WO2021239081A1 - 一种能够用于检测npc1l1突变基因分型的试剂、试剂盒及其使用方法、应用 - Google Patents

一种能够用于检测npc1l1突变基因分型的试剂、试剂盒及其使用方法、应用 Download PDF

Info

Publication number
WO2021239081A1
WO2021239081A1 PCT/CN2021/096565 CN2021096565W WO2021239081A1 WO 2021239081 A1 WO2021239081 A1 WO 2021239081A1 CN 2021096565 W CN2021096565 W CN 2021096565W WO 2021239081 A1 WO2021239081 A1 WO 2021239081A1
Authority
WO
WIPO (PCT)
Prior art keywords
npc1l1
probe
detecting
kit
genotyping
Prior art date
Application number
PCT/CN2021/096565
Other languages
English (en)
French (fr)
Inventor
欧阳冬生
李晓晖
李超鹏
侯利平
李湘
费云舟
陈亮
陈露露
王婷
Original Assignee
长沙都正生物科技股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 长沙都正生物科技股份有限公司 filed Critical 长沙都正生物科技股份有限公司
Publication of WO2021239081A1 publication Critical patent/WO2021239081A1/zh

Links

Images

Classifications

    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • 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
    • C12Q1/6876—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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • 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
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • the embodiment of the present invention relates to the field of biotechnology, in particular to a reagent, a kit, and a method of use and application thereof that can be used to detect NPC1L1 mutation genotyping.
  • Cholesterol is an important component of biofilms, and also a precursor for the synthesis of sterol hormones and bile acids. Excessive intake of cholesterol can cause many diseases, including cardiovascular and cerebrovascular diseases. Hypercholesterolemia is one of the most important risk factors for atherosclerotic cardiovascular diseases (including coronary heart disease, ischemic stroke, transient ischemic attack, atherosclerotic aortic disease, and peripheral artery disease). Cholesterol will effectively reduce the incidence and mortality of related cardiovascular diseases. The use of statins to reduce cholesterol levels has been recommended by a number of cardiovascular disease prevention guidelines as the core strategy for primary and secondary prevention of atherosclerotic cardiovascular disease. However, in clinical practice, many patients still fail to reach the target level of cholesterol after receiving statin treatment.
  • the Niemann-Pick C1-like 1 (NPC1L1) transporter on the intestinal mucosa is a key transmembrane transporter responsible for the absorption of cholesterol in the intestine.
  • Selective cholesterol absorption inhibitors can reduce cholesterol absorption and lower serum cholesterol levels by inhibiting the function of NPC1L1 protein.
  • Selective cholesterol absorption inhibitors provide new options for safer and effective reduction of serum cholesterol.
  • the IMPROVEIT study shows that the addition of ezetimibe to simvastatin in ACS patients can further reduce cardiovascular events.
  • Hezeimibe is a new type of cholesterol absorption inhibitor that is currently undergoing clinical research.
  • NPC1L1 protein is the main drug target of Hezemibe.
  • the detection methods for gene mutation include polymerase chain reaction linked restriction fragment length polymorphism (PCR-RFLP), direct sequencing method, qPCR TaqMan probe method, etc.
  • PCR-RFLP polymerase chain reaction linked restriction fragment length polymorphism
  • direct sequencing is a more accurate genotyping method, but it has the disadvantages of complicated operations, long sequencing cycles, and the need for special software to interpret sequencing results, which is inconvenient for clinical use.
  • the TaqMan probe method Compared with the traditional PCR-RFLP method, the TaqMan probe method has the following advantages: First, the TaqMan fluorescent probe has strong specificity and can identify single base mismatches, deletions or mutations in the target sequence; second, the TaqMan probe sensitivity High, reproducible, detection sensitivity up to 2 ⁇ 10 3 copies; third, TaqMan probes are time-saving, convenient, and high-throughput, which can integrate PCR amplification, fluorescence probe hybridization, and detection in one test tube Chemical; Fourth, the TaqMan probe avoids pollution, reduces errors, and has good safety.
  • the qPCR automatic detection method based on TaqMan probe method has great potential as a NPC1L1 genotyping detection method, and it is expected to become faster and more common.
  • One of the more economical methods Since there are multiple mutations in the NPC1L1 gene, if different genotypes can be identified, it will be able to better assist in predicting the effect of cholesterol absorption inhibitors.
  • a reagent capable of detecting NPC1L1 mutation genotyping is provided.
  • a kit for detecting genotyping of NPC1L1 mutations prepared by using the above reagents is provided.
  • a method for using the above kit for detecting genotyping of NPC1L1 mutations is provided.
  • an application of the above-mentioned reagent for detecting NPC1L1 mutation genotyping in preparing a detection reagent for predicting the curative effect of cholesterol absorption inhibitors is provided.
  • a reagent capable of detecting NPC1L1 mutation genotyping which comprises a specific primer set and a specific probe, wherein the specific primer set includes nucleotides The sequence of the primer 1 shown in SEQ ID No. 1 and the nucleotide sequence of the primer 2 shown in SEQ ID No. 2;
  • the specific probe includes probe 1 whose nucleotide sequence is shown in SEQ ID No. 3 and probe 2 whose nucleotide sequence is shown in SEQ ID No. 4.
  • the specific probe is a TaqMan probe, and the 5'end of the specific probe is labeled with a reporter fluorescent group.
  • the reporter fluorophore on probe 1 is different from the reporter fluorophore on probe 2.
  • the reporter fluorescent group includes but is not limited to 6-carboxy-fluorescein (6-carboxy-fluorescein, FAM), tetrachloro-6-carboxy-fluorescein (Tetrachlorofluorescein, TET), 2,7-dimethyl-4,5-dichloro-6-carboxyl fluorescein (2,7-dimethyl-4,5-dichloro-6-carboxyl fluorescein, JOE) and hexachloro-6-methyl fluorescein (Hexachlorofluorescein, HEX).
  • the reporter fluorophore is selected from FAM, TET, VIC, JOE, or HEX.
  • the 3'end of the specific probe is labeled with a quencher fluorescent group;
  • the quencher fluorescent group includes but is not limited to 6-carboxytetramethylrhodamine (Carboxytetramethylrhodamine, TAMRA), Non-Fluorescent Quencher (NFQ) and Black Hole Quencher (BHQ).
  • the fluorescent quenching group is selected from TAMRA, NFQ, or BHQ.
  • a minor Groove Binder (MGB) modification group is attached to the quenching fluorescent group.
  • the technical solution of the embodiment of the second aspect of the present invention is: a kit that can be used to detect NPC1L1 mutation genotyping, which includes the above-mentioned reagents.
  • the kit further includes UNG enzyme (Uracil-N-glycosylase, uracil-N-glycosylase) and dUTP (2'-deoxyuridine 5'-triphosphate).
  • UNG enzyme Uracil-N-glycosylase, uracil-N-glycosylase
  • dUTP 2,3-deoxyuridine 5'-triphosphate
  • the kit further includes a PCR master mix and a quality control plasmid
  • the quality control plasmid includes a synthetic plasmid with a nucleotide sequence comprising the wild-type gene sequence shown in SEQ ID No. 5 and a nuclear
  • the nucleotide sequence contains a synthetic plasmid with the mutant gene sequence shown in SEQ ID No.6.
  • the kit further includes a blank control, a positive control, and a negative control.
  • the positive control includes a positive plasmid; the negative control includes a negative plasmid; and the blank control is PCR-grade water.
  • the technical solution of the embodiment of the third aspect of the present invention is: a method of using a kit that can be used to detect NPC1L1 mutation genotyping, including the following steps:
  • step S2 Use the aforementioned kit that can be used to detect NPC1L1 mutation genotyping to process the genomic DNA sample obtained in step S1, and set the instrument reaction process and parameters for qPCR amplification detection;
  • the technical solution of the embodiment of the fourth aspect of the present invention is: an application of a kit that can be used to detect NPC1L1 mutation genotyping.
  • the above-mentioned genotyping kit for detecting NPC1L1 mutations is used to predict the efficacy of cholesterol absorption inhibitors.
  • the research is not for the purpose of disease diagnosis and treatment.
  • the application of the above-mentioned reagents that can be used to detect the genotyping of NPC1L1 mutations in the preparation of detection reagents for predicting the efficacy of cholesterol absorption inhibitors is provided.
  • the cholesterol absorption inhibitor is Hezemibe.
  • Figure 1 is an electrophoresis chart of the PCR product in Example 1 of the present invention.
  • Figure 2 is a qPCR map in Example 1 of the present invention.
  • Figure 3 is a sequencing map of the NPC1L1 g1679 CC type sample in Example 1 of the present invention.
  • Fig. 4 is a sequencing chart of the NPC1L1 g1679 GG type sample in Example 1 of the present invention.
  • Fig. 5 is a sequencing chart of the NPC1L1 g1679 GC type sample in Example 1 of the present invention.
  • FIG. 6 is a schematic diagram of the application process in Embodiment 2 of the present invention.
  • a reagent capable of detecting NPC1L1 mutation genotyping which comprises a specific primer set and a specific probe, wherein the specific primer set includes nucleotides The sequence of the primer 1 shown in SEQ ID No. 1 and the nucleotide sequence of the primer 2 shown in SEQ ID No. 2;
  • the specific probe includes probe 1 whose nucleotide sequence is shown in SEQ ID No. 3 and probe 2 whose nucleotide sequence is shown in SEQ ID No. 4.
  • the specific probe is a TaqMan probe, and the 5'end of the specific probe is labeled with a reporter fluorescent group.
  • the reporter fluorophore on probe 1 is different from the reporter fluorophore on probe 2.
  • the reporter fluorescent group includes but is not limited to 6-carboxy-fluorescein (6-carboxy-fluorescein, FAM), tetrachloro-6-carboxy-fluorescein (Tetrachlorofluorescein, TET), 2,7-dimethyl-4,5-dichloro-6-carboxyl fluorescein (2,7-dimethyl-4,5-dichloro-6-carboxyl fluorescein, JOE) and hexachloro-6-methyl fluorescein (Hexachlorofluorescein, HEX).
  • the reporter fluorophore is selected from FAM, TET, VIC, JOE, or HEX.
  • the 3'end of the specific probe is labeled with a quencher fluorescent group;
  • the quencher fluorescent group includes but is not limited to 6-carboxytetramethylrhodamine (Carboxytetramethylrhodamine, TAMRA), Non-Fluorescent Quencher (NFQ) and Black Hole Quencher (BHQ).
  • the fluorescent quenching group is selected from TAMRA, NFQ, or BHQ.
  • a minor Groove Binder (MGB) modification group is attached to the quenching fluorescent group.
  • the technical solution of the embodiment of the second aspect of the present invention is: a kit that can be used to detect NPC1L1 mutation genotyping, which includes the above-mentioned reagents.
  • the kit further includes UNG enzyme (Uracil-N-glycosylase, uracil-N-glycosylase) and dUTP (2'-deoxyuridine 5'-triphosphate).
  • UNG enzyme Uracil-N-glycosylase, uracil-N-glycosylase
  • dUTP 2,3-deoxyuridine 5'-triphosphate
  • the kit further includes a PCR master mix and a quality control plasmid
  • the quality control plasmid includes a synthetic plasmid with a nucleotide sequence comprising the wild-type gene sequence shown in SEQ ID No. 5 and a nuclear
  • the nucleotide sequence contains a synthetic plasmid with the mutant gene sequence shown in SEQ ID No.6.
  • the kit further includes a blank control, a positive control, and a negative control.
  • the positive control includes a positive plasmid; the negative control includes a negative plasmid; and the blank control is PCR-grade water.
  • the technical solution of the embodiment of the third aspect of the present invention is: a method of using a kit that can be used to detect NPC1L1 mutation genotyping, including the following steps:
  • step S2 Use the aforementioned kit that can be used to detect NPC1L1 mutation genotyping to process the genomic DNA sample obtained in step S1, and set the instrument reaction process and parameters for qPCR amplification detection;
  • the technical solution of the embodiment of the fourth aspect of the present invention is: an application of a kit that can be used to detect NPC1L1 mutation genotyping.
  • the above-mentioned genotyping kit for detecting NPC1L1 mutations is used to predict the efficacy of cholesterol absorption inhibitors.
  • the research is not for the purpose of disease diagnosis and treatment.
  • the application of the above-mentioned reagents that can be used to detect the genotyping of NPC1L1 mutations in the preparation of detection reagents for predicting the efficacy of cholesterol absorption inhibitors is provided.
  • the cholesterol absorption inhibitor is Hezemibe.
  • a genotyping kit that can be used to detect NPC1L1 mutations.
  • the kit includes the following reagents: a primer set and a specific probe.
  • the primer set includes an upstream primer and a downstream primer, wherein, The upstream reaction primer is primer 1 and the downstream reaction primer is primer 2, and the corresponding nucleotide sequences are SEQ ID No. 1 and SEQ ID No. 2 respectively; specific probes include those used to set NPC1L1 g1679 C>G (rs2072183)
  • the PCR probes for genotype discrimination are probe 1 and probe 2, respectively.
  • the probe nucleotide sequence corresponding to probe 1 is SEQ ID No. 3, and the probe nucleotide sequence corresponding to probe 2 is SEQ ID No.4.
  • the detection process includes the following steps:
  • the concentration and purity of the extracted DNA were measured using a micro-spectrophotometer (Nanodrop 2000) from Thermo Fisher Scientific, and the DNA was uniformly diluted to 50 ng/ ⁇ L. At the same time, DNA purity was detected by agarose gel electrophoresis.
  • ABI 7500 Quantitative Real-time PCR (Thermo Fisher Technology, USA) to perform fluorescent quantitative PCR reaction, where the upstream reaction primer is primer 1 and the downstream reaction primer is primer 2, primer 1 and primer 2
  • the corresponding nucleotide sequences are SEQ ID No. 1, SEQ ID No. 2; in order to distinguish NPC1L1 g1679 C>G (rs2072183) genotypes, a total of two fluorescent quantitative PCR probes were designed, namely probe 1 and Probe 2, the probe nucleotide sequence corresponding to probe 1 is SEQ ID No. 3, and the probe nucleotide sequence corresponding to probe 2 is SEQ ID No. 4.
  • reaction system is as follows:
  • reaction parameters are as follows:
  • the electrophoresis pattern of the PCR product is shown in Figure 1.
  • Select FAM and VIC channels and collect fluorescence in the annealing/extension phase.
  • Figure 2 shows the qPCR amplification map.
  • the probe of this embodiment is labeled with a fluorescent group, wherein the 5'end of the probe 1 is labeled with a FAM group, the 3'end is labeled with an MGB modification group, and the 5'end of the probe 2 is labeled with a VIC group. The 3'end is marked with an MGB modification group.
  • test results of the samples to be tested can be interpreted according to the following table:
  • FAM channel VIC channel Test results No fluorescent signal No fluorescent signal The test failed, it is recommended to do it again With fluorescent signal No fluorescent signal CC type With fluorescent signal With fluorescent signal GC type No fluorescent signal With fluorescent signal GG type
  • the scheme of the embodiment of the present invention can quickly judge the genotype result based on the fluorescent signal, and the operation and judgment are simple and convenient.
  • the detection samples of the embodiment of the present invention are detected by the Sanger sequencing method, Shenzhen Huada Gene is entrusted to perform Sanger sequencing on all DNA samples, and the sequencing results are compared with the reference sequence to determine the genotype.
  • Figure 3, Figure 4, and Figure 5 are the sequencing maps of NPC1L1 g1679 CC, NPC1L1 g1679 GG, and NPC1L1 g1679 GC samples, respectively.
  • the embodiments of the present invention have the same sensitivity and accuracy, and have the advantages of simpler, faster operation, easy-to-read results, and lower price.
  • a genotyping kit for detecting NPC1L1 mutations in predicting the efficacy of cholesterol absorption inhibitors includes: a specific primer set , Specific probe, UNG enzyme, PCR master mix and quality control plasmid.
  • the sequence of primers and probes are the same as in Example 1.
  • the quality control plasmid includes nucleotide sequence including the wild-type gene sequence shown in SEQ ID No.5
  • group A is atorvastatin (10mg) group 98 cases
  • group B is Hezemibu (HS -25) (20mg) group 105 cases
  • C group is HS-25 (20mg) and atorvastatin (10mg) combination group 99 cases
  • D group is HS-25 (10mg) group 96 cases
  • E group is HS -25 (10mg) and atorvastatin (10mg) combined use group of 100 cases
  • F group is the placebo group 99 cases.
  • Group B 0.0000 To 0.0000 0.1509 0.0000 0.0000
  • Group C 0.0000 0.0000 To 0.0000 0.9077 0.0000
  • Group D 0.0000 0.1509 0.0000 To 0.0000 0.0000
  • Group E 0.0000 0.0000 0.9077 0.0000 To 0.0000
  • Group F 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 To
  • the rs2072183 site mutation has a significant impact on the efficacy of Hezemibe: in the Hezemibe 20mg group (group B), CC, GC, The LDL_CH of GG type subjects decreased by 23.80%, 12.96%, 16.11% (p ⁇ 0.05); in the Hezemibu (10+20) mg group (B+D group), CC, GC, and GG types were tested The LDL_CH decreased by 20.55%, 11.77%, and 14.74% respectively (p ⁇ 0.05).
  • the 12-week decline ratio of LDL indicators compared to the baseline is divided into three levels of low, medium and high according to ⁇ 10%, 10%-25%, and >25%, and the analysis of variance and chi-square test are performed in each group.
  • Table 2 shows the P value of the overall ANOVA.
  • Group B Group C Group D Group E Group F Group B+D P value 0.9136 0.0128 0.2534 0.1885 0.4634 0.8827 0.0395
  • NPC1L1 can affect the efficacy of Hezetimibe.
  • the effect of rs2072183 genotype should be considered when Hezetimibe is used alone, and the effect on CC type Hezetimibe better.
  • the embodiment of the present invention will have a good application prospect in predicting the curative effect of cholesterol absorption inhibitors.
  • the kit for detecting the genotyping of NPC1L1 mutations designed by the embodiment of the present invention, it is possible to complete the evaluation of the efficacy of cholesterol absorption inhibitor drugs in different genotype groups. Therefore, the kit is used in the subsequent Hezemibu et al. The study of the efficacy of cholesterol absorption inhibitors also has a good effect.
  • NPC1L1 gene polymorphism can be used to predict the efficacy of cholesterol absorption inhibitors such as Hezemibu, and to develop related testing reagents to guide the precise medication of cholesterol absorption inhibitors.
  • the primers/probes of the embodiments of the present invention can be modified conventionally in the art, such as sulfurization, peptide nucleic acid formation, etc.
  • the above may cause a small amount of mismatches, but will not cause the entire DNA replication, synthesis and amplification The process cannot be carried out and will not cause significant fluctuations in the yield of PCR products. Therefore, a correct and sufficient fluorescent signal can be obtained. Therefore, for small changes in sequence, tagging, modification, etc., the new sequence generated should also include In the present invention.
  • the embodiment scheme of the present invention provides a reagent, method, kit for detecting NPC1L1 mutation genotyping based on the TaqMan probe method qPCR automatic detection method, and its application in predicting the curative effect of cholesterol absorption inhibitors.
  • This method has the advantages of high sensitivity, strong specificity, simple operation and low price.
  • the embodiment of the present invention is based on the NPC1L1 g1679 C>G (rs2072183) gene locus, which can quickly and effectively detect three genotypes at this locus, and can provide more effective information for predicting the curative effect of cholesterol absorption inhibitors.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种能够用于检测NPC1L1突变基因分型的试剂、试剂盒及其使用方法、应用，该检测NPC1L1突变基因分型的试剂，包括针对NPC1L1g1679C>G(rs2072183)基因位点的引物和探针，该探针为TaqMan探针。该试剂盒包含上述试剂，并使用TaqMan探针法qPCR全自动检测。在NPC1L1g1679C>G(rs2072183)基因位点的基因型中，CC型为对胆固醇吸收抑制剂敏感的族群,可作为医生用药的参考依据，指导治疗方式和用药。

Description

一种能够用于检测NPC1L1突变基因分型的试剂、试剂盒及其使用方法、应用
相关申请的交叉引用
本申请基于申请号为202010465791.0、申请日为2020年05月28日的中国专利申请提出，并要求该中国专利申请的优先权，该中国专利申请的全部内容在此引入本申请作为参考。
技术领域
本发明实施例涉及生物技术领域，具体涉及一种能够用于检测NPC1L1突变基因分型的试剂、试剂盒及其使用方法、应用。
背景技术
胆固醇是生物膜的重要组成成分，也是合成甾醇类激素和胆汁酸的前体。摄入过多的胆固醇会导致很多疾病，包括心脑血管疾病等。高胆固醇血症是动脉粥样硬化心血管疾病(包括冠心病、缺血性卒中、短暂脑缺血发作、动脉粥样硬化性主动脉疾病以及外周动脉疾病)极为重要的危险因素之一，降低胆固醇将有效降低相关心血管疾病的发病率和死亡率。应用他汀类药物降低胆固醇水平被多项心血管疾病防治指南推荐为动脉粥样硬化心血管疾病一级预防和二级预防的核心策略。但在临床实践中，许多患者接受他汀治疗后其胆固醇水平仍不能达到目标值。
肠黏膜上Niemann-Pick C1-like 1(NPC1L1)转运蛋白是负责肠道中胆固醇吸收的关键跨膜转运蛋白。选择性胆固醇吸收抑制剂可通过抑制NPC1L1蛋白功能，减少胆固醇吸收，降低血清胆固醇水平。选择性胆固醇吸收抑制剂为更安全、有效地减低血清胆固醇提供了新选择。IMPROVEIT研究表明，ACS患者在辛伐他汀基础上加用依折麦布能进一步降低心血管事件。
海泽麦布，是近来正在进行临床研究的一种新型胆固醇吸收抑制剂。NPC1L1蛋白是海泽麦布的主要药效靶点。NPC1L1编码基因存在丰富的遗传变异。其中，NPC1L1 g1679 C>G(rs2072183)是中国人群中较为常见的突变位点。研究已经证实，等位基因g1679 C(rs2072183)的携带者服用依折麦布后低密度脂蛋白降低更为显著(P＝0.028)。此外，NPC1L1基因突变对他汀类药物的反应也存在较大影响。目前研究表明，等位基因g1679 G(rs2072183)的携带者对他汀类药物的反应性明显降低。但尚未有通过检测该基因突变预测患者对海泽麦布等降脂药物疗效的报道，尤其是区分不同突变基因分型药效的报道。
目前，针对基因变异的检测方法有聚合酶链反应-限制性片段多态性(polymerase chain reaction linked restriction fragment length polymorphism，PCR-RFLP)、直接测序法、qPCR TaqMan探针法等。其中，PCR-RFLP法存在操作繁琐，电泳操作难以标准化，并且易产生PCR产物气溶胶造成环境污染和假阳性结果的缺陷，逐渐被临床淘汰。直接测序法是更为准确的基因分型方法，但存在：操作复杂，测序周期长，测序结果需要专门软件解读的缺陷，不便临床使用。而TaqMan探针法相较于传统的PCR-RFLP法优势在于：第一，TaqMan荧光探针特异性强，可识别靶序列中单个碱基的错配、缺失或突变；第二，TaqMan探针灵敏度高、重复性好，检测灵敏度可达2×10 3拷贝；第三，TaqMan探针省时、方便、高通量，可在一个试管内做到将PCR扩增、荧光探针杂交、检测一体化；第四，TaqMan探针避免污染、减少误差、安全性好。
综合考虑灵敏度、特异性、稳定性和操作自动化程度、通量高低等因素，基于TaqMan探针法qPCR全自动检测方法作为NPC1L1基因分型检测方法存在巨大潜力，将有望成为更快捷，更常见，更经济的方法之一。而由于NPC1L1基因存在多种突变方式，因此，若能识别出不同的基因分型将能够更好地辅助预测胆固醇吸收抑制剂的效果。
发明内容
有鉴于此，根据本发明第一方面的实施例：提供一种能够用于检测NPC1L1突变基因分型的试剂。
根据本发明第二方面的实施例：提供使用上述试剂制备的检测NPC1L1突变基因分型的试剂盒。
根据本发明第三方面的实施例：提供上述检测NPC1L1突变基因分型的试剂盒的使用方法。
根据本发明第四方面的实施例：提供一种上述检测NPC1L1突变基因分型的试剂在制备预测胆固醇吸收抑制剂疗效的检测试剂中的应用。
本发明第一方面的实施例的技术方案为：一种能够用于检测NPC1L1突变基因分型的试剂，其包含特异性引物组和特异性探针，其中所述特异性引物组包括核苷酸序列如SEQ ID No.1所示的引物1和核苷酸序列如SEQ ID No.2所示的引物2；
所述特异性探针包括核苷酸序列如SEQ ID No.3所示的探针1和核苷酸序列如SEQ ID No.4所示的探针2。
在一些实施例中，所述特异性探针为TaqMan探针，所述特异性探针的5’端标记有报告荧光基团。
在一些实施例中，所述探针1上的报告荧光基团与探针2上的报告荧光基团不同。
在一些实施例中，所述报告荧光基团包括但不限于6-羧基荧光素(6-carboxy-fluorescein，FAM)、四氯-6-羧基荧光素(Tetrachlorofluorescein，TET)、
Figure PCTCN2021096565-appb-000001
2,7-二甲基-4,5-二氯-6-羧基荧光素(2,7-dimethyl-4,5-dichloro-6-carboxyl fluorescein，JOE)和六氯-6-甲基荧光素(Hexachlorofluorescein，HEX)。在一些实施例中，所述报告荧光基团选自FAM、TET、VIC、JOE或HEX。
在一些实施例中，所述特异性探针的3’端标记有淬灭荧光基团；所述淬灭荧光基团包括但不限于6-羧基四甲基若丹明(Carboxytetramethylrhodamine，TAMRA)、非荧光淬灭基团(Non-Fluorescent Quencher，NFQ)和黑洞淬灭基团(Black Hole Quencher，BHQ)。在一些实施例中，所述淬灭荧光基团选自TAMRA、NFQ或BHQ。
在一些实施例中，所述淬灭荧光基团上连接有小沟结合粘结剂(Minor Groove Binder，MGB)修饰基团。
本发明第二方面的实施例的技术方案为：一种能够用于检测NPC1L1突变基因分型的试剂盒，其包含上述试剂。
在一些实施例中，所述试剂盒还包含UNG酶(Uracil-N-glycosylase，尿嘧啶-N-糖基化酶)和dUTP(2'-deoxyuridine 5'-triphosphate)。
在一些实施例中，所述试剂盒中还包含PCR预混液和质控质粒，所述质控质粒包括核苷酸序列包含如SEQ ID No.5所示的野生型基因序列的合成质粒和核苷酸序列包含如SEQ ID No.6所示的突变型基因序列的合成质粒。
在一些实施例中，所述试剂盒还包括空白对照、阳性对照和阴性对照，所述阳性对照包括阳性质粒；所述阴性对照包括阴性质粒；所述空白对照为PCR级水。
本发明第三方面的实施例的技术方案为：一种能够用于检测NPC1L1突变基因分型的试剂盒的使用方法，包括以下步骤：
S1、提取人体样本中的基因组DNA；
S2、使用上述能够用于检测NPC1L1突变基因分型的试剂盒处理步骤S1得到的基因组DNA样本，设置仪器反应流程和参数进行qPCR扩增检测；
S3、通过检测荧光变化进行分析。
本发明第四方面的实施例的技术方案为：一种能够用于检测NPC1L1突变基因分型的 试剂盒的应用。在一些情形中，使用上述用于检测NPC1L1突变基因分型试剂盒进行预测胆固醇吸收抑制制剂的疗效研究。
在一些实施例中，所述研究不以疾病诊断治疗为目的。
在一些实施例中提供了上述能够用于检测NPC1L1突变基因分型的试剂在制备预测胆固醇吸收抑制剂疗效的检测试剂中的应用。
在一些实施例中，所述胆固醇吸收抑制剂为海泽麦布。
本发明其他特征和相应的有益效果在说明书的后面部分进行阐述说明，且应当理解，至少部分有益效果从本发明说明书中的记载变得显而易见。
附图说明
图1为本发明实施例1中的PCR产物的电泳图谱；
图2为本发明实施例1中的qPCR图谱；
图3为本发明实施例1中的NPC1L1 g1679 CC型样本的测序图谱；
图4为本发明实施例1中的NPC1L1 g1679 GG型样本的测序图谱；
图5为本发明实施例1中的NPC1L1 g1679 GC型样本的测序图谱；
图6为本发明实施例2中的应用流程示意图。
具体实施方式
为详细说明本发明的技术内容、所实现目的及效果，以下结合实施方式并配合附图予以说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明第一方面的实施例的技术方案为：一种能够用于检测NPC1L1突变基因分型的试剂，其包含特异性引物组和特异性探针，其中所述特异性引物组包括核苷酸序列如SEQ ID No.1所示的引物1和核苷酸序列如SEQ ID No.2所示的引物2；
所述特异性探针包括核苷酸序列如SEQ ID No.3所示的探针1和核苷酸序列如SEQ ID No.4所示的探针2。
在一些实施例中，所述特异性探针为TaqMan探针，所述特异性探针的5’端标记有报告荧光基团。
在一些实施例中，所述探针1上的报告荧光基团与探针2上的报告荧光基团不同。
在一些实施例中，所述报告荧光基团包括但不限于6-羧基荧光素(6-carboxy-fluorescein，FAM)、四氯-6-羧基荧光素(Tetrachlorofluorescein，TET)、
Figure PCTCN2021096565-appb-000002
2,7-二甲基-4,5-二氯-6-羧基荧光素(2,7-dimethyl-4,5-dichloro-6-carboxyl fluorescein，JOE)和六氯-6-甲基荧光素(Hexachlorofluorescein，HEX)。在一些实施例中，所述报告荧光基团选自FAM、TET、VIC、JOE或HEX。
在一些实施例中，所述特异性探针的3’端标记有淬灭荧光基团；所述淬灭荧光基团包括但不限于6-羧基四甲基若丹明(Carboxytetramethylrhodamine，TAMRA)、非荧光淬灭基团(Non-Fluorescent Quencher，NFQ)和黑洞淬灭基团(Black Hole Quencher，BHQ)。在一些实施例中，所述淬灭荧光基团选自TAMRA、NFQ或BHQ。
在一些实施例中，所述淬灭荧光基团上连接有小沟结合粘结剂(Minor Groove Binder，MGB)修饰基团。
本发明第二方面的实施例的技术方案为：一种能够用于检测NPC1L1突变基因分型的试剂盒，其包含上述试剂。
在一些实施例中，所述试剂盒还包含UNG酶(Uracil-N-glycosylase，尿嘧啶-N-糖基化酶)和dUTP(2'-deoxyuridine 5'-triphosphate)。
在一些实施例中，所述试剂盒中还包含PCR预混液和质控质粒，所述质控质粒包括核苷酸序列包含如SEQ ID No.5所示的野生型基因序列的合成质粒和核苷酸序列包含如SEQ ID No.6所示的突变型基因序列的合成质粒。
在一些实施例中，所述试剂盒还包括空白对照、阳性对照和阴性对照，所述阳性对照包括阳性质粒；所述阴性对照包括阴性质粒；所述空白对照为PCR级水。
本发明第三方面的实施例的技术方案为：一种能够用于检测NPC1L1突变基因分型的试剂盒的使用方法，包括以下步骤：
S1、提取人体样本中的基因组DNA；
S2、使用上述能够用于检测NPC1L1突变基因分型的试剂盒处理步骤S1得到的基因组DNA样本，设置仪器反应流程和参数进行qPCR扩增检测；
S3、通过检测荧光变化进行分析。
本发明第四方面的实施例的技术方案为：一种能够用于检测NPC1L1突变基因分型的试剂盒的应用。在一些情形中，使用上述用于检测NPC1L1突变基因分型试剂盒进行预测胆固醇吸收抑制制剂的疗效研究。
在一些实施例中，所述研究不以疾病诊断治疗为目的。
在一些实施例中提供了上述能够用于检测NPC1L1突变基因分型的试剂在制备预测胆固醇吸收抑制剂疗效的检测试剂中的应用。
在一些实施例中，所述胆固醇吸收抑制剂为海泽麦布。
实施例1
根据本发明的一个实施例提供了一种能够用于检测NPC1L1突变基因分型试剂盒，该试剂盒中包含以下试剂：引物组和特异性探针，引物组包括上游引物和下游引物，其中， 上游反应引物为引物1和下游反应引物为引物2，对应的核苷酸序列分别为SEQ ID No.1、SEQ ID No.2；特异性探针包括用于将NPC1L1 g1679 C>G(rs2072183)基因型区分的PCR探针，分别为探针1和探针2，探针1对应的探针核苷酸序列为SEQ ID No.3，探针2对应的探针核苷酸序列为SEQ ID No.4。
其检测过程包括以下步骤：
S1、样本的提取：
临床收集100例健康志愿者，对每例患者静脉采集全血3ml，以EDTA抗凝管存储，并-20℃保存。对临床采集的样本提取DNA，采用天根生化科技(北京)有限公司血液基因组DNA提取试剂盒(DP348)，按照试剂盒说明书进行操作，该操作为本领域技术人员已知，不再概述。
所提取的DNA使用美国赛默飞世尔科技公司(Thermo Fisher Scientific)的微量分光光度计(Nanodrop 2000)进行浓度和纯度测定，并将DNA统一稀释至50ng/μL。同时通过琼脂糖凝胶电泳检测DNA纯度。
S2、荧光定量PCR反应：
1、引物和探针的设计
使用ABI 7500型实时荧光定量PCR(Quantitative Real-time PCR)(美国赛默飞世尔科技)进行荧光定量PCR反应，其中上游反应引物为引物1和下游反应引物为引物2，引物1和引物2分别对应的核苷酸序列为SEQ ID No.1、SEQ ID No.2；为将NPC1L1 g1679 C>G(rs2072183)基因型区分，共设计两段荧光定量PCR探针，分别为探针1和探针2，探针1对应的探针核苷酸序列为SEQ ID No.3，探针2对应的探针核苷酸序列为SEQ ID No.4。
2、PCR工作液的制备
委托生工生物工程(上海)股份有限公司公司合成上述设计的引物和探针，所合成的引物和探针按照合成报告单提示加入适量超纯水，溶解，制备成100μmol/L的母液，并进一步稀释成10μmol/L的工作液。
3、TaqMan荧光定量PCR检测过程
荧光定量PCR反应过程中，反应体系如下：
Figure PCTCN2021096565-appb-000003
Figure PCTCN2021096565-appb-000004
反应参数如下：
Figure PCTCN2021096565-appb-000005
PCR产物的电泳图谱如图1所示。选择FAM和VIC通道，在退火/延伸阶段采集荧光，图2为qPCR扩增图谱。本实施例的探针上标记有荧光基团，其中探针1的5’端标有FAM基团，3’端标记有MGB修饰基团，探针2的5’端标有VIC基团，3’端标记有MGB修饰基团。
S3、荧光定量PCR结果判断
可按照下表进行待测样本的检测结果解读：
FAM通道 VIC通道 检测结果
无荧光信号 无荧光信号 试验失败，建议重做
有荧光信号 无荧光信号 CC型
有荧光信号 有荧光信号 GC型
无荧光信号 有荧光信号 GG型
本发明实施例方案可通过荧光信号的情况快速判断基因型结果，操作及判断简便。
S4、验证灵敏性、准确性检测结果
本发明实施例检测样本采用Sanger测序法进行检测，委托深圳华大基因对所有DNA样本的进行Sanger测序，测序结果与参考序列进行比对以确定基因型。图3、图4和图5分别为NPC1L1 g1679 CC、NPC1L1 g1679 GG和NPC1L1 g1679 GC型样本的测序图谱。
将上述两种方法测得的基因分型统计结果进行比较，如下所示：
Figure PCTCN2021096565-appb-000006
Figure PCTCN2021096565-appb-000007
由上表结果可以看出，采用本发明实施例方案进行检测与Sanger直接测序法的结果完全吻合。
因此，从上表结果可知，本实施例实验方案对NPC1L1突变基因分型检查结果与Sanger测序完全一致，可见其特异性和准确度极高。相较于传统的直接测序法，本发明实施例方案有着相同的灵敏度和准确度，且具有操作更为简单、快速、结果易读且价格较低等优点。
实施例2
根据本发明的另一个实施例提供了一种能够用于检测NPC1L1突变基因分型试剂盒在预测胆固醇吸收抑制剂疗效研究中的应用，如图6所示；其中试剂盒包括：特异性引物组、特异性探针、UNG酶、PCR预混液和质控质粒，引物和探针的序列同实施例1，质控质粒包括核苷酸序列包含如SEQ ID No.5所示的野生型基因序列的合成质粒和核苷酸序列包含如SEQ ID No.6所示的突变型基因序列的合成质粒。
其具体步骤为：
通过随机、双盲、安慰剂对照、多中心参与的临床研究，入组中国汉族原发高固醇血症受试者720例(由于部分受试者脱落，故实际用于分析的受试者为598例)，按照用药方案，将受试者分6组，不同分组即代表不同的用药方案，其中A组为阿托伐他汀(10mg)组98例，B组为海泽麦布(HS-25)(20mg)组105例，C组为HS-25(20mg)和阿托伐他汀(10mg)联用组99例，D组为HS-25(10mg)组96例，E组为HS-25(10mg)和阿托伐他汀(10mg)联用组100例，F组为安慰剂组99例。通过比较LDL基线、年龄、性别等在各组之间的分布，证实各组之间LDL基线、年龄、性别等是均衡分布的，差异无显著的统计学意义(P值均大于0.05)，即受试者在试验前的分组是随机的，不存在偏好性。各分组受试者晨起空腹口服相当药物，每日一次，连用12周，以LDL_CH为临床指标观察疗效(LDC_CH相对于基线期的变化率)。将LDL_CH基线期测量值计为LDL_0，V4期(第12周)测量值计为LDL_12，V4期相对于基线期的变化率计算方法为：(LDL_12–LDL_0)/LDL_0。
分析不同组(用药方案)之间疗效差异，各指标各组之间两两比较，P值如表1所示：
表1各组间LDL_CH比较的P值
i/j A组 B组 C组 D组 E组 F组
A组   0.0000 0.0000 0.0000 0.0000 0.0000
B组 0.0000   0.0000 0.1509 0.0000 0.0000
C组 0.0000 0.0000   0.0000 0.9077 0.0000
D组 0.0000 0.1509 0.0000   0.0000 0.0000
E组 0.0000 0.0000 0.9077 0.0000   0.0000
F组 0.0000 0.0000 0.0000 0.0000 0.0000  
由表1可以看出，LDL_CH在大部分组间差异显著，仅在部分组间差异不显著，其中主要为C组和E组之间、B组和D组之间，说明大部分用药方案之间对LDL_CH的变化造成显著差异，并提示C组和E组之间用药效果一致、B组和D组之间用药效果一致。而在实际分组中，C组和E组均为HS25联合阿托法他汀用药(仅存在剂量差异)，B组和D组均为HS25单独用药(仅存在剂量差异)，这也提示将C组和E组联合分析，以及将B组和D组联合分析的可能性。
对受试者进行基因检测确定其基因型，取其样本后，采用实施例1能够用于检测NPC1L1突变基因分型试剂盒并且参照实施例1的检测过程进行。结果显示，中国原发性高胆固醇血症患者NPC1L1各种常见SNP中，rs2072183位点突变对海择麦布的疗效产生显著影响：海泽麦布20mg组(B组)中，CC、GC、GG型受试者的LDL_CH分别下降23.80％、12.96％、16.11％(p<0.05)；海泽麦布(10+20)mg组(B+D组)中，CC、GC、GG型受试者的LDL_CH分别下降20.55％、11.77％、14.74％(p<0.05)。
将LDL指标12周较基线的下降比例，按<10％、10％-25％、>25％划分为低、中和高三个等级，并在每组内分别进行方差分析和卡方检验，表2示出了总体ANOVA的P值。
表2各组中不同等级间比较的P值
  A组 B组 C组 D组 E组 F组 B+D组
P值 0.9136 0.0128 0.2534 0.1885 0.4634 0.8827 0.0395
通过结合药物基因组学的临床试验表明，NPC1L1的基因多态性可影响海泽麦布的药效，海泽麦布单用治疗时应考虑rs2072183基因型的影响，对于CC型海泽麦布疗效更优。本发明实施例方案在预测胆固醇吸收抑制剂的疗效研究中将具有良好的应用前景。
通过使用本发明实施例方案设计的检测NPC1L1突变基因分型的试剂盒，可以完成评价胆固醇吸收抑制剂药物在不同基因型族群中的疗效研究，因此，该试剂盒在之后的海泽麦布等胆固醇吸收抑制剂疗效的研究中同样具有良好的效果。
同时，NPC1L1基因多态性可用于预测海泽麦布等胆固醇吸收抑制剂疗效，开发相关检测试剂，用于指导胆固醇吸收抑制剂的精准用药。
此外，本领域技术人员公知，引物和探针上少数核苷酸残基的变化，尤其是引物5’端出现的少数核苷酸变化，或在引物5’端增加长段的标签序列(tag)，或者，对本发明实施例的引物/探针可以实施本领域常规的修饰，例如硫化、形成肽核酸等，以上，可能会造成少量的错配，但不会造成整个DNA复制合成与扩增过程无法进行，进而不会引起PCR产物产量的明显波动，因此，也能得到正确的足够的荧光信号，因此，对于序列的少量变动、加标签、修饰等，而产生的新序列，也应该包括在本发明中。
上述实施例中涉及的核苷酸序列信息汇总如下表3所示：
表3
Figure PCTCN2021096565-appb-000008
本发明实施例方案提供了一种基于TaqMan探针法qPCR全自动检测方法检测NPC1L1突变基因分型的试剂、方法、试剂盒及其在预测胆固醇吸收抑制制剂的疗效中的应用。该方法具有灵敏度高、特异性强、操作简便且价格较低等优点。同时，本发明实施例方案基于NPC1L1 g1679 C>G(rs2072183)基因位点，能够快速有效检测出该位点的三种基因型，能够为预测胆固醇吸收抑制制剂的疗效提供更有效的信息。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等同变换，或直接或间接运用在相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (10)

  1. 一种能够用于检测NPC1L1突变基因分型的试剂，其包含特异性引物组和特异性探针，其中所述特异性引物组包括核苷酸序列如SEQ ID No.1所示的引物1和核苷酸序列如SEQ ID No.2所示的引物2；
    所述特异性探针包括核苷酸序列如SEQ ID No.3所示的探针1和核苷酸序列如SEQ ID No.4所示的探针2。
  2. 根据权利要求1所述的能够用于检测NPC1L1突变基因分型的试剂，其中，所述特异性探针为TaqMan探针，所述探针1和探针2的5’端分别标记有报告荧光基团且所述探针1上的报告荧光基团与探针2上的报告荧光基团不同。
  3. 根据权利要求2所述的能够用于检测NPC1L1突变基因分型的试剂，其中，所述报告荧光基团选自FAM、TET、VIC、JOE或HEX。
  4. 一种能够用于检测NPC1L1突变基因分型的试剂盒，其包含如权利要求1～3中任一项所述的试剂。
  5. 根据权利要求4所述的能够用于检测NPC1L1突变基因分型的试剂盒，还包含UNG酶和dUTP。
  6. 根据权利要求4所述的能够用于检测NPC1L1突变基因分型的试剂盒，还包含PCR预混液和质控质粒，
    其中所述质控质粒包括核苷酸序列包含如SEQ ID No.5所示的野生型基因序列的合成质粒和核苷酸序列包含如SEQ ID No.6所示的突变型基因序列的合成质粒。
  7. 一种如权利要求4所述的能够用于检测NPC1L1突变基因分型的试剂盒的使用方法，包括以下步骤：
    S1、提取人体样本中的基因组DNA；
    S2、使用所述能够用于检测NPC1L1突变基因分型的试剂盒处理步骤S1得到的基因组DNA样本，设置仪器反应流程和参数进行qPCR扩增检测；
    S3、通过检测荧光变化进行分析NPC1L1的突变基因分型。
  8. 权利要求4所述的能够用于检测NPC1L1突变基因分型的试剂盒在预测胆固醇吸收抑制剂的疗效研究中的应用，其中所述研究不以疾病诊断治疗为目的。
  9. 一种如权利要求1所述的能够用于检测NPC1L1突变基因分型的试剂在制备预测胆固醇吸收抑制剂疗效的检测试剂中的应用。
  10. 根据权利要求9所述的应用，其特征在于：所述胆固醇吸收抑制剂为海泽麦布。
PCT/CN2021/096565 2020-05-28 2021-05-28 一种能够用于检测npc1l1突变基因分型的试剂、试剂盒及其使用方法、应用 WO2021239081A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010465791.0A CN111621553A (zh) 2020-05-28 2020-05-28 一种能够用于检测npc1l1突变基因分型的试剂及其应用
CN202010465791.0 2020-05-28

Publications (1)

Publication Number Publication Date
WO2021239081A1 true WO2021239081A1 (zh) 2021-12-02

Family

ID=72257128

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/096565 WO2021239081A1 (zh) 2020-05-28 2021-05-28 一种能够用于检测npc1l1突变基因分型的试剂、试剂盒及其使用方法、应用

Country Status (2)

Country Link
CN (1) CN111621553A (zh)
WO (1) WO2021239081A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111621553A (zh) * 2020-05-28 2020-09-04 长沙都正生物科技有限责任公司 一种能够用于检测npc1l1突变基因分型的试剂及其应用
CN113684310A (zh) * 2021-08-03 2021-11-23 宁夏回族自治区疾病预防控制中心 一种快速鉴别新型冠状病毒型别的试剂盒及方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006105537A2 (en) * 2005-03-30 2006-10-05 Schering Corporation Human niemann pick c1-like 1 gene (npc1l1) polymorphisms and methods of use thereof
CN109306377A (zh) * 2018-05-21 2019-02-05 上海迈浦生物科技有限公司 SNaPShot分型引物及其检测方法
CN109793718A (zh) * 2019-03-27 2019-05-24 董贵雨 一种海泽麦布口服固体药物组合物
CN109897896A (zh) * 2019-01-11 2019-06-18 江苏百世诺医疗科技有限公司 一种TaqMan-MGB探针技术检测影响降脂药物疗效基因的方法学建立
CN111621553A (zh) * 2020-05-28 2020-09-04 长沙都正生物科技有限责任公司 一种能够用于检测npc1l1突变基因分型的试剂及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006105537A2 (en) * 2005-03-30 2006-10-05 Schering Corporation Human niemann pick c1-like 1 gene (npc1l1) polymorphisms and methods of use thereof
CN109306377A (zh) * 2018-05-21 2019-02-05 上海迈浦生物科技有限公司 SNaPShot分型引物及其检测方法
CN109897896A (zh) * 2019-01-11 2019-06-18 江苏百世诺医疗科技有限公司 一种TaqMan-MGB探针技术检测影响降脂药物疗效基因的方法学建立
CN109793718A (zh) * 2019-03-27 2019-05-24 董贵雨 一种海泽麦布口服固体药物组合物
CN111621553A (zh) * 2020-05-28 2020-09-04 长沙都正生物科技有限责任公司 一种能够用于检测npc1l1突变基因分型的试剂及其应用

Also Published As

Publication number Publication date
CN111621553A (zh) 2020-09-04

Similar Documents

Publication Publication Date Title
Fernandez et al. Comparative study of three diagnostic approaches (FISH, STRs and MLPA) in 30 patients with 22q11. 2 deletion syndrome
CN111235272B (zh) 一次性检测肺癌多重基因突变的组合物及其应用
CN104232781B (zh) 一种检测HLA‑B*5801等位基因的TaqMan探针实时荧光PCR方法
WO2021239081A1 (zh) 一种能够用于检测npc1l1突变基因分型的试剂、试剂盒及其使用方法、应用
Zhou et al. A multiplex qPCR gene dosage assay for rapid genotyping and large-scale population screening for deletional α-thalassemia
CN117070607B (zh) 一种检测人脊髓性肌萎缩症smn1和smn2基因拷贝数的荧光定量pcr试剂盒
CN112941210A (zh) 结核分枝杆菌利福平和异烟肼耐药突变检测试剂盒及方法
Minucci et al. Competitive PCR-high resolution melting analysis (C-PCR-HRMA) for large genomic rearrangements (LGRs) detection: a new approach to assess quantitative status of BRCA1 gene in a reference laboratory
Abel et al. Genome-wide SNP association: identification of susceptibility alleles for osteoarthritis
CN110846408A (zh) 用于检测ttn基因突变的引物组合及其应用
CN115948532A (zh) 基于数字pcr技术的sma检测试剂盒
CN113136418B (zh) 一种检测y染色体微缺失的引物及探针的组合物、非诊断目的的检测方法及试剂盒
CN111394434B (zh) 一种TaqMan探针法的CHO宿主细胞DNA残留检测试剂盒及其应用
Dai et al. Rapid detection of twenty-nine common Chinese glucose-6-phosphate dehydrogenase variants using a matrix-assisted laser desorption/ionization-time of flight mass spectrometry assay on dried blood spots
CN111100924B (zh) 一种用于检测fmr1基因cgg重复数的质控品及其应用和含有该质控品的试剂盒
CN116004775A (zh) 一种人运动神经元拷贝数定量的引物探针组合物、试剂盒及方法
CN106636365A (zh) 用于检测agtr1基因的a1166c多态性位点的核酸、试剂盒及方法
CN108504731B (zh) 诊断脂质代谢相关疾病或阿尔茨海默病标志物的方法
Han et al. Rapid genotyping of 32 insertion/deletion panel for human identification using fluorogenic probes-based multiplex real-time PCR
Zhou et al. A quantitative assay to detect α-thalassemia deletions and triplications using multiplex nested real-time quantitative polymerase chain reaction
CN111690736A (zh) 一种华法林用药基因检测试剂盒及使用方法
CN110863044A (zh) 用于检测vcl基因突变的引物组合及其应用
WO2004092415A1 (ja) ミトコンドリアｄｎａ３２４３変異の検出法および定量法ならびにそのためのキット
CN116904584B (zh) 阿司匹林抵抗用药指导相关基因多态位点的试剂盒及其使用方法
US20220081707A1 (en) Diagnostic assay for a strain of neisseria meningitidis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21814050

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21814050

Country of ref document: EP

Kind code of ref document: A1