CN112501279A - Primer, kit and method for detecting PIEZO1 gene mutation - Google Patents

Primer, kit and method for detecting PIEZO1 gene mutation Download PDF

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CN112501279A
CN112501279A CN202011505289.4A CN202011505289A CN112501279A CN 112501279 A CN112501279 A CN 112501279A CN 202011505289 A CN202011505289 A CN 202011505289A CN 112501279 A CN112501279 A CN 112501279A
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刘赵玲
王淑一
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Changsha Aidikang Medical Laboratory Co Ltd
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Abstract

The invention relates to a primer and a method for detecting PIEZO1 gene mutation, which comprise a primer for amplifying a PIEZO1 gene exon sequence; sanger sequencing technology and sequencing primers were used. The invention can quickly detect the mutation of the PIEZO1 gene exon. The invention has accurate detection result, can assist in diagnosing hereditary erythrocytosis and has important reference significance for disease diagnosis.

Description

Primer, kit and method for detecting PIEZO1 gene mutation
Technical Field
The invention belongs to the field of molecular detection, and particularly relates to a primer, a kit and a detection method for detecting PIEZO1 gene mutation.
Background
Hereditary stomatocytosis (HSt) is a group of rare hereditary hemolytic anemias characterized by an abnormal ion-permeable function of the erythrocyte membrane and a morphological change of mature erythrocytes, in which different proportions of lipstick cells appear in the blood, which are red blood cells characterized by a wide transverse tear or lip-shape, and which can be seen in various acquired genetic diseases. In hereditary polycythemia lipstick, the proportion of the lipstick cells is more than 5% (normally less than 4%), and can reach 10-50%.
Hereditary polycythemia stomatitis includes dehydrated HSt, also known as hereditary neutrophilia (Hx), and hydrated HSt, Hx being the most common subtype of HSt. Hx diagnosis is mainly based on clinical features and peripheral red blood cell morphology, and diagnosis of the disease is delayed due to low morbidity and clinical phenotypic heterogeneity. In 2018 Kaufman et al reported that in recent years the incidence of Hx in the United states was 1/8000, about 6 times higher than the classical estimated incidence (1/5000), with a median age at diagnosis of about 51 years. Hst has a strong heterogeneity of clinical phenotype and limited diagnostic tools, and is prone to missed or misdiagnosed, leading to inappropriate splenectomy therapy, which increases the risk of serious thrombosis.
The PIEZO1 gene is one of the virulence genes for Hx. The PIEZO1 gene is located at 16q23q24 and comprises 51 exons, and the PIEZO1 protein coded by the gene mainly conducts monovalent ions (such as K)+、Na+、Li+、Cs+) And divalent ions (e.g. Ba)2+、ca2+、Mg2+、Mn2+). The PIEZO1 protein is one of the largest ion channels on the erythrocyte membrane to date, and also regulates the release of Adenosine Triphosphate (ATP) from erythrocytes. The PIEZO1 gene mutation can cause the delayed inactivation of PIEZO1 protein, the open state of an ion channel is prolonged, excessive potassium ion leakage and water loss in red blood cells are triggered, and the morphology of the red blood cells is changed. It is known that the PIEZO1 gene mutations related to Hx are found in total 17, and 15 (88.2%) are missense mutations. Hx patients from 29 independent families (133 patients total) were associated with a PIEZO1 gene mutation. Of the 133 patients, c.7367G was used>A、c.7463G>A. c.7479-7484dup are the most common of the three pathogenic gene mutations. The amino acid changes corresponding to the mutation sites reported so far are from amino acid 598 to amino acid 1358 and from amino acid 2000 to the cytoplasmic C-terminus of the PIEZO1 protein.
PIEZO1 mutation analysis has important significance for genetic consultation and prenatal diagnosis of hereditary hypererythrocytic disease, and the genetic mode of HSt is autosomal dominant inheritance. Therefore, if the disease-causing gene is clear, parents decide that the pregnancy should be subjected to relevant genetic counseling in time, prenatal molecular diagnosis is performed in the early stage of the pregnancy, and once an abnormal result occurs, whether treatment can be performed or not, how the treatment can be performed after the treatment and the like need to be clear, and feasible diagnosis and treatment measures are taken. Minimizing the birth of abnormal fetus and improving the population quality of newborn.
Disclosure of Invention
The invention aims to provide a kit for detecting PIEZO1 gene mutation, which can be used for rapidly detecting the PIEZO1 gene mutation in a patient by adopting a PCR (polymerase chain reaction) technology.
The primer for detecting PIEZO1 gene mutation is characterized by comprising a primer for amplifying PIEZO1 gene,
the base sequence is as follows:
Figure BDA0002844765890000021
Figure BDA0002844765890000031
further, the primers also comprise a sequencing primer for detecting the PIEZO1 gene, and the base sequence of the sequencing primer is as follows:
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG
further, the primers amplify corresponding exons as:
Figure BDA0002844765890000032
Figure BDA0002844765890000041
the invention also provides a method for detecting the PIEZO1 gene mutation condition, which comprises the following steps:
(1) extracting tissue DNA in blood;
(2) amplifying the DNA extracted in the step 1 by using PCR; wherein the PCR amplification primers are:
Figure BDA0002844765890000042
Figure BDA0002844765890000051
Figure BDA0002844765890000061
(3) sequencing the amplification product in the step 2; the sequencing primer base sequence is:
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG；
(4) and judging the sequencing result to determine whether the PIEZO1 gene is mutated.
The invention finally provides a kit for detecting PIEZO1 gene mutation sites, which comprises:
(i) blood DNA extraction reagent;
(ii) detecting a system PCR amplification reaction solution; comprises a primer for amplifying the PIEZO1 gene, and the base sequence of the primer is as follows:
Figure BDA0002844765890000062
Figure BDA0002844765890000071
Figure BDA0002844765890000081
(iii) sequencing system reagents; comprises a sequencing primer for detecting PIEZO1 gene, and the base sequence of the sequencing primer is as follows:
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG。
has the advantages that: the invention designs primers for amplifying exon sequences 1-21, 23-33 and 35-51 of PIEZO 1. By adding a joint, PCR products of 31 pairs of primers can be sequenced by using one sequencing primer; a stable amplification system is constructed by adopting a PCR technology. The amplification efficiency can be optimized by adjusting the reaction conditions such as primer concentration, annealing temperature and the like; the mutation types of the PIEZO1 gene are various and are distributed throughout the whole gene, so that the primer disclosed by the invention can amplify all the sequences of the PIEZO1 exons, and can ensure that the condition of missed detection can not occur no matter where the exons are mutated; compared with a fluorescent quantitative PCR method, the invention reduces the cost and the difficulty of detection because the fluorescent quantitative PCR method designs a plurality of probes aiming at different mutation types, thereby having high cost and great detection difficulty.
Drawings
FIG. 1 is a map of the PIEZO1 gene mapping on human chromosome 16.
FIGS. 2 and 3 are electrophoretograms of primers of exons 1 to 21, 23 to 33 and 35 to 52, M is Marker DL 2000, and as shown, the primers are amplified efficiently and bright, wherein the upper number of each band in FIGS. 2 and 3 represents the amplified exon, for example, "2" represents the amplification of exon 2 of PIEZO1 gene, and "8 to 10" represents the amplification of exons 8, 9 and 10 of PIEZO1 gene.
FIG. 4 shows a PIEZO 13 exon mutant sequencing screenshot demonstrating the complete mutation of exon 3 c.248 of the sample with T > C.
FIG. 5 shows a PIEZO 17 exon mutation type sequencing screenshot illustrating the complete mutation T > C in exon 7 c.749 of the sample.
FIG. 6 shows a PIEZO exon 110 mutant sequencing screenshot illustrating the complete G > C mutation in exon 10 c.1180 of the sample.
FIG. 7 shows a PIEZO exon 111 mutant sequencing screenshot demonstrating the complete mutation A > G in exon 11 c.1219 of the sample.
FIG. 8 shows a sequence screenshot of the PIEZO 120 exon mutation, illustrating the partial deletion mutation het _ delTCT of the 20 exons c.2972-2974 of the sample.
FIG. 9 shows a PIEZO exon 128 mutant sequencing screenshot illustrating the complete mutation T > C in exon 28 c.4014 of the sample.
FIG. 10 shows a PIEZO 137 exon mutation sequencing screenshot illustrating the partial G > GT mutation in exon 37 c.5214 of the sample.
FIG. 11 shows a sequence screenshot of a PIEZO 139 exon mutant demonstrating the complete mutation of exon 39 c.5538C > G to C > G.
FIG. 12 shows a sequence screenshot of the mutant of exon 145 PIEZO showing the complete mutation A > G in exon 45 c.6570 of the sample.
FIG. 13 shows a sequence screenshot of the PIEZO exon 145 mutant demonstrating the complete mutation G > C in exon 45 c.6642 of the sample.
FIG. 14 shows a PIEZO 149 exon mutant sequencing screenshot illustrating the complete mutation T > C in exon 49 c.7059 of the sample.
Detailed Description
The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings. It should be noted that the conventional conditions and methods not described in the examples are generally employed by those skilled in the art according to the routine procedures: such as OsOb and Kingston, fourth edition, or following the manufacturer's suggested procedures and conditions.
Example 1
A primer for detecting PIEZO1 gene mutation sites, which is designed to be an amplification primer designed aiming at PIEZO1 exons and comprises the following components:
the primer for amplifying the whole exon sequence of the PIEZO1 gene has the base sequence as follows:
Figure BDA0002844765890000091
Figure BDA0002844765890000101
Figure BDA0002844765890000111
a kit for detecting a mutation in the pinezo 1 gene, comprising:
(i) blood DNA extraction reagent;
(ii) detecting a system PCR reaction solution;
(iii) sequencing system reagents;
the tissue DNA extraction reagent can be purchased from commercial reagents such as Tiangen DNA extraction kit and the like.
The PCR amplification reaction solution of the detection system comprises: 2 times PCR Buffer; 2mM dNTPs; KOD FX DNA Polymerase (1U/. mu.l); the concentrations of the primers at the upstream and downstream of the exon sequences of PIEZO1 gene were 10. mu.M.
The sequencing system reagent comprises: sequencing purification solution (ExoI:0.6U, CIP:1.2U), EDTA (125mM), absolute ethanol, 75% ethanol, HIDI (highly deionized formamide), sequencing primers: the upstream and downstream primers for detecting the exon sequences of PIEZO1 gene were M13F (3.2. mu.M), M13R (3.2. mu.M), and Bigdye Terminator V3.1 (purchased from Applied Biosystems, USA), respectively.
Example 2
The operation flow of the blood/cell/tissue genome DNA extraction kit (Tiangen organism):
(1) extracting tissue DNA from blood: 1) mu.l of blood was taken and added to 900. mu.l of erythrocyte lysate, mixed by inversion, left at room temperature for 5 minutes, and mixed by inversion several times in the meantime. Centrifuge at 12,000 rpm for 1min, aspirate the supernatant, leave the leukocyte pellet, add 200. mu.l of buffer GA, and shake until thoroughly mixed. 2) Add 20. mu.l proteinase K solution and mix well. 3) Add 200. mu.l buffer GB, mix well by inversion, stand at 70 ℃ for 10 minutes, clear the solution, centrifuge briefly to remove beads on the inner wall of the tube cap. 4) Add 200. mu.l of absolute ethanol, mix well with shaking for 15 seconds, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cover. 5) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is put into a collecting pipe), centrifuging at 12,000 rpm for 30 s, pouring off waste liquid, and putting the adsorption column CB3 back into the collecting pipe. 6) Add 500. mu.l buffer GD (check whether absolute ethanol has been added before use) to adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, dump the waste and place adsorption column CB3 in the collection tube. 7) To the adsorption column CB3, 700. mu.l of a rinsing solution PW (previously used, whether or not absolute ethyl alcohol has been added) was added, and the mixture was centrifuged at 12,000 rpm for 30 seconds, and the waste liquid was discarded, and the adsorption column CB3 was put into a collection tube. 8) To the adsorption column CB3, 500. mu.l of a rinsing solution PW was added, and the mixture was centrifuged at 12,000 rpm for 30 seconds, and then the waste liquid was discarded. 9) The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000 rpm for 2 minutes, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material. 10) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 100 mu l of elution buffer TE into the middle part of the adsorption membrane, standing for 2-5 minutes at room temperature, centrifuging for 2 minutes at 12,000 rpm, and collecting the solution into the centrifuge tube.
(2) Reagent preparation: preparing X mul of PCR reaction liquid of a detection system according to the parts of detected people, and subpackaging 19 mul of PCR reaction liquid of each part of detected people:
19 μ l reaction X (n samples +1 blank control)
n is the number of detection samples.
(3) Sample adding: adding 1 mul DNA into the PCR reaction solution of the detection system; blank control was supplemented with 1. mu.l of physiological saline or nothing.
(4) Amplification: the detection is carried out on a conventional PCR instrument, and available instruments include ABI veriti (Applied Biosystems, USA) and the like. The reaction conditions were as follows:
Figure BDA0002844765890000121
the preparation method of the PCR amplification system reagent comprises the following steps:
Figure BDA0002844765890000122
wherein the primer F/R is selected from PIEZO1-1F/R, PIEZO1-2F/R, PIEZO1-3-4F/R, PIEZO1-5F/R, PIEZO1-6F/R, PIEZO1-7F/R, PIEZO1-8-10F/R, PIZEO1-11-12F/R, PIEZO1-13-15F/R, PIEZO1-16F/R, PIEZO1-17F/R, PIEZO1-18-19F/R, PIEZO1-20-21F/R, PIEZO1-23F/R, PIEZO1-24-25F/R, PIEZO1-26-27F/R, PIEZO1-28-29F/R, PIEZO1-30F/R, PIEZO1-31F/R, PIEZO1-32-33F/R, PIEZO1-35-36F/R, PIEZO1-37F/R, PIEZO1-38F/R, PIEZO1-39F/R, PIEZO1-40-42F/R, PIEZO 1-42F/R, PIEZO1-44F/R, PIEZO1-45-47F/R, PIEZO1-47-48F/R, PIEZO1-49-50F/R, PIEZO1-51F/R, wherein the specific base sequence is as follows:
Figure BDA0002844765890000123
Figure BDA0002844765890000131
(5) electrophoresis: electrophoresis on 1.5% agarose gel at 110V for 25min, and observation on a gel imaging system.
As shown in FIG. 2, the electropherogram of the amplification product obtained after the blood sample was amplified using the primer sequence F/R. The length of the amplified fragment is 649, 264, 600, 301, 349, 660, 747, 683, 495, 604, 700, 599, 244, 660, 546, 468, 228, 240, 476, 695, 417, 362, 401, 830, 619, 319, 708, 510, 472 and 698bp, and analysis of an electrophoretogram shows that the primers are all amplified effectively and have single bands.
(6) Sanger sequencing:
take 9. mu.l of PCR product and 2. mu.l of purification system. Purification was performed according to the following procedure:
Figure BDA0002844765890000132
Figure BDA0002844765890000141
mu.l of the purified product was mixed with the upper and lower sequencing primers, respectively, according to the following system:
Figure BDA0002844765890000142
reaction procedure:
Figure BDA0002844765890000143
and (3) a precipitation link:
adding 2 mu l of 125mmol EDTA into the product after the sequencing reaction, and standing for 5 min; adding 15 mul of absolute ethyl alcohol, and mixing evenly by vortex; centrifuging at 3700rpm for 30 min; inverting, centrifuging for 15sec, adding 50 μ l 70% ethanol, and mixing by vortex; centrifuging at 3700rpm for 15 min; inverting and centrifuging for 15sec, and placing on a metal bath at 95 ℃; denaturation test was performed after adding 10. mu.l Hi-Di.
After the denaturation procedure was completed, sequencing was performed using a sequencer (ABI 3730).
(7) And (5) judging a result: the sequencing results were compared to the TREX1 wild-type reference sequence (Genbank: NG-042229.1), respectively, and the results were reported as a function of the actual mutation.
Example 3
1 example of clinical samples were taken and genomic extraction, reagent formulation, amplification and sequencing were performed according to the reagents and methods of examples 1 and 2. Mu.l of sample was added to each PCR reaction solution. The electrophoresis results are shown in figures 2, 3 and 4, and show that the primers PIEZO1-1F/R, PIEZO1-2F/R, PIEZO1-3-4F/R, PIEZO1-5F/R, PIEZO1-6F/R, PIEZO1-7F/R, PIEZO1-8-10F/R, PIZEO1-11-12F/R, PIEZO1-13-15F/R, PIEZO1-16F/R, PIEZO1-17F/R, PIEZO1-18-19F/R, PIEZO1-20-21F/R, PIEZO1-23F/R, PIEZO1-24-25F/R, PIEZO1-26-27F/R, PIEZO1-28-29F/R, PIEZO1-30F/R, PIEZO1-31F/R, PIEZO1-32-33F/R, PIEZO1-35-36F/R, PIEZO1-37F/R, PIEZO1-38F/R, PIEZO1-39F/R, PIEZO1-40-42F/R, PIEZO1-42-43F/R, PIEZO1-44F/R, PIEZO1-45-47F/R, PIEZO1-47-48F/R, PIEZO1-49-50F/R, PIEZO1-51F/R can effectively amplify blood samples, and the bands are bright.
The sequencing results for this sample showed the presence of 11 mutation sites, each of which is shown in the following table:
Figure BDA0002844765890000151
FIG. 4 shows a PIEZO 13 exon mutant sequencing screenshot demonstrating the complete mutation of exon 3 c.248 of the sample with T > C.
FIG. 5 shows a PIEZO 17 exon mutation type sequencing screenshot illustrating the complete mutation T > C in exon 7 c.749 of the sample.
FIG. 6 shows a PIEZO exon 110 mutant sequencing screenshot illustrating the complete G > C mutation in exon 10 c.1180 of the sample.
FIG. 7 shows a PIEZO exon 111 mutant sequencing screenshot demonstrating the complete mutation A > G in exon 11 c.1219 of the sample.
FIG. 8 shows a sequence screenshot of the PIEZO 120 exon mutation, illustrating the partial deletion mutation het _ delTCT of the 20 exons c.2972-2974 of the sample.
FIG. 9 shows a PIEZO exon 128 mutant sequencing screenshot illustrating the complete mutation T > C in exon 28 c.4014 of the sample.
FIG. 10 shows a PIEZO 137 exon mutation sequencing screenshot illustrating the partial G > GT mutation in exon 37 c.5214 of the sample.
FIG. 11 shows a sequence screenshot of a PIEZO 139 exon mutant demonstrating the complete mutation of exon 39 c.5538C > G to C > G.
FIG. 12 shows a sequence screenshot of the mutant of exon 145 PIEZO showing the complete mutation A > G in exon 45 c.6570 of the sample.
FIG. 13 shows a sequence screenshot of the PIEZO exon 145 mutant demonstrating the complete mutation G > C in exon 45 c.6642 of the sample.
FIG. 14 shows a PIEZO 149 exon mutant sequencing screenshot illustrating the complete mutation T > C in exon 49 c.7059 of the sample.
As can be seen from the detection results, the primer disclosed by the invention already includes the target exon sequence, can amplify the PIEZO1 gene exon sequence, and has a completely accurate sequencing result. The primer provided by the invention can be used for accurately amplifying a target exon sequence of the PIEZO1 gene, whether the target exon sequence is a wild type or a mutant type.
Sequence listing
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<400> 24
aacagctatg accatgtcgg gctgcgaata cagagtt 37
<210> 25
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tgtaaaacga cggccagttc tccctgtttc tcaaacacct g 41
<210> 26
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aacagctatg accatgggca atgtccttgc ctcacc 36
<210> 27
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tgtaaaacga cggccagtgg accgggtgtc tcagg 35
<210> 28
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
aacagctatg accatgactt gtgagcagat ttgggg 36
<210> 29
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tgtaaaacga cggccagtgg tgggaggtgg gatttatg 38
<210> 30
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aacagctatg accatggcat ctgctccacg aagac 35
<210> 31
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tgtaaaacga cggccagttc accgtcatca tctccaag 38
<210> 32
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
aacagctatg accatgggcc tgagacagtg aggaac 36
<210> 33
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
tgtaaaacga cggccagtgt tgctggggtc gagtgc 36
<210> 34
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
aacagctatg accatggcct gccagacaga gggt 34
<210> 35
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tgtaaaacga cggccagtag acgggagcac tcacagc 37
<210> 36
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
aacagctatg accatggaag atagcaggcc aggcaga 37
<210> 37
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tgtaaaacga cggccagtgg gtctggctgg gagtc 35
<210> 38
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
aacagctatg accatgctga caccctctat gctgcc 36
<210> 39
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tgtaaaacga cggccagtcc cacccaactc ctgaatgg 38
<210> 40
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
aacagctatg accatggcca agagagacct cccac 35
<210> 41
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
tgtaaaacga cggccagtct gctctgcctg actacgc 37
<210> 42
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
aacagctatg accatgcatg acggcccgat ctgtt 35
<210> 43
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
tgtaaaacga cggccagtgt catgccttcg ggcag 35
<210> 44
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
aacagctatg accatgcagg tacttgacga ccaccg 36
<210> 45
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
tgtaaaacga cggccagtga cggccatcgt cttcac 36
<210> 46
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
aacagctatg accatgccct ggtggagagc acag 34
<210> 47
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
tgtaaaacga cggccagtca cctgtgctct ccaccag 37
<210> 48
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
aacagctatg accatgtctg agaaagaccc tccctg 36
<210> 49
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
tgtaaaacga cggccagtgt caggagccat gagccag 37
<210> 50
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
aacagctatg accatgggac cccatcagaa acagcc 36
<210> 51
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tgtaaaacga cggccagtca gagctcagca gccacc 36
<210> 52
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
aacagctatg accatgccag ggttcccgtc agg 33
<210> 53
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tgtaaaacga cggccagtaa cctcttcctc ttccaggg 38
<210> 54
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
aacagctatg accatggcag tgcctctgtc ttaggg 36
<210> 55
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
tgtaaaacga cggccagtgc agagctgagc gtatgacc 38
<210> 56
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
aacagctatg accatgccaa gtccaggacg aacctc 36
<210> 57
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tgtaaaacga cggccagtct ctgccctgtg aaagctg 37
<210> 58
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
aacagctatg accatgggtg ctggcaggtc agg 33
<210> 59
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tgtaaaacga cggccagtag ggctgtgcca ggttg 35
<210> 60
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
aacagctatg accatggggg aagctggtga gtcc 34
<210> 61
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
tgtaaaacga cggccagtgg ctacgggtga gtgagtg 37
<210> 62
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
aacagctatg accatgcaga gctgagcgtg aggac 35

Claims (5)

1. The primer for detecting PIEZO1 gene mutation is characterized by comprising a primer for amplifying PIEZO1 gene, wherein the base sequence of the primer is as follows:
PIEZO1-1F：TGTAAAACGACGGCCAGTGGTCTCCAGGCCCCAAC
PIEZO1-1R：AACAGCTATGACCATGTCATGCTTCAGGAATGCG
PIEZO1-2F：TGTAAAACGACGGCCAGTCCATCCTCTGTTGGCGACCTA
PIEZO1-2R：AACAGCTATGACCATGATTTCGGGACCCCTCATCTT
PIEZO1-3-4F：TGTAAAACGACGGCCAGTATCTCAGGCCTCTGGGCTC
PIEZO1-3-4R：AACAGCTATGACCATGGGTGGGAGTGTGGATGAGTC
PIEZO1-5F：TGTAAAACGACGGCCAGTGATTTCAGGCCCCAGGAAGT
PIEZO1-5R：AACAGCTATGACCATGCCTCAAAATGGCTGAGACCAC
PIEZO1-6F：TGTAAAACGACGGCCAGTGGAGAGGTGGCGTCAGTG
PIEZO1-6R：AACAGCTATGACCATGTGTCTCCACAGTCATCAGGG
PIEZO1-7F：TGTAAAACGACGGCCAGTTGATGACTGTGGAGACAGCG
PIEZO1-7R：AACAGCTATGACCATGGGCTAGACGAGCCAGGTG
PIEZO1-8-10F：TGTAAAACGACGGCCAGTGCCCACCTGGCTCGTCTA
PIEZO1-8-10R：AACAGCTATGACCATGCCGCACCCAGCCATACCTT
PIZEO1-11-12F：TGTAAAACGACGGCCAGTTTTTAGGGCATAGTCAGGGTGGG
PIZEO1-11-12R：AACAGCTATGACCATGCGGGCAAGTGGACATTGAACAG
PIEZO1-13-15F：TGTAAAACGACGGCCAGTCGGCTGACTGTGACACCC
PIEZO1-13-15R：AACAGCTATGACCATGGGGAAGTGCACGGGGTTT
PIEZO1-16F：TGTAAAACGACGGCCAGTGCCGTCTACACCTTCCAGTTC
PIEZO1-16R：AACAGCTATGACCATGTTGGTATGAGCAAATGCCCCTT
PIEZO1-17F：TGTAAAACGACGGCCAGTACAGGGCTTCTTCCAGCC
PIEZO1-17R：AACAGCTATGACCATGAGCTGGGCAGGCCAGAG
PIEZO1-18-19F：TGTAAAACGACGGCCAGTGTGCCTGAAGGTGGGTTGG
PIEZO1-18-19R：AACAGCTATGACCATGTCGGGCTGCGAATACAGAGTT
PIEZO1-20-21F：TGTAAAACGACGGCCAGTTCTCCCTGTTTCTCAAACACCTG
PIEZO1-20-21R：AACAGCTATGACCATGGGCAATGTCCTTGCCTCACC
PIEZO1-23F：TGTAAAACGACGGCCAGTGGACCGGGTGTCTCAGG
PIEZO1-23R：AACAGCTATGACCATGACTTGTGAGCAGATTTGGGG
PIEZO1-24-25F：TGTAAAACGACGGCCAGTGGTGGGAGGTGGGATTTATG
PIEZO1-24-25R：AACAGCTATGACCATGGCATCTGCTCCACGAAGAC
PIEZO1-26-27F：TGTAAAACGACGGCCAGTTCACCGTCATCATCTCCAAG
PIEZO1-26-27R：AACAGCTATGACCATGGGCCTGAGACAGTGAGGAAC
PIEZO1-28-29F：TGTAAAACGACGGCCAGTGTTGCTGGGGTCGAGTGC
PIEZO1-28-29R：AACAGCTATGACCATGGCCTGCCAGACAGAGGGT
PIEZO1-30F：TGTAAAACGACGGCCAGTAGACGGGAGCACTCACAGC
PIEZO1-30R：AACAGCTATGACCATGGAAGATAGCAGGCCAGGCAGA
PIEZO1-31F：TGTAAAACGACGGCCAGTGGGTCTGGCTGGGAGTC
PIEZO1-31R：AACAGCTATGACCATGCTGACACCCTCTATGCTGCC
PIEZO1-32-33F：TGTAAAACGACGGCCAGTCCCACCCAACTCCTGAATGG
PIEZO1-32-33R：AACAGCTATGACCATGGCCAAGAGAGACCTCCCAC
PIEZO1-35-36F：TGTAAAACGACGGCCAGTCTGCTCTGCCTGACTACGC
PIEZO1-35-36R：AACAGCTATGACCATGCATGACGGCCCGATCTGTT
PIEZO1-37F：TGTAAAACGACGGCCAGTGTCATGCCTTCGGGCAG
PIEZO1-37R：AACAGCTATGACCATGCAGGTACTTGACGACCACCG
PIEZO1-38F：TGTAAAACGACGGCCAGTGACGGCCATCGTCTTCAC
PIEZO1-38R：AACAGCTATGACCATGCCCTGGTGGAGAGCACAG
PIEZO1-39F：TGTAAAACGACGGCCAGTCACCTGTGCTCTCCACCAG
PIEZO1-39R：AACAGCTATGACCATGTCTGAGAAAGACCCTCCCTG
PIEZO1-40-42F：TGTAAAACGACGGCCAGTGTCAGGAGCCATGAGCCAG
PIEZO1-40-42R：AACAGCTATGACCATGGGACCCCATCAGAAACAGCC
PIEZO1-42-43F：TGTAAAACGACGGCCAGTCAGAGCTCAGCAGCCACC
PIEZO1-42-43R：AACAGCTATGACCATGCCAGGGTTCCCGTCAGG
PIEZO1-44F：TGTAAAACGACGGCCAGTAACCTCTTCCTCTTCCAGGG
PIEZO1-44R：AACAGCTATGACCATGGCAGTGCCTCTGTCTTAGGG
PIEZO1-45-47F：TGTAAAACGACGGCCAGTGCAGAGCTGAGCGTATGACC
PIEZO1-45-47R：AACAGCTATGACCATGCCAAGTCCAGGACGAACCTC
PIEZO1-47-48F：TGTAAAACGACGGCCAGTCTCTGCCCTGTGAAAGCTG
PIEZO1-47-48R：AACAGCTATGACCATGGGTGCTGGCAGGTCAGG
PIEZO1-49-50F：TGTAAAACGACGGCCAGTAGGGCTGTGCCAGGTTG
PIEZO1-49-50R：AACAGCTATGACCATGGGGGAAGCTGGTGAGTCC
PIEZO1-51F：TGTAAAACGACGGCCAGTGGCTACGGGTGAGTGAGTG
PIEZO1-51R：AACAGCTATGACCATGCAGAGCTGAGCGTGAGGAC。
2. the primer according to claim 1, further comprising a sequencing primer for detecting the PIEZO1 gene, the base sequence of which is:
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG。
3. a method for detecting the mutation condition of PIEZO1 gene, comprising the following steps:
(1) extracting tissue DNA in blood;
(2) amplifying the DNA extracted in the step (1) by using a PCR amplification primer to obtain an amplification product; the base sequence of the PCR amplification primer is as follows:
PIEZO1-1F：TGTAAAACGACGGCCAGTGGTCTCCAGGCCCCAAC
PIEZO1-1R：AACAGCTATGACCATGTCATGCTTCAGGAATGCG
PIEZO1-2F：TGTAAAACGACGGCCAGTCCATCCTCTGTTGGCGACCTA
PIEZO1-2R：AACAGCTATGACCATGATTTCGGGACCCCTCATCTT
PIEZO1-3-4F：TGTAAAACGACGGCCAGTATCTCAGGCCTCTGGGCTC
PIEZO1-3-4R：AACAGCTATGACCATGGGTGGGAGTGTGGATGAGTC
PIEZO1-5F：TGTAAAACGACGGCCAGTGATTTCAGGCCCCAGGAAGT
PIEZO1-5R：AACAGCTATGACCATGCCTCAAAATGGCTGAGACCAC
PIEZO1-6F：TGTAAAACGACGGCCAGTGGAGAGGTGGCGTCAGTG
PIEZO1-6R：AACAGCTATGACCATGTGTCTCCACAGTCATCAGGG
PIEZO1-7F：TGTAAAACGACGGCCAGTTGATGACTGTGGAGACAGCG
PIEZO1-7R：AACAGCTATGACCATGGGCTAGACGAGCCAGGTG
PIEZO1-8-10F：TGTAAAACGACGGCCAGTGCCCACCTGGCTCGTCTA
PIEZO1-8-10R：AACAGCTATGACCATGCCGCACCCAGCCATACCTT
PIZEO1-11-12F：TGTAAAACGACGGCCAGTTTTTAGGGCATAGTCAGGGTGGG
PIZEO1-11-12R：AACAGCTATGACCATGCGGGCAAGTGGACATTGAACAG
PIEZO1-13-15F：TGTAAAACGACGGCCAGTCGGCTGACTGTGACACCC
PIEZO1-13-15R：AACAGCTATGACCATGGGGAAGTGCACGGGGTTT
PIEZO1-16F：TGTAAAACGACGGCCAGTGCCGTCTACACCTTCCAGTTC
PIEZO1-16R：AACAGCTATGACCATGTTGGTATGAGCAAATGCCCCTT
PIEZO1-17F：TGTAAAACGACGGCCAGTACAGGGCTTCTTCCAGCC
PIEZO1-17R：AACAGCTATGACCATGAGCTGGGCAGGCCAGAG
PIEZO1-18-19F：TGTAAAACGACGGCCAGTGTGCCTGAAGGTGGGTTGG
PIEZO1-18-19R：AACAGCTATGACCATGTCGGGCTGCGAATACAGAGTT
PIEZO1-20-21F：TGTAAAACGACGGCCAGTTCTCCCTGTTTCTCAAACACCTG
PIEZO1-20-21R：AACAGCTATGACCATGGGCAATGTCCTTGCCTCACC
PIEZO1-23F：TGTAAAACGACGGCCAGTGGACCGGGTGTCTCAGG
PIEZO1-23R：AACAGCTATGACCATGACTTGTGAGCAGATTTGGGG
PIEZO1-24-25F：TGTAAAACGACGGCCAGTGGTGGGAGGTGGGATTTATG
PIEZO1-24-25R：AACAGCTATGACCATGGCATCTGCTCCACGAAGAC
PIEZO1-26-27F：TGTAAAACGACGGCCAGTTCACCGTCATCATCTCCAAG
PIEZO1-26-27R：AACAGCTATGACCATGGGCCTGAGACAGTGAGGAAC
PIEZO1-28-29F：TGTAAAACGACGGCCAGTGTTGCTGGGGTCGAGTGC
PIEZO1-28-29R：AACAGCTATGACCATGGCCTGCCAGACAGAGGGT
PIEZO1-30F：TGTAAAACGACGGCCAGTAGACGGGAGCACTCACAGC
PIEZO1-30R：AACAGCTATGACCATGGAAGATAGCAGGCCAGGCAGA
PIEZO1-31F：TGTAAAACGACGGCCAGTGGGTCTGGCTGGGAGTC
PIEZO1-31R：AACAGCTATGACCATGCTGACACCCTCTATGCTGCC
PIEZO1-32-33F：TGTAAAACGACGGCCAGTCCCACCCAACTCCTGAATGG
PIEZO1-32-33R：AACAGCTATGACCATGGCCAAGAGAGACCTCCCAC
PIEZO1-35-36F：TGTAAAACGACGGCCAGTCTGCTCTGCCTGACTACGC
PIEZO1-35-36R：AACAGCTATGACCATGCATGACGGCCCGATCTGTT
PIEZO1-37F：TGTAAAACGACGGCCAGTGTCATGCCTTCGGGCAG
PIEZO1-37R：AACAGCTATGACCATGCAGGTACTTGACGACCACCG
PIEZO1-38F：TGTAAAACGACGGCCAGTGACGGCCATCGTCTTCAC
PIEZO1-38R：AACAGCTATGACCATGCCCTGGTGGAGAGCACAG
PIEZO1-39F：TGTAAAACGACGGCCAGTCACCTGTGCTCTCCACCAG
PIEZO1-39R：AACAGCTATGACCATGTCTGAGAAAGACCCTCCCTG
PIEZO1-40-42F：TGTAAAACGACGGCCAGTGTCAGGAGCCATGAGCCAG
PIEZO1-40-42R：AACAGCTATGACCATGGGACCCCATCAGAAACAGCC
PIEZO1-42-43F：TGTAAAACGACGGCCAGTCAGAGCTCAGCAGCCACC
PIEZO1-42-43R：AACAGCTATGACCATGCCAGGGTTCCCGTCAGG
PIEZO1-44F：TGTAAAACGACGGCCAGTAACCTCTTCCTCTTCCAGGG
PIEZO1-44R：AACAGCTATGACCATGGCAGTGCCTCTGTCTTAGGG
PIEZO1-45-47F：TGTAAAACGACGGCCAGTGCAGAGCTGAGCGTATGACC
PIEZO1-45-47R：AACAGCTATGACCATGCCAAGTCCAGGACGAACCTC
PIEZO1-47-48F：TGTAAAACGACGGCCAGTCTCTGCCCTGTGAAAGCTG
PIEZO1-47-48R：AACAGCTATGACCATGGGTGCTGGCAGGTCAGG
PIEZO1-49-50F：TGTAAAACGACGGCCAGTAGGGCTGTGCCAGGTTG
PIEZO1-49-50R：AACAGCTATGACCATGGGGGAAGCTGGTGAGTCC
PIEZO1-51F：TGTAAAACGACGGCCAGTGGCTACGGGTGAGTGAGTG
PIEZO1-51R：AACAGCTATGACCATGCAGAGCTGAGCGTGAGGAC；
(3) sequencing the amplification product in the step (2) by using a pair of sequencing primers, wherein the base sequences of the pair of sequencing primers are as follows:
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG；
(4) and judging the sequencing result to determine whether the PIEZO1 gene is mutated.
4. A kit for detecting PIEZO1 gene mutation sites is characterized by comprising:
(i) blood DNA extraction reagent;
(ii) detecting a system PCR amplification reaction solution; comprises a primer for amplifying the PIEZO1 gene, and the base sequence of the primer is as follows:
PIEZO1-1F：TGTAAAACGACGGCCAGTGGTCTCCAGGCCCCAAC
PIEZO1-1R：AACAGCTATGACCATGTCATGCTTCAGGAATGCG
PIEZO1-2F：TGTAAAACGACGGCCAGTCCATCCTCTGTTGGCGACCTA
PIEZO1-2R：AACAGCTATGACCATGATTTCGGGACCCCTCATCTT
PIEZO1-3-4F：TGTAAAACGACGGCCAGTATCTCAGGCCTCTGGGCTC
PIEZO1-3-4R：AACAGCTATGACCATGGGTGGGAGTGTGGATGAGTC
PIEZO1-5F：TGTAAAACGACGGCCAGTGATTTCAGGCCCCAGGAAGT
PIEZO1-5R：AACAGCTATGACCATGCCTCAAAATGGCTGAGACCAC
PIEZO1-6F：TGTAAAACGACGGCCAGTGGAGAGGTGGCGTCAGTG
PIEZO1-6R：AACAGCTATGACCATGTGTCTCCACAGTCATCAGGG
PIEZO1-7F：TGTAAAACGACGGCCAGTTGATGACTGTGGAGACAGCG
PIEZO1-7R：AACAGCTATGACCATGGGCTAGACGAGCCAGGTG
PIEZO1-8-10F：TGTAAAACGACGGCCAGTGCCCACCTGGCTCGTCTA
PIEZO1-8-10R：AACAGCTATGACCATGCCGCACCCAGCCATACCTT
PIZEO1-11-12F：TGTAAAACGACGGCCAGTTTTTAGGGCATAGTCAGGGTGGG
PIZEO1-11-12R：AACAGCTATGACCATGCGGGCAAGTGGACATTGAACAG
PIEZO1-13-15F：TGTAAAACGACGGCCAGTCGGCTGACTGTGACACCC
PIEZO1-13-15R：AACAGCTATGACCATGGGGAAGTGCACGGGGTTT
PIEZO1-16F：TGTAAAACGACGGCCAGTGCCGTCTACACCTTCCAGTTC
PIEZO1-16R：AACAGCTATGACCATGTTGGTATGAGCAAATGCCCCTT
PIEZO1-17F：TGTAAAACGACGGCCAGTACAGGGCTTCTTCCAGCC
PIEZO1-17R：AACAGCTATGACCATGAGCTGGGCAGGCCAGAG
PIEZO1-18-19F：TGTAAAACGACGGCCAGTGTGCCTGAAGGTGGGTTGG
PIEZO1-18-19R：AACAGCTATGACCATGTCGGGCTGCGAATACAGAGTT
PIEZO1-20-21F：TGTAAAACGACGGCCAGTTCTCCCTGTTTCTCAAACACCTG
PIEZO1-20-21R：AACAGCTATGACCATGGGCAATGTCCTTGCCTCACC
PIEZO1-23F：TGTAAAACGACGGCCAGTGGACCGGGTGTCTCAGG
PIEZO1-23R：AACAGCTATGACCATGACTTGTGAGCAGATTTGGGG
PIEZO1-24-25F：TGTAAAACGACGGCCAGTGGTGGGAGGTGGGATTTATG
PIEZO1-24-25R：AACAGCTATGACCATGGCATCTGCTCCACGAAGAC
PIEZO1-26-27F：TGTAAAACGACGGCCAGTTCACCGTCATCATCTCCAAG
PIEZO1-26-27R：AACAGCTATGACCATGGGCCTGAGACAGTGAGGAAC
PIEZO1-28-29F：TGTAAAACGACGGCCAGTGTTGCTGGGGTCGAGTGC
PIEZO1-28-29R：AACAGCTATGACCATGGCCTGCCAGACAGAGGGT
PIEZO1-30F：TGTAAAACGACGGCCAGTAGACGGGAGCACTCACAGC
PIEZO1-30R：AACAGCTATGACCATGGAAGATAGCAGGCCAGGCAGA
PIEZO1-31F：TGTAAAACGACGGCCAGTGGGTCTGGCTGGGAGTC
PIEZO1-31R：AACAGCTATGACCATGCTGACACCCTCTATGCTGCC
PIEZO1-32-33F：TGTAAAACGACGGCCAGTCCCACCCAACTCCTGAATGG
PIEZO1-32-33R：AACAGCTATGACCATGGCCAAGAGAGACCTCCCAC
PIEZO1-35-36F：TGTAAAACGACGGCCAGTCTGCTCTGCCTGACTACGC
PIEZO1-35-36R：AACAGCTATGACCATGCATGACGGCCCGATCTGTT
PIEZO1-37F：TGTAAAACGACGGCCAGTGTCATGCCTTCGGGCAG
PIEZO1-37R：AACAGCTATGACCATGCAGGTACTTGACGACCACCG
PIEZO1-38F：TGTAAAACGACGGCCAGTGACGGCCATCGTCTTCAC
PIEZO1-38R：AACAGCTATGACCATGCCCTGGTGGAGAGCACAG
PIEZO1-39F：TGTAAAACGACGGCCAGTCACCTGTGCTCTCCACCAG
PIEZO1-39R：AACAGCTATGACCATGTCTGAGAAAGACCCTCCCTG
PIEZO1-40-42F：TGTAAAACGACGGCCAGTGTCAGGAGCCATGAGCCAG
PIEZO1-40-42R：AACAGCTATGACCATGGGACCCCATCAGAAACAGCC
PIEZO1-42-43F：TGTAAAACGACGGCCAGTCAGAGCTCAGCAGCCACC
PIEZO1-42-43R：AACAGCTATGACCATGCCAGGGTTCCCGTCAGG
PIEZO1-44F：TGTAAAACGACGGCCAGTAACCTCTTCCTCTTCCAGGG
PIEZO1-44R：AACAGCTATGACCATGGCAGTGCCTCTGTCTTAGGG
PIEZO1-45-47F：TGTAAAACGACGGCCAGTGCAGAGCTGAGCGTATGACC
PIEZO1-45-47R：AACAGCTATGACCATGCCAAGTCCAGGACGAACCTC
PIEZO1-47-48F：TGTAAAACGACGGCCAGTCTCTGCCCTGTGAAAGCTG
PIEZO1-47-48R：AACAGCTATGACCATGGGTGCTGGCAGGTCAGG
PIEZO1-49-50F：TGTAAAACGACGGCCAGTAGGGCTGTGCCAGGTTG
PIEZO1-49-50R：AACAGCTATGACCATGGGGGAAGCTGGTGAGTCC
PIEZO1-51F：TGTAAAACGACGGCCAGTGGCTACGGGTGAGTGAGTG
PIEZO1-51R：AACAGCTATGACCATGCAGAGCTGAGCGTGAGGAC。
5. the kit of claim 4, wherein the kit further comprises sequencing system reagents; comprises a sequencing primer for detecting PIEZO1 gene, and the base sequence of the sequencing primer is as follows:
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG。
CN202011505289.4A 2020-12-18 2020-12-18 Primer, kit and method for detecting PIEZO1 gene mutation Pending CN112501279A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007289142A (en) * 2006-03-31 2007-11-08 Otsuka Pharmaceut Factory Inc METHOD FOR MEASURING mRNA OF METABOLISM-ASSOCIATED ENZYME IN CYNOMOLGUS MONKEY, AND PROBE AND KIT FOR THE SAME
WO2015101175A1 (en) * 2013-12-31 2015-07-09 Berry Genomics Co., Ltd Method and kit for non-invasively detecting egfr gene mutaions
CN109576367A (en) * 2018-11-27 2019-04-05 福州艾迪康医学检验所有限公司 Detect primer, kit and the method for BCOR gene mutation
CN109593841A (en) * 2018-12-08 2019-04-09 南京艾迪康医学检验所有限公司 Detect method, kit, oligonucleotides and its application of F13A1 gene mutation
CN111057759A (en) * 2019-12-24 2020-04-24 北京艾迪康医学检验实验室有限公司 Primer, kit and method for detecting PAH gene mutation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007289142A (en) * 2006-03-31 2007-11-08 Otsuka Pharmaceut Factory Inc METHOD FOR MEASURING mRNA OF METABOLISM-ASSOCIATED ENZYME IN CYNOMOLGUS MONKEY, AND PROBE AND KIT FOR THE SAME
WO2015101175A1 (en) * 2013-12-31 2015-07-09 Berry Genomics Co., Ltd Method and kit for non-invasively detecting egfr gene mutaions
CN109576367A (en) * 2018-11-27 2019-04-05 福州艾迪康医学检验所有限公司 Detect primer, kit and the method for BCOR gene mutation
CN109593841A (en) * 2018-12-08 2019-04-09 南京艾迪康医学检验所有限公司 Detect method, kit, oligonucleotides and its application of F13A1 gene mutation
CN111057759A (en) * 2019-12-24 2020-04-24 北京艾迪康医学检验实验室有限公司 Primer, kit and method for detecting PAH gene mutation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AN SONG等: ""Low HbA1c With Normal Hemoglobin in a Diabetes Patient Caused by PIEZO1 Gene Variant: A Case Report"", 《CASE REPORT》, vol. 11, pages 2 *
李园等: ""PIEZO1基因突变遗传性口型红细胞增多症五例报道及文献复习"", 《中华血液学杂志》, vol. 40, no. 6, pages 518 - 521 *

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