CN111004845A - Primer combination for detecting AGL gene mutation and application thereof - Google Patents
Primer combination for detecting AGL gene mutation and application thereof Download PDFInfo
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Abstract
The invention discloses a method for detectingAGLA primer combination for gene mutation characterized in that: including for detectingAGLc.101G in gene>C mutation primer group and probe group; the invention uses the whole exon sequencing technology to obtain the new mutation of the pathogenic gene of the dilated cardiomyopathy and uses the fluorescent quantitative PCR technology to carry out the sequencing on the pathogenic geneAGL‑c.101G>C new mutation is detected simply, conveniently, quickly and accurately; the invention can detect a plurality of case samples at one time and establishes a new method for the clinical early molecular diagnosis and prevention of the dilated cardiomyopathy.
Description
Technical Field
The invention belongs to the field of medical molecular biology, relates to medical molecular diagnosis and biotechnology, and particularly relates to a primer composition for detecting AGL gene mutation, and application of the primer composition to diagnosis, prevention and treatment of Dilated Cardiomyopathy (DCM) in clinic.
Background
Dilated Cardiomyopathy (DCM), a genetic heterogeneous heart disease, is clinically manifested by left ventricular dilatation or biventricular enlargement, cardiac contractile dysfunction, thromboembolism and sudden cardiac death, is the most common cause of heart failure, and has no thorough treatment except heart transplantation. The disease rate is not less than 1/2500, about 30-50% is familial inheritance, and is called Familial Dilated Cardiomyopathy (FDCM). The mortality rate of DCM was high, and the mortality rate in 5 years was about 5-15%. Due to the clinical phenotype and other types of hereditary cardiomyopathy, such as hypertrophic cardiomyopathy, partial von-overlap of the proarrhythmic right ventricular cardiomyopathy can cause misdiagnosis of partial DCM patients, which is a great problem in the clinical evaluation of DCM.
The ACTC gene is one of six human actin genes, the encoded protein is quite conservative, one polarized end of the ACTC gene is connected with myosin through a transverse bridge, the other fixed end of the ACTC gene is connected with a Z band or a myoband disk and is an important component of a thin myofilament sarcomere, the gene is a common pathogenic gene of DCM and HCM which is first confirmed in the world, is positioned in 15q11-q14, 6 exons jointly encode 337 amino acid disabled α -cardiac actin and is the most common pathogenic gene of FDCM, the LMNA is positioned in a nuclear membrane and consists of two-dimensional matrix protein, and the LMNA gene encoding the lamin is positioned in 1q22, the pathogenic rate of related mutation sites accounts for about 5-10% of DCM patients, and the gene variation can not only cause the conduction obstruction of skeletal muscle and heart, but also cause malnutrition, familial partial fatty acid metabolic disorder and the like.
The research of gene mutation has become one of the hot spots of life science research at present, and in such a background, the detection technology thereof is also rapidly developed. The detection methods of gene mutation are many, mainly including single-strand conformation polymorphism, allele-specific PCR, nucleic acid molecule hybridization, TaqMan-MGB method, high-throughput sequencing technology and the like. Different methods of detecting gene mutations have their own advantages and disadvantages. The TaqMan-MGB fluorescent quantitative PCR technology has the characteristics of simplicity, convenience, rapidness and accuracy, provides a convenient technical means for mutation screening of the dilated cardiomyopathy patient, and is beneficial to prevention and treatment of dilated cardiomyopathy.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a primer group for detecting AGL gene mutation, which comprises a fluorescent PCR primer group and a TaqMan-MGB probe group for detecting c.101G > C mutation in AGL gene; the invention uses the whole exon sequencing technology to obtain the new mutation of the pathogenic gene of the dilated cardiomyopathy, and the AGL-c.101G > C new mutation is simply, quickly and accurately detected by the fluorescent quantitative PCR technology.
The nucleotide sequence of the primer group for detecting c.101G > C mutation in the AGL gene is shown as SEQ ID NO. 1 and SEQ ID NO. 2.
The nucleotide sequence of the probe set is shown as SEQ ID NO. 3 and SEQ ID NO. 4.
The invention also aims to apply the primer combination in the preparation of a detection reagent for detecting the AGL gene mutation of the dilated cardiomyopathy, wherein the detection reagent comprises a positive quality control product, and the positive quality control product comprises a wild type positive quality control product, a homozygous mutant positive quality control product and a heterozygous mutant positive quality control product; the reagent also comprises conventional components for detection: PCR enzyme premix, PCR amplification reaction mix enzyme, ROX correction dye, ddH2O, and the like.
The invention also aims to apply the primer combination to the preparation of a detection kit for detecting the AGL gene mutation of the dilated cardiomyopathy, wherein the kit also comprises the following conventional components for detection: PCR enzyme premix, PCR amplification reaction mix enzyme, ROX correction dye, ddH2O, and the like, and positive quality control substances, wherein the positive quality control substances comprise wild type, homozygous mutant type and heterozygous mutant type positive quality control substances.
In order to achieve the purpose, the invention adopts the following technical scheme:
1. screening the whole exon mutation of the human genome of a clinically diagnosed dilated cardiomyopathy proband by using a whole exon sequencing technology, and searching possible pathogenic variant sites related to the pathogenesis of the hereditary DCM;
(1) genome extraction: extracting peripheral venous blood of selected clinically diagnosed dilated cardiomyopathy predecessors, performing EDTA anticoagulation, extracting the whole genome by adopting a commercial Miniprep Kit (Axygen, USA), performing agarose gel electrophoresis, and determining the concentration and OD value, wherein the OD260/280 is 1.8-2.0;
(2) breaking the genome by an enzyme cutting method or a physical method, and recovering a target DNA fragment;
(3) carrying out end repair and tail end adding of A (adenine deoxynucleotide) on the target DNA fragment;
(4) adding a sequencing joint to the DNA fragment subjected to the adenine treatment in the previous step;
(5) carrying out fragment selection to effectively recover DNA fragments with sequencing joints successfully added at two ends, namely a library before capture, and carrying out library enrichment by PCR amplification;
(6) carrying out hybridization capture on the genome exon regions by using exon probes;
(7) amplifying and enriching the captured library by PCR;
(8) performing quality inspection and dilution of the library, mixing the library, and performing on-machine sequencing on Illumina PE 150;
(9) taking the hg19 genome of a human as a reference genome, performing quality evaluation and comparison analysis on a sequencing result, filtering sequencing data of the found variation sites by using a method shown in figure 1 after the comparison analysis, and finally determining a new variation site AGL-c.101G > C which is possibly related to the development of the dilated cardiomyopathy after Sanger sequencing verification; meanwhile, the site of the family of the patient is subjected to first-generation sequencing analysis, and the fact that the site has a family genetic phenomenon in the family is found;
2. in order to establish a simple, convenient, rapid and accurate method for detecting c.101G > C mutation in AGL gene, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer and a probe sequence, wherein the sequence is a real-time fluorescent PCR primer and a TaqMan-NGB probe sequence capable of detecting a new mutation site AGL-c.101G > C, and the nucleotide sequences are shown as SEQ ID NO 1 and SEQ ID NO 2, and as SEQ ID NO 3 and SEQ ID NO 4;
the 5 'end of the TaqMan-NGB probe sequence is marked with a luminescent reporter fluorophore (FAM, HEX or Cy5), and the 3' end is marked with a non-luminescent quencher;
in a second aspect, the present invention provides a reagent or kit comprising a real-time fluorescent PCR primer as described in the first aspect and a TaqMan-MGB probe sequence;
in a third aspect, the invention provides a primer and a probe as described in the first aspect or a kit as described in the second aspect for detecting a new mutation c.101G > C in an AGL gene causing dilated cardiomyopathy;
the detection method of the new mutation c.101G > C of the expanding cardiomyopathy related pathogenic AGL gene comprises the following steps:
1) extracting genomes of normal people, the dilated myocardial patients carrying the AGL-c.101G > C homozygous mutant and the AGL-c.101G > C heterozygous mutant;
the genome is from human peripheral blood, myocardial tissue, lymphoid organs, spleen, bone marrow or liver;
2) performing PCR amplification by taking the genome in the step 1) as a template to construct wild type, homozygous mutant type and heterozygous mutant type positive quality control products;
the amplification primers were as follows:
SEQ ID NO:1:5'-AAAGTAGTGCCAAAACAGC-3'
SEQ ID NO:2:5'-CACCTGACTTACCCTTGAA-3'
the reaction conditions for PCR amplification are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 1min for 40 cycles; extending for 5min at 72 ℃;
the specific steps for constructing the positive quality control product are as follows: purifying PCR amplification products, cloning the purified fragments onto a PMD-18T carrier, converting the purified fragments into escherichia coli JM109, selecting a single clone, sequencing and verifying wild type and homozygous mutant positive plasmids, and mixing the wild type and homozygous mutant positive plasmids according to the molar ratio of 1:1 to obtain the heterozygous mutant positive quality control product.
3) Carrying out real-time fluorescent PCR detection on the positive quality control product obtained in the step 2) by using the primer in the first aspect;
the reaction system of the real-time fluorescent PCR is as follows: PCR amplification reaction mix enzyme 5. mu.L, 50 XROX calibration dye 0.1. mu.L, upstream and downstream primers SEQ ID NO 1 and SEQ ID NO 2 each 0.3. mu.L,Taqman-MGB probe SEQ ID NO 30.5 mu L, Taqman-MGB probe SEQ ID NO 40.5 mu L genome 0.5 mu L, ddH2O 2.8μL；
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s outside the cycle, denaturation at 95 ℃ for 5s inside the cycle, annealing and extension at 56 ℃ for 34s and 45 cycles, and extension at 60 ℃ for 1min outside the cycle.
4) Extracting a genome of a sample to be detected, performing PCR real-time fluorescence PCR detection by using primers SEQ ID NO. 3 and SEQ ID NO. 4 and a Taqman-MGB probe set, comparing with the amplification result of the positive quality control product in the step 3), determining the mutation type of the detected sample, and further diagnosing whether the patient is the dilated cardiomyopathy carrying the mutation.
Compared with the prior art, the invention has the following beneficial effects:
a) the invention can detect a plurality of case samples at one time, has the characteristics of simplicity, convenience, rapidness, accuracy, economy and the like, and establishes a new method for the clinical early molecular diagnosis and prevention of the dilated cardiomyopathy;
b) the method can be used for prenatal diagnosis, guidance of prenatal and postnatal care, auxiliary and definite diagnosis, clinical intervention and family screening, and evaluation and management of family disease occurrence risk;
c) the method is convenient and quick, has low cost, and can detect a plurality of samples at one time by utilizing a 96-hole PCR reaction tube.
Drawings
FIG. 1 is a flow of data filtering analysis of whole exon sequencing results;
FIG. 2 is a family chart referred to in this patent;
FIG. 3 shows the sequencing results of Sanger of this family;
FIG. 4 is the PCR agarose gel electrophoresis chart of the positive quality control product AGL, c.101G > C bacteria liquid of the present invention;
FIG. 5 shows the detection of the positive quality control product AGL, c.101G by capillary electrophoresis;
FIG. 6 shows the detection of the positive quality control product AGL, c.101C by capillary electrophoresis;
FIG. 7 is an amplification curve of a sensitivity test of TaqMan-MGB of the genotype AGL, 101 GG;
FIG. 8 is a standard curve for sensitivity testing of TaqMan-MGB for AGL, 101CC genotype;
FIG. 9 is an amplification curve of a sensitivity test of TaqMan-MGB of the genotype AGL, 101 GG;
FIG. 10 is a standard curve for sensitivity testing of TaqMan-MGB for AGL, 101CC genotype;
FIG. 11 is an amplification curve of TaqMan-MGB specific detection of a new mutation site AGL, c.101G > C using a wild type as a template, wherein FAM represents a wild type probe and CY5 represents a mutant type probe;
FIG. 12 is an amplification curve of TaqMan-MGB specific detection of a new mutation site AGL, c.101G > C using homozygous mutant as a template, wherein FAM represents a wild-type probe and CY5 represents a mutant-type probe;
FIG. 13 is an amplification curve of TaqMan-MGB specific detection of a new mutation site AGL, c.101G > C using a hybrid mutant as a template, wherein FAM represents a wild-type probe and CY5 represents a mutant probe;
FIG. 14 is an amplification curve for TaqMan-MGB specific detection of a new mutation site AGL, c.101G > C using different genotypes as templates, wherein FAM represents a wild type probe and CY5 represents a mutant type probe;
FIG. 15 is a genotyping scattergram of real-time fluorescence detection of the AGL, c.101G > C mutation site in 15 patients with dilated cardiomyopathy of known genotypes.
Detailed Description
The present invention is further illustrated in detail below with reference to the drawings and examples, but the scope of the present invention is not limited to the above description, and reagents and methods used in the examples are, unless otherwise specified, conventional reagents and conventional methods.
Example 1: the whole exon sequencing technology is applied to carry out human genome whole exon mutation screening on 12 cases of dilated cardiomyopathy predecessors (collecting electrocardiogram and echocardiogram of DCM patients besides collecting demographic data of DCM patients) clinically diagnosed in the cardiovascular internal medicine of the first people hospital in Yunnan province, and possible pathogenic variation sites related to hereditary DCM pathogenesis are searched.
A. Genome extraction: for the selected clinically confirmed dilated cardiomyopathy predecessors, 1mL of peripheral venous blood was extracted, after anticoagulation with EDTA, the entire genome was extracted with a commercial Miniprep Kit (Axygen, USA), subjected to agarose gel electrophoresis, and the concentration and OD value were measured, with OD260/280 ranging from 1.8 to 2.0 being applicable.
B. Breaking the genome by an enzyme cutting method or a physical method, and recovering a target DNA fragment;
C. carrying out end repair and tail end adding of A (adenine deoxynucleotide) on the target DNA fragment;
D. adding a sequencing joint to the DNA fragment subjected to the adenine treatment in the previous step;
E. carrying out fragment selection to effectively recover DNA fragments with sequencing joints successfully added at two ends, namely a library before capture, and carrying out library enrichment by PCR amplification;
F. carrying out hybridization capture on the genome exon regions by using exon probes;
G. amplifying and enriching the captured library by PCR;
H. performing quality inspection and dilution of the library, mixing the library, and performing on-machine sequencing on Illumina PE 150;
I. taking the hg19 genome of a human as a reference genome, performing quality evaluation and comparison analysis on a sequencing result, filtering sequencing data of the found variation sites by using a method shown in figure 1 after the comparison analysis, and finally determining a new variation site AGL-c.101G > C which is possibly related to the development of the dilated cardiomyopathy after Sanger sequencing verification; meanwhile, the site of the patient's family is analyzed by one-generation sequencing (FIG. 2 and FIG. 3), and the site is found to have family genetic phenomena in the family.
Example 2: fluorescent quantitative PCR method for detecting c.101G > C mutation in AGL gene
(1) Taking the genome obtained in the step A of the embodiment 1 as a template, carrying out PCR amplification, and constructing wild type, homozygous mutation and heterozygous mutation positive quality control products;
wherein, the nucleic acid sequence of the primer amplified by the PCR is shown as follows, and the amplification length is 374 bp:
SEQ ID NO:5:5'-AAAGTAGTGCCAAAACAGC-3'
SEQ ID NO:6:5'-CACCTGACTTACCCTTGAA-3'
the reaction system is as follows: template DNA 1. mu.L, primer (3.2pmol/uL) 1. mu. L, PCR enzyme premix 10. mu. L, ddH2O 7μL。
The reaction conditions for PCR amplification were as follows:
after the PCR amplification reaction is finished, performing agarose gel electrophoresis, wherein the electrophoresis result is shown in FIG. 4, and the size of the target fragment is shown to be consistent with the expected size and is purified; purifying the PCR product by an agarose gel DNA purification kit, carrying out T-A cloning by a PMD-18T vector, screening out a positive cloning strain, and mutextracting a plasmid vector of the positive cloning strain; then, the plasmid vector containing the cloned target fragment was subjected to PCR amplification using ABI3130 apparatus, and the reaction system and amplification conditions were determined according to the conditions described above in this example
The mutation was analyzed by using the gene sequence of target gene AGL (GenBank:) as a template and using bioedit sequence analysis software.
As shown in the sequencing results of FIGS. 5 and 6, the wild-type and mutant-type positive quality control products were successfully constructed, and the heterozygous mutant-type positive quality control products were obtained by mixing the wild-type and homozygous mutant-type positive plasmids at a molar ratio of 1: 1.
(2) Carrying out real-time fluorescent PCR detection on the positive quality control product obtained in the step (1) by adopting the following primer composition, and verifying the sensitivity of the sequence of the primer-probe;
the primer and probe sequences are as follows:
the reaction system is as follows:
carrying out 10-fold gradient dilution on wild type and mutant type templates of AGL gene c.101G > C respectively, and detecting the sensitivity of a primer probe by using an ABI7500 real-time fluorescent PCR instrument, wherein the specific conditions are as follows: pre-denaturation at 95 ℃ for 30s outside the cycle, denaturation at 95 ℃ for 5s inside the cycle, annealing and extension at 56 ℃ for 34s and 45 cycles, and extension at 60 ℃ for 1min outside the cycle.
As shown in the amplification curves and the standard curves of FIGS. 7-10, the Ct value begins to increase correspondingly as the initial copy number of the template decreases, and the linear relationship of the standard curve is better (R)2>0.98), the results show that the primer probe has better sensitivity;
(3) repeatability verification of the primer-probe sequence described in step (2)
Respectively carrying out 3 times of repeated tests (batch and batch-to-batch repeated) on the wild type and mutant type probes of the variant site AGL-c.101G > C by using wild type and homozygous mutant type templates; referring to the step (2), the Ct value of the template is observed, the coefficient of variation (p) is calculated, the Standard Deviation (SD)/average (X), the sensitivity and the repeatability of the detection method are tested, and the results are shown in tables 1-2:
TABLE 1
TABLE 2
As can be seen from tables 1-2, both the intra-and inter-batch repeat variation coefficients are less than 2%, with better intra-and inter-batch repeatability.
(4) Verification of specificity of the primer-probe sequence described in step (2)
And (3) performing specificity verification by taking the wild type, homozygous mutant type and heterozygous mutant type positive plasmids of the variant locus AGL and c.101G > C as templates, adding a double-labeled probe, and performing TaqMan-MGB specificity experiment verification according to the reaction system and conditions in the step (2), wherein the results are shown in FIGS. 11-14.
The results show that: the curve of the wild-type sample response in FIG. 11 shows an increase in the fluorescence signal produced by the wild-type probe, whereas the homozygous mutation has no or only a very low fluorescence signal; the homozygous mutations in FIG. 12 show that only the mutant probes produce fluorescent signals; the heterozygous mutant samples in FIG. 13 enable both wild-type and mutant probes to exhibit relatively high fluorescence signals; more importantly, as shown in fig. 14, it is evident from the scattergram of the reaction results that samples of different genotypes are clustered individually.
Example 3: the primer group of the invention is adopted to carry out mutation detection on 15 cases of dilated cardiomyopathy patients with known (Sanger sequencing) genotypes at the AGL and c.101G & gtC mutation sites
The genotypes of 15 patients with dilated cardiomyopathy used in this example were determined by Sanger sequencing after PCR amplification using the primer sets SEQ ID NO. 5 and SEQ ID NO. 6, with reference to the reaction system and conditions of example 2, and 2% agarose gel electrophoresis to identify fragments of the desired size.
The genome of the whole blood samples of the 15 patients is taken as a template, the primer group is adopted to carry out real-time fluorescence quantitative PCR detection, the PCR amplification conditions and the system are shown in the step (2) of the example 2, and the result is shown in the figure 15; FIG. 15 is a genotyping scattergram showing the detection of mutation sites, wherein the detection results are consistent with those of Sanger sequencing with 100% accuracy, and the detection results are 9 cases of GG genotype, 4 cases of GC genotype and 2 cases of CC genotype.
In conclusion, the result analysis of the embodiment shows that the invention establishes a simple, convenient, rapid, accurate and economic genetic screening method for the new mutation site c.101G > C of the AGL of the dilated cardiomyopathy causing gene.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Sequence listing
<110> university of Kunming science
<120> primer combination for detecting AGL gene mutation and application thereof
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Claims (5)
1. For detectingAGLA primer combination for gene mutation characterized in that: including for detectingAGLc.101G in gene>C mutation primer group and probe group.
2. The method of claim 1 for detectingAGLA primer combination for gene mutation characterized in that: for detectingAGLc.101G in gene>The nucleotide sequences of the C-mutated primer set are shown in SEQ ID NO. 1 and SEQ ID NO. 2.
3. The method of claim 1 for detectingAGLA primer combination for gene mutation characterized in that: the nucleotide sequence of the probe set is shown as SEQ ID NO. 3 and SEQ ID NO. 4.
4. The method of any one of claims 1 to 3 for detectingAGLThe application of the gene mutation primer combination in preparing the detection reagent for detecting the dilated cardiomyopathy is characterized in that: also comprises a positive quality control product.
5. The method of any one of claims 1 to 3 for detectingAGLThe application of the gene mutation primer combination in preparing the detection kit for detecting the dilated cardiomyopathy is characterized in that: also comprises a positive quality control product.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012142159A2 (en) * | 2011-04-11 | 2012-10-18 | The Brigham And Women's Hospital, Inc | Structural mutations in titin cause dilated cardiomyopathy |
| CN103540645A (en) * | 2012-07-09 | 2014-01-29 | 解码（上海）生物医药科技有限公司 | Dilated cardiomyopathy susceptible detection kit (CTLA4 and other genes) |
| CN105671198A (en) * | 2016-04-21 | 2016-06-15 | 福州市传染病医院 | Reagent kit for detecting glycogen storage disease type III (GSD III) AGL gene mutation and application of reagent kit |
-
2019
- 2019-12-11 CN CN201911263516.4A patent/CN111004845A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012142159A2 (en) * | 2011-04-11 | 2012-10-18 | The Brigham And Women's Hospital, Inc | Structural mutations in titin cause dilated cardiomyopathy |
| CN103540645A (en) * | 2012-07-09 | 2014-01-29 | 解码（上海）生物医药科技有限公司 | Dilated cardiomyopathy susceptible detection kit (CTLA4 and other genes) |
| CN105671198A (en) * | 2016-04-21 | 2016-06-15 | 福州市传染病医院 | Reagent kit for detecting glycogen storage disease type III (GSD III) AGL gene mutation and application of reagent kit |
Non-Patent Citations (9)
| Title |
|---|
| DAGLI,A等: "Homo sapiens amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (AGL), RefSeqGene on chromosome 1", 《GENBANK》 * |
| ENSEMBLE: "rs746265668 SNP", 《ARCHIVE ENSEMBLE》 * |
| YUE ZHAO等: "Targeted next-generation sequencing of candidate genes reveals novel mutations in patients with dilated cardiomyopathy", 《INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE》 * |
| YUE ZHAO等: "Targeted Next-Generation Sequencing Reveals Hot Spots and Doubly Heterozygous Mutations in Chinese Patients with Familial Cardiomyopathy", 《BIOMED RESEARCH INTERNATIONAL》 * |
| 唐炳华主编: "《分子生物学》", 31 July 2017, 中国中医药出版社 * |
| 赵跃等: "原发性心肌病的分子遗传学特性", 《生命科学》 * |
| 陈旭华等: "对心脏再同步化治疗无反应的扩张型心肌病患者的分子遗传学特征研究", 《中国分子心脏病学杂志》 * |
| 陈晓帅等: "家族性扩张型心肌病一家系的致病基因研究", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
| 陈超等: "《新技术与精准医学》", 31 December 2017, 上海交通大学出版社 * |
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