CN112430655A - Detection primer and kit for detecting central precocity of children and application - Google Patents
Detection primer and kit for detecting central precocity of children and application Download PDFInfo
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Abstract
The invention relates to the technical field of children central precocity detection, in particular to a detection primer for detecting children central precocity, wherein the detection primer comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as SEQ ID No. 1-6; the nucleotide sequence of the downstream primer is shown as SEQ ID No. 7-12. The gene detection reagent comprises the detection primer. The gene detection kit comprises the detection primer. The invention solves the problems that the conventional detection of the central precocity of children in the prior art involves few gene loci and the high-throughput sequencing technology has clinical limitation. The simultaneous detection of a plurality of variable sites is realized through reasonable and ingenious primer sequence design, the convenient detection of 42 variable sites is realized, the detection period is short, the cost is low, and the clinical practical popularization and application are facilitated.
Description
Technical Field
The invention relates to the technical field of detection of central precocity of children, in particular to a detection primer, a kit and application for detecting central precocity of children.
Background
Adolescence is the transition period from growth and development of children to adults, and takes sexual maturity as a main mark. The gonadal development process is doubly regulated by the central nervous system and the endocrine system. The hypothalamus synthesizes Gonadotropin-releasing hormone (GnRH) to act on the pituitary gland in a pulsatile secretion manner, stimulating the anterior pituitary to release Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH), activating gonadal function, secreting Testosterone (Testosterone, T) and Estrogen (Estrogen, E), promoting reproductive organ maturation, initiating pubertal development, and beginning secondary sexual characteristics.
With the development of society, the change of life style, the change of living environment and dietary structure, the incidence rate of sexual precocity of children shows a trend of rising year by year, more than 10% of girls have breast development before the age of 8, and 1.65% of girls have menstruation before the age of 10.
Precocious puberty can be classified according to the etiology: central prematurity and peripheral prematurity. Peripheral Precocious Puberty (PPP) refers to an increase in endogenous or exogenous secretion of sex hormones, leading to an early development of reproductive organs and an early appearance of secondary sexual characteristics. Central precocity (CPP) is due to the early initiation of the hypothalamic-pituitary-gonadal axis, with a 20-fold higher incidence for girls than for boys.
Precocious puberty not only causes short stature and influences the normal development and mental health of children, but also causes the occurrence risk of breast cancer, endometrial cancer, obesity, diabetes, cardiovascular diseases and the like to be remarkably increased after the adults take the children. In addition, precocious puberty also increases the risk of developing behavioral disorders in adolescents. Therefore, the early diagnosis and timely intervention of the sexual precocity of the children can not only make the development of the youth return to the normal physiological process, but also reduce the risk of the occurrence of later-stage related diseases.
The time of puberty initiation varies greatly between individuals and is mainly related to the interaction of genetic factors, environment, nutrition, race, socioeconomic factors, etc., wherein the genetic factors play a key role and account for about 50-80%. To date, four mutant genes have been shown to be associated with CPP development: KISS1 gene, KISS1R gene, DLK1 gene and MKRN3 gene.
(1) KISS1 and KISS1R genes: the KISS1 gene is located on autosome 1q32, contains 4 exons and 3 introns, and encodes kisspeptin protein; the KISS1R gene, the G protein-coupled receptor 54 gene (GPR54), is located on autosomal 19p13.3, consists of 5 exons and 4 introns, and is a specific receptor for kisspeptin. kisspeptin is involved in regulating the activation process of puberty, and binds to GPR54 receptor on GnRH neurons, promoting pulsatile release of GnRH, thereby initiating puberty.
(2) MKRN3 gene: located on autosome 15q11.2, without introns, is a paternally expressed imprinted gene (silenced for methylation on the maternally derived allele and expressed on the paternally derived allele), and levels of MKRN3 are negatively correlated with gonadotropins, estradiol, and decrease before puberty, and regulate hypothalamic GnRH secretion in childhood.
(3) DLK1 gene: located on autosome 14q32.2, contains 5 exons, is a paternally expressed imprinted gene, and encodes Delta-Like1 protein consisting of 383 amino acids. In the embryonic stage, DLK1 is widely expressed and is involved in the growth and development of skeletal muscle; after birth, DLK1 expression is reduced and plays an important role in regulating puberty initiation.
Gene mutations or polymorphisms associated with CPP occurrence are up to 40-50, involving 4 genes. Therefore, it is technically difficult to detect these mutations with high efficiency, high sensitivity, and high specificity. Most of the current studies involve only individual sites of a single gene; the research of dozens of site detection can be simultaneously realized, and high-throughput sequencing technology is adopted, and has limitation on clinical applicability.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention aims to provide a detection primer for detecting central precocity of children, which is used for solving the problems of the prior art that the conventional detection for detecting central precocity of children involves few gene sites and the high throughput sequencing technology has clinical limitations, and also provides a gene detection reagent for detecting central precocity of children; meanwhile, the invention also provides a gene detection kit for detecting the central precocity of children; in addition, the invention also provides the application of the detection primer in preparing a gene detection kit for detecting the central precocity of children; meanwhile, the application of a detection primer in preparing a gene detection reagent for detecting the central precocity of children. The invention adopts a first-generation sequencing technology, simultaneously detects a plurality of variable sites in one sequencing reaction through reasonable and ingenious primer sequence design, realizes convenient detection of 42 variable sites, has short detection period and low cost, and is convenient for practical clinical popularization and application.
In order to attain the above and other related objects,
in a first aspect of the invention, a detection primer for detecting central precocity of children is provided, wherein the detection primer comprises an upstream primer and a downstream primer;
the upstream primer comprises MKRN3-1F, MKRN3-2F, MKRN3-3F, KISS1-F, KISS1R-F and DLK1-F, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 1-6;
the downstream primer comprises MKRN3-1R, MKRN3-2R, MKRN3-3R, KISS1-R, KISS1R-R, DLK1-R, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 7-12.
Primer sequence information is shown in table 7:
table 7
The invention adopts a first-generation sequencing technology, simultaneously detects a plurality of variable sites in one sequencing reaction through reasonable and ingenious primer sequence design, realizes convenient detection of 42 variable sites, has short detection period and low cost, and is convenient for practical clinical popularization and application. The detection of the pathogenic genes and the mutation sites related to the central precocity is beneficial to determining the pathogenesis of the central precocity, provides a basis of molecular medicine for implementing the genetic diagnosis and treatment scheme of the central precocity, and provides a tool for developing the precise medical diagnosis and treatment of the central precocity.
The gene mutation sites for central precocity detection are as follows:
(1) MKRN3 gene mutation site
c.89C>Tp.Pro30Leu，c.298A>T p.Ile100Phe，c.331G>T p.Glu111*，c.441-441delG p.His148Thr，c.475-476insC p.Ala162Gly，c.477-485del p.Pro160Cys，c.482-483insC p.Pro161Arg，c.482delC p.Pro161Arg，c.482insC p.Ala162Gly，c.587G>T p.Gly196Val，c.611T>C p.Ile204Thr，c.637delC p.Arg213Gly，c.675-676insA p.Gln226Thr，c.677A>C p.Gln226Pro，c.683-684insA p.Glu229Arg，c.699G>C p.Lys233Asn，c.737A>G p.Tyr246Cys，c.766-767delA p.Glu256Gly，c.802-803delAT p.Met268Val，c.841C>T p.Gln281*，c.935G>A p.Gly312Asp，c.943A>G p.Met315Val，c.982C>T p.Arg328Cys，c.1018T>G p.Cys340Gly，c.1034G>A p.Arg345His，c.1053-1056delACAG p.Arg351Ser，c.1095G>T p.Arg365Ser，c.1118C>T p.Pro373Leu，c.1171-1172insA p.Tyr391*，c.1188C>A p.Ser396Arg，c.1249T>A p.Phe417Ile，c.1260T>G p.His420Gln。
(2) Mutation site of KISS1 gene
c.220C>T p.Pro74Ser，c.242C>G/T p.Pro81Arg/Leu，c.268C>A/G/T p.His90Asn/Asp/Tyr，c.328C>A p.Pro110Thr。
(3) Mutation site of KISS1R gene
c.1091T>A/C p.Leu364His/Pro，c.1156C>T p.Arg386Cys，c.1157G>C p.Arg386Pro。
(4) DLK1 gene mutation site
c.479_479delC p.Pro160Leufs*50，c.594_594delC p.Gly199Alafs*11，c.810_810del T p.Val271Cysfs*14。
In a second aspect of the invention, a gene detection reagent for detecting central precocity of children is provided, which comprises the detection primer.
In a third aspect of the invention, a gene detection kit for detecting central precocity of children is provided, which comprises the detection primer.
In an embodiment of the present invention, a single-tube reaction solution in the gene detection kit includes the following components in parts by volume as shown in table 8:
table 8
| Name (R) | Volume ratio |
| 10×Buffer | 2.8～3.2 |
| Taq enzyme | 0.9～1.1 |
| MgCl2 | 1.8～2.2 |
| 10 μ M of forward primer | 1.3～1.7 |
| 10 μ M of downstream primer | 1.3～1.7 |
| 10 μ M deoxyribonucleoside triphosphate mixture | 2.8～3.2 |
| ddH2O | 15～17 |
Wherein 10 μ M of the forward primer and 10 μ M of the reverse primer are both prepared by sterile double distilled water (ddH)2O) the primers were diluted to 10. mu.M.
In one embodiment of the present invention, the single-tube reaction solution of the gene detection kit comprises the following components as shown in table 9:
table 9
| Name (R) | Volume of |
| 10×Buffer | 3μL |
| Taq enzyme | 1μL |
| MgCl2 | 2μL |
| 10 μ M of forward primer | 1.5μL |
| 10 μ M of downstream primer | 1.5μL |
| 10 μ M deoxyribonucleoside triphosphate mixture | 3μL |
| ddH2O | 16μL |
。
The third aspect of the invention provides an application of the detection primer in preparing a gene detection kit for detecting central precocity of children.
In one embodiment of the present invention, the method for detecting central precocity of children by using the gene detection kit comprises the following steps:
step one, carrying out PCR sample addition according to a single-tube reaction system shown in table 10:
table 10
| Name (R) | Volume ratio |
| 10×Buffer | 2.8～3.2 |
| Taq enzyme | 0.9～1.1 |
| MgCl2 | 1.8～2.2 |
| 10 μ M of forward primer | 1.3～1.7 |
| 10 μ M of downstream primer | 1.3～1.7 |
| Template DNA | 1.8～2.2 |
| 10 μ M deoxyribonucleoside triphosphate mixture | 2.8～3.2 |
| ddH2O | 15～17 |
；
The PCR reaction was carried out according to the set procedure, and the PCR reaction conditions are shown in Table 11:
table 11
And step two, performing electrophoresis on the products after the PCR reaction by using agarose gel, cutting the gel to recover PCR amplification products, sequencing the PCR amplification products by using a Sanger method, reading a sequencing result, and performing sequence comparison with a reference sequence.
In an embodiment of the present invention, the preparation process of the template DNA comprises:
A. adding 1-2.5 times of cell lysate CL into an anticoagulated whole blood sample, reversing and uniformly mixing, centrifuging, removing supernate, leaving cell nucleus precipitates, adding 0.3-0.4 times of buffer solution GS, and oscillating and uniformly mixing;
B. adding 0.03-0.04 time volume of protease K solution, oscillating and mixing uniformly, adding 0.3-0.4 time volume of buffer solution GB, oscillating and mixing uniformly, incubating at the constant temperature of 55-57 ℃ for 18-22 min, adding 0.3-0.4 time volume of absolute ethyl alcohol, fully reversing and mixing uniformly;
C. adding the solution obtained in the step B into an adsorption column CB3, centrifuging to remove waste liquid, adding a buffer solution GD with the volume of 0.9-1.0 time into the adsorption column CB3, centrifuging to remove the waste liquid, adding a rinsing liquid PW with the volume of 1.0-1.1 time into the adsorption column CB3, centrifuging to remove the waste liquid, finally adding a rinsing liquid PW with the volume of 1.0-1.1 time into the adsorption column CB3, centrifuging to remove the waste liquid, and placing the adsorption column CB3 into room temperature to dry the residual rinsing liquid in the adsorption column CB 3;
D. and C, transferring the adsorption column CB3 treated in the step C into a centrifugal tube, hanging and dropwise adding an elution buffer TB with the volume of 0.1-0.4 times of that of the adsorption film to the middle position of the adsorption film, standing at room temperature for 2-5 min, centrifuging, measuring OD260 and OD280 of the genomic DNA in the centrifugal tube by using an ultraviolet spectrophotometer, and storing the DNA with the ratio of OD260 to OD280 of the genomic DNA in the centrifugal tube of 1.7-1.9 in a refrigerator at-20 ℃ to obtain the template DNA.
In one embodiment of the present invention, the loading of PCR sample in the single-tube reaction system is shown in table 12:
table 12
| Name (R) | Volume of |
| 10×Buffer | 3μL |
| Taq enzyme | 1μL |
| MgCl2 | 2μL |
| 10 μ M of forward primer | 1.5μL |
| 10 μ M of downstream primer | 1.5μL |
| Template DNA | 2.0μL |
| 10 μ M deoxyribonucleoside triphosphate mixture | 3μL |
| ddH2O | 16μL |
；
The PCR reaction conditions are as in table 13:
table 13
In a third aspect of the invention, an application of the detection primer in preparing a gene detection reagent for detecting central precocity of children is provided.
As described above, the detection primer, the kit and the method for detecting central precocity of children of the present invention have the following beneficial effects: through a first-generation sequencing technology and reasonable and ingenious primer sequence design, a plurality of variable sites are simultaneously detected in one sequencing reaction, convenient detection of 42 variable sites is realized, the detection period is short, the cost is low, and the method is convenient to popularize and apply in clinical practice. The detection of the pathogenic genes and the mutation sites related to the central precocity is beneficial to determining the pathogenesis of the central precocity, provides a basis of molecular medicine for implementing the genetic diagnosis and treatment scheme of the central precocity, and provides a tool for developing the precise medical diagnosis and treatment of the central precocity.
Drawings
FIG. 1 is a partial diagram showing a sequence diagram of MKRN3 gene after PCR reaction using the gene detection kit of example 1.
FIG. 2 is a partial diagram showing a sequencing diagram of KISS1 gene after PCR reaction by the gene assaying kit in example 1.
FIG. 3 is a partial diagram showing a sequencing diagram of KISS1R gene after PCR reaction by the gene assaying kit in example 1.
FIG. 4 is a partial diagram showing a sequencing map of DLK1 gene after PCR reaction by the gene assaying kit in example 1.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Example 1
A gene detection kit for detecting central precocity of children comprises PCR reaction liquid, wherein detection primers of the PCR reaction liquid comprise an upstream primer and a downstream primer;
the upstream primer comprises MKRN3-1F, MKRN3-2F, MKRN3-3F, KISS1-F, KISS1R-F and DLK1-F, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 1-6;
the downstream primer comprises MKRN3-1R, MKRN3-2R, MKRN3-3R, KISS1-R, KISS1R-R, DLK1-R, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 7-12;
the single-tube reaction solution in the gene detection kit comprises the following components as shown in table 14:
table 14
The method for detecting the central precocity of the children by the gene detection kit comprises the following steps: step one, performing PCR sample addition according to a single-tube reaction system shown in table 15:
table 15
| Name (R) | Volume of |
| 10×Buffer | 3μL |
| Taq enzyme | 1μL |
| MgCl2 | 2μL |
| 10 μ M of forward primer | 1.5μL |
| 10 μ M of downstream primer | 1.5μL |
| Template DNA | 2μL |
| 10 μ M deoxyribonucleoside triphosphate mixture | 3μL |
| ddH2O | 16μL |
| Total volume | 30μL |
；
The PCR reaction was carried out according to the set procedure, and the PCR reaction conditions are shown in Table 16:
table 16
And step two, performing electrophoresis on the product after the PCR reaction by using 1.5% agarose gel, cutting the agarose gel and recovering the PCR amplification product, thereby ensuring the purity and integrity of the DNA. After recovery, the mixture was electrophoresed again to measure the concentration. Sequencing the PCR amplification product by a Sanger method, reading the sequencing result, and comparing the sequence with a reference sequence. The Sanger sequencing method is an internationally recognized sequencing gold standard, the accuracy of the gene sequencing by adopting the detection kit is basically 100%, and if the result of the gene sequencing is doubtful, the redetection can be carried out. The 42 variant sites are all CPP risk sites, and the mutation in any site means that the risk of CPP is increased.
Specifically, the preparation process of the template DNA comprises the following steps:
A. treatment of blood samples: adding 2.0 times volume of cell lysate CL into 600 mu L of anticoagulated whole blood sample, reversing and mixing uniformly, centrifuging at 12000rpm for 1 minute, removing supernatant, leaving cell nucleus precipitate, adding 200 mu L of buffer solution GS, oscillating and mixing uniformly;
B. adding 20 mu L of protease K solution, and fully and uniformly mixing; adding 200 mu L buffer solution GB, and fully and uniformly mixing; incubating at 56 deg.C for 20min, and mixing for several times; finally, 200 mu L of absolute ethyl alcohol is added, and the mixture is fully inverted and mixed evenly;
C. adding all the solution obtained in the step B into an adsorption column CB3, centrifuging at 12000rpm for 30 seconds, pouring the waste liquid in the collecting pipe, and putting the adsorption column CB3 back into the collecting pipe; adding 500 μ L buffer GD (added with anhydrous ethanol) into adsorption column CB3, centrifuging for 30 s at 12000rpm, pouring off waste liquid in the collection tube, and placing adsorption column CB3 back into the collection tube; adding 600 μ L of rinsing liquid PW (added with anhydrous ethanol) into adsorption column CB3, centrifuging for 30 s at 12000rpm, pouring off the waste liquid in the collection tube, and returning adsorption column CB3 to the collection tube; adding 600 μ L of rinsing solution PW (added with anhydrous ethanol) into adsorption column CB3, centrifuging for 2min at 12000rpm, and pouring off waste liquid; placing the adsorption column CB3 at room temperature for a plurality of minutes to completely dry the residual rinsing liquid in the adsorption column CB 3;
D. transferring the adsorption column CB3 treated in the step C into a 1.5mL centrifuge tube, suspending and dropwise adding 150 mu L of elution buffer TB into the middle position of an adsorption film, standing at room temperature for 3min, centrifuging for 2min at the rotating speed of 12000rpm, and collecting the solution into the centrifuge tube; the DNA product was measured for OD260 and OD280 by UV spectrophotometer, and the integrity and purity of the DNA was determined by the ratio of OD260/OD280, which was 1.8. And (3) storing the genome DNA in the centrifugal tube in a refrigerator at the temperature of-20 ℃ to avoid DNA degradation, thus obtaining the template DNA.
A partial map of the sequencing map of the MKRN3 gene after sequencing the PCR amplification products by Sanger's method is shown in FIG. 1. A partial map of the sequence chart of the KISS1 gene after sequencing the PCR amplification products by the Sanger method is shown in FIG. 2. A partial map of the sequencing map of the KISS1R gene after sequencing the PCR amplification products by the Sanger method is shown in FIG. 3. A partial map of the sequencing map of the DLK1 gene after sequencing the PCR amplification products by Sanger's method is shown in FIG. 4. The sequence read by the sequencing map is compared with the wild type sequence of the gene, so that whether the gene mutation exists or not and the complete information of the mutation site can be obtained.
Example 2
A gene detection kit for detecting central precocity of children comprises detection primers which are the same as those in embodiment 1;
the single-tube reaction solution in the gene detection kit comprises the following components as shown in table 17:
table 17
| Name (R) | Volume of |
| 10×Buffer | 3μL |
| Taq enzyme | 1μL |
| MgCl2 | 2μL |
| 10 μ M of forward primer | 1.4μL |
| 10 μ M of downstream primer | 1.4μL |
| 10 μ M deoxyribonucleoside triphosphate mixture | 2.8μL |
| ddH2O | 16μL |
| Total volume | 30μL |
。
The method for detecting the central precocity of the children by the gene detection kit comprises the following steps:
step one, performing PCR sample addition according to a single-tube reaction system shown in table 18:
table 18
| Name (R) | Volume of |
| 10×Buffer | 3μL |
| Taq enzyme | 1μL |
| MgCl2 | 2μL |
| 10 μ M of forward primer | 1.4μL |
| 10 μ M of downstream primer | 1.4μL |
| Template DNA | 1.8μL |
| 10 μ M deoxyribonucleoside triphosphate mixture | 2.8μL |
| ddH2O | 16μL |
| Total volume | 30μL |
；
The PCR reaction was carried out according to the set procedure, and the PCR reaction conditions are shown in Table 19:
table 19
And step two, performing electrophoresis on the product after the PCR reaction by using 1.5% agarose gel, cutting the agarose gel and recovering the PCR amplification product, thereby ensuring the purity and integrity of the DNA. After recovery, the mixture was electrophoresed again to measure the concentration. Sequencing the PCR amplification product by a Sanger method, reading the sequencing result, and comparing the sequence with a reference sequence.
Specifically, the preparation process of the template DNA comprises the following steps:
A. treatment of blood samples: adding 2.0 times volume of cell lysate CL into 600 mu L of anticoagulated whole blood sample, reversing and mixing uniformly, centrifuging at 12000rpm for 1 minute, removing supernatant, leaving cell nucleus precipitate, adding 200 mu L of buffer solution GS, oscillating and mixing uniformly;
B. adding 20 mu L of protease K solution, and fully and uniformly mixing; adding 200 mu L buffer solution GB, and fully and uniformly mixing; incubating at 56 deg.C for 20min, and mixing for several times; finally, 200 mu L of absolute ethyl alcohol is added, and the mixture is fully inverted and mixed evenly;
C. adding all the solution obtained in the step B into an adsorption column CB3, centrifuging at 12000rpm for 30 seconds, pouring the waste liquid in the collecting pipe, and putting the adsorption column CB3 back into the collecting pipe; adding 500 μ L buffer GD (added with anhydrous ethanol) into adsorption column CB3, centrifuging for 30 s at 12000rpm, pouring off waste liquid in the collection tube, and placing adsorption column CB3 back into the collection tube; adding 600 μ L of rinsing liquid PW (added with anhydrous ethanol) into adsorption column CB3, centrifuging for 30 s at 12000rpm, pouring off the waste liquid in the collection tube, and returning adsorption column CB3 to the collection tube; adding 600 μ L of rinsing solution PW (added with anhydrous ethanol) into adsorption column CB3, centrifuging for 2min at 12000rpm, and pouring off waste liquid; placing the adsorption column CB3 at room temperature for a plurality of minutes to completely dry the residual rinsing liquid in the adsorption column CB 3;
D. transferring the adsorption column CB3 treated in the step C into a 1.5mL centrifuge tube, suspending and dropwise adding 100 mu L of elution buffer TB into the middle position of an adsorption film, standing at room temperature for 5min, centrifuging for 2min at the rotating speed of 12000rpm, and collecting the solution into the centrifuge tube; the DNA product was measured for OD260 and OD280 by UV spectrophotometer, and the integrity and purity of the DNA was determined by the ratio of OD260/OD280, which was 1.7. And (3) storing the genome DNA in the centrifugal tube in a refrigerator at the temperature of-20 ℃ to avoid DNA degradation, thus obtaining the template DNA.
The Sanger sequencing method is an internationally recognized sequencing gold standard, the accuracy of the gene sequencing by adopting the detection kit is basically 100%, and if the result of the gene sequencing is doubtful, the redetection can be carried out.
In conclusion, the invention simultaneously detects a plurality of variable sites in one sequencing reaction through a first-generation sequencing technology and reasonable and ingenious primer sequence design, realizes convenient detection of 42 variable sites, has short detection period and low cost, and is convenient for practical clinical popularization and application. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
SEQUENCE LISTING
<110> Taizhou Zer-Wei inspection of medicine Co
<120> detection primer and kit for detecting children central precocity and application
<130> 2019.08.19
<160> 12
<170>PatentIn version 3.5
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ccggaagtaggtaggaacac ac 22
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tgcagcgtggtatggacaag 20
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tctggcaaacattctaaatcaaagc 25
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<213> Artificial sequence (Artificial)
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cctacgcgcttaagacctgg 20
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<213> Artificial sequence (Artificial)
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Claims (10)
1. A detection primer for detecting central precocity of children, which is characterized by comprising an upstream primer and a downstream primer;
the upstream primer comprises MKRN3-1F, MKRN3-2F, MKRN3-3F, KISS1-F, KISS1R-F and DLK1-F, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 1-6;
the downstream primer comprises MKRN3-1R, MKRN3-2R, MKRN3-3R, KISS1-R, KISS1R-R, DLK1-R, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 7-12.
2. A gene detection reagent for detecting central precocity of children, which is characterized in that: comprising the detection primer of claim 1.
3. A gene detection kit for detecting central precocity of children is characterized in that: comprising the detection primer of claim 1.
4. The gene detection kit for detecting the central precocity of children according to claim 3, wherein a single-tube reaction solution in the gene detection kit comprises the following components in parts by volume as shown in Table 1:
table 1
。
5. The gene detection kit for detecting central precocity of children according to claim 2 or 3, wherein the single-tube reaction solution in the gene detection kit comprises the following components as shown in Table 2:
table 2
6. Use of the detection primer of claim 1 in the preparation of a gene detection kit for detecting central precocity of children.
7. The use of claim 6, wherein the method for detecting the central precocity of children by using the gene detection kit comprises the following steps:
step one, carrying out PCR sample adding according to a single-tube reaction system shown in table 3:
table 3
；
The PCR reaction was carried out according to the set procedure, the PCR reaction conditions are shown in Table 4:
table 4
And step two, performing electrophoresis on the products after the PCR reaction by using agarose gel, cutting the gel to recover PCR amplification products, sequencing the PCR amplification products by using a Sanger method, reading a sequencing result, and performing sequence comparison with a reference sequence.
8. The use of claim 8, wherein the template DNA is prepared by:
A. adding 1-2.5 times of cell lysate CL into an anticoagulated whole blood sample, reversing and uniformly mixing, centrifuging, removing supernate, leaving cell nucleus precipitates, adding 0.3-0.4 times of buffer solution GS, and oscillating and uniformly mixing;
B. adding 0.03-0.04 time volume of protease K solution, oscillating and mixing uniformly, adding 0.3-0.4 time volume of buffer solution GB, oscillating and mixing uniformly, incubating at the constant temperature of 55-57 ℃ for 18-22 min, adding 0.3-0.4 time volume of absolute ethyl alcohol, fully reversing and mixing uniformly;
C. adding the solution obtained in the step B into an adsorption column CB3, centrifuging to remove waste liquid, adding a buffer solution GD with the volume of 0.9-1.0 time into the adsorption column CB3, centrifuging to remove the waste liquid, adding a rinsing liquid PW with the volume of 1.0-1.1 time into the adsorption column CB3, centrifuging to remove the waste liquid, finally adding a rinsing liquid PW with the volume of 1.0-1.1 time into the adsorption column CB3, centrifuging to remove the waste liquid, and placing the adsorption column CB3 into room temperature to dry the residual rinsing liquid in the adsorption column CB 3;
D. and C, transferring the adsorption column CB3 treated in the step C into a centrifugal tube, hanging and dropwise adding an elution buffer TB with the volume of 0.1-0.4 times of that of the adsorption film to the middle position of the adsorption film, standing at room temperature for 2-5 min, centrifuging, measuring OD260 and OD280 of the genomic DNA in the centrifugal tube by using an ultraviolet spectrophotometer, and storing the DNA with the ratio of OD260 to OD280 of the genomic DNA in the centrifugal tube of 1.7-1.9 in a refrigerator at-20 ℃ to obtain the template DNA.
9. The use of claim 8, wherein the single-tube reaction system is used for PCR loading as shown in Table 5:
table 5
；
The PCR reaction conditions are as in table 6:
table 6
10. Use of the detection primer of claim 1 in the preparation of a gene detection reagent for detecting central precocity in children.
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