WO2021057667A1 - 一种检测由6个cd36突变基因所导致gpiv缺失的基因分型试剂盒 - Google Patents

一种检测由6个cd36突变基因所导致gpiv缺失的基因分型试剂盒 Download PDF

Info

Publication number
WO2021057667A1
WO2021057667A1 PCT/CN2020/116567 CN2020116567W WO2021057667A1 WO 2021057667 A1 WO2021057667 A1 WO 2021057667A1 CN 2020116567 W CN2020116567 W CN 2020116567W WO 2021057667 A1 WO2021057667 A1 WO 2021057667A1
Authority
WO
WIPO (PCT)
Prior art keywords
mutant
gpiv
seq
wild
type
Prior art date
Application number
PCT/CN2020/116567
Other languages
English (en)
French (fr)
Inventor
李丽兰
吴国光
Original Assignee
南宁中心血站(南宁输血医学研究所)
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 南宁中心血站(南宁输血医学研究所) filed Critical 南宁中心血站(南宁输血医学研究所)
Publication of WO2021057667A1 publication Critical patent/WO2021057667A1/zh
Priority to US17/673,769 priority Critical patent/US20220170103A1/en

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
    • C12Q1/6876—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
    • C12Q2600/156—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
    • C12Q2600/16—Primer sets for multiplex assays
    • 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

Definitions

  • the invention belongs to the field of biomedical detection, and particularly relates to a genotyping kit for human GPIV deletion caused by a CD36 mutant gene.
  • Platelet glycoprotein IV also known as CD36, GP88, GPIIIb FAT or SCARB3, belongs to the class B scavenger receptor transmembrane glycoprotein family.
  • the protein molecule can be extensively glycosylated, with a molecular mass of about 88,000D.
  • the human GPIV protein consists of 472 amino acids. GPIV protein is widely distributed in a variety of cells and tissues in the human body, including platelets, monocytes/macrophages, microvascular endothelial cells, brain microglia, astrocytes, cardiac and skeletal muscles, adipocytes, and trees Dendritic cells, retinal epithelial cells and breast and kidney tissues.
  • thrombin receptor Its main functions include acting as thrombin receptor, collagen receptor, long-chain fatty acid receptor, etc.; it also participates in inducing cell apoptosis, removing oxidized low-density lipoprotein in plasma, and enhancing the adhesion of abnormal red blood cells.
  • a variety of physiological and pathological processes have played an important role. A large number of studies have proved that: GPIV is involved in hemostasis, thrombosis, platelet alloimmune diseases, hypercholesterolemia, obesity, peripheral atherosclerosis, arterial hypertension, and myocardium Disease, diabetes, malaria, early senile dementia (Alzheimer’s disease), cancer and other diseases.
  • GPIV loss in platelets or monocytes. According to the phenotype of GPIV loss, it can be divided into two categories: type I loss, which does not express GPIV on platelets and monocytes; type II loss, and GPIV is only expressed on platelets , But expressed in monocytes.
  • type I loss which does not express GPIV on platelets and monocytes
  • type II loss and GPIV is only expressed on platelets , But expressed in monocytes.
  • the mutation of the CD36 gene encoding GPIV is an important reason for the deletion of human GPIV antigen.
  • the CD36 gene encoding GPIV is located at q11.2 on chromosome 7. It has 15 exons and is more than 32,000 bp in length.
  • the GPIV protein coding region is located in exon 3 to exon 14.
  • Carrying out the detection and screening of the polymorphisms of GPIV deletion caused by these 6 CD36 mutant genes in the population will help to comprehensively detect the distribution characteristics and countermeasures of individuals with GPIV deletion caused by these 6 GPIV mutant genes in the population.
  • PCR- SSP sequence-specific primer-polymerase chain reaction
  • PCR-RFLP restriction fragment length polymorphism polymerase chain reaction
  • TaqMan probe technology to detect the occurrence of individuals with GPIV deletion by analyzing the existence of gene mutations and comparison with wild-type
  • the detection technology used is cumbersome and non-specific, and cannot achieve the purpose of specific and high-throughput screening and identification of individuals with GPIV deletion caused by specific mutant genes.
  • the present invention aims to invent a GPIV caused by a specific CD36 mutant gene.
  • the genotyping kits for missing individuals can perform specific and high-throughput detection and identification of the 6 CD36 mutant genes that lead to GPIV deletion.
  • the present invention aims to create a genotyping kit for detecting GPIV deletion caused by the discovered CD36 mutant gene based on the genetic basis of the CD36 gene encoding human GPIV and the newly discovered CD36 mutant gene that causes the deletion of GPIV.
  • PCR-SSP sequence-specific primer polymerase chain reaction
  • CD36 mutant genes C275T (Thr92Met), 730G>A (Asp244Asn), Exon-10+2T>G (Change in splicing site) ), 1123C>T (Pro375Ser), 1229T>C (Ile410Thr) and 1332ints TGAT (frameshift at AA 445) caused by genotyping detection and identification of individuals with GPIV deletion, providing a simple and quick genotyping kit.
  • a genotyping kit for detecting GPIV deletion caused by 6 CD36 mutant genes characterized in that the kit includes: wild-type sequence-specific primers for detecting each mutation site that causes GPIV deletion and the specificity of the mutant Primers and public primers; the mutation sites are: C275T (Thr92Met), 730G>A (Asp244Asn), Exon-10+2T>G (Change in splicing site), 1123C>T (Pro375Ser), 1229T> C (Ile410Thr), 1332ints TGAT (frameshift at AA445).
  • the mutation sites are: C275T (Thr92Met), 730G>A (Asp244Asn), Exon-10+2T>G (Change in splicing site), 1123C>T (Pro375Ser), 1229T> C (Ile410Thr), 1332ints TGAT (frameshift at AA445).
  • each genotyping detection system for GPIV deletion caused by CD36 mutant gene includes wild-type sequence-specific PCR primers and mutant sequence-specific PCR primers for the mutation sites of GPIV deletion caused by CD36 mutant gene.
  • the genotyping detection system for detecting the GPIV deletion caused by each CD36 mutant gene is performed by two PCR reactions to amplify the wild-type sequence and the mutant sequence at each mutation site, respectively, and prepare the gene analysis of each mutation site.
  • Type PCR amplification PCR reaction system all PCR reactions for detection of mutation sites can be amplified under the same PCR amplification conditions, and the PCR amplified products are finally electrophoresed in a 2% agarose gel to observe and analyze the detection results .
  • the PCR used in the present invention refers to the primer information used in the genotyping detection system for GPIV deletion caused by each CD36 mutant gene as shown in Table 1:
  • Table 1 PCR-SSP primer sequences and internal reference primers in the genotyping system of 6 CD36 mutant genes leading to GPIV deletion
  • each PCR reaction adds forward primer CRP I and reverse primer CRP II PCR to amplify internal reference fragments; divided into three according to different mutant types
  • a PCR reaction system, its composition and configuration method are as follows:
  • Table 2 The composition of the PCR amplification reaction system (final volume 10 ⁇ L/reaction)
  • Proper magnesium ion concentration is one of the basic conditions to obtain good PCR reaction effect. Magnesium ion is necessary for Taq DNA polymerase, primer annealing, template and PCR product melting temperature, product specificity, primer dimerization Body formation, etc. have an impact. When the magnesium ion concentration is too low, the enzyme activity is significantly reduced; when the magnesium ion concentration is too high, the enzyme catalyzes non-specific amplification.
  • the present invention has studied the magnesium ion concentration of different mutant amplification systems, and found that when the final magnesium ion concentration of the C275T and Exon-10 (+2T>G) amplification systems is 2mM, T1229C and 1332ints TGAT amplification The final magnesium ion concentration of the system is 3.5 mM, and the amplification effect is best when the final magnesium ion concentration of the G730A and C1123T amplification systems is 1.5 mM.
  • the applicable PCR amplification reaction system for each mutant SNP site is shown in Table 3.
  • PCR amplification is carried out by a PCR machine. All PCR reactions can be amplified under the same PCR amplification cycle conditions.
  • the cycle parameters are:
  • the PCR amplified products were finally electrophoresed in a 2% agarose gel.
  • the beneficial effects of the present invention Based on the molecular basis that leads to human GPIV deletion and the principle of sequence-specific primer polymerase chain reaction (PCR-SSP) technology, the present invention creates a CD36 mutant gene that leads to human GPIV deletion.
  • PCR-SSP sequence-specific primer polymerase chain reaction
  • CD36 mutant genes C275T (Thr92Met), 730G>A (Asp244Asn), Exon-10+2T>G (Change in splicing site), 1123C>T (Pro375Ser), 1229T >C (Ile410Thr), 1332ints TGAT (frameshift at AA 445) for genotyping of individuals with GPIV deletion caused by CD36 mutation gene, provides a simple and quick genotyping for the genotyping of individuals with GPIV deletion caused by CD36 mutation gene
  • the detection method provides an experimental basis for population surveys for the genotyping detection and identification of GPIV deletions, and for understanding the polymorphisms that lead to GPIV deletions in various populations.
  • sequence-specific primers for the wild-type site and sequence-specific primers for the mutant site are respectively designed, and
  • the common primers for typing the mutation polymorphism site, the genotyping detection system for each CD36 mutation gene SNP site that causes GPIV deletion are completed by two PCR reactions, respectively targeting the wild-type SNP site of the mutation gene
  • Amplification of human C-reactive protein (CRP) DNA sequence fragments by designing a pair of PCR primers, as an internal reference for each PCR reaction; by exploring the optimal annealing temperature and adjusting the Mg 2+ ion concentration, etc.
  • PCR-specific amplification and genotyping can be performed on the SNP sites of the CD36 mutant genes that cause GPIV deletions, and the PCR amplification of all the SNP sites of the mutant genes that cause GPIV deletions described in the present invention can be performed at the same Complete under the conditions of the amplification cycle.
  • This kit is suitable for genotyping detection and identification of individuals with GPIV deletion caused by 6 CD36 mutant genes, auxiliary diagnostic testing of anti-GPIV platelet alloimmune diseases, and population genetics and anthropology of GPIV deletion caused by CD36 mutant genes And advanced chemistry and other applied and basic research work.
  • Figure 1 is the genotyping amplification reaction effect diagram of the C275T mutation site in 6 samples of Example 1 (S1-1 is C275T mutant heterozygote, S1-2 ⁇ S1-6 are 275C wild-type homozygote. Note: W is the wild-type lane and M is the mutant-type lane).
  • Figure 2 is the genotyping amplification reaction effect diagram of the G730A mutation site of 6 samples of Example 2 (S2-1 is the G730A mutant heterozygote, and S2-2 ⁇ S2-6 are the 730G wild-type homozygous. Note: W is the wild-type lane and M is the mutant-type lane).
  • Figure 3 is the genotyping amplification reaction effect diagram of the Exon-10+2T>G mutation site of 8 samples of Example 3 (S3-1 ⁇ S3-3 are Exon-10+2T>G mutant heterozygotes, S3-4 ⁇ S3-8 are Exon-10+2T wild-type homozygotes. Note: W is the wild-type lane and M is the mutant-type lane).
  • Figure 4 is the genotyping amplification reaction effect diagram of the C1123T mutation site in 6 samples of Example 4 (S4-1 is C1123T mutant heterozygote, S4-2 ⁇ S4-6 are 1123C wild-type homozygote. Note: W is the wild-type lane and M is the mutant-type lane).
  • FIG. 5 is the genotyping amplification reaction effect diagram of the T1229C mutation site of 7 samples of Example 5 (S5-1 ⁇ S5-3 are T1229C mutant heterozygotes, S5-4 ⁇ S5-7 are 1229T wild-type pure Zygote. Note: W is the wild-type lane, M is the mutant lane).
  • Figure 6 is the genotyping amplification reaction effect diagram of the 7 samples of Example 6 at 1332ints TGAT mutation site (S6-1 ⁇ S6-2 are 1332ints TGAT mutant heterozygotes, S6-4 ⁇ S6-7 are 1332 wild Type homozygous. Note: W is the wild type lane, M is the mutant type lane).
  • Figure 7 is the amplified electrophoresis diagram of the G730A mutation site at different final magnesium ion concentrations (Note: W is wild-type lane, M is mutant lane; S2-1 genotype is 730G/A mutant heterozygote, use this kit
  • the genotyping test results should be positive for the W and M lanes.
  • the S2-2 ⁇ S2-6 genotype is 730G/G homozygous.
  • the genotyping test results using this kit should be W lanes.
  • Figure 8 is the amplified electrophoresis diagram of the C1123T mutation site at different final magnesium ion concentrations (Note: W is wild-type lane, M is mutant lane; S4-1 genotype is 1123C/T mutant heterozygote, use this kit
  • the genotyping test results should be positive for the W and M lanes, the S4-2 ⁇ S4-6 genotype is 1123C/C homozygous, and the genotyping test results using this kit should be W lanes.
  • Figure 9 is the amplified electrophoresis diagram of T1229C at different final magnesium ion concentrations (W is wild-type lane, M is mutant lane; S5-1 and S5-2 genotypes are 1229T/C mutant heterozygotes, use this kit to do
  • the genotyping test results should be positive for the W and M lanes.
  • the S5-4 ⁇ S5-6 genotypes are 1229T/T homozygotes.
  • the genotyping test results using this kit should be W lanes.
  • Figure 10 is the amplified electrophoresis diagram of 1332ints TGAT at different final magnesium ion concentrations (W is wild-type lane, M is mutant lane; S6-1 genotype is 1332ints TGAT mutant heterozygote, use this kit for genotyping detection
  • W wild-type lane
  • M mutant lane
  • S6-1 genotype 1332ints TGAT mutant heterozygote
  • the result should be that the target bands in the W and M lanes are both positive, and the S6-4 genotype is 1332 homozygous without TGAT insertion.
  • a total of 20 PCR primers of the present invention were commissioned to synthesize by Shanghai Jierui Biological Engineering Co., Ltd.
  • Amplification enzymes are carried out using Taq DNA Polmerase produced by TaKaRa.
  • This example provides a genotyping kit for human GPIV deletion caused by the CD36 mutant gene of the present invention, and genotyping detection of individuals with GPIV deletion caused by the CD36 mutant gene [C275T (Thr92Met)].
  • sequence-specific primers should be GPIV-275a; in the 275T mutant PCR amplification reaction system, the sequence-specific primers should be GPIV-275b.
  • the PCR amplification cycle parameters are:
  • This example provides a genotyping kit for human GPIV deletion caused by the CD36 mutant gene of the present invention, and genotyping detection of individuals with GPIV deletion caused by the CD36 mutant gene [G730A (Asp244Asn)].
  • sequence-specific primers should be GPIV-730a; in the 730A mutant PCR amplification reaction system, the sequence-specific primers should be GPIV-730b.
  • the PCR amplification cycle parameters are the same as in Example 1.
  • This example provides a genotyping kit for human GPIV deletion caused by the CD36 mutant gene of the present invention.
  • individuals with GPIV deletion caused by the CD36 mutant gene [Exon-10+2T>G (Change in splicing site)] Perform genotyping testing.
  • Exon-10+2T wild-type sequence-specific primer GPIV-E10(+2)a forward primer
  • Exon-10+2G mutant-type sequence-specific primer GPIV-E10(+2)b of the present invention Forward primer
  • the public reverse primer GPIV-E10(+2)c the public reverse primer GPIV-E10(+2)c.
  • the 3 cases of Exon-10+2T>G mutant samples confirmed by DNA sequencing (all Exon-10+2T>G mutant heterogeneous Zygotic type) and 5 Exon-10+2T wild-type samples were subjected to PCR amplification of Exon-10+2T>G mutation sites.
  • PCR amplification reaction system was added with C-reactive protein PCR amplification forward primer CRP I and reverse primer CRP II, as internal reference, PCR amplification was carried out on ABI 9700 PCR instrument, PCR amplification reaction system A was selected, and the composition was:
  • sequence-specific primers are selected GPIV-E10(+2)a; in Exon-10+2G mutant PCR amplification reaction system, sequence-specific primers are selected GPIV-E10(+2)b.
  • the PCR amplification cycle parameters are the same as in Example 1.
  • This example provides a genotyping kit for human GPIV deletion caused by the CD36 mutant gene of the present invention, and genotyping detection of individuals with GPIV deletion caused by the CD36 mutant gene [C1123T (Pro375Ser)].
  • sequence-specific primers should be GPIV-1123a; in the 1123T mutant PCR amplification reaction system, the sequence-specific primers should be GPIV-1123b.
  • the PCR amplification cycle parameters are the same as in Example 1.
  • This example provides a genotyping kit for human GPIV deletion caused by the CD36 mutant gene of the present invention, and genotyping detection of individuals with GPIV deletion caused by the CD36 mutant gene [T1229C (Ile410Thr)].
  • sequence-specific primers should be GPIV-1229a; in the 1229C mutant PCR amplification reaction system, the sequence-specific primers should be GPIV-1229b.
  • the PCR amplification cycle parameters are the same as in Example 1.
  • This example provides a genotyping kit for human GPIV deletion caused by the CD36 mutant gene of the present invention, and genotyping detection of individuals with GPIV deletion caused by the CD36 mutant gene [1332ints TGAT (frameshift at AA 445)] .
  • sequence-specific primers should be GPIV-1332a; in the 1332ints TGAT mutant PCR amplification reaction system, the sequence-specific primers should be GPIV-1332b.
  • the PCR amplification cycle parameters are the same as in Example 1.
  • Example 7 In the genotyping detection system where different CD36 mutant genes cause GPIV deletion, the influence of the final concentration of magnesium ions in the PCR reaction on the detection results.
  • T1229C PCR reaction [Mg 2+ ] final 2.0mM
  • the target band was successfully amplified, and the result was more accurate, as shown in Figure 9.
  • S6-1 genotype is 1332ints TGAT mutation heterozygote
  • the genotyping test result using this kit should be positive for the W and M lanes
  • the S6-4 genotype is 1332ints homozygous without TGAT insertion, use this kit
  • the genotyping test result of the kit should be that the target band of W lane is positive and the target band of M lane is negative.

Landscapes

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

Abstract

提供一种检测由6个CD36基因突变导致GPIV缺失个体的基因分型试剂盒，所述的6个CD36基因突变的位点分别为：C275T(Thr92Met)、730G＞A(Asp244Asn)、Exon-10+2T＞G(剪接位点改变)、1123C＞T(Pro375Ser)、1229T＞C(Ile410Thr)和1332ints TGAT(AA 445移码)。该试剂盒适用于由6个CD36基因突变所导致的GPIV缺失个体的分型检测和鉴定及抗-GPIV血小板同种免疫疾病的辅助诊断检测。

Description

一种检测由6个CD36突变基因所导致GPIV缺失的基因分型试剂盒 技术领域
本发明属于生物医学检测领域，特别是涉及一种由CD36突变基因导致人类GPIV缺失的基因分型试剂盒。
背景技术
血小板糖蛋白IV(GPIV)，又名CD36、GP88、GPIIIb FAT或SCARB3，属于B类清道夫受体跨膜糖蛋白家族。该蛋白分子能被广泛糖基化，分子质量约为88000D。人类GPIV蛋白由472个氨基酸组成。GPIV蛋白在人体内广泛分布于多种细胞和组织中，包括血小板、单核/巨噬细胞、微血管内皮细胞、大脑小胶质细胞、星形胶质细胞、心肌和骨骼肌、脂肪细胞、树突状细胞、视网膜上皮细胞和乳腺及肾脏组织。其主要的功能包括作为凝血酶受体，胶原蛋白受体，长链脂肪酸受体等；还参与诱导细胞凋亡，去除血浆中的氧化低密度脂蛋白，增强异常形态红细胞的黏附等功能，在多种生理病理过程中发挥了重要的作用，大量的研究已证明：GPIV涉及止血、血栓、血小板同种免疫性疾病、高胆固醇血症、肥胖症、外周动脉粥样硬化、动脉高血压、心肌病、糖尿病、疟疾、早老年型痴呆(阿尔茨海默病)、癌症等疾病的形成。
部分个体在血小板或和单核细胞可出现GPIV缺失，按表现型GPIV缺失可分为2类：I型缺失，血小板和单核细胞上均不表达GPIV；II型缺失，GPIV仅在血小板不表达，但在单核细胞表达。编码GPIV的CD36基因变异，是导致人类GPIV抗原缺失的重要原因。编码GPIV的CD36基因位于第7号染色体的q11.2，有15个外显子，长度超过32000bp。GPIV蛋白编码区位于外显子3至外显子14，，由于人群多态性原因，不同人群中导致GPIV缺失的基因突变有所不同，如在日本人以268C＞T、329-330delAC等突变多见(见文献Yanai H，Chiba H，Fujiwara H，et al.Phenotype-genotype correlation in GPIVdeficiency types I and II.Thromb Haemost，2000，84(3)：436-441.)。而本申请人在中国广西人群中发现了6个导致GPIV缺失的新突变基因，分别为C275T(Thr92Met)、730G＞A(Asp244Asn)、Exon-10+2T＞G(Change in splicing site)、1123C＞T(Pro375Ser)、1229T＞C(Ile410Thr)和1332ints TGAT(frameshift at AA 445)。在人群中开展由这6个CD36突变基因所导致GPIV缺失的多态性的检测和筛查，将有助于全面检测人群中这6个GPIV突变基因所导致的GPIV缺失个体的分布特点和对个体生理及病理功能的影响，掌握人群中导致GPIV缺失的基因多态性情况，及遗传学特征。
为检查人群中所存在的GPIV缺失个体，目前文献报道中大多以血清学或基因测序的方式对GPIV缺失个体进行基因分型检测，也有部分报道以序列特异性引物-聚合酶链反应(PCR-SSP)、限制性片段长度多态性聚合酶链反应(PCR-RFLP)、实时荧光定量PCR TaqMan探针技术，通过分析基因突变的存在以及与野生型的对比情况，来检测GPIV缺失个体的发生，所采用的检测技术繁琐而且是非特异性，不能达到对由特异性的突变基因所导致的GPIV缺失个体进行特异性的、高通量的筛查和鉴定的目的，更不能对本发明中所描述的发生在人群中的6个CD36突变基因所导致的GPIV缺失个体做针对性的、群体性的正确检测、分 析和研究，因此，本发明旨在发明一种由特定的CD36突变基因所导致GPIV缺失个体的基因分型试剂盒，对所发现的6个CD36突变基因导致GPIV缺失个体做特异性、高通量地检测和鉴定。
发明内容
本发明旨在基于编码人类GPIV的CD36基因遗传学基础和新发现的导致GPIV缺失的CD36突变基因，创建一种检测由所发现的CD36突变基因导致GPIV缺失的基因分型试剂盒，该试剂盒以序列特异性引物聚合酶链反应(PCR-SSP)方法为基础，为由6个CD36突变基因：C275T(Thr92Met)、730G＞A(Asp244Asn)、Exon-10+2T＞G(Change in splicing site)、1123C＞T(Pro375Ser)、1229T＞C(Ile410Thr)和1332ints TGAT(frameshift at AA 445)导致的GPIV缺失个体的基因分型检测鉴定，提供了一种简便快捷的基因分型试剂盒。
为实现上述目的，本发明采取的技术方案为：
一种检测由6个CD36突变基因导致GPIV缺失的基因分型试剂盒，其特征在于，所述试剂盒包括：检测导致GPIV缺失的各突变位点的野生型序列特异性引物和突变型的特异性引物，以及公共引物；所述突变位点分别为：C275T(Thr92Met)、730G＞A(Asp244Asn)、Exon-10+2T＞G(Change in splicing site)、1123C＞T(Pro375Ser)、1229T＞C(Ile410Thr)、1332ints TGAT(frameshift at AA445)。
上述试剂盒的使用方法如下：
①通过一对PCR引物扩增人类C反应蛋白(CRP)DNA序列片段，作为每个PCR反应的内参照；
②每种由CD36突变基因导致的GPIV缺失的基因分型检测体系，均包含分别针对CD36突变基因导致GPIV缺失的基因突变位点的野生型序列特异性PCR引物和突变型序列特异性PCR引物，以及1条公共引物，对由6个CD36突变基因：C275T(Thr92Met)、730G＞A(Asp244Asn)、Exon-10+2T＞G(Change in splicing site)、1123C＞T(Pro375Ser)、1229T＞C(Ile410Thr)和1332ints TGAT(frameshift at AA 445)导致的GPIV缺失个体进行基因分型检测。
③检测每种CD36突变基因导致的GPIV缺失的基因分型检测体系，均由2个PCR反应分别对各自突变位点的野生型序列和突变型序列进行扩增，分别配制各突变位点基因分型PCR扩增的PCR反应体系，所有突变位点检测的PCR反应均可在同一PCR扩增条件下完成扩增，PCR扩增产物最后于2％琼脂糖凝胶中电泳，观察及分析检测结果。
本发明中采用的PCR针对每种CD36突变基因导致GPIV缺失的基因分型检测体系中所采用的引物信息如表1所示：
表1：6种CD36突变基因导致GPIV缺失的基因分型体系中PCR-SSP引物序列以及内参引物
Figure PCTCN2020116567-appb-000001
Figure PCTCN2020116567-appb-000002
对每种CD36突变基因型进行PCR，对各导致GPIV缺失的突变基因的单核苷酸多态性(SNP)位点，分别使用野生型引物a和公共引物c特异性扩增野生型目的片段，使用突变型引物b和公共引物c特异性扩增突变型目的片段；每个PCR反应均加入正向引物CRP I和反向引物CRP II PCR扩增内参照片段；根据不同突变型分为三个PCR反应体系，其组成及配置方法如下表2：
表2：PCR扩增反应体系组成为(终体积10μL/反应)
Figure PCTCN2020116567-appb-000003
适合的镁离子浓度是获得良好PCR反应效果的基本条件之一，镁离子是Taq DNA聚合酶所必需的，对引物的退火、模板与PCR产物的解链温度、产物的特异性、引物二聚体的形成等均有影响。当镁离子浓度过低时，酶活力显著降低；镁离子浓度过高时，酶催化非特异性的扩增。因此，本发明对不同突变型的扩增体系的镁离子浓度进行了研究，发现当C275T和Exon-10(+2T＞G)扩增体系的镁离子终浓度为2mM，T1229C和1332ints TGAT扩增体系的镁离子终浓度为3.5mM，G730A和C1123T扩增体系的镁离子终浓度为1.5mM时扩增效果最好。各突变SNP位点适用的PCR扩增反应体系如表3所示。
表3：各突变基因的SNP位点适用的PCR扩增反应体系
PCR扩增反应体系 适用突变型别
反应体系A([Mg 2+]终＝2mM) C275T，Exon-10(+2T＞G)
反应体系B([Mg 2+]终＝3.5mM) T1229C，1332ints TGAT
反应体系C([Mg 2+]终＝1.5mM) G730A，C1123T
PCR扩增通过PCR仪进行，所有的PCR反应均可在同一PCR扩增循环条件下完成扩增，其循环参数为：
95℃                5min
以下条件扩增25个循环：
95℃                30sec
68℃-0.4℃/循环     30sec
72℃                30sec
以下条件扩增15个循环：
95℃                30sec
54℃                30sec
72℃                30sec
最后以下条件延伸和保存：
72℃    5min
12℃     ∞
PCR扩增产物最后于2％琼脂糖凝胶中进行电泳。
本发明的有益效果：本发明基于导致人类GPIV缺失的分子基础和序列特异性引物聚合酶链反应(PCR-SSP)技术原理，创建了一种CD36突变基因导致人类GPIV缺失的GPIV缺失个体基因分型方法，其贡献在于，它可针对6个CD36突变基因：C275T(Thr92Met)、730G＞A(Asp244Asn)、Exon-10+2T＞G(Change in splicing site)、1123C＞T(Pro375Ser)、1229T＞C(Ile410Thr)、1332ints TGAT(frameshift at AA 445)所导致的GPIV缺失个体进行基因分型检测，为CD36突变基因导致的GPIV缺失个体的基因分型检测提供了一种简便快捷的基因分型检测方法，为GPIV缺失的基因分型检测鉴定，及为了解各人群中导致GPIV缺失的多态性情况提供了人群调查的实验基础。
本发明的方法中，针对各导致GPIV缺失的CD36突变基因的多态性(SNP)位点，分别设计针对野生型位点的序列特异性引物和突变型位点的序列特异性引物，以及可完成该突变多态性位点分型的公共引物，每个导致GPIV缺失的CD36突变基因SNP位点的基因分型检测体系，由2个PCR反应完成，分别针对该突变基因SNP位点的野生型及突变型进行扩增；通过设计一对PCR引物扩增人类C反应蛋白(CRP)DNA序列片段，作为每个PCR反应的内参照；通过探索最佳退火温度及调节Mg 2+离子浓度等条件，可对导致GPIV缺失的CD36突变基因SNP位点进行PCR特异性扩增和基因分型，并且使本发明中描述的所有导致GPIV缺失的突变基因SNP位点的PCR扩增均可在同一扩增循环条件下完成。该试剂盒适合于6个CD36突变基因导致GPIV缺失个体的基因分型检测和鉴定，抗-GPIV血小板同种免疫疾病的辅助诊断检测，以及CD36突变基因导致的GPIV缺失的群体遗传学、人类学及进化学等应用和基础研究工作。
附图说明
图1是实施例1的6例样本C275T突变位点的基因分型扩增反应效果图(S1-1为C275T突变型杂合子，S1-2～S1-6为275C野生型纯合子。注：W为野生型泳道，M为突变型泳道)。
图2是实施例2的6例样本G730A突变位点的基因分型扩增反应效果图(S2-1为G730A突变型杂合子，S2-2～S2-6为730G野生型纯合子。注：W为野生型泳道，M为突变型泳道)。
图3是实施例3的8例样本Exon-10+2T＞G突变位点的基因分型扩增反应效果图(S3-1～S3-3为Exon-10+2T＞G突变型杂合子，S3-4～S3-8为Exon-10+2T野生型纯合子。注：W为野生型泳道，M为突变型泳道)。
图4是实施例4的6例样本C1123T突变位点的基因分型扩增反应效果图(S4-1为C1123T突变型杂合子，S4-2～S4-6为1123C野生型纯合子。注：W为野生型泳道，M为突变型泳道)。
图5是实施例5的7例样本T1229C突变位点的基因分型扩增反应效果图(S5-1～S5-3为T1229C突变型杂合子，S5-4～S5-7为1229T野生型纯合子。注：W为野生型泳道，M为突变型泳道)。
图6是实施例6的7例样本1332ints TGAT突变位点的基因分型扩增反应效果图(S6-1～S6-2为1332ints TGAT突变型杂合子，S6-4～S6-7为1332野生型纯合子。注：W为野生型泳道，M为突变型泳道)。
图7是G730A突变位点在不同镁离子终浓度的扩增电泳图(注：W为野生型泳道，M为突变型泳道；S2-1基因型为730G/A突变杂合子，使用本试剂盒做基因分型检测结果应为W和M泳道目的条带均为阳性，S2-2～S2-6基因型是730G/G纯合子，使用本试剂盒做基因分型检测结果应为W泳道目的条带均为阳性，M泳道目的条带阴性，[Mg 2+]终＝1.5mM的PCR反应体系扩增结果更优且准确)。
图8是C1123T突变位点在不同镁离子终浓度的扩增电泳图(注：W为野生型泳道，M为突变型泳道；S4-1基因型为1123C/T突变杂合子，使用本试剂盒做基因分型检测结果应为W和M泳道目的条带均为阳性，S4-2～S4-6基因型是1123C/C纯合子，使用本试剂盒做基因分型检测结果应为W泳道目的条带均为阳性，M泳道目的条带阴性，[Mg 2+]终＝1.5mM的PCR反应体系扩增结果更优且准确)。
图9是T1229C在不同镁离子终浓度的扩增电泳图(W为野生型泳道，M为突变型泳道；S5-1和S5-2基因型为1229T/C突变杂合子，使用本试剂盒做基因分型检测结果应为W和M泳道目的条带均为阳性，S5-4～S5-6基因型是1229T/T纯合子，使用本试剂盒做基因分型检测结果应为W泳道目的条带均为阳性，M泳道目的条带阴性，[Mg 2+]终＝3.5mM的PCR反应体系扩增结果更为准确)。
图10是1332ints TGAT在不同镁离子终浓度的扩增电泳图(W为野生型泳道，M为突变型泳道；S6-1基因型为1332ints TGAT突变杂合子，使用本试剂盒做基因分型检测结果应为W和M泳道目的条带均为阳性，S6-4基因型是1332无TGAT插入纯合子，使用本试剂盒做基因分型检测结果应为W泳道目的条带均为阳性，M泳道目的条带阴性，[Mg 2+]终＝3.5mM的PCR反应体系扩增结果准确)。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点，下面结合具体实施例及其附图对本发明做进一步的详细描述。
本发明的PCR引物共20条，委托上海捷瑞生物工程有限公司合成。扩增酶采用TaKaRa公司生产的Taq DNA Polmerase进行。
实施例1
本实施例给出了通过本发明的CD36突变基因导致人类GPIV缺失的基因分型试剂盒，对由CD36突变基因【C275T(Thr92Met)】导致的GPIV缺失的个体进行基因分型检测。
首先，取本发明中的275C野生型序列特异性引物GPIV-275a(反向引物)、275T突变型序列特异性引物GPIV-275b(反向引物)和公共正向引物GPIV-275c，对经DNA测序检测确认的1例C275T突变型样本(为C275T突变杂合子型)和5例275C野生型样本进行C275T突变位点的PCR扩增，每个PCR扩增反应体系均加入C反应蛋白的PCR扩增正向引物CRP I和反向引物CRP II，作为内参照，PCR扩增于ABI 9700型PCR仪进行，选择PCR扩增反应体系A，组成为：
Figure PCTCN2020116567-appb-000004
Figure PCTCN2020116567-appb-000005
(备注：在275C野生型PCR扩增反应体系，序列特异性引物选择GPIV-275a；在275T突变型PCR扩增反应体系，序列特异性引物选择GPIV-275b。)
PCR扩增循环参数为：
95℃             5min
以下条件扩增25个循环：
95℃             30sec
68℃-0.4℃/循环  30sec
72℃             30sec
以下条件扩增15个循环：
95℃             30sec
54℃             30sec
72℃             30sec
最后以下条件延伸和保存：
72℃             5min
12℃             ∞
取8μl PCR产物，于2％琼脂糖凝胶中电泳[琼脂糖凝胶含5％的绿如蓝荧光染料(北京天恩泽基因科技有限公司)]，在凝胶成像系统中观察特异性PCR产物条带，PCR产物条带清晰、特异，结果如图1所示，样本S1-1为C275T突变型杂合子，因此，在突变型泳道(M)扩增出目的条带，而S1-2～6为275C野生型纯合子，在突变型泳道(M)未扩增出目的条带。
实施例2
本实施例给出了通过本发明的CD36突变基因导致人类GPIV缺失的基因分型试剂盒，对由CD36突变基因【G730A(Asp244Asn)】导致的GPIV缺失的个体进行基因分型检测。
首先，取本发明中的730G野生型序列特异性引物GPIV-730a(正向引物)、730A突变型序列特异性引物GPIV-730b(正向引物)和公共反向引物GPIV-730c，对经DNA测序检测确认的1例G730A突变型样本(为G730A突变杂合子型)和5例730G野生型样本进行G730A突变位点的PCR扩增，每个PCR扩增反应体系均加入C反应蛋白的PCR扩增正向引物CRP I和反向引物CRP II，作为内参照，PCR扩增于ABI 9700型PCR仪进行，选择PCR扩增反应体系C，组成为：
Figure PCTCN2020116567-appb-000006
(备注：在730G野生型PCR扩增反应体系，序列特异性引物选择GPIV-730a；在730A突变型PCR扩增反应体系，序列特异性引物选择GPIV-730b。)
PCR扩增循环参数与实施例1相同。
取8μl PCR产物，于2％琼脂糖凝胶中电泳[琼脂糖凝胶含5％的绿如蓝荧光染料(北京天恩泽基因科技有限公司)]，在凝胶成像系统中观察特异性PCR产物条带，PCR产物条带清晰、特异，结果如图2所示，样本S2-1为G730A突变型杂合子，因此，在突变型泳道(M)扩增出目的条带，而S2-2～6为730G野生型纯合子，在突变型泳道(M)未扩增出目的条带。
实施例3
本实施例给出了通过本发明的CD36突变基因导致人类GPIV缺失的基因分型试剂盒，对由CD36突变基因【Exon-10+2T＞G(Change in splicing site)】导致的GPIV缺失的个 体进行基因分型检测。
首先，取本发明中Exon-10+2T野生型序列特异性引物GPIV-E10(+2)a(正向引物)、Exon-10+2G突变型序列特异性引物GPIV-E10(+2)b(正向引物)和公共反向引物GPIV-E10(+2)c，对经DNA测序检测确认的3例Exon-10+2T＞G突变型样本(均为Exon-10+2T＞G突变杂合子型)和5例Exon-10+2T野生型样本进行Exon-10+2T＞G突变位点的PCR扩增，每个PCR扩增反应体系均加入C反应蛋白的PCR扩增正向引物CRP I和反向引物CRP II，作为内参照，PCR扩增于ABI 9700型PCR仪进行，选择PCR扩增反应体系A，组成为：
Figure PCTCN2020116567-appb-000007
(备注：在Exon-10+2T野生型PCR扩增反应体系，序列特异性引物选择GPIV-E10(+2)a；在Exon-10+2G突变型PCR扩增反应体系，序列特异性引物选择GPIV-E10(+2)b。)
PCR扩增循环参数与实施例1相同。
取8μl PCR产物，于2％琼脂糖凝胶中电泳[琼脂糖凝胶含5％的绿如蓝荧光染料(北京天恩泽基因科技有限公司)]，在凝胶成像系统中观察特异性PCR产物条带，PCR产物条带清晰、特异，结果如图3所示，样本S3-1～3为Exon-10+2T＞G突变型杂合子，因此，在突变型泳道(M)扩增出目的条带，而S3-4～8为Exon-10+2T野生型纯合子，在突变型泳道(M)未扩增出目的条带。
实施例4
本实施例给出了通过本发明的CD36突变基因导致人类GPIV缺失的基因分型试剂盒，对由CD36突变基因【C1123T(Pro375Ser)】导致的GPIV缺失的个体进行基因分型检测。
首先，取本发明中的1123C野生型序列特异性引物GPIV-1123a(反向引物)、1123T突变型序列特异性引物GPIV-1123b(反向引物)和公共正向引物GPIV-1123c，对经DNA测序检测确认的1例C1123T突变型样本(为C1123T突变杂合子型)和5例1123C野生型样本进行C1123T突变位点的PCR扩增，每个PCR扩增反应体系均加入C反应蛋白的PCR扩增正向引物CRP I和反向引物CRP II，作为内参照，PCR扩增于ABI 9700型PCR仪进行，选择PCR扩增反应体系C，组成为：
Figure PCTCN2020116567-appb-000008
(备注：在1123C野生型PCR扩增反应体系，序列特异性引物选择GPIV-1123a；在1123T突变型PCR扩增反应体系，序列特异性引物选择GPIV-1123b。)
PCR扩增循环参数与实施例1相同。
取8μl PCR产物，于2％琼脂糖凝胶中电泳[琼脂糖凝胶含5％的绿如蓝荧光染料(北京天恩泽基因科技有限公司)]，在凝胶成像系统中观察特异性PCR产物条带，PCR产物条带清晰、特异，结果如图4所示，样本S4-1为C1123T突变型杂合子，因此，在突变型泳道(M)扩增出目的条带，而S4-2～6为1123C野生型纯合子，在突变型泳道(M)未扩增出目的条带。
实施例5
本实施例给出了通过本发明的CD36突变基因导致人类GPIV缺失的基因分型试剂盒，对由CD36突变基因【T1229C(Ile410Thr)】导致的GPIV缺失的个体进行基因分型检测。
首先，取本发明中的1229T野生型序列特异性引物GPIV-1229a(反向引物)、1229C突变型序列特异性引物GPIV-1229b(反向引物)和公共正向引物GPIV-1229c，对经DNA测序 检测确认的3例T1229C突变型样本(均为T1229C突变杂合子型)和4例1229T野生型样本进行T1229C突变位点的PCR扩增，每个PCR扩增反应体系均加入C反应蛋白的PCR扩增正向引物CRP I和反向引物CRP II，作为内参照，PCR扩增于ABI 9700型PCR仪进行，选择PCR扩增反应体系B，组成为：
Figure PCTCN2020116567-appb-000009
(备注：在1229T野生型PCR扩增反应体系，序列特异性引物选择GPIV-1229a；在1229C突变型PCR扩增反应体系，序列特异性引物选择GPIV-1229b。)
PCR扩增循环参数与实施例1相同。
取8μl PCR产物，于2％琼脂糖凝胶中电泳[琼脂糖凝胶含5％的绿如蓝荧光染料(北京天恩泽基因科技有限公司)]，在凝胶成像系统中观察特异性PCR产物条带。PCR产物条带清晰、特异，结果如图5所示，样本S5-1～3为T1229C突变型杂合子，因此，在突变型泳道(M)扩增出目的条带，而S5-4～7为1229T野生型纯合子，在突变型泳道(M)未扩增出目的条带。
实施例6
本实施例给出了通过本发明的CD36突变基因导致人类GPIV缺失的基因分型试剂盒，对由CD36突变基因【1332ints TGAT(frameshift at AA 445)】导致的GPIV缺失的个体进行基因分型检测。
首先，取本发明中的1332野生型序列特异性引物GPIV-1332a(正向引物)、1332ints TGAT突变型序列特异性引物GPIV-1332b(正向引物)和公共反向引物GPIV- 1332c，对经DNA测序检测确认的2例1332ints TGAT突变型样本(均为1332ints TGAT突变杂合子型)和5例1332野生型样本进行1332突变位点的PCR扩增，每个PCR扩增反应体系均加入C反应蛋白的PCR扩增正向引物CRP I和反向引物CRP II，作为内参照，PCR扩增于ABI 9700型PCR仪进行，选择PCR扩增反应体系B，组成为：
Figure PCTCN2020116567-appb-000010
(备注：在1332野生型PCR扩增反应体系，序列特异性引物选择GPIV-1332a；在1332ints TGAT突变型PCR扩增反应体系，序列特异性引物选择GPIV-1332b。)
PCR扩增循环参数与实施例1相同。
取8μl PCR产物，于2％琼脂糖凝胶中电泳[琼脂糖凝胶含5％的绿如蓝荧光染料(北京天恩泽基因科技有限公司)]，在凝胶成像系统中观察特异性PCR产物条带，PCR产物条带清晰、特异，结果如图6所示，样本S6-1～2为1332ints TGAT突变型杂合子，因此，在突变型泳道(M)扩增出目的条带，而S6-3～7为1332野生型纯合子，在突变型泳道(M)未扩增出目的条带。
实施例7不同CD36突变基因导致GPIV缺失的基因分型检测体系中，PCR反应中镁离子终浓度的对检测结果的影响。
1、不同镁离子终浓度对G730A扩增影响
G730A PCR反应中的[Mg 2+]终＝2.0mM时，在确认该位点基因型为野生型730G/G纯合子的部分样本(S2-2～5)中730A突变基因的PCR反应扩增在目的条带位置出现非特异性扩增产物，造成假阳性结果，影响结果判断；而当[Mg 2+]终＝1.5mM时，该非特异性条带明显 减弱或消失，而不影响野生型目的条带的扩增，对携带有该突变基因的样本突变位点PCR扩增亦能取得良好检测效果，能获得正确结果判读，如图7所示。
2、不同镁离子终浓度对C1123T扩增影响
C1123T PCR反应中的[Mg 2+]终＝2.0mM时，在确认该位点基因型为野生型1123C/C纯合子的部分样本(S4-2～6)中1123T突变基因的PCR反应扩增在目的条带位置出现较弱的非特异性扩增产物，且一些样本扩增效果不理想，影响结果判断；而当[Mg 2+]终＝1.5mM时，该非特异性条带明显减弱或消失，而不影响野生型目的条带的扩增，对携带有该突变基因的样本突变位点PCR扩增亦能取得良好检测效果，能获得正确结果判读，如图8所示。
3、不同镁离子终浓度对T1229C扩增影响
T1229C PCR反应中的[Mg 2+]终＝2.0mM时，T1229C突变杂合子样本DNA(S5-1、S5-2)和野生型样本DNA(S5-4～S5-6)，不管是突变位点还是野生型位点虽然均不能得到PCR反应扩增结果。当PCR反应中的[Mg 2+]终＝3.5mM时，目的条带得到成功扩增，结果判读更为准确，如图9所示。
4、不同镁离子终浓度对1332ints TGAT扩增影响
S6-1基因型为1332ints TGAT突变杂合子，使用本试剂盒做基因分型检测结果应为W和M泳道目的条带均为阳性；S6-4基因型是1332无TGAT插入纯合子，使用本试剂盒做基因分型检测结果应为W泳道目的条带均为阳性和M泳道目的条带为阴性
1332ints TGAT PCR反应中的[Mg 2+]终＝2.0mM时，1332ints TGAT突变杂合子样本DNA(S6-1)和野生型样本DNA(S6-4)，不管是突变位点还是野生型位点虽然均不能得到PCR反应扩增结果。当PCR反应中的[Mg 2+]终＝3.5mM时，成功扩增出的目的条带，获得更好的检测效果，结果判读更为准确，如图10所示。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (7)

  1. 一种检测由6个CD36突变基因所导致GPIV缺失个体的基因分型试剂盒，其特征在于，所述试剂盒包括：检测由6个CD36突变基因所导致的GPIV缺失的各个基因位点的野生型序列特异性引物和突变型序列的特异性引物，以及公共引物；所述的CD36突变基因的突变位点分别为：275C＞T、730G＞A、Exon-10+2T＞G、1123C＞T、1229T＞C和1332 ints TGAT；所述275C＞T突变位点的野生型序列特异性引物如SEQ ID NO.1所示，275C＞T突变位点的突变型序列的特异性引物如SEQ ID NO.2所示，公共引物如SEQ ID NO.3所示；
    所述突变位点730G＞A的野生型序列特异性引物如SEQ ID NO.4所示，突变型序列的特异性引物如SEQ ID NO.5所示，公共引物如SEQ ID NO.6所示；
    所述突变位点Exon-10+2T＞G的野生型序列特异性引物如SEQ ID NO.7所示，突变型序列的特异性引物如SEQ ID NO.8所示，公共引物如SEQ ID NO.9所示；
    所述突变位点1123C＞T的野生型序列特异性引物如SEQ ID NO.10所示，突变型序列的特异性引物如SEQ ID NO.11所示，公共引物如SEQ ID NO.12所示；
    所述突变位点1229T＞C的野生型序列特异性引物如SEQ ID NO.13所示，突变型的序列特异性引物如SEQ ID NO.14所示，公共引物如SEQ ID NO.15所示；
    所述突变位点1332 ints TGAT的野生型序列特异性引物如SEQ ID NO.16所示，突变型序列的特异性引物如SEQ ID NO.17所示，公共引物如SEQ ID NO.18所示；所述试剂盒还包括CRP内参，所述内参正向引物序列如SEQ ID NO.19所示，所述内参反向引物序列如SEQ ID NO.20所示。
  2. 如权利要求1所述的检测由6个CD36突变基因所导致GPIV缺失的基因分型试剂盒，其特征在于，还包括MgCl 2试剂，其在PCR反应体系中的终浓度为1.5～3.5mM。
  3. 如权利要求2所述的检测由6个CD36突变基因所导致GPIV缺失的基因分型试剂盒，其特征在于，在检测275C＞T和Exon-10+2T＞G突变位点的各野生型和突变型的PCR扩增反应体系中镁离子终浓度为2mM，PCR扩增反应体系终体系为10μL。
  4. 如权利要求2所述的检测由6个CD36突变基因所导致GPIV缺失的基因分型试剂盒，其特征在于，在检测1229T＞C和1332 ints TGAT突变位点的各野生型和突变型的PCR扩增反应体系中镁离子终浓度为3.5mM，PCR扩增反应体系终体系为10μL。
  5. 如权利要求2所述的检测由6个CD36突变基因所导致GPIV缺失的基因分型试剂盒，其特征在于，在检测730G＞A和1123C＞T突变位点的野生型和突变型的PCR扩增反应体系中镁离子终浓度为1.5mM，PCR扩增反应体系终体系为10μL。
  6. 如权利要求1所述的检测由6个CD36突变基因所导致GPIV缺失的基因分型试剂盒，其特征在于，还包括2.5mM的dNTPs试剂，10×Buffer、DNA聚合酶、10mg/ml的甲酚红、50％的甘油。
  7. 如权利要求1所述的检测由6个CD36突变基因所导致GPIV缺失的基因分型试剂盒，其特征在于，检测权利要求1所述的突变位点进行PCR扩增的反应程序为：
    95℃                    5min
    以下条件扩增25个循环：
    95℃                    30sec
    68℃-0.4℃/循环         30sec
    72℃                    30sec
    以下条件扩增15个循环：
    95℃                   30sec
    54℃                   30sec
    72℃                   30sec
    最后以下条件延伸和保存：
    72℃                   5min
    12℃                   ∞。
PCT/CN2020/116567 2019-09-25 2020-09-21 一种检测由6个cd36突变基因所导致gpiv缺失的基因分型试剂盒 WO2021057667A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/673,769 US20220170103A1 (en) 2019-09-25 2022-02-16 Genotyping kit for detection of six cd36 mutant genes that encode gpiv deficiency

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910908778.5A CN110684834B (zh) 2019-09-25 2019-09-25 一种检测由6个cd36突变基因所导致gpⅳ缺失的基因分型试剂盒
CN201910908778.5 2019-09-25

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/673,769 Continuation-In-Part US20220170103A1 (en) 2019-09-25 2022-02-16 Genotyping kit for detection of six cd36 mutant genes that encode gpiv deficiency

Publications (1)

Publication Number Publication Date
WO2021057667A1 true WO2021057667A1 (zh) 2021-04-01

Family

ID=69110573

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/116567 WO2021057667A1 (zh) 2019-09-25 2020-09-21 一种检测由6个cd36突变基因所导致gpiv缺失的基因分型试剂盒

Country Status (3)

Country Link
US (1) US20220170103A1 (zh)
CN (1) CN110684834B (zh)
WO (1) WO2021057667A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110684834B (zh) * 2019-09-25 2020-09-15 南宁中心血站(南宁输血医学研究所) 一种检测由6个cd36突变基因所导致gpⅳ缺失的基因分型试剂盒
CN114381518A (zh) * 2020-10-05 2022-04-22 复旦大学附属华山医院 一种用于快速检测胶质瘤突变位点及分型的引物和试剂盒

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434564A (zh) * 2016-11-01 2017-02-22 南宁输血医学研究所 导致cd36缺失的cd36突变基因真核稳定表达细胞株的建立方法
CN110684834A (zh) * 2019-09-25 2020-01-14 南宁中心血站(南宁输血医学研究所) 一种检测由6个cd36突变基因所导致gpⅳ缺失的基因分型试剂盒

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434564A (zh) * 2016-11-01 2017-02-22 南宁输血医学研究所 导致cd36缺失的cd36突变基因真核稳定表达细胞株的建立方法
CN110684834A (zh) * 2019-09-25 2020-01-14 南宁中心血站(南宁输血医学研究所) 一种检测由6个cd36突变基因所导致gpⅳ缺失的基因分型试剂盒

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE 24 August 2019 (2019-08-24), ANONYMOUS: "Homo sapiens CD36 molecule (CD36), RefSeqGene on chromosome 7.", XP055795813, retrieved from GENBANK Database accession no. NG_008192 *
LI, LILAN ET AL.: "A Novel CD36 Mutation T538C (Trp180Arg)Results in CD36 Deficiency and Establishment of a Genotyping Method for the Novel Mutation based on Sequence-Specific Primer PCR)", ZHONGHUA YIXUE YICHUANXUE ZAZHI - CHINESE JOURNAL OF MEDICAL GENETICS, vol. 33, no. 5, 31 October 2016 (2016-10-31), pages 619 - 624, XP009527053, ISSN: 1003-9406, DOI: 10.3760/cma.j.issn.1003-9406.2016.05.008 *
Y. YANG, B.R. LUO, M. HU, D.M. ZHAO, W.J. JING: "Association of CD36 gene single nucleotide polymorphism with gestational diabetes mellitus in Chinese Han population", CLINICAL AND EXPERIMENTAL OBSTETRICS & GYNECOLOGY, vol. 45, no. 2, 31 December 2018 (2018-12-31), pages 266 - 271, XP009527051, ISSN: 0390-6663, DOI: 10.12891/ceog3844.2018 *
YUAN, YAN ET AL.: "Relationship Between the Polymorphism of rs17154181 and rs1761667 Sites of CD36 Gene and Macroangiopathy in Patients With Type 2 Diabetes Mellitus in Guangxi Province)", CHINESE GENERAL PRACTICE, vol. 18, no. 20, 31 July 2015 (2015-07-31), pages 2421 - 2425, XP055795806, ISSN: 1007-9572 *
ZHONG, ZHOULIN ET AL.: "Characteristic and Structure Analysis of CD36 Deficiency in Han, Zhuang and Yao Population from Guangxi Region in China", CHINESE JOURNAL OF BLOOD TRANSFUSION, vol. 27, no. 1, 31 January 2014 (2014-01-31), pages 14 - 17, XP055795794, ISSN: 1004-549X *

Also Published As

Publication number Publication date
CN110684834A (zh) 2020-01-14
US20220170103A1 (en) 2022-06-02
CN110684834B (zh) 2020-09-15

Similar Documents

Publication Publication Date Title
US20200270692A1 (en) Predicting age-related macular degeneration with single nucleotide polymorphisms within or near the genes for complement component c2, factor b, plekha1, htra1, prelp, or loc387715
US20110207124A1 (en) Genetic Alterations Associated with Autism and the Autistic Phenotype and Methods of Use Thereof for the Diagnosis and Treatment of Autism
WO2021057667A1 (zh) 一种检测由6个cd36突变基因所导致gpiv缺失的基因分型试剂盒
JP4031199B2 (ja) ヒト気道トリプシン様酵素遺伝子多型による疾病体質解析
CN111662973B (zh) 与慢性阻塞性肺疾病易感性辅助诊断相关的snp位点及其应用
CN115772214B (zh) F8突变体蛋白、f8基因突变体、检测f8基因突变体的引物组合、试剂及应用
US8137916B2 (en) Susceptibility gene for alzheimer's disease
CN115927356B (zh) Slc45a2致病突变基因、致病突变体及在制备眼皮肤白化病ⅳ型诊断试剂盒中的应用
CN104894261B (zh) 一种预测雷珠单抗治疗年龄相关性黄斑变性疗效的试剂盒
EP2126113A1 (en) Diagnostic marker and platform for drug design in myocardial infarction and heart failure
WO2002006467A1 (fr) Methode de detection d'erreurs du metabolisme lipidique
CN115927354B (zh) 一种sh3tc2基因致病突变体及其在制备腓骨肌萎缩症4c型诊断试剂盒中的应用
EP1199372A2 (en) Polymorphisms in the human P2X7 gene
US9708663B2 (en) Use of SAA1B/B homozygote in the prognosis and diagnosis of liver cirrhosis
KR102072504B1 (ko) Dock8 유전자 단일염기다형성을 이용한 아토피 피부염 진단용 조성물과 검출 방법
US20050084849A1 (en) Diagnostic polymorphisms for the ecnos promoter
Morkūnienė et al. Are TGFA, TGFB3, GABRB3, RARA and BCL3 loci associated with nonsyndromic orofacial clefts? A Lithuanian study
US20210310073A1 (en) A genetic predisposition to liver disease
EP2149612A1 (en) Genetic markers of response to efalizumab
JP5111763B2 (ja) 気管支喘息の遺伝的素因検査方法
CN111321218A (zh) 迟发性耳聋GJB2基因pV37I突变的检测试剂盒
WO2009034661A1 (en) Method for diagnosis and induction of resistance to virus
MORKORKŪNIENĖ et al. Are TGFA, TGFB3, GABRB3, RARA and BCL3 loci associated with nonsyndromic orofacial clefts? A Lithuanian study
Pelo et al. IMPROVEMENT OF MOLECULAR TECHNOLOGIES AND PRENATAL DIAGNOSIS OF CYSTIC FIBROSIS
WO2005033339A1 (ja) アレルギー疾患の遺伝的素因マーカーとしてのヒトトル様レセプター３遺伝子多型およびその使用

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: 20867553

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: 20867553

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 18/05/2022)

122 Ep: pct application non-entry in european phase

Ref document number: 20867553

Country of ref document: EP

Kind code of ref document: A1