CN113481289A - Primer composition for detecting sideroblastic red blood cell anemia and application thereof - Google Patents
Primer composition for detecting sideroblastic red blood cell anemia and application thereof Download PDFInfo
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Abstract
The invention provides a primer composition for detecting an iron granulocyte erythrocytic anemia gene and application thereof, and relates to the technical field of molecular biology. The gene group of SA-related gene mutation provided by the invention can be used for detecting 342 gene mutation sites of 2 genes at one time by combining a high-throughput sequencing technology, has the advantages of wide coverage, high detection efficiency, simplicity and convenience in operation, high accuracy, high sensitivity and the like, solves the technical defects of low detection accuracy and limitation of a detection method at present, can realize auxiliary diagnosis of SA in addition to bone marrow morphology and blood routine examination, particularly has special advantages in predicting the subsequent treatment direction and effect of diseases, and can make up the defects of clinical SA in guidance aspects such as molecular diagnosis, disease evolution, treatment, prognosis, medication and the like.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a primer composition for detecting an iron granulocyte erythrocytic anemia gene and application thereof.
Background
Sideroblastic Anemia (SA) is a rare heterogeneous group of diseases caused by dysheme synthesis and poor iron utilization due to different etiologies, and is characterized by the presence of a large number of annular sideroblastic cells in the bone marrow, ineffective hematopoiesis in the red line, increased tissue iron, and the presence of different proportions of hypopigmented red blood cells in the peripheral blood. Sideroblasts can be divided into two categories, hereditary and acquired, and different types of SA treatment strategies and outcome differ. Hereditary sideroblastic erythrocytic anemia is mostly X-chromosome heredity, the anemia degrees of patients are very different, some patients with mild anemia are easy to miss diagnosis without family survey, and some patients with hereditary SA caused by ALAS2 can benefit by applying pyridol with pharmacological dose. Acquired iron granulocytic anemia can be further classified into primary and secondary. Secondary iron granulocytic anemia can be caused by drug administration, alcohol drinking, and poisoning by lead and zinc. The primary iron particle anemia mainly comprises Myelodysplastic syndrome with ring-shaped iron particle blasts (MDS-RS) and the like, and the disease can progress into acute leukemia. Occasionally, myelodysplastic syndrome occurs in patients with iron granulocytic anemia with significant familial morbidity. Iron overload is a common complication of SA, especially in the late stages of the disease, and serious individuals can be life threatening and die. In addition, complications such as hepatosplenomegaly, diabetes, skin pigmentation, arrhythmia, and immunologic hypofunction can also be seen.
At present, no clear diagnostic guideline reference exists for the diagnosis of SA, and the clinical performance and laboratory examination items lack specificity. Patients have a clear family history that aids in the diagnosis of genetic SA. Hereditary SA is of a younger age, and most patients develop anemia after birth or during infancy, and the anemia develops in about 10-20 years of age, and occasionally develops after 50-60 years of age; hereditary SA is usually microcytic hypopigmentation anemia, and occasionally macrocellular anemia; meanwhile, the serum of the hereditary SA patient is higher in iron. Therefore, indicators such as the age of onset, the nature of anemia, and iron metabolism of a patient are important indicators for the diagnosis of hereditary SA. Because bone marrow iron staining shows a large number of annular iron granulocyte juvenile cells, indexes such as age, small cell or large cell anemia and iron metabolism have no definite specificity, and genetic SA and acquired SA, particularly MDS-RS, are difficult to identify clinically. While gene mutation analysis is an important standard for genetic SA diagnosis, 30-70% of patients with genetic SA can be detected at present.
The current methods for detecting SA mainly relate to blood routine, cell morphology, molecular biology and the like. The molecular biological experiment method mainly comprises nested PCR, first-generation sequencing and the like. The PCR method cannot intuitively obtain a specific mutation sequence, and the first-generation sequencing method cannot detect all exon regions of a plurality of genes at one time due to the influence of sequencing flux. The second-generation sequencing is used as a high-flux detection method, can accurately measure the exon sequences of a plurality of genes at one time, and enables accurate treatment to be possible. At present, the research on the aspect of gene detection and identification of SA by using a second-generation sequencing method is flexible, and the detection of SA at the gene diagnosis level is particularly important.
The patent CN108192880A discloses a human delta-aminolevulinic acid synthase mutant protein and application thereof, a gene chip, a monoclonal antibody and an ELISA kit are prepared according to the delta-aminolevulinic acid synthase mutant protein, and a guiding effect is provided for gene diagnosis of X-linked hereditary sideroblasts anemia and X-linked erythropoietic hematoporphyria, but the method only aims at the human delta-aminolevulinic acid synthase mutant protein, and the detection rate is low.
Therefore, a kit and a method for detecting SA with higher accuracy and accuracy are needed to make up for the deficiency of clinical SA in guidance such as molecular diagnosis, disease progression, treatment, prognosis, and medication.
Disclosure of Invention
Aiming at the defects, the invention provides a primer composition for detecting an iron granulocyte erythroblast anemia gene and application thereof. The primer composition disclosed by the invention is used for detecting the iron granulocyte erythroblast anemia related gene, has the advantages of simplicity and convenience in operation, high accuracy and high sensitivity, and can make up for the defects of clinical SA in the aspects of guidance such as molecular diagnosis, disease evolution, treatment, prognosis, medication and the like.
In order to achieve the above object, the technical solution of the present invention is as follows:
in one aspect, the invention provides a primer composition for detecting sideroblasts anemia genes, wherein the sideroblasts anemia genes are one or more of ALAS2 or SF3B 1.
Specifically, the mutation sites of the sideroblasts anemia gene are shown in the following table 1.
TABLE 1 iron granulocyte erythroblast anemia Gene mutation sites
More specifically, the primer compositions are shown in table 2 below.
TABLE 2 primer compositions
In particular, the primers cover the entire exon sequences of the entire gene.
In another aspect, the invention provides an application of the primer composition in preparation of a product for detecting the sideroblasts anemia gene.
In another aspect, the invention provides a product for detecting iron granulocyte erythroblast anemia genes, which comprises the primer composition.
Specifically, the product also comprises DNA polymerase, Buffer and ddH2O、dNTP。
Specifically, the product is an independent reagent or a kit.
In another aspect, the invention provides an application of the primer composition or the product in detecting sideroblasts anemia genes.
In still another aspect, the present invention provides a method for detecting a sideroblasts anemia gene, which is a non-disease diagnosis and treatment method, comprising detecting a sideroblasts anemia gene in a test sample using the above primer composition or product.
Specifically, the method comprises the following steps:
(1) extracting genome DNA;
(2) multiplex PCR: amplifying the genomic DNA obtained in the step (1) by using the primer composition;
(3) building a library;
(4) library sequencing and data processing.
Compared with the prior art, the invention has the advantages that:
(1) the invention provides a primer composition for detecting an iron granulocyte erythroblast anemia gene, which can realize auxiliary diagnosis of SA in addition to bone marrow morphology and blood routine examination by utilizing the gene group related to SA mutation provided by the invention and combining a high-throughput sequencing technology, particularly has special advantages for predicting the subsequent treatment direction and effect of diseases, and can make up the defects of clinical SA in the aspects of guidance such as molecular diagnosis, disease evolution, treatment, prognosis, medication and the like.
(2) The primer composition disclosed by the invention is used for detecting the genes related to the iron granulocyte erythroblast anemia, has the advantages of simplicity and convenience in operation, high accuracy, high sensitivity and the like, and solves the technical defects of low monitoring accuracy and limitation of a detection method at present.
(3) The primer composition can detect 342 gene mutation sites of the 2 genes at one time, and has wide coverage and high detection efficiency.
Drawings
FIG. 1 is a schematic diagram of nucleotide mutation of ALAS2 gene c.1355G > A.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
The examples, where no specific techniques or conditions are indicated, are carried out according to the techniques or conditions described in the literature of the art (for example, see J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, scientific Press, ed. by Huang Pe, et al) or according to the instructions of the product.
Example 1 Ferro-granulocyte erythroblast anemia Gene, mutation site and primer composition thereof
(1) The dbSNP database (http:// www.ncbi.nlm.nih.gov/snp /), the thousand human genome database 1000Genomes (http:// www.1000genomes.org /), the exome integration database ExAC (http:// ExAC. broadinstruction. org /) and the human gene mutation database HGMD (http:// www.hgmd.cf.ac.uk/ac/index. php), and the functional prediction tool Polyphen (http:// genetics. bw. harvard. edu/pph2/) and SIFT (http:// SIFT. jcvvi. org /), were used to screen for pathogenic mutation sites using the following principles: (1) filtering out mutations which do not affect the sequence of the protein product according to the position and the type of the genome in which the mutations are positioned; (2) annotating the proportion of each mutation in the population using 1000Genomes data and ExAC data, and not considering a polymorphic site if the proportion is 1% or less; (3) searching a human gene mutation database, and inquiring whether a mutation is recorded in HGMD and appears in SA and other related diseases; (4) predicting whether the mutation affects the protein function by using protein function prediction software PolyPhen-2 and SIFT; (5) if a mutation satisfies at least two of the three "non-polymorphic", "described by SA", and "affecting protein function" after (2), (3), and (4), it is judged as possibly associated with the disease; (6) if one mutation is recorded in the HGMD although not satisfying (5), the mutation is judged as a mutation with unknown meaning; if a mutation is well documented in the literature as being highly correlated with disease, it is directly identified as a hotspot mutation.
The following genes were obtained by final screening: (1) ALAS2 gene: hereditary sideroblasts anemia is mostly X-chromosome associated heredity and is caused by mutation of 5-aminoketoglutaric acid synthase (ALAS 2) gene; (2) SF3B1 gene: RNA splice genes, the mutations of which are closely related to Myelodysplastic syndrome with ring-shaped sideroblasts (MDS-RS).
Specific mutation sites are shown in table 3 below.
TABLE 3 sites of mutations
(2) The primer compositions designed based on the above genes and mutation sites and the concentrations thereof are shown in Table 4 below.
TABLE 4 primer compositions
(3) Performing PCR amplification of a composite system: the primers are mixed into a reaction system, and the most appropriate primer concentration is selected under the condition of ensuring certain template concentration, so that each pair of primers in the composite system can be optimally amplified.
Example 2A kit for detecting SA and method of use thereof
1. Amplification primers in multiplex amplification system
In the multiplex amplification system, the forward and reverse amplification primers for each gene and mutation site are shown in table 4 above, and in order to make the amplification efficiency of each gene and mutation site as uniform as possible, the concentration of each pair of primers in the multiplex system is adjusted, and the final adjusted primer concentration is shown in table 4 above.
DNA extraction: and extracting the whole genome DNA of the peripheral blood or bone marrow sample by using a DNA extraction kit.
3. The extracted DNA was subjected to multiplex PCR amplification according to the primer composition provided in example 1.
Among them, the multiplex amplification system is shown in Table 5 below.
TABLE 5 multiplex amplification System
| Composition (I) | Volume of |
| AgriSeqTM Amplification Mix | 2μL |
| Ion AmpliSeqTM Primer Pool | 5μL |
| Diluted DNA sample (10 ng/. mu.L) | 3μL |
The DNA was derived from the sample to be tested, and the Primer pool was a mixture of the above primers, and the concentrations of the primers are shown in Table 4.
The multiplex amplification reaction procedure was as follows: 99 ℃ for 2 min; 99 ℃, 15s, 62 ℃, 4min, 2 cycles; 99 ℃, 15s, 60 ℃, 8min, 14 cycles; storing at 10 ℃.
4. And preparing the amplified product into a DNA library for sequencing by a sequencing platform by using the kit, and completing library construction.
4.1 the amplified product is digested. The cleavage system is shown in Table 6 below.
TABLE 6 enzyme digestion System
| Step 3 composite amplification product | 20μL |
| Pre-ligation | 2μL |
The digestion reaction procedure was as follows: 50 ℃ for 20 min; 20min at 55 ℃; at 60 deg.C for 20 min; storing at 10 ℃. And after the reaction is finished, purifying the product by a magnetic bead method.
Wherein Pre-ligation is available from ThermoFisher scientific under the product number A34141.
4.2 end repair & Add A. The reaction system is shown in table 7 below.
TABLE 7 end repair & Add A
| Enzyme digestion product | 40μL |
| End Repair&A-Tailing Enzyme | 4μL |
| End Repair&A-Tailing Buffer | 6μL |
The reaction procedure was as follows: 20 ℃ for 30 min; 30min at 65 ℃; and (4) storing at 4 ℃.
Wherein, End Repair & A-Tailing Enzyme and End Repair & A-Tailing Buffer are available from Naonda, cat # 1002103.
4.3 connecting joints. The reaction system is shown in Table 8 below.
TABLE 8 Joint connection
| End repair and A-tailing reaction product | 50μL |
| IDT UDI joint (15 μ M) | 2μL |
| Ligation Buffer | 26μL |
| DNALigase | 2μL |
The reaction procedure was as follows: 20 ℃ for 20 min. And after the reaction is finished, purifying the product by a magnetic bead method.
Among them, IDT UDI linker (15. mu.M), Ligation Buffer, and DNA ligand were purchased from Naonda, cat # 1003227, and ligand # 1002103.
4.4 library enrichment. The reaction system is shown in table 9 below.
TABLE 9 library enrichment
The amplification reaction procedure was as follows: at 98 ℃ for 2 min; 98 ℃, 15s, 60 ℃, 30s, 72 ℃, 30s, 7 cycles; 72 ℃ for 2 min; and (4) storing at 4 ℃. And after the reaction is finished, purifying the product by a magnetic bead method.
Among these, 2 XHiFi PCR Master Mix was purchased from Naonda, cat # 1002103.
5. The DNA library was template prepared for sequencing, off-line data was obtained, and biological analysis was performed on the off-line data.
Example 3 reproducibility test
One example of a patient diagnosed with SA by clinical symptoms and relevant laboratory tests was selected, in which the nucleotide mutation of ALAS2 gene c.1355g > a was detected by first-generation sequencing, and the amino acid mutation was p.r452h.
The primer composition and the kit are adopted to repeatedly detect the sample for three times, and the specific steps and the detection method are as follows:
DNA extraction: whole genome DNA of bone marrow samples was extracted using Tiangen DNA extraction kit (cat # DP 318-03).
2. The extracted DNA was subjected to multiplex PCR amplification according to the primer compositions and kits provided in examples 1 and 2.
3. The amplified product is prepared into a DNA Library for the Ion Torrent sequencing platform to sequence, and the specific operation steps are detailed in the instruction book of a Life company Kit (name: Ion AmpliSeqTM Library Kit, cat number: 4480441).
4. Preparing a template for the obtained DNA library, and then sequencing to obtain the following data: carrying out high-throughput sequencing on the constructed library on an Ion Torrent platform after water-in-oil treatment, and carrying out the water-in-oil treatment method and the high-throughput sequencing method by referring to a high-throughput sequencer Ion Torrent and an instruction book of a matched device One Touch thereof.
5. Bioinformatics analysis was performed on the off-line data: first, low quality sequencing fragments were filtered using Ion Report software (v4.6, Thermo Fisher, Carlsbad, Calif., USA) and aligned to human reference genome hg19(http:// hgdownload. cse. ucsc. edu/downloads. html), and mutation sites were detected using the Torrent Variant Caller (v4.6.0.7) subroutine, with the software parameters using default settings. This software has been optimized for handling error types specific to Ion Torrent sequencing platforms. The mutations found, including SNPs and indels, were finally annotated using ANNOVAR (http:// innovar. openbioinformatics. org/en/test /) software, including the location of the mutation in the genome, the associated gene, the exon numbering of the gene, the nucleotide level variation, the corresponding protein level variation, and the mutations in the dbSNP database (http:// www.ncbi.nlm.nih.gov/SNP /), the thousand human genome database 1000Genomes (http:// www.1000genomes.org /), the exome integration database ExAC (http:// ExAC. branched. organization. org /) and the human gene mutation database HGMD (http:// www.hgmd.cf.ac.uk/ac/index. php), the Polyphen (http:// genetic genetics. bwh. apparatus. e. /) and SIFT:/. JO.f..
As shown in the following Table 10, the three detection results are positive for the mutation of the ALAS2 gene c.1355G > A nucleotide, and the detection results are shown in figure 1, which indicates that the primer composition and the kit have good repeatability.
TABLE 10 results of repeated measurements
| The result of the detection | For the first time | For the second time | The third time |
| Mutant genes | ALAS2 | ALAS2 | ALAS2 |
| Nucleotide changes | c.1355G>A | c.1355G>A | c.1355G>A |
| Amino acid changes | p.R452H | p.R452H | p.R452H |
| Mutation position | NM_000032:Exon9 | NM_000032:Exon9 | NM_000032:Exon9 |
| Sequencing reads number | 893751322 | 897641696 | 890480291 |
| Target area coverage | 98.56％ | 98.56％ | 98.55％ |
| Mean depth of sequencing (X) | 1091 | 1096 | 1087 |
| Uniformity of | 98.56％ | 98.55％ | 98.53％ |
Example 4 specific assay
4.1. The DNA samples extracted in example 3 were amplified using the amplification system and amplification procedure given in example 2 using the primers and concentrations given in example 1, sequenced using Ion Torrent, and further analyzed.
4.2. The primer concentrations given in example 1 were all modified to 0.05. mu.M, and the DNA samples extracted in example 3 were amplified using the same amplification system and amplification procedure, sequenced using Ion Torrent, and further analyzed.
4.3. The primer concentrations given in example 1 were all modified to 0.2. mu.M, and the DNA samples extracted in example 3 were amplified using the same amplification system and amplification procedure, sequenced using Ion Torrent, and further analyzed.
The results of the tests of 4.1, 4.2 and 4.3 are shown in Table 11 below.
TABLE 11 results of specific detection
As can be seen from the results of Table 7, the results of sequencing reads, sequencing depth and uniformity were poor when the primer concentration was too low or too high.
Example 5 sensitivity detection
According to the kit provided in example 2, DNA samples extracted in example 3 at different concentrations were amplified, sequenced using Ion Torrent, and further analyzed. Wherein, the concentrations of the DNA templates are respectively as follows: 5 ng/. mu.L, 2.5 ng/. mu.L, 1.25 ng/. mu.L, 0.625 ng/. mu.L, 0.3125 ng/. mu.L, 0.15625 ng/. mu.L.
The results are shown in Table 8 below:
TABLE 8 sensitivity test results
From the results, it is found that: the multiplex amplification system provided by the invention can be used for accurately detecting a sample with the concentration of 0.15625 ng/mu L, the sensitivity is 0.64%, the sensitivity is far better than 10% of first-generation sequencing, and the recognition capability is strong.
Example 6 accuracy testing
30 patients diagnosed as SA by clinical symptoms and relevant laboratory examination in the Kanghuamei medical diagnosis center are selected, 3mL of peripheral blood of each patient is collected to extract genomic DNA to be tested, and the test is approved by the central ethical committee and approved by the patients.
The kit and the method for using the kit provided in example 2 are used for sample detection, and the detection results are shown in the following table 9.
TABLE 9 accuracy test results
As can be seen from the above table, SA was detected in all of the 30 patients, 19 patients with hereditary sideroblasts anemia due to mutation of ALAS2 gene, and 11 patients with acquired sideroblasts anemia due to mutation of SF3B1 gene, and the results were consistent with the final clinical diagnosis. Therefore, the detection kit can be used for effectively diagnosing the patient with the sideroblasts anemia, and has the characteristics of quick and accurate diagnosis and large amount of acquired information.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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<210> 29
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 29
gctcatgccc aggtaatcat tactac 26
<210> 30
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 30
acgaattctt ttcatcctca tatctgctc 29
<210> 31
<211> 28
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 31
tgtaaacaaa gaaaggacag tcatgagt 28
<210> 32
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 32
tctgtacatg agcatttcat cagtaattga 30
<210> 33
<211> 27
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 33
acaacatgca ttcaagttga ctaaaga 27
<210> 34
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 34
tcacgtaatc agcaatgagt attctcttc 29
<210> 35
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 35
gtgtatcacc tcgtccagga gta 23
<210> 36
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 36
ttctgtgtgg gtgtgtgaaa taaaattttt 30
<210> 37
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 37
gtttgaacac aaacatctac agcagt 26
<210> 38
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 38
tgtagtcagg attttaacag cttgca 26
<210> 39
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 39
tcacccaatg gcctttgcaa ta 22
<210> 40
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 40
cttcagcctt tctgaagagt agtaagtttt 30
<210> 41
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 41
cacccaaaca tctgttgctg tt 22
<210> 42
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 42
agcagcagat attgatcata aacttgaaga 30
<210> 43
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 43
tcatttcctc atcaggagac tggaa 25
<210> 44
<211> 28
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 44
tgtgttaaag cctttatgga agggtatc 28
<210> 45
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 45
tgcttttata ttataaacgc aaaccaagac 30
<210> 46
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 46
caccagccat gaacatggct a 21
<210> 47
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 47
catcttcaag ttcagttgca gcaa 24
<210> 48
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 48
gggagttctt ttggaaagtg aaattaagaa 30
<210> 49
<211> 28
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 49
ctactggatt tctagctctt cctctatg 28
<210> 50
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 50
ccggaaagtc agagatgtat attggaaaa 29
<210> 51
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 51
aaaacaagaa aaagtcttat gtaaccagca 30
<210> 52
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 52
gaactgctgt tgtcatgaag acttg 25
<210> 53
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 53
ccaggagtct gatcagctgt tt 22
<210> 54
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 54
cgctaatggt agaaaaatac attgaaagca 30
<210> 55
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 55
gttctgttga ctgtggtata tcattaagca 30
<210> 56
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 56
gcttagacat cacactgtca atagatcttt 30
<210> 57
<211> 28
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 57
ttacggcata aatgtagtct tttcccat 28
<210> 58
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 58
acagcttgtt gacccatttg ttttt 25
<210> 59
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 59
acaggctgtg tgtgtacctc ta 22
<210> 60
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 60
cgagacacac tggtattaag attgtacaa 29
<210> 61
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 61
ctcattggtg gtgttccatt cttaattttt 30
<210> 62
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 62
ccatctggaa atcttccatt tttaaaacct 30
<210> 63
<211> 31
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 63
ttttaaaaat ctttaactta caggcagtgg g 31
<210> 64
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 64
gctattggac catgtagaat gttgc 25
<210> 65
<211> 27
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 65
cataactgcc tgaattacat gaggaga 27
<210> 66
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 66
cttataagac cgcatcttaa aggacttttt 30
<210> 67
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 67
cctggaaata gctaagagaa tggaatgac 29
<210> 68
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 68
gagtatgact cctgaacagc ttcag 25
<210> 69
<211> 28
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 69
ggaaagaatt accatctgca aaaggatc 28
<210> 70
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 70
tgtgctttat ggtgttctga tttttgttt 29
<210> 71
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 71
tatggacgaa ctaagtcatc aagtttgtac 30
<210> 72
<211> 28
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 72
aaaccacacc tattactctg ctcttttt 28
<210> 73
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 73
atctgactag ctggtgtttc atcc 24
<210> 74
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 74
tcaaacaact ttaaatgggt caccatttt 29
<210> 75
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 75
gggaagaagt aagaatttga tgcaaaagt 29
<210> 76
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 76
gtgcagtcat aaaccaaatg aatatgtctt 30
<210> 77
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 77
agaaaccata gaaaaaggca gaatcctag 29
<210> 78
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 78
ctctaggtgg cagttctgtc ac 22
<210> 79
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 79
aaaaagaaac ctaacagtct ctcaatcac 29
<210> 80
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 80
cagggcagat aaatcagttg aacc 24
<210> 81
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 81
attttgctaa ttgaatacaa agtggccaa 29
<210> 82
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 82
gacattgcta agtaaaagga aagtgaacaa 30
<210> 83
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 83
tccagatggc tggtcattaa cac 23
<210> 84
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 84
gatgttgtct ccaagcaaat aaaaacctta 30
<210> 85
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 85
agacagcaaa accttgtgtc aaaaag 26
<210> 86
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 86
tcagaagaag ccaggatatc atgc 24
<210> 87
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 87
tcctaagact ccaggctagc aa 22
<210> 88
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 88
tgttaaaatc gctttccttc ttgtttgaat 30
<210> 89
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 89
caagatggca cagcccataa ga 22
<210> 90
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 90
actcatgact gtcctttctt tgtttacat 29
<210> 91
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 91
cccaaaagca gctggttatt tatacg 26
<210> 92
<211> 28
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 92
gctcaaaaat atgggatcct acacctag 28
<210> 93
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 93
aggtaattgg tggatttacc tttcct 26
<210> 94
<211> 27
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 94
ggcatagtta aaacctgtgt ttggttt 27
<210> 95
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 95
aatgaagaga atactcattg ctgattacgt 30
<210> 96
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 96
ggatgcagaa tatgccaact actatactag 30
<210> 97
<211> 28
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 97
cattccaata atcagaaggt cagtggtt 28
<210> 98
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 98
gctattcgta gagccacagt caac 24
<210> 99
<211> 28
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 99
gcgatttctc tcatcaattt ctctttcc 28
<210> 100
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 100
tttttccttt gtggtattct gtgtactatt 30
<210> 101
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 101
tctgttcttg gaaagcataa agaataccat 30
<210> 102
<211> 28
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 102
ccttggaaaa gcagtctaaa aggttttt 28
<210> 103
<211> 27
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 103
tttttcttgt ccttctacct ggaaaga 27
<210> 104
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 104
aggcggacca tgataatttc cc 22
<210> 105
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 105
tatgaacctc ttacggcaaa gatgac 26
<210> 106
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 106
caagagcgtc atttacttgt gaaagttatt 30
<210> 107
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 107
acccaagcag actaatacag tcca 24
<210> 108
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 108
agcctccatc aaaacgaaaa cg 22
<210> 109
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 109
gcaacaaaca tgacaattta acaaactgg 29
<210> 110
<211> 27
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 110
aatcaacact tagtccagaa gagcaaa 27
<210> 111
<211> 33
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 111
aaatctttaa cttacctgta aacaatattg caa 33
<210> 112
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 112
cttgttgaac tatgtatggc ccaatg 26
<210> 113
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 113
ccataaattc tcagaacaaa gcatttcca 29
<210> 114
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 114
aagtgacagc agatttgctg gata 24
<210> 115
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 115
cgtcctggga accaatgtag at 22
<210> 116
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 116
cagcttaaga gcagtgtaat tagtaggttc 30
<210> 117
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 117
acaattatgt ccaatgagac agttctacc 29
<210> 118
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 118
gactcctgga gccagtaaaa ga 22
Claims (10)
1. A primer composition for detecting a sideroblasts anemia gene, comprising: the sideroblasts anemia gene is one or more of ALAS2 or SF3B 1.
2. The primer composition of claim 1, wherein: the mutation sites of the sideroblasts anemia gene are shown in the following table:
3. the primer composition of claim 2, wherein said primer composition is as shown in the following table:
4. use of the primer composition of any one of claims 1 to 3 for the preparation of a product for detecting sideroblastic anemia gene.
5. A product for detecting sideroblastic red blood cell anemia gene is characterized in that: the product comprises the primer composition of any one of claims 1 to 3.
6. The product of claim 5, wherein: the product also comprises DNA polymerase, Buffer and ddH2O、dNTP。
7. The product of claim 6, wherein: the product is an independent reagent or a kit.
8. Use of the primer composition of any one of claims 1 to 3 or the product of claim 5 for detecting a sideroblasts anemia gene.
9. A method for detecting sideroblastic red blood cell anemia gene, said method is a non-disease diagnosis and treatment method, characterized in that: the method comprises detecting sideroblastic anemia gene in a test sample using the primer composition of any one of claims 1-3 or the product of claim 5.
10. The method of claim 9, wherein: the method comprises the following steps:
(1) extracting genome DNA;
(2) multiplex PCR: amplifying the genomic DNA obtained in step (1) with the primer composition according to any one of claims 1 to 3;
(3) building a library;
(4) library sequencing and data processing.
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| CN109468372A (en) * | 2018-11-21 | 2019-03-15 | 中国人民解放军陆军军医大学第附属医院 | Primer combination, method and the kit in a variety of hereditary metabolic disorders hepatopathy targetings library are constructed based on high-flux sequence |
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|---|---|
| CN113481289B (en) | 2022-03-29 |
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