CN114763571A - Ferrochelatase gene genetic variation diagnostic kit - Google Patents
Ferrochelatase gene genetic variation diagnostic kit Download PDFInfo
- Publication number
- CN114763571A CN114763571A CN202110038334.8A CN202110038334A CN114763571A CN 114763571 A CN114763571 A CN 114763571A CN 202110038334 A CN202110038334 A CN 202110038334A CN 114763571 A CN114763571 A CN 114763571A
- Authority
- CN
- China
- Prior art keywords
- gene
- dna
- fech
- ferrochelatase
- diagnostic kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010057394 Ferrochelatase Proteins 0.000 title claims abstract description 28
- 238000009007 Diagnostic Kit Methods 0.000 title claims abstract description 13
- 230000007614 genetic variation Effects 0.000 title claims abstract description 10
- 239000002299 complementary DNA Substances 0.000 claims abstract description 16
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 108700024394 Exon Proteins 0.000 claims abstract description 8
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims description 5
- 239000013522 chelant Substances 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 238000003745 diagnosis Methods 0.000 claims description 3
- 108020004414 DNA Proteins 0.000 abstract description 35
- 230000001717 pathogenic effect Effects 0.000 abstract description 14
- 101150063237 FECH gene Proteins 0.000 abstract description 8
- 108091026890 Coding region Proteins 0.000 abstract description 5
- 102000053602 DNA Human genes 0.000 abstract description 3
- 238000010008 shearing Methods 0.000 abstract description 3
- 102100030771 Ferrochelatase, mitochondrial Human genes 0.000 abstract 2
- 101000843611 Homo sapiens Ferrochelatase, mitochondrial Proteins 0.000 abstract 2
- 102000004169 proteins and genes Human genes 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 36
- 230000035772 mutation Effects 0.000 description 15
- 238000001514 detection method Methods 0.000 description 11
- 238000003752 polymerase chain reaction Methods 0.000 description 11
- 230000003321 amplification Effects 0.000 description 10
- 239000012634 fragment Substances 0.000 description 10
- 238000003199 nucleic acid amplification method Methods 0.000 description 10
- 238000003776 cleavage reaction Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 230000007017 scission Effects 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 108091093088 Amplicon Proteins 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 206010064571 Gene mutation Diseases 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 102000003875 Ferrochelatase Human genes 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 238000013399 early diagnosis Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000007918 pathogenicity Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 238000010805 cDNA synthesis kit Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229960001484 edetic acid Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000010437 erythropoiesis Effects 0.000 description 2
- 230000000913 erythropoietic effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 101100532034 Drosophila melanogaster RTase gene Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 206010034972 Photosensitivity reaction Diseases 0.000 description 1
- 208000037062 Polyps Diseases 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 208000037340 Rare genetic disease Diseases 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 230000036211 photosensitivity Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
Images
Classifications
-
- 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
- C12Q1/6876—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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a diagnostic kit for genetic variation of a ferrochelatase gene, which contains a primer designed autonomously, overcomes the problem that partial exons in an FECH gene are difficult to amplify, can successfully amplify to obtain an exon DNA gene sequence of a coding region of a FECH mature protein and an inclusion region containing important known shearing sites at the upstream and the downstream of the exon DNA gene sequence, can detect full-length cDNA of the FECH, and achieves the aim of comprehensively detecting potential pathogenic regions of the FECH gene.
Description
Technical Field
The invention relates to the field of genetic metabolism molecular biology, in particular to a ferrochelatase gene genetic variation diagnostic kit.
Background
Erythropoiesis protoporphyrinosis (EPP) caused by mutations in the Ferrochelatase gene (FECH) is a rare genetic disease, and an effective treatment means is not yet available, and the first drug named "afealanotide (16 mg) approved by FDA in 2019 is used for treating erythropoiesis protoporphyrinosis, but the effect is limited. Early diagnosis, however, can block the disease from continuing to develop and prevent serious complications, thereby significantly reducing the fatality rate. However, the conventional detection means are also extremely limited, and the early diagnosis rate of erythropoietic protoporphyrinemia is not high, and the effect of reducing the mortality of erythropoietic protoporphyrinemia by early intervention therapy has not yet been achieved.
The presence of EPP is usually determined by detecting FECH gene mutation, but EPP patients have a very low ferrochelatase gene mutation carrying rate, resulting in a lower actual detection rate. Moreover, the genetic pathogenesis of the gene is special, the pathogenic molecule is based on Trans-co-inheritance (Trans-co-inheritance), T to C variation (IVS3-48T/C) of IVS3-48 positions needs to be detected from the FECH gene, and an effective kit technology for detecting FECH pathogenic intron regions and whole exons is not available up to now.
Disclosure of Invention
In order to solve the problems, the invention provides a ferrochelatase gene genetic variation diagnostic kit. It comprises primers for amplifying the following genes: all exons of the ferrous chelate enzyme gene and intron regions of 50-150bp near the upstream and downstream sides of the exons, and the primer sequences are shown in the following table:
preferably, the diagnostic kit provided by the present invention further comprises primers for amplifying the full-length cDNA of the ferrochelatase gene, the primer sequences are shown in the following table:
compared with the prior art, the invention has the advantages that:
the invention solves the technical problems of the prior art that the early diagnosis and the prevention blocking of diseases caused by ferrochelatase gene (FECH) are lacked. The ferrochelase gene genetic variation diagnostic kit contains the autonomously designed primer, overcomes the problem that partial exons in the FECH gene are difficult to amplify, can successfully amplify to obtain the exon DNA gene sequence of the FECH mature protein coding region and the intron regions containing important known shearing sites at the upstream and downstream, can detect the transcript of the FECH, achieves the aim of comprehensively detecting the potential pathogenic region of the FECH gene, can be used as a confirmation kit for identifying and diagnosing erythrocyte generating protoporphyrinemia suspected patients with similar symptoms of systems such as photosensitivity, nerves, blood, digestion and the like or home screening, and has wide application prospect.
Drawings
FIG. 1 is an electrophoretogram of an amplicon obtained after amplification by the primers of the present invention;
FIG. 2 is a graph comparing the sequence of an amplification product of a sample with the internationally recognized reference sequence NG 008175;
FIG. 3 is a map of an intron fragment containing a pathogenic cleavage site and an exon-internal mutant fragment of sample 1;
FIG. 4 is a map of an intron fragment containing a pathogenic cleavage site and an exon-internal mutant fragment of sample 2;
FIG. 5 is a map of intron fragments and cDNA fragments containing pathogenic cleavage sites of sample 3;
FIG. 6 is a map of intron fragments and cDNA fragments containing pathogenic cleavage sites of sample 3.
Detailed Description
The advantages of the invention are explained in detail below with reference to the drawings and the embodiments.
The invention adopts Polymerase Chain Reaction (PCR) and utilizes self-developed primers to amplify a whole blood sample so as to obtain an exon of an FECH gene, an intron of about 50-150bp at the upstream and the downstream of the FECH gene and full-length cDNA thereof, and judges according to the exon, the intron and the cDNA genes so as to determine whether ferrochelatase gene mutation exists in the sample. The reagents and instruments used in the specific embodiments of the invention are as follows:
1. Reagent
QIAamp Blood Mini kit from QIAGEN, germany, cat No.: 51106 and preserving at room temperature for 250 persons/box;
protease K, 1 bottle, and storing at 4 ℃;
Buffer
AllPrep DNA/RNA Mini kit from QIAGEN, cat #: 80204, 50 persons/box, storing at room temperature;
from Promega
Polymerase, 100U/piece, preservation at-20 ℃;
dNTP mix of TAKARA 2.5mM each, 1.28 ml/piece, stored at-20 ℃;
PrimeScript of TAKARATMII 1stStrand cDNA synthesis kit, preservation at-20 ℃;
100 g of agarose from Shanghai workers per bottle, and storing at room temperature;
the DL2000 DNA Marker kit from TAKARA, stored at-20 deg.C;
primer sequences were designed autonomously (see table 1).
TABLE 1 PCR primers and cDNA sequences, amplification conditions and amplicon lengths
2. Instrument for measuring the position of a moving object
Eppendorf 5415D centrifuge from Eppendorf corporation;
nanodrop 2000 spectrophotometer by Thermo scientific;
ProFlex of ABITMA 3X 32-well PCR system thermocycler;
Bio-Rad electrophoreser from Bio-Rad;
tanon 3500 gel imaging system from Tanon corporation.
3. Detection conditions and procedures
Extraction of DNA
Respectively taking 200 mul of EDTA (ethylene diamine tetraacetic acid) anticoagulation whole blood sample to be detected, 20 mul of proteinase K and 200 mul of Buffer AL, mixing and oscillating for 15 seconds, fully and uniformly mixing, and placing in a 56 ℃ water bath for 30 minutes; adding 200 mul of absolute ethyl alcohol (96-100%), oscillating for 15 seconds, and centrifuging for 1 minute in a centrifugal machine at the frequency of 10000 rpm; transferring the mixture into QIAamp Spin Column, and centrifuging at 10000 rpm for 1 min; replacing the collecting pipe; adding Buffer AW 1500. mu.l, centrifuging at 10000 rpm for 1 min; replacing the collecting pipe; adding Buffer AW 2500 μ l, centrifuging at 12000 rpm for 3 min, and replacing with 1.5ml centrifuge tube; adding 200 μ l of Buffer AE, standing at room temperature for 5 min, centrifuging at 8000 rpm for 3 min to obtain genome DNA, and storing at 4 deg.C; the concentration of the extracted genomic DNA was quantified using a spectrophotometer.
Extraction of RNA：
The procedure was carried out according to the instruction manual of AllPrep DNA/RNA Mini kit (cat. No. 80204) from QIAGEN.
Synthesis of cDNA
Using PrimeScriptTMII 1st Strand cDNA Synthesis kit (Takara, Japan) the following reaction mixture was prepared in a 0.6ml centrifuge tube: oligo dT Primer (50. mu.M), dNTP mix (10mM each) 1. mu.l, template RNA Total RNA of 5. mu.g or less, and an appropriate amount of RNase free dH2O were added to the Mixture in a Total amount of 10. mu.l, and the Mixture was incubated at 65 ℃ for 5 minutes, followed by rapid cooling on ice to denature the template RNA. 4. mu.l of 5 XPrimeScript II buffer, 0.5. mu.l (20U) of RNase Inhibitor (40U/. mu.l), 1. mu.l (200U) of PrimeScript II RTase (200U/. mu.l) and an appropriate amount of RNase free dH2O were added to the tube to a total amount of 20. mu.l, the mixture was mixed slowly and mixed, left at 42 ℃ for 60 minutes, then at 95 ℃ for 5 minutes to inactivate the reverse transcriptase, and then the mixture was rapidly cooled on ice.
PCR reaction
Table 2: PCR reaction system
| DNA templateOr cDNA template | 3μl |
| 5×Buffer | 5μl |
| dNTP Mixture | 1μl |
| GoTaq DNA polymerase | 0.25μl |
| Upstream primer | 0.5μl |
| Downstream primer | 0.5μl |
| Double-steaming water supplement | The total volume is 25 mu l |
And (3) PCR reaction conditions:
95℃2min
95 ℃ for 30s (35 cycles)
55 ℃ for 30s (35 cycles)
72℃35s
72℃6min
Quality control
Controlling the ratio of A260/A280 of the DNA sample to be between 1.6 and 2.0 according to the quantitative requirement;
An experimental room for preparing a PCR reaction system and identifying PCR reaction products is strictly separated from a working room of a PCR instrument;
negative controls without DNA template were performed under the same conditions for each primer pair tested for each batch.
Primer validity detection
5 mul of PCR reaction product is taken to be observed in 1% agarose gel electrophoresis, wherein no band appears in negative control, and a band with a corresponding size appears in a detection sample, so that the experiment is judged to be effective. The results of electrophoresis experiments on the amplicons obtained after amplification of the primers of the invention are shown in FIG. 1: from left to right in the figure, the first lane DL2000 is a nucleic acid molecular weight standard (bands from top to bottom correspond to 1500bp,1000bp,750bp,500bp,250bp and 100bp, respectively), followed by a negative control, the corresponding amplicon (from 1 to 10-11-1), respectively. As can be seen from the electrophoretic graphical representation of FIG. 1, the amplicons of the present invention all showed the corresponding molecular weights, thus preliminarily verifying that the primers were effective.
A known ferrochelatase gene sample without mutation is amplified by the primer according to the amplification experimental conditions and the method, the sequence of the obtained amplification product is compared with an internationally recognized reference sequence NG 008175, and the result is shown in figure 2. As can be seen from FIG. 2, the primers of the present invention amplified to obtain the corresponding positions of each exon + intron corresponding to the standard Sequence of the internationally recognized ferrochelatase gene (Reference Sequence: NG-008175.1), further confirming the successful design of the primers.
Determination of mutations
And (4) sending the PCR amplification positive product to a sequencing company for sequencing, and taking a primer related to the tested project as a sequencing primer.
Sequencing results were analyzed using Vector NTI suite 11.0(Invitrogen), Chromas and CodonCode Aligner software, the relevant test items were aligned using the respective genomic DNA and reading frame DNA sequences as reference sequences, the disease mutation databases were referenced to databases such as HGMD (http:// www.hgmd.cf.ac.uk/ac/index. php), OMIM (http:// www.ncbi.nlm.nih.gov/OMIM), dSNP, and the new mutations were predicted for pathogenicity using tools such as Mutatentaster (http:// www.mutationtaster.org /), Polypen-2(http:// genetics. bw. harvah. u/pph2/index. shtml), SIFT, and the like.
Clinical experiments
Example 1
Taking a family sample 1 with known genetic variation of the ferrochelatase gene, and carrying out PCR amplification by using the diagnostic kit according to the detection conditions and the method to obtain the exon from 1 to 10-11-1 of the ferrochelatase gene of the family sample 1 and the intron region of 50-150bp upstream or downstream of the exon. As can be seen from FIG. 3, the presence of pathogenic cleavage sites IVS3-48T/C in the intron region of the ferrochelatase gene of the amplified family sample 1 indicates that the sample 1 is a pathogenic patient of genetic variation of the ferrochelatase gene; and the serious insertion deletion mutation also exists on the coding region segment of the sample 1, and further proves that the ferrochelatase gene mutation exists in the sample 1, and the diagnosis result of the diagnosis kit of the invention is consistent with the actual condition of the sample.
Example 2
Taking an unknown sample 2, and carrying out PCR amplification by using the diagnostic kit of the invention according to the detection conditions and the method to obtain exons of the ferrochelatase gene of the unknown sample 2 from 1 to 10-11-1 and intron regions of the upstream or downstream thereof from 50-150 bp. As can be seen from FIG. 4, the pathogenic cleavage sites IVS3-48T/C were present in the amplified intron region of the ferrochelatase gene of the unknown sample 2. The same amplification and sequencing are respectively carried out on the DNA of the parents of the unknown sample 2, and the experimental result of figure 4 shows that the pathogenic shearing sites IVS3-48T/C exist in the intron region of the ferrous chelate gene of the unknown sample 2, the phenomenon is the same as that of the parent, the pathogenic gene of the unknown sample 2 comes from the parent, the coding region of the unknown sample 2 has serious insertion deletion mutation, and the deletion mutation can be found to be new mutation through database retrieval. Therefore, the gene detection result of the unknown sample 2 is consistent with the actual condition of clinical families.
Example 3
Taking an unknown sample 3, and carrying out PCR amplification by using the diagnostic kit according to the detection conditions and the method to obtain the exon from 1 to 10-11-1 of the ferrous chelate enzyme gene of the unknown sample 3 and the intron region of 50-150bp upstream or downstream of the exon; meanwhile, RNA is extracted from an unknown sample 3, a cDNA fragment of the ferrochelatase gene is obtained through reverse transcription, and the full-length cDNA of the ferrochelatase gene is obtained through amplification by the primer which is independently developed. As can be seen from FIG. 5, the pathogenicity cleavage sites IVS3-48T/C and the single site mutation of the coding region exist in the amplified intron region of the ferrochelatase gene of the unknown sample 3, and the single site mutation of the coding region is known to be a novel pathogenicity mutation by database search. However, comparison of cDNA sequencing with standard sequences revealed that the transcripts were intact, demonstrating that changes in the level of DNA did not affect transcript integrity. Therefore, the unknown sample 3 can be judged to be a mild patient, and the early detection of the invention can realize the control of the disease condition, which is consistent with the actual clinical condition.
Example 4
Taking an unknown sample 4, and carrying out PCR amplification by using the diagnostic kit according to the detection conditions and the method to obtain the exon from 1 to 10-11-1 of the ferrous chelate enzyme gene of the unknown sample 4 and the intron region of 50-150bp upstream or downstream of the exon; meanwhile, RNA is extracted from an unknown sample 4, a cDNA fragment of the ferrochelatase gene is obtained through reverse transcription, and the full-length cDNA of the ferrochelatase gene is obtained through amplification by the autonomously developed primer. As can be seen from FIG. 6, the cleavage sites IVS3-48 exist in the intron region of the ferrochelatase gene of the unknown sample 4 obtained by amplification, the signal is T, no nested peak exists, the T/T homozygous wild type mutation can be judged, and meanwhile, the known pathogenic mutation or other unknown mutation does not exist in the exon region. comparison of the cDNA sequencing with the standard sequence revealed that the transcript was intact. The possibility that the unknown sample 4 is a patient sample can therefore be excluded, consistent with clinical practice.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Sequence listing
<110> Renjin Hospital affiliated to Shanghai university of transportation medical school
<120> ferrochelatase gene genetic variation diagnostic kit
<141> 2021-01-12
<160> 20
<170> SIPOSequenceListing 1.0
<210> 2
<211> 20
<212> DNA
<213> Artificial
<400> 2
cctgacttcg ctagtttggc 20
<210> 2
<211> 20
<212> DNA
<213> Artificial
<400> 2
aaggaccatg ggctttcctc 20
<210> 3
<211> 19
<212> DNA
<213> Artificial
<400> 3
tgtcagtcat gttggtctc 19
<210> 4
<211> 19
<212> DNA
<213> Artificial
<400> 4
tgtgtctatt ggtgtctgc 19
<210> 5
<211> 19
<212> DNA
<213> Artificial
<400> 5
gcggagtgaa gactgatat 19
<210> 6
<211> 20
<212> DNA
<213> Artificial
<400> 6
taaccattac cagatacgca 20
<210> 7
<211> 21
<212> DNA
<213> Artificial
<400> 7
gcttaatctt gttaggctct c 21
<210> 8
<211> 18
<212> DNA
<213> Artificial
<400> 8
taacgcctgg agacctaa 18
<210> 9
<211> 18
<212> DNA
<213> Artificial
<400> 9
tatgccagcc aagaacag 18
<210> 10
<211> 19
<212> DNA
<213> Artificial
<400> 10
atggactgac ctgaactct 19
<210> 11
<211> 22
<212> DNA
<213> Artificial
<400> 11
catctactaa gtggtctctg ta 22
<210> 12
<211> 20
<212> DNA
<213> Artificial
<400> 12
aatgctcttc tggtcatagg 20
<210> 13
<211> 18
<212> DNA
<213> Artificial
<400> 13
ctgagaggct ggactgtt 18
<210> 14
<211> 20
<212> DNA
<213> Artificial
<400> 14
tagttagcac caagtctgag 20
<210> 15
<211> 19
<212> DNA
<213> Artificial
<400> 15
gtcagcacat agtcagagg 19
<210> 16
<211> 19
<212> DNA
<213> Artificial
<400> 16
gagaatgagg acaccgtac 19
<210> 17
<211> 21
<212> DNA
<213> Artificial
<400> 17
ggaaggacat aagaattgag g 21
<210> 18
<211> 18
<212> DNA
<213> Artificial
<400> 18
cggaggtatc tggaggtt 18
<210> 19
<211> 20
<212> DNA
<213> Artificial
<400> 19
cgggccgagg ctgcccaggc 20
<210> 20
<211> 19
<212> DNA
<213> Artificial
<400> 20
cggggtccac cggcggggg 19
Claims (2)
1. A diagnosis kit for genetic variation of ferrochelatase gene is characterized by comprising primers for amplifying the following genes: all exons of the ferrous chelate enzyme gene and intron regions of 50-150bp near the upstream and downstream sides of the exons, and the primer sequences are shown in the following table:
2. the diagnostic kit of claim 1, further comprising primers for amplifying the full-length cDNA of the ferrochelatase gene, the primer sequences being as set forth in the following table:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110038334.8A CN114763571A (en) | 2021-01-12 | 2021-01-12 | Ferrochelatase gene genetic variation diagnostic kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110038334.8A CN114763571A (en) | 2021-01-12 | 2021-01-12 | Ferrochelatase gene genetic variation diagnostic kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114763571A true CN114763571A (en) | 2022-07-19 |
Family
ID=82364400
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110038334.8A Pending CN114763571A (en) | 2021-01-12 | 2021-01-12 | Ferrochelatase gene genetic variation diagnostic kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114763571A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160122767A1 (en) * | 2013-06-13 | 2016-05-05 | Université Paris Diderot - Paris 7 | Methods and pharmaceutical compositions for the treatment of erythropoietic protoporphyria |
-
2021
- 2021-01-12 CN CN202110038334.8A patent/CN114763571A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160122767A1 (en) * | 2013-06-13 | 2016-05-05 | Université Paris Diderot - Paris 7 | Methods and pharmaceutical compositions for the treatment of erythropoietic protoporphyria |
Non-Patent Citations (6)
| Title |
|---|
| CATERINA AURIZI等: "Heterogeneity of mutations in the ferrochelatase gene in Italian patients with erythropoietic protoporphyria", MOLECULAR GENETICS AND METABOLISM * |
| S D WHATLEY等: "Autosomal recessive erythropoietic protoporphyria in the United Kingdom: prevalence and relationship to liver disease", J MED GENET * |
| TSUYOSHI TAHARA等: "The low expression allele (IVS3-48C) of the ferrochelatase gene leads to low enzyme activity associated with erythropoietic protoporphyria", INT J HEMATOL * |
| X WANG等: "A novel splicing mutation in the ferrochelatase gene responsible for erythropoietic protoporphyria", BIOCHIM BIOPHYS ACTA . * |
| XIAO-FEI KONG等: "Identification of a ferrochelatase mutation in a Chinese family with erythropoietic protoporphyria", J HEPATOL. * |
| 许亚丽;胡扬;温辉;任志军;: "中国北方汉族男子FECH基因IVS3-48T/C多态与有氧运动能力表型指标的关联性研究", 中国运动医学杂志 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104805206B (en) | The kit and its detection method of detection TERT gene promoter mutation | |
| KR20190116989A (en) | PCR primer set for HLA gene, and sequence analysis method using the same | |
| CN113637776A (en) | Primer group special for Diego blood group gene sequencing, kit and method | |
| CN105420233B (en) | HBB gene mutation and HLA typing detection kit | |
| CN113265471A (en) | Method for detecting sheep FASN gene single nucleotide polymorphism and application of method in meat quality early screening | |
| CN110241227B (en) | Method for detecting sheep SPATA6 gene single nucleotide polymorphism and application | |
| CN113265473B (en) | SNP molecular marker influencing birth weight of alpine merino sheep and application thereof | |
| Tatarskyy et al. | Allelic polymorphism of F2, F5 and MTHFR genes in population of Ukraine | |
| CN108753952A (en) | A kind of gene parting detecting reagent for 10 common mutations sites of mankind SLC25A13 genes | |
| CN116287321B (en) | SNP site of H blood group system antigen deletion related to immune hemolytic transfusion reaction, application and reagent | |
| CN110734974B (en) | SNP locus combination and detection primer for cancer chemotherapy drugs | |
| CN114763571A (en) | Ferrochelatase gene genetic variation diagnostic kit | |
| US7794982B2 (en) | Method for identifying gene with varying expression levels | |
| EP2053133A2 (en) | Improvements in induced heteroduplex generators | |
| CN110029162B (en) | SNP marker for detecting susceptibility of systemic lupus erythematosus in non-coding gene region and application thereof | |
| KR102063486B1 (en) | Association of RNF213 single nucleotide polymorphism with the risk of Moyamoya disease in a Korean population | |
| CN105899675B (en) | Major histocompatibility complex single nucleotide polymorphisms | |
| CN116656828A (en) | Application of gene ERBB2 locus g.3974028A > G mutation in preparation of breast cancer early-stage screening kit | |
| Watling et al. | Loss of heterozygosity analysis of chromosomes 9, 10 and 17 in gliomas in families | |
| CN112410441A (en) | Method for identifying anti-cysticercosis trait of bee colony by using SNP marker KZ 288479.1-95621 | |
| Oruč et al. | Assessment of relatedness between neurocan gene as bipolar disorder susceptibility locus and schizophrenia | |
| CN114381513B (en) | Biomarker related to X-linked lymphoproliferative disorder and application thereof | |
| CN115011702B (en) | Allele of RhD blood type gene RHD56C & gtG and detection thereof | |
| WO2002066639A1 (en) | Brain aneurysm-sensitive gene | |
| CN109266760B (en) | Method for detecting fertility of alpine merino based on INHA gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |