CN113549683B - Eye and throat distal myopathy biomarker, detection method and application thereof - Google Patents
Eye and throat distal myopathy biomarker, detection method and application thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention relates to the field of biological medicine, in particular to a biological marker for remote myopathy of the eye and throat, a detection method and application thereof, and provides application of a GIPC1 gene in preparing products for diagnosing, detecting or treating the remote myopathy of the eye and throat, and researches show that repeated abnormal amplification of GGC or CGG in a 5' UTR region of the GIPC1 gene is brand-new pathogenic mutation of OPDM, and the mutation is the most common pathogenic cause in OPDM patients in China at present, so that the GIPC1 gene has application in preparing the products for diagnosing, detecting or treating the remote myopathy of the eye and throat, is the most common pathogenic gene in OPDM patients in China, and provides an important basis for gene diagnosis, gene therapy and pathogenesis research of the remote myopathy of the eye and throat in China.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to an eye-throat distal myopathy biomarker, a detection method and application thereof.
Background
Remote myopathy (Oculopharyngodistal myopathy, OPDM [ MIM:164310 ]) is a rare, early adult onset hereditary neuromuscular disease. The typical clinical manifestations of the disease are young people's morbidity, slowly progressive symmetric ptosis, extraocular muscle paralysis, facial muscle weakness, choking with drinking water and dysphagia, and the like, and muscle weakness of the distal limb. For a long time, the pathogenic genes of the disease are unknown, and the lack of genetic diagnosis indexes leads to the frequent misdiagnosis and diagnosis delay of OPDM. Therefore, searching for pathogenic genes of OPDM has important implications for diagnosis, gene therapy and research of pathogenesis of the disease.
Currently, in the research of OPDM pathogenic genes, only the dynamic mutation of CGG repeated abnormal amplification of 5' UTR region of LRP12 gene reported for the first time by Japan Tsuji S team in 2019 in Nature Genetics is the pathogenic mutation of OPDM. However, this causative gene was found to be 25% of those in OPDM of Japan (Noncoding CGG repeat expansions in neuronal intranuclear inclusion disease, oculopharyngodistal myopathy and an overlapping disease. Nat Genet 2019,51 (8): 1222-1232,Ishiura H,Shibata S,Yoshimura J, etc.), and the applicant examined 24 Chinese OPDM families and found that only 1 OPDM patient had been amplified abnormally by repeated amplification of CGG trinucleotide of the LRP12 gene. Therefore, there is an urgent need to develop a new pathogenic gene widely existing in OPDM patients in china, which will provide an important basis for gene diagnosis, gene therapy and pathogenesis research of myopathy patients far from the eye pharynx in china.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide an eye-throat distal myopathy biomarker, a detection method and application thereof.
For this purpose, the invention provides the following technical scheme:
in a first aspect, the present invention provides the use of the GIPC1 gene in the manufacture of a product for diagnosing, detecting or treating myopathy at the far end of the eye and pharynx.
The product comprises a detection kit, an oligonucleotide or gene editing kit designed for the GIPC1 gene, and the like.
In the described use, the GGC or CGG sequence of the 5'UTR region (5' untranslated region) of the GIPC1 gene is dynamically mutated.
In the application, the dynamic mutation refers to the abnormality of GGC or CGG sequence repetition times, and can be 88-164 times.
In a second aspect, the present invention provides an eye-pharynx distal myopathy biomarker comprising the GIPC1 gene described above.
In a third aspect, a primer for detecting a remote myopathy biomarker in the oculopharynx, the primer being designed based on the GIPC1 gene.
Preferably, a qualitative detection primer is included, said primer being (a) or (b) as follows:
(a) Primer GIPC1-F: as shown in SEQ ID NO. 1; the primer GIPC1-R is shown as SEQ ID NO. 2; the primer GIPC1-linker-R is shown as SEQ ID NO. 3;
(b) At least one nucleotide is substituted, deleted or added in the nucleotide sequences of the primer GIPC1-F, the primer GIPC1-R and/or the primer GIPC 1-linker-R.
Further, the 5' end of one of the qualitative detection primers is labeled with a fluorescent reporter group. The fluorophore may be FAM (6-carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), JOE (2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein), HEX (hexachloro-6-methylfluorescein) or VIC.
Preferably, the kit further comprises a quantitative detection primer, wherein the primer is as follows (c) or (d):
(c) Primer GIPC1-AL-F: as shown in SEQ ID NO. 4; the primer GIPC1-AL-R is shown as SEQ ID NO. 5;
(d) At least one nucleotide is substituted, deleted or added in the nucleotide sequence of the primer GIPC1-AL-F and/or the primer GIPC 1-AL-R.
Further, the 5' end of one of the quantitative detection primers is marked with a fluorescent reporter group. The fluorophore may be FAM (6-carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), JOE (2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein), HEX (hexachloro-6-methylfluorescein) or VIC.
In a fourth aspect, the invention provides a kit for detecting the remote myopathy biomarker of the eye and throat, which comprises the primer for detecting the remote myopathy biomarker of the eye and throat.
Further, the method comprises the steps of qualitatively detecting the PCR reaction system and/or quantitatively detecting the PCR reaction system; the qualitative detection PCR reaction system and/or the quantitative detection PCR reaction system respectively contain Prime STAR GXL DNA polymerase, 1 XPrimeSTAR GXL buffer, dATP, dTTP, dCTP, 7-Deaza-dGTP, dimethyl sulfoxide and/or betaine.
Preferably, the method comprises the steps of qualitatively detecting a PCR reaction system: contains 0.25U Prime STAR GXL DNA polymerase, 1 XPimeSTAR GXL buffer, 200. Mu.M dATP, 200. Mu.M dTTP, 200. Mu.M dCTP, 200. Mu.M 7-Deaza-dGTP,5% dimethyl sulfoxide and 1M betaine by volume.
Preferably, the method comprises the steps of quantitatively detecting a PCR reaction system: contains 0.25U Prime STAR GXL DNA polymerase, 1 XPimeSTAR GXL buffer, 200. Mu.M dATP, 200. Mu.M dTTP, 200. Mu.M dCTP, 200. Mu.M 7-Deaza-dGTP,5% dimethyl sulfoxide and 1M betaine by volume.
In a fifth aspect, the invention provides a method for detecting a myopathy biomarker at the far end of the eye and throat, comprising PCR amplification using the primer or the kit.
Further, the method also comprises a qualitative detection PCR reaction system and/or a quantitative detection PCR reaction system; the qualitative detection PCR reaction system and/or the quantitative detection PCR reaction system respectively contain Prime STAR GXL DNA polymerase, 1 XPrimeSTAR GXL buffer, dATP, dTTP, dCTP, 7-Deaza-dGTP, dimethyl sulfoxide and/or betaine.
Preferably, the PCR reaction system is tested qualitatively, with a total reaction volume of 20. Mu.l: contains 0.25U Prime STAR GXL DNA polymerase, 1 XPimeSTAR GXL buffer, 200. Mu.M dATP, 200. Mu.M dTTP, 200. Mu.M dCTP, 200. Mu.M 7-Deaza-dGTP,5% dimethyl sulfoxide by volume, 1M betaine, 0.3. Mu.M primer GIPC1-F, 0.3. Mu.M primer GIPC1-linker-R, 0.1. Mu.M primer GIPC1-R and 100ng genomic DNA.
Preferably, the PCR reaction system is quantitatively detected with a total reaction volume of 20. Mu.l: contains 0.25U Prime STAR GXL DNA polymerase, 1 XPimeSTAR GXL buffer, 200. Mu.M dATP, 200. Mu.M dTTP, 200. Mu.M dCTP, 200. Mu.M 7-Deaza-dGTP,5% dimethyl sulfoxide, 1M betaine, 0.3. Mu.M primer GIPC1-AL-F, 0.3. Mu.M primer GIPC1-AL-R, and 100ng of genomic DNA.
Also included are qualitative and/or quantitative PCR reaction conditions; the qualitative detection PCR reaction conditions and/or the quantitative detection PCR reaction conditions are as follows: reacting for 10 minutes at 98 ℃; then 9 cycles were performed: reacting at 98 ℃ for 30 seconds, 66 ℃ for 15 seconds, and each cycle is reduced by 1 ℃ and reacted at 72 ℃ for 4 minutes; this is followed by 28 cycles: reacting at 98 ℃ for 30 seconds, 58 ℃ for 15 seconds, and 72 ℃ for 4 minutes; finally, the step of extension is carried out at 72 ℃ for 10 minutes.
In a sixth aspect, the invention provides the use of said primers or said kit for the preparation of a product for the diagnosis, detection or treatment of myopathy at the far end of the eye and pharynx.
The technical scheme of the invention has the following advantages:
1. the application of the GIPC1 gene in preparing products for diagnosing, detecting or treating the eye-throat remote myopathy provided by the invention is that the applicant detects 38 OPDM patient cases of 24 Chinese OPDM families, finds that the GGC repeated abnormal amplification in the 5' UTR region of the GIPC1 gene positioned on chromosome 19 is a new pathogenic gene of OPDM, the pathogenic gene accounts for 50% of detected patients, and the GGC repeated times of the GIPC1 gene of the OPDM patients are 88-164 times, and the GGC repeated times of the normal human control is 12-32 times. Therefore, the repeated abnormal amplification of GGC or CGG in the 5' UTR region of GIPC1 is a brand-new pathogenic mutation of OPDM, and the mutation is the most common pathogenic cause in OPDM patients in China at present, and in conclusion, the GIPC1 gene has the application in preparing products for diagnosing, detecting or treating the remote-type myopathy of the oculopharynx, is the most common pathogenic gene in OPDM patients in China, and provides an important basis for gene diagnosis, gene therapy and pathogenesis research of the remote-type myopathy of the oculopharynx in China.
2. The invention provides an eye-pharynx distal myopathy biomarker which comprises a GIPC1 gene, wherein the GIPC1 gene can be used as the eye-pharynx distal myopathy biomarker for diagnosing, detecting or treating the eye-pharynx distal myopathy.
3. The invention provides a primer for detecting an eye-throat remote myopathy biomarker, which is characterized in that the qualitative detection primer is (a) or (b), wherein (a) is a primer GIPC1-F: as shown in SEQ ID NO. 1; the primer GIPC1-R is shown as SEQ ID NO. 2; the primer GIPC1-linker-R is shown as SEQ ID NO. 3; (b) At least one nucleotide is substituted, deleted or added in the nucleotide sequences of the primer GIPC1-F, the primer GIPC1-R and/or the primer GIPC 1-linker-R; the primer is designed for detecting the remote myopathy biomarker of the eye and throat, has high specificity and is used for qualitatively detecting the remote myopathy biomarker of the eye and throat.
4. The invention provides a primer for detecting an eye-throat remote myopathy biomarker, which is used for quantitatively detecting the primer (c) or (d), wherein (c) is a primer GIPC1-AL-F: as shown in SEQ ID NO. 4; the primer GIPC1-AL-R is shown as SEQ ID NO. 5; (d) At least one nucleotide is substituted, deleted or added in the nucleotide sequence of the primer GIPC1-AL-F and/or the primer GIPC 1-AL-R; the primer is designed for detecting the far-end type myopathy biomarker of the eye and the throat, has high specificity and is used for quantitatively detecting the repeated amplification times of GGC in the far-end type myopathy biomarker of the eye and the throat.
5. The invention provides a method for detecting eye and throat remote myopathy biomarkers, which comprises the steps of RP-PCR (Repeat-primed polymerase chain reaction), a PCR reaction system, a PCR detection kit, a detection kit and a detection kit, wherein the PCR reaction system comprises 0.25U Prime STAR GXL DNA polymerase, 1 x PrimeSTAR GXL buffer, 200 mu M dATP,200 mu M dTTP,200 mu M dCTP,200 mu M7-Deaza-dGTP, 5% dimethyl sulfoxide, 1M betaine, 0.3 mu M primer GIPC1-F,0.3 mu M primer GIPC1-linker-R,0.1 mu M primer GIPC1-R and 100ng genome DNA; the reaction system is added with the dimethyl sulfoxide with the volume percentage of 5%, so that the DNA amplification with high GC content is facilitated, the concentration ratio of the primers GIPC1-F, GIPC1-linker-R to the primers GIPC1-R is adjusted to be 3:3:1, and meanwhile, the success rate, the stability and the product signal of PCR amplification are improved under the synergistic effect of betaine and the dimethyl sulfoxide.
6. The invention provides a method for detecting eye pharyngeal remote myopathy biomarkers, which qualitatively detects PCR reaction conditions as follows: reacting for 10 minutes at 98 ℃; then 9 cycles were performed: reacting at 98 ℃ for 30 seconds, 66 ℃ for 15 seconds, and each cycle is reduced by 1 ℃ and reacted at 72 ℃ for 4 minutes; this is followed by 28 cycles: reacting at 98 ℃ for 30 seconds, 58 ℃ for 15 seconds, and 72 ℃ for 4 minutes; finally, the step of extending is carried out at 72 ℃ for 10 minutes; the reaction condition is that the PCR process of tool-down is adopted to amplify, the signal of amplified products is enhanced, the amplifying time is shorter, and the stability and success rate are higher.
7. The invention provides a method for detecting eye pharyngeal remote myopathy biomarker, the quantitative detection method is AL-PCR (Fluorescence amplicon length analysis polymerase chain reaction), the PCR reaction system contains 0.25U Prime STAR GXL DNA polymerase, 1 x PrimeSTAR GXL buffer, 200. Mu.M each dATP, dTTP, dCTP and 7-Deaza-dGTP,5% dimethyl sulfoxide, 1M betaine, 0.3. Mu.M primer GIPC1-AL-F, 0.3. Mu.M primer GIPC1-AL-R, and 100ng genomic DNA; the reaction system is added with 5% dimethyl sulfoxide, which is more conducive to DNA amplification with high GC content, and simultaneously adjusts the concentration ratio of the primer GIPC1-AL-F, GIPC1-AL-R to 1:1, and simultaneously improves the success rate, stability and product signal of PCR amplification under the synergistic effect of betaine and dimethyl sulfoxide.
8. The invention provides a method for detecting eye pharyngeal remote myopathy biomarkers, which quantitatively detects PCR reaction conditions as follows: reacting for 10 minutes at 98 ℃; then 9 cycles were performed: reacting at 98 ℃ for 30 seconds, 66 ℃ for 15 seconds, and each cycle is reduced by 1 ℃ and reacted at 72 ℃ for 4 minutes; this is followed by 28 cycles: reacting at 98 ℃ for 30 seconds, 58 ℃ for 15 seconds, and 72 ℃ for 4 minutes; finally, the step of extending is carried out at 72 ℃ for 10 minutes; the reaction condition is that the PCR process of tool-down is adopted to amplify, the signal of amplified products is enhanced, the amplifying time is shorter, and the stability and success rate are higher.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a qualitative detection of a myopathy biomarker of the distal end of the eye and throat in OPDM patients in example 5 of the invention;
FIG. 2 is a qualitative measurement of a remote myopathy biomarker for normal human eyes and pharynx in example 5 of the present invention;
FIG. 3 is a quantitative determination of the far-end myopathy biomarker in the oculopharynx of OPDM patients in example 6 of the invention;
FIG. 4 is a quantitative determination result of a remote myopathy biomarker for normal human eyes and pharynx in example 6 of the present invention;
FIG. 5 is a qualitative detection of the far-end myopathy biomarkers in the eye and throat of 38 OPDM patients in example 7 of the invention; FIGS. 5 (a) -5 (f) are, respectively, qualitative detection results of the far-end myopathy biomarkers in the eye and throat of a portion of 38 OPDM patients;
FIG. 6 is a quantitative determination of the far-end myopathy biomarker in the oculopharynx of 19 OPDM patients with positive qualitative detection results in example 7 of the invention; FIGS. 6 (a) -6 (d) are the quantitative detection results of the far-end myopathy biomarkers of the eye and throat in 19 OPDM patients with positive partial qualitative detection results, respectively;
FIG. 7 is a quantitative determination result of a far-end type myopathy biomarker for the eye pharynx in 1 case of 550 cases of normal persons in example 7 of the present invention;
FIG. 8 is a statistical chart of quantitative detection results of far-end myopathy biomarkers of eye-pharynx in 550 normal persons in example 7 of the invention.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
The primers in the following examples were synthesized by Beijing Tian Yihui Yuan Biotech Co.
Example 1 qualitative detection primer design
In this example, qualitative detection primers were designed based on the far-end myopathy biomarkers of the eye and throat, such as GIPC1-F: as shown in SEQ ID NO. 1; GIPC1-R is shown as SEQ ID NO. 2; GIPC1-linker-R is shown as SEQ ID NO. 3.
Further, a fluorescent reporter group is labeled on the 5 'end of one of the qualitative detection primers, which may be FAM (6-carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), JOE (2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein), HEX (hexachloro-6-methylfluorescein) or VIC, and in this embodiment, a FAM fluorescent group is labeled on the 5' end of primer GIPC 1-F.
Example 2 quantitative detection primer design
The quantitative detection primer is designed based on the eye-pharynx distal myopathy biomarker in the embodiment, and is as follows: as shown in SEQ ID NO. 4; GIPC1-AL-R is shown as SEQ ID NO. 5.
Further, a fluorescent reporter group is labeled on the 5 'end of one of the quantitative detection primers, which may be FAM (6-carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), JOE (2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein), HEX (hexachloro-6-methylfluorescein) or VIC, and in this embodiment, a VIC fluorophore is labeled on the 5' end of the primer GIPC 1-AL-F.
Example 3A kit for detecting a remote myopathy biomarker in the oculopharynx
This example provides a kit for detecting a remote myopathy biomarker in the oculopharynx, comprising the primers of example 1, packaged separately.
Further, the method also comprises an independently packaged qualitative detection PCR reaction system, which is as follows:
contains 0.25U Prime STAR GXL DNA polymerase, 1 XPimeSTAR GXL buffer, 200. Mu.M dATP, 200. Mu.M dTTP, 200. Mu.M dCTP, 200. Mu.M 7-Deaza-dGTP,5% dimethyl sulfoxide, and 1M betaine.
Example 4A kit for detecting a remote myopathy biomarker in the oculopharynx
This example provides a kit for detecting a remote myopathy biomarker in the oculopharynx, comprising the primers of example 2, packaged separately.
Further, the quantitative detection PCR reaction system of independent package is also included, as follows: contains 0.25U Prime STAR GXL DNA polymerase, 1 XPimeSTAR GXL buffer, 200. Mu.M dATP, 200. Mu.M dTTP, 200. Mu.M dCTP, 200. Mu.M 7-Deaza-dGTP,5% dimethyl sulfoxide, and 1M betaine.
Example 5A method of detecting a remote myopathy biomarker in the oculopharynx
The embodiment provides a method for detecting an eye-throat remote myopathy biomarker, which comprises the following steps of:
(1) Extracting the genome DNA of the OPDM patient and the normal person respectively, and extracting the genome DNA by adopting a root DP 349-blood genome DNA extraction system;
(2) The following PCR reaction system was prepared by using the genomic DNAs of OPDM patients and normal persons obtained in the step (1) as templates, respectively, using the kit of example 3, and the total reaction volume was 20. Mu.l: contains 0.25U Prime STAR GXL DNA polymerase, 1 XPimeSTAR GXL buffer, 200. Mu.M dATP, 200. Mu.M dTTP, 200. Mu.M dCTP, 200. Mu.M 7-Deaza-dGTP,5% dimethyl sulfoxide, 1M betaine, 0.3. Mu.M primer GIPC1-F, 0.3. Mu.M primer GIPC1-linker-R, 0.1. Mu.M primer GIPC1-R and 100ng genomic DNA;
the PCR conditions were as follows: reacting for 10 minutes at 98 ℃; then 9 cycles were performed: reacting at 98 ℃ for 30 seconds, 66 ℃ for 15 seconds, and each cycle is reduced by 1 ℃ and reacted at 72 ℃ for 4 minutes; this is followed by 28 cycles: reacting at 98 ℃ for 30 seconds, 58 ℃ for 15 seconds, and 72 ℃ for 4 minutes; finally, the step of extending is carried out at 72 ℃ for 10 minutes;
(3) The PCR products obtained in step (2) were separated by capillary electrophoresis using 3500xl gene analyzer and data analyzed using GeneMapper software, respectively. The analysis results of OPDM patients are shown in FIG. 1, and the jagged peaks can be seen in FIG. 1, which indicate that the detection of the remote myopathy biomarker of the hypopharynx, i.e., the GGC repeated abnormal amplification of the GIPC1 gene, was detected. As a result of analysis of normal persons, as shown in FIG. 2, no jagged peak was seen, indicating that no repeated abnormal amplification of GGC was detected in the GIPC1 gene, and it was identified as normal persons.
Example 6A method of detecting a remote myopathy biomarker in the oculopharynx
The embodiment provides a method for detecting an eye-throat remote myopathy biomarker, which comprises the following steps of:
(1) Extracting the genome DNA of the patient with the confirmed diagnosis of OPDM and the normal person respectively, and extracting the genome DNA by adopting a root DP 349-blood genome DNA extraction system;
(2) The following PCR reaction system was prepared using the kit of example 4 with the genomic DNA as a template, and the total reaction volume was 20. Mu.l: contains 0.25U Prime STAR GXL DNA polymerase, 1 XPimeSTAR GXL buffer, 200. Mu.M dATP, 200. Mu.M dTTP, 200. Mu.M dCTP, 200. Mu.M 7-Deaza-dGTP,5% dimethyl sulfoxide, 1M betaine, 0.3. Mu.M primer GIPC1-AL-F, 0.3. Mu.M primer GIPC1-AL-R, and 100ng genomic DNA;
the PCR conditions were as follows: reacting for 10 minutes at 98 ℃; then 9 cycles were performed: reacting at 98 ℃ for 30 seconds, 66 ℃ for 15 seconds, and each cycle is reduced by 1 ℃ and reacted at 72 ℃ for 4 minutes; this is followed by 28 cycles: reacting at 98 ℃ for 30 seconds, 58 ℃ for 15 seconds, and 72 ℃ for 4 minutes; finally, the step of extending is carried out at 72 ℃ for 10 minutes;
(3) The PCR product obtained in step (2) was separated by capillary electrophoresis using 3500xl gene analyzer and analyzed by data analysis using GeneMapper software. The analysis results of OPDM patients are shown in FIG. 3, and it can be seen from FIG. 3 that 115 GGC repeats exist in the far-end myopathy biomarker of the oculopharynx, and the GGC repeats are located between 88 and 164 times in OPDM patients, and can be identified as OPDM patients. As a result of analysis of normal persons, it was found from the figure that GGC repeats were within the range of normal persons (12-32 times) and were identified as normal persons, as shown in FIG. 4.
Example 7
This example, through clinical and skeletal muscle disease examination, 38 OPDM patients from 24 families were diagnosed and collected and identified according to the methods of example 5 and example 6, the results of the tests according to example 5 are shown in fig. 5, and fig. 5 (a) -5 (f) are qualitative test results of the far-end myopathy biomarkers of the eye pharynx of the 38 OPDM patients, respectively; according to the detection results of example 6, as shown in fig. 6, the quantitative detection results of the far-end myopathy biomarkers of the eye and throat of 19 OPDM patients with positive qualitative detection results are shown in fig. 6, and the quantitative detection results of the far-end myopathy biomarkers of the eye and throat of 19 OPDM patients with positive partial qualitative detection results are shown in fig. 6 (a) -6 (d), respectively; the detection results of 19 OPDM patients of 12 families showed jagged peaks as seen in fig. 5 (a) -5 (f), and the detection results were positive, and the number of GGC repeats was 88-164 in the GIPC1 gene 5' utr region of 19 OPDM patients of 12 families as seen in fig. 6 (a) -6 (d).
In this example, 550 normal persons were also identified according to the method of example 6, wherein the detection result of one normal person is shown in FIG. 7, and the result of counting the number of GGC repeats in the 5'UTR region of the GIPC1 gene of 550 normal persons is shown in FIG. 8, and it can be seen from the figure that the number of GGC repeats in the 5' UTR region of the GIPC1 gene of the normal person is 12 to 32.
From the above, it can be seen that the far-end myopathy biomarker for the eye and throat of the present invention can be used for diagnosing the far-end myopathy of the eye and throat, accounting for 50% of the detected patients, and can be used for preparing products for diagnosing OPDM by detecting whether abnormal repetition (whether jagged peak) of 5' utr region GGC occurs in GIPC1 gene or not by the detection method of example 5 of the present invention and quantitatively detecting the number of repetitions of GIPC1 mutation by the detection method of example 6.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
SEQUENCE LISTING
<110> Deng Jianwen
<120> an oculopharyngeal distal myopathy biomarker, detection method and use thereof
<130> HA202001092
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 27
<212> DNA
<213> primer (GIPC 1-F)
<400> 1
cagacacatc cttctcgcag aggccac 27
<210> 2
<211> 39
<212> DNA
<213> primer (GIPC 1-R)
<400> 2
caggaaacag ctatgaccgg cggaggcagc ggcggcggc 39
<210> 3
<211> 18
<212> DNA
<213> primer (GIPC 1-linker-R)
<400> 3
caggaaacag ctatgacc 18
<210> 4
<211> 23
<212> DNA
<213> primer (GIPC 1-AL-F)
<400> 4
cacatccttc tcgcagaggc cac 23
<210> 5
<211> 23
<212> DNA
<213> primer (GIPC 1-AL-R)
<400> 5
gaagacgcgg attggctgcg agc 23
Claims (6)
1. Use of a detection primer of GIPC1 gene in the preparation of a product for diagnosing or detecting myopathy of the distal end of the eye and pharynx, characterized in that the detection primer is a qualitative detection primer or a quantitative detection primer as follows:
the qualitative detection primers are as follows:
primer GIPC1-F: as shown in SEQ ID NO. 1; the primer GIPC1-R is shown as SEQ ID NO. 2; and a primer GIPC1-linker-R shown in SEQ ID NO. 3;
the quantitative detection primers are as follows:
primer GIPC1-AL-F: as shown in SEQ ID NO. 4; and the primer GIPC1-AL-R is shown as SEQ ID NO. 5.
2. The use according to claim 1, characterized in that the 5' utr region GGC or CGG sequence of the GIPC1 gene is dynamically mutated.
3. The use according to claim 1, wherein the product for detecting myopathy at the far end of the eye and throat comprises a kit comprising the qualitative or quantitative detection primer.
4. The use according to claim 3, wherein the kit further comprises a qualitative detection PCR reaction system and/or a quantitative detection PCR reaction system; the qualitative detection PCR reaction system and/or the quantitative detection PCR reaction system respectively contain Prime STAR GXL DNA polymerase, 1 XPrimeSTAR GXL buffer, dATP, dTTP, dCTP, 7-Deaza-dGTP, dimethyl sulfoxide and/or betaine.
5. The use according to claim 4, wherein the qualitative detection PCR reaction system has a total reaction volume of 20 μl: contains 0.25U Prime STAR GXL DNA polymerase, 1 XPimeSTAR GXL buffer, 200. Mu.M dATP, 200. Mu.M dTTP, 200. Mu.M dCTP, 200. Mu.M 7-Deaza-dGTP,5% dimethyl sulfoxide by volume, 1M betaine, 0.3. Mu.M primer GIPC1-F, 0.3. Mu.M primer GIPC1-linker-R, 0.1. Mu.M primer GIPC1-R and 100ng genomic DNA;
and/or, the quantitative detection PCR reaction system has a total reaction volume of 20 μl: contains 0.25U Prime STAR GXL DNA polymerase, 1 XPimeSTAR GXL buffer, 200. Mu.M each dATP, dTTP, dCTP and 7-Deaza-dGTP,5% dimethyl sulfoxide, 1M betaine, 0.3. Mu.M primer GIPC1-AL-F, 0.3. Mu.M primer GIPC1-AL-R, and 100ng genomic DNA.
6. Use of the kit of claim 3 for the preparation of a product for diagnosing or detecting myopathy distal to the eye and throat.
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