CN113215245A - Gene detection kit for personalized medicine for children asthma and application thereof - Google Patents

Abstract

The invention belongs to the field of gene detection, and particularly relates to a gene detection kit for personalized medicine for children asthma and application thereof. The kit provided by the invention mainly comprises a primer mixed solution, a multiple PCR library amplification buffer solution, a digestion buffer solution, a connection buffer solution, ligase, a specific joint, a HiFi library amplification buffer solution, a positive/negative quality control product, a report system and an instruction book. The kit provided by the invention can be used for detecting common asthma-relieving medicines for children, 31 genes and 41 gene polymorphism sites are provided; realizes the relevant gene detection and report analysis for the prediction of the curative effect and adverse reaction of the asthma medicament of children and provides recommendation for the medication of clinicians.

Description

Gene detection kit for personalized medicine for children asthma and application thereof

Technical Field

The invention relates to the field of gene detection, in particular to a gene detection kit for detecting personalized medicine for children asthma and application thereof.

Background

Bronchial asthma (asthma for short) is a heterogeneous disease characterized by chronic airway inflammation, and is mainly clinically manifested by respiratory symptoms such as wheezing, shortness of breath, chest distress, cough and the like which are continuously changed and aggravated along with time, and is accompanied by reversible expiratory airflow limitation.

The data of the investigation showed that the average incidence of adult asthma worldwide was 4.3%, while the incidence of childhood asthma was 11.6% in children aged 6-7 and 13.7% in children aged 13-14. In China, 2010 survey data show that the total asthma incidence rate of children aged 0-14 years in urban areas is 3.02%, and the increase is 52.8% compared with 2000, wherein the increase of big cities is more remarkable. Meanwhile, the clinical control condition of asthma is not ideal, and data show that the uncontrolled rate of European adult asthma reaches 45 percent; the uncontrolled rate of Asian children asthma is higher and reaches 53.4 percent. In China, the control rate of asthma patients older than 14 years old is only 28.5%, and the control rate of asthma in children is not more than 10%. In addition, despite the standardized diagnosis, 3-4% of childhood asthma can continue to adult, and 30-50% of childhood asthma recurs in adult. Many children asthma patients are delayed and not cured because of improper treatment or untimely final development of adult asthma, and some patients even completely lose physical activity. Severe asthma attacks, if not treated in a timely manner, can even be life threatening. Asthma is the most common chronic lung disease threatening public health in the world at present, has great influence on life of asthma patients, causes loss to sustainable development of world economy to a certain extent, and brings negative influence on social stability.

The etiology and pathogenesis of asthma are very complex and are the result of a combination of immunological, genetic, and environmental effects. Medical consensus has recognized that childhood asthma has two peaks around 5 years of age. Most asthma patients begin with occult attacks, while children often suffer from asthma due to age, physiological defecation, immune function, allergen exposure, environment, etc., and the asthma-causing factors are complex and difficult to be examined compared with adult asthma, so that it is more desirable to improve the accuracy of diagnosis.

Through Genome-wide association study (GWAS), it has been found that there are over 1000 asthma-associated candidate genes, and about 50 genes are currently validated in the human population. Variations in these genes can affect the occurrence, severity and response of asthma to various therapeutic agents. At present, different individuals clinically have great difference in response to drugs, up to 40% of asthma patients do not respond to drug treatment, and in addition, some patients have obvious drug toxic and side effects after using the drugs, so that the death risk is increased.

Therefore, a kit capable of screening therapeutic drugs is urgently needed in the market, so that toxic and side effects and adverse reactions of patients caused by ineffective drugs are avoided, and the first cure effectiveness is improved.

Disclosure of Invention

In order to solve the problems that the existing detection products cover fewer drugs and genes and are not accurate enough in prediction, and a new effective treatment scheme is needed for patients; the invention provides a gene detection kit for detecting individualized medication of childhood asthma and application thereof based on the facts that the medication has more related genes and sites, interpretation is complex, interpretation is not beneficial to clinicians or detection results are utilized, and the like.

In order to achieve the purpose, the invention adopts the technical scheme that:

the first object of the present invention is: provides a gene detection kit for personalized medicine for children asthma, which comprises the following components: primer mixed liquor, multiplex PCR library amplification buffer solution, digestion buffer solution, connection buffer solution, ligase, specific joint, HiFi library amplification buffer solution, positive/negative quality control product, report system and instruction book.

Preferably, the library primer mixture contains 31 genes and 41 gene polymorphism sites.

Preferably, the 31 genes are ABCB1, ADRB2, BCL2L11, BMP7, CA10, CLOCK, COL22A1, CRHR1, CRHR2, CTLA4, CTNNB1, CXCL12, DCAF4, DOK5, DROSHA, TSPYL1, DUSP1, FKBP5, GATA3, GLCCI1, GSTA1, HCG22, HDAC1, LINC00251, PNPLA3, PYGL, SERPIPI 1, ST13, TAAR6, TBX21, TBXT, respectively.

Preferably, the site information and corresponding primer information of the 41 gene polymorphic sites for amplifying the 31 genes are shown in tables 1 and 2 below, and the primers are mixed in equal volumes to form a library primer mixture.

TABLE 1 amplification of information on polymorphic sites of 41 genes of 31 genes

Preferably, the specific joints are index p5 and index p 7; the sequence of the index p5 is shown as SEQ ID NO. 83; the sequence of the index p7 is shown in SEQ ID NO. 84.

5’-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’(SEQ ID NO.83)

5’-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’(SEQ ID NO.84)

Wherein NNNNNNNN (AAGTCTCT, CCAGCGCT, ATGAACCT).

Preferably, the process of setting the system in the computer is to digitize the influence of different genotypes of a single gene on the 'response' and 'adverse reaction risk' of the drug, analyze the response and the adverse reaction of the drug in sequence to obtain the response and the adverse reaction score of the patient to the drug, and predict the medication condition of the patient by synthesizing the response and the adverse reaction score of the drug.

Preferably, the algorithm is established by relying on CPIC, PharmGKB, expert consensus, medical guidelines and drug gene information of drug instructions.

Another object of the invention is: provides the application of the gene detection kit for the personalized medicine for children asthma in the medicine detection of the personalized medicine related to the children asthma, and the detection process is as follows:

s1, designing a multiple PCR amplification primer according to the polymorphism sequence of the related gene locus of the children asthma treatment medicine;

s2, preparing a library construction kit;

s3, extracting genome DNA from samples such as blood samples, buccal swabs, dried blood slices and the like;

s4, constructing a library for sequencing by using the genome DNA of the sample to be tested;

s5, performing high-throughput sequencing on the qualified gene library obtained in the step S4 to obtain sequencing data;

s6, analyzing the sequencing data result obtained in the step S5, and providing a detection report by combining a report system.

Preferably, the gene-related medicaments are common antiasthmatic medicaments for children, and comprise salmeterol, corticosteroids, salbutamol and terbutaline.

The scheme of primer design provided by the invention comprises the following steps:

1. gene screening:

according to the invention, 31 gene loci with high correlation degree of epilepsy clinical treatment drugs are screened out to design a basic amplification primer group by referring to PharmGKB, CPIC, FDA and other known database information according to epilepsy treatment clinical first-line drugs recommended by domestic and foreign clinical guidelines.

2. Design of primers the present invention designs primers following the following ideas:

(1) the primers are not bound to each other nor to regions other than the target fragment on the template DNA.

(2) The combination of the primers needs to solve the problem that different amplification fragments compete with each other, and the high-abundance template is prevented from being completely covered, so that the low-abundance template falls into the background completely.

(3) The design of the primers adopts a joint PCR amplification system, and certain interference exists between the primers of each group of PCR in a mixed system, so that a large number of primers must be designed for experimental screening according to the requirement of a detection method, and interference and balance among a plurality of groups of primers are considered through professional primer design software to adjust the primers.

3. Genes related to childhood asthma include ABCB1, ADRB2, BCL2L11, BMP7, CA10, CLOCK, COL22A1, CRHR1, CRHR2, CTLA4, CTNNB1, CXCL12, DCAF4, DOK5, DROSHA, TSPYL1, DUSP1, FKBP5, GATA3, GLCCI1, GSTA1, HCG22, HDAC1, LINC00251, PNPLA3, PYGL, SERPINE1, ST13, TAAR6, TBX21, TBXT.

The gene comprises the following 41 gene polymorphic sites, and amplification primers are shown as SEQ ID NO.1-SEQ ID NO. 82:

4. primer sequence information:

the primer sequence is designed and screened by the invention, the primers are synthesized by Shanghai Jielii bioengineering GmbH, each primer is prepared into 10 mu M working solution by using sterilized purified water, the signal value of each primer in PCR amplification reaction is used as the index of each primer concentration in the primer mixture, and the mixture is mixed into the mixed solution with each primer concentration of 0.01-0.05 mu M according to experimental investigation. The primer mixture contained 41 designed pairs of primers. The length of each primer is 18-32bp, the Tm value is 60 ℃, the difference is not more than 1 ℃, and the amplification product is 250bp at 100 ℃. The specific primer sequence is SEQ NO.1-SEQ NO. 82.

The report system of the invention has the following technical characteristics:

1. the reporting system embodied by the present invention is implemented by a computer-implemented reporting system having a processor executing specific instructions in a computer program to complete the overall reporting process in a mode that receives the patient's genotype and outputs the corresponding recommended dosage of the drug after analysis according to a system-implemented algorithm. Specifically, the report system of the invention is a set of system designed based on 41 gene polymorphism sites detected by the kit and the effect of different gene sites on the treatment effect of different drugs, and assisted by the clinical index of the individual to be tested, and can efficiently and accurately realize the goal of providing personalized medication guidance for asthma patients.

2. The algorithm used by the report system is based on drug gene information from reliable sources such as CPIC, PharmGKB, expert consensus, diagnosis and treatment guidelines and drug instructions to establish a basic algorithm model, the algorithm model is further optimized by referring to the conventional use condition of clinical drugs, and the final algorithm is determined after a large amount of theoretical data analysis and experimental verification. The core of the method is that the curative effect of the therapeutic drug is digitally scored according to the genetic polymorphism carried by the patient, and personalized dose recommendation is provided for the patient to take the drug according to the scoring result. The algorithm analysis design is based on the influence of different gene polymorphisms on the curative effect of the medicine, and the recommendation of the medicine dosage is divided into 3 types: 1) the medicines can be used according to the medicine specification, namely the medicine has good curative effect on the tested genotype patients and low adverse reaction risk; 2) medicines need to be used under the guidance of doctors, namely the medicines have good curative effect on patients with the tested genotype but have certain probability of adverse reaction; 3) the use of drugs is avoided, namely the drug has no obvious curative effect on the tested genotype patients or the toxic risk is greater than the benefit of the curative effect.

3. It should be noted that although the report system of the present invention has taken into account the situations such as past medical history of the patient, clinical response of family members to the drug, etc. when analyzing the effect of genotype on the therapeutic effect of the drug, the specific medication needs to be combined with the actual situations of the patient such as past medical history, whether there is a surgical history, whether there is an allergic condition, etc. Therefore, the report provided by the invention is only responsible for the received sample, the correctness and accuracy of sample detection and analysis are ensured, and the specific medication guidance suggestion needs to consult the attending physicians.

Compared with the prior art, the invention has the advantages that:

(1) the invention provides a gene detection kit for personalized medicine for children asthma, which can provide accurate and reliable biological genetic information for clinicians by analyzing the influence of 31 genes and 41 gene polymorphism sites related to the children asthma on the curative effect and adverse reaction of children asthma medicines, and has the advantages of simplicity, convenience, high efficiency, low cost and the like.

(2) The invention comprises a report analysis system matched with gene detection, can accurately and effectively analyze a sequencing result, and provides reliable reference for the personalized medication guidance of the asthma of children.

(3) The invention adopts NGS technology to realize high-throughput, rapid and accurate detection of hundreds of different gene mutations, and can save time and cost for multiple detections compared with the existing detection kit which can only detect a single gene or dozens of genes at one time on the market.

(4) The gene detection method for the medicine for treating the asthma in children is mainly used for library construction and analysis reports, and by adopting the library construction method, a sequencing library covering gene loci of the medicine for treating the asthma in children can be simply and quickly constructed, and the mutation conditions of 31 related genes are detected. Meanwhile, the established report system can provide a treatment scheme of clinical children common antiasthmatic medicines such as salmeterol, glucocorticoid, salbutamol, terbutaline and the like according to the detection result and by combining the specific situation of the patient, and recommend a treatment scheme with high feasibility for the patient.

Drawings

FIG. 1 is a graph of library fragment distribution;

FIG. 2 is a graph showing the results of type GG at rs37973 site of GLCCI1 gene;

FIG. 3 is a graph showing the results of type AA at locus rs3824662 of the GATA3 gene;

FIG. 4 shows the result of AG type at rs7142143 of PYGL gene.

Detailed Description

The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative and not intended to limit the present invention, and all technical solutions similar or equivalent to the present invention are within the protection scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The specific linker is VAHTSTM AmpSeq Adapters for Illumina cargo number # NA 111; the Agencour AMPure XP magnetic bead is VAHTS^TMDNA Clean Beads cat # N411; digestion buffer, ligation buffer, ligase were purchased from VAHTS^TM(ii) a The PCR primer mixed solution is synthesized by Shanghai Czeri bioengineering GmbH; the DNA extraction kit is purchased from Tiangen Biochemical technology (Beijing) Co., Ltd., Cat number: DP318 Standard: 50/200 parts by weight; the Miseq reagent kit v3-500cycles sequencing reagent was purchased from Illumina, Inc., with the trade name MS-102-(ii) a The Miseq second generation sequencer was purchased from Illumina.

Experiment one: preparation of gene detection kit for personalized medicine for children asthma

1.1 the main components: primer mixed liquor, multiple PCR library amplification buffer solution, digestion buffer solution, connection buffer solution, ligase, a special-shaped joint, HiFi library amplification buffer solution, a positive/negative quality control product, a report system and an instruction book.

1.2 sources of materials: the joint being VAHTS^TMAmpSeq Adapters for Illumina cargo # NA 111; the magnetic beads are VAHTS^TMDNA Clean Beads cat # N411; digestion buffer, ligation buffer, ligase were all from VAHTS^TM。

1.3 matched instruments: general PCR instrument, Miseq second generation sequencer.

1.4 according to experimental groping, the amplification primers are designed SEQ NO.1-SEQ NO.82 primers, the primers are synthesized by Shanghai Jieli bioengineering GmbH, each primer is prepared into 10 mu M working solution by using sterilized purified water, the signal value of each primer in PCR amplification reaction is used as the concentration of each primer in a primer mixture to determine an index, and according to the experimental groping, the primers are mixed into mixed solution with the concentration of each primer of 0.01-0.05 mu M.

1.5. Sample requirements: the invention is suitable for genome DNA extracted from samples such as blood samples, oral swabs, dried blood slices and the like; the DNA content of the detected sample is 1-100 ng; frozen DNA samples should be stored below-20 ℃ and repeated freezing and thawing is avoided.

FIG. 1 shows the distribution of the library fragments constructed according to the present invention, wherein the numerical labels in FIG. 1 from left to right are: 69. 94, 113, 119, 131, 141, 159, 165, 172, 179, 194, 212, 217, 236, 242, 267, 284, 291, 322, 338, 415, 440, 474, 532, 730, 1000, > 5 k. As can be seen from FIG. 1, the library fragments exhibit multiple peaks in the range of 190bp to 350bp, which proves that the samples meet the requirements.

1.6 description of quality control products: the positive quality control substance (Hela cell DNA) can detect corresponding genotype, and the negative quality control substance (H) is used as a control₂O) no signal detection.

Experiment two: 2 blood samples were tested using the kit and reporter system of the present invention

The invention can be used for detecting various samples such as blood, oral swab, dried blood sheet and the like, and 2 anticoagulation samples are taken as examples to explain the specific use of the reagent kit in the experiment I.

2.1 sample DNA extraction

1-2ml of 2 anticoagulated blood samples were collected and subjected to DNA extraction using a blood genome DNA extraction kit (centrifugal column type) manufactured by Tiangen Biochemical technology (Beijing) Co., Ltd. (product number: DP318 standard: 50/200 parts) in accordance with the procedures described in the specification.

The concentration (more than or equal to 1 ng/mu L) and the purity (1.6-2.0) of the extracted DNA are measured by using an ultramicro ultraviolet spectrophotometer.

2.2 library construction

And (3) adopting a kit in the first experiment to construct a library for qualified sample DNA in quality inspection, and specifically comprising the following steps:

2.2.1 the library building reagent composition of the present invention, as shown in Table 2 below:

TABLE 2 library-establishing reagent composition table of the present invention

The joint being VAHTS^TMAmpSeq Adapters for Illumina cargo number NA 121;

the magnetic bead is VAHTSTM DNA Clean Beads cargo number # N411;

2.2.2 library construction procedure was as follows:

(1) PCR amplification reaction

Preparing a multiple PCR reaction mixed solution according to the following reaction conditions:

10 mu L of primer mixed solution, 4 mu L of multiplex PCR amplification buffer solution, 1-100 ng of template DNA, and supplementing sterile ultrapure water to 20 mu L of total reaction system.

PCR amplification was performed under the following program conditions to obtain PCR products:

99 deg.C, 2min, (99 deg.C, 15s, 60 deg.C, 4min)28 cycles, 72 deg.C, 10min, 4 deg.C hold.

(2) Digestion of partial primer sequences

After amplification is completed, a digestion buffer is added to the PCR product in the previous step, which is as follows:

20. mu.L of PCR product and 2. mu.L of digestion buffer.

Digestion was performed according to the following procedure:

50℃10min，55℃10min，60℃20min，10℃hold。

(3) joint connection

The reaction mixture was prepared as follows:

mu.L of the digestion product in the previous step, 6. mu.L of ligation buffer, 1. mu.L of linker, ligase

mu.L, 30. mu.L in total.

Ligation was performed as follows:

22℃30min，72℃10min，10℃hold。

(4) library purification

a. Prepared before purification, VAHTSTM DNA Clean Beads were vortexed and equilibrated to room temperature. Sufficient fresh 80% ethanol was prepared, requiring about 400 μ L per sample. Before this purification step, the sample was filled to a volume of 60. mu.l with sterile water.

b. Vortex the magnetic beads to mix well, add 60 μ L (1 ×) magnetic beads to the PCR reaction system, and gently blow 10 times with a pipette to ensure the uniformity of the whole system. The library was bound to magnetic beads by incubation at room temperature for 8 min.

c. The reaction tube was briefly centrifuged and placed on a magnetic rack to separate the beads from the liquid.

d. The PCR tube was kept on the magnetic rack and after the solution was clarified (about 5min), the supernatant was carefully discarded, taking care not to disturb the beads.

e. Keep the PCR tube on the magnetic frame, add 200. mu.L freshly prepared 80% ethanol, take care not to disturb the beads when adding ethanol, and carefully remove the supernatant after 30sec of incubation.

f. Repeat step e, rinse twice altogether.

g. The samples were collected to the bottom of the PCR tube by brief centrifugation and placed on a magnetic rack for 30sec, and any residual ethanol was aspirated off with a pipette. And opening the cover and drying in air for 3-5 min.

h. After the magnetic beads were air-dried, the PCR tube was removed from the magnetic stand, 22. mu.L of enzyme-free water was added to cover the magnetic beads, and the magnetic beads were pipetted and mixed well.

i. Incubate at room temperature for 2 min. If the magnetic beads are dry and cracked, the incubation time is suitably prolonged.

j. The PCR tube was collected by brief centrifugation and placed in a magnetic rack, and the beads and liquid were separated until the solution was clear.

k. Carefully pipette 20. mu.L of the supernatant into a new EP tube.

(5) Library amplification:

preparing reaction mixed solution, performing library PCR amplification:

20 mu L of purified library, 25 mu L of HiFi library amplification buffer solution, 5 mu L of library primer mixture, and amplification reaction conditions:

95 ℃ for 3min, (98 ℃ for 20s, 60 ℃ for 15s, 72 ℃ for 30s)5 cycles, 72 ℃ for 10min, 4 ℃ hold.

(6) Library purification:

purification was performed using 120. mu.L (1.2X) of magnetic beads, as in the previous purification step.

(7) And (4) performing quality inspection and quality evaluation on the library.

Using fragment analysis instruments

GX Touch^TMAnalyzing the fragments, and performing fragment quality inspection on the constructed library. The quality test results are shown in FIG. 1: the library fragments are multimodal in the range of 190bp-350 bp. The results show that the library has neither small fragment linkers nor large fragment tailing peaks.

(8) Sequencing library mixing

And quantifying each library by using a qubit respectively, diluting each library according to the dilution denaturation requirement of the library before Miseq sequencing according to the quantification result, wherein the total volume is 20 mu L after mixing, and the final concentration is in the range of 30-60pM so as to ensure the template content balance from each sample in a template mixture for subsequent sequencing.

2.3 sequencing and data analysis

The Miseq reagent kit v2-500cycles sequencing reagent is a product of Illumina, and the catalog number is MS-102-2003. The Miseq second generation sequencer is a product of Illumina. The same library was sequenced using PE 250.

And after the sequencing is finished, the instrument automatically generates a fastq file, and the data analysis is carried out on the fastq file by using the STRait Razor 2.6 software. BAM files were analyzed with IGV _2.3.72 and sequencing data for each locus was visualized by Integrated Genome Viewer (IGV) v 2.3.72. Data processing and BAM and BAI creation tools were performed using SAMtools and PICARD. Data extraction and variant calling was performed with GATK. Microsoft Excel and RStudio v1.2.1335 for data processing and statistical analysis.

And (3) data analysis: the sample sequencing depth is more than 500X, Q30 is more than 90%, the coverage rate is more than 100%, and the quality control requirement is met. The results of the sample gene mutation analysis are shown in table 4 below:

TABLE 3 table of analysis results of sample gene mutation

Experiment three: 20 clinical samples are tested by using the kit and the report system

In this case, 20 blood samples of known genotypes were tested using a pilot kit.

The specific operation steps are the same as those of experiment two: the sequencing depth of the sample is more than 500X, Q30 is more than 90%, the coverage rate is 100%, and the quality control requirement is met.

Sequencing the detection sites of the genes ABCB1, BMP7 and CRHR1, as shown in FIG. 2, FIG. 3 and FIG. 4, each sequencing map reflects that this site has only one peak, indicating homozygosity; if the locus is heterozygous, 2 peaks are corresponding to the locus; wherein, fig. 2 is a sequencing chart of rs1045642 of the sample 2, fig. 3 is a sequencing chart of rs79085477 of the sample 9, and fig. 3 is a sequencing chart of rs7142143 of the sample 11.

According to the analysis result, an experimental conclusion can be obtained: in 20 samples, ABCB 1rs 1045642 heterozygous mutation 4 cases and homozygous mutation 2 cases; BMP7 rs79085477 heterozygous mutation case 1, homozygous mutation case 1; 3 cases of CRHR 1rs 1876828 heterozygous mutation and 2 cases of homozygous mutation; 1 case of FKBP5 rs4713916 heterozygous mutation and 3 cases of homozygous mutation; 2 heterozygous mutations of DROSHA rs 639172; GLCCI1rs37973 heterozygous mutations in 3 cases, and homozygous mutations in 1 case; 1 example of PYGL rs7142143 heterozygous mutation, 1 example of homozygous mutation; TBXT rs2305089 heterozygous mutation 3 cases, homozygous mutation 2 cases, and the same as Sanger sequencing method.

Experiment four: 1 case of drug recommendation by using the kit and the report system

3.1 case background

Lijiang, male, born in 2018 in 9 months, and visited a doctor in a hospital before 24 months in 2018 in 9 months because of repeated asthma symptoms, and the disease is diagnosed as bronchiolitis, intravenous drip cephalosporin, atomization and the like, and the disease is cured in one week. Asthma attacks again in 12 months in 2018, the asthma attacks are more serious than the asthma attacks in the first time, and the intravenous drip cephalosporins are cured for one week. The third asthma attack in 7 months in 2019 smells the wheezing sound, and the treatment with prednisone and glucocorticoid improves one week. Similar cases occur 2 times in 2 months in 2020, and the treatment is similar, asthma is treated, but asthma is not treated. Asthma appears due to cold in 5 months in 2020, outpatient cephalosporin and triple atomization (Link Shu, Borikoni and Eletrol) do not relieve symptoms, and severe attack at night is seen again in hospital. Physical examination data: breathing 50 times/min, heart rate 136 times/min, body temperature 36.5 deg.C, and wheezing sound of both lungs; electrocardiographic monitoring SO 292%, WBC and CRP are in normal range, lung slice: mild emphysema. The preliminary diagnosis is: acute attack of bronchial asthma. The clinician uses salbutamol and cephalosporin to treat asthma, but the sick children have nausea, slight emesis, dysphoria and other adverse reactions.

3.2 results of the assay

And (3) carrying out gene detection analysis on the patient by adopting the kit in the first experiment, and carrying out the specific steps of the second experiment. Wherein, the salbutamol scoring standard is as follows: response score: high: not less than 7; the method comprises the following steps: 4-6; low: less than or equal to 3; risk scoring: high: not less than 5; the method comprises the following steps: 3-4; low: less than or equal to 2. The following scoring table was obtained:

TABLE 4 salbutamol drug Gene detection assay

3.3 report analysis

The detection result shows that the genotype of the infant patient scores 4 points for the overall response capability of the infant patient to the salbutamol and 3 points for the overall risk occurrence probability, and the analysis is carried out according to the grading standard of the salbutamol: the genotype infant patient has general response to salbutamol and has the probability of generating adverse reactions with intermediate risk, belongs to the medicines which need to be avoided, and recommends the treatment of changing the medicine.

Finally, it should be noted that the above-described embodiments are described to facilitate understanding and use of the invention by those of ordinary skill in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Sequence listing

<110> Guangzhou-in-one Biotechnology Ltd

<120> gene detection kit for personalized medicine for children asthma and application thereof

<130> 2021.05.14

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<213> rs1876828-R

<400> 24

cactgtggga gtggacag 18

<210> 25

<211> 27

<212> DNA

<213> rs2032582-F

<400> 25

gcagtaggga gtaacaaaat aacactg 27

<210> 26

<211> 28

<212> DNA

<213> rs2032582-R

<400> 26

gaaatgaaaa tgttgtctgg acaagcac 28

<210> 27

<211> 32

<212> DNA

<213> rs2229109-F

<400> 27

atttatcact gtaccttaac ttcttttcga ga 32

<210> 28

<211> 28

<212> DNA

<213> rs2229109-R

<400> 28

ctgtttagaa gccaagtatt gacagcta 28

<210> 29

<211> 18

<212> DNA

<213> rs2240017-F

<400> 29

gcatcgtgga gccgggtt 18

<210> 30

<211> 16

<212> DNA

<213> rs2240017-R

<400> 30

cgctcgtccg cgtcct 16

<210> 31

<211> 24

<212> DNA

<213> rs2241843-F

<400> 31

gagacacggc ttttcctttt actc 24

<210> 32

<211> 18

<212> DNA

<213> rs2241843-R

<400> 32

agggaatcgt gcggatgg 18

<210> 33

<211> 23

<212> DNA

<213> rs2267715-F

<400> 33

gacctgagtt tccggattta cag 23

<210> 34

<211> 18

<212> DNA

<213> rs2267715-R

<400> 34

ggtcttggag cagttggg 18

<210> 35

<211> 23

<212> DNA

<213> rs2284220-F

<400> 35

caggttgtgt agagaacacc caa 23

<210> 36

<211> 25

<212> DNA

<213> rs2284220-R

<400> 36

gagccatctt acccagagtt atttc 25

<210> 37

<211> 23

<212> DNA

<213> rs2305089-F

<400> 37

cttctcacct cctcgttctg ata 23

<210> 38

<211> 25

<212> DNA

<213> rs2305089-R

<400> 38

gctgaactcc ttgcataagt atgag 25

<210> 39

<211> 31

<212> DNA

<213> rs2523864-F

<400> 39

ttacatacat acgtttccta gctctaactt c 31

<210> 40

<211> 26

<212> DNA

<213> rs2523864-R

<400> 40

cagagctctg gctcctcttt gtatta 26

<210> 41

<211> 21

<212> DNA

<213> rs255100-F

<400> 41

ggtcatgatg gtgtggcagt a 21

<210> 42

<211> 20

<212> DNA

<213> rs255100-R

<400> 42

caggcactgt caatggctag 20

<210> 43

<211> 27

<212> DNA

<213> rs37973-F

<400> 43

cctgctattc agtgttattg tcttgga 27

<210> 44

<211> 26

<212> DNA

<213> rs37973-R

<400> 44

gaacttctgg tgatcaggag aaatgt 26

<210> 45

<211> 27

<212> DNA

<213> rs3824662-F

<400> 45

ctttttcaag aactgagaag agccgtt 27

<210> 46

<211> 26

<212> DNA

<213> rs3824662-R

<400> 46

cattccaaaa gattcttagc ctaggg 26

<210> 47

<211> 20

<212> DNA

<213> rs3828743-F

<400> 47

cccccaacaa ctcggatctg 20

<210> 48

<211> 18

<212> DNA

<213> rs3828743-R

<400> 48

aggcgacaca ggtgatgg 18

<210> 49

<211> 25

<212> DNA

<213> rs3873352-F

<400> 49

cagggtggaa aaagtttgag agaag 25

<210> 50

<211> 21

<212> DNA

<213> rs3873352-R

<400> 50

ctgaacacct caggagaagc a 21

<210> 51

<211> 21

<212> DNA

<213> rs3957357-F

<400> 51

ctggctcgac aactgaattc c 21

<210> 52

<211> 18

<212> DNA

<213> rs3957357-R

<400> 52

tccagtaggt ggcccctt 18

<210> 53

<211> 29

<212> DNA

<213> rs4135385-F

<400> 53

ctaagcattt gtgtaatgtt ggagttact 29

<210> 54

<211> 26

<212> DNA

<213> rs4135385-R

<400> 54

tcagcagtct agataaacat gcacaa 26

<210> 55

<211> 27

<212> DNA

<213> rs4553808-F

<400> 55

cactctatca tgatcatggg tttagct 27

<210> 56

<211> 26

<212> DNA

<213> rs4553808-R

<400> 56

taacaaccta atgggcactt cctaat 26

<210> 57

<211> 24

<212> DNA

<213> rs4713916-F

<400> 57

caaccctaac ctctctggac tcct 24

<210> 58

<211> 26

<212> DNA

<213> rs4713916-R

<400> 58

ttgtagagat tatttaatca ttcagt 26

<210> 59

<211> 23

<212> DNA

<213> rs6092-F

<400> 59

ctttccattg ctctaggatg cag 23

<210> 60

<211> 18

<212> DNA

<213> rs6092-R

<400> 60

ctcaccccga agtctgag 18

<210> 61

<211> 21

<212> DNA

<213> rs639174-F

<400> 61

tttcctgact tcttctagtc t 21

<210> 62

<211> 22

<212> DNA

<213> rs639174-R

<400> 62

gggcaacaga gcaagactcc tt 22

<210> 63

<211> 18

<212> DNA

<213> rs6988229-F

<400> 63

actcccaccc tcagtcgt 18

<210> 64

<211> 20

<212> DNA

<213> rs6988229-R

<400> 64

tctccggatg tgtggtcatc 20

<210> 65

<211> 18

<212> DNA

<213> rs7142143-F

<400> 65

ccagcctgcg taacagga 18

<210> 66

<211> 28

<212> DNA

<213> rs7142143-R

<400> 66

atatcagctg aaaataacat ctctccct 28

<210> 67

<211> 23

<212> DNA

<213> rs7160796-F

<400> 67

gttagtacaa agcttgcgcc tga 23

<210> 68

<211> 24

<212> DNA

<213> rs7160796-R

<400> 68

gcatgcctat aatcccagca gtta 24

<210> 69

<211> 30

<212> DNA

<213> rs724710-F

<400> 69

ttttgttttg ttttgttctg atgcagcttc 30

<210> 70

<211> 28

<212> DNA

<213> rs724710-R

<400> 70

aagaaaacat cattaccctc cttgcata 28

<210> 71

<211> 27

<212> DNA

<213> rs738409-F

<400> 71

gaaggatcag gaaaattaaa agggtgc 27

<210> 72

<211> 21

<212> DNA

<213> rs738409-R

<400> 72

cttaccacgc ctctgaagga a 21

<210> 73

<211> 32

<212> DNA

<213> rs7772821-F

<400> 73

ttttacccat ggtttaggaa agcaataaaa gt 32

<210> 74

<211> 31

<212> DNA

<213> rs7772821-R

<400> 74

cttggtaatt ttaaaggtat cctgaacttc g 31

<210> 75

<211> 27

<212> DNA

<213> rs7793837-F

<400> 75

ttcagttttc tcacaggtat gtacatc 27

<210> 76

<211> 27

<212> DNA

<213> rs7793837-R

<400> 76

ctcaagtccc caaattgata atgacct 27

<210> 77

<211> 23

<212> DNA

<213> rs79085477-F

<400> 77

gaaaccctgt tctagaacag aag 23

<210> 78

<211> 25

<212> DNA

<213> rs79085477-R

<400> 78

tcacattcag tccctcactc attta 25

<210> 79

<211> 17

<212> DNA

<213> rs881152-F

<400> 79

gaggctgacc tccggga 17

<210> 80

<211> 20

<212> DNA

<213> rs881152-R

<400> 80

ggaagcccct ttcggttcag 20

<210> 81

<211> 26

<212> DNA

<213> rs967676-F

<400> 81

gagtaatagt ttggcacttc tctctt 26

<210> 82

<211> 25

<212> DNA

<213> rs967676-R

<400> 82

agacctgcac tgtacacata aaatc 25

<210> 83

<211> 70

<212> DNA

<213> sequence of index p5 (index p5 sequence)

<400> 83

aatgatacgg cgaccaccga gatctacacn nnnnnnnaca ctctttccct acacgacgct 60

cttccgatct 70

<210> 84

<211> 66

<212> DNA

<213> sequence of index p7 (index p7 sequence)

<400> 84

caagcagaag acggcatacg agatnnnnnn nngtgactgg agttcagacg tgtgctcttc 60

cgatct 66

Claims (10)

1. A gene detection kit for personalized medicine for childhood asthma is characterized by comprising the following components: primer mixed liquor, multiplex PCR library amplification buffer solution, digestion buffer solution, connection buffer solution, ligase, specific joint, HiFi library amplification buffer solution, positive/negative quality control product, report system and instruction book.

2. The gene detection kit of claim 1, wherein the library primer mixture contains 31 genes and 41 gene polymorphic sites.

3. The gene detection kit of claim 2, wherein the 31 genes are ABCB1, ADRB2, BCL2L11, BMP7, CA10, CLOCK, COL22A1, CRHR1, CRHR2, CTLA4, CTNNB1, CXCL12, DCAF4, DOK5, DROSHA, TSPYL1, DUSP1, FKBP5, GATA3, GLCCI1, GSTA1, HCG22, HDAC1, LINC00251, PNPLA3, GL, SERPINE1, ST13, TAAR6, TBX21, PYTBXT.

4. The gene detection kit of claim 2, wherein the primer information for amplifying 41 gene polymorphic sites of the 31 genes is shown in SEQ ID No.1 to SEQ ID No.82, and each primer is mixed in equal volume to form a library primer mixture.

5. The gene detection kit according to claim 1, wherein the specific linkers are index p5 and index p 7; the sequence of the index p5 is shown as SEQ ID NO. 83; the sequence of the index p7 is shown in SEQ ID NO. 84.

6. The gene assaying kit according to claim 1, wherein the reporting system is implemented by setting a system in a computer to complete the entire reporting process in a mode of receiving the genotype of the patient and outputting the adjustment recommendation table of the therapeutic agent after the genotype is analyzed according to the system setting algorithm.

7. The gene detection kit of claim 6, wherein the process of setting up the system in the computer is to digitize the effects of different genotypes of a single gene on the "response" and the "adverse reaction risk" of a drug, analyze the response and the adverse reaction of the drug in turn to obtain the response and the adverse reaction score of a patient to the drug, and predict the medication condition of the patient by synthesizing the response and the adverse reaction score of the drug.

8. The gene detection kit of claim 6, wherein the algorithm is established based on the drug gene information of CPIC, PharmGKB, expert consensus, medical guidelines, and drug instructions.

9. The use of the kit of claim 1 for the detection of the administration of a personalized gene related drug in childhood asthma, wherein the detection process comprises:

s1, designing a multiple PCR amplification primer according to the polymorphism sequence of the related gene locus of the children asthma treatment medicine;

s2, preparing a library construction kit;

s3, extracting genome DNA from samples such as blood samples, buccal swabs, dried blood slices and the like;

s4, constructing a library for sequencing by using the genome DNA of the sample to be tested;

s5, performing high-throughput sequencing on the qualified gene library obtained in the step S4 to obtain sequencing data;

s6, analyzing the sequencing data result obtained in the step S5, and providing a detection report by combining a report system.

10. The use according to claim 9, wherein the gene-related drug is a childhood antiasthmatic drug comprising salmeterol, a corticosteroid, salbutamol, terbutaline.

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