection reagent provided by the invention also has the following characteristics: wherein the DNA polymerase is one or more of GoldStar TaqMan Mix, TaqPath ProAmp Master Mix and TaqMan GTXpress Master Mix.
The detection reagent provided by the invention also has the following characteristics: wherein the blank control sample is ddH2O。
The detection reagent provided by the invention also has the following characteristics: wherein, the positive control sample is genomic DNA positive to HLA-B1502. In a preferred embodiment of the invention, the concentration of the genomic DNA is 1 ng/. mu.L.
In another aspect, the invention further provides a use of a detection reagent in the preparation of a kit for medication guidance of an epileptic patient, wherein the detection reagent is the detection reagent.
The invention also provides a kit for detecting the HLA-B1502 genotype for the medication guidance of carbamazepine, which comprises the detection reagent.
The kit provided by the invention detects the genotype of HLA-B1502 based on a fluorescent quantitative PCR TaqMan MGB probe method. The kit comprises 3 sets of primers and probes of MIX reaction solution, and the concentration can adopt the conventional acceptable concentration in the field. Preferably, the concentration of primers and probes is not more than 2. mu. mol/L, for example, the concentration of primers is 0.02 to 1.2. mu. mol/L and the concentration of probes is 0.01 to 0.6. mu. mol/L. More preferably, the concentration of the primer is 50 to 1000 nmol/L, e.g., 50nmol/L, 80nmol/L, 100nmol/L, 150nmol/L, 180nmol/L, 200nmol/L, 250nmol/L, 300nmol/L, 350nmol/L, 400nmol/L, 450nmol/L, 500nmol/L, 550nmol/L, 600nmol/L, 700nmol/L, 800nmol/L, 900nmol/L, and the like. The concentration of the probe may be 50-200 nmol/L. For example, 60nmol/L, 80nmol/L, 90nmol/L, 100nmol/L, 110nmol/L, 120nmol/L, 130nmol/L, 140nmol/L, 150nmol/L, 160nmol/L, 180nmol/L, and the like.
In the invention, the concentration of the primer and the probe can be adjusted according to different amplification systems to obtain a better detection result, improve the accuracy, the sensitivity and the resolution and reduce the occurrence of false negative and false positive. For example, the MIX1 reaction solution contains primer 1 and primer 2 at concentrations of 0.2-0.7. mu. mol/L, and contains primer 7 and primer 8 at concentrations of 0.2-1.2. mu. mol/L; in the MIX2 reaction solution, the concentration of the primer 3 and the primer 4 is 0.3-0.6 mu mol/L, and the concentration of the primer 7 and the primer 8 is 0.2-1.2 mu mol/L; the MIX3 reaction solution contained primer 5 and primer 6 at concentrations of 0.2-0.8. mu. mol/L, and primer 7 and primer 8 at concentrations of 0.2-1.2. mu. mol/L.
The kit provided by the invention also has the following characteristics: wherein, when the kit is used, in the MIX1 reaction solution, the concentration of the probe 1 is 0.09-0.12 mu mol/L, and the concentration of the probe 4 is 0.1-0.5 mu mol/L; in the MIX2 reaction liquid, the concentration of the probe 2 is 0.12-0.15 mu mol/L, and the concentration of the probe 4 is 0.1-0.5 mu mol/L; in the MIX3 reaction solution, the concentration of the probe 3 is 0.14-0.20. mu. mol/L, and the concentration of the probe 4 is 0.1-0.5. mu. mol/L.
The invention also provides a using method of the kit for non-diagnostic treatment, wherein the kit is the kit, and the kit detects the HLA-B1502 locus genotype based on a fluorescence quantitative PCR TaqMan MGB probe method.
The use method provided by the invention also has the following characteristics: the sample to be detected by the kit is a biological sample containing genomic DNA, such as whole blood or oral mucosa exfoliative cells from an isolated body.
Preferably, the present invention provides a method of use wherein the number of amplification cycles is 38-45, and the overall efficiency and specificity of detection is better, e.g., 38, 39, 40, 41, 42, 43, 44, 45. Preferably, the conditions for performing the PCR amplification reaction are:
(1) UNG enzyme treatment at 50 deg.C for 5min,
(2) pre-denaturation at 95 ℃ for 10min,
(3) the denaturation is carried out for 15s at the temperature of 95 ℃,
(4) the extension is carried out for 45s at the temperature of 60 ℃,
(3) 40 cycles of (4).
Preferably, in the use method provided by the invention, when the temperature for annealing the MIX (reaction mixed liquid, including MIX1, MIX2 and MIX 3) reaction liquid is 59-61 ℃, the specificity and the sensitivity are better overall, for example, the annealing temperature is 59 ℃, 60 ℃ and 61 ℃. The annealing time is in the range of 30-60 seconds, and the specific signal value can be improved, for example, the annealing time is 30s, 45s and 60 s.
The use method provided by the invention is also characterized in that the result judgment is as follows:
when the Ct values of the VIC channel and the FAM channel are less than or equal to 38, the amplification curve has an obvious exponential amplification period, and the result is read as positive according to a positive control result; when the VIC and FAM channels have no Ct value or the Ct value is larger than 38, the result is read as blank according to the blank control result; when the reference positive control result is positive and the reference blank control result is blank, the genotyping result of the test sample is determined as shown in table 7.
The invention has the following functions and effects: the invention provides a detection reagent for detecting HLA-B1502 genotype, application of the detection reagent in preparing a kit for medication guidance of epileptics, a corresponding kit and a using method thereof. The kit can be widely applied to detection of HLA-B1502 alleles, guides clinical application of CBZ drugs, and avoids the possibility that HLA-B1502 genotype patients can cause SJS/TEN after using the CBZ drugs. The detection reagent provided by the invention comprises a primer group which can be specifically and accurately used for typing the HLA-B1502 gene for detection, so that the detection reagent can be used for evaluation basis of medication guidance of epileptics and development guidance of related medicines.
Drawings
Figure 1 is a representation of the results for combination 1 in comparative example 1.
Figure 2 is a representation of the results for combination 2 in comparative example 1.
Figure 3 is a representation of the results for combination 3 in comparative example 1.
Figure 4 is a representation of the results for combination 1 of comparative example 2.
Figure 5 is a representation of the results of another reaction of combination 1 of comparative example 2.
Figure 6 is a representation of the results for combination 2 of comparative example 2.
Figure 7 is a representation of the results for combination 3 of comparative example 2.
Figure 8 is a representation of the results for combination 1 of comparative example 3.
Figure 9 is a representation of the results for combination 2 of comparative example 3.
Figure 10 is a representation of the results for combination 3 of comparative example 3.
Detailed Description
The following describes specific embodiments of the present invention. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.
The methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
Example 1
This example specifically illustrates a primer set and a kit that can be used for a gene group for medication guidance for epileptic patients.
This example provides a test reagent for the detection of HLA-B1502 genotype.
The detection reagent comprises: the nucleic acid amplification system of the kit comprises MIX1 reaction liquid and primers 1 and 2, 7 and 8 for amplifying oligonucleotide nucleic acid; MIX2 reaction solution and primer 3 and primer 4, primer 7 and primer 8 thereof for amplifying oligonucleotide nucleic acid; MIX3 reaction solution and primer 5 and primer 6, primer 7 and primer 8 thereof for amplifying oligonucleotide nucleic acid; wherein, the nucleotide sequences of the primer 1-primer 8 are respectively shown as SEQ ID NO.1-SEQ ID NO.8 (Table 1).
The detection reagent further comprises: the kit is used for detecting a fluorescent detection probe 1 and a fluorescent detection probe 4 which are used for MIX1 reaction liquid, detecting a fluorescent detection probe 2 and a fluorescent detection probe 4 which are used for MIX2 reaction liquid, and detecting a fluorescent detection probe 3 and a fluorescent detection probe 4 which are used for MIX3 reaction liquid, wherein the probe 1, the probe 2 and the probe 3 are respectively as follows: FAM marker at 5 'end and MGB marker at 3' end; the probe 4 is: 5 'end VIC mark and 3' end MGB mark. The nucleotide sequences of probes 1-4 are shown in SEQ ID NO.9-SEQ ID NO.12, respectively (Table 2).
Wherein, the 5 ' end VIC mark and the 5 ' end FAM mark respectively indicate that the 5 ' end is marked with different fluorescent reporter groups, and the 3 ' end MGB mark indicates that the 3 ' end is marked with fluorescent group quenching.
The detection reagent of the present embodiment further comprises a DNA polymerase, a blank control sample, and a positive control sample. Wherein, the DNA polymerase can be one or more of GoldStar TaqMan Mix, TaqPath ProAmp Master Mix and TaqMan GTXpress Master Mix.
The blank control sample is used for system error correction in the process of detecting the HLA-B1502 locus genotype, namely, whether a reaction system is polluted or not is detected, the reaction detection result is ensured to be accurate and reliable, and in particular, ddH is adopted2O as a blank control sample.
The positive control sample is used for quality control of the PCR reaction system in the process of detecting the HLA-B1502 locus genotype, in the embodiment, the positive control sample is the genomic DNA containing the gene HLA-B1502, and the concentration of the genomic DNA is 1 ng/muL or the similar concentration.
The quality control of the PCR reaction system is also used for quality control purposes to monitor and detect whether the reaction system can be normally used.
The detection reagent provided by the embodiment can be applied to preparation of a kit for medication guidance of epileptic patients, so that the embodiment also provides a kit for detecting the genotype of HLA-B1502 locus for medication guidance of epileptic.
In the kit provided by the embodiment, the HLA-1502 locus genotype is detected based on a fluorescent quantitative PCR TaqMan MGB probe method, and in the use of the kit, the MIX1 reaction liquid contains 0.2-0.7 mu mol/L of primer 1 and primer 2, 0.2-1.2 mu mol/L of primer 7 and primer 8, preferably, the concentration of primer 1 and primer 2 is 0.4 mu mol/L, and the concentration of primer 7 and primer 8 is 0.25 mu mol/L; in the MIX2 reaction solution, the concentration of the primer 3 and the primer 4 is 0.3-0.6. mu. mol/L, the concentration of the primer 7 and the primer 8 is 0.2-1.2. mu. mol/L, preferably, the concentration of the primer 3 and the primer 4 is 0.4. mu. mol/L, and the concentration of the primer 7 and the primer 8 is 0.25. mu. mol/L; in the MIX3 reaction solution, the concentration of the primer 5 and the primer 6 is 0.2-0.8. mu. mol/L, the concentration of the primer 7 and the primer 8 is 0.2-1.2. mu. mol/L, preferably, the concentration of the primer 1 and the primer 2 is 0.7. mu. mol/L, and the concentration of the primer 7 and the primer 8 is 0.25. mu. mol/L; in addition, in the use of the kit, in the MIX1 reaction solution, the concentration of the probe 1 is 0.09-0.12 mu mol/L, the concentration of the probe 4 is 0.1-0.5 mu mol/L, preferably, the concentration of the probe 1 is 0.11 mu mol/L, and the concentration of the probe 4 is 0.25 mu mol/L; in the MIX2 reaction solution, the concentration of the probe 2 is 0.12-0.15 mu mol/L, the concentration of the probe 4 is 0.1-0.5 mu mol/L, preferably, the concentration of the probe 2 is 0.14 mu mol/L, and the concentration of the probe 4 is 0.25 mu mol/L; in the MIX3 reaction solution, the concentration of probe 3 is 0.14-0.20. mu. mol/L, the concentration of probe 4 is 0.1-0.5. mu. mol/L, preferably, the concentration of probe 3 is 0.18. mu. mol/L, and the concentration of probe 4 is 0.25. mu. mol/L.
Wherein, the working principle of FQ-PCR (real-time fluorescence quantitative PCR) is as follows: the probe used in the invention is a TaqMan probe marked by VIC/FAM at the 5 'end and MGB at the 3' end, and the two ends of the probe are respectively marked with a fluorescence reporter group (R) and a fluorescence quenching group (Q). When the probe is complete, namely in a random state and a PCR product-free hybridization state, the fluorescence emitted by the reporter group is absorbed by the quencher group, and the existence of the fluorescence cannot be detected. In the FQ-PCR amplification process, when a specific PCR product and a TaqMan MGB probe are subjected to hybridization reaction, the 5' -end exonuclease activity of the DNA polymerase with strong tolerance cuts the bases of the TaqMan MGB probe one by one, and the fluorescence released by the reporter group can be detected by a fluorometer built in a PCR instrument. After one cycle of PCR, the fluorescent signal is the same as the target fragment, and has a synchronous exponential amplification process, and the strength of the fluorescent signal represents the copy number of the template DNA.
In this embodiment, the use method of the kit is as follows:
the method for detecting the HLA-B1502 locus genotype based on the fluorescent quantitative PCR TaqMan MGB probe method specifically comprises the following steps:
and 3, genotyping: a TaqMan MGB probe method (Life, TaqPath promoter Master Mix) is applied to carry out genotyping on three systems of MIX1 reaction liquid, MIX2 reaction liquid and MIX3 reaction liquid, and the adopted primer group and probe group are as above.
A. Preparation of different MIX reaction solution systems, the final volume of each reaction system was 25. mu.L.
A1, MIX1 reaction solution preparation details are shown in Table 3.
A2, MIX2 reaction solution preparation details see Table 4.
A3, MIX3 reaction solution preparation details see Table 5.
Each nucleic acid amplification system detects the corresponding template as follows: a sample to be tested, a blank control sample and a positive control sample. For the detection of each MIX reaction solution, corresponding primers and probes shown in tables 3, 4 and 5 are added, and then different templates are respectively added for parallel reaction, so as to respectively obtain a reference positive control result, a reference blank control result and a genotyping result. Wherein the adding concentration of the sample to be detected is 5-15 ng/muL, and the adding concentration of the positive control sample is 1 ng/muL.
B. FQ-PCR reaction amplification procedure (Table 6)
C. And (4) interpretation of results:
when the Ct values of the VIC channel and the FAM channel are less than or equal to 38, the amplification curve has an obvious exponential amplification period, and the result is read as positive according to a positive control result;
when the channel VIC and the channel FAM have no Ct value or the Ct value is more than 38, the amplification curve is indicated to be absent, or the amplification curve is a straight line or a slight oblique line and has no obvious exponential growth period, and the channel VIC and the channel FAM are interpreted as blank according to blank control results;
when the reference positive control result is positive and the reference blank control result is blank, the genotyping result of the sample to be tested is determined as shown in table 7:
note: "/" indicates not considered.
Example 2
In this example, for some cases of epilepsy, by using the method of detecting HLA-B1502 using PCR-SBT kit as gold standard and using the detection reagent, corresponding kit and corresponding method provided in example 1, samples of cases were taken, and HLA-B1502 sites were detected, and the results of genotyping detection are shown in Table 8.
The above results show that: the genotyping results obtained using the kit of example 1 were 100% identical to those obtained using the PCR-SBT kit.
It can be seen that the detection reagent, the corresponding kit and the method of example 1 are used for detection, and the reliability of the detection result of the site of HLA-B1502 is high, which shows that the detection reagent, the corresponding kit and the using method of example 1 can accurately detect the gene of the site 1502 of the human leukocyte antigen, and therefore, can be accurately used for epileptic patients as the evaluation basis of the medication guidance of carbamazepine, the research and development of corresponding drugs, and the like.
Moreover, compared with the genotyping technology such as gene sequencing and the like, the real-time fluorescence quantitative PCR detection method for detecting the HLA-B1502 locus genotyping has the following advantages:
1. the specificity is strong: the designed primer pairs respectively aim at specific sequences of HLA-B1502 and can specifically amplify corresponding genomic DNA; probes designed respectively aiming at the amplification products of the MIX reaction solution are added, and the MGB can effectively distinguish single base difference;
2. the detection process is a closed tube reaction, so that the possibility of pollution and result deviation is greatly reduced;
3. the operation is quick and simple, and the result can be finished within 3-4 hours from the sample submission. The gene sequencing method has complicated detection steps: the method comprises the steps of specimen inspection → DNA extraction → PCR amplification → verification of PCR products (electrophoresis) → purification of PCR products → gene sequencing → result analysis, wherein the electrophoresis process of the two PCR products has high pollution probability and is not suitable for large-scale development in hospitals;
4. the interpretation result is clear and objective;
5. high throughput, 30 samples can be detected in a single plate (96 well plate);
6. the PCR product is safe, does not contain toxic and harmful substances in the whole system, does not need post-treatment of the PCR product, and is harmless to operators and the environment.
In a word, compared with genotyping technologies such as gene sequencing and the like, the kit provided in example 1 is used for genotyping of HLA-B1502 sites, and has the advantages of strong specificity, high sensitivity, simple and rapid operation, high throughput, accurate interpretation result, simplicity, convenience and the like, so that the problems of time consumption, complex procedures, easy pollution and the like in the existing genotyping detection technology can be solved. Moreover, the primers and probes in the kit of example 1 are repeatedly optimized and tested, so that the HLA-B1502 gene can be rapidly, accurately, cheaply and highly-throughput typed, the sensitivity is high, the specificity is strong, the kit is suitable for common samples such as blood, oral mucosa cast-off cells and the like, the type of the HLA-B1502 gene site in human blood or other histiocytes can be rapidly, cheaply and accurately detected at high throughput, and for the typing technology, the kit is the method with the best cost performance at present, and is more convenient for clinical or health detection large-scale promotion.
Examples effects and effects
The detection reagent for detecting the genotype of the HLA-B1502 locus, the application of the detection reagent in preparing the kit for the medication guidance of the epileptic, the corresponding kit and the use method thereof, provided by the embodiment 1, can be used for the evaluation basis of the medication guidance of the epileptic and the development guidance of related medicines because the detection reagent comprises the primer probe which can be specifically and accurately used for the genotyping detection of the HLA-B1502 locus.
In addition, the application method of the kit provided in embodiment 1, with the primers and probes designed by the present invention, can select the real-time fluorescence quantitative PCR detection with simple and rapid operation, high throughput and accurate interpretation result, which is more convenient for large-scale clinical or health detection.
Comparative example 1
The results of screening the MIX1 reaction solution using different primer probe combinations are shown in Table 9.
The primer probe sequence information is shown in Table 10.
Comparative example 2
And (3) screening MIX2 reaction liquid by adopting different primer probe combinations. The results are shown in Table 11 and FIGS. 4-7.
The primer probe sequence information is shown in Table 12.
Comparative example 3
And (3) screening MIX3 reaction liquid by adopting different primer probe combinations. The results are shown in Table 13 and FIGS. 8-10.
The primer probe sequence information is shown in Table 14.
Sequence listing
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Claims (12)
1. A detection reagent of human leukocyte antigen B site 1502 gene is characterized in that,
the detection reagent comprises MIX1 reaction liquid, MIX2 reaction liquid and MIX3 reaction liquid;
the MIX1 reaction solution contains a primer 1 and a primer 2 which can be combined with oligonucleotide nucleic acid;
the MIX2 reaction solution contains a primer 3 and a primer 4 which can be combined with oligonucleotide nucleic acid;
the MIX3 reaction solution contains a primer 5 and a primer 6 which can be combined with oligonucleotide nucleic acid;
the fluorescent detection system used in conjunction with the MIX1 reaction solution nucleic acid amplification system includes a probe 1 for hybridizing with an amplification product of the nucleic acid amplification system;
the fluorescent detection system used in conjunction with the MIX2 reaction solution nucleic acid amplification system includes a probe 2 for hybridizing with an amplification product of the nucleic acid amplification system;
the fluorescent detection system used in conjunction with the MIX3 reaction solution nucleic acid amplification system includes a probe 3 for hybridizing with an amplification product of the nucleic acid amplification system;
in the MIX1 reaction solution, the nucleotide sequences of the primer 1 and the primer 2 are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
in the MIX2 reaction solution, the nucleotide sequences of the primer 3 and the primer 4 are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4;
in the MIX3 reaction solution, the nucleotide sequences of the primer 5 and the primer 6 are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6;
the nucleotide sequences of the probe 1, the probe 2 and the probe 3 are respectively shown as SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO. 11.
2. The detection reagent according to claim 1, wherein the 5 'ends of probe 1, probe 2, probe 3 and probe 4 are labeled with different fluorescent reporter groups, and the 3' ends of probe 1, probe 2, probe 3 and probe 4 are labeled with fluorescent quencher groups.
3. The detection reagent according to claim 1,
the detection reagent also comprises DNA polymerase, wherein the DNA polymerase is one or more of GoldStar TaqMan Mix, TaqPath ProAmp Master Mix and TaqMan GTXpress Master Mix.
4. The detection reagent according to claim 1, wherein the MIX1, MIX2 and MIX3 reaction solution further contains a primer 7, a primer 8 and a probe 4;
the primer 7 and the primer 8 are used for amplifying the reference gene, and the probe 4 identifies the reference gene amplified by the primer 7 and the primer 8.
5. The detection reagent according to claim 4,
the nucleotide sequences of the primer 7 and the primer 8 are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8;
the nucleotide sequence of the probe 4 is shown as SEQ ID NO. 12.
6. Use of a detection reagent for the preparation of a kit for non-disease diagnostic or therapeutic purposes for use in medication guidance for epileptic patients, wherein the detection reagent is according to any one of claims 1 to 5.
7. A kit for detecting the genotype of an HLA-B1502 locus, said kit comprising: the detection reagent according to any one of claims 1 to 5.
8. The kit of claim 7, wherein the kit comprises the detection reagent of claim 5;
in the kit, the MIX1 reaction solution contains 0.2-0.7 mu mol/L of primer 1 and primer 2, and 0.2-1.2 mu mol/L of primer 7 and primer 8; in the MIX2 reaction solution, the concentration of the primer 3 and the primer 4 is 0.3-0.6 mu mol/L, and the concentration of the primer 7 and the primer 8 is 0.2-1.2 mu mol/L; the MIX3 reaction solution contained primer 5 and primer 6 at concentrations of 0.2-0.8. mu. mol/L, and primer 7 and primer 8 at concentrations of 0.2-1.2. mu. mol/L.
9. The kit according to claim 7,
the kit comprises the detection reagent of claim 5;
in the kit, in a fluorescence detection system of MIX1 reaction liquid, the concentration of a probe 1 is 0.09-0.12 mu mol/L, and the concentration of a probe 4 is 0.1-0.5 mu mol/L; in the fluorescence detection system of the MIX2 reaction solution, the concentration of the probe 2 is 0.12-0.15 mu mol/L, and the concentration of the probe 4 is 0.1-0.5 mu mol/L; in the fluorescence detection system of the MIX3 reaction solution, the concentration of the probe 3 is 0.14-0.20 mu mol/L, and the concentration of the probe 4 is 0.1-0.5 mu mol/L.
10. A method for detecting HLA-B1502 gene locus based on fluorescence quantitative PCR TaqMan MGB probe method is characterized in that,
the method comprises the following steps:
step 1, obtaining a sample to be detected: the sample to be detected is a biological sample containing genome DNA;
step 2, separating and extracting genome DNA, and adjusting the concentration and purity of the DNA to the concentration and purity acceptable by genotyping detection;
step 3, genotyping using the detection reagent of any one of claims 1 to 5 or the kit of any one of claims 6 to 9;
step 4, determining the genotype of the HLA-B1502 locus according to the typing result in the step 3;
and (4) interpretation of results:
when the Ct values of the VIC channel and the FAM channel are less than or equal to 38, the amplification curve has an obvious exponential amplification period, and the result is read as positive according to a positive control result;
when the channel VIC and the channel FAM have no Ct value or the Ct value is more than 38, the amplification curve is indicated to be absent, or the amplification curve is a straight line or a slight oblique line and has no obvious exponential growth period, and the channel VIC and the channel FAM are interpreted as blank according to blank control results;
when the result of the reference positive control is positive and the result of the reference blank control is blank, the genotyping results of the samples to be tested are determined as shown in Table 7.
11. The method of claim 10,
the genotyping method for three systems of MIX1 reaction liquid, MIX2 reaction liquid and MIX3 reaction liquid by using a TaqMan MGB probe method comprises the following steps:
preparation of different nucleic acid amplification systems: each MIX reaction solution contains DNA polymerase, dNTPs, PCR buffer solution, primers and probes; the MIX1 reaction solution contains a primer 1, a primer 2, a primer 7 and a primer 8 which can be combined with the oligonucleotide; the MIX2 reaction solution contains a primer 3, a primer 4, a primer 7 and a primer 8 which can be combined with the oligonucleotide; the MIX3 reaction solution contains a primer 5, a primer 6, a primer 7 and a primer 8 which can be combined with the oligonucleotide; the fluorescent detection system used in conjunction with the MIX1 reaction solution nucleic acid amplification system comprises probe 1 and probe 4 for hybridizing with the amplification product of the nucleic acid amplification system; the fluorescent detection system used with the MIX2 reaction solution nucleic acid amplification system comprises probe 2 and probe 4 for hybridizing with the amplification product of the nucleic acid amplification system; the fluorescent detection system used in conjunction with the MIX3 reaction solution nucleic acid amplification system comprises probe 3 and probe 4 for hybridizing with the amplification product of the nucleic acid amplification system;
FQ-PCR reaction: the PCR amplification procedure comprises UNG enzyme treatment, pre-denaturation, denaturation and extension, and the cycle number of the denaturation and extension is 40 times.
12. The method of claim 11, wherein the annealing temperature is 59-61 ℃ and the annealing time is 30-60 seconds.
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| CN114350786A (en) * | 2022-01-10 | 2022-04-15 | 常州先趋医疗科技有限公司 | For detecting HLA-B15: primer group of 02 allele and application thereof |
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