CN111676284A - Monitoring method for biological medicine for neuromuscular diseases - Google Patents
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Abstract
The invention relates to the technical field of biological medicines, in particular to a method for monitoring biological medicines for neuromuscular diseases, which comprises the following steps: A. extracting genome DNA; B. preparing a capture library; C. high-throughput sequencing: sequencing on a computer, analyzing data to obtain related information of pathogenic sites, and performing PCR amplification twice before and after by using different primer sequences so as to generate more primers with different labels; D. and (3) analysis and summary: comparing the obtained sequencing data with a reference sequence of a human genome, analyzing the condition of each mutation site of the detected gene to obtain mutation sites of genes related to the neuromuscular disease, and summarizing the action mechanism and index data of effective components in the used biological targeted medicine; E. transverse comparison; the invention not only improves the accuracy of biomedical monitoring for the neuromuscular disease, but also improves the monitoring efficiency of the biomedical monitoring for the neuromuscular disease.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a monitoring method for biomedicine for neuromuscular diseases.
Background
The hereditary neuromuscular disease is a hereditary disease which is manifested by motor dysfunction, mainly comprises hereditary myopathy, peripheral neuropathy, motor neuropathy, neuromuscular junction disease and the like, and can be subdivided into a plurality of subclasses, and the clinical manifestations of various subclasses are greatly overlapped. Therefore, conventional muscle biopsy, immunohistochemistry, immunofluorescence staining, etc. examinations are only suitable for the definite diagnosis of partial subtypes, and for this reason, the relevant personnel design a second-generation sequencing technology for targeted capture for enhanced biomedical monitoring of neuromuscular diseases.
However, the existing monitoring method for the biomedicine of the neuromuscular disease still has certain problems, and the specific problems are as follows:
1. in the conventional monitoring method, the quantitative and amplification treatment of a DNA sample is lacked, so that the capacity and the detection scheme of the same sample are limited, and a plurality of groups of pathogenic genes of the same disease are difficult to screen, so that the number of detection results is small, and the error is increased;
2. because the number of pathogenic genes of the hereditary neuromuscular disease is large, the comparison work aiming at the final result still needs more manual participation in the prior art, the actual monitoring efficiency is low, and much time is wasted in scientific research.
Disclosure of Invention
The invention aims to provide a biomedical monitoring method for neuromuscular diseases, which has the advantages of accurate and reliable monitoring result and high monitoring efficiency and solves the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a monitoring method for biomedicine of neuromuscular diseases comprises the following steps:
A. extracting genome DNA: extracting the genomic DNA of a patient, carrying out quantitative operation on the extracted genomic DNA, and then respectively filling the genomic DNA into culture dishes with unified specifications;
B. preparation of a capture library: fragmenting genome DNA, repairing the end of the fragmented genome DNA, adding a base A to the end of 3 ', connecting a product of adding the base A to the end of 3' with an amplification joint, carrying out PCR amplification on the connected product, enriching effective products, capturing a target region in an enriched template by using a probe, separating the captured target fragment, obtaining a captured library by adding a complete joint, carrying out PCR amplification on the captured target sequence, and carrying out quantitative operation on the library;
C. high-throughput sequencing: sequencing on a computer, analyzing data to obtain related information of pathogenic sites, and performing PCR amplification twice before and after by using different primer sequences so as to generate more primers with different labels;
D. and (3) analysis and summary: comparing the obtained sequencing data with a reference sequence of a human genome, analyzing the condition of each mutation site of the detected gene to obtain mutation sites of genes related to the neuromuscular disease, and summarizing the action mechanism and index data of effective components in the used biological targeted medicine;
E. transverse alignment: and extracting related detection results from the related documents through a semantic analysis algorithm to generate comprehensive performance evaluation of the used biological targeting medicine.
Preferably, the extraction method in the step a comprises purification column purification, magnetic bead purification or phenol chloroform extraction.
Preferably, the culture dish in step A is made of glass.
Preferably, the quantitative method in step A and step B comprises quantitative device quantitative method based on fluorescence quantitative principle, Q-PCR quantitative method and electrophoresis quantitative method.
Preferably, the DNA fragmentation method in step B-r comprises ultrasonication, transposase cleavage and restriction endonuclease cleavage.
Preferably, in steps B-c, the captured target sequence is PCR amplified by using DNA polymerase.
Preferably, the probes in step B-fifthly include probes for detecting CLCN1, SCN4A, CACNA1S, KCNJ2, TIA1, GNE, MYH7, NEB, FLNC, SEPN1, POMT2, VCP, DMD, MYOT, LMNA, CAV3, CAPN3, DYSF, SGCG, SGCA, SGCB, SGCD, TCAP, TRIM32, POMT1, ANO5, FKTN, BAG3, AGL, DAG1, PLEC and EMD.
Preferably, the primer sequence used in the first PCR amplification in step C is as follows,
Primer F：
5’-ACACTCTCTTTCCCTACACGACGCTCTTCCGATCT-3’；
Primer R：
5’-GTACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’。
preferably, the primer sequence used in the second PCR amplification in step C is as follows,
TrueSeq Universal Primer：
5’
-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’；
TrueSeq Primer-Index X：
5’-
CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’。
preferably, theNNNNNNIn part, a variety of different base combinations are used.
Preferably, the specific process in step E is to use a computer (with a corresponding algorithm written in the processor in advance) to calculate comprehensive score data of a plurality of biomedical products of the same product category, sort according to the score, compare the existing test data with public data of the biomedical products, and finally reorder and display the compared results.
Preferably, the externally displayed index is any one or more of literature information of related products, use condition information of related products, and test result information of related products in multiple groups of biomedical experimental methods.
Compared with the prior art, the invention has the following beneficial effects:
1. the method is used for amplifying the capacity of the same sample by carrying out quantitative operation for multiple times during extraction of genome DNA and preparation of a capture library, and is used for carrying out different analyses on the same sample by carrying out PCR amplification for multiple times during preparation of the capture library and high-throughput sequencing, so that the accuracy of biomedical monitoring for the neuromuscular disease is improved;
2. the mutation sites of genes related to the neuromuscular disease and the action mechanism and index data of effective components in the used biological targeted medicine are obtained through analysis and summary, and are transversely compared through a semantic analysis algorithm to generate comprehensive performance evaluation of the used biological targeted medicine, so that the test time is saved, and the monitoring efficiency of the biomedicine for the neuromuscular disease is improved.
Drawings
FIG. 1 is a process flow diagram of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Please refer to fig. 1, in which:
a monitoring method for biomedicine of neuromuscular diseases comprises the following steps:
A. extracting genome DNA: extracting the genomic DNA of a patient, carrying out quantitative operation on the extracted genomic DNA, and then respectively filling the genomic DNA into culture dishes with unified specifications;
B. preparation of a capture library: fragmenting genome DNA, repairing the end of the fragmented genome DNA, adding a base A to the end of 3 ', connecting a product of adding the base A to the end of 3' with an amplification joint, carrying out PCR amplification on the connected product, enriching effective products, capturing a target region in an enriched template by using a probe, separating the captured target fragment, obtaining a captured library by adding a complete joint, carrying out PCR amplification on the captured target sequence, and carrying out quantitative operation on the library;
C. high-throughput sequencing: sequencing on a computer, analyzing data to obtain related information of pathogenic sites, and performing PCR amplification twice before and after by using different primer sequences so as to generate more primers with different labels;
D. and (3) analysis and summary: comparing the obtained sequencing data with a reference sequence of a human genome, analyzing the condition of each mutation site of the detected gene to obtain mutation sites of genes related to the neuromuscular disease, and summarizing the action mechanism and index data of effective components in the used biological targeted medicine;
E. transverse alignment: and extracting related detection results from the related documents through a semantic analysis algorithm to generate comprehensive performance evaluation of the used biological targeting medicine.
The extraction method in the step A comprises purification column purification, magnetic bead purification or phenol chloroform extraction.
The culture dishes in the step A are all made of glass materials.
The quantitative method in the step A and the step B comprises quantitative device quantitative method based on the fluorescent quantitative principle, Q-PCR quantitative method and electrophoresis quantitative method.
And the DNA fragmentation method in the step B-I comprises ultrasonic crushing, transposase enzyme digestion and restriction enzyme digestion.
And B- ((c)) all adopting DNA polymerase to carry out PCR amplification on the captured target sequence.
The probes in step B-fifthly include probes for detecting CLCN1, SCN4A, CACNA1S, KCNJ2, TIA1, GNE, MYH7, NEB, FLNC, SEPN1, POMT2, VCP, DMD, MYOT, LMNA, CAV3, CAPN3, DYSF, SGCG, SGCA, SGCB, SGCD, TCAP, TRIM32, POMT1, ANO5, FKTN, BAG3, AGL, DAG1, PLEC and EMD.
The primer sequence adopted by the first PCR amplification in the step C is as follows,
Primer F：
5’-ACACTCTCTTTCCCTACACGACGCTCTTCCGATCT-3’；
PrimerR：
5’-GTACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’。
the primer sequence adopted by the second PCR amplification in the step C is as follows,
TrueSeq Universal Primer：
5’
-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’；
TrueSeq Primer-Index X：
5’-
CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’。
the above-mentionedNNNNNNIn part, a variety of different base combinations are used.
The specific process in the step E is to use a computer (corresponding algorithm is written in the processor in advance) to respectively calculate comprehensive score data of a plurality of biomedical products of the same product category, sort according to the score, compare the existing test data with public data of the biomedical products, and finally reorder and display the compared results.
The externally displayed index is any one or combination of multiple kinds of literature information of the related product, use condition information of the related product and test result information of the related product in multiple groups of biological medicine experimental methods.
Control group one:
the content of the comparison group is basically the same as that of the embodiment, and the same parts are not repeated, except that: and (4) eliminating the PCR amplification treatment in the step B and the step C, and directly carrying out on-machine sequencing on the capture library.
Control group two:
the content of the comparison group is basically the same as that of the embodiment, and the same parts are not repeated, except that: and D, comparing and summarizing the evaluation by adopting manual tests one by one without adopting the step E.
Table one:
although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (12)
1. A monitoring method aiming at biomedicine of neuromuscular disease is characterized in that: the method comprises the following steps:
A. extracting genome DNA: extracting the genomic DNA of a patient, carrying out quantitative operation on the extracted genomic DNA, and then respectively filling the genomic DNA into culture dishes with unified specifications;
B. preparation of a capture library: fragmenting genome DNA, repairing the end of the fragmented genome DNA, adding a base A to the end of 3 ', connecting a product of adding the base A to the end of 3' with an amplification joint, carrying out PCR amplification on the connected product, enriching effective products, capturing a target region in an enriched template by using a probe, separating the captured target fragment, obtaining a captured library by adding a complete joint, carrying out PCR amplification on the captured target sequence, and carrying out quantitative operation on the library;
C. high-throughput sequencing: sequencing on a computer, analyzing data to obtain related information of pathogenic sites, and performing PCR amplification twice before and after by using different primer sequences so as to generate more primers with different labels;
D. and (3) analysis and summary: comparing the obtained sequencing data with a reference sequence of a human genome, analyzing the condition of each mutation site of the detected gene to obtain mutation sites of genes related to the neuromuscular disease, and summarizing the action mechanism and index data of effective components in the used biological targeted medicine;
E. transverse alignment: and extracting related detection results from the related documents through a semantic analysis algorithm to generate comprehensive performance evaluation of the used biological targeting medicine.
2. The method for monitoring biomedical drugs for neuromuscular diseases according to claim 1, characterized in that: the extraction method in the step A comprises purification column purification, magnetic bead purification or phenol chloroform extraction.
3. The method for monitoring biomedical drugs for neuromuscular diseases according to claim 1, characterized in that: the culture dishes in the step A are all made of glass materials.
4. The method for monitoring biomedical drugs for neuromuscular diseases according to claim 1, characterized in that: the quantitative method in the step A and the step B comprises quantitative device quantitative method based on the fluorescent quantitative principle, Q-PCR quantitative method and electrophoresis quantitative method.
5. The method for monitoring biomedical drugs for neuromuscular diseases according to claim 1, characterized in that: and the DNA fragmentation method in the step B-I comprises ultrasonic crushing, transposase enzyme digestion and restriction enzyme digestion.
6. The method for monitoring biomedical drugs for neuromuscular diseases according to claim 1, characterized in that: and B- ((c)) all adopting DNA polymerase to carry out PCR amplification on the captured target sequence.
7. The method for monitoring biomedical drugs for neuromuscular diseases according to claim 1, characterized in that: the probes in step B-fifthly include probes for detecting CLCN1, SCN4A, CACNA1S, KCNJ2, TIA1, GNE, MYH7, NEB, FLNC, SEPN1, POMT2, VCP, DMD, MYOT, LMNA, CAV3, CAPN3, DYSF, SGCG, SGCA, SGCB, SGCD, TCAP, TRIM32, POMT1, ANO5, FKTN, BAG3, AGL, DAG1, PLEC and EMD.
8. The method for monitoring biomedical drugs for neuromuscular diseases according to claim 1, characterized in that: the primer sequence adopted by the first PCR amplification in the step C is as follows,
Primer F：
5’-ACACTCTCTTTCCCTACACGACGCTCTTCCGATCT-3’；
Primer R：
5’-GTACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’。
9. the method for monitoring biomedical drugs for neuromuscular diseases according to claim 1, characterized in that: the primer sequence adopted by the second PCR amplification in the step C is as follows,
TrueSeq Universal Primer：
5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’；
TrueSeq Primer-Index X：
5’-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’。
10. the method for monitoring biomedical drugs for neuromuscular diseases according to claim 9, characterized in that: the above-mentionedNNNNNNIn part, a variety of different base combinations are used.
11. The method for monitoring biomedical drugs for neuromuscular diseases according to claim 1, characterized in that: the specific process in the step E is to use a computer (corresponding algorithm is written in the processor in advance) to respectively calculate comprehensive score data of a plurality of biomedical products of the same product category, sort according to the score, compare the existing test data with public data of the biomedical products, and finally reorder and display the compared results.
12. The method for biomedical monitoring for neuromuscular diseases according to claim 11, wherein: the externally displayed index is any one or combination of multiple kinds of literature information of the related product, use condition information of the related product and test result information of the related product in multiple groups of biological medicine experimental methods.
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| CN105861700A (en) * | 2016-05-17 | 2016-08-17 | 湖州昂朴医学检验有限公司 | High throughput test method for neuromuscular disorders |
| CN107177670A (en) * | 2017-05-31 | 2017-09-19 | 上海昂朴生物科技有限公司 | A kind of method of high flux detection Parkinson's Disease-causing gene mutation |
| CN110607363A (en) * | 2019-11-01 | 2019-12-24 | 上海昂朴生物科技有限公司 | Nucleic acid group for high-throughput detection of diabetes pathogenic gene mutation, kit and application thereof |
| CN111009286A (en) * | 2018-10-08 | 2020-04-14 | 深圳华大因源医药科技有限公司 | Method and apparatus for microbiological analysis of host samples |
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| CN105861700A (en) * | 2016-05-17 | 2016-08-17 | 湖州昂朴医学检验有限公司 | High throughput test method for neuromuscular disorders |
| CN107177670A (en) * | 2017-05-31 | 2017-09-19 | 上海昂朴生物科技有限公司 | A kind of method of high flux detection Parkinson's Disease-causing gene mutation |
| CN111009286A (en) * | 2018-10-08 | 2020-04-14 | 深圳华大因源医药科技有限公司 | Method and apparatus for microbiological analysis of host samples |
| CN110607363A (en) * | 2019-11-01 | 2019-12-24 | 上海昂朴生物科技有限公司 | Nucleic acid group for high-throughput detection of diabetes pathogenic gene mutation, kit and application thereof |
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