CN107365846B - Kit for rapidly detecting common β thalassemia mutation allele of Chinese population - Google Patents
Kit for rapidly detecting common β thalassemia mutation allele of Chinese population Download PDFInfo
- Publication number
- CN107365846B CN107365846B CN201710664619.6A CN201710664619A CN107365846B CN 107365846 B CN107365846 B CN 107365846B CN 201710664619 A CN201710664619 A CN 201710664619A CN 107365846 B CN107365846 B CN 107365846B
- Authority
- CN
- China
- Prior art keywords
- 6fam
- tamra
- ivs
- kit
- thalassemia
- 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.)
- Active
Links
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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for rapidly detecting common β thalassemia mutant allele of Chinese population, which comprises deletion type β thalassemia gene HPFH-SEA andGγ+(Aγδβ)0designed primer aiming at the poor allele β of non-deletion type β‑90、β‑29、β‑28、βCap+40‑43、βInt M、βInt CD、βCD14‑15、βCD17、βCD26、β27/28、βIVS‑I‑1、βIVS‑I‑5、βCD37、βCD41‑42、βCD43、βCD71–72、βCD95And βIVS‑II‑654The kit can quickly and accurately detect the common β thalassemia mutation allele of Chinese people.
Description
Technical Field
The invention relates to a kit for detecting thalassemia, in particular to a kit for rapidly detecting common β thalassemia mutant alleles of Chinese people.
Background
The method is characterized in that thalassemia is a monogenic hereditary disease with high incidence in tropical and subtropical regions, severe β thalassemia patients need conventional blood transfusion and deferrization treatment and have low quality of life, Guangxi and Guangdong are regions with high incidence of thalassemia, and about 7 percent of people are carriers of β thalassemia genes.
The existing research (Pontangrong, Longfoal, Yesheng, et al. analysis of mutation of thalassemia genes in the population of northern gulf of Guangxi. J. Youngen and Genetime, 2016(1):39-42.) shows that the main type of β thalassemia genes in the population of Guangxi is β-90、β-29、β-28、βCap+40-43、βInt M、βInt CD、βCD14-15、βCD17、βCD26、β27/28、βIVS-I-1、βIVS-I-5、βCD37、βCD41-42、βCD43、βCD71–72、βCD95、βIVS-II-654、HPFH-SEA、Gγ+(Aγδβ)0(DBT for short) allele. The abbreviation and HGVS (Human Genome Variation Society) name of the aforementioned non-deleted thalassemia allele are shown in Table 1 below:
table 1:
in the diagnosis of thalassemia in China, the methods for detecting the genotype of thalassemia are generally Gap-PCR and PCR-RDB. The principle of Gap-PCR diagnosis of thalassemia is that a sample is amplified by designing a cross breakpoint primer, an amplification product is analyzed by agarose gel electrophoresis, and the PCR-RDB principle can complete the analysis by completing the processes of amplification, hybridization and the like. As the detection conditions and the operation methods of the two methods are different, the two methods need to be separately and independently operated, and the PCR-RDB has the disadvantages of complicated operation, short amplified fragment and easy generation of PCR product pollution. The detection range of the kit developed at the earlier stage is limited, so that the adjustment of the detection range and the improvement of the detection method can provide support for the prevention and treatment of the thalassemia.
Disclosure of Invention
The invention aims to solve the technical problem of providing a kit for rapidly detecting common β thalassemia mutant alleles of Chinese people, and the kit can realize β of β thalassemia by using a single-tube multiplex PCR technology-90、β-29、β-28、βCap+40-43、βInt M、βInt CD、βCD14-15、βCD17、βCD26、β27/28、βIVS-I-1、βIVS-I-5、βCD37、βCD41-42、βCD43、βCD71–72、βCD95、βIVS-II-654、HPFH-SEA、Gγ+(Aγδβ)0The combined detection of the allele and AMEL locus can be carried out in a sequencing tube for fragment analysis detection in one step.
The kit for rapidly detecting the common β thalassemia mutant allele of Chinese people comprises deletion type β thalassemia gene HPFH-SEA andGγ+(Aγδβ)0designed primer aiming at the poor allele β of non-deletion type β-90、β-29、β-28、βCap+40-43、βInt M、βInt CD、βCD14-15、βCD17、βCD26、β27/28、βIVS-I-1、βIVS-I-5、βCD37、βCD41-42、βCD43、βCD71–72、βCD95And βIVS-II-654Designed primer aiming at the poor allele β of non-deletion type β-28、βCD17、βCD26、β27/28、βIVS-I-1、βCD41-42、βCD43、βCD71–72And βIVS-II-654The designed normal control primer and the AMXY primer for detecting the sex have the following specific primer names and sequences:
Beta1-F：5’-TCCTAAGCCAGTGCCAGAAGAGC-3’；
Beta2-R：5’-CCTGAGACTTCCACACTGATGC-3’；
Beta3-R：5’-AGTTGGACTTAGGGAACAAAGGAAC-3’；
AMXY-6FAM-F：5’-6-FAM-CCCTGGGCTCTGTAAAGAATAGTG-3’；
AMXY-R：5’-ATCAGAGCTTAAACTGGGAAGCTG-3’；
CD37M-6FAM-R：5’-6FAM-GGACTCAAAGAACCTCTGGGACT-3’；
IntM-6FAM-R：5’-6FAM-CAAAAATCCTCAGGAGTCAGTTGCACT-3’；
-90M-6FAM-R：5’-6FAM-GTAGATTGGCCAACCCAAGGGTA-3’；
-28M-6FAM-R：5’-6FAM-CAATAGATGGCTCTGCCCTGACTAC-3’；
-29M-6FAM-R：5’-6FAM-GATGGCTCTGCCCTGCCTTTC-3’；
IntCD-6FAM-R：5’-6FAM-CCTCAGGAGTCAGATGCATCC-3’；
CD17M-6FAM-R：5’-6FAM-CAACTTCATCCACGTTCGCCTA-3’；
Cap+40-43M-6FAM-R：5’-6FAM-GATGCACCATGGTGTCTGAGG-3’；
CD14-15(+G)M-6FAM-R：5’-6FAM-CCACGTTCACCTTGCTCTACCA-3’；
CD26M-6FAM-R：5’-6FAM-ACCAACCTGCACAGGGTCTT-3’；
IVS-Ⅰ-1M-6FAM-R：5’-6FAM-CTGTCTTGTAACCTTGATGCCAAA-3’；
IVS-Ⅰ-5M-6FAM-R：5’-6FAM-AACTTAAACCTGTCTTGTAACCTTGACAG-3’；
CD27/28(+C)M-6FAM-R：5’-6FAM-TTTTGATACCAACCTGACCAGGAGC-3’；
CD71-72(+A)M-6FAM-F：5’-6FAM-CAAGAAAGTGCTCGGTGCCTGTAA-3’；
CD95M-6FAM-F：5’-6FAM-GTGAGCTGCACTGTGACGAAG-3’；
CD43M-6FAM-F：5’-6FAM-CCTTGGACCCAGAGGTTCGTTT-3’；
CD41-42M-6FAM-F：5’-6FAM-CAAAAAAAAAACAAAACTACCCTTGGACCCAGATGTTG-3’；
654M-6FAM-F：5’-6FAM-CAGTGATAATTTCTGGGTTGAGGT-3’；
-28N-TAMRA-R：5’-TAMRA-TAGATGGCTCTGCCCTGACATT-3’；
CD17N-TAMRA-R：5’-TAMRA-CAACTTCATCCACGTTCACCAT-3’；
CD26N-TAMRA-R：5’-TAMRA-ACCAACCTGCTCAGGGACTC-3’；
IVS-Ⅰ-1N-TAMRA-R：5’-TAMRA-CTGTCTTGTAACCTTGATACCATC-3’；
CD27/28N-TAMRA-R：5’-TAMRA-AAAATGATACCAACCTGCCCAGCGC-3’；
CD71-72N-TAMRA-F：5’-TAMRA-CAAGAAAGTGCTCGGTGCCTTGAG-3’；
43/41-42N-TAMRA-F：5’-TAMRA-CCTTGGACCCAGAGGTTCCTTG-3’；
654N-TAMRA-F：5’-TAMRA-CAGTGATAAGTTCTGGGTTAAGGC-3’；
HPFH-F：5’-AAAACCAGCCTCATGGTAGCAGAATC-3’；
HPFH-M-FAM-R：5’-6FAM-TGGTATCTGCAGCAGTTGCC-3’；
DBT-M-FAM-F：5’-6FAM-CCAGAAATTGCCTCATGTCTCT-3’；
DBT-R：5’-CACATATAAAATGCTGCTAATGCTTCATTAC-3’；
in the above primers, "M" represents a mutation site, "N" represents a normal control site, FAM represents hydroxyfluorescein, and TAMRA represents carboxytetramethylrhodamine. The fluorescence labeling combination of the above primers can be replaced by other fluorescence dyes according to the type and setting of the sequencer, for example, FAM, HEX, TA MRA and ROX are one combination of E5 dyes in ABI sequencer, or FAM (blue), VIC (green), NED (yellow) and PET (red) in G5 setting, or other fluorescence dye combination with spectrum correction can be selected. Only one of the combinations is listed in the application, but the combination is not limited to the above one, and the combination can be realized by other dye combinations; the fluorescence labeling chosen is also not limited to the above colors, and any combination of distinguishable spectrally corrected fluorescence colors can be used.
The kit for rapidly detecting the common β thalassemia mutant allele of Chinese people also comprises some conventional and necessary components in the conventional kit, such as buffer solution, enzyme solution and Mg2+(MgCl2) And dNTPs. Specifically, the enzyme solution is a Taq polymerase system, including a hot start enzyme system which can be used for a hydrolysis probe method, and the like; the buffer is a conventional PCR buffer. When GoldStar Taq DNA Polymerase produced by Kangji century was used as the enzyme solution, the buffer is preferably a buffer compatible with GoldStar Taq DNA Polymerase.
The method for rapidly detecting the common β thalassemia mutation allele of Chinese people by adopting the kit comprises the following steps:
1) extracting sample genome DNA, and preparing a DNA template by adopting the conventional method;
2) preparing a reaction system, which specifically comprises the following steps:
taking DNA to be detected, each primer, PCR buffer solution, enzyme solution and MgCl2dNTP, water and DNA template to prepare a reaction system; wherein, the concentration of each component is usually the DNA to be detected: 20 ng-50 ng; each primer is as follows: 0.037-0.445 mu mol/L; mg (magnesium)2+: 1.6-1.8 mmol/L; the final volume of the reaction system may be 10. mu.L, 20. mu.L or 25. mu.L;
3) sample detection: performing PCR reaction, namely taking 0.4 mu L of PCR product, mixing a molecular weight internal reference of a sequencing system, mixing 10 mu L of sequencing loading carrier deionized formamide (HiDi), uniformly mixing, then loading by using a sequencer for sequencing, and detecting the result by using a fragment analysis method;
4) data analysis and result judgment:
data reading and analysis are carried out by using GeneMapper software, the allele corresponding to the PCR product, the fluorescence color and the product length of the final PCR product are respectively shown in tables 2 and 3, wherein the allele and the fluorescence color and the product length of the final PCR product in table 2 are of a normal control site, and the allele and the fluorescence color and the product length of the mutation site in table 3 are:
table 2:
table 3:
the interpretation of each sample genotype can be done after one sequencing reaction. The specific interpretation method is as follows:
m represents mutation site, N represents normal control site, 9 non-deletion β poor alleles β-28、βCD17、βCD26、β27/28、βIVS-I-1、βCD41-42、βCD43、βCD71–72、βIVS-II-654Designing normal control SNP detection primer (β)CD41-42、βCD431 normal control primer was used in total, so 8 normal control primers for the poor allele β were used in total).
A, setting a detection site for normal control, and when a color signal and a length signal corresponding to M are not detected, the sample does not carry the mutation site genes; when signals corresponding to M, N are detected simultaneously, the allele of the sample at the detection site is mutant heterozygote; if only the color and length signals corresponding to M are detected, but the color and length signals corresponding to N are not detected, the sample is a non-deletion homozygote at the detection site.
B, no normal control non-deletion type thalassemia detection site is arranged, and when a color signal and a length signal corresponding to M are not detected, the sample does not carry the mutation site genes; the sample carries the mutation locus gene when detecting the color and length signals corresponding to M, and the heterozygote mutation of the locus is generally carried out when detecting the color and length signals corresponding to M because the proportion of the non-deletion type thalassemia genes not provided with the normal control in the population is low, but the heterozygote mutation is preferably confirmed by sequencing.
C, the deficient thalassemia determination method is that when the product length detection signal corresponding to the fluorescence value of the deficient thalassemia is detected, the sample is determined to carry the deficient thalassemia, but the normal control sites of the non-deficient thalassemia are required to be combined, and if the normal reference site of 1 non-deficient β thalassemia allele is positive, the β gene is required to be combined and considered.
Compared with the prior art, the invention is characterized in that:
1. the kit can realize single-tube pair of HPFH-SEA of β thalassemia,Gγ+(Aγδβ)0、β-90、β-29、β-28、βCap+40-43、βInt M、βInt CD、βCD14-15、βCD17、βCD26、β27/28、βIVS-I-1、βIVS-I-5、βCD37、βCD41-42、βCD43、βCD71–72、βCD95And βIVS-II-654The genotype is adopted for detection, extra agarose electrophoresis and hybridization operations are not needed, and PCR product pollution is avoided.
2. The kit disclosed by the invention has high sensitivity, stability and accuracy for detecting the detection sites, and higher specificity; the primer has good specificity and experimental repeatability;
3. the adoption of the fluorescent marker has lower requirements on the DNA concentration of a tested sample, and can adopt hair with hair follicles, a mouth swab and the like to extract DNA and complete detection;
4. the kit can detect 96 samples at the same time, and an automatic detection platform reduces the manual participation and reduces the manual operation errors;
5. the kit has the characteristics of rapid detection (only 4-5 hours are needed for extracting a result from DNA), accuracy, simple and convenient operation, wide detection range and low cost;
6. the detection range of the kit is suitable for detection mechanisms for detecting the thalassemia allelic variation of Chinese population.
Detailed Description
The present invention will be better understood from the following detailed description of specific examples, which should not be construed as limiting the scope of the present invention.
Example 1: detection result of the kit in known genotype sample
1. Composition of the kit
(1) Primer and method for producing the same
Designed according to the published sequence of the β -globin gene cluster (NG-000007.3) at NCBI, where HPFH-SEA andGγ+(Aγδβ)0design of primers by Gap-PCR β-90、β-29、β-28、βCap+40-43、βInt M、βInt CD、βCD14-15、βCD17、βCD26、β27/28、βIVS-I-1、βIVS-I-5、βCD37、βCD41-42、βCD43、βCD71–72、βCD95And βIVS-II-654The primers were designed by AS-PCR, wherein β-28、βCD17、βCD26、β27/28、βIVS-I-1、βCD41-42、βCD43、βCD71–72、βIVS-II-654Designing normal control SNP detection primers and sex detection AMXY primers, wherein the specific primer names and sequences are shown in the following table 4:
table 4:
in Table 4, Beta1-F, Beta2-R, Beta3-R is the common primer end for amplification of the poor allele of non-deletion type β, the mutation end comprises the detection end for amplification of 18 poor alleles of non-deletion type β and the detection end of the reference normal site of 9 poor alleles of non-deletion type β, wherein 43/41-42N-TAMRA-F is the common reference site of CD43M and CD41-42M (i.e. β M)CD41-42、βCD431 normal control primer was used in total, so 8 normal control primers for the poor allele β were set in total).
The final composition concentrations of each primer in the kit are shown in table 5 below:
table 5:
in Table 5, "M" represents a mutation site, "N" represents a normal control site, FAM represents hydroxyfluorescein, TAMRA represents carboxytetramethylrhodamine
(2) Other components:
golstar Taq DNA Polymerase, buffers and dNTPs compatible with Golstar Taq DNA Polymerase were purchased from the kang century, MgCl2Purchased from Life Technology.
The PCR reaction system was prepared as follows in Table 6:
table 6:
2. method of implementation
The PCR reaction system added with the template DNA is placed on a general PCR instrument, and the PCR reaction program comprises 10 minutes at 95 ℃, 30 seconds at 94 ℃, 30 seconds at 62.5 ℃, 30 seconds at 72 ℃, 28-35 cycles and 60 minutes extension at 62 ℃.
Sample treatment: DNA is extracted by the universal DNA kit and diluted to 20-50 ng/mu L by double distilled water for later use.
Sample detection: running a PCR program in a PCR reaction system of a sample to be detected with a known genotype, taking 0.4 mu L of PCR product after the running of the PCR program is finished, mixing a molecular weight internal reference liz500 of a sequencing system, mixing 10 mu L of deionized formamide (HiDi) of a sequencing loading carrier, uniformly mixing, loading and sequencing by adopting a sequencer, and detecting a result by adopting a fragment analysis method.
3. Sample source: all samples were derived from DNA samples genotyped by conventional Gap-PCR techniques or sequencing techniques.
4. Data analysis and result judgment: the results were read and analyzed using GeneMapper software, and the genotype was judged by synthesizing the results of the detection, which are shown in Table 7 below, and were the same as those of the conventional Gap-PCR technique or sequencing technique.
Table 7:
SEQUENCE LISTING
<110> Huang, De Zhen
<120> kit for rapidly detecting common β dixian mutant allele of Chinese population
<130>2017
<160>35
<170>PatentIn version 3.5
<210>1
<211>23
<212>DNA
<213> Artificial sequence
<400>1
tcctaagcca gtgccagaag agc 23
<210>2
<211>22
<212>DNA
<213> Artificial sequence
<400>2
cctgagactt ccacactgat gc 22
<210>3
<211>25
<212>DNA
<213> Artificial sequence
<400>3
agttggactt agggaacaaa ggaac 25
<210>4
<211>24
<212>DNA
<213> Artificial sequence
<400>4
ccctgggctc tgtaaagaat agtg 24
<210>5
<211>24
<212>DNA
<213> Artificial sequence
<400>5
atcagagctt aaactgggaa gctg 24
<210>6
<211>23
<212>DNA
<213> Artificial sequence
<400>6
ggactcaaag aacctctggg act 23
<210>7
<211>27
<212>DNA
<213> Artificial sequence
<400>7
caaaaatcct caggagtcag ttgcact 27
<210>8
<211>23
<212>DNA
<213> Artificial sequence
<400>8
gtagattggc caacccaagg gta 23
<210>9
<211>25
<212>DNA
<213> Artificial sequence
<400>9
caatagatgg ctctgccctg actac 25
<210>10
<211>21
<212>DNA
<213> Artificial sequence
<400>10
gatggctctg ccctgccttt c 21
<210>11
<211>21
<212>DNA
<213> Artificial sequence
<400>11
cctcaggagt cagatgcatc c 21
<210>12
<211>22
<212>DNA
<213> Artificial sequence
<400>12
caacttcatc cacgttcgcc ta 22
<210>13
<211>21
<212>DNA
<213> Artificial sequence
<400>13
gatgcaccat ggtgtctgag g 21
<210>14
<211>22
<212>DNA
<213> Artificial sequence
<400>14
ccacgttcac cttgctctac ca 22
<210>15
<211>20
<212>DNA
<213> Artificial sequence
<400>15
accaacctgc acagggtctt 20
<210>16
<211>24
<212>DNA
<213> Artificial sequence
<400>16
ctgtcttgta accttgatgc caaa 24
<210>17
<211>29
<212>DNA
<213> Artificial sequence
<400>17
aacttaaacc tgtcttgtaa ccttgacag 29
<210>18
<211>25
<212>DNA
<213> Artificial sequence
<400>18
ttttgatacc aacctgacca ggagc 25
<210>19
<211>24
<212>DNA
<213> Artificial sequence
<400>19
caagaaagtg ctcggtgcct gtaa 24
<210>20
<211>21
<212>DNA
<213> Artificial sequence
<400>20
gtgagctgca ctgtgacgaa g 21
<210>21
<211>22
<212>DNA
<213> Artificial sequence
<400>21
ccttggaccc agaggttcgt tt 22
<210>22
<211>38
<212>DNA
<213> Artificial sequence
<400>22
caaaaaaaaa acaaaactac ccttggaccc agatgttg 38
<210>23
<211>24
<212>DNA
<213> Artificial sequence
<400>23
cagtgataat ttctgggttg aggt 24
<210>24
<211>22
<212>DNA
<213> Artificial sequence
<400>24
tagatggctc tgccctgaca tt 22
<210>25
<211>22
<212>DNA
<213> Artificial sequence
<400>25
caacttcatc cacgttcacc at 22
<210>26
<211>20
<212>DNA
<213> Artificial sequence
<400>26
accaacctgc tcagggactc 20
<210>27
<211>24
<212>DNA
<213> Artificial sequence
<400>27
ctgtcttgta accttgatac catc 24
<210>28
<211>25
<212>DNA
<213> Artificial sequence
<400>28
aaaatgatac caacctgccc agcgc 25
<210>29
<211>24
<212>DNA
<213> Artificial sequence
<400>29
caagaaagtg ctcggtgcct tgag 24
<210>30
<211>22
<212>DNA
<213> Artificial sequence
<400>30
ccttggaccc agaggttcct tg 22
<210>31
<211>24
<212>DNA
<213> Artificial sequence
<400>31
cagtgataag ttctgggtta aggc 24
<210>32
<211>26
<212>DNA
<213> Artificial sequence
<400>32
aaaaccagcc tcatggtagc agaatc 26
<210>33
<211>20
<212>DNA
<213> Artificial sequence
<400>33
tggtatctgc agcagttgcc 20
<210>34
<211>22
<212>DNA
<213> Artificial sequence
<400>34
ccagaaattg cctcatgtct ct 22
<210>35
<211>31
<212>DNA
<213> Artificial sequence
<400>35
cacatataaa atgctgctaa tgcttcatta c 31
Claims (2)
1. A kit for rapidly detecting common β thalassemia mutant alleles in Chinese population is characterized by comprising deletion type β thalassemia gene HPFH-SEA and mutant genesGγ+(Aγδβ)0Designed primer aiming at the poor allele β of non-deletion type β-90、β-29、β-28、βCap+40-43、βIntM、βIntCD、βCD14-15、βCD17、βCD26、β27/28、βIVS-I-1、βIVS-I-5、βCD37、βCD41-42、βCD43、βCD71–72、βCD95And βIVS-II-654Designed primer aiming at the poor allele β of non-deletion type β-28、βCD17、βCD26、β27/28、βIVS-I-1、βCD41-42、βCD43、βCD71–72And βIVS-II-654The designed normal control primer and the AMXY primer for detecting the sex have the following specific primer names and sequences:
Beta1-F：5’-TCCTAAGCCAGTGCCAGAAGAGC-3’；
Beta2-R：5’-CCTGAGACTTCCACACTGATGC-3’；
Beta3-R：5’-AGTTGGACTTAGGGAACAAAGGAAC-3’；
AMXY-6FAM-F：5’-6-FAM-CCCTGGGCTCTGTAAAGAATAGTG-3’；
AMXY-R：5’-ATCAGAGCTTAAACTGGGAAGCTG-3’；
CD37M-6FAM-R：5’-6FAM-GGACTCAAAGAACCTCTGGGACT-3’；
IntM-6FAM-R：5’-6FAM-CAAAAATCCTCAGGAGTCAGTTGCACT-3’；
-90M-6FAM-R：5’-6FAM-GTAGATTGGCCAACCCAAGGGTA-3’；
-28M-6FAM-R：5’-6FAM-CAATAGATGGCTCTGCCCTGACTAC-3’；
-29M-6FAM-R：5’-6FAM-GATGGCTCTGCCCTGCCTTTC-3’；
IntCD-6FAM-R：5’-6FAM-CCTCAGGAGTCAGATGCATCC-3’；
CD17M-6FAM-R：5’-6FAM-CAACTTCATCCACGTTCGCCTA-3’；
Cap+40-43M-6FAM-R：5’-6FAM-GATGCACCATGGTGTCTGAGG-3’；
CD14-15(+G)M-6FAM-R：5’-6FAM-CCACGTTCACCTTGCTCTACCA-3’；
CD26M-6FAM-R：5’-6FAM-ACCAACCTGCACAGGGTCTT-3’；
IVS-Ⅰ-1M-6FAM-R：5’-6FAM-CTGTCTTGTAACCTTGATGCCAAA-3’；
IVS-Ⅰ-5M-6FAM-R：5’-6FAM-AACTTAAACCTGTCTTGTAACCTTGACAG-3’；
CD27/28(+C)M-6FAM-R：5’-6FAM-TTTTGATACCAACCTGACCAGGAGC-3’；
CD71-72(+A)M-6FAM-F：5’-6FAM-CAAGAAAGTGCTCGGTGCCTGTAA-3’；
CD95M-6FAM-F：5’-6FAM-GTGAGCTGCACTGTGACGAAG-3’；
CD43M-6FAM-F：5’-6FAM-CCTTGGACCCAGAGGTTCGTTT-3’；
CD41-42M-6FAM-F：5’-6FAM-CAAAAAAAAAACAAAACTACCCTTGGACCCAGATGTTG-3’；
654M-6FAM-F：5’-6FAM-CAGTGATAATTTCTGGGTTGAGGT-3’；
-28N-TAMRA-R：5’-TAMRA-TAGATGGCTCTGCCCTGACATT-3’；
CD17N-TAMRA-R：5’-TAMRA-CAACTTCATCCACGTTCACCAT-3’；
CD26N-TAMRA-R：5’-TAMRA-ACCAACCTGCTCAGGGACTC-3’；
IVS-Ⅰ-1N-TAMRA-R：5’-TAMRA-CTGTCTTGTAACCTTGATACCATC-3’；
CD27/28N-TAMRA-R：5’-TAMRA-AAAATGATACCAACCTGCCCAGCGC-3’；
CD71-72N-TAMRA-F：5’-TAMRA-CAAGAAAGTGCTCGGTGCCTTGAG-3’；
43/41-42N-TAMRA-F：5’-TAMRA-CCTTGGACCCAGAGGTTCCTTG-3’；
654N-TAMRA-F：5’-TAMRA-CAGTGATAAGTTCTGGGTTAAGGC-3’；
HPFH-F：5’-AAAACCAGCCTCATGGTAGCAGAATC-3’；
HPFH-M-FAM-R：5’-6FAM-TGGTATCTGCAGCAGTTGCC-3’；
DBT-M-FAM-F：5’-6FAM-CCAGAAATTGCCTCATGTCTCT-3’；
DBT-R：5’-CACATATAAAATGCTGCTAATGCTTCATTAC-3’；
in the above primers, "M" represents a mutation site, "N" represents a normal control site, FAM represents hydroxyfluorescein, and TAMRA represents carboxytetramethylrhodamine.
2. The kit for rapidly detecting the common β diji mutant allele according to claim 1, wherein the kit further comprises buffer solution, enzyme solution and Mg2+And dNTPs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710664619.6A CN107365846B (en) | 2017-08-04 | 2017-08-04 | Kit for rapidly detecting common β thalassemia mutation allele of Chinese population |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710664619.6A CN107365846B (en) | 2017-08-04 | 2017-08-04 | Kit for rapidly detecting common β thalassemia mutation allele of Chinese population |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107365846A CN107365846A (en) | 2017-11-21 |
| CN107365846B true CN107365846B (en) | 2020-04-14 |
Family
ID=60310211
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710664619.6A Active CN107365846B (en) | 2017-08-04 | 2017-08-04 | Kit for rapidly detecting common β thalassemia mutation allele of Chinese population |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107365846B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255225A (en) * | 2013-05-27 | 2013-08-21 | 钦州市妇幼保健院 | Thalassemia gene detection method based on fluorescence labeling quantitation PCR (Polymerase Chain Reaction) technology |
| CN106399478A (en) * | 2016-08-31 | 2017-02-15 | 桂林优利特医疗电子有限公司 | Kit for rapidly detecting alpha/beta-thalassemia through fluorescent probe PCR method |
| CN106868187A (en) * | 2015-11-04 | 2017-06-20 | 深圳市瀚海基因生物科技有限公司 | Multiple PCR primer, kit and purposes |
| CN106939350A (en) * | 2017-04-28 | 2017-07-11 | 深圳市龙华区人民医院 | Detection kit, detection method and its application of thalassemia associated gene mutation |
-
2017
- 2017-08-04 CN CN201710664619.6A patent/CN107365846B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255225A (en) * | 2013-05-27 | 2013-08-21 | 钦州市妇幼保健院 | Thalassemia gene detection method based on fluorescence labeling quantitation PCR (Polymerase Chain Reaction) technology |
| CN106868187A (en) * | 2015-11-04 | 2017-06-20 | 深圳市瀚海基因生物科技有限公司 | Multiple PCR primer, kit and purposes |
| CN106399478A (en) * | 2016-08-31 | 2017-02-15 | 桂林优利特医疗电子有限公司 | Kit for rapidly detecting alpha/beta-thalassemia through fluorescent probe PCR method |
| CN106939350A (en) * | 2017-04-28 | 2017-07-11 | 深圳市龙华区人民医院 | Detection kit, detection method and its application of thalassemia associated gene mutation |
Non-Patent Citations (2)
| Title |
|---|
| 广西北部湾地区人群地中海贫血基因突变分析;庞婉容等;《中国优生与遗传杂志》;20160125;第24卷(第1期);第39-42页 * |
| 应用PCR对脆性X综合征进行产前筛查与诊断;杨丹彤等;《中国优生与遗传杂志》;20120325;第20卷(第3期);摘要，第34页第二节第3小节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107365846A (en) | 2017-11-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105586427B (en) | Primers, kit and method for detecting human BRCA1 and BRCA2 gene mutation | |
| US11834707B2 (en) | Nucleic acid amplification blocker for detecting low-abundance mutation sequence and application thereof | |
| CN113073150A (en) | Digital PCR detection kit for novel coronavirus and variant thereof | |
| CN104212904A (en) | Kit for quickly detecting polymorphism of human CYP2C19 gene and using method of kit | |
| CN105039514A (en) | Detection kit for human BRAF gene V600E mutation | |
| CN113913530B (en) | Molecular marker related to sheep body height and application thereof | |
| CN108642152A (en) | The kit and method of Genotyping detection | |
| CN115058543B (en) | Primer group and kit for identifying Delta variant strain and Omicron variant strain | |
| WO2012051800A1 (en) | Plasmid standard for use in quantitative assays using fluorescent quantitative pcr | |
| CN108642138B (en) | Method and kit for detecting genetic information of folate metabolism related gene | |
| CN107245528B (en) | Rapid detection kit for common thalassemia mutant genes of Chinese population | |
| CN115976182A (en) | Primer probe set and kit for detecting spinal muscular atrophy pathogenic gene SMN1 | |
| US10851408B2 (en) | Methods and kits for detecting gene mutations | |
| CN110923325A (en) | Primer Blocker group, kit and method for detecting EGFR gene mutation | |
| CN108060213B (en) | Probe and kit for detecting SNP (single nucleotide polymorphism) sites by recombinase-mediated isothermal amplification method based on probe guidance | |
| CN107365846B (en) | Kit for rapidly detecting common β thalassemia mutation allele of Chinese population | |
| CN111394441B (en) | Primer group, kit and method for amplification of vitamin A related genes | |
| CN107267648B (en) | Kit for rapidly detecting common mutant genes of thalassemia | |
| CN110819709A (en) | Method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction) | |
| CN110438224B (en) | Primer, kit and detection method for UGT1A1 gene polymorphism detection | |
| CN114959030A (en) | Application of reagent for detecting HCG9 gene methylation in preparation of product for diagnosing bladder cancer | |
| CN114134212A (en) | Primer probe combination and kit for plasmid quantification | |
| CN110616260B (en) | Deletion type beta thalassemia detection primer based on SNP analysis, kit and application | |
| CN111471768A (en) | PCR primer group and kit for detecting JAK2V617F and CA L R ninth exon gene mutation | |
| CN114457151B (en) | Detection kit for detecting phenylalanine hydroxylase gene mutation and detection method 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 | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20191009 Address after: No. 301, 302, 304, 401a1, building 1, Shenzhen Biomedical Innovation Industrial Park, No. 14, Jinhui Road, Kengzi street, Pingshan District, Shenzhen City, Guangdong Province Applicant after: Yaneng Biotechnology (Shenzhen) Co., Ltd. Address before: East Pearl Street of Qinzhou city the Guangxi Zhuang Autonomous Region 535099 No. 92 Applicant before: Huang De Zhen |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |