WO2020177433A1 - 一种用于指导人精神疾病用药的试剂盒及其检测方法 - Google Patents

一种用于指导人精神疾病用药的试剂盒及其检测方法 Download PDF

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WO2020177433A1
WO2020177433A1 PCT/CN2019/124304 CN2019124304W WO2020177433A1 WO 2020177433 A1 WO2020177433 A1 WO 2020177433A1 CN 2019124304 W CN2019124304 W CN 2019124304W WO 2020177433 A1 WO2020177433 A1 WO 2020177433A1
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seq
kit
extension
primers
sap
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PCT/CN2019/124304
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French (fr)
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何熲
石忆湘
魏宁
黄成琛
何宗顺
顾孝平
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上海康黎医学检验所有限公司
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6872—Methods for sequencing involving mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of reagent kits, and more specifically to a reagent kit for guiding the medication of human mental diseases and a detection method thereof.
  • the selection and dosage adjustment of drugs are guided through genetic testing of drug efficacy and adverse reactions to achieve the purpose of individualized treatment. It is particularly important for clinicians to choose drugs, dose adjustments, clinical monitoring and reasonable control of adverse reactions.
  • FDA U.S. Food and Drug Administration
  • more than 200 drugs have been recommended for pharmacogenomic biomarker testing.
  • the top three are anti-tumor drugs, psychotropic drugs, and Cardiovascular drugs.
  • a large number of clinical data have also shown the importance and necessity of individualized pharmacy in psychiatric departments.
  • drug-related genetic testing for psychotropic drugs has not been widely used in clinical practice.
  • pharmacogenomics With the establishment of a new clinical model, pharmacogenomics will play an increasingly important role in guiding clinical rational use of drugs, and its clinical application and popularization will become a trend.
  • the cost and time period of detection cannot meet clinical needs: the cost of whole-genome resequencing is about 10,000 yuan per sample, and the whole exome Sequencing each sample costs 3000-4000 yuan, because there are many gene loci detected and subsequent biological information analysis takes a long time, so it is basically impossible to get results within two months for these two technologies, and the clinical spirit Genetic testing of diseases requires timely feedback. Doctors and patients hope to get the results within a week at most. Although the genotyping technology based on fluorescence quantitative PCR technology can meet the timeliness, the throughput is low.
  • the purpose of the present invention is to provide a kit for guiding the medication of human mental illness and its detection method, so as to solve the problem of the high accuracy of the whole genome resequencing and whole exome sequencing technology in the prior art, but the detection cost Higher, longer time period, so there is still a lack of appropriate products for genetic testing and medication guidance for mental diseases.
  • the present invention adopts the following technical solutions:
  • kits A for guiding the medication of human mental illness can simultaneously type 16 gene loci, and the kit A includes a kit for amplifying 16 16 pairs of amplification primers for each gene fragment, the sequences of the 16 pairs of amplification primers SEQ ID No. 1 to 32 are as follows:
  • the kit A also includes 16 extension primers for identifying mutations of these 16 gene fragments, and the sequence of the extension primers SEQ ID No. 33 to 48 is specifically as follows:
  • reaction system of PCR amplification is as follows:
  • reaction system extended by iPLEX is as follows:
  • the kit further includes a SAP reaction system, and the SAP reaction system is as follows:
  • the molar concentrations of the 16 pairs of amplification primers in the amplification primer mixture are equivalent, that is, the amount of each amplification primer is substantially equal.
  • the molar concentration ratio of the 16 extension primers is preferably: SEQ ID No. 33: SEQ ID No. 34: SEQ ID No. 35: SEQ ID No. 36: SEQ ID No .37: SEQ ID No. 38: SEQ ID No. 39: SEQ ID No. 40: SEQ ID No. 41: SEQ ID No. 42: SEQ ID No. 43: SEQ ID No. 44: SEQ ID No.
  • kits B for guiding the medication of human mental illness.
  • the kit B can simultaneously type 16 gene loci, and the kit B includes 16 pairs of amplification primers with 16 gene fragments added, the sequences of the 16 pairs of amplification primers are as follows:
  • the kit B also includes 16 extension primers, and the sequence of the extension primers is as follows:
  • SNP_ID Extension primer rs1041983 No.33:CAATGTTAGGAGGGTATTTTTA rs1058164 No.34:GAAGAGAGCAGAGGCGCTTCTCCGT rs1065852 No.35:TGGGCTGCACGCTAC rs1079597 No.55:CCCCGTGCTGTCAGAATCACCTATTC rs1135840 No.56:GGTTTGCTTTCCTGGTGA rs1360780 No.38:GCTTTCACATAAGCAAAGTTA rs1799930 No.39:GACTCAAAATCTTCAATTGTT rs1799978 No.
  • this kit B is based on kit A, and further uses SEQ ID No. 49 to replace SEQ ID No. 3, SEQ ID No. 50 to replace SEQ ID No. 4, and SEQ ID No. 51 to replace SEQ ID No. 17, SEQ ID No. 52 replaces SEQ ID No. 18, SEQ ID No. 53 replaces SEQ ID No. 21, SEQ ID No. 54 replaces SEQ ID No. 22, SEQ ID No. 55 replaces SEQ ID No. 36, SEQ ID No. 56 replaces SEQ ID No. 37.
  • the kit B can basically achieve similar typing effect by replacing part of the primer sequence on the basis of kit A, and the interference between the primers is also very low, and it also has good performance. Application prospects.
  • the molar concentration ratio of the 16 extension primers is preferably: SEQ ID No. 33: SEQ ID No. 34: SEQ ID No. 35: SEQ ID No. 55: SEQ ID No .56: SEQ ID No. 38: SEQ ID No. 39: SEQ ID No. 40: SEQ ID No. 41: SEQ ID No. 42: SEQ ID No. 43: SEQ ID No. 44: SEQ ID No.
  • kits provided by the present invention it is preferable to use a time-of-flight mass spectrometer for detection.
  • the present invention preferably adopts MassArray introduced by Agena, a time-of-flight mass spectrometer, which ensures the accuracy and sensitivity of detection results, is simple and feasible, and its advantages are as follows:
  • Strict quality control, mass spectrometry technology is a "one-tube operation", that is, the reaction system always reacts in one test tube during the biochemical experiment, avoiding human errors caused by multiple transfers.
  • a detection method of the kit is not used for the diagnosis and treatment of diseases, but is only used for individual identification.
  • the specific steps of the detection method are as follows: 1) PCR amplification : Configure the PCR reaction mixture according to the above PCR amplification reaction system, divide it into 96-well plates, take DNA samples into the 96-well plates, and put them into the thermal cycler.
  • the amplification procedure is as follows: denature at 95°C for 2 minutes; 95 Denaturation at °C for 30s, annealing at 56°C for 30s, extension at 72°C for 60s, a total of 45 cycles; the last extension at 72°C for 5min; 2) SAP reaction: configure SAP reaction mixture, the reaction system is as follows: SAP buffer 0.17 ⁇ L, SAP enzyme 0.5U , Make up to 2 ⁇ L of pure water; add 2 ⁇ L of SAP reaction mixture to each well of step 1) and put it into the thermal cycler.
  • the amplification procedure is as follows: 37°C, 40min; 85°C, 5min; 4°C incubation ; 3) Extension reaction: Take out the SAP reaction plate and centrifuge at 2000 rpm for 1 to 2 minutes; configure the iPLEX extension reaction solution according to the above iPLEX extension reaction system, take 2 ⁇ L iPLEX extension reaction solution and add them to the centrifuged well plates, and put them into the amplification
  • the instrument performs the following thermal cycle to accurately type 16 gene loci at the same time.
  • the thermal cycle procedure is as follows:
  • kit A and kit B are provided.
  • the 16 loci combinations used are the inventors read through a large amount of literature, collect evidence, rank the evidence, and then combine the genotypes in the self-built Chinese population gene mutation database The frequency data is finalized.
  • the 16 loci we selected are not only supported by abundant clinical trial evidence, but also suitable for the genetic characteristics of the Chinese population (the loci are in The mutation frequency in the Chinese population is above 3%).
  • the sites involved in the present invention include CYP2D6*10 (including rs1135840, rs1065852, and rs1058164 three sites), CYP2C19*2 (rs4244285 site), CYP1A2*1F (rs762551 site), CYP1A2*1C (rs2069514) Locus), CYP3A5*3 (rs776746 locus), NAT2*6 (rs1799930 locus), NAT2*13 (rs1041983 locus), DRD2 (rs1079597 locus), DRD2 (rs1799978 locus), DRD2 (rs1800497 locus) ), MC4R (rs489693), UGT2B15*2 (rs1902023 locus), FKBP5 (rs1360780 locus), and HTR2A (rs7997012 locus).
  • CYP2D6*10 including rs1135840, r
  • SSRI serotonin reuptake inhibitors
  • tricyclic psychotropic drugs are metabolized through CYP2D6 or mainly through CYP2D6.
  • CYP2D6*10 is a frequent mutation in the Chinese population that leads to weakened activity. The weakening of CYP2D6 activity will increase the blood concentration and peak value of drugs metabolized by CYP2D6, which may affect its therapeutic effect and toxic side effects.
  • Part of SSRI and tricyclic psychotropic drugs are metabolized by CYP2C19 or partly metabolized by CYP2C19.
  • CYP2C19*2 is a common mutation with reduced activity in the Chinese population.
  • CYP1A2*1F and CYP1A2*1C are important mutation types in East Asian populations. CYP1A2*1F mutation will increase the metabolic activity of CYP1A2 enzyme, while CYP1A2*1C will weaken the metabolic activity of CYP1A2. The change of CYP1A2 activity will change the blood concentration state of the drugs metabolized by it.
  • the anti-anxiety drug alprazolam is mainly metabolized by CYP3A5, and CYP3A5*3 is the main inactive mutation in the East Asian population.
  • the loss of CYP3A5 activity significantly affects the metabolism of alprazolam and changes its plasma concentration.
  • NAT2 is N-acetyltransferase, an important metabolic enzyme of clonazepam. Mutations in NAT2*6 and *13 cause the clonazepam metabolism to be significantly slowed down, thereby affecting the therapeutic effect and side effects of clonazepam.
  • DRD2 is a dopamine receptor and the target of many anti-schizophrenia drugs.
  • the polymorphisms of Rs1079597, rs1800497 and rs1799978 can affect the efficacy of a variety of anti-schizophrenia and related treatment drugs.
  • MC4R is a melanocyte stimulating hormone receptor, which plays an important role in controlling weight gain. Weight gain is a common side effect of second-generation anti-schizophrenia drugs, and the single nucleotide polymorphism site rs489693 of MC4R is associated with the weight gain side-effects of multiple anti-schizophrenics. Its polymorphism can help predict schizophrenia drugs. Side effects of weight gain.
  • UGT2B15 is the main metabolizing enzyme of benzodiazepines.
  • UGT2B15*2 mutation leads to reduced enzyme activity, which will slow down the metabolism of lorazepam, oxazepam and other drugs.
  • FKBP5 is an immunophilic protein that can affect the therapeutic effects of a variety of SSRI drugs.
  • the rs1360780 polymorphism of FKBP5 can help predict the efficacy of multiple SSRI drugs.
  • HTR2A is the serotonin receptor and the target of many SSRI drugs. A number of studies have shown that the polymorphism of HTR2A rs7997012 can help predict the therapeutic effect of SSRI drugs. Therefore, the accurate typing results of the present invention for these 16 gene loci will provide an important basis for doctors' clinical individualized medication.
  • test results obtained according to this kit only represent CYP2D6*10 (including rs1135840, rs1065852, and rs1058164 three sites), CYP2C19*2 (rs4244285 site), CYP1A2*1F (rs762551 site), CYP1A2 *1C (rs2069514 locus), CYP3A5*3 (rs776746 locus), NAT2*6 (rs1799930 locus), NAT2*13 (rs1041983 locus), DRD2 (rs1079597 locus), DRD2 (rs1799978 locus), DRD2 (rs1800497 locus), MC4R (rs489693), UGT2B15*2 (rs1902023 locus), FKBP5 (rs1360780 locus), HTR2A (rs7997012 locus) these 16 gene locus typing results, for the doctor's clinical personalized medicine for reference.
  • the inventors After the 16 gene locus combinations were determined, the inventors further designed special primers for each gene locus, and then tried different primer combinations several times, and finally selected that most samples can reach 16 loci accurately. At the same time, the blank control has no primer combination with abnormal extension, and finally kit A with very significant application effect is obtained.
  • the ability to accurately type 16 sites at the same time without abnormal extension is very important for obtaining a qualified kit, and it is two requirements that must be met at the same time.
  • kit A After further research on kit A, the inventor found that, on the basis of being basically the same as kit A, by only replacing the amplification primers of three gene loci in kit A, and two genes The extension primers at the sites were replaced, and a kit B was obtained.
  • kit B can also accurately type most samples at 16 sites, and the blank control did not exhibit abnormal extensions. Surprisingly, The typing effect of kit B is slightly better than kit A.
  • kits are complementary and indispensable. If they are replaced at will, it is likely to cause huge differences in typing effects. In fact, Any replacement primer sequence does not necessarily achieve the same good typing effect, and unexpected extensions may occur, and these results are unwilling to be seen by those skilled in the art. In fact, the inventors of this application have conducted research on the theoretical knowledge recorded in scientific and technological literature, and through repeated experimental exploration and verification by themselves, finally obtained the kit A, which has good typing effect and clinical guidance effect. B.
  • kit A and kit B have substantially the same primer sequence, and use the same working principle to achieve accurate typing of the 16 sites adopted in the present invention, kit A and kit B should belong to the same The inventive concept has the same specific technical features and solves the same technical problems, and should have unity.
  • the technology used in this kit has 16 times the throughput of fluorescent quantitative PCR, which can solve the efficiency problem when the sample volume is large.
  • the detection cost for 16 sites is about 3,000 yuan (including labor, reagents, consumables, and instrument losses), and the detection cost of this kit can be controlled within 1,000.
  • the present invention performs genotyping based on time-of-flight mass spectrometry technology. It is to perform mid-throughput sequencing on carefully selected mutation sites of some genes that are most relevant to psychiatric medications, thus greatly reducing the time period and detection cost, and still Obtain highly reliable test results, so that the technology has practical value in clinical use.
  • the typing based on multiplex PCR and mass spectrometry sequencing technology can accurately identify the base of the mutation position, so its typing accuracy is compared with the typing based on non-sequencing technology It has been greatly improved.
  • the kit and detection method provided by the present invention greatly reduce the experimental cost and cycle, so Has very important practical application value.
  • Figure 1 is a typing effect diagram of 15 human DNA samples detected using the kit A provided by the present invention, in which, except for the bottom hole on the right side which is NTC, the remaining holes are from left to right and from top to bottom. Samples 1-15;
  • Figure 2 is a graph of the overall typing results of NTC using kit A provided by the present invention.
  • Figure 3 is a typing effect diagram of 15 human DNA samples detected using the kit B provided by the present invention, in which, except for the bottom hole on the right side which is NTC, the remaining holes are from left to right and from top to bottom. Samples 1-15;
  • Figure 4 is a graph of the overall typing results of NTC using kit B provided by the present invention.
  • Figure 5 is a typing effect diagram of 15 human DNA samples using the primer combination C provided by the present invention, in which, except for the bottom hole on the right side which is NTC, the other holes are from left to right and from top to bottom. Samples 1-15;
  • Figure 6 is a graph showing the overall typing results of NTC using the primer combination C provided by the present invention.
  • Fig. 7 is a typing effect diagram of 15 human DNA samples detected by using the primer combination D provided by the present invention, in which, except for the bottom hole on the right side which is NTC, the remaining holes are from left to right and from top to bottom. Samples 1-15;
  • Figure 8 is a graph showing the typing results of the entire NTC using the primer combination D provided by the present invention.
  • the detection method adopted in the present invention is multiplex PCR combined with nucleic acid flight mass spectrometry.
  • 16 target sequences are amplified simultaneously in a system by multiplex PCR, and then specific extension primers for each SNP sequence are added to extend 1 base at the SNP site.
  • the sample analyte prepared above is co-crystallized with the chip matrix and excited by a strong laser in the vacuum tube of the mass spectrometer.
  • the nucleic acid molecules are desorbed into singly charged ions.
  • the ion flight time in the electric field is inversely proportional to the ion mass.
  • the main components of the kit include: 10XPCR buffer, MgCl 2 solution, dNTP solution, HS Taq, 3-Pt Calibrant, SAP Buffer, SAP Enzyme, iPlex Buffer Plus-10x, iPlex Termination Mix, iPlex Enzyme, amplification primer mix Solution, extension primer mixture, ddH 2 O, positive control substance, dry resin, chip board.
  • This kit is stored at -20°C, with a shelf life of 9 months.
  • This product is suitable for genomic DNA extracted from oral mucosal cells, oral exfoliated cells, blood, tissues and dried blood tablets.
  • the ratio of A 260 / A 280 of DNA should be between 1.8 and 2.0.
  • Frozen DNA samples should be below -20°C and avoid repeated freezing and thawing.
  • PCR I area take out the reagents (kits) from the refrigerator at -20°C, melt them on ice (4°C), and then take out the amplification primers from the refrigerator at 4°C, vortex and shake for 10s and then centrifuge briefly, and set aside .
  • the SAP reaction mixture is shown in the following table:
  • Klenow enzyme buffer 0.2 ⁇ L (from NEB, buffer 2) Klenow enzyme 1U (made from NEB) Extension primer mix 0.94 ⁇ L Reaction stop solution 0.2 ⁇ L (produced from NEB) Pure water Fill up to 2 ⁇ L
  • the following program is for a 96-well plate, please adjust the program according to the actual number of wells. Wear gloves and goggles.
  • the standard product can detect the corresponding genotype
  • the blank control NTC only detects the extension primer signal
  • the weak positive control can detect the corresponding positive signal, the test result is valid, otherwise the test The result is invalid.
  • This method may be affected by the DNA quality of the test sample. If the DNA quality of the test sample is poor, false negative results may occur.
  • test results are only for clinical medication reference, used to guide individualized medication, and cannot be used as the only basis for clinical medication.
  • This product can detect at least 10ng human genomic DNA with A 260 /A 280 purity between 1.80-2.00.
  • kit A provided by the present invention
  • a kit A for guiding medication for human mental illness can simultaneously type 16 gene loci, and the kit includes 16 pairs of amplification primers SEQ ID for amplifying 16 gene fragments No. 1 ⁇ SEQ ID No. 32, and 16 extension primers SEQ ID No. 33 ⁇ SEQ ID No. 48, please refer to the sequence table for specific sequences.
  • the molar concentration of the 16 pairs of amplification primers in the amplification primer mixture is equivalent, that is, the amount of each amplification primer is basically equal, and the molar concentration of the 16 extension primers
  • concentration ratio is preferably as shown in the following table:
  • kit B provided by the present invention
  • kit A On the basis of kit A, the present invention has carried out further research and development on it, and further provides kit B.
  • kit B For the specific sequence, please refer to the sequence table.
  • the molar concentration of the 16 pairs of amplification primers in the amplification primer mixture is equivalent, that is, the amount of each amplification primer is basically equal, and the molar concentration of the 16 extension primers
  • concentration ratio is preferably as shown in the following table:
  • the kit B provided by the present invention the mutual interference between the primers is also very small, and the typing effect on 15 samples is also better, and even better than the typing effect of the kit A, which proves Therefore, the kit B and the kit A can be successfully applied to guide the medication of human mental diseases.
  • the present invention also designed a comparison primer combination C, whose amplification primers are based on kit A, and further replaced SEQ ID No. 53 with SEQ ID No. 21, SEQ ID No. 54 replaces SEQ ID No. 22, and the extension primer is exactly the same as the extension primer of kit A.
  • the specific sequence is shown in the following table:
  • rs1058164 No. 3 ACGTTGGATGGTGTTCCTGGCGCGCTATG
  • No. 4 ACGTTGGATGACTGCTCCAGCGACTTCTTG
  • rs1065852 No.5 ACGTTGGATGTGGTGGACCTGATGCACCG
  • No. 6 ACGTTGGATGAGTCCACATGCAGCAGGTTG rs1079597
  • No. 7 ACGTTGGATGATACATGATCCTAAGGGCAG No.
  • the present invention also designs a comparison primer combination D, whose amplification primers are based on kit A, and further replace SEQ ID No. 49 with SEQ ID No. 3, SEQ ID No. 50 replaces SEQ ID No. 4, SEQ ID No. 51 replaces SEQ ID No. 17, SEQ ID No. 52 replaces SEQ ID No. 18, SEQ ID No. 53 replaces SEQ ID No. 21, SEQ ID No. .54 replaces SEQ ID No. 22, the extension primer is based on kit A, and further uses SEQ ID No. 57 to replace SEQ ID No. 34, SEQ ID No. 56 replaces SEQ ID No. 37, SEQ ID No. 58 Replace SEQ ID No. 41.
  • SEQ ID No. 49 with SEQ ID No. 3
  • SEQ ID No. 50 replaces SEQ ID No. 4
  • SEQ ID No. 51 replaces SEQ ID No. 17
  • SEQ ID No. 52 replaces SEQ ID No. 18
  • SEQ ID No. 53 replaces SEQ ID No. 21
  • the typing effect is shown in Figure 7.
  • the lower right corner is NTC, and the others are test samples 1-15.
  • the redder the NTC the lower the background of mutual interference between the primers, while the original color picture is light yellow, so the higher the background of mutual interference.
  • the primer combination D changes 6 amplification primer sequences and 3 extension primer sequences relative to kit A, and changes 3 extension primers relative to kit B.
  • the mutual interference between the primers is significantly higher than that of kit B.
  • Both A and Kit B have increased, and the typing effect of 15 samples has also become significantly worse. It can be seen that the amplification primers and extension primers in Kit A and Kit B are complementary to each other If it is replaced arbitrarily, the corresponding typing effect will vary greatly.
  • Fig. 8 it is a graph of the typing results of the entire NTC of the primer combination, and multiple primers have the phenomenon of extension. It can be seen that the abnormal extension phenomenon occurred in the kit due to partial replacement of the amplification primer and extension primer.
  • the mutual interference between the primers of kit A and kit B is extremely small, and accurate typing of 16 sites can be achieved for most samples at the same time, and the blank control has no abnormal extension.
  • the present invention is based on The typing of multiplex PCR and mass spectrometry sequencing technology can accurately identify the base of the mutation position, so the accuracy of detection typing has been greatly improved compared with non-sequencing-based typing.
  • the kit provided by the present invention also greatly reduces the experimental cost and cycle, so it has very important practical application value.
  • kits A and B show that the amplification primers and extension primers in a kit are complementary to each other. If they are replaced at will, The corresponding typing effects will also vary greatly. Any replacement primer sequence may not necessarily achieve good typing effects, and there will be no accidental extension.
  • kit A, B Since kit A and kit B have substantially the same primer sequence, and use the same working principle, so as to achieve the accurate typing of the 16 sites adopted in the present invention, kit A and kit B should belong to The same inventive concept has the same specific technical features and solves the same technical problems, so it should have unity.
  • CYP2C19 participates in the metabolism of drugs such as amitriptyline and clomipramine, the lack of detection of this gene locus will result in drugs such as amitriptyline and clomipramine that cannot be classified correctly. This proves that the lack of primers at any gene locus will affect the detection effect of the kit.
  • Sertraline was added to 100mg/d, and clonazepam was added to 3mg (once every night). Measure the concentration of sertraline.
  • Treatment for 10 weeks The patient spends most of the time resting in the hotel and does not want to go out. My sleep has improved, my sleep is light and I cannot get up in the morning. The contact is slightly passive, lacks initiative, doesn't talk much, lowers his head and looks down, saying that he feels a little better than last week, but he is unwilling to go out. He is still depressed, lacks pleasure, lacks motivation, and lacks confidence. Treatment: 1. Detect the drug concentration of sodium valproate and sertraline. 2. Change Sertraline 100mg (twice a day).
  • Treatment 1. Test sertraline, sodium valproate, quetiapine drug concentration 2. Betaloc 12.5mg (twice a day)
  • Treatment Maintain the original plan.
  • Case 2 Patient Wang xx, male, 66 years old, 23 years of suspicion, occasional emotional agitation, and impulsive behavior, 47 years of epilepsy history, and increased impulsivity in recent January. She lived in Shanghai for the second time on September 12, 2016.
  • Olanzapine was reduced to 5mg (twice a day). Give Antan 2mg (twice a day).
  • the patient After that, the patient enters the geriatric ward, and his diet and daily life are taken care of by nursing staff.
  • Test report August 12, 2017, sodium valproate concentration, 62.2 ⁇ g/mL; August 30, 2017, sodium valproate concentration, 47.3 ⁇ g/mL.
  • Case 3 Patient Xu, male, 30 years old, mother provided medical history. It is said that the patient had no obvious cause of onset on the third day of 2003. At that time, he felt that the glasses were inappropriate. He repeatedly adjusted and changed glasses, and changed 8 pairs a month. Glasses. For this reason, he has visited Shanghai Mental Health Center many times, diagnosed anxiety, obsessive-compulsive disorder, analfenil, clozapine, and doxepin. The effect is not good, often loses temper for no reason, is sensitive, and suspects that others speak The voice is loud, sometimes the bath lasts more than an hour. In April 2005, he lived in the Shanghai Mental Health Center for 2 months.
  • the patient was admitted to a mental hospital for the fifth time, diagnosed with schizophrenia (with obsessive-compulsive symptoms), and maintained the previous regimen upon admission: olanzapine 10 mg/d, chlorpromazine 200 mg/d, sertraline hydrochloride Dispersible tablets (Vitadine) 150mg/d treatment.
  • Olanzapine 54.6ng/mL
  • Sertraline 209ng/mL
  • Chlorpromazine 33.1ng/mL.
  • the genetic test results are as follows:

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Abstract

一种用于指导人精神疾病用药的试剂盒及其检测方法，该试剂盒可以通过一个反应同时对16个基因位点进行分型，包括用于扩增16个基因片段的16对扩增引物，该16对扩增引物的序列具体为：SEQ ID No.1～SEQ ID No.32，所述试剂盒还包括16条延伸引物，该延伸引物的序列具体为：SEQ ID No.33～SEQ ID No.48。根据上述试剂盒及其检测方法，基于多重PCR和质谱测序技术的分型，可以精准地检测出精神疾病用药相关的人类基因组突变，相对于全基因组重测序和全外显子组测序，上述试剂盒及其检测方法还大大降低了实验成本及周期，因此具有非常重要的实际应用价值。

Description

一种用于指导人精神疾病用药的试剂盒及其检测方法 技术领域
本发明涉及试剂盒领域，更具体地涉及一种用于指导人精神疾病用药的试剂盒及其检测方法。
背景技术
精神疾病已经成为我国的高发疾病，据统计我国成年人中有超过17％的人深受抑郁症、精神分裂症等精神疾病的困扰。药物治疗是目前精神疾病的主要临床手段，但是仍然处于试误法(Trial and error)的阶段，药物疗效和不良反应也呈现出显著的个体差异。在精神分裂症患者中，只有33％～50％的患者在足量足疗程的抗精神分裂症药物治疗下可以获得临床症状的完全缓解。导致药物治疗出现巨大个体差异的原因，除了传统上的病理、生理、性别、年龄、身高、体重、依从性等方面外，遗传因素是影响药物反应差异的重要因素。近年来，药物基因组学得到了飞速发展，通过对药物疗效和不良反应的相关基因检测指导药物的选择和剂量调整，达到个体化治疗的目的。对于临床医生来说如何合理地选择药物、剂量调整、临床监测以及合理地控制不良反应显得尤为重要。目前，在美国食品药品监督管理局(FDA)批准的药品说明书中，已有超过200种药物被推荐进行药物基因组生物标记物的检测，占据前3位的分别是抗肿瘤药物、精神类药物和心血管类药物。大量的临床数据也都显示了精神科开展个体化药学的紧要性和必要性，但在中国，精神类药物的药物相关基因检测一直没有在临床得到广泛的应用,随着个体化用药基因学检测的临床模式的建立，药物基因组学在指导临床合理用药中将发挥越来越重要的作用，其临床应用和普及将成为趋势。
目前市场上从事基因检测的公司很多，但大多数是针对肿瘤，使用的主流检测技术都是基于高通量的全基因组重测序(即对人基因组的全部约30亿个碱基全部重新测定，并和参考基因组进行比较，以确定变异位点)或全外显子组测序(测定人基因组上所有基因中可以表达为蛋白质的部分，即外显子的序列)技术；近年来使用大、中、小型基因位点组成的panel技术在肿瘤基因检测领域也开始得到一定程度的应用。相比之下，针对精神疾病进行基因检测及用药指导的产品不是很多。全基因组重测序和全外显子组测序技术虽然准确性较高，但检测成本和时间周期还不能满足临床需求：全基因组重测序每个样本的费用在1万元左右，全外显子组测序每个样本也要3000-4000元，因为检测的基因位点多，后续的生物信息分析需要较长时间，因此这两项技术都基本不可能在两个月内拿到结果，而临床精神疾病的基因检测需要反馈及时，医生及病人希望最多一周之内拿到结果。基于荧光定量PCR技术的基因分型技术虽然在时效性上可以满足，但是通量较低，每个反应仅能检测一个样本的一个位点，16个位点需要16个检测反应才能实现，这无疑也增加了检测成本。在样本量较多的情况下，荧光定量PCR技术的检测时间将会被大大延长，因而会丧失检测时效性的优势。
发明内容
本发明的目的是提供一种用于指导人精神疾病用药的试剂盒及其检测方法，从而解决现有技术中全基因组重测序和全外显子组测序技术虽然准确性较高，但检测成本较高，时间周期较长，因此仍然缺乏适当的针对精神疾病进行基因检测及用药指导的产品的问题。
为了解决上述技术问题，本发明采用以下技术方案：
根据本发明的第一方面，提供一种用于指导人精神疾病用药的试剂盒A，该试剂盒A可同时对16个基因位点进行分型，所述试剂盒A包括用于扩增16个基因片段的16对扩增引物，所述16对扩增引物的序列SEQ ID No.1～32具体如下：
基因位点 上游引物5’-3’ 下游引物5’-3’
rs1041983 No.1:ACGTTGGATGAGACCACAATGTTAGGAGGG No.2:ACGTTGGATGCAGGAGAAGGTGAACCATGC
rs1058164 No.3:ACGTTGGATGGTGTTCCTGGCGCGCTATG No.4:ACGTTGGATGACTGCTCCAGCGACTTCTTG
rs1065852 No.5:ACGTTGGATGTGGTGGACCTGATGCACCG No.6:ACGTTGGATGAGTCCACATGCAGCAGGTTG
rs1079597 No.7:ACGTTGGATGATACATGATCCTAAGGGCAG No.8:ACGTTGGATGTCCACAGTGCTGTCAGAATC
rs1135840 No.9:ACGTTGGATGACTAGGTACCCCATTCTAGC No.10:ACGTTGGATGCCATGGTGTCTTTGCTTTCC
rs1360780 No.11:ACGTTGGATGAGGCACAGAAGGCTTTCACA No.12:ACGTTGGATGTGCCAGCAGTAGCAAGTAAG
rs1799930 No.13:ACGTTGGATGTGGAGACGTCTGCAGGTATG No.14:ACGTTGGATGCCTGCCAAAGAAGAAACACC
rs1799978 No.15:ACGTTGGATGTTTTGAGGCGGGAACGCAAC No.16:ACGTTGGATGAGGACCCAGCCTGCAATCA
rs1800497 No.17:ACGTTGGATGACACAGCCATCCTCAAAGTG No.18:ACGTTGGATGTGTGCAGCTCACTCCATCCT
rs1902023 No.19:ACGTTGGATGCCATCTATCGAGAATTTTCAG No.20:ACGTTGGATGCAATGCCAGTAAATCATCTGC
rs2069514 No.21:ACGTTGGATGATCTTGGCTCACCGCAACCT No.22:ACGTTGGATGTGGTGCACACCTGTAATTCC
rs4244285 No.23:ACGTTGGATGCACTTTCCATAAAAGCAAGG No.24:ACGTTGGATGGCAATAATTTTCCCACTATC
rs489693 No.25:ACGTTGGATGCCAGATTTGGTCAATACAGG No.26:ACGTTGGATGATACCTGCCACGCTGTAAAC
rs762551 No.27:ACGTTGGATGCTAAGCTCCATCTACCATGC No.28:ACGTTGGATGGAATCTTGAGGCTCCTTTCC
rs776746 No.29:ACGTTGGATGCAGCTTAACGAATGCTCTAC No.30:ACGTTGGATGGTAATGTGGTCCAAACAGGG
rs7997012 No.31:ACGTTGGATGCTTTCTTAGTGACATTTGCC No.32:ACGTTGGATGCCTTCCAAGAATCCTGGATG
；
所述试剂盒A还包括用于鉴定这16个基因片段突变的16条延伸引物，所述延伸引物的序列SEQ ID No.33～48具体如下：
SNP_ID 延伸引物
rs1041983 No.33:CAATGTTAGGAGGGTATTTTTA
rs1058164 No.34:GAAGAGAGCAGAGGCGCTTCTCCGT
rs1065852 No.35:TGGGCTGCACGCTAC
rs1079597 No.36:TGTGATCAGATTCGCCTTT
rs1135840 No.37:TCATAGGGGGATGGG
rs1360780 No.38:GCTTTCACATAAGCAAAGTTA
rs1799930 No.39:GACTCAAAATCTTCAATTGTT
rs1799978 No.40:CCCCCCACACCCAGAGTAA
rs1800497 No.41:GGGGCCTCAAAGTGCTGGTC
rs1902023 No.42:ATTTGCAGAAGAGAATCTTCCAAAT
rs2069514 No.43:GTCAACCTCCGCCTCTC
rs4244285 No.44:GTAAGTAATTTGTTATGGGTTCC
rs489693 No.45:CCCATTCTGTTGTCATTAGTTCC
rs762551 No.46:ATCTACCATGCGTCCTG
rs776746 No.47:TTACAGAGCTCTTTTGTCTTTCA
rs7997012 No.48:TGCCATTATCTTCAAAGACTTAATT
其中，PCR扩增的反应体系如下：
Figure PCTCN2019124304-appb-000001
iPLEX延伸的反应体系如下：
Figure PCTCN2019124304-appb-000002
根据本发明提供的上述试剂盒A，该试剂盒还包括SAP反应体系，所述SAP反应体系如下：
SAP缓冲液    0.17μL
SAP酶        0.5U
纯水         补齐至2μL。
根据本发明提供的上述试剂盒A，所述扩增引物混合液中16对扩增引物的摩尔浓度相当，即各扩增引物的用量是基本相等的。
根据本发明提供的上述试剂盒A，所述16条延伸引物的摩尔浓度比值优选为：SEQ ID No.33﹕SEQ ID No.34﹕SEQ ID No.35﹕SEQ ID No.36﹕SEQ ID No.37﹕SEQ ID No.38﹕SEQ ID No.39﹕SEQ ID No.40﹕SEQ ID No.41﹕SEQ ID No.42﹕SEQ ID No.43﹕SEQ ID No.44﹕SEQ ID No.45﹕SEQ ID No.46﹕SEQ ID No.47﹕SEQ ID No.48＝(1.64～1.67)﹕(1.84～1.88)﹕(0.98～1.02)﹕(1.38～1.41)﹕(1.03～1.07)﹕(1.55～1.58)﹕(1.53～1.57)﹕(1.77～1.81)﹕(1.48～1.51)﹕(1.84～1.88)﹕(1.15～1.19)﹕(1.70～1.74)﹕(1.67～1.70)﹕(1.17～1.20)﹕(1.69～1.72)﹕(1.82～1.86)。
最优选地，16条延伸引物的摩尔浓度比值为：SEQ ID No.33﹕SEQ ID No.34﹕SEQ ID No.35﹕SEQ ID No.36﹕SEQ ID No.37﹕SEQ ID No.38﹕SEQ ID No.39﹕SEQ ID No.40﹕SEQ ID No.41﹕SEQ ID No.42﹕SEQ ID No.43﹕SEQ ID No.44﹕SEQ ID No.45﹕SEQ ID No.46﹕SEQ ID No.47﹕SEQ ID No.48＝1.66﹕1.87﹕1.00﹕1.39﹕1.05﹕1.56﹕1.55﹕1.79﹕1.50﹕1.85﹕1.16﹕1.73﹕1.69﹕1.19﹕1.70﹕1.84。
根据本发明的第二方面，还提供另一种用于指导人精神疾病用药的试剂盒B，该试剂盒B可同时对16个基因位点进行分型，所述试剂盒B包括用于扩增16个基因片段的16对扩增引物，所述16对扩增引物的序列具体如下：
基因位点 上游引物5’-3’ 下游引物5’-3’
rs1041983 No.1:ACGTTGGATGAGACCACAATGTTAGGAGGG No.2:ACGTTGGATGCAGGAGAAGGTGAACCATGC
rs1058164 No.49:ACGTTGGATGATCACCCACCGGAGTGGTT No.50:ACGTTGGATGGGAGACCAGGGGGAGCAT
rs1065852 No.5:ACGTTGGATGTGGTGGACCTGATGCACCG No.6:ACGTTGGATGAGTCCACATGCAGCAGGTTG
rs1079597 No.7:ACGTTGGATGATACATGATCCTAAGGGCAG No.8:ACGTTGGATGTCCACAGTGCTGTCAGAATC
rs1135840 No.9:ACGTTGGATGACTAGGTACCCCATTCTAGC No.10:ACGTTGGATGCCATGGTGTCTTTGCTTTCC
rs1360780 No.11:ACGTTGGATGAGGCACAGAAGGCTTTCACA No.12:ACGTTGGATGTGCCAGCAGTAGCAAGTAAG
rs1799930 No.13:ACGTTGGATGTGGAGACGTCTGCAGGTATG No.14:ACGTTGGATGCCTGCCAAAGAAGAAACACC
rs1799978 No.15:ACGTTGGATGTTTTGAGGCGGGAACGCAAC No.16:ACGTTGGATGAGGACCCAGCCTGCAATCA
rs1800497 No.51:ACGTTGGATGACATGATGCCCTGCTTTCGG No.52:ACGTTGGATGACACAGCCATCCTCAAAGTG
rs1902023 No.19:ACGTTGGATGCCATCTATCGAGAATTTTCAG No.20:ACGTTGGATGCAATGCCAGTAAATCATCTGC
rs2069514 No.53:ACGTTGGATGTTGAGACAGAATCTTGCTCT No.54:ACGTTGGATGGAAGCTGGGGCATGACAATT
rs4244285 No.23:ACGTTGGATGCACTTTCCATAAAAGCAAGG No.24:ACGTTGGATGGCAATAATTTTCCCACTATC
rs489693 No.25:ACGTTGGATGCCAGATTTGGTCAATACAGG No.26:ACGTTGGATGATACCTGCCACGCTGTAAAC
rs762551 No.27:ACGTTGGATGCTAAGCTCCATCTACCATGC No.28:ACGTTGGATGGAATCTTGAGGCTCCTTTCC
rs776746 No.29:ACGTTGGATGCAGCTTAACGAATGCTCTAC No.30:ACGTTGGATGGTAATGTGGTCCAAACAGGG
rs7997012 No.31:ACGTTGGATGCTTTCTTAGTGACATTTGCC No.32:ACGTTGGATGCCTTCCAAGAATCCTGGATG
所述试剂盒B还包括16条延伸引物，所述延伸引物的序列具体如下：
SNP_ID 延伸引物
rs1041983 No.33:CAATGTTAGGAGGGTATTTTTA
rs1058164 No.34:GAAGAGAGCAGAGGCGCTTCTCCGT
rs1065852 No.35:TGGGCTGCACGCTAC
rs1079597 No.55:CCCCGTGCTGTCAGAATCACCTATTC
rs1135840 No.56:GGTTTGCTTTCCTGGTGA
rs1360780 No.38:GCTTTCACATAAGCAAAGTTA
rs1799930 No.39:GACTCAAAATCTTCAATTGTT
rs1799978 No.40:CCCCCCACACCCAGAGTAA
rs1800497 No.41:GGGGCCTCAAAGTGCTGGTC
rs1902023 No.42:ATTTGCAGAAGAGAATCTTCCAAAT
rs2069514 No.43:GTCAACCTCCGCCTCTC
rs4244285 No.44:GTAAGTAATTTGTTATGGGTTCC
rs489693 No.45:CCCATTCTGTTGTCATTAGTTCC
rs762551 No.46:ATCTACCATGCGTCCTG
rs776746 No.47:TTACAGAGCTCTTTTGTCTTTCA
rs7997012 No.48:TGCCATTATCTTCAAAGACTTAATT
根据上述表格可知，该试剂盒B是在试剂盒A的基础上，进一步采用SEQ ID No.49替换SEQ ID No.3，SEQ ID No.50替换SEQ ID No.4，SEQ ID No.51替换SEQ ID No.17，SEQ ID No.52替换SEQ ID No.18，SEQ ID No.53替换SEQ ID No.21，SEQ ID No.54替换SEQ ID No.22，SEQ ID No.55替换SEQ ID No.36，SEQ ID No.56替换SEQ ID No.37得到的。根据本发明的研究发现，该试剂盒B在相对试剂盒A的基础上，通过替换部分引物序列，可以基本实现相似的分型效果，并且各引物之间的干扰同样很低，同样具有良好的应用前景。
根据本发明提供的上述试剂盒B，所述16条延伸引物的摩尔浓度比值优选为：SEQ ID No.33﹕SEQ ID No.34﹕SEQ ID No.35﹕SEQ ID No.55﹕SEQ ID No.56﹕SEQ ID No.38﹕SEQ ID No.39﹕SEQ ID No.40﹕SEQ ID No.41﹕SEQ ID No.42﹕SEQ ID No.43﹕SEQ ID No.44﹕SEQ ID No.45﹕SEQ ID No.46﹕SEQ ID No.47﹕SEQ ID No.48＝(1.65～1.69)﹕(2.72～2.75)﹕(0.98～1.02)﹕(1.38～1.41)﹕(1.46～1.50)﹕(1.55～1.58)﹕(1.53～1.57)﹕(1.77～1.81)﹕(1.48～1.51)﹕(1.84～1.88)﹕(1.15～1.19)﹕(1.70～1.74)﹕(2.10～2.13)﹕(1.17～1.20)﹕(1.69～1.72)﹕(1.82～1.86)。
最优选地，16条延伸引物的摩尔浓度比值为：SEQ ID No.33﹕SEQ ID No.34﹕SEQ ID No.35﹕SEQ ID No.55﹕SEQ ID No.56﹕SEQ ID No.38﹕SEQ ID No.39﹕SEQ ID No.40﹕SEQ ID No.41﹕SEQ ID No.42﹕SEQ ID No.43﹕SEQ ID No.44﹕SEQ ID No.45﹕SEQ ID No.46﹕SEQ ID No.47﹕SEQ ID No.48＝1.66﹕2.73﹕1.00﹕1.39﹕1.48﹕1.56﹕1.55﹕1.79﹕1.50﹕1.85﹕1.16﹕1.73﹕2.12﹕1.19﹕1.70﹕1.84。
申请号为CN201811318666.6的中国专利的内容，也通过引用全文合并于本申请。
根据本发明所提供的试剂盒，均优选采用飞行时间质谱仪进行检测。本发明优选采用美国Agena公司推出的MassArray，即一种飞行时间质谱仪，确保了检测结果的准确性和灵敏度，简单可行，其优势具体如下：
(1)准确性高，直接检测待测物分子量，准确度超过99.9％；还可以检测PCR实验失败或三等位基因的存在；
(2)灵敏度高，检测窗口内，任何pmol级别的物质都能被检测出来；
(3)通量高，一张芯片上可同时完成384个样品的多重检测，每个反应孔可实现多达30重反应，每次最多能进行数万个基因型分析；
(4)灵活，一张芯片上，样本的数量和位置可以自由选择，同时样本和SNP位点的配对可以自 由选择；
(5)质控严格，质谱技术是“一管式操作”，即反应体系在生化学实验过程中始终在一个试管内反应，避免了多次转移造成的人为误差。
(6)操作简单，完全改变传统测序等技术在基因检测中价格昂贵，耗时长，操作繁琐等劣势。
根据本发明的第三方面，还提供一种试剂盒的检测方法，所述检测方法不用于疾病的诊断和治疗，仅用于个体鉴别，所述检测方法的具体步骤如下：1)PCR扩增：按照上述PCR扩增的反应体系配置PCR反应混合液，分装至96孔板中，取DNA样本分别加入96孔板中，放入扩增仪，扩增程序如下：95℃变性2min；95℃变性30s，56℃退火30s，72℃延伸60s，共45个循环；72℃最后延伸5min；2)SAP反应：配置SAP反应混合液，反应体系如下：SAP缓冲液0.17μL，SAP酶0.5U，纯水补齐至2μL；取2μL SAP反应混合液分别加至步骤1)的每个孔中，放入扩增仪，扩增程序如下：37℃，40min；85℃，5min；4℃保温；3)延伸反应：取出SAP反应板，2000rpm离心1～2min；按照上述iPLEX延伸的反应体系配置iPLEX延伸反应液，取2μL iPLEX延伸反应液分别加至离心后的孔板中，放入扩增仪，进行以下热循环以同时对16个基因位点进行精准分型，热循环程序如下：
Figure PCTCN2019124304-appb-000003
根据本发明提供试剂盒A、试剂盒B，其中所采用的16个位点组合是发明人通过大量文献阅读，搜集证据，对证据进行评级，然后结合自建中国人群基因突变型数据库中基因型频率数据才最终确定的。虽然根据现有的资料记载，可以用于指导精神疾病用药指导的基因位点很多，但是我们选取的16个位点是既具有丰富的临床试验证据支持，又适合中国人群遗传特征(位点在中国人群中的突变频率在3％以上)的位点。
具体地，本发明所涉及的位点包括CYP2D6*10(包括rs1135840,rs1065852,rs1058164三个位点)，CYP2C19*2(rs4244285位点)，CYP1A2*1F(rs762551位点)，CYP1A2*1C(rs2069514位点)，CYP3A5*3(rs776746位点)，NAT2*6(rs1799930位点)，NAT2*13(rs1041983位点)，DRD2(rs1079597位点)，DRD2(rs1799978位点)，DRD2(rs1800497位点)，MC4R(rs489693)，UGT2B15*2(rs1902023位点)，FKBP5(rs1360780位点)，以及HTR2A(rs7997012位点)。
其中，多种选择性5-羟色胺再吸收抑制剂(SSRI)类和三环类精神药物经由CYP2D6或者主要经由CYP2D6代谢。而CYP2D6*10是中国人群多发的导致活性减弱的突变。CYP2D6活性的减弱会致使经CYP2D6代谢的药物的血药浓度和峰值的升高，进而可能影响其治疗效果和毒副作用。部分SSRI和三环类精神药物经CYP2C19代谢或部分经CYP2C19代谢。CYP2C19*2是中国人群中常见的活性降低的突变。CYP2C19活性的减弱将会导致经由CYP2C19代谢的药物血药浓度和峰值的升高。部分SSRI类和三环类精神药物经由CYP1A2代谢。CYP1A2*1F和CYP1A2*1C是东亚人群中重要的突变类型。CYP1A2*1F突变会增强CYP1A2酶的代谢活性，而CYP1A2*1C则会减弱CYP1A2的代谢活性。CYP1A2活性的改变将会改变经由其代谢的药物的血药浓度状态。抗焦虑药物阿普唑仑主要经由CYP3A5代谢，而CYP3A5*3是东亚人群中主要的无活性突变。CYP3A5活性的丧失显著影响阿普唑仑代谢，改变其血药浓度。NAT2是N-乙酰转移酶，是氯硝西泮的重要代谢酶。NAT2*6和*13突变导致氯硝西泮代谢明显减慢，从而影响氯硝西泮的治疗效果和毒副作用。DRD2是多巴胺受体，是多种抗精神分裂类药物的作用靶点。Rs1079597，rs1800497和rs1799978三个位点的多态性可以影响到多种抗精神分裂症及相关治疗的药物疗效。MC4R是黑色细胞刺激素受体，在控制体重增加中起重要作用。体重增加是第二代抗精神分裂药物的常见副作用，而MC4R的单核苷酸多态性位点rs489693与多种抗精神分裂药物的体重增加副作用相关，其多态性可以帮助预测精神分裂药物的体重增加副作用。UGT2B15是苯二氮卓类药物的主要代谢酶，UGT2B15*2突变导致其酶活性降低，将导致劳拉西泮， 奥沙西泮等药物的代谢变慢。FKBP5是一种亲免蛋白，可以影响多种SSRI类药物的治疗效果。FKBP5的rs1360780位点多态性可以帮助预测多种SSRI类药物的疗效。HTR2A是5羟色胺受体，是许多SSRI类药物的作用靶点。多项研究表明，HTR2A rs7997012位点多态性可以帮助预测SSRI类药物的治疗效果。因此，本发明针对这16个基因位点的精确分型结果将为医生临床个体化用药提供重要依据。
应当理解的是，根据本试剂盒获得的检测结果仅代表CYP2D6*10(包括rs1135840,rs1065852,rs1058164三个位点)，CYP2C19*2(rs4244285位点)，CYP1A2*1F(rs762551位点)，CYP1A2*1C(rs2069514位点)，CYP3A5*3(rs776746位点)，NAT2*6(rs1799930位点)，NAT2*13(rs1041983位点)，DRD2(rs1079597位点)，DRD2(rs1799978位点)，DRD2(rs1800497位点)，MC4R(rs489693)，UGT2B15*2(rs1902023位点)，FKBP5(rs1360780位点)，HTR2A(rs7997012位点)这16个基因位点的分型结果，为医生临床个体化用药提供参考。
在16个基因位点组合确定后，发明人进一步针对每个基因位点设计专用引物，然后通过多次尝试不同的引物组合，最终选择出对绝大部分样本都可以达到16个位点精准分型，同时空白对照又没有出现异常延伸的引物组合，最终才得到具有非常显著的应用效果的试剂盒A。而既可以同时对16个位点进行精准分型，同时又不会出现异常延伸对于获得一个合格的试剂盒来说是非常重要，是必须同时满足的两个要求。
此外，发明人在针对试剂盒A做进一步的研究之后发现，在与试剂盒A基本一致的基础上，通过仅对试剂盒A中三个基因位点的扩增引物进行替换，以及两个基因位点的延伸引物进行替换，获得了一种试剂盒B，该试剂盒B同样可以对绝大部分样本实现16个位点精准分型，同时空白对照又没有出现异常延伸，出乎意料的是该试剂盒B的分型效果相对试剂盒A还稍好一些。
应当理解的是，一个试剂盒中的各条扩增引物以及延伸引物之间均为相辅相成的作用，缺一不可，如果对其进行任意的替换则很有可能导致分型效果的千差万别，事实上，任意的替换引物序列并不一定能实现同样良好的分型效果，并且还可能出现意外的延伸，而这些结果都是本领域技术人员不愿意看到的。事实上，本申请的发明人正是通过对科技文献上记载的理论知识进行研究，并且经过自身反复的实验摸索以及验证才最终获得了具有良好的分型效果以及临床指导效果的试剂盒A、B。由于试剂盒A、试剂盒B具有基本大致相同的引物序列，并且采用了相同的工作原理实现对本发明所采取的16个位点的精准分型，因此试剂盒A、试剂盒B应当属于同一个发明构思，具有相同的特定技术特征，解决了相同的技术问题，应当具有单一性。
总之，本试剂盒所采用的技术在通量上是荧光定量PCR的16倍，可以很好的解决在样本量较多时的效率问题。在成本方面据测算，使用荧光定量PCR，16个位点的检测成本约在3千元(包括人工和试剂耗材以及仪器损耗等成本)左右，而本试剂盒的检测成本可以控制在1000以内。其次，本发明基于飞行时间质谱技术进行基因分型，是对精心挑选的和精神疾病用药最为相关的一些基因的变异位点进行中通量测序，因此可以大大降低时间周期和检测成本，而且仍然得到高度可靠的检测结果，从而使得该技术在临床上具有实际使用的价值。
根据本发明提供的上述试剂盒及其检测方法，基于多重PCR和质谱测序技术的分型，可以精确地识别突变位置的碱基，因此其分型的精准性相比非基于测序技术的分型有了的极大提高，而相对于其它基于测序技术的方法(全基因组重测序和全外显子组测序)，本发明所提供的试剂盒及其检测方法大大降低了实验成本及周期，因此具有非常重要的实际应用价值。
附图说明
图1是采用本发明提供的试剂盒A针对15个人源DNA样本进行检测的分型效果图，其中，除了右侧最下面一个孔为NTC，其余孔从左往右、从上至下依次为样本1-15；
图2是采用本发明提供的试剂盒A的NTC全体分型结果图；
图3是采用本发明提供的试剂盒B针对15个人源DNA样本进行检测的分型效果图，其中，除了右侧最下面一个孔为NTC，其余孔从左往右、从上至下依次为样本1-15；
图4是采用本发明提供的试剂盒B的NTC全体分型结果图；
图5是采用本发明提供的引物组合C针对15个人源DNA样本进行检测的分型效果图，其中，除了右侧最下面一个孔为NTC，其余孔从左往右、从上至下依次为样本1-15；
图6是采用本发明提供的引物组合C的NTC全体分型结果图；
图7是采用本发明提供的引物组合D针对15个人源DNA样本进行检测的分型效果图，其中，除了右侧最下面一个孔为NTC，其余孔从左往右、从上至下依次为样本1-15；
图8是采用本发明提供的引物组合D的NTC全体分型结果图。
具体实施方式
以下结合具体实施例，对本发明做进一步说明。应理解，以下实施例仅用于说明本发明而非用于限制本发明的范围。
实施例1有关试剂盒的详情说明
1.1检验原理
本发明采用的检测方法为多重PCR结合核酸飞行质谱。首先通过多重PCR在一个体系内同时扩增16个目标序列，然后加入针对每个SNP序列的特异延伸引物，在SNP位点上，延伸1个碱基。然后将上述制备的样品分析物与芯片基质共结晶后在质谱仪的真空管经强激光激发，核酸分子解吸附为单电荷离子，电场中离子飞行时间与离子质量成反比，通过检测核酸分子在真空管中的飞行时间而获得样品分析物的精确分子量，从而检测出SNP位点信息。
1.2主要组成成分
该试剂盒的主要组成成分包括：10XPCR buffer，MgCl 2溶液，dNTP溶液，HS Taq，3-Pt Calibrant，SAP Buffer，SAP Enzyme，iPlex Buffer Plus–10x，iPlex Termination Mix，iPlex Enzyme，扩增引物混合液，延伸引物混合液，ddH 2O，阳性对照品，干燥树脂，芯片板。
1.3储存条件及有效期
本试剂盒保存于-20℃，保质期9个月。
1.4配套仪器
通用PCR仪；点样仪Massarray Nanodispenser，型号RS1000(Agena Bioscience)；质谱仪Massarray Analyser Four，型号Massarray Analyser Four System 96/24 Genotyping(Agena Bioscience)。
1.5样本要求
本品适用于从口腔黏膜细胞、口腔脱落细胞、血液、组织和干血片中提取的基因组DNA，要求DNA的A 260/A 280比值应介于1.8到2.0之间。冷冻DNA样本应在-20℃以下，并且避免反复冻融。
1.6检验方法
1.6.1 PCR反应
1)在PCRⅠ区，从-20℃冰箱中将各试剂(盒)取出，置于冰上(4℃)融解，再从4℃冰箱取出扩增引物，均涡旋震荡10s后简短离心，备用。
2)按下表依次加入相关试剂组分配置PCR反应混合液，并做好标记、分装至96孔板中，3μl/孔；分装完后从PCRⅠ区通过传递窗传至Ⅱ区；
10X PCR buffer 0.5μL
Mg 2+ 2μM(终浓度)
dNTP 500μM(终浓度)
扩增引物混合液 0.5ul(终浓度0.1μM，各引物之间量比为1)
Taq酶 1U
水 补齐至5μL
3)在PCRⅡ区，从-20℃冰箱取出DNA模板，冰上(4℃)熔解，涡旋震荡10s后简短离心，吸出一定量DNA稀释至5ng/μl，备用。
4)向96孔板中每孔加入2μl 5ng/μl DNA模板，盖上管盖，涡旋震荡10s后简短离心，从PCR Ⅱ区通过传递窗传至Ⅲ区，再从PCRⅢ区通过传递窗传至Ⅳ区，每次实验时必须设定空白对照(2μl ddH 2O)、阴性对照(2μL DNA提取洗脱液)和阳性对照。
5)将96孔板放入扩增仪，运行程序：pcr，具体程序如下：
Figure PCTCN2019124304-appb-000004
1.6.2 SAP反应
1)PCR反应结束后，按下表在1.5mL EP管中配制SAP混液。表2的数字是以一块96孔板加上38％过剩额计算出来的。该配置过程在PCRⅠ区完成。
SAP反应混合液如下表所示：
SAP缓冲液 0.17μL CutSmart buffer(生产商NEB)
SAP酶 0.5U(生产商NEB)
水 补齐至2μL
2)将配制好的SAP混液由PCRⅠ区传递至Ⅳ区，加2μl SAP混液到每一个孔里(加混液后总体积：7μL)。
3)把板用膜(用life的或其它公司质量较好的膜)封好，涡旋震荡和离心(4000rpm 5秒)。
4)将板放上PCR仪进行以下程序：
37℃  40分钟，
85℃  5分钟，
4℃ 保温。
1.6.3延伸反应
1)取出SAP反应板2000rpm离心1min。
2)根据下表在1.5mL管中配制iPLEX延伸混液，表3的数字是以一块96孔板加上38％过剩额计算出来的。请根据实际反应的数量调整数字。该配置过程在PCRⅠ区完成。
Klenow酶缓冲液 0.2μL(产自NEB，缓冲液2)
Klenow酶 1U(产自NEB)
延伸引物混合液 0.94μL
反应终止液 0.2μL(产自NEB)
纯水 补齐至2μL
3)将iPLEX延伸混液由PCRⅠ区传递至Ⅳ区，每一个孔加入2μl iPLEX延伸混液并混匀(加混液后总体积：9μL)。
4)把板用膜封好，涡旋震荡和离心(4000rpm 5秒)。
5)将96孔板放上PCR仪进行以下热循环：
Figure PCTCN2019124304-appb-000005
1.6.4调节(样本脱盐)
以下程序为一块96孔板而设，请根据实际孔的数量调节程序。戴上手套和护眼镜。
1)把洁净树脂(Resin)铺平在96/15mg凹坑板(dimple plate)上，风干最少10分钟。
注意：先用勺子将树脂铺放在板上，然后用刮板刮从左至右或从右至左刮树脂，使96个孔都被树脂填满，当96个孔都被填满后再用刮板轻轻地刮一遍并将表面残留的树脂刮下去，防止干扰下步贴膜。当树脂由深黄变为淡黄色，即表明树脂已经干的差不多了。
2)在样本板的每一个有样本的孔里加入41μl水，封膜(用普通的膜即可)，然后离心。
3)加入15mg洁净树脂(Resin)：轻轻将样本板凌空反转，放在已放树脂的凹坑板上，一定要孔对孔！然后将凹坑板连样本板一起反转(过程中两块板不可水平移动)，让树脂掉到孔里。
4)用膜(用普通的膜即可)把板封好，放在旋转器上颠倒摇匀15分钟。
5)以3200g(标准板离心机的4000rpm)将板离心5分钟。
1.6.5点样
按照Massarray Nanodispenser操作说明将PCR产物转移至芯片板。
1.6.6质谱分析
1)从点样仪中取出芯片板。
2)用镊子将芯片板转移至Massarray Analyser Four芯片槽，芯片印有文字的一面朝外，并用镊子将芯片向左下方方向挤压。
3)按照Massarray Analyser Four操作说明，进行飞行质谱检测及结果读出。
1.6.7检验结果的解释
1)试剂盒有效性判定：标准品可以检测对应基因型，空白对照品(NTC)仅检出延伸引物信号，弱阳性对照可以检出对应阳性信号时，此次检测结果有效，否则此次检测结果无效。
1.6.8检验方法的局限性
1)本方法可能受到检测样本DNA质量的影响，如果检测样本DNA质量较差，可能出现假阴性结果。
2)本检测结果仅供临床用药参考，用于指导个体化用药，不能作为临床用药的唯一依据。
3)当本品检出对应位点的基因型为野生型时，不能排除该基因还有其它位点的突变。
1.6.9产品性能指标
本产品最低可以检出A 260/A 280纯度在1.80-2.00之间的10ng人类基因组DNA。
实施例2不同引物组合对样本的分型效果比较
下面以相同的15个人源DNA模板为样本，依次采用本发明提供的试剂盒A，试剂盒B，以及对比引物组合C，D对这15个样本进行基因分型，结果如下：
2.1本发明提供的试剂盒A的分型效果
根据本发明提供的一种用于指导人精神疾病用药的试剂盒A，可同时对16个基因位点进行分型，试剂盒包括用于扩增16个基因片段的16对扩增引物SEQ ID No.1～SEQ ID No.32，以及16条延伸引物SEQ ID No.33～SEQ ID No.48，具体序列参见序列表所示。
经过实验验证，本试剂盒A在使用中，优选地，扩增引物混合液中16对扩增引物的摩尔浓度相当，即各扩增引物的用量是基本相等的，而16条延伸引物的摩尔浓度比值优选如下表所示：
SNP_ID 最佳比例 低 高
rs1041983 1.66 1.64 1.67
rs1058164 1.87 1.84 1.88
rs1065852 1.00 0.98 1.02
rs1079597 1.39 1.38 1.41
rs1135840 1.05 1.03 1.07
rs1360780 1.56 1.55 1.58
rs1799930 1.55 1.53 1.57
rs1799978 1.79 1.77 1.81
rs1800497 1.50 1.48 1.51
rs1902023 1.85 1.84 1.88
rs2069514 1.16 1.15 1.19
rs4244285 1.73 1.70 1.74
rs489693 1.69 1.67 1.70
rs762551 1.19 1.17 1.20
rs776746 1.70 1.69 1.72
rs7997012 1.84 1.82 1.86
采用该试剂盒A对上述15个人源DNA模板进行分析，其分型效果如图1所示，其中，右下角为NTC，其它为测试样本1-15，绿色越深，代表分型效果越好，NTC越红(原彩图中为深红色)，说明其引物相互干扰的本底越低。
该结果证明，根据本发明提供的试剂盒A，各引物之间相互干扰极小，对15个样本的分型效果也 较好，从而证明该试剂盒A可成功应用于指导人精神疾病的用药。
如图2所示，为NTC全体分型结果图，基本没有出现异常延伸的情况。由此可见，该试剂盒同时也避免了异常延伸的情况。
2.2本发明提供的试剂盒B的分型效果
在试剂盒A的基础上，本发明对其进行了进一步的研究和开发，进一步提供了试剂盒B，具体序列参见序列表所示。
经过实验验证，本试剂盒B在使用中，优选地，扩增引物混合液中16对扩增引物的摩尔浓度相当，即各扩增引物的用量是基本相等的，而16条延伸引物的摩尔浓度比值优选如下表所示：
SNP_ID 最佳比例 低 高
rs1041983 1.66 1.65 1.69
rs1058164 2.73 2.72 2.75
rs1065852 1.00 0.98 1.02
rs1079597 1.39 1.38 1.41
rs1135840 1.48 1.46 1.50
rs1360780 1.56 1.55 1.58
rs1799930 1.55 1.53 1.57
rs1799978 1.79 1.77 1.81
rs1800497 1.50 1.48 1.51
rs1902023 1.85 1.84 1.88
rs2069514 1.16 1.15 1.19
rs4244285 1.73 1.70 1.74
rs489693 2.12 2.10 2.13
rs762551 1.19 1.17 1.20
rs776746 1.70 1.69 1.72
rs7997012 1.84 1.82 1.86
采用该试剂盒B对上述15个人源DNA模板进行分析，其分型效果如图3所示，其中，右下角为NTC，其它为测试样本1-15，绿色越深，代表分型效果越好，NTC越红(原彩图中为深红色)，说明其引物相互干扰的本底越低。
该结果证明，根据本发明提供的试剂盒B，各引物之间相互干扰也非常小，对15个样本的分型效果也较好，甚至相对试剂盒A的分型效果更好一些，因而证明了该试剂盒B与试剂盒A同样可成功应用于指导人精神疾病的用药。
如图4所示，为NTC全体分型结果图，基本没有出现异常延伸的情况。由此可见，该试剂盒同时也避免了异常延伸的情况。
2.3对比引物组合C的分型效果
在针对相同的16个基因位点的基础上，本发明还设计了对比引物组合C，其扩增引物是在试剂盒A基础上，进一步采用SEQ ID No.53替换SEQ ID No.21，SEQ ID No.54替换SEQ ID No.22，延伸引物与试剂盒A的延伸引物一模一样。具体序列如下表所示：
基因位点 上游引物5’-3’ 下游引物5’-3’
rs1041983 No.1:ACGTTGGATGAGACCACAATGTTAGGAGGG No.2:ACGTTGGATGCAGGAGAAGGTGAACCATGC
rs1058164 No.3:ACGTTGGATGGTGTTCCTGGCGCGCTATG No.4:ACGTTGGATGACTGCTCCAGCGACTTCTTG
rs1065852 No.5:ACGTTGGATGTGGTGGACCTGATGCACCG No.6:ACGTTGGATGAGTCCACATGCAGCAGGTTG
rs1079597 No.7:ACGTTGGATGATACATGATCCTAAGGGCAG No.8:ACGTTGGATGTCCACAGTGCTGTCAGAATC
rs1135840 No.9:ACGTTGGATGACTAGGTACCCCATTCTAGC No.10:ACGTTGGATGCCATGGTGTCTTTGCTTTCC
rs1360780 No.11:ACGTTGGATGAGGCACAGAAGGCTTTCACA No.12:ACGTTGGATGTGCCAGCAGTAGCAAGTAAG
rs1799930 No.13:ACGTTGGATGTGGAGACGTCTGCAGGTATG No.14:ACGTTGGATGCCTGCCAAAGAAGAAACACC
rs1799978 No.15:ACGTTGGATGTTTTGAGGCGGGAACGCAAC No.16:ACGTTGGATGAGGACCCAGCCTGCAATCA
rs1800497 No.17:ACGTTGGATGACACAGCCATCCTCAAAGTG No.18:ACGTTGGATGTGTGCAGCTCACTCCATCCT
rs1902023 No.19:ACGTTGGATGCCATCTATCGAGAATTTTCAG No.20:ACGTTGGATGCAATGCCAGTAAATCATCTGC
rs2069514 No.53:ACGTTGGATGTTGAGACAGAATCTTGCTCT No.54:ACGTTGGATGGAAGCTGGGGCATGACAATT
rs4244285 No.23:ACGTTGGATGCACTTTCCATAAAAGCAAGG No.24:ACGTTGGATGGCAATAATTTTCCCACTATC
rs489693 No.25:ACGTTGGATGCCAGATTTGGTCAATACAGG No.26:ACGTTGGATGATACCTGCCACGCTGTAAAC
rs762551 No.27:ACGTTGGATGCTAAGCTCCATCTACCATGC No.28:ACGTTGGATGGAATCTTGAGGCTCCTTTCC
rs776746 No.29:ACGTTGGATGCAGCTTAACGAATGCTCTAC No.30:ACGTTGGATGGTAATGTGGTCCAAACAGGG
rs7997012 No.31:ACGTTGGATGCTTTCTTAGTGACATTTGCC No.32:ACGTTGGATGCCTTCCAAGAATCCTGGATG
SNP_ID 延伸引物
rs1041983 No.33:CAATGTTAGGAGGGTATTTTTA
rs1058164 No.34:GAAGAGAGCAGAGGCGCTTCTCCGT
rs1065852 No.35:TGGGCTGCACGCTAC
rs1079597 No.36:TGTGATCAGATTCGCCTTT
rs1135840 No.37:TCATAGGGGGATGGG
rs1360780 No.38:GCTTTCACATAAGCAAAGTTA
rs1799930 No.39:GACTCAAAATCTTCAATTGTT
rs1799978 No.40:CCCCCCACACCCAGAGTAA
rs1800497 No.41:GGGGCCTCAAAGTGCTGGTC
rs1902023 No.42:ATTTGCAGAAGAGAATCTTCCAAAT
rs2069514 No.43:GTCAACCTCCGCCTCTC
rs4244285 No.44:GTAAGTAATTTGTTATGGGTTCC
rs489693 No.45:CCCATTCTGTTGTCATTAGTTCC
rs762551 No.46:ATCTACCATGCGTCCTG
rs776746 No.47:TTACAGAGCTCTTTTGTCTTTCA
rs7997012 No.48:TGCCATTATCTTCAAAGACTTAATT
采用该引物组合C对上述15个人源DNA模板进行分析，其分型效果如图5所示，其中，右下角为NTC，其它为测试样本1-15，绿色越深，代表分型效果越好，NTC越红，说明其引物相互干扰的本底越低，而原彩图中为浅黄色，因此说明相互干扰的本底较高。
该结果证明，该引物组合C虽然相对试剂盒A仅改变了一对扩增引物，但是各引物之间的相互干扰明显较试剂盒A有所增高，对15个样本的分型效果也明显变差，由此可以看出，试剂盒A中各扩 增引物以及延伸引物之间均为相辅相成的作用，如果对其进行任意替换则相应的分型效果也会相差巨大。
如图6所示，为该引物组合的NTC全体分型结果图，部分引物有延伸现象。由此可见，该试剂盒由于扩增引物的部分替换导致了异常延伸现象的发生。
2.4对比引物组合D的分型效果
在针对相同的16个基因位点的基础上，本发明还设计了对比引物组合D，其扩增引物是在试剂盒A基础上，进一步采用SEQ ID No.49替换SEQ ID No.3，SEQ ID No.50替换SEQ ID No.4，SEQ ID No.51替换SEQ ID No.17，SEQ ID No.52替换SEQ ID No.18，SEQ ID No.53替换SEQ ID No.21，SEQ ID No.54替换SEQ ID No.22，延伸引物是在试剂盒A基础上，进一步采用SEQ ID No.57替换SEQ ID No.34，SEQ ID No.56替换SEQ ID No.37，SEQ ID No.58替换SEQ ID No.41。具体序列如下表所示：
基因位点 上游引物5’-3’ 下游引物5’-3’
rs1041983 No.1:ACGTTGGATGAGACCACAATGTTAGGAGGG No.2:ACGTTGGATGCAGGAGAAGGTGAACCATGC
rs1058164 No.49:ACGTTGGATGATCACCCACCGGAGTGGTT No.50:ACGTTGGATGGGAGACCAGGGGGAGCAT
rs1065852 No.5:ACGTTGGATGTGGTGGACCTGATGCACCG No.6:ACGTTGGATGAGTCCACATGCAGCAGGTTG
rs1079597 No.7:ACGTTGGATGATACATGATCCTAAGGGCAG No.8:ACGTTGGATGTCCACAGTGCTGTCAGAATC
rs1135840 No.9:ACGTTGGATGACTAGGTACCCCATTCTAGC No.10:ACGTTGGATGCCATGGTGTCTTTGCTTTCC
rs1360780 No.11:ACGTTGGATGAGGCACAGAAGGCTTTCACA No.12:ACGTTGGATGTGCCAGCAGTAGCAAGTAAG
rs1799930 No.13:ACGTTGGATGTGGAGACGTCTGCAGGTATG No.14:ACGTTGGATGCCTGCCAAAGAAGAAACACC
rs1799978 No.15:ACGTTGGATGTTTTGAGGCGGGAACGCAAC No.16:ACGTTGGATGAGGACCCAGCCTGCAATCA
rs1800497 No.51:ACGTTGGATGACACAGCCATCCTCAAAGTG No.52:ACGTTGGATGTGTGCAGCTCACTCCATCCT
rs1902023 No.19:ACGTTGGATGCCATCTATCGAGAATTTTCAG No.20:ACGTTGGATGCAATGCCAGTAAATCATCTGC
rs2069514 No.53:ACGTTGGATGTTGAGACAGAATCTTGCTCT No.54:ACGTTGGATGGAAGCTGGGGCATGACAATT
rs4244285 No.23:ACGTTGGATGCACTTTCCATAAAAGCAAGG No.24:ACGTTGGATGGCAATAATTTTCCCACTATC
rs489693 No.25:ACGTTGGATGCCAGATTTGGTCAATACAGG No.26:ACGTTGGATGATACCTGCCACGCTGTAAAC
rs762551 No.27:ACGTTGGATGCTAAGCTCCATCTACCATGC No.28:ACGTTGGATGGAATCTTGAGGCTCCTTTCC
rs776746 No.29:ACGTTGGATGCAGCTTAACGAATGCTCTAC No.30:ACGTTGGATGGTAATGTGGTCCAAACAGGG
rs7997012 No.31:ACGTTGGATGCTTTCTTAGTGACATTTGCC No.32:ACGTTGGATGCCTTCCAAGAATCCTGGATG
SNP_ID 延伸引物
rs1041983 No.33:CAATGTTAGGAGGGTATTTTTA
rs1058164 No.57:GTTGCGCAAGGTGGA
rs1065852 No.35:TGGGCTGCACGCTAC
rs1079597 No.36:TGTGATCAGATTCGCCTTT
rs1135840 No.56:GGTTTGCTTTCCTGGTGA
rs1360780 No.38:GCTTTCACATAAGCAAAGTTA
rs1799930 No.39:GACTCAAAATCTTCAATTGTT
rs1799978 No.40:CCCCCCACACCCAGAGTAA
rs1800497 No.58:GGGCTGGGCGCCTGCCT
rs1902023 No.42:ATTTGCAGAAGAGAATCTTCCAAAT
rs2069514 No.43:GTCAACCTCCGCCTCTC
rs4244285 No.44:GTAAGTAATTTGTTATGGGTTCC
rs489693 No.45:CCCATTCTGTTGTCATTAGTTCC
rs762551 No.46:ATCTACCATGCGTCCTG
rs776746 No.47:TTACAGAGCTCTTTTGTCTTTCA
rs7997012 No.48:TGCCATTATCTTCAAAGACTTAATT
采用该引物组合D对上述15个人源DNA模板进行分析，其分型效果如图7所示，其中，右下角为NTC，其它为测试样本1-15，绿色越深，代表分型效果越好，NTC越红，说明其引物相互干扰的本底越低，而原彩图中为浅黄色，因此说明相互干扰的本底较高。
然而该结果证明，该引物组合D相对试剂盒A改变了6条扩增引物序列以及3条延伸引物序列，相对试剂盒B改变了3条延伸引物，各引物之间的相互干扰明显较试剂盒A、试剂盒B均有所增高，对15个样本的分型效果也明显变差，由此可以看出，试剂盒A、试剂盒B中各扩增引物以及延伸引物之间均为相辅相成的作用，如果对其进行任意替换则相应的分型效果也会相差巨大。
如图8所示，为该引物组合的NTC全体分型结果图，多个引物有延伸现象。由此可见，该试剂盒由于扩增引物和延伸引物的部分替换导致了异常延伸现象的发生。
综上所述，试剂盒A与试剂盒B各引物之间相互干扰极小，可以对绝大部分样本同时实现16个位点的精准分型，同时空白对照又没有出现异常延伸，本发明基于多重PCR和质谱测序技术的分型，可以精确地识别突变位置的碱基，因此检测分型的精准性相比非基于测序技术的分型有了极大提高。而相对于其它基于测序技术的方法(全基因组重测序和全外显子组测序)，本发明所提供的试剂盒还大大降低了实验成本及周期，因此具有非常重要的实际应用价值。
然而，通过试剂盒A、B与对比引物组合C、D的分型效果比较可知，一个试剂盒中的各条扩增引物以及延伸引物之间均为相辅相成的作用，如果对其进行任意替换则相应的分型效果也会相差巨大，任意的替换引物序列并不一定能实现良好的分型效果，并且不出现意外延伸，而本申请的发明人正是通过理论以及反复的实验验证才最终获得了试剂盒A、B。由于试剂盒A、试剂盒B具有基本大致相同的引物序列，并且采用了相同的工作原理，从而实现对本发明所采取的16个位点的精准分型，因此试剂盒A、试剂盒B应当属于同一个发明构思，具有相同的特定技术特征，解决了相同的技术问题，应当具有单一性。
实施例3试剂盒A的实验例子
实验例子1：试剂盒检测得到如下结果：
Figure PCTCN2019124304-appb-000006
Figure PCTCN2019124304-appb-000007
根据上面的结果，我们做出如下判读：
Figure PCTCN2019124304-appb-000008
因此，我们可以对下列药物的使用情况进行推荐，如下表所示：
Figure PCTCN2019124304-appb-000009
实验例子2：
试剂盒检测得到如下结果：
Figure PCTCN2019124304-appb-000010
Figure PCTCN2019124304-appb-000011
根据上面的结果，我们做出如下判读：
Figure PCTCN2019124304-appb-000012
因此，我们可以对下列药物的使用情况进行推荐，如下表所示：
Figure PCTCN2019124304-appb-000013
实验例子3：
试剂盒检测得到如下结果：
Figure PCTCN2019124304-appb-000014
根据上面的结果，我们做出如下判读：
Figure PCTCN2019124304-appb-000015
Figure PCTCN2019124304-appb-000016
因此，我们可以对下列药物的使用情况进行推荐，如下表所示：
Figure PCTCN2019124304-appb-000017
实验例子4：
假如剔除检测CYP2C19*2这个位点的相关引物，试剂盒检测得到如下结果：
Figure PCTCN2019124304-appb-000018
根据上面的结果，我们做出如下判读：
Figure PCTCN2019124304-appb-000019
Figure PCTCN2019124304-appb-000020
由于CYP2C19参与了阿米替林、氯丙咪嗪等药物的代谢，所以缺少了对该基因位点的检测，将导致阿米替林、氯丙咪嗪等药物无法正确归类。由此证明，任何一个基因位点的引物缺失，都将影响该试剂盒的检测效果。
实施例4具体案例
我们将试剂盒A应用于人精神疾病用药的指导，并且获得了非常好的临床治疗效果，详细说明如下：
案例一
患者王xx，21岁，湖北武汉人。于2017-09-14在父母陪同下首次来上海医院就诊。该患者2015年9月去温哥华留学，但不能适应，兴趣低落，1周后就退学回国，之后仍觉情绪持续低落、失眠、紧张、担心、自杀意念强烈。2015年10月曾在武汉人民医院精神科住院治疗一月，诊断“抑郁症”，给予文拉法辛、米氮平等治疗，合并10次MECT(电休克治疗)及RTMS(经颅磁刺激)治疗，病情仍无明显改善。此后去武汉心理医院、北京安定医院就诊，服用来士普、舍曲林、希德、麦普替林、米氮平、碳酸锂、帕可、乐孚亭、奥氮平、氯氮平、舒思、艾司唑仑、氯硝西泮等，最多的时候一天服用5种药，来士普最高服用到30mg/d，麦普替林最高服用到300mg/d，效果仍不佳。2016年10月曾服用30粒奥氮平自杀，被家人发现。近半年服用米氮平15mg(每天一次)，麦普替林50mg(每晚一次)，情绪低迷，睡眠不好，常有自杀意念。因10月份将去英国留学，家人担心不能适应学校生活，来上海市徐汇区精神卫生中心寻求帮助。既往史、个人史、家族史、体检：均无特殊。精神检查：意识清，定向全，仪态尚整，接触较被动，交谈时一直低着头，不正视医生，没有目光的交流，注意力尚集中，有问有答，对答切题，说话声音轻。能向医生介绍自己的爱好，参加国际登山队攀登尼泊尔境内8516米的洛子峰。谈及自己的病情，称“睡觉不好，吃了药也睡不着”，“情绪低落，什么都不想做，都没有意思”，“不愿意交朋友”，觉得自卑，没有信心，情绪低落，没有愉快感，有自杀观念，否认有行动，否认有情绪特别好的时候，未引出幻觉妄想，活动减少，睡眠差，智能好，自知力完整。HAMD量表测定，总分33分，诊断：抑郁症。处理：1、米氮平15mg(每晚一次，自备)，阿普唑仑0.4mg qn，舍曲林50mg qd。停用帕可、麦普替林。同时使用抗抑郁及精神分裂药物基因检测。
2017-09-18日治疗第4日就诊：近4天睡眠减药后时间缩短，醒得早，白天精神显得萎靡。情绪状态与之前差不多。治疗：盐酸舍曲林片(左洛复)50mg(每天一次)；氯硝西泮片(基)2mg(每晚一次)。停用米氮平及阿普唑仑。
2017-09-21治疗1周：患者情绪有改善，但是睡眠仍不好。能主动与医生交流，思维流畅，交谈中有适当的动作，表情放松，情绪仍偏低，否认有消极观念，自知力存在。
我们采用试剂盒A对该病患的DNA样本进行基因检测，检测得到如下结果：
Figure PCTCN2019124304-appb-000021
根据上面的结果，我们做出如下判读：
Figure PCTCN2019124304-appb-000022
Figure PCTCN2019124304-appb-000023
根据以上判读结果，我们做出了以下个体化用药指导建议：
Figure PCTCN2019124304-appb-000024
处理：舍曲林加至100mg/d，氯硝安定加至3mg(每晚一次)。测舍曲林浓度。
2017-09-28治疗2周：患者情绪较上周又有改善，但是睡眠仍不好，睡眠浅，早上没睡醒，起不来。加用思瑞康200mg(每晚一次)。接触时能与医生聊聊自己的兴趣爱好，谈及自己的一些特殊经历，感觉不错，面带笑容。情绪尚可，但是缺乏动力，自知力存在。实验室报告：2017.9.25舍曲林浓度报告：24.6ng/ml；生化全套、血常规、T3、T4、PRL(-)；处理：舍曲林加至125mg/d，氯硝安定3mg(每晚一次)改善睡眠，思瑞康200mg(每晚一次)；测舍曲林药物浓度。
2017-11-08治疗8周，父亲陪同：患者在英国就学1个月时间中，能坚持服用舍曲林150mg/d，但情绪低落，睡眠差，时有自杀的想法。遂办理休学回国。接触时患者坐着不说话，接触被动，一问一答，没有主动性言语，回答寥寥数字，经常用“是”、“没有”作答。愁眉不展，情绪低落，称自己没有愉快感，在英国时候不愿意与人交往，社交回避，称有过想死的念头，但是没有行为，自知力存在。实验室：9.29舍曲林浓度报告：41.1ng/ml。处理：检测舍曲林浓度。
2017-11-14治疗9周：患者病情稍有改善，能和朋友一起外出散步，服药后无不良反应。实验室报告：11.10舍曲林浓度报告：85ng/ml。处理：丙戊酸钠缓释片(德巴金)0.5(每天一次)；阿普唑仑0.8mg(每晚一次),舍曲林150mg/d，思瑞康200mg(每晚一次)。
2017-11-24治疗10周：患者大部分时间在宾馆休息，不愿外出。睡眠有好转，睡眠浅，早上起不来。接触略微被动，缺乏主动性，话不多，低着头，眼睛看下方，称自我感觉比上周好一点，但是不愿意出门，仍有抑郁情绪，没有愉快感，缺乏动力，没有自信。处理：1.检测丙戊酸钠、舍曲林药物浓度。2.改舍曲林100mg(每天两次)。
2017-12-1治疗11周：病情演变：近来有时有点烦躁，心慌感。情绪自我感觉有所提高。实验室报告：丙戊酸钠，54.1μg/mL；舍曲林，66.9ng/mL。
处理：1、检测舍曲林、丙戊酸钠、喹硫平药物浓度2、倍他乐克12.5mg(每天两次)
2017-12-7治疗第12周，母亲陪同。病情演变：本周情绪有明显改善，3天前跟一位登上好友一起外出爬山10km，2天前与朋友一起自驾去杭州来回，自觉目前状态较满意。实验室报告：舍曲林，118ng/mL；丙戊酸钠，μg/mL；喹硫平，59.4ng/mL。
处理：维持原方案。
案例二：患者汪xx，男，66岁，因猜疑、时有情绪激惹、冲动行为23年，癫痫病史47年，近1月冲动加剧。于2016年9月12日第2次住上海就诊。入院诊断：癫痫性精神病。入院处理：原方案：苯妥英钠0.4/d，丙戊酸镁缓释片0.25(每天三次)，氯硝西泮0-1mg-0-1mg。增加：奥氮平(悉敏)5mg(每天两次)。
至2016.10.13在病区表现安静，平时生活自理，经常与病友打牌，无明显猜疑被害，同时也无明显癫痫发作情况。治疗方案改：奥氮平为10mg bid(每天两次)。
2016年11月后几个月内，患者行走不稳，逐渐出现四肢扭转痉挛，躯干也出现不停扭转，扭转幅度较大，出现摔跤3次。
2017.3.5病区讨论后：奥氮平减为5mg(每天两次)。予安坦2mg(每天两次)。
调整治疗后患者无明显改善，仍旧不停扭转。
2017年5月19日晚餐后起身时突然绊倒，头面部着地，鼻部流鲜血伴肿胀，导致鼻骨骨折，予保守治疗。
2017年5月25日头颅CT检查：无异常。
此后患者进入老年病房，饮食起居由护工照顾。
2017年6月12日患者坐在圈椅内摔跤，经翻阅录像发现患者伸手去碰门，连圈椅一起偏向左侧倒在地上，当时予防跌倒约束。
2017年6月-8月患者坐在老年护理椅中，经常时间定向不清，地点及人物定向可，反映显迟钝，有时一句话要反复询问时才予相应的回答，情绪显烦躁、偏低，合作不佳，说话时口齿含糊，不能自行站立，肢体不停扭动，扭动幅度大，稍不慎就摔倒，吃饭时拿不住碗筷，饭菜经常弄到地上及身上。
2017年8月11日头颅CT：1.左侧额叶可疑稍高密度影；2.左侧颞顶部皮下血肿；3.老年脑；4.右侧鼻翼下缘骨折可能；5.附见双侧上颌窦炎。
2017年8月17日组织讨论后认为：患者有意识障碍、行动不协调、多次摔跤，与目前联合用药有关，故适当减少药物剂量。丙戊酸镁缓释片从0.25(每天两次)减至0.25(每天一次)；奥氮平5mg(每天两次)减至5mg(每晚一次)。同时进行抗精神病药基因检测。
2017年8月24日查房时患者坐在圈椅中，扭动幅度有减小，表现情绪尚稳定，接触一般，基本有问有答，偶有答非所问，夜间仍时有自语或吵闹，白天不能回忆。根据主任查房意见，苯妥英钠从0.15-0.1-0.15减至0.1(每天三次)。
2017年8月31日主任查房：患者近日较前明显改善，查房时表现接触可，见到医生能主动表达要求，能在工作人员搀扶下走路，有少许有不自主扭动，白天情绪平稳，夜间时睡时醒，有时自语吵闹。
2017年9月17日基因检测结果：
Figure PCTCN2019124304-appb-000025
Figure PCTCN2019124304-appb-000026
根据上面的结果，我们做出如下判读：
Figure PCTCN2019124304-appb-000027
根据以上判读结果，我们做出了以下个体化用药指导建议：
Figure PCTCN2019124304-appb-000028
检测报告：2017年8月12日，丙戊酸钠浓度，62.2μg/mL；2017年8月30日，丙戊酸钠浓度，47.3μg/mL。
调整治疗：停奥氮平5mg(每天一次)，予喹硫平0.2(每晚一次)
2017年10月下旬-12月，患者目前在老年病室中表现安静，行为有序，对答切题，无身体扭动，夜间无明显吵闹，行走时需人搀扶以防跌倒。
案例三：患者徐某，男，30岁，母亲提供病史，据称患者2003年初三时，无明显诱因起病，当时觉得眼镜不合适，反复调整眼镜、换眼镜，曾一个月换8副眼镜，为此曾至上海市精神卫生中心多次就诊，诊断焦虑症、强迫症，安拿芬尼、氯氮平、多虑平等药物，疗效不佳，常无故发脾气，敏感，嫌别人讲话声音大，有时洗澡达一个多小时，于2005年4月住上海市精神卫生中心2个月，诊断“强迫症”，治疗不详。后在上海市精神卫生中心门诊治疗，用“西酞普兰”等治疗，疗效仍欠佳，仍有反复洗手、洗澡，反复检查等。一直未再上学，长期在家。
2011年9月-2016年患者表现基本雷同，经常反复做一些奇怪的事情，如：要求将喝过的可乐瓶、塑料袋、旧抹布等垃圾收集在家中不许人扔掉，有时还要反复寻找查看，买东西前脚买好，后脚不满意就一定要母亲退掉才满意，否则就会发脾气，反复要买自行车零件，吃饭掉地上的米饭粒，一定要仔细的全部找回，在家里的反复要求想住院，将家中没用的肥皂边角料收集起来，反复检查，不许家人丢弃；纠缠家人买电视机，且要和以前的一模一样，连新旧程度都要一样。经常打母亲、打父亲，发脾气。为此先后4次住院。诊断“强迫症”及“精神分裂症”，给予奥氮平、喹硫平、氯氮平、氯丙嗪、艾斯西酞普兰、氟西汀、舍曲林等治疗，合并数个疗程的MECT治疗。疗效均不理想。
至2017年5月，患者第五次人住精神病院，诊断精神分裂症(伴发强迫症状)，入院时维持以前方案：奥氮平10mg/d、氯丙嗪200mg/d、盐酸舍曲林分散片(唯他停)150mg/d治疗。
检测报告：
奥氮平，54.6ng/mL；舍曲林，209ng/mL；氯丙嗪，33.1ng/mL。
患者病情依旧，常有强迫性行为，有时因病友不想与他打牌或者聊天，患者反复询问对方“你为什么不想打牌了，到底什么意思”，诸如此类问题，要上百次问对方，要其反复解释，患者仍不能接受，有时还会情绪失控打人。2017年7月给予抗精神分裂病药和抗抑郁药基因检测。
基因检测结果如下：
Figure PCTCN2019124304-appb-000029
根据上面的结果，我们做出如下判读：
Figure PCTCN2019124304-appb-000030
根据以上判读结果，我们给出个体化用药指导建议如下：
Figure PCTCN2019124304-appb-000031
8月份逐步换用氯米帕明150mg(每天两次)、喹硫平200mg-400mg治疗，同时合并12次MECT治疗，换药后患者激越冲动及强迫现象有所好转，近4月来未再与其他病友发生纠纷口角等，能服从病房的管理，仍存在部分强迫性的观念，对住院比较安心。
以上所述的，仅为本发明的较佳实施例，并非用以限定本发明的范围，本发明的上述实施例还可以做出各种变化。凡是依据本发明申请的权利要求书及说明书内容所作的简单、等效变化与修饰，皆落入本发明专利的权利要求保护范围。本发明未详尽描述的均为常规技术内容。

Claims (10)

  1. 一种用于指导人精神疾病用药的试剂盒，该试剂盒可同时对16个基因位点进行分型，所述试剂盒包括用于扩增16个基因片段的16对扩增引物，所述16对扩增引物的序列具体如下：
    基因位点 上游引物5’-3’ 下游引物5’-3’ rs1041983 No.1:ACGTTGGATGAGACCACAATGTTAGGAGGG No.2:ACGTTGGATGCAGGAGAAGGTGAACCATGC rs1058164 No.3:ACGTTGGATGGTGTTCCTGGCGCGCTATG No.4:ACGTTGGATGACTGCTCCAGCGACTTCTTG rs1065852 No.5:ACGTTGGATGTGGTGGACCTGATGCACCG No.6:ACGTTGGATGAGTCCACATGCAGCAGGTTG rs1079597 No.7:ACGTTGGATGATACATGATCCTAAGGGCAG No.8:ACGTTGGATGTCCACAGTGCTGTCAGAATC rs1135840 No.9:ACGTTGGATGACTAGGTACCCCATTCTAGC No.10:ACGTTGGATGCCATGGTGTCTTTGCTTTCC rs1360780 No.11:ACGTTGGATGAGGCACAGAAGGCTTTCACA No.12:ACGTTGGATGTGCCAGCAGTAGCAAGTAAG rs1799930 No.13:ACGTTGGATGTGGAGACGTCTGCAGGTATG No.14:ACGTTGGATGCCTGCCAAAGAAGAAACACC rs1799978 No.15:ACGTTGGATGTTTTGAGGCGGGAACGCAAC No.16:ACGTTGGATGAGGACCCAGCCTGCAATCA rs1800497 No.17:ACGTTGGATGACACAGCCATCCTCAAAGTG No.18:ACGTTGGATGTGTGCAGCTCACTCCATCCT rs1902023 No.19:ACGTTGGATGCCATCTATCGAGAATTTTCAG No.20:ACGTTGGATGCAATGCCAGTAAATCATCTGC rs2069514 No.21:ACGTTGGATGATCTTGGCTCACCGCAACCT No.22:ACGTTGGATGTGGTGCACACCTGTAATTCC rs4244285 No.23:ACGTTGGATGCACTTTCCATAAAAGCAAGG No.24:ACGTTGGATGGCAATAATTTTCCCACTATC rs489693 No.25:ACGTTGGATGCCAGATTTGGTCAATACAGG No.26:ACGTTGGATGATACCTGCCACGCTGTAAAC rs762551 No.27:ACGTTGGATGCTAAGCTCCATCTACCATGC No.28:ACGTTGGATGGAATCTTGAGGCTCCTTTCC rs776746 No.29:ACGTTGGATGCAGCTTAACGAATGCTCTAC No.30:ACGTTGGATGGTAATGTGGTCCAAACAGGG rs7997012 No.31:ACGTTGGATGCTTTCTTAGTGACATTTGCC No.32:ACGTTGGATGCCTTCCAAGAATCCTGGATG
    ；
    所述试剂盒还包括用于鉴定这16个基因片段突变的16条延伸引物，所述延伸引物的序列具体如下：
    SNP_ID 延伸引物 rs1041983 No.33:CAATGTTAGGAGGGTATTTTTA rs1058164 No.34:GAAGAGAGCAGAGGCGCTTCTCCGT rs1065852 No.35:TGGGCTGCACGCTAC rs1079597 No.36:TGTGATCAGATTCGCCTTT
    rs1135840 No.37:TCATAGGGGGATGGG rs1360780 No.38:GCTTTCACATAAGCAAAGTTA rs1799930 No.39:GACTCAAAATCTTCAATTGTT rs1799978 No.40:CCCCCCACACCCAGAGTAA rs1800497 No.41:GGGGCCTCAAAGTGCTGGTC rs1902023 No.42:ATTTGCAGAAGAGAATCTTCCAAAT rs2069514 No.43:GTCAACCTCCGCCTCTC rs4244285 No.44:GTAAGTAATTTGTTATGGGTTCC rs489693 No.45:CCCATTCTGTTGTCATTAGTTCC rs762551 No.46:ATCTACCATGCGTCCTG rs776746 No.47:TTACAGAGCTCTTTTGTCTTTCA rs7997012 No.48:TGCCATTATCTTCAAAGACTTAATT
    ；
    所述试剂盒的PCR扩增的反应体系如下：
    Figure PCTCN2019124304-appb-100001
    所述试剂盒的iPLEX延伸的反应体系如下：
    Figure PCTCN2019124304-appb-100002
  2. 根据权利要求1所述的试剂盒，其特征在于，所述试剂盒还包括SAP反应体系，所述SAP反应体系如下：
    SAP缓冲液   0.17μL
    SAP酶       0.5U
    纯水        补齐至2μL。
  3. 根据权利要求1所述的试剂盒，其特征在于，所述扩增引物混合液中16对扩增引物的摩尔浓度相当。
  4. 根据权利要求1所述的试剂盒，其特征在于，所述16条延伸引物的摩尔浓度比值为：SEQ IDNo.33﹕SEQ ID No.34﹕SEQ ID No.35﹕SEQ ID No.36﹕SEQ ID No.37﹕SEQ ID No.38﹕SEQ ID No.39﹕SEQ ID No.40﹕SEQ ID No.41﹕SEQ ID No.42﹕SEQ ID No.43﹕SEQ ID No.44﹕SEQ ID No.45﹕SEQ ID No.46﹕SEQ ID No.47﹕SEQ ID No.48＝(1.64～1.67)﹕(1.84～1.88)﹕(0.98～1.02)﹕(1.38～1.41)﹕(1.03～1.07)﹕(1.55～1.58)﹕(1.53～1.57)﹕(1.77～1.81)﹕(1.48～1.51)﹕(1.84～1.88)﹕(1.15～1.19)﹕(1.70～1.74)﹕(1.67～1.70)﹕(1.17～1.20)﹕(1.69～1.72)﹕(1.82～1.86)。
  5. 根据权利要求4所述的试剂盒，其特征在于，所述16条延伸引物的摩尔浓度比值为：SEQ ID  No.33﹕SEQ ID No.34﹕SEQ ID No.35﹕SEQ ID No.36﹕SEQ ID No.37﹕SEQ ID No.38﹕SEQ ID No.39﹕SEQ ID No.40﹕SEQ ID No.41﹕SEQ ID No.42﹕SEQ ID No.43﹕SEQ ID No.44﹕SEQ ID No.45﹕SEQ ID No.46﹕SEQ ID No.47﹕SEQ ID No.48＝1.66﹕1.87﹕1.00﹕1.39﹕1.05﹕1.56﹕1.55﹕1.79﹕1.50﹕1.85﹕1.16﹕1.73﹕1.69﹕1.19﹕1.70﹕1.84。
  6. 一种用于指导人精神疾病用药的试剂盒，其特征在于，所述试剂盒是在根据权利要求1所述的试剂盒的基础上，进一步采用SEQ ID No.49替换SEQ ID No.3，SEQ ID No.50替换SEQ ID No.4，SEQ ID No.51替换SEQ ID No.17，SEQ ID No.52替换SEQ ID No.18，SEQ ID No.53替换SEQ ID No.21，SEQ ID No.54替换SEQ ID No.22，SEQ ID No.55替换SEQ ID No.36，SEQ ID No.56替换SEQ ID No.37。
  7. 根据权利要求6所述的试剂盒，其特征在于，所述16条延伸引物的摩尔浓度比值为：SEQ ID No.33﹕SEQ ID No.34﹕SEQ ID No.35﹕SEQ ID No.55﹕SEQ ID No.56﹕SEQ ID No.38﹕SEQ ID No.39﹕SEQ ID No.40﹕SEQ ID No.41﹕SEQ ID No.42﹕SEQ ID No.43﹕SEQ ID No.44﹕SEQ ID No.45﹕SEQ ID No.46﹕SEQ ID No.47﹕SEQ ID No.48＝(1.65～1.69)﹕(2.72～2.75)﹕(0.98～1.02)﹕(1.38～1.41)﹕(1.46～1.50)﹕(1.55～1.58)﹕(1.53～1.57)﹕(1.77～1.81)﹕(1.48～1.51)﹕(1.84～1.88)﹕(1.15～1.19)﹕(1.70～1.74)﹕(2.10～2.13)﹕(1.17～1.20)﹕(1.69～1.72)﹕(1.82～1.86)。
  8. 根据权利要求7所述的试剂盒，其特征在于，所述16条延伸引物的摩尔浓度比值为：SEQ ID No.33﹕SEQ ID No.34﹕SEQ ID No.35﹕SEQ ID No.55﹕SEQ ID No.56﹕SEQ ID No.38﹕SEQ ID No.39﹕SEQ ID No.40﹕SEQ ID No.41﹕SEQ ID No.42﹕SEQ ID No.43﹕SEQ ID No.44﹕SEQ ID No.45﹕SEQ ID No.46﹕SEQ ID No.47﹕SEQ ID No.48＝1.66﹕2.73﹕1.00﹕1.39﹕1.48﹕1.56﹕1.55﹕1.79﹕1.50﹕1.85﹕1.16﹕1.73﹕2.12﹕1.19﹕1.70﹕1.84。
  9. 根据权利要求1或6所述的试剂盒，其特征在于，所述试剂盒采用飞行时间质谱仪进行检测。
  10. 一种根据上述权利要求1-9所述的试剂盒的检测方法，所述检测方法不用于疾病的诊断和治疗，仅用于个体鉴别，其特征在于，所述检测方法的具体步骤如下：
    1)PCR扩增：按照上述PCR扩增的反应体系配置PCR反应混合液，分装至96孔板中，取DNA样本分别加入96孔板中，放入扩增仪，扩增程序如下：95℃变性2min；95℃变性30s，56℃退火30s，72℃延伸60s，共45个循环；72℃最后延伸5min；
    2)SAP反应：配置SAP反应混合液，反应体系如下：SAP缓冲液0.17μL，SAP酶0.5U，纯水补齐至2μL；取2μL SAP反应混合液分别加至步骤1)的每个孔中，放入扩增仪，扩增程序如下：37℃，40min；85℃，5min；4℃保温；
    3)延伸反应：取出SAP反应板，2000rpm离心1～2min；按照上述iPLEX延伸的反应体系配置iPLEX延伸反应液，取2μL iPLEX延伸反应液分别加至离心后的孔板中，放入扩增仪，进行以下热循环以同时对16个基因位点进行精准分型，热循环程序如下：
    Figure PCTCN2019124304-appb-100003
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