CN110863044A - Primer combination for detecting VCL gene mutation and application thereof - Google Patents
Primer combination for detecting VCL gene mutation and application thereof Download PDFInfo
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Abstract
The invention discloses a method for detectingVCLPrimer combination for gene mutation, which includes primer combination for detectingVCLc.439G in gene>A mutated 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 geneVCL‑c.439G>The new mutation A is simply, conveniently, quickly and accurately detected; 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 method for detectingVCLThe primer combination of gene mutation and the application of the primer combination in the diagnosis, prevention and treatment of Dilated Cardiomyopathy (DCM) in the auxiliary clinic.
Background
Dilated Cardiomyopathy (DCM) is a common hereditary heart disease and is the main cause of heart failure and heart transplantation, the pathological feature of dilated cardiomyopathy is left ventricle or double ventricles, and the cause of heart contraction dysfunction, the biggest characteristic of the dilated cardiomyopathy is clinical genetic heterogeneity, mainly caused by gene mutation, and the first pathogenic variant site is discovered in 1990. The incidence of dilated cardiomyopathy is about 1/2700 as reported by Olmstead county, 1975 to 1984, and more relevant evidence suggests that dilated cardiomyopathy does not fall below 1/2500 worldwide, plus more potential patients, perhaps even 1/250, with incidence rates potentially greater than 1/500 for hypertrophic cardiomyopathy. The familial genetic screening shows that about 20-35% of dilated cardiomyopathy cases have a familial aggregation phenomenon and become familial dilated cardiomyopathy. The genetic modes of the dilated cardiomyopathy comprise mitochondrial DNA inheritance, sex chromosome inheritance and autosomal recessive inheritance besides autosomal dominant inheritance. The number of pathogenic genes related to the development of dilated cardiomyopathy is more than 50.
The main pathogenic genes of the dilated cardiomyopathy comprise a lamin gene and a mitochondrial oxidative phosphorylation related gene besides a sarcomere gene. The pathogenic mechanism of dilated cardiomyopathy has not been clarified so far, and there are the following hypotheses (1) Ca2+The hypothesis of abnormal regulation of sensitivity: the interaction between myosin and actin is via intracellular Ca2+And finally the normal contraction of the muscle cells. After mutation of sarcomere gene in most DCM patients, Ca pairs of troponin complex and myosin light chain in sensing system are reduced2+May lead to a reduction in the contractile capacity of the titin; (2) LMNA protein-mediated AKT-Mtor signaling pathway: the LMNA gene is located at lq22, and the A-type lamin coded by the LMNA gene is located in the nuclear membrane, is highly conserved and plays an important role in maintaining nuclear stability, chromatin structure and gene expression. Many evidences indicate that through constructing LMNA-DCM transgenic mice, protein kinase B-mammalian target protein rapamycin (AKT-mTOR) signal pathways are activated, cell metabolism is disordered, autophagy function is damaged, cell function is insufficient in LMNA mutant mice compared with wild type mice, and therefore LMNA-DCM is generated. It has also been found that the ERK1/2 pathway of LMNA transgenic mice is activated, and when inhibitors of ERK1/2 phosphorylation are used to act on LMNA miceIn mice, the degree of myocardial fibrosis of the mice is reduced, and the cardiac function is improved. The gene detection and the family screening of the dilated cardiomyopathy can provide important guidance for clinical diagnosis, and mainly comprise the following steps: 1) prenatal diagnosis, guiding prepotency; 2) assisting in definite diagnosis and carrying out clinical intervention; 3) and (4) family screening, and performing family disease occurrence risk assessment and management.
At present, genetic disease gene mutation detection methods are more, such as: restriction fragment length polymorphism, single-strand conformation polymorphism, high-resolution dissolution curve analysis, a fluorescent quantitative PCR detection method, PCR amplification direct sequencing and high-throughput sequencing. The methods have advantages and disadvantages, the capillary electrophoresis technology is the gold standard for gene mutation detection, but the method is difficult to popularize from laboratories to clinics due to the defects of high requirements on equipment, high price and the like, and the fluorescence quantitative PCR has the characteristics of rapidness, simplicity, convenience, economy, accuracy and the like.
CN 104388595A discloses a real-time fluorescent quantitative PCRMGB-TaqMan probe detection method for porcine circovirus type 2, which utilizes a fluorescent quantitative PCR technology and adopts an MGB probe to establish a porcine circovirus type 2 (PCV 2) detection method. However, MGB probes are not currently used for detection of DCM.
Disclosure of Invention
In view of the deficiencies of the prior art, the present invention provides a method for detectingVCLPrimer set for gene mutation, which comprises primer set for detectingVCLc.439G in gene>A fluorescent PCR primer set and a TaqMan-MGB probe set for mutation A; 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 geneVCL-c.439G>The new mutation A is detected simply, quickly and accurately.
The method is used for detectingVCLc.439G in gene>The primer group of the A mutation is the nucleotide sequence shown in SEQ ID NO. 3 and SEQ ID NO. 4.
The probe set is a nucleotide sequence shown as SEQ ID NO. 5 and SEQ ID NO. 6.
The invention also aims to apply the primer combination to the preparation and detection of the dilated cardiomyopathyVCLThe gene being mutatedIn the detection reagent, the detection reagent comprises a positive quality control product, and the positive quality control product comprises a wild type, a homozygous mutant type and a heterozygous mutant type 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 the primer combination for detecting the dilated cardiomyopathyVCLIn the detection kit for gene mutation, the kit further comprises 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 human hg19 genome as a reference genome, performing quality evaluation and comparison analysis on a sequencing result, filtering sequencing data of the found variant sites by using a method shown in figure 1 after the comparison analysis, and finally determining a new variant site possibly related to the development of the dilated cardiomyopathy after Sanger sequencing verificationVCL-c. 439G>A; 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. to establish simple, rapid and accurate detectionVCLc.439G in gene>The invention adopts the following technical scheme:
in a first aspect, the invention provides a primer and probe sequence that is capable of detecting new mutation sitesVCL-c.439G>The real-time fluorescent PCR primer and TaqMan-NGB probe sequence of A have nucleotide sequences shown as SEQ ID NO. 3 and SEQ ID NO. 4, and nucleotide sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6;
the 5 'end of the TaqMan-NGB probe sequence is marked with a luminescent reporter fluorophore (FAM, HEX or Cy 5), 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 present invention provides a primer and probe according to the first aspect or a kit according to the second aspect
Used for treating dilated cardiomyopathyVCLNew mutation of gene c.439G>Detecting A;
the invention relates to dilated cardiomyopathy related diseasesVCLNew mutation of gene c.439G>The detection method of A is as follows:
1) extracting normal people and carryingVCL- c.439G>A homozygous mutant and carrierVCL- c.439G>A genome of a heterozygous mutant type dilated myocardial patient;
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'-TAGAATGAAAATATGTTGAATGCTGCACATC-3'
SEQ ID NO:2: 5'-GGCTCCAACATGGACAATCCTTA-3'；
the reaction conditions for PCR amplification are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 seconds, annealing at 57 ℃ for 30 seconds, and extension at 72 ℃ for 1 minute for 40 cycles; extension at 72 ℃ for 5 min;
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 mixed enzyme 5 muL, 50 XROX correction dye 0.1 muL, upstream and downstream primers SEQ ID NO 3 and SEQ ID NO 4 are respectively 0.3 mu L, Taqman-MGB probe SEQ ID NO 50.5 mu L, Taqman-MGB probe SEQ ID NO 60.5 muL, genome 0.5 muL and ddH2O is 2.8 muL;
the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s outside the cycle, annealing and extension at 95 ℃ for 5s and 58 ℃ for 34s and 45 cycles inside the cycle, 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 spectrogram referred to in the present application;
FIG. 3 shows the sequencing results of Sanger of this family;
FIG. 4 shows the positive quality control product of the present inventionVCL，c. 439G>A, a PCR agarose gel electrophoresis picture of bacteria liquid;
FIG. 5 shows a positive control substance of the present inventionVCLC, 439G capillary electrophoresis detection;
FIG. 6 shows the positive quality control product of the present inventionVCLC, 439A capillary electrophoresis detection;
FIG. 7 is a drawing showingVCLAmplification curve for sensitivity test of TaqMan-MGB of 439GG genotype;
FIG. 8 is a drawing showingVCLA standard curve for sensitivity testing of TaqMan-MGB of 439GG genotype;
FIG. 9 is a schematic view ofVCLAmplification curve for sensitivity test of TaqMan-MGB of genotype 439 AA;
FIG. 10 is a drawing showingVCLA standard curve for sensitivity testing of TaqMan-MGB of genotype 439 AA;
FIG. 11 shows novel mutation sites using wild type as templateVCL，c. 439G>A, an amplification curve for specific detection of TaqMan-MGB, wherein FAM represents a wild-type probe, and HEX represents a mutant-type probe;
FIG. 12 shows novel mutation sites using homozygous mutants as templatesVCL，c. 439G>A, an amplification curve for specific detection of TaqMan-MGB, wherein FAM represents a wild-type probe, and HEX represents a mutant-type probe;
FIG. 13 shows novel mutation sites using hybrid mutants as templatesVCL，c. 439G>A, an amplification curve for specific detection of TaqMan-MGB, wherein FAM represents a wild-type probe, and HEX represents a mutant-type probe;
FIG. 14 shows novel mutation sites using different genotypes as templatesVCL，c. 439G>A, distribution of a scatter diagram of TaqMan-MGB specificity detection;
FIG. 15 is a graph showing the results of 16 patients with dilated cardiomyopathy of known genotypesVCL，c. 439G>A mutation site real-time fluorescence detection gene typing scatter diagram.
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 human hg19 genome as a reference genome, performing quality evaluation and comparison analysis on a sequencing result, filtering sequencing data of the found variant sites by using a method shown in figure 1 after the comparison analysis, and finally determining a new variant site possibly related to the development of the dilated cardiomyopathy after Sanger sequencing verificationVCL-c. 439G>A; 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: detection ofVCLc.439G in gene>Fluorescent quantitative PCR method for mutation A
(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 256 bp:
SEQ ID NO:1 5'-TAGAATGAAAATATGTTGAATGCTGCACATC-3'
SEQ ID NO:2 5'-GGCTCCAACATGGACAATCCTTA-3'
the reaction system is as follows: 1 mu L of template DNA and 1 mu L, PCR enzyme premixed solution 10 mu L, ddH of primer (3.2 pmol/uL)2O 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 an ABI3130 apparatus, and the reaction system and amplification conditions were the same as those described in the present example.
With a target geneVCL(GenBank:) as a template, and the mutation was analyzed 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:
are respectively provided withVCLGene c, 439G>A, carrying out 10-fold gradient dilution on wild type and mutant type templates, 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, annealing and extension at 95 ℃ for 5s and 58 ℃ for 34s and 45 cycles inside the cycle, 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)
The mutant site is matched with wild type and homozygous mutant templateVCL-c.439G>The wild type and mutant type probes of a are respectively subjected to 3 times of repeated tests (batch-to-batch and batch-to-batch), the reaction system and the reaction conditions refer to the step (2), the Ct value of the template is observed, the variation coefficient is calculated, the variation coefficient (p) = Standard Deviation (SD)/mean (X), the sensitivity and the repeatability of the detection method are tested, and the results are shown in tables 1-2:
TABLE 1
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)
Specificity verification by mutation sitesVCL，c. 439G>And (3) taking the wild type, homozygous mutant type and heterozygous mutant type positive plasmids of the A as templates, simultaneously adding a double-labeled probe, and carrying out TaqMan-MGB specificity experimental verification on the positive plasmids according to the reaction system and conditions in the step (2), wherein the results are shown in figures 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: 16 patients with dilated cardiomyopathy from known (Sanger sequencing) genotypes were subjected to the primer set of the inventionVCL，c. 439G>Mutation detection of A mutation site
The genotypes of 16 patients with dilated cardiomyopathy used in this example were determined by Sanger sequencing after PCR amplification using the primer sets SEQ ID NO:1 and SEQ ID NO:2, with reference to the reaction system and conditions in example 2, and 2% agarose gel electrophoresis to identify fragments of the desired size.
The genome of the whole blood samples of the 16 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 that the detection results of 10 cases of GG genotype, 4 cases of GA genotype and 2 cases of AA genotype are consistent with those of Sanger sequencing with 100% accuracy.
In conclusion, it can be seen from the results analysis of the examples that the present invention establishes the causative gene of the dilated cardiomyopathyVCLNew mutation site c.439G>A is a simple, rapid, accurate and economical genetic screening method.
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.
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Claims (5)
1. For detectingVCLGene mutation primerIn combination, characterized in that: including for detectingVCLc.439G in gene>Primer set and probe set of mutation A.
2. The method of claim 1 for detectingVCLA primer combination for gene mutation characterized in that: for detectingVCLc.439G in gene>The nucleotide sequences of the primer group with the mutation A are shown as SEQ ID NO. 3 and SEQ ID NO. 4.
3. The method of claim 1 for detectingVCLA primer combination for gene mutation characterized in that: the nucleotide sequences of the probe sets are shown as SEQ ID NO. 5 and SEQ ID NO. 6.
4. The method of any one of claims 1 to 3 for detectingVCLThe 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 detectingVCLThe 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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