CN107385036B - Detection primer group for T239C, C298T and C893A sites of ARR3 gene, kit and application - Google Patents
Detection primer group for T239C, C298T and C893A sites of ARR3 gene, kit and application Download PDFInfo
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Abstract
The invention discloses a detection primer group, a kit and application of T239C, C298T and C893A sites of ARR3 genes. The method is based on the Sanger sequencing principle, has the characteristics of high sensitivity, stability and accuracy, can be used for simultaneously detecting three high-myopia-caused mutation sites of the ARR3 gene, is used for early screening and auxiliary diagnosis of high myopia, provides basis for early prevention and treatment of patients, and has good market application prospect.
Description
The technical field is as follows:
the invention belongs to the technical field of biology, and particularly relates to a detection primer group for T239C, C298T and C893A sites of ARR3 genes, a kit and application.
Background art:
myopia is a common cause of impaired vision, with complications of high myopia (myopia above-6.00D diopters) (glaucoma, cataract, vitreous opacity, retinal detachment, etc.) being the second leading cause of blindness; the prevalence of myopia in Chinese is much higher than that of other races, and is affected for life. More seriously, with the advance of urbanization and the change of life style, the incidence of myopia is increasing year by year, especially for asian people living in economically developed areas. A large number of evidences show that the high myopia is closely related to heredity, the familial hereditary high myopia, especially the early high myopia before the school age, is slightly influenced by environmental factors and is mostly caused by gene mutation, the myopia degrees of patients are progressively increased, the retinal choroidopathy is aggravated year by year, and generations are transmitted, so that heavy psychological and economic burdens are brought to the patients. If the detection of the pathogenic gene mutation is carried out on the patients with the high myopia, the detection is the basis of the early diagnosis and the prevention.
The existing detection kit for the pathogenic genes of the early-onset high myopia only detects six pathogenic sites of the ZNF644 gene (CN102732607A), but the six pathogenic sites are detected in a small number of patients, the pathogenic mutation of the majority of the early-onset high myopia patients is unknown, and mutation detection needs to be carried out on more pathogenic genes. Therefore, more mutation sites which can cause early-onset high myopia are found and developed into a detection kit, and the method has important significance for accurate diagnosis and treatment of the early-onset high myopia.
The invention content is as follows:
the invention aims to provide a detection primer group, a kit and application of T239C, C298T and C893A sites of ARR3 genes, which have the advantages of good specificity, high sensitivity and strong stability.
The first purpose of the invention is to provide a detection primer group of T239C, C298T and C893A sites of ARR3 gene, which is shown as follows:
for ARR3 gene T239C and C298T sites:
ARR 3-1F: 5'-GCCAGCCTTCGATCATATTACAT-3' (shown in SEQ ID NO. 1);
ARR 3-1R: 5'-TGGCGATGAGGCTGTGGAGGTTA-3' (shown in SEQ ID NO. 2);
for the C893A site of ARR3 gene:
ARR 3-2F: 5'-AGGATTTCCCACCAGAACTAAT-3' (shown in SEQ ID NO. 3);
ARR 3-2R: 5'-GGCTTTACTGAACATGAAACCT-3' (shown in SEQ ID NO. 4).
The second purpose of the invention is to provide a detection kit for T239C and C298T sites and/or C893A sites of ARR3 genes, which comprises the upstream and downstream primers for T239C and C298T sites of ARR3 genes and/or the upstream and downstream primers for C893A sites of ARR3 genes.
Preferably, the detection kit further comprises 2 Master Mix and ddH2O, positive quality control material and negative quality control material.
Preferably, the positive quality control product comprises plasmid DNA containing a sequence shown by SEQ ID NO.5, plasmid DNA containing a sequence shown by SEQ ID NO.6 and plasmid DNA containing a sequence shown by SEQ ID NO. 7.
The third purpose of the invention is to provide a method for detecting T239C, C298T and C893A sites of ARR3 genes, which comprises the following steps:
(1) extracting the genome DNA of a sample to be detected;
(2) performing PCR amplification on the genomic DNA extracted in the step (1) by using the upstream and downstream primers aiming at the T239C and the C298T sites of the ARR3 gene and the upstream and downstream primers aiming at the C893A site of the ARR3 gene respectively;
(3) sequencing the PCR amplification product, and then comparing with a wild type ARR3 gene sequence to determine whether a gene mutation site exists.
The sequencing method is preferably a Sanger sequencing method.
According to the Sanger sequencing principle, when a DNA template is copied in the presence of DNA polymerase, primers and four types of deoxynucleotide triphosphates (dNTPs), four types of dideoxynucleoside triphosphates (ddNTPs) marked by fluorescent stains are respectively introduced into a reaction system according to a certain proportion. Since ddNTPs lack the 3' -OH group required for extension, when ddNTPs are incorporated into the end of a chain, the chain stops extending. Thus, a series of DNA fragments with ddNTP as the 3' end and different lengths are generated in each reaction tube. After the reaction is terminated, gel electrophoresis is carried out in the capillary to separate DNA fragments with different lengths, the lengths of the adjacent fragments are different by one base, the mobility in the capillary electrophoresis is different, the laser detector can detect fluorescent molecules one by one and synchronously image on a CCD camera, and analysis software can automatically convert different fluorescence into DNA sequences, thereby achieving the purpose of DNA sequencing.
The reaction system of the PCR amplification is preferably 20 mu L, and the reaction system comprises 60ng of genomic DNA, 10 mu L of 2 Master Mix, 0.3 mu L of 10 mu M upstream primer, 0.3 mu L of 10 mu M downstream primer and ddH2Make up to 20. mu.L of O.
The reaction procedure of the PCR amplification is preferably as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 45s, and repeating for 30 cycles; extension at 72 ℃ for 5 min.
The fourth purpose of the invention is to provide the application of the detection primer group or the detection kit in detecting early-onset high myopia.
The invention designs a set of detection primer group aiming at three mutation sites of ARR3 gene T239C, C298T and C893A, develops a kit containing the detection primer group and a detection method using the kit. The kit adopts a classic Sanger dideoxy chain termination sequencing method, and the method has the advantages of accurate, visual and visible result, high repeatability, strong specificity and high sensitivity, and is a gold standard for gene diagnosis.
The method is based on the Sanger sequencing principle, has the characteristics of high sensitivity, stability and accuracy, can be used for simultaneously detecting three high-myopia-caused mutation sites of the ARR3 gene, is used for early screening and auxiliary diagnosis of high myopia, provides basis for early prevention and treatment of patients, and has good market application prospect.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
1. design of specific primers
Specific primers were designed based on the upstream and downstream sequences of the ARR3 Gene (GeneBank sequence number: Gene ID: 407; NC-000023) T239C (i.e., the 202 nd base from the 5 ' end of the sequence shown in SEQ ID NO.5 was T/C), C298T (i.e., the 261 th base from the 5 ' end of the sequence shown in SEQ ID NO.6 was C/T) and C893A (i.e., the 293 th base from the 5 ' end of the sequence shown in SEQ ID NO.7 was C/A) sites in the NCBI database, and the designed specific primers were shown in Table 1.
TABLE 1 specific primer sequences
2. Genomic DNA extraction
The genomic DNA of peripheral blood leukocytes of 200 normal persons and 200 early-onset patients with high myopia were extracted by phenol/chloroform method or kit extraction method, respectively. The method comprises the following specific steps:
2.1 Add 200. mu.L of Proteinase K (20mg/mL) solution to a 15mL centrifuge tube;
2.2 treatment materials: extracting a blood sample, directly adding 3mL of the blood sample, and uniformly mixing;
2.3 adding 3.6mL of buffer GE into the centrifuge tube filled with the blood sample, oscillating for 30sec and mixing uniformly;
standing at 2.465 deg.C for 10min, and shaking every 3min to assist cracking. Centrifuging briefly to collect water droplets on the inner wall of the tube cover;
2.5 adding 3mL of absolute ethyl alcohol into the sample, and uniformly mixing, wherein flocculent precipitates can appear at the moment;
2.6 transferring the solution and half of the flocculent precipitate obtained in the previous step into an adsorption column CB5 (the adsorption column is placed into a 15mL collection tube), centrifuging for 3min at 3,000rpm (1,850 Xg), pouring the waste liquid, and placing the adsorption column CB5 back into the collection tube;
2.7 transferring the solution remained in the step 2.6 into the same adsorption column, and repeating the operation of the step 2.6;
2.8 adding 2mL of buffer GD (firstly checking whether absolute ethyl alcohol is added before using) into the adsorption column CB5, centrifuging for 1min at 5,000rpm (4,500 Xg), pouring the waste liquid, and putting the adsorption column CB5 back into the collection tube;
2.9 to adsorption column CB5, 2mL of buffer PW (check before use whether or not absolute ethanol has been added) is added, centrifuged at 5,000rpm (-4,500 Xg) for 1min, the waste liquid is discarded, adsorption column CB5 is returned to the collection tube, and the collected DNA is ready for use.
3. Preparation of plasmids
Cloning SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 to a PMD19-T vector respectively, and then sequencing and verifying clones to obtain a mutant plasmid PMD19-T1, a mutant plasmid PMD19-T2 and a mutant plasmid PMD19-T3, cloning SEQ ID NO.8 and SEQ ID NO.9 to a PMD19-T vector respectively, and then sequencing and verifying clones to obtain a wild type plasmid PMD19-T4 and a wild type plasmid PMD 19-T5; plasmid DNA was extracted.
4. PCR amplification
4.1 PCR amplification was performed using the genomic DNA extracted in step 2 as a template and the primer pairs ARR3-1F/R and ARR3-2F/R shown in Table 1, respectively. Positive controls were mutant plasmid PMD19-T1 and mutant plasmid PMD19-T2, respectively. The negative control was PMD 19-T. A blank control was performed using 2. mu.L of purified water as a template.
4.2PCR reaction System: the total is 20. mu.L, containing 30 ng/. mu.L of genomic DNA 2. mu.L, 2 MasterMix 10. mu.L, 10. mu.M forward primer 0.3. mu.L, 10. mu.M reverse primer 0.3. mu.L, ddH2O7.4 μ L; PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 45s, and repeating30 cycles; extension at 72 ℃ for 5 min.
4.3 detection of PCR amplification product: taking 3 μ L of PCR amplification product, performing electrophoresis in 20g/L agarose gel (containing 0.5 μ g/mLEB) at 50V for 45-60min, and directly observing the result under an ultraviolet lamp: amplifying by using ARR3-1F/R as a primer, wherein genome DNA and positive control of 200 normal persons and 200 early-onset high myopia patients amplify a band of about 873 bp; ARR3-2F/R is used as a primer for amplification, and genome DNA and positive control of 200 normal persons and 200 early-onset high myopia patients amplify a band of about 400 bp.
4.4PCR amplification product purification:
the method comprises the following operation steps: before use, anhydrous ethanol is added into a rinsing liquid PW, and the volume of the anhydrous ethanol is added according to a label on a bottle. All centrifugation steps were performed using a bench top centrifuge at room temperature.
4.4.1 column equilibration step: 500. mu.L of the equilibrium solution BL was added to the adsorption column CB3 (the adsorption column was put into the collection tube), and the tube was centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to discard the waste liquid from the collection tube, and the adsorption column was returned to the collection tube. (please use the column treated the day).
4.4.2 estimate the volume of the PCR reaction solution, 5 times the volume of the binding solution PB was added thereto and mixed well.
4.4.3 the solution from the previous step was added to an adsorption column CB3 (adsorption column placed in collection tube), left at room temperature for 2min, centrifuged at 12,000rpm (-13,400 Xg) for 30-60sec, the waste liquid in the collection tube was decanted, and adsorption column CB3 was placed in the collection tube. Note that: the volume of the adsorption column is 800. mu.L, and if the volume of the sample is more than 800. mu.L, the sample can be added in batches.
4.4.4 to the adsorption column CB3, 600 uL of rinsing liquid PW is added, the mixture is kept stand for 2 to 5min, centrifuged at 12,000rpm (-13,400 Xg) for 30 to 60sec, the waste liquid in the collection tube is poured off, and the adsorption column CB3 is placed in the collection tube.
4.4.5 repeat operation 4.4.4.
4.4.6 the centrifugation and adsorption column CB3 was returned to the collection tube and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to remove the rinse as much as possible. The column was left at room temperature for several minutes and thoroughly dried to prevent residual rinse from affecting the next experiment. And collecting the purified product.
4.4.7 sequencing reaction: the purified DNA amplification product sample, the positive control, and the negative control were added to the reaction mixture in a predetermined order, each 2. mu.L.
Sequencing PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 10s, annealing at 61 ℃ for 4min, and repeating 30 cycles.
4.5 sequencing amplification product purification:
4.5.1 mu.L of the sequencing reaction amplification product and 35. mu.L of 70% absolute ethanol were added to a 96-well plate and centrifuged at 4,000rpm for 30min, which in turn was 500rpm in the 96-well plate for 30sec short.
4.5.2 standing at room temperature for 40min to dry.
4.6 sequencing on machine:
preparing a machine sample: the air-dried sample is added with Hi-Di deionized formamide 10 mu L, denatured at 95 ℃ for 5min, and then immediately placed on ice for cooling for 5 min. Then placed on the sample plate base of a sequencer for sequencing.
According to the Sanger sequencing principle, when a DNA template is copied in the presence of DNA polymerase, primers and four types of deoxynucleotide triphosphates (dNTPs), four types of dideoxynucleoside triphosphates (ddNTPs) marked by fluorescent stains are respectively introduced into a reaction system according to a certain proportion. Since ddNTPs lack the 3' -OH group required for extension, when ddNTPs are incorporated into the end of a chain, the chain stops extending. Thus, a series of DNA fragments with ddNTP as the 3' end and different lengths are generated in each reaction tube. After the reaction is terminated, gel electrophoresis is carried out in the capillary to separate DNA fragments with different lengths, the lengths of the adjacent fragments are different by one base, the mobility in the capillary electrophoresis is different, the laser detector can detect fluorescent molecules one by one and synchronously image on a CCD camera, and analysis software can automatically convert different fluorescence into DNA sequences, thereby achieving the purpose of DNA sequencing.
And (3) analyzing a sequencing result: the raw data were derived and the sequenced sequences were aligned with the original normal sequence (Gene ID:407 NC-000023, the wild type ARR3 Gene sequence) using "DNAStar" software.
The results show that: no mutation occurred in T239C, C298T and C893A sites of 200 normal persons, and all the normal persons were negative; in 200 patients with early-onset high myopia, 1 patient had a T239C site mutation, 1C 298T site mutation, and 1 patient had a C893A site mutation, and we further analyzed family members of the above three patients in the same manner, and found that: 4 of 8 members of the T239C family all had high myopia patients with the site mutation, and 4 other normal people did not have the site mutation; 10 of 12 members of the C298T family all had high myopia patients with the site mutation, and 2 other normal people had no site mutation; 15 of the 30 members of the C893A family all carried this mutation in patients with high myopia, and 15 other normal individuals did not carry this mutation. The mutation of the three sites of T239C, C298T and C893A is proved to be obviously related to the early-onset high myopia.
Example 2:
1. sensitivity detection
1.1 plasmid dilution
Mutant plasmids PMD19-T1, PMD19-T2 and PMD19-T3, wild-type plasmids PMD19-T4 and PMD19-T5 are respectively diluted to a final concentration of 5 ng/muL, and then the mutant plasmids and the corresponding wild-type plasmids are mixed according to the volume ratio of 0.2:9.8, 0.5:9.5, 1:9, 1.5:8.5, 2:8, 3:7 and 4:6 to prepare positive samples with mutation abundances of 2%, 5%, 10%, 15%, 20%, 30% and 40% (Table 2). The PCR amplification and sequencing were performed as described in example 1, using positive samples of different mutation abundances as templates. The results are shown in Table 3. The preliminary results show that the detection sensitivity of the detection kit (Sanger sequencing method) is 10% of the mutation abundance by combining the sequencing map and judgment of Table 3.
TABLE 2 plasmid information for mutant plasmids
TABLE 3 sensitivity test results (Forward and reverse test)
| Mutant plasmid mutant abundance | 2％ | 5％ | 10％ | 15％ | 20％ | 30％ | 40％ |
| T239C | -/- | -/- | +/+ | +/+ | +/+ | +/+ | +/+ |
| C298T | -/- | -/- | +/+ | +/+ | +/+ | +/+ | +/+ |
| C893A | -/- | -/- | +/+ | +/+ | +/+ | +/+ | +/+ |
1.2 sensitivity verification
And (4) qualified standard: and detecting the positive sample with 10% mutation abundance for 10 times, wherein at least 9 detection results meet the detection lower limit, namely the detection rate is greater than or equal to 90%.
Verification of 10% detection sensitivity of mutant abundance
And (2) taking the mutant plasmids PMD19-T1/PMD19-T4, PMD19-T2/PMD19-T4 and PMD19-T3/PMD19-T5 with the mutation abundance of 10% prepared in the step 1.1, and carrying out gene mutation detection on the mutant plasmids by using the detection kit (Sanger sequencing method) disclosed by the invention. The sequencing was repeated 10 times with a detection rate of 100% (table 4). And the sensitivity verification is passed.
TABLE 4 sensitivity verification detection results (mutation abundance 10%)
| Detection sample (mutation abundance 10%) | Number of detections | Number of times of detection |
| T239C | 10 | 10 |
| C298T | 10 | 10 |
| C893A | 10 | 10 |
2. And (3) specificity test:
2.1 methods for specificity verification: the primer pair ARR3-1F/R shown in Table 1 was matched in human, cynomolgus, wild boar, domestic pig and white rhinoceros genome sequences by electronic simulated PCR. The primer pair ARR3-2F/R shown in Table 1 was matched in human, wild boar, domestic pig and white rhinoceros genome sequences using electronic mock PCR.
The results show that: the ARR3-1F/R primer can generate specific products in the human genome sequence, and amplification products are not generated in the genome sequences of macaques, wild boars, domestic pigs and white rhinoceros; the ARR3-2F/R primer can generate specific products in human genome sequences, and amplification products are not generated in genome sequences of macaques, wild boars, domestic pigs and white rhinoceros. Therefore, the detection kit of the present invention has high specificity.
3. And (3) repeatability test:
3.1 repeatability test method: the mutant plasmids PMD19-T1, PMD19-T2 and PMD19-T3 were diluted to 10% and 30% abundance of two mutations as templates according to the method of step 2.1, PCR amplification was performed according to the method of example 1, sequencing was repeated 10 times, and the repetition rate of positive results was calculated (see Table 5).
TABLE 5 repeat sequencing results of samples with two different mutation abundances at ARR3 Gene 3 site
3.2 conclusion: comprehensive sequencing graphs and a table 5 show that the repetition rate of positive results of 10 repeated sequencing of 10 times of each of 10% and 30% of mutation abundances of the 3 site of the ARR3 gene is 100%, the detection repeatability of the amplification primers and the PCR reaction conditions is good, and stable detection can be realized by comparing samples with 10% of mutation abundances.
Sequence listing
<110> Zhongshan ophthalmic center of Zhongshan university
<120> detection primer group of T239C, C298T and C893A sites of ARR3 gene, kit and application
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gaagctcaag aaggacaaca agggagaggg ttccccattt cttttgtatt tgtttgtatt 420
cttttctact tcttttctga tctccttttt gtcccctaga tggtgaccaa cctgccctgt 480
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cagctaagga aagaggtcag gcattttctt ataggcccat gaggaaggga ggacagcact 660
ggaaatgagc tctctttgcc cttgtccctt tacagccctg tgggattgac tttgaagtga 720
agagtttctg tgctgaaaac ccagaggaga cagtctccaa gaggtattct ttggttgtcc 780
ccaaaaatcc ctgcctccag cctctgccag gaaacagatc ctgctctcat gacagaaata 840
gccccacttc taacctccac agcctcatcg cca 873
<210>9
<211>400
<212>DNA
<213> human
<400>9
aggatttccc accagaacta atttctccca gtcttcagtt aaacatgtta cctctcttgg 60
atactccagt aggttcttgt ataatcttgt acaagatact cttgggagta aaagtatgct 120
tagtcagcaa gcccagccta aattcttctc ttgttcttct tttgtaggga gactgtagct 180
gctaattcca gcttctccca gagctttgca gtaaccccaa tcctggctgc cagctgccag 240
aaacggggcc tggcactgga tggcaaactt aagcatgaag ataccaacct ggcctctagc 300
acaatgtaag ctcaaatata actctcagcc tccatttcct acccctcaac cattccacat 360
gctacattta cagctctgag gtttcatgtt cagtaaagcc 400
Claims (4)
1. A detection primer group of T239C, C298T and C893A sites of ARR3 genes is characterized in that the detection primer group is shown as follows:
for ARR3 gene T239C and C298T sites:
ARR3-1F：5’-GCCAGCCTTCGATCATATTACAT-3’；
ARR3-1R：5’-TGGCGATGAGGCTGTGGAGGTTA-3’；
for the C893A site of ARR3 gene:
ARR3-2F：5’-AGGATTTCCCACCAGAACTAAT-3’；
ARR3-2R：5’-GGCTTTACTGAACATGAAACCT-3’。
2. a kit for detecting T239C and C298T sites and/or C893A sites of ARR3 gene, which comprises the upstream and downstream primers of claim 1 for T239C and C298T sites of ARR3 gene and/or for C893A site of ARR3 gene.
3. The test kit of claim 2, further comprising 2 Master Mix, ddH2O, positive quality control material and negative quality control material.
4. The detection kit according to claim 3, wherein the positive quality control product comprises plasmid DNA comprising a sequence shown by SEQ ID No.5, plasmid DNA comprising a sequence shown by SEQ ID No.6 and plasmid DNA comprising a sequence shown by SEQ ID No. 7.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710598012.2A CN107385036B (en) | 2017-07-20 | 2017-07-20 | Detection primer group for T239C, C298T and C893A sites of ARR3 gene, kit and application |
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|---|---|---|---|
| CN201710598012.2A CN107385036B (en) | 2017-07-20 | 2017-07-20 | Detection primer group for T239C, C298T and C893A sites of ARR3 gene, kit and application |
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| CN107385036B true CN107385036B (en) | 2020-08-11 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732607A (en) * | 2011-03-07 | 2012-10-17 | 四川省医学科学院（四川省人民医院） | Kit for detecting high myopia |
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- 2017-07-20 CN CN201710598012.2A patent/CN107385036B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732607A (en) * | 2011-03-07 | 2012-10-17 | 四川省医学科学院（四川省人民医院） | Kit for detecting high myopia |
Non-Patent Citations (1)
| Title |
|---|
| X-linked heterozygous mutations in ARR3 cause female-limited early onset high myopia;Xueshan Xiao等;《Molecular Vision》;20161026;第22卷;摘要 * |
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| CN107385036A (en) | 2017-11-24 |
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