CN106834475B - Detection kit for intelligent genes - Google Patents
Detection kit for intelligent genes Download PDFInfo
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- CN106834475B CN106834475B CN201710090107.3A CN201710090107A CN106834475B CN 106834475 B CN106834475 B CN 106834475B CN 201710090107 A CN201710090107 A CN 201710090107A CN 106834475 B CN106834475 B CN 106834475B
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
Abstract
The invention discloses a detection kit of an intelligent gene, which comprises a PCR reaction reagent, a specific primer for gene locus SNP detection and a fluorescent probe, wherein the gene locus comprises JMJD1C gene locus rs7923609 and LRRC14 gene locus rs 2721173.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a detection kit for intelligent genes.
Background
Wisdom (i.e., cognitive ability) is the ability of the human brain to process, store and extract information, i.e., the ability of a person to grasp the composition, performance, relationship with other objects, development motivation, development direction and basic rules of things. The abilities of perception, memory, attention, thinking and imagination are all considered cognitive abilities, which are the most important psychological conditions for a person to successfully complete an activity. The intellectual characteristics of people have significant influence on the social and economic conditions, and the enhancement of the intelligence of people has also been found to be related to the increase of wealth and life expectancy.
The development of molecular biology techniques and the research of genetic medicine in recent years have shown that the development and perfection of intellectual development and brain functions in humans are influenced and restricted by genetic and environmental factors, of which about 40-50% are influenced by genetic factors. Because the genetic factors can not be changed by people and the environmental factors can be created artificially, the external environments (social environment, natural environment and the like) can be created for children according to the genetic factors of the children, the advantages are enhanced, the education is performed according to the materials, and the personalized education culture is performed.
At present, the detection technology of wisdom-related genes is mainly a direct sequencing method, but the method has the disadvantages of higher cost, fussy operation and long period.
Disclosure of Invention
The invention aims to provide a detection kit for intelligent genes, which can finish the detection of different genotypes of two sites in a PCR system by using a simple, quick, low-price and easily-interpreted detection method without open-tube operation, thereby reducing the possibility of PCR product pollution. In particular, the amount of the solvent to be used,
a detection kit for intelligent genes comprises a PCR reagent for gene detection, wherein the PCR reagent comprises a specific Primer and a fluorescent probe for gene locus SNP detection, the gene locus detected by the specific Primer comprises one or two of JMJD1C gene locus rs7923609 and LRRC14 gene locus rs2721173, the Primer and the probe are designed by adopting Primer 5 software according to the gene JMJD1C (rs7923609) and LRRC14(rs2721173) sequences disclosed by a NCBI Genbank database as a template, the length of a target fragment is controlled to be about 100bp, and the probe sequence is ensured to comprise polymorphic sites, and the detection kit specifically comprises the following steps:
① when detecting the locus rs7923609 of the JMJD1C gene,
a forward primer: 5'-CAAACAAGCATTCTCACTTC-3' SEQ ID NO.1,
reverse primer: 5'-CATAGTTTGCTTAGCCAGTT-3' SEQ ID NO.2,
JMJD1C fluorescent probe: 5'-TAATGACAATATCCCGTTGC-3' SEQ ID NO. 5;
② when detecting the LRRC14 gene locus rs2721173,
a forward primer of 5'-AGGTTGAAAAGACCGAGAC-3' SEQ ID NO.3,
reverse primer 5'-TTTAGGCCAGTGGTAAGG-3' SEQID NO.4,
LRRC14 fluorescent probe: 5'-TCCGTGCTTGGTGACGCTCTG-3' SEQ ID NO. 6.
The primers and probes of the invention may also be the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 6.
Further, the JMJD1C fluorescent probe and the LRRC14 fluorescent probe are double-labeled probes respectively having a fluorescent group at the 5 'terminal region and a quenching group at the 3' terminal region.
Further, said 5' end region employs: a fluorophore label selected from FAM, TET, VIC, HEX, ROX, JOE, CY3 or CY 5; the 3' terminal region was: BHQ, DABCYL or TAMRA.
Further, the concentration of each primer and probe in the PCR reagent is as follows: the final concentration of the JMJD1C forward primer is 0.08-0.15 mu M, the final concentration of the JMJD1C reverse primer is 0.45-0.13 mu M, and the final concentration of the JMJD1C fluorescent probe is 0.04-0.25 mu M; the final concentration of the LRRC14 forward primer is 0.45-0.13. mu.M, the final concentration of the LRRC14 reverse primer is 0.08-0.15. mu.M, and the final concentration of the LRRC14 fluorescent probe is 0.04-0.25. mu.M.
Further, the kit also comprises one or more of PCR buffer solution, a negative reference substance, a positive reference substance, a nucleic acid DNA extraction reagent, Taq polymerase required by PCR reaction and dNTP.
Further, the detection program of the kit is 5min at 37 ℃ and 15min at 95 ℃; then collecting fluorescence at 95 ℃ for 20sec, 51 ℃ for 30sec, and 72 ℃ for 30sec for 40 cycles; and (3) melting curve analysis: fluorescence was collected at 95 ℃ for 30sec, 40 ℃ for 30sec, and 40-75 ℃.
The invention has the following advantages:
1. the technical scheme of the invention designs a novel detection primer and a corresponding fluorescent probe for detecting the gene polymorphism of human intelligent genes JMJD1C (rs7923609) and LRRC14(rs2721173), the PCR detection specificity is very high, highly specific amplification can be realized, an asymmetric PCR technology is adopted for PCR amplification, a melting peak generated by a melting curve is analyzed by combining a real-time fluorescent PCR technology, and different genotypes are conveniently read by the existence or non-existence of a specific melting peak.
2. The detection primers and the probes in the technical scheme of the invention are low in price, and the single gene can realize the detection of the gene polymorphism only by a single fluorescent probe on the premise of not influencing the specificity.
3. According to the technical scheme, detection of different genotypes can be completed only by a single tube in the experimental process, and the operation is simple; and the subsequent analysis of PCR products is not needed, the detection cost is low, the period is short, the efficiency is high, and meanwhile, the false positive risk caused by PCR product pollution is reduced.
Drawings
The 1 st PCR tube in FIG. 1 shows JMJD1C-GG type melting peak with Tm of 53.9 in FAM channel;
FIG. 2 shows that the 1 st PCR tube shows LRRC14-AA type melting peak with Tm of 52.8 ℃ in the HEX channel;
FIG. 3 shows the JMJD1C-AA type melting peak with Tm of 48.4 ℃ in the FAM channel of the 2 nd PCR tube;
FIG. 4 shows that the 2 nd PCR tube shows LRRC14-GG type melting peak with Tm of 63.3 ℃ in the HEX channel;
FIG. 5 shows JMJD1C-AG type melting peaks at Tm of 48.8 ℃ and 55.2 ℃ in the FAM channel in the 3 rd PCR tube;
FIG. 6 shows the 3 rd PCR tube showing the LRRC14-GA type melting peaks at the Tm of 53.1 ℃ and 62.9 ℃ in the HEX channel;
FIG. 7 shows JMJD1C-AG type melting peaks with Tm of 47.7 ℃ and Tm of 53.7 ℃ in the FAM channel in the 4 th PCR tube;
FIG. 8 shows the melting peaks of LRRC14-GA type at Tm of 53.4 ℃ and 63.4 ℃ in the 4 th PCR tube in the HEX channel;
FIG. 9 negative control takes care of melting peaks in the FAM channel;
FIG. 10 negative control takes care of melting peaks in the HEX channel.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Detection of gene polymorphism of intelligent genes JMJD1C (rs7923609) and LRRC14(rs2721173)
DNA extraction
The method for extracting DNA from a human saliva sample by adopting a Chelex-100 method comprises the following specific steps:
A. adding 300 mu L of saliva into a 1.5mL centrifuge tube, centrifuging at 13000rpm for 3min, removing the supernatant, adding 500 mu L of sterilized double distilled water into the precipitate, violently shaking, standing at room temperature for 15min, centrifuging at 13000rpm for 3min, removing the supernatant, and collecting the precipitate;
B. adding 100 μ L of nucleic acid extractive solution into the precipitate, wherein the nucleic acid extractive solution contains particulate matter, shaking thoroughly before each absorption to ensure that the particles are sucked out together, oscillating repeatedly on oscillator, and keeping the temperature at 56 deg.C for more than 30 min;
C. taking out, oscillating sufficiently, keeping the temperature at 100 ℃ for 8min, taking out, oscillating sufficiently, centrifuging at 13,000rpm for 3min, directly using the supernatant for PCR amplification, storing at 4 ℃ for one week, and storing in a refrigerator at-20 ℃ for later use.
The sample may also comprise one of body fluid, tissue sample or exfoliated cells, such as a blood filter disc, hair follicle, oral mucosa scrape or whole blood sample, etc., the blood sample is preserved below-20 ℃ for no more than one year, and the sample may be pretreated accordingly depending on the sampling mode and the sample to be tested.
2. Design of primers and fluorescent probes
According to the gene JMJD1C (rs7923609) and LRRC14(rs2721173) sequences disclosed by the NCBI's Genbank database as templates, Primer 5 software is adopted to design primers and probes, the primers are designed on the upstream and downstream of polymorphic sites, the length of a target fragment is controlled to be about 100bp, and the probe sequences are ensured to contain the polymorphic sites, specifically as follows:
primer: JMJD1C forward primer: 5'-CAAACAAGCATTCTCACTTC-3' the flow of the air in the air conditioner,
JMJD1C reverse primer: 5'-CATAGTTTGCTTAGCCAGTT-3' the flow of the air in the air conditioner,
LRRC14 forward primer: 5'-AGGTTGAAAAGACCGAGAC-3' the flow of the air in the air conditioner,
LRRC14 reverse primer: 5'-TTTAGGCCAGTGGTAAGG-3' the flow of the air in the air conditioner,
and (3) probe: JMJD1C fluorescent probe: 5'-TAATGACAATATCCCGTTGC-3' the flow of the air in the air conditioner,
LRRC14 fluorescent probe: 5'-TCCGTGCTTGGTGACGCTCTG-3' are provided.
The 5 'terminal region of the JMJD1C fluorescent probe is marked by FAM fluorescent group, and the 3' terminal region is marked by BHQ quenching group; the 5 'terminal region of the LRRC14 fluorescent probe is labeled with HEX fluorescent group, and the 3' terminal region is labeled with BHQ quenching group.
The primers and probes were synthesized by Biotechnology engineering (Shanghai) Inc. After the probe is quantified, the sample is stored at minus 20 ℃ in a dark place.
PCR amplification test
The template used for PCR amplification is the genome DNA of a person carrying the homozygous mutant genotype, the homozygous wild genotype or the heterozygous mutant genotype of the JMJD1C gene and the homozygous mutant genotype, the homozygous wild genotype or the heterozygous mutant genotype of the LRRC14 gene.
The reaction system is as follows: PCR buffer (2 × buffer) 10. mu.L, JMJD1C forward primer (10. mu.M) 0.2. mu.L, reverse primer (10. mu.M) 2. mu.L, fluorescent probe (10. mu.M) 0.3. mu.L; LRRC14 forward primer (10. mu.M) 1. mu.l, reverse primer (10. mu.M) 0.2. mu.l, fluorescent probe (10. mu.M) 0.1. mu.l, template 2. mu.l, Taq enzyme (5U/. mu.L) 0.3. mu.l, dNTP (10mM) 0.4. mu.l, ddH2O 3.5μL。
The reaction system comprises five PCR reaction tubes, wherein a JMJD1C gene homozygous mutant genotype and a LRRC14 gene homozygous mutant genotype template (JMJD1C-GG and LRRC14-AA) are placed in the first PCR reaction tube, a JMJD1C gene homozygous wild genotype and a JRC 14 gene homozygous wild genotype template (JMJD1C-AA and LRRC14-GG) are placed in the second PCR reaction tube, a JMJD1C gene heterozygous mutant genotype and an LRRC14 gene heterozygous mutant genotype template (JMJD1C-AG and LRRC14-GA) are placed in the third PCR reaction tube, the fourth PCR reaction tube is a positive control, the heterozygous mutation positive control is used as a template, and both the JMJD1C gene and the LRRC14 gene are heterozygous mutant genotypes (JMJD1C-AG and LRRC14-GA), the fifth PCR reaction tube is used as a double-steamed water template. Wherein the template concentration in each PCR reaction tube is the same.
The reaction procedure was as follows:
amplification was performed using a SLAN8.0 fluorescent quantitative PCR instrument according to the following protocol:
and (3) amplification reaction conditions: 5min at 37 ℃ and 15min at 95 ℃; then 95 ℃ 20sec, 51 ℃ 30sec, 72 ℃ 30sec (fluorescence collection), 40 cycles;
melting curve conditions: 95 ℃ for 30sec, 40-75 ℃ (fluorescence collected). The fluorescence channel is selected from FAM (465-510nm) and HEX (533-580 nm).
4. Analysis of detection results
The melting curves of the five PCR reaction tubes in the embodiment are respectively shown in the attached drawings 1-10, and the results show that each reaction tube containing the template has specific amplification, and Tm values of each PCR product can be well distinguished.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Applicant's name Gene technology (Beijing) Ltd
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Claims (5)
1. A detection kit for a smart gene, characterized in that: the kit comprises a PCR reagent for gene detection, the PCR reagent comprises a specific primer and a fluorescent probe for gene locus SNP detection, the gene locus detected by the specific primer comprises one or two of JMJD1C gene locus rs7923609 and LRRC14 gene locus rs2721173, wherein:
① when detecting the locus rs7923609 of the JMJD1C gene,
a forward primer: 5'-CAAACAAGCATTCTCACTTC-3' SEQ ID NO.1,
reverse primer: 5'-CATAGTTTGCTTAGCCAGTT-3' SEQ ID NO.2,
JMJD1C fluorescent probe: 5'-TAATGACAATATCCCGTTGC-3' SEQ ID NO. 5;
② when detecting the LRRC14 gene locus rs2721173,
a forward primer of 5'-AGGTTGAAAAGACCGAGAC-3' SEQ ID NO.3,
reverse primer 5'-TTTAGGCCAGTGGTAAGG-3' SEQ ID NO.4,
LRRC14 fluorescent probe: 5'-TCCGTGCTTGGTGACGCTCTG-3' SEQ ID NO. 6;
the concentration of each primer and each probe in the PCR reagent is as follows: the final concentration of the JMJD1C forward primer is 0.08-0.15 mu M, the final concentration of the JMJD1C reverse primer is 0.45-0.13 mu M, and the final concentration of the JMJD1C fluorescent probe is 0.04-0.25 mu M; the final concentration of the LRRC14 forward primer is 0.45-0.13. mu.M, the final concentration of the LRRC14 reverse primer is 0.08-0.15. mu.M, and the final concentration of the LRRC14 fluorescent probe is 0.04-0.25. mu.M.
2. The kit for detecting a smart gene according to claim 1, wherein: the JMJD1C fluorescent probe and the LRRC14 fluorescent probe are double-labeled probes respectively having a fluorescent group at the 5 'terminal region and a quenching group at the 3' terminal region.
3. The kit for detecting a smart gene according to claim 2, wherein: the 5' end region adopts: a fluorophore label selected from FAM, TET, VIC, HEX, ROX, JOE, CY3 or CY 5; the 3' terminal region was: BHQ, DABCYL or TAMRA.
4. The kit for detecting a smart gene according to claim 1, wherein: the kit also comprises one or more of PCR buffer solution, negative reference substance, positive reference substance, nucleic acid DNA extraction reagent, Taq polymerase required by PCR reaction and dNTP.
5. The kit for detecting a smart gene according to claim 1, wherein: the detection program of the kit is 5min at 37 ℃ and 15min at 95 ℃; then collecting fluorescence at 95 ℃ for 20sec, 51 ℃ for 30sec, and 72 ℃ for 30sec for 40 cycles; and (3) melting curve analysis: fluorescence was collected at 95 ℃ for 30sec, 40 ℃ for 30sec, and 40-75 ℃.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296621A (en) * | 2015-10-27 | 2016-02-03 | 智海生物工程（北京）有限公司 | Primer pair, fluorescence probe and kit for detecting polymorphism of MTHFR gene |
| CN105506118A (en) * | 2016-01-08 | 2016-04-20 | 智海生物工程（北京）股份有限公司 | Primer pair used for detecting CYP2C19 genotypes, fluorescent probe, reagent kit and method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296621A (en) * | 2015-10-27 | 2016-02-03 | 智海生物工程（北京）有限公司 | Primer pair, fluorescence probe and kit for detecting polymorphism of MTHFR gene |
| CN105506118A (en) * | 2016-01-08 | 2016-04-20 | 智海生物工程（北京）股份有限公司 | Primer pair used for detecting CYP2C19 genotypes, fluorescent probe, reagent kit and method |
Non-Patent Citations (3)
| Title |
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| A review of intelligence GWAS hits: Their relationship to country IQ and the issue of spatial autocorrelation;Davide Piffer;《Intelligence》;20150903;43-50 * |
| Common genetic variants associated with cognitive performance identified using the proxy-phenotype method;Cornelius A. Rietveld等;《PNAS》;20140923;第111卷(第38期);13790–13794 * |
| Genome-wide association study of cognitive functions and educational attainment in UK Biobank (N=112151);G Davies等;《Molecular Psychiatry》;20160405;1-10 * |
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