CN110157790A - 一种不明原因心脏性猝死的快速基因筛查试剂盒 - Google Patents
一种不明原因心脏性猝死的快速基因筛查试剂盒 Download PDFInfo
- Publication number
- CN110157790A CN110157790A CN201910308341.8A CN201910308341A CN110157790A CN 110157790 A CN110157790 A CN 110157790A CN 201910308341 A CN201910308341 A CN 201910308341A CN 110157790 A CN110157790 A CN 110157790A
- Authority
- CN
- China
- Prior art keywords
- seq
- primer sequence
- site
- kit
- gene
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 238000012216 screening Methods 0.000 title claims abstract description 41
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 38
- 206010049418 Sudden Cardiac Death Diseases 0.000 title claims abstract description 26
- 108091006736 SLC22A5 Proteins 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 15
- 108091093088 Amplicon Proteins 0.000 claims abstract description 6
- 230000003321 amplification Effects 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 44
- 239000002585 base Substances 0.000 claims description 30
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 238000004925 denaturation Methods 0.000 claims description 7
- 230000036425 denaturation Effects 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 102200001392 rs386134211 Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 102200001734 rs11568520 Human genes 0.000 claims description 5
- 102220002474 rs121908893 Human genes 0.000 claims description 5
- 102220007465 rs386134209 Human genes 0.000 claims description 5
- 102200001327 rs60376624 Human genes 0.000 claims description 5
- 102200001739 rs72552725 Human genes 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- 239000012498 ultrapure water Substances 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 3
- 230000004044 response Effects 0.000 claims description 3
- 102200001742 rs202088921 Human genes 0.000 claims description 3
- 102220055175 rs727504159 Human genes 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 238000004153 renaturation Methods 0.000 claims description 2
- 102220002467 rs72552727 Human genes 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims description 2
- 238000011144 upstream manufacturing Methods 0.000 claims 12
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 239000012264 purified product Substances 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 3
- 230000004217 heart function Effects 0.000 abstract description 2
- 230000000869 mutational effect Effects 0.000 abstract description 2
- 102100036924 Solute carrier family 22 member 5 Human genes 0.000 description 34
- 206010042434 Sudden death Diseases 0.000 description 21
- 208000016505 systemic primary carnitine deficiency disease Diseases 0.000 description 20
- 208000033897 Systemic primary carnitine deficiency Diseases 0.000 description 17
- 108010064675 calcium dependent sulfhydryl protease Proteins 0.000 description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 15
- 201000010099 disease Diseases 0.000 description 13
- 238000001514 detection method Methods 0.000 description 10
- 206010064571 Gene mutation Diseases 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000003745 diagnosis Methods 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 230000000747 cardiac effect Effects 0.000 description 7
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 102000004310 Ion Channels Human genes 0.000 description 5
- 229960004203 carnitine Drugs 0.000 description 5
- 238000007403 mPCR Methods 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 206010049993 Cardiac death Diseases 0.000 description 4
- 206010011906 Death Diseases 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 108010007577 Exodeoxyribonuclease I Proteins 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 208000031229 Cardiomyopathies Diseases 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 208000024556 Mendelian disease Diseases 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 206010003119 arrhythmia Diseases 0.000 description 2
- 230000006793 arrhythmia Effects 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 208000030159 metabolic disease Diseases 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000006916 protein interaction Effects 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 108091071337 20 family Proteins 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 206010019842 Hepatomegaly Diseases 0.000 description 1
- 206010020575 Hyperammonaemia Diseases 0.000 description 1
- 208000013016 Hypoglycemia Diseases 0.000 description 1
- 206010028372 Muscular weakness Diseases 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- 108091006735 SLC22A2 Proteins 0.000 description 1
- 108010038615 Solute Carrier Family 22 Member 5 Proteins 0.000 description 1
- 102100021543 Solute carrier family 22 member 16 Human genes 0.000 description 1
- 101710102937 Solute carrier family 22 member 16 Proteins 0.000 description 1
- 102100032417 Solute carrier family 22 member 2 Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000021024 autosomal recessive inheritance Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015978 inherited metabolic disease Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000036473 myasthenia Effects 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 238000009609 prenatal screening Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000002336 repolarization Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
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
本发明公开了一种不明原因心脏性猝死的快速基因筛查试剂盒,该试剂盒包含了针对SLC22A5基因的12个SNP位点的特异性多重PCR引物对,所述引物对通过扩增能够包含不同SNP的扩增子,每段扩增子至少含有一个SNP位点,所述特异性多重PCR引物对的核苷酸序列如SEQ NO.1~24所示,且所述试剂盒还包含SNP的SNaPshot单碱基延伸引物。本试剂盒利用多重PCR技术和SNaPshot微测序技术相结合的方法,可在一个反应内筛查与心脏结构和功能高度相关的SLC22A5基因的12个突变位点,大大节省了时间和经济成本。
Description
技术领域
本发明属于生物基因检测技术领域,具体涉及一种不明原因心脏性猝死的快速基因筛查试剂盒。
背景技术
猝死(sudden death)是指机体潜在的疾病或重要器官急性功能障碍导致的急速、意外的自然性死亡。在全球,每年猝死人数占自然死亡人数的10%甚至更多,严重威胁着社区人群的生命安全。心脏性猝死(sudden cardiac death,SCD)是指由于心脏原因所致的突然死亡,约占全部猝死的50%,居猝死病因分布的首位。SCD主要病因有冠状动脉性心脏病、心肌炎、心肌病等,但在法医病理学实践中,部分猝死案例并无明显器质性病变,病因不明,被称之为不明原因猝死(sudden unexplained death,SUD),其法医学死因鉴定已成为一个亟待解决的难题;同时,具有这类猝死风险的高危患者的筛选诊断问题也成为临床医学的难题。
目前针对SUD诊断领域的研究主要集中于探寻心脏离子通道病与重要离子通道蛋白编码基因以及与离子通道蛋白相互作用蛋白编码基因的突变与猝死的关联性。但有相当数量的SUD无法用心脏离子通道病来解释死因,其真正死因无法被揭示。例如一种遗传代谢性疾病——原发性肉碱缺乏症,在临床上较不常见,且临床表现不典型,可以以猝死为首发且唯一的表现。然而,在法医病理学及临床医学领域,对本病与恶性心律失常、猝死的关系均认识不足,未将本病列入SUD的病因诊断和筛查之列,造成部分SUD难于探寻到准确病因,给其死因鉴定及其高危近亲属的猝死防治带来困难。
原发性肉碱缺乏症(Primary Carnitine Deficiency Syndrome,CDSP)是SLC22A5基因突变导致新型有机阳离子转运体2(organic carnitine transporter 2,OCTN2)功能缺陷所致的一种脂肪酸β氧化代谢疾病,遗传方式为常染色体隐性遗传。CDSP临床表现个体差异大,主要表现为代谢异常(低酮型低血糖、高血氨等)、心肌受累(心肌病、心律失常等)、肌无力、肝肿大等,非典型表现包括发育迟缓、易疲劳甚至猝死等。其中心脏是CDSP患者最易受累的器官,因此CDSP所引起的猝死大多是SCD,且可先于器质性心脏病变出现,这种情况下,如死者生前无明显临床症状,又未确诊为CDSP,真正死因很难被发现。
CDSP是为数不多可治疗的遗传性代谢疾病,SLC22A5基因突变为目前唯一已知病因,多数患者在婴幼儿期确诊,血浆游离肉碱浓度明显下降(参考值:>10μM)即可确诊,存在SLC22A5基因突变可辅助诊断。目前唯一且有效的治疗方法为口服左旋肉碱,需终身服药。因此通过早期SLC22A5基因筛查来规避本病导致的不良后果(特别是猝死)就显得尤为重要。但我国目前并未将本病列入产前筛查及新生儿筛查常规项目,这就给本病患者带来了极大的健康及生命安全隐患。
现有猝死分子病医学诊断及筛查试剂盒不能发现CDSP这一病因引起的不明原因猝死的分子病因;且绝大多数的筛查试剂盒对猝死病因诊断的精确性不够(只筛查多态位点),未定位到引起猝死的遗传突变。现有猝死分子病因学诊断及筛查试剂盒主要针对心脏离子通道及其相互作用蛋白编码基因的筛查,没有包括CDSP相关致病基因的检测;且绝大部分猝死筛查试剂盒仅针对多态位点进行检测,未精确定位致病性突变的检测。
因此,为了协助早期治疗CDSP、预防SCD和完善猝死易感性风险评估体系,开发相关快速基因突变检测试剂盒是亟待解决的问题。
发明内容
针对上述现有技术,本发明提供了一种心脏性猝死快速基因筛查试剂盒,本试剂盒是基于与心脏结构、功能相关的SLC22A5基因的12个变异点作为不明原因猝死筛查的位点,设计了引物混合物,实现了多位点的基因扩增。本方法采用多重PCR技术和SNaPshot微测序技术相结合的方法来解决位于SLC22A5基因上SCD相关的12个突变位点的检测问题。
本发明是通过以下技术方案实现的:一种不明原因心脏性猝死的快速基因筛查试剂盒,包含针对SLC22A5基因的12个SNP位点的特异性多重PCR引物对,所述引物对通过扩增能够包含不同SNP的扩增子,每段扩增子至少含有一个SNP位点,所述特异性多重PCR引物对的核苷酸序列如SEQ NO.1~24所示,且所述试剂盒还包含SNP的SNaPshot单碱基延伸引物。
进一步地,所述不明原因心脏性猝死的快速基因筛查试剂盒,包含检测SLC22A5基因的12个SNP位点,所述SNP位点的rs编号表1所示:
表1:SLC22A5基因的12个SNP位点
序号 | 氨基酸突变类型 | rs编号 |
1 | F17L | rs11568520 |
2 | N32S | rs72552725 |
3 | P46S | rs202088921 |
4 | C113Y | 727504159 |
5 | W132X | r886041277 |
6 | R254X | rs121908893 |
7 | V275X | rs765954398 |
8 | S280F | rs386134209 |
9 | W283C | rs386134211 |
10 | R289X | rs756260416 |
11 | Y396X | rs756015838 |
12 | S467C | rs60376624 |
进一步地,所述不明原因心脏性猝死的快速基因筛查试剂盒,扩增SLC22A5基因12个SNP位点特异性PCR多重引物对的核苷酸序列如表2所示:
表2:SLC22A5基因的12个SNP位点的特异性多重引物对
进一步地,所述不明原因心脏性猝死的快速基因筛查试剂盒,其SNP的SNaPshot单碱基延伸引物的核苷酸序列如表3所示。
表3:SLC22A5基因的12个SNP位点的SNaPshot单碱基延伸引物
rs编号 | 延伸引物序列(5'to3') | 序列号 |
rs11568520 | gagcaggaagaagatgaggcgctg | SEQ NO.25 |
rs72552725 | tgactgactgactgactgactacggaggacaggccggtgaagcca | SEQ NO.26 |
rs20208892 | gactgacttgtcctccgtgttcctgatagcgacc | SEQ NO.27 |
rs727504159 | tgactgactgactgactgactgactgactgaactcccagccatccaga | SEQ NO.28 |
rs886041277 | gactgactgactgactgactgactgactgactgactccagtcgtcctcacacaccaggttc | SEQ NO.29 |
rs121908893 | gactcgccaccagcagcatccgccagtctc | SEQ NO.30 |
rs765954398 | ctgactgactgactgactgactgactgactgactgggtgctatgcgtggcactctggt | SEQ NO.31 |
rs386134209 | gactgactgactgactgactgactgactgactgactgacttccctgagagatgagccatcggggg | SEQ NO.32 |
rs386134211 | tgactgactgactgagtttcatccctgagtccccccgatg | SEQ NO.33 |
rs756260416 | gactgactgactgactgactgactgactgactccgatggctcatctctcagggac | SEQ NO.34 |
rs756015838 | actgactgactgactgactgactgactgaagcagttcacaaagatgtcccca | SEQ NO.35 |
rs60376624 | actgactgactgactgacttgctgcccaggcgggatgctgtg | SEQ NO.36 |
进一步地,所述不明原因心脏性猝死的快速基因筛查试剂盒,还包括扩增试剂,所述扩增试剂包括PCR缓冲液、dNTP混合液、Taq酶和超纯水。
进一步地,应用所述不明原因心脏性猝死的快速基因筛查试剂盒进行PCR扩增的反应体系为10μl,所述反应体系包括1μl的DNA模板、1μl引物混合物、5μl的2×Taq PCR预混液、3μl超纯水。
进一步地,应用所述不明原因心脏性猝死的快速基因筛查试剂盒进行PCR扩增的程序如下:
95℃,3min预变性;
95℃,30sec变性,56℃,30sec退火,72℃,30sec,复性,热循环45次;
72℃,2min,延伸。
进一步地,应用所述不明原因心脏性猝死的快速基因筛查试剂盒进行SNaPshot单碱基延伸的反应体系为5μl,所述反应体系为0.5μl SNaPshot Mix、3μl多重PCR反应纯化产物、1μl的单碱基延伸引物、0.5μl超纯水。
进一步地,应用所述不明原因心脏性猝死的快速基因筛查试剂盒进行SNaPshot单碱基延伸的反应程序如下:
95℃,2min预变性;
95℃,10sec;52℃,5sec;60℃,30sec,共40个循环;
4℃forever。
本发明的有益效果:本试剂盒综合检测与分析受检人群是否携带与心脏结构和功能高度相关的SLC22A5基因突变,及时筛查出具猝死风险的CDSP患者,给予临床干预,避免SCD的发生,弥补了现有试剂盒仅针对于心脏离子通道及其相互作用蛋白编码基因筛查诊断的局限性。
本发明的试剂盒操作简便,节约成本。目前临床上对SLC22A5基因的检测多采用全外显子组测序,无目的性,价格昂贵。本试剂盒利用多重PCR技术和SNaPshot微测序技术相结合的方法,可在一个反应内筛查与心脏结构和功能高度相关的SLC22A5基因的12个突变位点,大大节省了时间和经济成本。
附图说明
图1为应用实施例中该家系中成员外周血样本的sanger测序SLC22A5基因1种突变峰图;
图2为应用实施例中健康人群Sanger测序SLC22A5基因峰图;
具体实施方式
下面结合具体实施例及附图对本发明做进一步的说明,以助于理解本发明的内容。
实施例1本发明试剂盒的操作步骤
1、Multiplex PCR及其产物纯化处理:
(1)多重PCR反应
以样本中提取的DNA为模板,通过Thermal Cycler2720PCR仪进行多重PCR反应,获得Multiplex PCR产物;所述样本包括健康个体构成的对照组、CDSP可疑家系构成的CDSP组。
所述样本中提取DNA的方法为:采用TIANGEN TGuide M16自动核酸提取仪(OSE-M16),配合TIANGE TGuide大体积血液基因组DNA提取试剂盒(1-3ml)提取人体静脉血DNA,对于提取的DNA按照光密度值法进行分析,确定基因组DNA的浓度。
多重PCR体系包含如SEQ NO.1~24所示的12对引物,体系组成成分如下:
2×Taq PCR Master Mix | 5μl |
Primer Mix | 1ul |
DNA template | 1μl |
ddH<sub>2</sub>O | 3ul |
多重PCR体系的扩增程序如下:
(2)多重PCR产物纯化
采用虾碱酶纯化法:虾碱酶MIX(EX-SAP)的主要功能成分为SAP及ExoI,SAP酶将残余dNTPs去磷酸化,ExoI降解游离单链引物。
多重PCR产物纯化处理的组成成分,其纯化体系如下:
消化体系组分 | 体积(ul) |
ddH<sub>2</sub>O | 0.75 |
SAP(1U/ul) | 0.5 |
ExoI(5U/ul) | 0.15 |
10×SAP buffer | 0.6 |
PCR产物 | 4 |
总体积 | 6 |
纯化步骤:将上述纯化体系在PCR仪上进行消化孵育,其条件为:37℃,40min后85℃,5min。
2、SNaPshot单碱基延伸反应(SBE)及其纯化处理:
(1)SNaPshot单碱基延伸反应(SBE):对Multiplex PCR产物通过如SEQ NO.25~36所示的引物进行SNaPshot单碱基延伸反应。
SNaPshot单碱基延伸反应的组成成分,体积和扩增程序如下:
SBE体系如下:
组分 | 体积(ul) |
SNaPshot Mix | 0.5 |
Pooled PCR Products | 3 |
Pooled Primers | 1 |
dH<sub>2</sub>O | 0.5 |
总体积 | 5 |
SBE反应程序:95℃2min→(95℃10sec→52℃5sec→60℃30sec)×40循环→4℃forever
(2)SBE产物纯化:
向SBE反应产物中加入2ul SAP Mix,配制组分如下:
组分 | 体积(ul) |
水 | 0.9 |
SAP(1U/ul) | 0.5 |
10×SAP buffer | 0.6 |
总计 | 2 |
在PCR仪上,程序:37℃40min→75℃15min。
3、SNaPshot单碱基延伸产物的结果分析:取2ul消化后的SBE反应产物加入到8ul含有0.4%LIZ120的去离子甲酰胺中,95℃变性5min,然后放置-20℃骤冷后利用ABI3730进行测序,完成对延伸引物的检测。
实施例2应用实施例
为了检测本试剂盒的应用效能,对不明原因猝死死者1名,经常规基因检测未发现携带重要离子通道蛋白编码基因以及与离子通道蛋白相互作用蛋白编码基因突变,及其他任何已证实为猝死相关基因突变。经本试剂盒检测,结果显示死者携带SLC22A5突变p.F17L与p.R254X。经了解家族史发现家族中另存在2名不明原因猝死者,高度怀疑为CDSP家系,提示死者真正死因可能为CDSP。利用本试剂盒检测上述CDSP疑似家系共20人和健康人群的外周血样本。
检测结果:该家系中20名家族成员外周血样本均出现阳性结果,提示均携带至少一种SLC22A5基因变异。附图1为该疑似CDSP家系中,经本试剂盒检测后,发现携带一杂合突变(图中箭头所示)。附图2为健康人群样本测序结果,经本试剂盒检测后,未检测到SLC22A5基因的突变,均为纯合野生型。
经后续临床检查,有3名成员出现血游离肉碱浓度下降,其中1名心电图检查表现为早期复极综合征,其他2名出现血游离肉碱浓度显著下降,心电图结果均表现为短QT综合征,临床确诊为CDSP。上述3名成员在接受临床治疗后,血游离肉碱水平维持在正常水平,心电图异常改变消失。
在本应用实例中,该家系2名血游离肉碱浓度显著下降的成员虽无器质性病变,却已出现短QT综合征的心电图改变,短QT综合征与SCD高度相关,结合猝死家族史,此2名家族成员有高度猝死风险,使用本试剂盒进行筛查后,及时接受临床治疗,避免了这一不良后果的发生。
以上所述实施方式仅表达了本发明的其中一种或几种实施方式,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。
SEQUENCE LISTING
<110> 中山大学
<120> 一种不明原因心脏性猝死的快速基因筛查试剂盒
<130> 2019.4.9
<160> 36
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> 人工合成
<400> 1
agtgggacag tgtggttgc 19
<210> 2
<211> 19
<212> DNA
<213> 人工合成
<400> 2
catgcgggac tacgacgag 19
<210> 3
<211> 19
<212> DNA
<213> 人工合成
<400> 3
catgcgggac tacgacgag 19
<210> 4
<211> 19
<212> DNA
<213> 人工合成
<400> 4
agtgggacag tgtggttgc 19
<210> 5
<211> 20
<212> DNA
<213> 人工合成
<400> 5
tgtcctccgt gttcctgata 20
<210> 6
<211> 20
<212> DNA
<213> 人工合成
<400> 6
tagacgtcct gactgaactc 20
<210> 7
<211> 20
<212> DNA
<213> 人工合成
<400> 7
tagacgtcct gactgaactc 20
<210> 8
<211> 20
<212> DNA
<213> 人工合成
<400> 8
tccgtgttcc tgatagcgac 20
<210> 9
<211> 20
<212> DNA
<213> 人工合成
<400> 9
gctatgaaaa acaggatggg 20
<210> 10
<211> 20
<212> DNA
<213> 人工合成
<400> 10
acaaggagat tgtgagtggg 20
<210> 11
<211> 20
<212> DNA
<213> 人工合成
<400> 11
agggattcat gggttgttgc 20
<210> 12
<211> 20
<212> DNA
<213> 人工合成
<400> 12
tctctacgtt aggagtgtgc 20
<210> 13
<211> 20
<212> DNA
<213> 人工合成
<400> 13
agggattcat gggttgttgc 20
<210> 14
<211> 20
<212> DNA
<213> 人工合成
<400> 14
tctctacgtt aggagtgtgc 20
<210> 15
<211> 20
<212> DNA
<213> 人工合成
<400> 15
caaccttatt cccacctatg 20
<210> 16
<211> 20
<212> DNA
<213> 人工合成
<400> 16
ggtcagtctg tccctctcac 20
<210> 17
<211> 20
<212> DNA
<213> 人工合成
<400> 17
accttattcc cacctatggc 20
<210> 18
<211> 20
<212> DNA
<213> 人工合成
<400> 18
ttacctcact cgggtcaaag 20
<210> 19
<211> 20
<212> DNA
<213> 人工合成
<400> 19
ttacctcact cgggtcaaag 20
<210> 20
<211> 20
<212> DNA
<213> 人工合成
<400> 20
accttattcc cacctatggc 20
<210> 21
<211> 20
<212> DNA
<213> 人工合成
<400> 21
accatagatg cacatggtcc 20
<210> 22
<211> 20
<212> DNA
<213> 人工合成
<400> 22
tgggctattt tgggctttcg 20
<210> 23
<211> 20
<212> DNA
<213> 人工合成
<400> 23
cagttagtac ttccatcccg 20
<210> 24
<211> 20
<212> DNA
<213> 人工合成
<400> 24
atttggctac agtcctggtg 20
<210> 25
<211> 24
<212> DNA
<213> 人工合成
<400> 25
gagcaggaag aagatgaggc gctg 24
<210> 26
<211> 45
<212> DNA
<213> 人工合成
<400> 26
tgactgactg actgactgac tacggaggac aggccggtga agcca 45
<210> 27
<211> 34
<212> DNA
<213> 人工合成
<400> 27
gactgacttg tcctccgtgt tcctgatagc gacc 34
<210> 28
<211> 48
<212> DNA
<213> 人工合成
<400> 28
tgactgactg actgactgac tgactgactg aactcccagc catccaga 48
<210> 29
<211> 61
<212> DNA
<213> 人工合成
<400> 29
gactgactga ctgactgact gactgactga ctgactccag tcgtcctcac acaccaggtt 60
c 61
<210> 30
<211> 30
<212> DNA
<213> 人工合成
<400> 30
gactcgccac cagcagcatc cgccagtctc 30
<210> 31
<211> 58
<212> DNA
<213> 人工合成
<400> 31
ctgactgact gactgactga ctgactgact gactgggtgc tatgcgtggc actctggt 58
<210> 32
<211> 65
<212> DNA
<213> 人工合成
<400> 32
gactgactga ctgactgact gactgactga ctgactgact tccctgagag atgagccatc 60
ggggg 65
<210> 33
<211> 40
<212> DNA
<213> 人工合成
<400> 33
tgactgactg actgagtttc atccctgagt ccccccgatg 40
<210> 34
<211> 55
<212> DNA
<213> 人工合成
<400> 34
gactgactga ctgactgact gactgactga ctccgatggc tcatctctca gggac 55
<210> 35
<211> 52
<212> DNA
<213> 人工合成
<400> 35
actgactgac tgactgactg actgactgaa gcagttcaca aagatgtccc ca 52
<210> 36
<211> 42
<212> DNA
<213> 人工合成
<400> 36
actgactgac tgactgactt gctgcccagg cgggatgctg tg 42
Claims (10)
1.一种不明原因心脏性猝死的快速基因筛查试剂盒,其特征在于,所述试剂盒包含针对SLC22A5基因的12个SNP位点的特异性多重PCR引物对,所述引物对通过扩增能够包含不同SNP的扩增子,每段扩增子至少含有一个SNP位点,所述特异性多重PCR引物对的核苷酸序列如SEQ NO.1~24所示;所述试剂盒还包含SNP的SNaPshot单碱基延伸引物。
2.如权利要求1所述的不明原因心脏性猝死的快速基因筛查试剂盒,其特征在于,所述SNP位点的rs编号如下:rs11568520、rs72552725、rs202088921、727504159、r886041277、rs121908893、rs765954398、rs386134209、rs386134211、rs756260416、rs756015838、rs60376624。
3.如权利要求2所述的不明原因心脏性猝死的快速基因筛查试剂盒,其特征在于,扩增SLC22A5基因rs11568520位点的上游引物序列如SEQ NO.1所示,下游引物序列如SEQ NO.2所示;
扩增SLC22A5基因rs72552725位点的上游引物序列如SEQ NO.3所示,下游引物序列如SEQ NO.4所示;
扩增SLC22A5基因rs202088921位点的上游引物序列如SEQ NO.5所示,下游引物序列如SEQ NO.6所示;
扩增SLC22A5基因727504159位点的上游引物序列如SEQ NO.7所示,下游引物序列如SEQ NO.8所示;
扩增SLC22A5基因r886041277位点的上游引物序列如SEQ NO.9所示,下游引物序列如SEQ NO.10所示;
扩增SLC22A5基因rs121908893位点的上游引物序列如SEQ NO.11所示,下游引物序列如SEQ NO.12所示;
扩增SLC22A5基因rs765954398位点的上游引物序列如SEQ NO.13所示,下游引物序列如SEQ NO.14所示;
扩增SLC22A5基因rs386134209位点的上游引物序列如SEQ NO.15所示,下游引物序列如SEQ NO.16所示;
扩增SLC22A5基因rs386134211位点的上游引物序列如SEQ NO.17所示,下游引物序列如SEQ NO.18所示;
扩增SLC22A5基因rs756260416位点的上游引物序列如SEQ NO.19所示,下游引物序列如SEQ NO.20所示;
扩增SLC22A5基因rs756015838位点的上游引物序列如SEQ NO.21所示,下游引物序列如SEQ NO.22所示;
扩增SLC22A5基因rs60376624位点的上游引物序列如SEQ NO.23所示,下游引物序列如SEQ NO.24所示.
4.如权利要求2所述的不明原因心脏性猝死的快速基因筛查试剂盒,其特征在于,所述SNP的SNaPshot单碱基延伸引物核苷酸序列如SEQ NO.25~36所示。
5.如权利要求4所述的不明原因心脏性猝死的快速基因筛查试剂盒,其特征在于,所述rs386134211位点的SNaPshot单碱基延伸引物序列如SEQ NO.25所示;所述rs72552725位点的SNaPshot单碱基延伸引物序列如SEQ NO.26所示;所述rs202088921位点的SNaPshot单碱基延伸引物序列如SEQ NO.27所示;所述rs727504159位点的SNaPshot单碱基延伸引物序列如SEQ NO.28所示;所述rs886041277位点的SNaPshot单碱基延伸引物序列如SEQ NO.29所示;所述rs121908893位点的SNaPshot单碱基延伸引物序列如SEQ NO.30所示;所述rs765954398位点的SNaPshot单碱基延伸引物序列如SEQ NO.31所示;所述rs386134209位点的SNaPshot单碱基延伸引物序列如SEQ NO.32所示;所述rs386134211位点的SNaPshot单碱基延伸引物序列如SEQ NO.33所示;所述rs756260416位点的SNaPshot单碱基延伸引物序列如SEQ NO.34所示;所述rs756015838位点的SNaPshot单碱基延伸引物序列如SEQ NO.35所示;所述rs60376624位点的SNaPshot单碱基延伸引物序列如SEQ NO.36所示。
6.如权利要求1-5任一项所述的不明原因心脏性猝死的快速基因筛查试剂盒,其特征在于,还包括扩增试剂,所述扩增试剂包括PCR缓冲液、dNTP混合液、Taq酶和超纯水。
7.如权利要求6所述的不明原因心脏性猝死的快速基因筛查试剂盒,其特征在于,应用所述试剂盒进行PCR扩增的反应体系为10μl,所述反应体系包括1μl的DNA模板、1μl引物混合物、5μl的2×Taq PCR预混液、3μl超纯水。
8.如权利要求7所述的不明原因心脏性猝死的快速基因筛查试剂盒,其特征在于,应用所述试剂盒进行PCR扩增的程序如下:
95℃,3min预变性;
95℃,30sec变性,56℃,30sec退火,72℃,30sec,复性,热循环45次;
72℃,2min,延伸。
9.如权利要求6所述的不明原因心脏性猝死的快速基因筛查试剂盒,其特征在于,应用所述试剂盒进行SNaPshot单碱基延伸的反应体系为5μl,所述反应体系包括0.5μlSNaPshot Mix、3μl多重PCR反应纯化产物、1μl的单碱基延伸引物、0.5μl超纯水。
10.如权利要求9所述的不明原因心脏性猝死的快速基因筛查试剂盒,其特征在于,应用所述试剂盒进行SNaPshot单碱基延伸的反应程序如下:
95℃,2min预变性;
95℃,10sec;52℃,5sec;60℃,30sec,共40个循环;
4℃ forever。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910308341.8A CN110157790A (zh) | 2019-04-17 | 2019-04-17 | 一种不明原因心脏性猝死的快速基因筛查试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910308341.8A CN110157790A (zh) | 2019-04-17 | 2019-04-17 | 一种不明原因心脏性猝死的快速基因筛查试剂盒 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110157790A true CN110157790A (zh) | 2019-08-23 |
Family
ID=67639453
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910308341.8A Pending CN110157790A (zh) | 2019-04-17 | 2019-04-17 | 一种不明原因心脏性猝死的快速基因筛查试剂盒 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110157790A (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113215243A (zh) * | 2021-05-19 | 2021-08-06 | 北京华诺奥美医学检验实验室有限公司 | 一种用于检测slc22a5基因高频致病变异的荧光定量pcr探针引物组和试剂盒 |
CN114317714A (zh) * | 2021-12-31 | 2022-04-12 | 北京华诺奥美基因医学检验实验室有限公司 | 用于检测slc22a5基因九种高频致病变异的荧光定量pcr引物、探针及试剂盒 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005026724A2 (en) * | 2003-09-13 | 2005-03-24 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with organic cation transporter slc22a5 (slc22a5) |
US20050265995A1 (en) * | 2003-11-13 | 2005-12-01 | Stephen Tomlinson | Tissue targeted complement modulators |
CN105297145A (zh) * | 2015-11-06 | 2016-02-03 | 艾吉泰康生物科技(北京)有限公司 | 一种遗传代谢病的筛查方法和试剂盒 |
-
2019
- 2019-04-17 CN CN201910308341.8A patent/CN110157790A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005026724A2 (en) * | 2003-09-13 | 2005-03-24 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with organic cation transporter slc22a5 (slc22a5) |
US20050265995A1 (en) * | 2003-11-13 | 2005-12-01 | Stephen Tomlinson | Tissue targeted complement modulators |
CN105297145A (zh) * | 2015-11-06 | 2016-02-03 | 艾吉泰康生物科技(北京)有限公司 | 一种遗传代谢病的筛查方法和试剂盒 |
Non-Patent Citations (4)
Title |
---|
FANG-YUAN LI等: "Molecular Spectrum of SLC22A5 (OCTN2) Gene Mutations Detected in 143 Subjects Evaluated for Systemic Carnitine Deficiency", 《HUMAN MUTATION》 * |
LIANSHU HAN等: "Analysis of genetic mutations in Chinese patients with systemic primary carnitine deficiency", 《EUROPEAN JOURNAL OF MEDICAL GENETICS》 * |
SHIBBANI K.等: "Primary carnitine deficiency: novel mutations and insights into the cardiac phenotype", 《CLINICAL GENETICS》 * |
饶姣等: "原发性肉碱缺乏症性心肌病患儿基因突变及家系分析", 《中华儿科杂志》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113215243A (zh) * | 2021-05-19 | 2021-08-06 | 北京华诺奥美医学检验实验室有限公司 | 一种用于检测slc22a5基因高频致病变异的荧光定量pcr探针引物组和试剂盒 |
CN114317714A (zh) * | 2021-12-31 | 2022-04-12 | 北京华诺奥美基因医学检验实验室有限公司 | 用于检测slc22a5基因九种高频致病变异的荧光定量pcr引物、探针及试剂盒 |
CN114317714B (zh) * | 2021-12-31 | 2024-05-14 | 北京华诺奥美基因医学检验实验室有限公司 | 用于检测slc22a5基因九种高频致病变异的荧光定量pcr引物、探针及试剂盒 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111560428B (zh) | 检测线粒体DNA rs3937033单核苷酸多态性的物质的用途 | |
CN111676283B (zh) | 与高原肺水肿发生相关的线粒体dna单核苷酸多态性的应用 | |
EP2787085B1 (en) | Use of hla-b*1301 allele | |
CN110157790A (zh) | 一种不明原因心脏性猝死的快速基因筛查试剂盒 | |
CN104962655B (zh) | 一种与卵巢癌易感性相关的分子标记及检测引物和试剂盒 | |
CN108004313B (zh) | 早发冠心病致病基因及其体外检测的试剂、制剂或试剂盒和应用 | |
CN105802969A (zh) | lncRNA ENST00000581794.1及制剂或诊断剂或药物或试剂盒和应用 | |
CN108823303A (zh) | 一种检测试剂盒及其评估精神分裂症遗传风险的用途 | |
CN111549137B (zh) | 胃癌辅助诊断相关的遗传分子标志物及其应用 | |
CN107385076B (zh) | 一种甲状腺功能减退致病基因突变及基于此基因突变的诊断试剂 | |
CN112501306A (zh) | 一种用于CpG岛甲基化表型检测的试剂盒及其应用 | |
Villar et al. | Cardiac Dysfunction in Mitochondrial Disease–Clinical and Molecular Features– | |
CN111041086B (zh) | Rpl13基因先天性心脏病易感snp位点及其应用 | |
CA2579885C (en) | Association of protein polymorphisms with coronary heart disease | |
RU2630648C2 (ru) | Способ диагностики заболевания, сопровождающегося повышенной гибелью клеток, и набор для его осуществления | |
CN111088358B (zh) | 结直肠癌分子标志物组合、其用途及引物组和检测试剂盒 | |
CN111560430B (zh) | 检测rs1766位点多态性的试剂及其应用 | |
KR102565803B1 (ko) | 고혈압에 대한 정보 제공 방법 및 이를 이용한 키트 | |
RU2657821C1 (ru) | Способ выявления у детей ранних нарушений физиологической функции сердца в условиях контаминации фенолом | |
CN108410889B (zh) | 一种用于癫痫辅助诊断的atp1a2突变基因及应用 | |
CN109554466B (zh) | 一种用于检测糖尿病的试剂盒 | |
Zhang et al. | Risk of sudden coronary death based on genetic background in Chinese Han population | |
RU2650867C1 (ru) | Способ определения генетической предрасположенности к развитию панического расстройства | |
Renugadevi et al. | Molecular Genetic Testing for Carrier-Prenatal Diagnosis and Computational Analysis of Oculocutaneous Albinism Type 1 | |
CN112063711A (zh) | 基于stim1基因插入缺失多态性位点的心源性猝死易感性检测试剂盒 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |