CN117925629A - Detection kit for novel locus of familial dilated cardiomyopathy LMNA gene - Google Patents
Detection kit for novel locus of familial dilated cardiomyopathy LMNA gene Download PDFInfo
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- 101150077556 LMNA gene Proteins 0.000 title claims abstract description 66
- 201000011257 dilated cardiomyopathy 1B Diseases 0.000 title claims abstract description 36
- 208000004996 familial dilated cardiomyopathy Diseases 0.000 title claims abstract description 36
- 238000001514 detection method Methods 0.000 title claims abstract description 33
- 238000012163 sequencing technique Methods 0.000 claims abstract description 36
- 230000035772 mutation Effects 0.000 claims abstract description 34
- 239000012472 biological sample Substances 0.000 claims abstract description 23
- 239000000523 sample Substances 0.000 claims abstract description 23
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 21
- 101001003584 Homo sapiens Prelamin-A/C Proteins 0.000 claims abstract description 19
- 206010064571 Gene mutation Diseases 0.000 claims abstract description 16
- 238000000605 extraction Methods 0.000 claims abstract description 15
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 14
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 14
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 14
- 238000001712 DNA sequencing Methods 0.000 claims abstract description 11
- 238000012545 processing Methods 0.000 claims abstract description 11
- 238000012360 testing method Methods 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 10
- 102100026531 Prelamin-A/C Human genes 0.000 claims description 8
- 239000011324 bead Substances 0.000 claims description 8
- 239000008280 blood Substances 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 239000012670 alkaline solution Substances 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 5
- 230000009089 cytolysis Effects 0.000 claims description 5
- 238000007405 data analysis Methods 0.000 claims description 5
- 238000007781 pre-processing Methods 0.000 claims description 5
- 239000003761 preservation solution Substances 0.000 claims description 5
- 238000007480 sanger sequencing Methods 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 5
- 238000010801 machine learning Methods 0.000 claims description 4
- 102200027627 rs267607259 Human genes 0.000 claims description 4
- 239000012535 impurity Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000005457 optimization Methods 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000013473 artificial intelligence Methods 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims description 2
- 238000012165 high-throughput sequencing Methods 0.000 claims description 2
- 230000004543 DNA replication Effects 0.000 claims 1
- 230000002068 genetic effect Effects 0.000 abstract description 4
- 206010056370 Congestive cardiomyopathy Diseases 0.000 description 6
- 201000010046 Dilated cardiomyopathy Diseases 0.000 description 6
- 208000031229 Cardiomyopathies Diseases 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000009223 counseling Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000010448 genetic screening Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 108010047294 Lamins Proteins 0.000 description 1
- 206010042434 Sudden death Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000009091 contractile dysfunction Effects 0.000 description 1
- 238000013135 deep learning Methods 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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Abstract
The invention belongs to the technical fields of genomics and biomedicine, and discloses a detection kit for new loci of LMNA genes of familial dilated cardiomyopathy and application thereof. Sample collection means for collecting a biological sample; nucleic acid extraction reagents for extracting DNA from a biological sample; PCR primers for specifically amplifying a target region of the LMNA gene; a DNA sequencing reagent for sequencing PCR amplified products and determining mutation of new loci of LMNA genes; data analysis software for reading the sequencing results and reporting the type and location of LMNA gene mutations. The invention can more accurately diagnose familial dilated cardiomyopathy by detecting the new site mutation of the LMNA gene, and helps doctors to better understand the genetic risk of patients. The invention reduces the error of operators and improves the reliability and reproducibility of the test through standardized and automatic processing.
Description
Technical Field
The invention belongs to the technical fields of genomics and biomedicine, and particularly relates to a detection kit for a novel locus of an LMNA gene of familial dilated cardiomyopathy and application thereof.
Background
Dilated Cardiomyopathy (DCM) is one of the primary cardiomyopathies and refers to heart failure that eventually results from a complete contractile dysfunction of the heart without any abnormal load on the heart. In many cases, DCM is hereditary, and if at least two members of the family are ill, or there is sudden death of unknown origin in first-degree relatives under the age of 35, this type is approximately 20-48% of all cases, familial Dilated Cardiomyopathy (FDCM). FDCM pathogenesis is associated with multiple gene mutations, where mutations in the LMNA gene have been shown to be closely related to FDCM development. Type a and lamin genes (LMNA) are one of the most common genes responsible for DCM. LMNA encodes intermediate silk proteins and polymerizes around the nucleus to form scaffolds for Nuclear Layers (NL). Patients with LMNA mutations exhibit dysfunction in specific tissues. LMNA-associated cardiomyopathy accounts for 5-10% of familial DCM and 2-5% of sporadic DCM, and more than 160 different mutations in the LMNA gene have been identified as the etiology of cardiomyopathy. However, detection methods for new site mutations of LMNA gene are limited, and thus development of a new detection kit and application are required to diagnose FDCM more accurately.
Through the above analysis, the problems and defects existing in the prior art are as follows:
(1) Current methods for detecting LMNA gene mutations may be limited, resulting in insufficient recognition of new site mutations.
(2) Since 160 different mutations are known to exist in the LMNA gene, existing detection kits may not cover all of these mutations, and thus some critical genetic information may be missed.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a detection kit for new loci of LMNA genes of familial dilated cardiomyopathy and application thereof.
The present invention is realized in such a way that a novel gene mutation site, in particular
LMNA:NM_001257374:exon5:c.485C>A:p.A162D。
Further, the LMNA mutant gene is characterized in that the base C at the 485 th base is mutated to the base A at the 5 th exon of the NM-001257374 transcript, and the reference genome version is GRCh38.
Base sequence:
Atggggaactctgagggctgcaataccaagaaggagggtgacctgatagctgctcaggctcggctgaaggacctggaggctctgctgaactccaaggaggccgcactgagcactgctctcagtgagaagcgcacgctggagggcgagctgcatgatctgcggggccaggtggccaagcttgaggcagccctaggtgaggccaagaagcaacttcaggatgagatgctgcggcgggtggatgctgagaacaggctgcagaccatgaaggaggaactggacttccagaagaacatctacagtgaggagctgcgtgagaccaagcgccgtcatgagacccgactggtggagattgacaatgggaagcagcgtgagtttgagagccggctggcggatgcgctgcaggaactgcgggcccagcatgaggaccaggtggagcagtataagaaggagctggagaagacttattctgccaagctggacaatgc/Acaggcagtctgctgagaggaacagcaacctggtgggggctgcccacgaggagctgcagcagtcgcgcatccgcatcgacagcctctctgcccagctcagccagctccagaagcagctggcagccaaggaggcgaagcttcgagacctggaggactcactggcccgtgagcgggacaccagccggcggctgctggcggaaaaggagcgggagatggccgagatgcgggcaaggatgcagcagcagctggacgagtaccaggagcttctggacatcaagctggccctggacatggagatccacgcctaccgcaagctcttggagggcgaggaggagaggctacgcctgtcccccagccctacctcgcagcgcagccgtggccgtgcttcctctcactcatcccagacacagggtgggggcagcgtcaccaaaaagcgcaaactggagtccactgagagccgcagcagcttctcacagcacgcacgcactagcgggcgcgtggccgtggaggaggtggatgaggagggcaagtttgtccggctgcgcaacaagtccaatgaggaccagtccatgggcaattggcagatcaagcgccagaatggagatgatcccttgctgacttaccggttcccaccaaagttcaccctgaaggctgggcaggtggtgacgatctgggctgcaggagctggggccacccacagcccccctaccgacctggtgtggaaggcacagaacacctggggctgcgggaacagcctgcgtacggctctcatcaactccactggggaagaagtggccatgcgcaagctggtgcgctcagtgactgtggttgaggacgacgaggatgaggatggagatgacctgctccatcaccaccacggctcccactgcagcagctcgggggaccccgctgagtacaacctgcgctcgcgcaccgtgctgtgcgggacctgcgggcagcctgccgacaaggcatctgccagcggctcaggagcccaggtgggcggacccatctcctctggctcttctgcctccagtgtcacggtcactcgcagctaccgcagtgtggggggcagtgggggtggcagcttcggggacaatctggtcacccgctcctacctcctgggcaactccagcccccgaacccagagcccccagaactgcagcatcatacaagagatgggaatgaggtgggaggtggaagaagggagaagaaaggtgagtttgagctgccttccctag
Protein sequence:
MGNSEGCNTKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGEAKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQREFESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNA/dRQSAERNSNLVGAAHEELQQSRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRLSPSPTSQRSRGRASSHSSQTQGGGSVTKKRKLESTESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKRQNGDDPLLTYRFPPKFTLKAGQVVTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRTALINSTGEEVAMRKLVRSVTVVEDDEDEDGDDLLHHHHGSHCSSSGDPAEYNLRSRTVLCGTCGQPADKASASGSGAQVGGPISSGSSASSVTVTRSYRSVGGSGGGSFGDNLVTRSYLLGNSSPRTQSPQNCSIIQEMGMRWEVEEGRRKVSLSCLP
Sanger sequencing primer:
forward primer GCTATGCCTTCTGGGGATCAG
Reverse primer GGGGTATCACCTGCTTCTGGAG
Another object of the present invention is to provide a kit for detecting a novel site of LMNA gene of familial dilated cardiomyopathy, which comprises:
sample collection means for collecting a biological sample;
nucleic acid extraction reagents for extracting DNA from a biological sample;
PCR primers for specifically amplifying a target region of the LMNA gene;
a DNA sequencing reagent for sequencing PCR amplified products and determining mutation of new loci of LMNA genes;
data analysis software for reading the sequencing results and reporting the type and location of LMNA gene mutations.
Further, the biological sample is blood or saliva.
Further, the nucleic acid extraction reagent includes a lysis-binding solution, a removing solution and an alkaline solution, a preservation solution, a magnetic bead suspension, a washing solution, and an eluent.
Further, sanger sequencing or second generation sequencing was performed to determine mutations at new sites of the LMNA gene.
The invention also aims to provide a detection method of a novel locus of the LMNA gene of familial dilated cardiomyopathy, which comprises the following steps:
Step one, collecting a biological sample;
step two, extracting DNA from the biological sample;
Step three, specifically amplifying a target region of the LMNA gene;
Step four, sequencing PCR amplification products, and determining mutation of new loci of LMNA genes;
and fifthly, reading the sequencing result and reporting the type and the position of the LMNA gene mutation.
Further, the method for extracting DNA specifically comprises the following steps:
Step 1, fully mixing the obtained biological sample with preservation solution, and centrifuging to obtain supernatant 1;
step 2, adding impurity removing liquid into the obtained supernatant 1, adding alkaline solution until no flocculent precipitate is formed, and centrifuging to obtain supernatant 2;
And 3, simultaneously adding a lysis binding solution and a magnetic bead suspension into the obtained supernatant 2 under normal temperature to form a DNA-magnetic bead complex, and then adding a washing solution for washing and eluting to obtain DNA.
The invention further aims at providing an application of the detection kit for the novel locus of the LMNA gene of the familial dilated cardiomyopathy in detecting the novel locus of the LMNA gene of the familial dilated cardiomyopathy.
In combination with the technical scheme and the technical problems to be solved, the technical scheme to be protected has the following advantages and positive effects:
the detection kit for the novel locus of the LMNA gene of the familial dilated cardiomyopathy provided by the invention has the remarkable technical progress that:
1. Detection efficiency is improved: by using a specially designed PCR primer and a DNA sequencing reagent, the detection kit can rapidly and accurately detect the new site mutation of the LMNA gene. This reduces the time and resources required in the laboratory so that more samples can be processed and analyzed at the same time.
2. Detection precision is improved: by using data analysis software, sequencing results can be automatically read, so that human errors are reduced, and the precision and reliability of mutation detection are improved.
3. The application range is enlarged: the detection kit can be used for research and clinical diagnosis, and can also be used for large-scale genetic screening, for example, for early detection and prevention of diseases.
4. Helping to personalize the medical treatment: by identifying new site mutations in the LMNA gene, more personalized diagnostic and therapeutic regimens can be provided for each patient, helping to improve the therapeutic efficacy and quality of life for the patient.
These remarkable technological advances make this detection kit have important application value in diagnosis and research of genetic diseases.
Secondly, the intelligent detection kit for the novel locus of the LMNA gene of the familial dilated cardiomyopathy adopts automatic equipment and an artificial intelligent algorithm, and performs automatic and intelligent processing on the steps of sample acquisition, DNA extraction, PCR amplification, sequencing and the like, so that the detection process is more accurate, efficient and stable. Particularly, the intelligent processing of the data analysis part can greatly improve the accuracy and efficiency of reading the sequencing result, reduce human errors and improve the reliability of the detection result.
The signal and data processing process provided by the invention enables the data processing to be more systematic and scientific through the steps of data acquisition, preprocessing, analysis, interpretation, feedback and the like, and can comprehensively and deeply analyze and utilize the data, discover and utilize the internal rules thereof, so as to optimize the use of the kit. And particularly, a machine learning algorithm is utilized to analyze the data, so that potential modes and rules in the data can be identified, and a basis is provided for optimizing the use of the kit. Finally, the interpretation result is automatically fed back to the user, so that the user can know the detection result more conveniently, and meanwhile, continuous optimization can be performed according to the feedback and the service condition of the user, and the user experience and performance of the kit are improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a block diagram of a detection kit for novel loci of LMNA gene of familial dilated cardiomyopathy provided by the embodiment of the invention;
FIG. 2 is a flowchart of a method for detecting a novel locus of LMNA gene of familial dilated cardiomyopathy provided by the embodiment of the invention;
FIG. 3 is a flowchart of a method for extracting DNA according to an embodiment of the present invention;
FIG. 4 shows a novel sequencing map of the mutation sites of the gene provided by the embodiment of the invention;
FIG. 5 is a family chart provided by an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention provides an LMNA mutant gene, which is characterized in that a base C at a 485 th base is mutated into a base A at an exon 5 of an NM_001257374 transcript, and a reference genome version is GRCh38.
Base sequence:
Atggggaactctgagggctgcaataccaagaaggagggtgacctgatagctgctcaggctcggctgaaggacctggaggctctgctgaactccaaggaggccgcactgagcactgctctcagtgagaagcgcacgctggagggcgagctgcatgatctgcggggccaggtggccaagcttgaggcagccctaggtgaggccaagaagcaacttcaggatgagatgctgcggcgggtggatgctgagaacaggctgcagaccatgaaggaggaactggacttccagaagaacatctacagtgaggagctgcgtgagaccaagcgccgtcatgagacccgactggtggagattgacaatgggaagcagcgtgagtttgagagccggctggcggatgcgctgcaggaactgcgggcccagcatgaggaccaggtggagcagtataagaaggagctggagaagacttattctgccaagctggacaatgc/Acaggcagtctgctgagaggaacagcaacctggtgggggctgcccacgaggagctgcagcagtcgcgcatccgcatcgacagcctctctgcccagctcagccagctccagaagcagctggcagccaaggaggcgaagcttcgagacctggaggactcactggcccgtgagcgggacaccagccggcggctgctggcggaaaaggagcgggagatggccgagatgcgggcaaggatgcagcagcagctggacgagtaccaggagcttctggacatcaagctggccctggacatggagatccacgcctaccgcaagctcttggagggcgaggaggagaggctacgcctgtcccccagccctacctcgcagcgcagccgtggccgtgcttcctctcactcatcccagacacagggtgggggcagcgtcaccaaaaagcgcaaactggagtccactgagagccgcagcagcttctcacagcacgcacgcactagcgggcgcgtggccgtggaggaggtggatgaggagggcaagtttgtccggctgcgcaacaagtccaatgaggaccagtccatgggcaattggcagatcaagcgccagaatggagatgatcccttgctgacttaccggttcccaccaaagttcaccctgaaggctgggcaggtggtgacgatctgggctgcaggagctggggccacccacagcccccctaccgacctggtgtggaaggcacagaacacctggggctgcgggaacagcctgcgtacggctctcatcaactccactggggaagaagtggccatgcgcaagctggtgcgctcagtgactgtggttgaggacgacgaggatgaggatggagatgacctgctccatcaccaccacggctcccactgcagcagctcgggggaccccgctgagtacaacctgcgctcgcgcaccgtgctgtgcgggacctgcgggcagcctgccgacaaggcatctgccagcggctcaggagcccaggtgggcggacccatctcctctggctcttctgcctccagtgtcacggtcactcgcagctaccgcagtgtggggggcagtgggggtggcagcttcggggacaatctggtcacccgctcctacctcctgggcaactccagcccccgaacccagagcccccagaactgcagcatcatacaagagatgggaatgaggtgggaggtggaagaagggagaagaaaggtgagtttgagctgccttccctag
Protein sequence:
GGGATCAGSANGER sequencing primers:
forward primer GCTATGCCTTCTGGGGATCAG
Reverse primer GGGGTATCACCTGCTTCTGGAG
The intelligent improvement and treatment process of the detection kit for the novel locus of the familial dilated cardiomyopathy LMNA gene provided by the invention can comprise the following steps:
1. Sample collection: the biological sample is collected by using the sample collection tool, and the process can be automatically operated by an unmanned plane or a robot, so that the efficiency and the stability are improved.
2. Nucleic acid extraction: the extraction of DNA from biological samples using nucleic acid extraction reagents can be performed by automated equipment, reducing human error.
PCR amplification: the target region of the LMNA gene was amplified specifically using PCR primers. This step can be performed by an automated PCR apparatus to ensure consistent processing conditions for each sample.
DNA sequencing: the PCR amplification product was sequenced using DNA sequencing reagents to determine the mutation at the new site of the LMNA gene. This step can be performed by high throughput sequencing equipment, enabling large-scale, efficient sequencing.
5. Data analysis: sequencing results were read using data analysis software and the type and location of LMNA gene mutations were reported. This step may be performed by artificial intelligence algorithms, such as deep learning, machine learning, etc., which may improve the accuracy and efficiency of interpretation.
In this improvement, the signal and data processing may include the steps of:
1. and (3) data acquisition: the device automatically collects and records various data such as temperature, time, concentration and the like in the sample processing process.
2. Data preprocessing: the collected data is subjected to preprocessing operations such as cleaning, formatting, standardization and the like for subsequent analysis.
3. Data analysis: the data is analyzed using a machine learning algorithm to identify potential patterns and rules to optimize the use of the kit.
4. Data interpretation: based on the analysis results, sequencing data are read, and mutation types and positions of new loci of the LMNA gene are determined.
5. And (3) result feedback: and automatically feeding back the interpretation result to the user, and simultaneously carrying out continuous optimization according to the feedback and the use condition of the user.
Through the steps, the intelligent improvement of the detection kit for the new locus of the LMNA gene of the familial dilated cardiomyopathy and the processing of signals and data can be realized.
The two specific embodiments provided by the invention, corresponding implementation schemes are also provided:
application example 1:
the test kit is used in diagnosing Familial Dilated Cardiomyopathy (FDCM) cases.
1. An oral swab sample is collected from a suspected FDCM patient using a sample collection tool.
2. DNA is extracted from the oral swab sample using a nucleic acid extraction reagent.
3. Specific regions of the LMNA gene were amplified using PCR primers.
4. And sequencing the PCR product by using a DNA sequencing reagent to obtain the sequence information of the LMNA gene.
5. Finally, the sequencing result is read by using data analysis software, and the type and the position of the LMNA gene mutation are reported, so that whether the FDCM related LMNA gene mutation exists or not is determined.
Application example 2:
the detection kit is used in large-scale population genetic screening to early discover familial dilated cardiomyopathy.
1. Oral swabs or blood samples are collected from the population involved in the screening using a sample collection tool.
2. DNA is extracted from the collected sample using a nucleic acid extraction reagent.
3. Specific regions of the target LMNA gene were amplified using PCR primers.
4. And sequencing the PCR product by using a DNA sequencing reagent to obtain the sequence information of the LMNA gene.
5. Finally, the sequencing result is read by using data analysis software, and the type and the position of the LMNA gene mutation are reported, so that early warning and intervention are carried out before the disease is developed.
Both embodiments make full use of all components of the detection kit, demonstrating its practical operation and potential value in different application scenarios.
Aiming at the problems existing in the prior art, the invention provides a detection kit for new loci of LMNA genes of familial dilated cardiomyopathy and application thereof.
As shown in FIG. 1, the detection kit for the novel locus of the LMNA gene of the familial dilated cardiomyopathy provided by the embodiment of the invention comprises the following components:
sample collection means for collecting a biological sample;
nucleic acid extraction reagents for extracting DNA from a biological sample;
PCR primers for specifically amplifying a target region of the LMNA gene;
a DNA sequencing reagent for sequencing PCR amplified products and determining mutation of new loci of LMNA genes;
data analysis software for reading the sequencing results and reporting the type and location of LMNA gene mutations.
Further, the biological sample is blood or saliva.
Further, the nucleic acid extraction reagent includes a lysis-binding solution, a removing solution and an alkaline solution, a preservation solution, a magnetic bead suspension, a washing solution, and an eluent.
Further, sanger sequencing or second generation sequencing was performed to determine mutations at new sites of the LMNA gene.
As shown in FIG. 2, the method for detecting the novel locus of the LMNA gene of the familial dilated cardiomyopathy provided by the embodiment of the invention comprises the following steps:
Step one, collecting a biological sample;
step two, extracting DNA from the biological sample;
Step three, specifically amplifying a target region of the LMNA gene;
Step four, sequencing PCR amplification products, and determining mutation of new loci of LMNA genes;
and fifthly, reading the sequencing result and reporting the type and the position of the LMNA gene mutation.
As shown in fig. 3, the method for extracting DNA provided in the embodiment of the present invention specifically includes:
Step 1, fully mixing the obtained biological sample with preservation solution, and centrifuging to obtain supernatant 1;
step 2, adding impurity removing liquid into the obtained supernatant 1, adding alkaline solution until no flocculent precipitate is formed, and centrifuging to obtain supernatant 2;
And 3, simultaneously adding a lysis binding solution and a magnetic bead suspension into the obtained supernatant 2 under normal temperature to form a DNA-magnetic bead complex, and then adding a washing solution for washing and eluting to obtain DNA.
1. A blood sample is collected from a patient.
2. DNA is extracted from a blood sample using a nucleic acid extraction reagent.
3. The target region of the LMNA gene was amplified using specific PCR primers.
4. Sanger sequencing was performed to determine mutations at new sites in the LMNA gene.
5. Sequencing results were analyzed using specially developed data analysis software.
6. The mutation types and positions are reported for diagnosis and genetic counseling of familial dilated cardiomyopathy.
Example 2:
1. a blood sample is collected from a patient.
2. DNA is extracted from a blood sample using a nucleic acid extraction reagent.
3. The target region of the LMNA gene was amplified using specific PCR primers.
4. Second generation sequencing was performed to determine mutations at new sites of the LMNA gene.
5. Sequencing results were analyzed using specially developed data analysis software.
6. The mutation types and positions are reported for diagnosis and genetic counseling of familial dilated cardiomyopathy.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (10)
1. A novel gene mutation site is specifically:
LMNA:NM_001257374:exon5:c.485C>A:p.A162D。
2. the mutation site of claim 1 wherein base C is mutated to base a at base 485 at exon 5 of nm_001257374 transcript.
3. A kit for detecting a novel site of LMNA gene of familial dilated cardiomyopathy prepared by using the mutation site of gene according to claim 1, comprising:
sample collection means for collecting a biological sample;
nucleic acid extraction reagents for extracting DNA from a biological sample;
PCR primers for specifically amplifying a target region of the LMNA gene;
a DNA sequencing reagent for sequencing PCR amplified products and determining mutation of new loci of LMNA genes;
the data analysis software is used for reading the sequencing result and reporting the type and the position of the LMNA gene mutation;
The automatic device comprises an unmanned plane or a robot for automatic sample collection, the automatic device is used for nucleic acid extraction and PCR amplification, and the high-throughput sequencing device is used for DNA sequencing;
And the artificial intelligence algorithm is used for optimizing data analysis and improving the accuracy and efficiency of interpretation.
4. The intelligent detection kit according to claim 1, wherein the signal and data processing process of the detection kit comprises:
the data acquisition module is used for automatically acquiring and recording various data in the sample processing process by the equipment;
The data preprocessing module is used for performing preprocessing operations such as cleaning, formatting, standardization and the like on the acquired data;
The data analysis module analyzes the data by utilizing a machine learning algorithm and identifies potential modes and rules so as to optimize the use of the kit;
The data interpretation module is used for interpreting sequencing data based on the analysis result and determining the mutation type and the position of the new LMNA gene locus;
the result feedback module automatically feeds back the interpretation result to the user, and simultaneously carries out continuous optimization according to the feedback of the user and the use condition;
sanger sequencing or second generation sequencing was performed to determine mutations at new sites of the LMNA gene.
5. The method for detecting the novel locus of the LMNA gene of the familial dilated cardiomyopathy is characterized by comprising the following steps:
Step one, collecting a biological sample;
step two, extracting DNA from the biological sample;
Step three, specifically amplifying a target region of the LMNA gene;
Step four, sequencing PCR amplification products, and determining mutation of new loci of LMNA genes;
and fifthly, reading the sequencing result and reporting the type and the position of the LMNA gene mutation.
6. The method for detecting a novel site of LMNA gene of familial dilated cardiomyopathy according to claim 5, wherein the method for extracting DNA comprises:
Step 1, fully mixing the obtained biological sample with preservation solution, and centrifuging to obtain supernatant 1;
step 2, adding impurity removing liquid into the obtained supernatant 1, adding alkaline solution until no flocculent precipitate is formed, and centrifuging to obtain supernatant 2;
And 3, simultaneously adding a lysis binding solution and a magnetic bead suspension into the obtained supernatant 2 under normal temperature to form a DNA-magnetic bead complex, and then adding a washing solution for washing and eluting to obtain DNA.
The use of a kit for detecting a novel site of LMNA gene for familial dilated cardiomyopathy according to any one of claims 1 to 4.
The claims are a very important part of the present invention at the time of writing a patent application, as they define the scope of the invention.
7. A detection kit for detecting a new site mutation of the LMNA gene of familial dilated cardiomyopathy, the detection kit comprising: sample collection means for collecting a biological sample; nucleic acid extraction reagents for extracting DNA from a biological sample; PCR primers for specifically amplifying a target region of the LMNA gene; a DNA sequencing reagent for sequencing PCR amplified products and determining mutation of new loci of LMNA genes; data analysis software for reading the sequencing results and reporting the type and location of LMNA gene mutations.
8. The test kit of claim 7, wherein the sample collection means comprises means for collecting an oral swab or blood sample.
9. The test kit according to claim 7, wherein the PCR primer is designed to pair with a specific region of the LMNA gene to direct the DNA replication process, and to replicate only the specific region of the LMNA gene.
10. The test kit of claim 7, wherein the data analysis software is designed to compare the sequencing results to known LMNA gene sequences, look for any differences, and report the specific type and location of the mutation.
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