CN111235268A - SNP locus genotype detection reagent and application in corresponding kit and kit - Google Patents
SNP locus genotype detection reagent and application in corresponding kit and kit Download PDFInfo
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Abstract
The invention provides a SNP locus genotype detection reagent, application in a corresponding kit and the kit, wherein the detection reagent comprises: the primer group comprises a primer pair 1 for specifically amplifying a rs2239063 site of a gene CACNA1C to obtain a first amplification product, a primer pair 2 for specifically amplifying a rs 169921385 site of a gene SLC1A1 to obtain a second amplification product, and a primer pair 3 for specifically amplifying a rs17022006 site of a gene CNTN4 to obtain a third amplification product, wherein the nucleotide sequences of an upstream primer and a downstream primer of the primer pair 1 are respectively shown as SEQ ID No.1 and SEQ ID No.2, the nucleotide sequences of the upstream primer and the downstream primer of the primer pair 2 are respectively shown as SEQ ID No.3 and SEQ ID No.4, and the nucleotide sequences of the upstream primer and the downstream primer of the primer pair 3 are respectively shown as SEQ ID No.5 and SEQ ID No. 6.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a detection reagent for SNP locus genotype detection, application of the detection reagent in preparation of a kit for medication guidance of schizophrenic patients, a corresponding kit and a use method thereof.
Background
Mental diseases have become global high-incidence diseases, and more than 17% of adults in China are seriously affected by mental diseases such as depression and schizophrenia according to statistics (Phillips. 2009). Schizophrenia (SCZ) is one of the most common and serious mental disorders, and is prevalent in all people worldwide, with schizophrenia (muesser.2004) currently occurring in about 1% of the world. Schizophrenia not only causes great pain to the patient himself and his family members, but also causes heavy economic burden to the family, medical health and the whole society. At the Chinese disease burden problem seminar, mental disease has been listed as the first place of disease burden, accounting for 13%.
The research shows that 30-50% of patients have poor response to anti-schizophrenia drugs, and individual differences exist, heredity is the main reason of the differences, detection of drug efficacy-related sites can predict the efficacy of the drugs, Yueyawa and the like (2018) are published in Lancet Psychiatry 3030 cases of clinical research shows that rs2239063 site of CACNA1C, rs 921385 site of SLC1A1 and rs 17006 site of CNTN 2 are respectively related to olanzapine, risperidone and aripiprazole in treating schizophrenia diseases with obvious efficacy-related sites, CACNA1C is a subunit gene coding Cav1.2-gated L-type voltage-gated calcium channel, and multiple researches show that the drug efficacy-related genes are remarkably related to schizophrenia (Kristin.2010) and the gene transporter gene is used for detecting schizophrenia, and the most frequently used in the clinical application of drugs for schizophrenia, and the drug transporter gene is used in the field of the drug addiction, the field of schizophrenia, the field application of the drugs for the field of the drugs, namely the drug addiction, the field of the drugs, the field of the.
The method can be used for the genotyping of CACNA1C rs2239063, SLC1A1 rs 169921385 and CNTN4rs17022006 sites, and comprises a gene sequencing method, a denaturing high performance liquid chromatography method, a high-resolution melting curve method and the like. The method is suitable for high-throughput multi-site detection, is time-consuming, complex to operate, high in requirement on operators, low in sensitivity, easy to cause cross contamination among samples and not suitable for rapid clinical detection; and the amount of data information obtained by sequencing is large, much information is useless for patients, result interpretation also needs some professional knowledge, and the cost is relatively high. The denaturing high performance liquid chromatography has the advantages of high automation degree and high flux, but the equipment is expensive and is not suitable for common medical institutions. The high-resolution melting curve method is rapid, simple, convenient, economical and practical, but has special requirements on equipment, can be used only by installing a machine with special software and sensitive temperature, and has certain difficulty in clinical popularization.
Disclosure of Invention
The invention provides a detection reagent for SNP locus genotype detection, application of the detection reagent in preparation of a kit for medication guidance of schizophrenic patients, a corresponding kit and a using method thereof, so as to detect the genotypes of an rs2239063 locus of CACNA1C, an rs 169921385 locus of SLC1A1 and an rs17022006 locus of CNTN4 more accurately, conveniently and comprehensively at low cost.
The invention provides a detection reagent for detecting SNP locus genotype, wherein the SNP loci are rs2239063 locus of gene CACNA1C, rs 169921385 locus of gene SLC1A1 and rs17022006 locus of gene CNTN4 respectively, the detection reagent is characterized by comprising the following components in percentage by weight: the primer group comprises a primer pair 1 for specifically amplifying a rs2239063 site of a gene CACNA1C to obtain a first amplification product, a primer pair 2 for specifically amplifying a rs 169921385 site of a gene SLC1A1 to obtain a second amplification product, and a primer pair 3 for specifically amplifying a rs17022006 site of a gene CNTN4 to obtain a third amplification product, wherein nucleotide sequences of an upstream primer and a downstream primer of the primer pair 1 are respectively shown as SEQ ID No.1 and SEQ ID No.2, nucleotide sequences of an upstream primer and a downstream primer of the primer pair 2 are shown as SEQ ID No.3 and SEQ ID No.4, and nucleotide sequences of an upstream primer and a downstream primer of the primer pair 3 are shown as SEQ ID No.5 and SEQ ID No. 6.
The detection reagent provided by the present invention is also characterized by further comprising: a probe population comprising a first probe set for hybridizing to the first amplification product, a second probe set for hybridizing to the second amplification product, and a third probe set for hybridizing to the third amplification product.
The detection reagent provided by the invention also has the following characteristics: wherein, all include respectively in first probe group, the second probe group and the third probe group: a first TaqMan probe marked by 5 'end VIC and 3' end MGB, and a second TaqMan probe marked by 5 'end FAM and 3' end MGB.
The detection reagent provided by the invention also has the following characteristics: the nucleotide sequences of the first TaqMan probe and the second TaqMan probe of the first probe set are respectively shown as SEQ ID No.7 and SEQ ID No.8, the nucleotide sequences of the first TaqMan probe and the second TaqMan probe of the second probe set are respectively shown as SEQ ID No.9 and SEQ ID No.10, and the nucleotide sequences of the first TaqMan probe and the second TaqMan probe of the third probe set are respectively shown as SEQ ID No.11 and SEQ ID No. 12.
The detection reagent provided by the present invention is also characterized by further comprising: a DNA polymerase; a negative control sample for system error correction in the SNP locus genotype detection process; and the positive control sample is used for the quality control of a PCR reaction system in the SNP locus genotype detection process.
The detection reagent provided by the invention also has the following characteristics: wherein the DNA polymerase is one or more of GoldStar TaqMan Mix, TaqPath ProAmp Multiplex Master Mix, TaqMan GTXpress Master Mix.
The detection reagent provided by the invention also has the following characteristics: wherein the negative control sample is ddH2O。
The detection reagent provided by the invention also has the following characteristics: wherein the content of the first and second substances,
the positive control sample is genomic DNA containing rs2239063 site AC genotype of gene CACNA1C, rs 169921385 site AG genotype of SLC1A1 and rs17022006 site GA genotype of CNTN4, and the concentration of the genomic DNA is 1 ng/muL.
The invention also provides an application of the detection reagent in preparing a kit for medication guidance of schizophrenic patients, which is characterized in that: wherein the detection reagent is the detection reagent.
The invention also provides a kit for detecting the genotype of the SNP locus used for guidance of schizophrenia medication, which is characterized by comprising: the detection reagent described above.
The invention also provides a kit, which is also characterized in that: the kit detects the SNP locus genotypes based on a fluorescence quantitative PCRtaqMan MGB probe method, and in use, the concentration of an upstream primer and the concentration of a downstream primer in a PCR reaction amplification system aiming at each SNP locus genotype are both 0.2-1.2 mu M.
The kit provided by the invention also has the following characteristics: wherein the concentration of the upstream primer and the concentration of the downstream primer are both 0.7. mu.M.
The kit provided by the invention also has the following characteristics: wherein, when the kit is used, the concentration of the first TaqMan probe and the second TaqMan probe in a PCR reaction system aiming at the genotype of each SNP locus is 0.1-0.8 mu M.
The kit provided by the invention also has the following characteristics: wherein the concentration of the first TaqMan probe and the second TaqMan probe is 0.25 mu M.
The invention also provides a method for using the kit for non-diagnostic treatment, which is characterized in that: the kit is used for detecting the SNP locus genotype based on a fluorescent quantitative PCR TaqMan MGB probe method.
The use method provided by the invention also has the following characteristics: the sample to be detected by the kit is a biological sample containing genomic DNA, and the biological sample is from whole blood or oral mucosa exfoliative cells.
The use method provided by the invention also has the following characteristics: wherein, the concentration of the upstream primer and the downstream primer in the PCR reaction amplification system aiming at the genotype of each SNP locus is 0.2-1.2 mu M.
The use method provided by the invention is also characterized in that the concentration of the upstream primer and the concentration of the downstream primer are both 0.7 mu M.
The use method provided by the invention also has the following characteristics: wherein, the concentration of the first TaqMan probe and the second TaqMan probe in the PCR reaction amplification system aiming at the genotype of each SNP locus is 0.1-0.8 mu M.
The use method provided by the invention also has the following characteristics: wherein the concentration of the first TaqMan probe and the second TaqMan probe is 0.25 mu M.
The use method provided by the invention also has the following characteristics: wherein, the conditions for carrying out the PCR amplification reaction are as follows:
(1) UNG enzyme treatment at 50 deg.C for 5min,
(2) pre-denaturation at 95 ℃ for 10min,
(3) the denaturation is carried out for 15s at the temperature of 95 ℃,
(4) the extension is carried out for 1min at the temperature of 60 ℃,
(3) 40 cycles of (4).
The use method provided by the invention is also characterized in that the result judgment is as follows:
when Ct values of VIC and FAM channels are less than or equal to 32, the amplification curve has an obvious exponential amplification period, and the amplification curve is judged to be positive according to a positive control result; when the channel VIC and the channel FAM have no Ct value or the Ct value is more than or equal to 38, judging the channel VIC and the channel FAM to be negative according to a negative control result; when the reference positive control result is positive and the reference negative control result is negative, the genotyping result of the test sample is determined as shown in table 5.
The detection reagent for SNP locus genotype detection, the application of the detection reagent in preparing the kit for medication guidance of schizophrenic patients, the corresponding kit and the use method thereof, provided by the invention, have the advantages that the primer group included in the detection reagent can be specifically and accurately used for genotyping of rs2239063 locus of CACNA1C, rs 169921385 locus of SLC1A1 and rs17022006 locus of CNTN4, so that the detection reagent can be used for evaluation basis of medication guidance of schizophrenic patients and research and development guidance of related medicines.
Detailed Description
The following describes specific embodiments of the present invention. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.
The methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
The SNP site genotypes, i.e., single nucleotide polymorphisms, referred to in the examples are as follows: mainly refers to DNA sequence polymorphism caused by single nucleotide variation on genome level, SNP is the most common variation form in human genome DNA sequence, and is estimated to account for a very small part of millions of SNPs, which have great influence on diseases and drug treatment.
Example 1
This example specifically illustrates a primer set and a kit that can be used for a gene group for drug administration guidance of a schizophrenia patient.
The embodiment provides a detection reagent for SNP locus genotype detection, wherein the targeted SNP loci are rs2239063 locus of gene CACNA1C, rs 169921385 locus of gene SLC1A1 and rs17022006 locus of gene CNTN4 respectively.
The detection reagent comprises: the primer group comprises a primer pair 1 for specifically amplifying a rs2239063 site of a gene CACNA1C to obtain a first amplification product, a primer pair 2 for specifically amplifying a rs 169921385 site of a gene SLC1A1 to obtain a second amplification product, and a primer pair 3 for specifically amplifying a rs17022006 site of a gene CNTN4 to obtain a third amplification product, wherein nucleotide sequences of an upstream primer and a downstream primer of the primer pair 1 are respectively shown as SEQ ID No.1 and SEQ ID No.2, nucleotide sequences of an upstream primer and a downstream primer of the primer pair 2 are shown as SEQ ID No.3 and SEQ ID No.4, and nucleotide sequences of an upstream primer and a downstream primer of the primer pair 3 are shown as SEQ ID No.5 and SEQ ID No.6 (Table 1).
The detection reagent further comprises: a probe group comprising a first probe group for hybridizing to the first amplification product, a second probe group for hybridizing to the second amplification product, and a third probe group for hybridizing to the third amplification product, wherein each of the first probe group, the second probe group, and the third probe group comprises: a first TaqMan probe marked by 5 'end VIC and 3' end MGB, and a second TaqMan probe marked by 5 'end FAM and 3' end MGB. That is, each probe set includes the first TaqMan probe and the second TaqMan probe described above. Specifically, the nucleotide sequences of the first TaqMan probe and the second TaqMan probe of the first probe set are respectively shown as SEQ ID No.7 and SEQ ID No.8, the nucleotide sequences of the first TaqMan probe and the second TaqMan probe of the second probe set are respectively shown as SEQ ID No.9 and SEQ ID No.10, and the nucleotide sequences of the first TaqMan probe and the second TaqMan probe of the third probe set are respectively shown as SEQ ID No.11 and SEQ ID No.12 (Table 2).
Wherein, the 5 ' end VIC mark and the 5 ' end FAM mark respectively indicate that the 5 ' end is marked with different fluorescent reporter groups, and the 3 ' end MGB mark indicates that the 3 ' end is marked with fluorescent group quenching.
The detection reagent of the embodiment further comprises: DNA polymerase, negative control sample, positive control sample.
Wherein the DNA polymerase can be one or more of GoldStar TaqMan Mix, TaqPath ProAmp MultiplexMaster Mix, TaqMan GTXpress Master Mix.
The negative control sample is used for correcting system errors in the SNP locus genotype detection process, namely, detecting whether a reaction system is polluted or not, and ensuring that the reaction detection result is accurate and reliable, and particularly, ddH is adopted2O as a negative control sample.
In this embodiment, the positive control sample is genomic DNA containing an rs2239063 site AC genotype of gene CACNA1C, an rs 1691385 site AG genotype of SLC1a1, and an rs17022006 site GA genotype of CNTN4, and the concentration of the genomic DNA is 1ng/μ L.
The quality control of the PCR reaction system is also used for quality control purposes to monitor and detect whether the reaction system can be normally used.
The detection reagent provided by the embodiment can be applied to the preparation of a kit for medication guidance of schizophrenic patients, so that the kit for detecting the genotype of the SNP sites for medication guidance of the schizophrenic patients is also provided by the embodiment, and the SNP sites are the sites.
In the kit provided by the embodiment, the SNP locus genotype is detected based on a fluorescent quantitative PCR TaqMan MGB probe method, and in use, the concentration of an upstream primer and the concentration of a downstream primer in a PCR reaction system aiming at each SNP locus genotype are both 0.2-1.2 mu M, and preferably, the concentration of the upstream primer and the concentration of the downstream primer are both 0.7 mu M. In addition, in the kit, in a PCR reaction system aiming at each SNP locus genotype, the concentration of the first TaqMan probe and the concentration of the second TaqMan probe are both 0.1-0.8 mu M, and preferably, the concentration of the first TaqMan probe and the concentration of the second TaqMan probe are both 0.25 mu M.
Wherein, the QPCR (real-time fluorescence quantification) working principle is as follows: the probe used in the invention is a TaqMan probe marked by VIC/FAM at the 5 'end and MGB at the 3' end, and the two ends of the probe are respectively marked with a fluorescence reporter group (R) and a fluorescence quenching group (Q). When the probe is complete, namely in a random state and a PCR product-free hybridization state, the fluorescence emitted by the reporter group is absorbed by the quencher group, and the existence of the fluorescence cannot be detected. In the qPCR amplification process, when a specific PCR product and a TaqMan MGB probe are subjected to hybridization reaction, the 5' -end exonuclease activity of the DNA polymerase with strong tolerance cuts the bases of the TaqMan MGB probe one by one, and the fluorescence released by the reporter group can be detected by a fluorometer which is arranged in a PCR instrument. After one cycle of PCR, the fluorescent signal is the same as the target fragment, and has a synchronous exponential amplification process, and the strength of the fluorescent signal represents the copy number of the template DNA. Therefore, the method is a good genotyping method.
In this embodiment, the method of using the kit is as follows:
the method for detecting the SNP locus genotype based on the fluorescent quantitative PCR TaqMan MGB probe method specifically comprises the following steps:
step 1, obtaining a sample to be detected:
the sample to be tested is a biological sample containing genome DNA,
the biological sample is derived from whole blood or oral mucosa exfoliated cells, and specifically, in the present embodiment, genomic DNA is extracted from whole blood or oral mucosa exfoliated cells as template DNA;
step 2, identifying the DNA concentration and purity:
identifying by using an ultraviolet spectrophotometer, calculating the DNA purity by using the ratio of OD260nm/OD280nm, wherein the ratio is 1.7-2.0, and preferably, using ddH (ddH) for DNA2Diluting the O to 5-15 ng/mu L;
and 3, genotyping:
the TaqMan MGB probe method (Life, TaqPath promoter Master Mix) is used for genotyping three sites of CACNA1Crs2239063, SLC1A1 rs 169921385 and CNTN4rs17022006, and the adopted primer group and probe group are as above.
A. The specific PCR amplification system for each SNP locus genotype detection is shown in Table 3.
The corresponding templates for genotype detection of each SNP locus are divided into: a sample to be tested, a negative control sample and a positive control sample. And adding an upstream primer, a downstream primer, a first TaqMan probe and a second TaqMan probe corresponding to the upstream primer, the downstream primer, the first TaqMan probe and the second TaqMan probe in tables 1 and 2 for genotype detection of each SNP locus, and respectively adding different templates for parallel reaction to respectively obtain a reference positive control result, a reference negative control result and a genotyping result. Wherein the adding concentration of the sample to be detected is 5-15 ng/muL, and the adding concentration of the positive control sample is 1 ng/muL.
In the finally obtained PCR reaction system, the concentrations of the upstream primer and the downstream primer are both 0.2-1.2 mu M, and preferably both 0.7 mu M; the first TaqMan probe and the second TaqMan probe are contained at concentrations of 0.1-0.8. mu.M, preferably 0.25. mu.M, respectively.
B. qPCR reaction amplification procedure (Table 4)
C. And (4) interpretation of results:
when the Ct values of the VIC channel and the FAM channel are less than or equal to 32, the amplification curve has an obvious exponential amplification period, and the result is read as positive according to a positive control result;
when the channel VIC and the FAM have no Ct value or the Ct value is more than or equal to 38, the amplification curve is indicated to be absent, or the amplification curve is a straight line or a slight oblique line and has no obvious exponential growth period, and the result is judged to be negative according to a negative control result;
when the reference positive control result is positive and the reference negative control result is negative, the genotyping results of the test samples are determined as shown in table 5:
the detection reagent for SNP locus genotype detection, the application of the detection reagent in preparing the kit for medication guidance of schizophrenic patients, the corresponding kit and the use method thereof, which are provided by the embodiment 1, can be used for the evaluation basis of the medication guidance of schizophrenic patients and the research and development guidance of related medicines because the primer group included in the detection reagent can be specifically and accurately used for genotyping of the rs2239063 site of CACNA1C, the rs 169921385 site of SLC1A1 and the rs17022006 site of CNTN 4.
In addition, the application method of the kit provided in embodiment 1, by using the primer group and the probe group designed by the present invention, can select the real-time fluorescence quantitative PCR assay with simple and rapid operation, high throughput and accurate interpretation result, which is more convenient for large-scale clinical or health detection.
Example 2
In this example, a generation sequencing method as gold standard and the detection reagent, the corresponding kit and the corresponding method provided in example 1 are respectively adopted for some cases of schizophrenia, samples of cases are obtained, and the rs2239063 site of the gene CACNA1C, the rs 169921385 site of the gene SLC1a1 and the rs17022006 site of the gene CNTN4 are detected, and the genotyping detection results are shown in table 6.
The above results show that: the genotyping results obtained using the kit of example 1 were 100% identical to the genotyping results obtained by first-generation sequencing.
It can be seen that the detection reagent, the corresponding kit and the method of embodiment 1 are used for detection, and the detection result of the variation of the SNP site is highly reliable, which shows that the detection reagent, the corresponding kit and the use method of embodiment 1 can accurately detect the genotyping of the rs2239063 site of CACNA1C, the rs 169921385 site of gene SLC1a1 and the rs17022006 site of gene CNTN4, so that the reagent, the corresponding kit and the use method can be accurately used for evaluation basis of medication guidance, research and development of corresponding drugs, and the like of schizophrenia patients.
Moreover, by adopting the advantages of real-time fluorescence quantitative PCR, compared with the genotyping technologies such as gene sequencing and the like, the detection of the genotyping of the locus CACNA1C rs2239063, SLC1A1 rs 169921385 and CNTN4rs17022006 also has the following advantages:
1. the specificity is strong: the designed primer pairs are respectively directed at specific sequences of CACNA1C rs2239063, SLC1A1 rs 169921385 and CNTN4rs17022006, and can specifically amplify corresponding genome DNA; probes designed aiming at the amplification products of CACNA1C rs2239063, SLC1A1 rs 169921385 and CNTN4rs17022006 respectively increase the single base difference of MGB which can be effectively distinguished;
2. the detection sensitivity of the kit can reach 1 percent and is far higher than the sensitivity of 15 percent of gene sequencing;
3. the detection process is a closed tube reaction, so that the possibility of pollution and result deviation is greatly reduced;
4. the operation is quick and simple, and the result can be completed within 3 hours from the sample submission. The gene sequencing method has complicated detection steps: the method comprises the steps of specimen inspection → DNA extraction → PCR amplification → verification of PCR products (electrophoresis) → purification of PCR products → gene sequencing → result analysis, wherein the electrophoresis process of the two PCR products has high pollution probability and is not suitable for large-scale development in hospitals;
5. the interpretation result is clear and objective;
6. high flux;
7. the PCR product is safe, does not contain toxic and harmful substances in the whole system, does not need post-treatment of the PCR product, and is harmless to operators and the environment.
In a word, compared with genotyping technologies such as gene sequencing and the like, the kit provided in the embodiment 1 for genotyping of CACNA1Crs2239063, SLC1a1 rs 169921385 and CNTN4rs17022006 sites has the advantages of strong specificity, high sensitivity, simple and rapid operation, high flux, accurate interpretation result, simplicity and convenience and the like, so that the problems of time consumption, complex program, easy pollution and the like in the existing genotyping detection technology can be solved. Moreover, the kit of embodiment 1 can rapidly, accurately, cheaply and high-flux perform typing on the genes CACNA1C rs2239063, SLC1A1 rs 16994 and CNTN4rs17022006, has high sensitivity and strong specificity, is suitable for common samples such as blood, oral mucosa cast-off cells and the like, can rapidly, cheaply and accurately detect different types of gene loci of CACNA1C rs2239063, SLC1A1 rs 169921385 and CNTN4rs17022006 in human blood or other tissue cells at high flux, is the method with the best cost performance for typing technology at present, and is more convenient for clinical or health detection large-scale implementation.
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aatcgaagca ccaaata 17
Claims (14)
1. A detection reagent for detecting SNP locus genotypes, the SNP loci are rs2239063 locus of gene CACNA1C, rs 169921385 locus of gene SLC1A1 and rs17022006 locus of gene CNTN4 respectively, which is characterized by comprising:
a primer group, which comprises a primer pair 1 for specifically amplifying the rs2239063 site of the gene CACNA1C to obtain a first amplification product, a primer pair 2 for specifically amplifying the rs 169921385 site of the gene SLC1A1 to obtain a second amplification product, and a primer pair 3 for specifically amplifying the rs17022006 site of the gene CNTN4 to obtain a third amplification product,
wherein the nucleotide sequences of the upstream primer and the downstream primer of the primer pair 1 are respectively shown as SEQ ID NO.1 and SEQ ID NO.2,
the nucleotide sequences of the upstream primer and the downstream primer of the primer pair 2 are shown as SEQ ID NO.3 and SEQ ID NO.4,
the nucleotide sequences of the upstream primer and the downstream primer of the primer pair 3 are shown as SEQ ID NO.5 and SEQ ID NO. 6.
2. The detection reagent according to claim 1, further comprising:
a probe population comprising a first probe set for hybridizing to the first amplification product, a second probe set for hybridizing to the second amplification product, and a third probe set for hybridizing to the third amplification product.
3. The detection reagent according to claim 2, wherein:
wherein, each of the first probe group, the second probe group and the third probe group respectively comprises: a first TaqMan probe marked by 5 'end VIC and 3' end MGB, and a second TaqMan probe marked by 5 'end FAM and 3' end MGB.
4. The detection reagent according to claim 3, wherein:
wherein the nucleotide sequences of the first TaqMan probe and the second TaqMan probe of the first probe set are respectively shown as SEQ ID NO.7 and SEQ ID NO.8,
the nucleotide sequences of the first TaqMan probe and the second TaqMan probe of the second probe set are respectively shown as SEQ ID NO.9 and SEQ ID NO.10,
the nucleotide sequences of the first TaqMan probe and the second TaqMan probe of the third probe set are respectively shown as SEQ ID NO.11 and SEQ ID NO. 12.
5. The detection reagent according to any one of claims 1 to 4, further comprising:
a DNA polymerase;
a negative control sample for system error correction in the SNP locus genotype detection process;
and the positive control sample is used for controlling the quality of a PCR reaction system in the SNP locus genotype detection process.
6. The detection reagent according to claim 5, wherein:
wherein the DNA polymerase is one or more of GoldStar TaqMan Mix, TaqPath ProAmp MultiplexMaster Mix and TaqMan GTXpress Master Mix.
7. The detection reagent according to claim 5, wherein:
wherein the negative control sample is ddH2O。
8. The detection reagent according to claim 5, wherein:
wherein the positive control sample is genomic DNA containing rs2239063 site AC genotype of gene CACNA1C, rs 1691385 site AG genotype of SLC1A1 and rs17022006 site GA genotype of CNTN4, and the concentration of the genomic DNA is 1 ng/muL.
9. Use of a detection reagent for the manufacture of a kit for use in medication guidance for schizophrenic patients, characterized in that:
wherein the detection reagent is the detection reagent according to any one of claims 1 to 8.
10. A kit for detecting a genotype of an SNP site for schizophrenia medication guidance, comprising:
the detection reagent according to any one of claims 1 to 8.
11. The kit of claim 10, wherein:
the kit detects the SNP locus genotype based on a fluorescent quantitative PCR TaqMan MGB probe method, and in use, the concentration of an upstream primer and the concentration of a downstream primer in a PCR reaction amplification system aiming at each SNP locus genotype are both 0.2-1.2 mu M.
12. The kit of claim 11, wherein:
wherein the concentration of the upstream primer and the concentration of the downstream primer are both 0.7 mu M.
13. The kit according to claim 10 or 12, characterized in that:
wherein, when the kit is used, the concentration of the first TaqMan probe and the second TaqMan probe in a PCR reaction system aiming at the genotype of each SNP locus is 0.1-0.8 mu M.
14. The kit of claim 13, wherein:
wherein the concentration of the first TaqMan probe and the second TaqMan probe is 0.25 mu M.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113913521A (en) * | 2021-11-05 | 2022-01-11 | 复旦大学 | Genotyping detection kit and application thereof |
| CN115579056A (en) * | 2022-08-24 | 2023-01-06 | 南方医科大学南方医院 | Gene group for evaluating schizophrenia molecular typing and diagnostic product and application thereof |
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2020
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113913521A (en) * | 2021-11-05 | 2022-01-11 | 复旦大学 | Genotyping detection kit and application thereof |
| CN113913521B (en) * | 2021-11-05 | 2024-05-14 | 复旦大学 | Genotyping detection kit and application thereof |
| CN115579056A (en) * | 2022-08-24 | 2023-01-06 | 南方医科大学南方医院 | Gene group for evaluating schizophrenia molecular typing and diagnostic product and application thereof |
| CN115579056B (en) * | 2022-08-24 | 2024-05-31 | 南方医科大学南方医院 | Gene group for evaluating molecular typing of schizophrenia, and diagnostic product and application thereof |
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Effective date of registration: 20221223 Address after: 11 / F, building 1, 2555 xiupu Road, Pudong New Area, Shanghai, 201319 Patentee after: Shanghai Enyuan Biotechnology Co.,Ltd. Patentee after: Shanghai Enyuan Medical Laboratory Co.,Ltd. Address before: 11 / F, building 1, 2555 xiupu Road, Pudong New Area, Shanghai, 201319 Patentee before: Shanghai Enyuan Biotechnology Co.,Ltd. |