CN116121360A - Kit for detecting DBA pathogenic gene set and detection method - Google Patents

Kit for detecting DBA pathogenic gene set and detection method Download PDF

Info

Publication number
CN116121360A
CN116121360A CN202310026668.2A CN202310026668A CN116121360A CN 116121360 A CN116121360 A CN 116121360A CN 202310026668 A CN202310026668 A CN 202310026668A CN 116121360 A CN116121360 A CN 116121360A
Authority
CN
China
Prior art keywords
primer
primer combination
gene
dba
combination
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310026668.2A
Other languages
Chinese (zh)
Inventor
汝昆
李静
蔺亚妮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin Jiankang Huamei Medical Diagnosis Technology Co ltd
Original Assignee
Tianjin Jiankang Huamei Medical Diagnosis Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Jiankang Huamei Medical Diagnosis Technology Co ltd filed Critical Tianjin Jiankang Huamei Medical Diagnosis Technology Co ltd
Priority to CN202310026668.2A priority Critical patent/CN116121360A/en
Publication of CN116121360A publication Critical patent/CN116121360A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the technical field of biomedical detection, and provides a kit and a detection method for detecting a DBA pathogenic gene set, wherein the DBA pathogenic gene set comprises mutation sites of one or more mutant genes in RPL11, RPL15, RPL26, RPL27, RPL35A, RPL, RPS10, RPS17, RPS19, RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, TSR2 or GATA1, and the primer combination comprises a forward primer and a reverse primer. The primer combination is designed aiming at mutant genes, has the advantages of wide coverage and high detection efficiency, realizes auxiliary detection of DBA outside bone marrow morphology and blood routine examination by utilizing a high-throughput sequencing technology, accurately determines the genotype of DBA pathogenicity, has obvious advantages for guiding the subsequent treatment direction of diseases, and makes up the defects of clinical DBA in the aspects of molecular diagnosis, disease evolution, treatment, prognosis, medication and other guidance. The method 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 the existing detection method.

Description

Kit for detecting DBA pathogenic gene set and detection method
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a kit and a detection method for detecting DBA pathogenic gene sets.
Background
Diamond Blackfan Anemia (DBA) is the second most common congenital bone marrow failure syndrome following Fanconi anemia, and is characterized by red blood cell dysgenesis, physical malformation, and susceptibility to cancer. DBA usually occurs in infancy, with most patients presenting with clinical manifestations within one year of age. About 50% of DBA patients have physical abnormalities including short stature, craniofacial, skeletal, upper limb, cardiac system, genitourinary system defects, and the like. In addition, DBA patients have an increased risk of susceptibility to cancer, with a cumulative incidence of cancer of about 20% at age 46, far higher than normal populations, and high risk cancers including Acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), colon adenocarcinoma, osteosarcoma, etc.
The clinical manifestations of DBA patients and laboratory examination projects have lacked specificity. Patients have a clear family history to aid in diagnosis of DBA. Most patients are less than 1 year old in age, and most have large cell anemia, reticulocyte depletion, erythrocyte precursor cell depletion in bone marrow, elevated erythrocyte Adenosine Deaminase Activity (ADA), persistent hemoglobin F (HbF), and physical deformity. However, given the vast diversity in their clinical manifestations, and the fact that most patients are new variants, there is no relevant positive family history, diagnosing DBA with traditional methods is a challenge. Gene mutation analysis is an important standard for DBA diagnosis, and 80% of DBA patients can be detected at present.
The current method for detecting DBA mainly relates to blood convention, bone marrow cell morphology, molecular biology and the like. The experimental method of the molecular biology mainly comprises nested PCR, first-generation sequencing and the like. The PCR method cannot intuitively obtain a specific mutant sequence, whereas the first-generation sequencing method is affected by the sequencing flux, and cannot detect all exon regions of multiple genes at one time. And second generation sequencing is used as a high-flux detection method, so that the exon sequences of a plurality of genes can be accurately measured at one time, and accurate treatment is possible. However, the current second generation sequencing method lacks a convenient detection method in DBA gene detection and determination of pathogenic genes.
Disclosure of Invention
Aiming at the defects and actual requirements of DBA gene detection in the prior art, the invention provides a kit and a detection method for detecting DBA pathogenic gene sets, and solves the problems that MPN detection methods in the prior art cannot detect more related gene mutations at the same time and judgment of related gene mutations is difficult.
In a first aspect, the invention provides a set of DBA pathogenic genes comprising a mutation site of one or more of the mutant genes RPL11, RPL15, RPL26, RPL27, RPL35A, RPL5, RPS10, RPS17, RPS19, RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, TSR2 or GATA 1.
Further, the set of DBA pathogenic genes includes the mutation sites of RPL11, RPL15, RPL26, RPL27, RPL35A, RPL5, RPS10, RPS17, RPS19, RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, TSR2 and GATA1 mutant genes.
Further, the mutation sites of the mutant genes are shown in Table 1.
TABLE 1 mutation sites of mutant genes
Figure SMS_1
/>
Figure SMS_2
In a second aspect, the invention provides a primer combination for detecting the DBA pathogenic gene assembly according to claim 1, wherein the primer combination comprises a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO.1-SEQ ID NO.89, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.90-SEQ ID NO. 178.
Further, the primer combination includes primer combination a, primer combination B, primer combination C, primer combination D, primer combination E, primer combination F, primer combination G, primer combination H, primer combination I, primer combination J, primer combination K, primer combination L, primer combination M, primer combination N, primer combination O, primer combination P, primer combination Q for amplifying the mutation sites of RPS 11, RPL15, RPL26, RPL27, RPL35A, RPL5, RPS10, RPS17, RPS19, RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, TSR2, and GATA1 mutant genes, respectively;
the nucleotide sequences of the primers in the primer combination A are shown in the table 2,
TABLE 2 primer nucleotide sequences in primer combination A
Gene Primer sequences SEQ ID NO
RPL11 CTAGCACTACCCACAAGGGAGAA 1
RPL11 GGATGGCGACGGATAAAGGAAA 90
RPL11 AGCAGGATCAAGGTGAAAAGGAG 2
RPL11 CAGCAGGTAAATCAAGAAACAACCAAG 91
RPL11 TGGATGAATGCTTAATGTCTCTTTAAGTCA 3
RPL11 CTACATGTGTTACTAAAGGAGTGCTGAT 92
The nucleotide sequences of the primers in the primer combination B are shown in the table 3,
TABLE 3 primer nucleotide sequences in primer combination B
Figure SMS_3
/>
Figure SMS_4
The primer nucleotide sequences in the primer combination C are shown in Table 4,
TABLE 4 primer nucleotide sequences in primer combination C
Gene Primer sequences SEQ ID NO
RPL26 AAGAACGGATGGCTGCTGATTA 18
RPL26 TGACACTAGCCATAAATTTATTTCCCAGTT 107
RPL26 GTGCGTAAAATATCCATCAAGACAACG 19
RPL26 GGAAGCTGGATGAGATTTTGGTCATTTA 108
RPL26 TAGGGAGTGTCCTAAATCACCACA 20
RPL26 GGCTTTCCCTTGTGTTTTAGTGATTATTTA 109
RPL26 GCCCTTGATTTAAGAAAGCAACAGATA 21
RPL26 ATCTTTGGTAAACTTACAGAACCGGAA 110
The primer nucleotide sequences in the primer combination D are shown in Table 5,
TABLE 5 primer nucleotide sequences in primer combination D
Gene Primer sequences SEQ ID NO
RPL27 GCTCTGGGCATCTTTGGCTTTA 22
RPL27 GAAACAGGCCTTGAATTCCAAGC 111
RPL27 GCCTAAATAGGCCCTCAGTGAA 23
RPL27 ATTATTCAACAGAGCAGGGAGAAGAAAT 112
RPL27 CCAAGGTCAAGTTTGAAGAGAGGTAA 24
RPL27 GCCCACCCAGTGTTTCCTATTC 113
The primer nucleotide sequences in the primer combination E are shown in Table 6,
TABLE 6 primer nucleotide sequences in primer combination E
Gene Primer sequences SEQ ID NO
RPL35A CGAACTCCTACATGAAACTCCCA 25
RPL35A CCACCCAAAAGTTACTCAGAGCA 114
RPL35A CCACTAGAACATGTAATTGGTAGCCTT 26
RPL35A CCCTTGTACTCCAAAAATGCTGAG 115
RPL35A GGCGGAGTCGAAAGATGTTTAATC 27
RPL35A CCAACATCTCCTCCATCCTGTTC 116
The nucleotide sequences of the primers in the primer combination F are shown in Table 7,
TABLE 7 primer nucleotide sequences in primer combination F
Gene Primer sequences SEQ ID NO
RPL5 GGGACTGATGGCAGCTACTAAA 28
RPL5 GTTTTACCATGTCTGGAGTTACGCT 117
RPL5 GAAACCTGGGATTCTACAAGTGACA 29
RPL5 TGTAGAAAGGATACTCCACTGCATCT 118
RPL5 TGAAACCAGCATTTACATTGGTTTCTTG 30
RPL5 GTCCAGGCACCAAGGTCTTAAA 119
RPL5 CCTCTCTCTTTCACACGTCACT 31
RPL5 CTGGCATACGGGCAAGAAAAGG 120
RPL5 GACTTTTGGCCCTATGGTAGTAGG 32
RPL5 TGAGCTACCCGATCCTTCTTCT 121
RPL5 TTTATTTTAGTACCAAACGATTCCCTGGT 33
RPL5 CCCAAAACAAGTAATAAACCTATTTCCATAAAT 122
The primer nucleotide sequences in the primer combination G are shown in Table 8,
TABLE 8 primer nucleotide sequences in primer set G
Gene Primer sequences SEQ ID NO
RPS10 CCTCGGGACTTGAGAGACTGTAA 34
RPS10 GTTCAAGGCAAGTTCAAAAAGGGA 123
RPS10 TCTTCTTAGGCATCAACATCTGCAA 35
RPS10 GCATGTTGTCTACGGTTTTGTGTG 124
RPS10 TTGACAGGACCCACCCATACTA 36
RPS10 GGAGTGGTACTTGGCTTTTCCTG 125
RPS10 GGGAAGATCCCTCCATCCCATT 37
RPS10 TCAGAAGTGCTCTTGACAATCGT 126
RPS10 AGCCGATGGAACTCGAACG 38
RPS10 AGATCCGCCAGTTCTTCAGG 127
The primer nucleotide sequences in the primer combination H are shown in Table 9,
gene Primer sequences SEQ ID NO
RPS17 CCTTTGGAAGCCAAGAACCCAT 39
RPS17 GTATTTATATATGCACATGTGCTCAGTACCA 128
RPS17 CGGGTCCTTGGTAAAAGAGGAA 40
RPS17 GCGGTCCCATTAAAAATCAGATGG 129
RPS17 AAAAACATCAAAGTGCAATGAAGATACTC 41
RPS17 GATCATGATTCCCAAATCTAGTGGCTT 130
RPS17 GCACCTGAATCCCAGACAAGTG 42
RPS17 CACAGATCTCACCAGTAACTGCT 131
RPS17 TACTAGCTGGGTGACCTTGGATAATAA 43
RPS17 CTATCCTTGTGCAGCTCCTCTT 132
RPS17 AGGGAGTCTCAGGCTAACGAAA 44
RPS17 CCGGGTCATCATAGAAAAGTACTACAC 133
RPS17 CCGGCTAAACAGTGCCG 45
RPS17 AGCTTTAACAGGCTTCGCCT 134
TABLE 9 primer nucleotide sequences in primer combination H
The primer nucleotide sequences in the primer combination I are shown in the table 10,
table 10 primer nucleotide sequences in primer combination I
Gene Primer sequences SEQ ID NO
RPS19 CTGAGACCTTGATCAAGACCCTT 46
RPS19 TCAAGGCTACTCCATCAATGCAG 135
RPS19 GGCAGCCTTCCTCAAAAAGTGA 47
RPS19 CTGTGCCCAGAGTACAAAGAGAG 136
RPS19 CCCTAGGTTCCCTGTCACAGTT 48
RPS19 GTTTCCTTCTCCCGGAGTGTAA 137
RPS19 GGCACAGCATAGTTGTGTTGAG 49
RPS19 CAGAGGAGACAGGGAAGTATGGT 138
RPS19 GAAAAGGACCAAGATGGGTAAGCA 50
RPS19 TGCTGTTTGCTTTTCTGCAGAATC 139
The nucleotide sequences of the primers in the primer combination J are shown in Table 11,
table 11 primer nucleotide sequences in primer set J
Figure SMS_5
/>
Figure SMS_6
The nucleotide sequences of the primers in the primer combination K are shown in Table 12,
table 12 primer nucleotide sequences in primer combination K
Gene Primer sequences SEQ ID NO
RPS26 AGGATGGTGGTCAAGTTCTTTGG 60
RPS26 CCATGAAAATAATGTGTGTCACTTCACA 149
RPS26 CGCCGTTTTCCTAACAGTTTTCC 61
RPS26 GCTTTAAGATACTGGGATCCTCACA 150
RPS26 GCGTATACCGGAAAACTGCATC 62
RPS26 CACCGGGAGAATCCTTAGGAGA 151
The primer nucleotide sequences in the primer combination L are shown in Table 13,
TABLE 13 primer nucleotide sequences in primer combination L
Gene Primer sequences SEQ ID NO
RPS27 TGGATGATGAGTTGGGCATAAGTG 63
RPS27 TCACTGTTAAGACCGAAAAATCCTAAGAAA 152
RPS27 GGGCTATTTTCGACCATCCCAT 64
RPS27 CAGCTAACACTACAACATTTTCCAACTG 153
The primer nucleotide sequences in the primer combination M are shown in Table 14,
table 14 primer nucleotide sequences in primer combination M
Gene Primer sequences SEQ ID NO
RPS28 GGCAGAGTCTACATACAGAGACTGA 65
RPS28 GCCACTCAAGAAACCTGGAGAAA 154
RPS28 GCGGTGCTAGAACGTCCTATAA 66
RPS28 CTTGGTGACCTGGAGGAAAGAAG 155
The primer nucleotide sequences in the primer combination N are shown in Table 15,
table 15 primer nucleotide sequences in primer combination N
Gene Primer sequences SEQ ID NO
RPS29 TCAGAATTACCACTCCAGGGAAGA 67
RPS29 GTCTGAGTGCGGAGTCTTGTAA 156
RPS29 GAAGTTGCAGAGAGCTGAGCT 68
RPS29 CTGTAAAAACAAAAACAACAAAAACCAGAGACC 157
RPS29 CAGGAGGCTTGAACCTAGGTTG 69
RPS29 GGGCATCTACTCAATGAAAAACCATGATA 158
RPS29 CCTAGCAGAGGTTATGCAAGCA 70
RPS29 GGAAATGAGGAAGACATGAAAGCAGA 159
RPS29 CATTAGCCACACGAACAACTGT 71
RPS29 GCAACAAATCTTACTGTGTTGAGATCACT 160
RPS29 CGTGGCCTCCTCTACTTGAGAT 72
RPS29 CGTTTCCTTTGAGCACTGCATTC 161
The primer nucleotide sequences in the primer combination O are shown in Table 16,
table 16 primer nucleotide sequences in primer combination O
Gene Primer sequences SEQ ID NO
RPS7 GGGCACAATTTCACGAAACTATTAGG 73
RPS7 CACACAGGACGCCACTGATAAT 162
RPS7 GACCACAACGTTTACTTTCTGAAGC 74
RPS7 AATGCAGTCTAAATACAAGCAATTTTAGCA 163
RPS7 AAAGCGTTACCAAGACACTCGT 75
RPS7 CCTGACTCCAAAAGGCGTTTTC 164
RPS7 CCTGCTCTCCGACAGAACTTTT 76
RPS7 GCTTTCTCCTGGGAGAACTGAA 165
RPS7 GTCGGTCCTGCTGTTCGT 77
RPS7 GGCAAGTCATCATCAACCGTAAC 166
The nucleotide sequences of the primers in the primer combination P are shown in Table 17,
TABLE 17
Figure SMS_7
/>
Figure SMS_8
The primer nucleotide sequences in the primer combination Q are shown in Table 18.
Table 18 primer nucleotide sequences in primer combination Q
Gene Primer sequences SEQ ID NO
GATA1 TTCTGTCCTCGCAGGTTAATCC 83
GATA1 ACAGCCACTCAATGGAGTTACC 172
GATA1 GCGCTGACCCTAGACTGATTTT 84
GATA1 CTCTGTCTTCAAAGTCTCCAGGAA 173
GATA1 GGTGGTGTCCTTCTCCTCTTGTA 85
GATA1 GGCATTGTTACACACTCCACTCA 174
GATA1 ACAGGCCACTACCTATGCAAC 86
GATA1 GGGAAGATGTGGCTTCTTGTACAG 175
GATA1 GTGTCCTCCACACCAGAATCAG 87
GATA1 GGTATTCTGACCTAGCCAAGGATCT 176
GATA1 CCTATCAGCCTTGGAGGCTTTC 88
GATA1 TCTTTTTCCCTTTTCCAGATGCCTT 177
GATA1 GGTACTGCCCATCTCTACCAAG 89
GATA1 CAGAGACTTGGGTTGTCCAGAA 178
In a third aspect, the invention provides a kit for detecting a set of DBA pathogenic genes, the kit comprising reagents for extracting whole genomic DNA from a sample, a multiplex amplification system and reagents for constructing a DNA library.
Further, the multiplex amplification system comprises primers in the primer combination and an amplification premix, wherein the working concentration of the primers in the primer combination is 0.1 mu M.
In a fourth aspect, the present invention provides a detection method for detecting a set of DBA pathogenic genes, the detection method comprising the steps of:
s1, extracting sample whole genome DNA;
s2, amplifying a target sequence of a DBA pathogenic gene;
s3, constructing a DNA library;
s4, performing on-machine sequencing to obtain an original sequencing signal file;
s5, performing biological analysis on the original sequencing signal file.
Further, the biological analysis in step S5 includes mutation detection annotations, the content of which includes one or more of a position of a mutation in a genome, an associated gene, a gene exon number, a nucleotide level variation, and a corresponding protein level variation.
The invention has the advantages that:
1) The invention provides a primer composition for detecting DBA pathogenic gene sets, a comprehensive DBA pathogenic gene set is obtained by setting a screening method of pathogenic mutation sites, and the primer composition is designed according to the characteristics of mutant genes, so that the primer composition has the advantages of wide coverage and high detection efficiency, realizes auxiliary detection of DBA outside bone marrow morphology and blood routine examination by using a high-throughput sequencing technology, accurately determines the genotype of DBA pathogenic, has obvious advantages for guiding the subsequent treatment direction of diseases, and makes up the defects of clinical DBA in the aspects of molecular diagnosis, disease evolution, treatment, prognosis, medication and other guidance.
2) The kit for detecting the DBA pathogenic gene set and the detection method provided by the invention have the advantages of simplicity and convenience in operation, high accuracy, high sensitivity and the like, and the technical defects of low monitoring accuracy and limitation of the detection method at present are overcome.
Drawings
FIG. 1 is a schematic diagram showing the detection result of the mutation of c.3G > T nucleotide of the RPS19 gene.
FIG. 2 is a schematic diagram showing the detection result of the mutation of c.82C > T nucleotide of RPS26 gene.
Detailed Description
The invention is described in further detail with examples given only for the purpose of illustration and not for the purpose of limiting the scope of the invention. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
EXAMPLE 1 screening of mutation sites of DBA pathogenic Gene
The database of dbSNP (http:// www.ncbi.nlm.nih.gov/snp /), the database of thousand genome 1000genome (http:// www.1000genomes.org /), the database of exome integration ExAC (http:// ExAC. Broadenstitute. Org /) and the database of human gene mutation HGMD (http:// www.hgmd.cf.ac.uk/ac/index. Php) are used, as well as the tools of functional prediction PolyPhen (http:// genetics. Bwh. Harvard. U/pph2 /) and SIFT (http:// SIFT. Jcvi. Org /), and the following method steps are used to screen for pathogenic mutation sites:
step 1: according to the position and type of the mutation sites, filtering out a plurality of mutation sites which do not affect the protein product sequence to obtain a plurality of mutation sites to be screened;
step 2: first annotation, annotating the proportion of each mutation site to be screened in the population by using 1000genome data and ExAC data, and marking the mutation site as a non-polymorphism site if the proportion is less than or equal to 1%; if the proportion is more than 1%, marking as polymorphic sites;
step 3: a second annotation for searching whether each mutation site to be screened is recorded in HGMD (human gene mutation database) and appears in DBA-related diseases, if yes, the mutation site is marked as recorded in DBA, and if not, the mutation site is marked as not recorded in DBA;
step 4: thirdly, predicting whether each mutation site to be screened affects the protein function of the corresponding mutant gene by using protein function prediction software PolyPhen-2 and SIFT, if so, marking the mutation site as affecting the protein function, and if not, marking the mutation site as not affecting the protein function;
step 5: judging the pathogenicity of each mutation site to be screened according to the first annotation, the second annotation and the third annotation, judging the pathogenicity to be relevant when the annotation of the mutation site to be screened is a polymorphism site, DBA records or affects two or three of protein functions, and judging the pathogenicity to be of unknown significance when the annotation of the mutation site to be screened is a DBA records;
step 6: whether the mutation site to be screened with unknown query meaning is recorded in the scientific literature to be directly related to DBA or not is judged to be related to pathogenicity if the mutation site to be screened is recorded in the scientific literature, and if the mutation site to be screened is not recorded in the scientific literature to be directly related to DBA, the mutation site to be screened is judged to be related to pathogenicity if the mutation site is not recorded in the scientific literature to be directly related to DBA.
Finally screening to obtain the following genes: (1) RPS19 gene: mutations associated with Diamond Blackfan anemia type 1; (2) RPS24 gene: mutations associated with Diamond Blackfan anemia type 3; (3) RPS17 Gene: mutations associated with Diamond Blackfan anemia type 4; (4) RPL35A Gene: mutations associated with Diamond Blackfan anemia type 5; (5) RPL5 gene: mutations associated with Diamond Blackfan anemia type 6; (6) RPL11 Gene: mutations associated with Diamond Blackfan anemia type 7; (7) RPS7 gene: mutations associated with Diamond Blackfan anemia type 8; (8) RPS10 gene: its mutation is associated with Diamond Blackfan anemia type 9; (9) RPS26 gene: its mutation is associated with Diamond Blackfan anemia type 10; (10) RPL26 gene: mutations associated with Diamond Blackfan anemia type 11; (11) RPL15 gene: mutations associated with Diamond Blackfan anemia type 12; (12) RPS29 gene: its mutation is associated with Diamond Blackfan anemia type 13; (13) TSR2 Gene: mutations associated with Diamond Blackfan anemia type 14; (14) RPS28 gene: its mutation is associated with Diamond Blackfan anemia type 15; (15) RPL27 gene: mutations associated with Diamond Blackfan anemia type 16; (16) RPS27 gene: its mutation is associated with Diamond Blackfan anemia type 17; (17) GATA1 Gene: mutations are associated with X-linked anemia with or without neutropenia and/or platelet abnormalities.
The mutation sites corresponding to each mutant gene are shown in Table 1.
Example 2 primer combinations for detection of DBA pathogenic Gene sets
Primer combinations were designed based on the mutation sites in the DBA pathogenic gene set of example 1, and included primer combination a, primer combination B, primer combination C, primer combination D, primer combination E, primer combination F, primer combination G, primer combination H, primer combination I, primer combination J, primer combination K, primer combination L, primer combination M, primer combination N, primer combination O, primer combination P, primer combination Q for amplifying the mutation sites of RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, TSR2 and GATA1 mutant genes, respectively, with specific nucleotide sequences as shown in tables 2-18.
Example 3A kit for detecting DBA pathogenic Gene set
The kit comprises a reagent for extracting the whole genome DNA of a sample, a multiplex amplification system and a reagent for constructing a DNA library;
the composite amplification system comprises a primer combination and an amplification premix for detecting the DBA pathogenic gene set, wherein the primer combination comprises a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO.1-SEQ ID NO.89, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.90-SEQ ID NO. 178.
The working concentration of each primer in the primer set was 0.1. Mu.M.
The DNA library constructing reagent is used for preparing the amplified product obtained by the multiplex amplification system into a DNA library which can be sequenced by a sequencing platform.
Example 4 detection method for detecting DBA pathogenic Gene set
S1, extracting sample whole genome DNA; the whole genomic DNA of the peripheral blood or bone marrow sample was extracted using the reagent for extracting whole genomic DNA of the sample in example 3, to obtain a sample DNA to be detected.
S2, amplifying a target sequence of a DBA pathogenic gene; the sample DNA to be detected extracted in S1 was subjected to multiplex amplification using the multiplex amplification system in example 2.
S2-1, a multiplex amplification system was prepared, and the multiplex amplification system is shown in Table 19 below.
Table 19 composite amplification System
Composition of the components Volume of
AgriSeq TM Amplification Mix 2μL
Ion AmpliSeq TM Primer Pool 5μL
Diluted DNA sample (10 ng/. Mu.L) 3μL
The DNA sample is the DNA of the sample to be detected obtained in the step S1, the Primer Pool is the Primer mixture in the Primer combination, and the concentration of each Primer is 0.10 mu M.
The conditions of the composite amplification reaction are as follows: 99 ℃ for 2min;99 ℃,15s,62 ℃,4min,2cycles;99 ℃,15s,60 ℃,8min,14cycles; storing at 10 ℃.
S3, constructing a DNA library, and preparing an amplification product into the DNA library for sequencing by a sequencing platform by adopting a DNA library constructing reagent in the kit.
S3-1, performing enzyme digestion on the amplified product. The cleavage system is shown in Table 20 below.
Table 20 enzyme digestion system
Step 2 Complex amplification product 20μL
Pre-ligation 2μL
The conditions of the enzyme digestion reaction are as follows: 50 ℃ for 20min;55 ℃ for 20min;60 ℃ for 20min; storing at 10 ℃. And (3) purifying the product by a magnetic bead method after the reaction is finished.
Wherein, pre-ligation was purchased from ThermoFisher scientific under the designation A34111.
S3-2, end repair and A addition. The reaction system is shown in Table 21 below.
TABLE 21 end repair & addition A
Cleavage product 40μL
End Repair&A-Tailing Enzyme 4μL
End Repair&A-Tailing Buffer 6μL
The reaction procedure was as follows: 20 ℃ for 30min;65 ℃ for 30min;4 ℃, and storing.
Wherein the End Repair & A-tagging Enzyme and the End Repair & A-tagging Buffer are purchased from Naonda, cat. No. 1002103.
S3-3, joint connection. The reaction system is shown in Table 22 below.
Meter 22 joint connection
Figure SMS_9
Figure SMS_10
The reaction conditions were as follows: 20 ℃ for 20min. And (3) purifying the product by a magnetic bead method after the reaction is finished.
Among them, IDT UDI linker (15. Mu.M), ligation Buffer, DNA Ligation were purchased from Naonda, accession number 1003227, ligation accession number 1002103.
S3-4, library enrichment. The reaction system is shown in Table 23 below.
TABLE 23 library enrichment
2X HiFi PCR Master Mix 25μL
Amplification Primer Mix(10μM) 5μL
Adapter-ligated library 20μL
The amplification reaction procedure was as follows: 98 ℃ for 2min;98 ℃,15s,60 ℃,30s,72 ℃,30s,7cycles;72 ℃ for 2min;4 ℃, and storing. And (3) purifying the product by a magnetic bead method after the reaction is finished.
Of these, 2X HiFi PCR Master Mix was purchased from nafida under the product number 1002103.
S4, preparing a template of the DNA library for sequencing to obtain an original sequencing signal file
S5, performing biological analysis on the original sequencing signal file, including data analysis and mutation detection annotation.
Example 5 repeatability test
2 patients diagnosed with DBA by clinical symptoms and related laboratory tests are selected, and the patients are respectively rpS19 gene c.3G > T nucleotide mutation, the amino acid mutation is p.M1I and rpS26 gene c.82C > T nucleotide mutation, and the amino acid mutation is p.R28X by first-generation sequencing detection.
The test was repeated three times on samples of 2 patients according to this example using the test method of example 4, and the specific steps and test method were as follows:
DNA extraction: total genomic DNA of a patient bone marrow sample was extracted using the Tiangen DNA extraction kit (cat# DP 318-03).
2. The extracted DNA was subjected to multiplex PCR amplification according to the primer combinations and the kit provided in example 2 and example 3.
3. The amplified product was prepared into a DNA library for sequencing by Ion Torrent sequencing platform, and specific operation steps are shown in the instruction of Life company kit (name: ion AmpliSeqTM Library Kit, cat#: 4480441).
4. Preparing templates of the obtained DNA library, and sequencing to obtain the following machine data: and carrying out high-flux sequencing on the constructed library on a Ion Torrent platform after water-in-oil treatment, wherein the water-in-oil treatment method and the high-flux sequencing method are carried out by referring to the use instruction of a high-flux sequencer Ion Torrent and a matched device One Touch thereof.
5. Bioinformatics analysis is carried out on the off-machine data: first, low quality sequencing fragments were filtered using Ion Report software (v 4.6, thermo Fisher, carlsbad, calif., USA) and qualified sequencing fragments were aligned to human reference genome hg19 (http:// hgdownload. Cse. Ucsc. Edu/downloads. Html), mutation sites were detected using Torrent Variant Caller (v 4.6.0.7) subroutine, and software parameters were set by default. This software has been optimized to handle error types specific to Ion Torrent sequencing platforms. Finally, the mutations found include SNP and Indel were annotated using ANNOVAR (http:// ANNOVAR. Openbioiformat. Org/en/latest /) software, the annotation content included the position of the mutation in the genome, the associated gene, the gene exon numbering, nucleotide level variation, corresponding protein level variation, and the mutation in dbSNP database (http:// www.ncbi.nlm.nih.gov/SNP /), thousand genome database 1000genome (http:// www.1000genomes.org /), exome integration database ExAC (http:// ExAC. Broadinstitute. Org /) and human gene mutation database HGMD (http:// www.hgmd.cf.ac.uk/ac/index. Php), the annotation of the mutation in the database of the dbSNP (http:// www.ncbi.nlm.nih.gov/snd. Edu/pph2 /), and SIFT (http:// hvt. Jvi /) functions, etc.
Wherein, as shown in Table 24-1 and Table 24-2, the three detection results of Table 24-1 are positive for the mutation of the nucleotide c.3G > T of the RPS19 gene, and the detection results are shown in FIG. 1; the three detection results of the table 24-2 are all the nucleotide mutations of the RPS26 gene c.82C > T, and the detection results are shown in the figure 2, which shows that the primer combination and the kit have better repeatability.
TABLE 24-1 patient-repeatability test results
Detection result First time Second time Third time
Mutant genes RPS19 RPS19 RPS19
Nucleotide changes c.3G>T c.3G>T c.3G>T
Amino acid changes p.M1I p.M1I p.M1I
Mutation position NM_001022:Exon2 NM_001022:Exon2 NM_001022:Exon2
Sequencing reads number 894761267 895632678 892453258
Target area coverage 98.56％ 98.56％ 98.55％
Average sequencing depth (X) 1088 1092 1089
Uniformity of 98.65％ 98.63％ 98.66％
TABLE 24-2 patient two-fold test results
Detection result First time Second time Third time
Mutant genes RPS26 RPS26 RPS26
Nucleotide changes c.82C>T c.82C>T c.82C>T
Amino acid changes p.R28X p.R28X p.R28X
Mutation position NM_001029:Exon2 NM_001029:Exon2 NM_001029:Exon2
Sequencing reads number 804367769 811893039 801859345
Target area coverage 98.56％ 98.56％ 98.55％
Average sequencing depth (X) 962 971 959
Uniformity of 98.63％ 98.64％ 98.63％
EXAMPLE 6 specific assay
4-1 Using the primer combinations and concentrations given in example 3, the DNA samples extracted in example 5 were amplified using the amplification system and amplification procedure given in example 4, sequenced using Ion Torrent, and further analyzed.
4-2 the DNA samples extracted in example 5 were amplified using the same amplification system and amplification procedure with the primer concentrations given in example 3 all modified to 0.05. Mu.M, sequenced using Ion Torrent, and further analyzed.
4-3 the DNA samples extracted in example 5 were amplified using the same amplification system and amplification procedure with the primer concentrations given in example 3 all modified to 0.2. Mu.M, sequenced using Ion Torrent, and further analyzed.
The results of the above-mentioned tests of 4-1, 4-2 and 4-3 are shown in Table 25-1 and Table 25-2.
TABLE 25-1 patient-specific test results
Detection result 4-1 4-2 4-3
Mutant genes RPS19 RPS19 RPS19
Nucleotide changes c.3G>T c.3G>T c.3G>T
Amino acid changes p.M1I p.M1I p.M1I
Mutation position NM_001022:Exon2 NM_001022:Exon2 NM_001022:Exon2
Sequencing reads number 894761267 616158752 655647351
Target area coverage 98.56％ 73.35％ 75.33％
Average sequencing depth (X) 1088 723 786
Uniformity of 98.65％ 79.92％ 84.35％
TABLE 25-2 patient bispecific assay results
Figure SMS_11
/>
Figure SMS_12
As can be seen from the results of the tests in Table 25-1 and Table 25-2, the primer concentration was too low or too high, and the sequencing reads number, sequencing depth and uniformity were poor.
Example 7 sensitivity detection
The kit provided in example 3 was used to amplify DNA samples of different concentrations extracted in example 5, sequenced using Ion Torrent, and further analyzed. Wherein, the DNA template concentration is respectively: 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 test results are shown in Table 26 below:
table 26 sensitivity test results
Figure SMS_13
/>
Figure SMS_14
From the results, it can be seen that: the complex amplification system provided by the invention can accurately detect samples with the concentration of 0.15625 ng/. Mu.L, has the sensitivity of 0.64%, is far better than 10% of the first-generation sequencing, and has strong recognition capability.
Example 8 accuracy testing
A total of 20 patients diagnosed with DBA by clinical symptoms and related laboratory tests at the medical diagnostic center, see Kang Huamei, were selected, 3mL of each patient was collected and genomic DNA was extracted for testing, and the test was approved by the central ethics committee and approved by the patient.
Sample detection was performed according to the kit and the detection method provided in example 3 and example 4, and the detection results are shown in table 27 below.
Table 9 accuracy test results
Figure SMS_15
/>
Figure SMS_16
As can be seen from the above table, DBA was detected in the above 20 patients, 13 patients with Diamond Blackfan anemia having RPS19 gene mutation, 1 patient with Diamond Blackfan anemia having RPL35A gene mutation, 2 patients with Diamond Blackfan anemia having RPL11 gene mutation, 2 patients with Diamond Blackfan anemia having RPL5 gene mutation, 1 patient with Diamond Blackfan anemia having RPS10 gene mutation, and 1 patient with Diamond Blackfan anemia having RPS26 gene mutation, and the results were consistent with the clinical final diagnosis. Therefore, the detection kit can be used for effectively diagnosing the patient with the Diamond Blackfan anemia, and has the characteristics of rapid and accurate diagnosis and large information acquisition.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (9)

  1. A DBA pathogenic gene set, characterized in that the DBA pathogenic gene set comprises mutation sites of one or more mutant genes of RPL11, RPL15, RPL26, RPL27, RPL35A, RPL, RPS10, RPS17, RPS19, RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, TSR2, GATA 1.
  2. 2. The set of DBA pathogenic genes according to claim 1, wherein the set of DBA pathogenic genes comprises mutation sites of RPL11, RPL15, RPL26, RPL27, RPL35A, RPL, RPS10, RPS17, RPS19, RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, TSR2 and GATA1 mutant genes.
  3. 3. The set of DBA pathogenic genes according to any one of claims 1-2, wherein the mutation sites of the mutated genes are as follows:
    Figure FDA0004044881890000011
    />
    Figure FDA0004044881890000021
    。
  4. 4. a primer combination for detecting the DBA pathogenic gene set according to claim 1, wherein the primer combination comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID No.1-SEQ ID No.89, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.90-SEQ ID No. 178.
  5. 5. A primer combination for detecting a DBA pathogenic gene set according to claim 3, wherein the primer combination comprises primer combination a, primer combination B, primer combination C, primer combination D, primer combination E, primer combination F, primer combination G, primer combination H, primer combination I, primer combination J, primer combination K, primer combination L, primer combination M, primer combination N, primer combination O, primer combination P, primer combination Q for amplifying the mutation sites of RPS 11, RPL15, RPL26, RPL27, RPL35A, RPL, RPS10, RPS17, RPS19, RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, TSR2 and GATA1 mutant genes, respectively;
    the primer nucleotide sequences in the primer combination A are shown in the following table:
    gene Primer sequences SEQ ID NO RPL11 CTAGCACTACCCACAAGGGAGAA 1 RPL11 GGATGGCGACGGATAAAGGAAA 90 RPL11 AGCAGGATCAAGGTGAAAAGGAG 2 RPL11 CAGCAGGTAAATCAAGAAACAACCAAG 91 RPL11 TGGATGAATGCTTAATGTCTCTTTAAGTCA 3 RPL11 CTACATGTGTTACTAAAGGAGTGCTGAT 92
    ；
    The primer nucleotide sequences in the primer combination B are shown in the following table:
    Figure FDA0004044881890000031
    />
    Figure FDA0004044881890000041
    the primer nucleotide sequences in the primer combination C are shown in the following table:
    gene Primer sequences SEQ ID NO RPL26 AAGAACGGATGGCTGCTGATTA 18 RPL26 TGACACTAGCCATAAATTTATTTCCCAGTT 107 RPL26 GTGCGTAAAATATCCATCAAGACAACG 19 RPL26 GGAAGCTGGATGAGATTTTGGTCATTTA 108 RPL26 TAGGGAGTGTCCTAAATCACCACA 20 RPL26 GGCTTTCCCTTGTGTTTTAGTGATTATTTA 109 RPL26 GCCCTTGATTTAAGAAAGCAACAGATA 21 RPL26 ATCTTTGGTAAACTTACAGAACCGGAA 110
    ；
    The primer nucleotide sequences in the primer combination D are shown in the following table:
    gene Primer sequences SEQ ID NO RPL27 GCTCTGGGCATCTTTGGCTTTA 22 RPL27 GAAACAGGCCTTGAATTCCAAGC 111 RPL27 GCCTAAATAGGCCCTCAGTGAA 23 RPL27 ATTATTCAACAGAGCAGGGAGAAGAAAT 112 RPL27 CCAAGGTCAAGTTTGAAGAGAGGTAA 24 RPL27 GCCCACCCAGTGTTTCCTATTC 113
    ；
    The primer nucleotide sequences in the primer combination E are shown in the following table:
    gene Primer sequences SEQ ID NO RPL35A CGAACTCCTACATGAAACTCCCA 25 RPL35A CCACCCAAAAGTTACTCAGAGCA 114 RPL35A CCACTAGAACATGTAATTGGTAGCCTT 26 RPL35A CCCTTGTACTCCAAAAATGCTGAG 115 RPL35A GGCGGAGTCGAAAGATGTTTAATC 27 RPL35A CCAACATCTCCTCCATCCTGTTC 116
    ；
    The primer nucleotide sequences in the primer combination F are shown in the following table:
    gene Primer sequences SEQ ID NO RPL5 GGGACTGATGGCAGCTACTAAA 28 RPL5 GTTTTACCATGTCTGGAGTTACGCT 117 RPL5 GAAACCTGGGATTCTACAAGTGACA 29 RPL5 TGTAGAAAGGATACTCCACTGCATCT 118 RPL5 TGAAACCAGCATTTACATTGGTTTCTTG 30 RPL5 GTCCAGGCACCAAGGTCTTAAA 119 RPL5 CCTCTCTCTTTCACACGTCACT 31 RPL5 CTGGCATACGGGCAAGAAAAGG 120 RPL5 GACTTTTGGCCCTATGGTAGTAGG 32 RPL5 TGAGCTACCCGATCCTTCTTCT 121 RPL5 TTTATTTTAGTACCAAACGATTCCCTGGT 33 RPL5 CCCAAAACAAGTAATAAACCTATTTCCATAAAT 122
    ；
    The primer nucleotide sequences in the primer combination G are shown in the following table:
    gene Primer sequences SEQ ID NO RPS10 CCTCGGGACTTGAGAGACTGTAA 34 RPS10 GTTCAAGGCAAGTTCAAAAAGGGA 123 RPS10 TCTTCTTAGGCATCAACATCTGCAA 35 RPS10 GCATGTTGTCTACGGTTTTGTGTG 124 RPS10 TTGACAGGACCCACCCATACTA 36 RPS10 GGAGTGGTACTTGGCTTTTCCTG 125 RPS10 GGGAAGATCCCTCCATCCCATT 37 RPS10 TCAGAAGTGCTCTTGACAATCGT 126 RPS10 AGCCGATGGAACTCGAACG 38 RPS10 AGATCCGCCAGTTCTTCAGG 127
    ；
    The primer nucleotide sequences in the primer combination H are shown in the following table:
    gene Primer sequences SEQ ID NO RPS17 CCTTTGGAAGCCAAGAACCCAT 39 RPS17 GTATTTATATATGCACATGTGCTCAGTACCA 128 RPS17 CGGGTCCTTGGTAAAAGAGGAA 40 RPS17 GCGGTCCCATTAAAAATCAGATGG 129 RPS17 AAAAACATCAAAGTGCAATGAAGATACTC 41 RPS17 GATCATGATTCCCAAATCTAGTGGCTT 130 RPS17 GCACCTGAATCCCAGACAAGTG 42 RPS17 CACAGATCTCACCAGTAACTGCT 131 RPS17 TACTAGCTGGGTGACCTTGGATAATAA 43 RPS17 CTATCCTTGTGCAGCTCCTCTT 132 RPS17 AGGGAGTCTCAGGCTAACGAAA 44 RPS17 CCGGGTCATCATAGAAAAGTACTACAC 133 RPS17 CCGGCTAAACAGTGCCG 45 RPS17 AGCTTTAACAGGCTTCGCCT 134
    ；
    The primer nucleotide sequences in the primer combination I are shown in the following table:
    gene Primer sequences SEQ ID NO RPS19 CTGAGACCTTGATCAAGACCCTT 46 RPS19 TCAAGGCTACTCCATCAATGCAG 135 RPS19 GGCAGCCTTCCTCAAAAAGTGA 47 RPS19 CTGTGCCCAGAGTACAAAGAGAG 136 RPS19 CCCTAGGTTCCCTGTCACAGTT 48 RPS19 GTTTCCTTCTCCCGGAGTGTAA 137 RPS19 GGCACAGCATAGTTGTGTTGAG 49 RPS19 CAGAGGAGACAGGGAAGTATGGT 138 RPS19 GAAAAGGACCAAGATGGGTAAGCA 50 RPS19 TGCTGTTTGCTTTTCTGCAGAATC 139
    ；
    The primer nucleotide sequences in the primer combination J are shown in the following table:
    Figure FDA0004044881890000061
    />
    Figure FDA0004044881890000071
    the primer nucleotide sequences in the primer combination K are shown in the following table:
    gene Primer sequences SEQ ID NO RPS26 AGGATGGTGGTCAAGTTCTTTGG 60 RPS26 CCATGAAAATAATGTGTGTCACTTCACA 149 RPS26 CGCCGTTTTCCTAACAGTTTTCC 61 RPS26 GCTTTAAGATACTGGGATCCTCACA 150 RPS26 GCGTATACCGGAAAACTGCATC 62 RPS26 CACCGGGAGAATCCTTAGGAGA 151
    ；
    The primer nucleotide sequences in the primer combination L are shown in the following table:
    gene Primer sequences SEQ ID NO RPS27 TGGATGATGAGTTGGGCATAAGTG 63 RPS27 TCACTGTTAAGACCGAAAAATCCTAAGAAA 152 RPS27 GGGCTATTTTCGACCATCCCAT 64 RPS27 CAGCTAACACTACAACATTTTCCAACTG 153
    ；
    The primer nucleotide sequences in the primer combination M are shown in the following table:
    gene Primer sequences SEQ ID NO RPS28 GGCAGAGTCTACATACAGAGACTGA 65 RPS28 GCCACTCAAGAAACCTGGAGAAA 154 RPS28 GCGGTGCTAGAACGTCCTATAA 66 RPS28 CTTGGTGACCTGGAGGAAAGAAG 155
    ；
    The primer nucleotide sequences in the primer combination N are shown in the following table:
    gene Primer sequences SEQ ID NO RPS29 TCAGAATTACCACTCCAGGGAAGA 67 RPS29 GTCTGAGTGCGGAGTCTTGTAA 156 RPS29 GAAGTTGCAGAGAGCTGAGCT 68 RPS29 CTGTAAAAACAAAAACAACAAAAACCAGAGACC 157 RPS29 CAGGAGGCTTGAACCTAGGTTG 69 RPS29 GGGCATCTACTCAATGAAAAACCATGATA 158 RPS29 CCTAGCAGAGGTTATGCAAGCA 70 RPS29 GGAAATGAGGAAGACATGAAAGCAGA 159 RPS29 CATTAGCCACACGAACAACTGT 71 RPS29 GCAACAAATCTTACTGTGTTGAGATCACT 160 RPS29 CGTGGCCTCCTCTACTTGAGAT 72 RPS29 CGTTTCCTTTGAGCACTGCATTC 161
    ；
    The primer nucleotide sequences in the primer combination O are shown in the following table:
    gene Primer sequences SEQ ID NO RPS7 GGGCACAATTTCACGAAACTATTAGG 73 RPS7 CACACAGGACGCCACTGATAAT 162 RPS7 GACCACAACGTTTACTTTCTGAAGC 74 RPS7 AATGCAGTCTAAATACAAGCAATTTTAGCA 163 RPS7 AAAGCGTTACCAAGACACTCGT 75 RPS7 CCTGACTCCAAAAGGCGTTTTC 164 RPS7 CCTGCTCTCCGACAGAACTTTT 76 RPS7 GCTTTCTCCTGGGAGAACTGAA 165 RPS7 GTCGGTCCTGCTGTTCGT 77 RPS7 GGCAAGTCATCATCAACCGTAAC 166
    ；
    The primer nucleotide sequences in the primer combination P are shown in the following table:
    Figure FDA0004044881890000081
    />
    Figure FDA0004044881890000091
    the primer nucleotide sequences in the primer combination Q are as follows:
    gene Primer sequences SEQ ID NO GATA1 TTCTGTCCTCGCAGGTTAATCC 83 GATA1 ACAGCCACTCAATGGAGTTACC 172 GATA1 GCGCTGACCCTAGACTGATTTT 84 GATA1 CTCTGTCTTCAAAGTCTCCAGGAA 173 GATA1 GGTGGTGTCCTTCTCCTCTTGTA 85 GATA1 GGCATTGTTACACACTCCACTCA 174 GATA1 ACAGGCCACTACCTATGCAAC 86 GATA1 GGGAAGATGTGGCTTCTTGTACAG 175 GATA1 GTGTCCTCCACACCAGAATCAG 87 GATA1 GGTATTCTGACCTAGCCAAGGATCT 176 GATA1 CCTATCAGCCTTGGAGGCTTTC 88 GATA1 TCTTTTTCCCTTTTCCAGATGCCTT 177 GATA1 GGTACTGCCCATCTCTACCAAG 89 GATA1 CAGAGACTTGGGTTGTCCAGAA 178
    。
  6. 6. A kit comprising a primer combination for detecting DBA pathogenic gene pool according to claim 4, wherein the kit comprises reagents for extracting whole genomic DNA from a sample, a multiplex amplification system and reagents for constructing a DNA library.
  7. 7. The kit for detecting a DBA pathogenic gene set according to claim 5, wherein the composite amplification system comprises primers in the primer combination, wherein the primer working concentration in the primer combination is 0.1 μm.
  8. 8. A detection method using a kit for detecting a DBA pathogenic gene set according to claim 6, characterized in that the detection method comprises the steps of:
    s1, extracting sample whole genome DNA;
    s2, amplifying a target sequence of a DBA pathogenic gene;
    s3, constructing a DNA library;
    s4, performing on-machine sequencing to obtain an original sequencing signal file;
    s5, performing biological analysis on the original sequencing signal file.
  9. 9. The method according to claim 7, wherein the biological analysis in step S5 includes mutation detection comments, the content of which includes one or more of mutation position in genome, associated genes, gene exon numbering, nucleotide level variation, and corresponding protein level variation.
CN202310026668.2A 2023-01-09 2023-01-09 Kit for detecting DBA pathogenic gene set and detection method Pending CN116121360A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310026668.2A CN116121360A (en) 2023-01-09 2023-01-09 Kit for detecting DBA pathogenic gene set and detection method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310026668.2A CN116121360A (en) 2023-01-09 2023-01-09 Kit for detecting DBA pathogenic gene set and detection method

Publications (1)

Publication Number Publication Date
CN116121360A true CN116121360A (en) 2023-05-16

Family

ID=86307649

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310026668.2A Pending CN116121360A (en) 2023-01-09 2023-01-09 Kit for detecting DBA pathogenic gene set and detection method

Country Status (1)

Country Link
CN (1) CN116121360A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116987809A (en) * 2023-07-24 2023-11-03 广东海洋大学 Bojioocyst algae reference gene and its amplification primer and application

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116987809A (en) * 2023-07-24 2023-11-03 广东海洋大学 Bojioocyst algae reference gene and its amplification primer and application
CN116987809B (en) * 2023-07-24 2024-04-05 广东海洋大学 Bojioocyst algae reference gene and its amplification primer and application

Similar Documents

Publication Publication Date Title
CN106834502B (en) A kind of spinal muscular atrophy related gene copy number detection kit and method based on gene trap and two generation sequencing technologies
AU2014346680A1 (en) Targeted screening for mutations
CN110229897A (en) MED12 gene mutation detection kit and its application
CN113450871A (en) Method for identifying sample identity based on low-depth sequencing
CN108319817B (en) Method and device for processing circulating tumor DNA repetitive sequence
CN116121360A (en) Kit for detecting DBA pathogenic gene set and detection method
CN105803054A (en) Kit and use thereof in detection of orofacial clefts related genes
CN109295219B (en) The biomarker and its detection kit of the congenital fibrinogen defect of one group of detection
CN109055547B (en) Biomarker for evaluating aortic dissection risk and application thereof
CN105838720B (en) PTPRQ gene mutation body and its application
JP2017512324A (en) Mutation analysis in high-throughput sequencing applications
CN112662754B (en) Methods of using compositions for predicting the probability of occurrence of small ear deformities
CN109097465B (en) Application of SNP (single nucleotide polymorphism) site of CLIP3 gene
KR20220125708A (en) Next-generation sequencing-based target gene RNA sequencing panel and analysis algorithm
CN115948541A (en) Primer composition for detecting CDA gene and application thereof
CN116064782A (en) Primer composition for detecting PNH gene and application thereof
CN113444784B (en) Severe congenital neutropenia detection primer composition and application
CN113265409B (en) TIMM21 mutant gene, primer, kit and method for detecting same and application thereof
CN109097464B (en) Application of SNP (single nucleotide polymorphism) site of CFAP43 gene
CN116694753A (en) Primer composition, kit and method for detecting congenital platelet defect
CN113265405B (en) SAMM50 mutant gene, primer, kit and method for detecting same, and use thereof
CN115798584B (en) Method for simultaneously detecting forward and reverse mutation of EGFR gene T790M and C797S
CN109457019B (en) KCNH2 gene SCD related SNP detection kit and detection method
WO2024007971A1 (en) Analysis of microbial fragments in plasma
CN109097466B (en) SNP (Single nucleotide polymorphism) site related to aortic dissection disease and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination