CN113881761A - Primer and probe for identifying sex of embryo chromosome and application thereof - Google Patents
Primer and probe for identifying sex of embryo chromosome and application thereof Download PDFInfo
- Publication number
- CN113881761A CN113881761A CN202111341135.0A CN202111341135A CN113881761A CN 113881761 A CN113881761 A CN 113881761A CN 202111341135 A CN202111341135 A CN 202111341135A CN 113881761 A CN113881761 A CN 113881761A
- Authority
- CN
- China
- Prior art keywords
- seq
- embryo
- probe
- probes
- sex
- 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
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
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
-
- 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
- C12Q1/6851—Quantitative 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
- 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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- 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
-
- 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
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of sex identification, and particularly discloses a primer and a probe for identifying sex of embryo chromosomes and application thereof. The sequences of the primer and the probe for identifying the sex of the embryo chromosome are shown as SEQ ID NO 1-12. The primer and the probe for identifying the sex of the embryo chromosome can realize accurate identification of the sex of the embryo chromosome before transplantation under the condition of not damaging the embryo, and the identification method is simple, short in period, high in detection flux and high in sensitivity, and greatly reduces the cost and difficulty of sex identification of the embryo before planting compared with the traditional high-flux gene sequencing (NGS) method. Meanwhile, the identification process does not cause any damage to the embryo, and has important application value in related technologies of embryo transplantation and genetic detection.
Description
Technical Field
The invention relates to the technical field of sex identification, in particular to a primer and a probe for identifying sex of embryo chromosomes and application thereof.
Background
The technique of tube baby is a common name of artificial assisted pregnancy technique such as in vitro fertilization-embryo transplantation, is a comprehensive technique combining embryology, endocrine, genetics and micromanipulation, and is most effective in the method for treating infertility. The method is characterized in that sperms and ova are placed in vitro, various technologies are utilized to fertilize the ova, and the ova are cultured for several days and then are moved into a uterus, so that female is fertilized. The method can solve the problem of infertility, avoid the occurrence of sex-linked genetic diseases, and reduce social burden. For people with family history of the associated genetic diseases, the genetic test (PGT) before embryo implantation is carried out, and embryos which do not carry pathogenic variation are selected for transplantation, so that the repeated appearance of the associated genetic diseases in the family can be avoided. However, the invasive nature of the PGT technique to the embryo and the complexity of the technique limit its wide clinical application.
In recent years, with the discovery of free genomic DNA in embryo culture fluid and blastocoel fluid, new possibilities are opened for non-invasive pre-embryo genetic testing of assisted reproductive technologies. However, the concentration of free DNA in embryo culture is very low, and clinicians often use high throughput gene sequencing (NGS) to detect free DNA in embryo culture and analyze the chromosomal copy number of blastocysts. Although the NGS method can analyze the variation of embryo chromosomes, the application of the NGS method cannot avoid the defects of high detection cost, overlong report period, complicated analysis process and the like. At present, there is no other accurate method for detecting chromosome sex before embryo transplantation except NGS.
Disclosure of Invention
Aiming at the problems of the existing embryo preimplantation chromosome sex detection, the invention provides the primer and the probe for identifying the embryo chromosome sex and the application thereof.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a primer and probe for identifying the sex of an embryo chromosome comprising:
an upstream primer SEQ ID NO 1, a downstream primer SEQ ID NO 2 and a probe SEQ ID NO 3 which are designed according to the gene SRY;
an upstream primer SEQ ID NO. 4, a downstream primer SEQ ID NO. 5 and a probe SEQ ID NO. 6 which are designed according to the specific region of the Y chromosome; an upstream primer SEQ ID NO. 7, a downstream primer SEQ ID NO. 8 and a probe SEQ ID NO. 9;
specific primers and probes of an internal standard gene beta-actin: an upstream primer SEQ ID NO 10, a downstream primer SEQ ID NO 11 and a probe SEQ ID NO 12;
the fluorescent groups carried by the probe SEQ ID NO. 3 and the probe SEQ ID NO. 9 are different from the fluorescent groups carried by the probe SEQ ID NO. 6 and the probe SEQ ID NO. 12.
Compared with the prior art, the primer and the probe for identifying the sex of the chromosome of the embryo can realize the accurate identification of the sex of the chromosome of the embryo before transplantation by only utilizing the free DNA in the embryo culture solution under the condition of not damaging the embryo. The four groups of primers and probes can carry out multiple fluorescent quantitative PCR detection on free DNA in the embryo culture solution under the same fluorescent quantitative RCR reaction condition, compared with the prior method, the detection process effectively ensures the accuracy of the detection result under the condition of not damaging the embryo, and has the advantages of short detection period, simple method, high detection flux and high sensitivity.
Preferably, the probe SEQ ID NO. 3 and the probe SEQ ID NO. 9 carry FAM fluorophore; the probe SEQ ID NO. 6 and the probe SEQ ID NO. 12 carry a VIC fluorophore.
The probe SEQ ID NO 3 is specifically: 5 '-FAM-GATGACTGTACGAAAGCCAG-BHQ 1-3';
the probe SEQ ID NO 9 is specifically: 5 '-FAM-ATTCTTGTAGTACCTATGGGA-BHQ 1-3';
the probe SEQ ID NO 6 is specifically: 5 '-VIC-ATGACGAGCACATAACTTTA-BHQ 3-3';
the probe SEQ ID NO:12 is specifically: 5 '-YIC-TGCTGGGCGCCAGGGTGGC-BHQ 3-3';
the invention also provides a method for identifying the sex of the chromosome of the embryo by using the primer and the probe, which comprises the following steps:
a. extracting free DNA in the embryo culture solution;
b. in a fluorescent quantitative PCR reaction system A, the free DNA is taken as a template, the primer and the probe SEQ ID NO 1-6 are added, and fluorescent quantitative multiple PCR reaction is carried out; in a fluorescent quantitative PCR reaction system B, the free DNA is taken as a template, the primer and the probe SEQ ID NO. 7-12 are added, and fluorescent quantitative multiplex PCR reaction is carried out;
c. detecting fluorescent quantitative multiplex PCR reaction by using fluorescent detection channels corresponding to fluorescent groups carried by the probes SEQIDNO 3, 6, 9 and 12 respectively; if the probes SEQ ID NO. 3 and 12 detect fluorescence signals and at least one of the probes SEQ ID NO. 6 and 9 detects fluorescence signals, the embryo contains Y chromosome; if the probes SEQ ID NO 3, 6 and 9 have NO fluorescence signal detection and the probes SEQ ID NO 12 have NO fluorescence signal detection, the embryo does not contain Y chromosome; if NO fluorescent signal is detected in any of the probes SEQ ID NO. 3, 6, 9, 12, the detection fails.
Compared with the prior art, the method for identifying the sex of the embryo chromosome by using the primers and the probes, provided by the invention, has the advantages of simple operation, short detection period, low cost, no need of high-throughput gene sequencing, capability of completing the identification process by using a fluorescent quantitative PCR instrument, simple result judgment method, capability of saving a large amount of expensive instruments and equipment and fussy test operation, and easiness in popularization and application.
Preferably, in step b, the fluorescent quantitative PCR reaction system a is 23ul, and includes:
the fluorescent quantitative PCR reaction system B is 23ul and comprises:
the concentrations of the primers and the probes in the fluorescent quantitative PCR reaction system A and the fluorescent quantitative PCR reaction system B are both 10 uM; the concentration of the template is more than or equal to 10 ng/ul.
Preferably, the conditions of the fluorescent quantitative multiplex PCR reaction are as follows: pre-denaturation: 95 deg.C for 5 min; pre-amplification: 94 ℃, 20s, 57 ℃, 20s, 72 ℃, 60s, said pre-amplification for a total of 4 cycles; detection and amplification: the detection amplification was carried out for 35 cycles at 94 ℃ for 15s, 56 ℃ for 60 s.
The invention also provides a kit for identifying the sex of the chromosome of the embryo before implantation, which comprises the primer and the probe.
Preferably, the kit further comprises a PCR buffer and a DNA polymerase.
Drawings
FIG. 1 is a graph showing the amplification of FAM channel detection when embryos contain Y chromosome in example 2 of the present invention;
FIG. 2 is a graph showing the amplification of the VIC channel detection when the embryo contains Y chromosome in example 2 of the present invention;
FIG. 3 is a graph showing the amplification of the VIC channel detection in the case where the embryo does not contain the Y chromosome in example 2 of the present invention;
FIG. 4 is a graph showing the amplification of the FAM channel detection when the embryo does not contain the Y chromosome in example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
Primers and probes for the identification of the chromosomal sex of an embryo comprising:
an upstream primer SEQ ID NO 1, a downstream primer SEQ ID NO 2 and a probe SEQ ID NO 3 which are designed according to the gene SRY;
an upstream primer SEQ ID NO. 4, a downstream primer SEQ ID NO. 5 and a probe SEQ ID NO. 6 which are designed according to the specific region of the Y chromosome; an upstream primer SEQ ID NO. 7, a downstream primer SEQ ID NO. 8 and a probe SEQ ID NO. 9;
specific primers and probes of an internal standard gene beta-actin: an upstream primer SEQ ID NO. 10, a downstream primer SEQ ID NO. 11 and a probe SEQ ID NO. 12.
Wherein, the probe SEQ ID NO. 3 and the probe SEQ ID NO. 9 carry FAM fluorophore; probe SEQ ID NO 6 and probe SEQ ID NO 12 carry a VIC fluorophore.
The method for identifying the sex of the embryo chromosome by using the primer and the probe comprises the following steps:
a. for assisted reproduction assisted pregnancy patients, fertilized eggs obtained by adopting a single sperm intracytoplasmic injection mode are cleaned by blastocyst culture solution, embryos cultured on the 3 rd day are transferred to newly prepared 10ul blastocyst culture microdroplets to be continuously cultured to the 5 th day, the blastocysts are completely expanded, the embryos forming the blastocysts are taken out for cryopreservation, 10ul of the rest embryo culture solution is collected as a sample to be detected, and free DNA in the sample to be detected is extracted. By using
The micro-genome rapid amplification kit increases the copy number of free DNA of a sample to be detected by a whole genome DNA in-vitro amplification technology, and then uses a TIANGEN common DNA product purification kit to purify an amplification product and remove residual DNA polymerase, primers, dNTP and Mg in a system2+And the like, and the purified DNA is used as a template for detection;
the purification method comprises the following steps:
using a DNA purification column to remove residual DNA polymerase, primers, dNTP and Mg in the whole genome amplification reaction system2+And the specific steps are as follows:
1) adding 500ul of balance liquid into the DNA purification column, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and replacing the adsorption column into the collection tube again;
2) taking a new 1.5ml enzyme-free centrifuge tube, adding 500ul of binding solution, adding DNA to be purified into the binding solution, and fully and uniformly mixing to obtain a mixed solution;
3) adding the mixed solution into the adsorption column prepared in the step (1), standing at room temperature for 2min, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and replacing the adsorption column into the collection tube again;
4) adding 600ul of rinsing liquid into the adsorption column, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and replacing the adsorption column into the collection tube;
5) repeating the operation (4) to sufficiently wash the product;
6) placing the adsorption column back into the collecting tube, centrifuging at 12000rpm for 2min, removing rinsing liquid, standing at room temperature for 5min, and air drying the adsorption column completely;
7) and (3) placing the dried adsorption column into a new 1.5ml enzyme-free centrifuge tube, suspending and dripping 30ul of eluent into the center of the adsorption membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 2min, and collecting the purified DNA solution.
8) Taking out 2ul of each sample, detecting the DNA concentration of the sample, and taking the sample with the concentration more than or equal to 10ng/ul as a qualified sample which can be used as a template for carrying out fluorescence quantitative PCR detection.
b. In a fluorescent quantitative PCR reaction system A, taking the DNA as a template, adding primers and probes SEQ ID NO 1-6, and carrying out fluorescent quantitative multiplex PCR reaction; in a fluorescent quantitative PCR reaction system B, taking the DNA as a template, adding primers and probes SEQ ID NO. 7-12, and carrying out fluorescent quantitative multiplex PCR reaction; the PCR reaction systems A and B simultaneously carry out a fluorescent quantitative PCR reaction program;
c. monitoring the fluorescent quantitative multiplex PCR reaction process by using FAM and VIC fluorescent detection channels respectively; if the probes SEQ ID NO. 3 and 12 detect fluorescence signals and at least one of the probes SEQ ID NO. 6 and 9 detects fluorescence signals, the embryo contains Y chromosome; if the probes SEQ ID NO 3, 6 and 9 have NO fluorescence signal detection and the probes SEQ ID NO 12 have NO fluorescence signal detection, the embryo does not contain Y chromosome; if NO fluorescent signal is detected in any of the probes SEQ ID NO. 3, 6, 9, 12, the detection fails.
The fluorescent quantitative PCR reaction system A is 23ul and comprises:
the fluorescent quantitative PCR reaction system B is 23ul and comprises:
the concentration of the primers and the concentration of the probes in the fluorescent quantitative PCR reaction system A and the fluorescent quantitative PCR reaction system B are both 10 uM;
the conditions of the fluorescent quantitative multiplex PCR reaction are as follows: pre-denaturation: 95 deg.C for 5 min; pre-amplification: 94 ℃, 20s, 57 ℃, 20s, 72 ℃, 60s, said pre-amplification for a total of 4 cycles; detection and amplification: the detection amplification was carried out for 35 cycles at 94 ℃ for 15s, 56 ℃ for 60 s.
Setting a positive control and a negative control in the fluorescent quantitative PCR detection process respectively: the positive control is normal male genome DNA; the negative control was deionized water. The primers, the probe, the buffer solution, the Taq enzyme and the fluorescent quantitative PCR reaction program added in the positive control and the negative control are consistent with the sample to be detected.
Example 2
8 in vitro fertilized embryo samples (patient donated voluntarily) were selected, and the presence or absence of the Y chromosome in the embryo culture solution was examined using the primers and probes and the detection method of example 1, to identify the chromosomal sex of the embryo. The fluorescence signal of the fluorescence quantitative PCR when the embryo contains the Y chromosome is: FAM channel has 2 amplification curves as shown in fig. 1, and VIC channel has at least 1 amplification curve as shown in fig. 2, i.e., the sex of embryo is male; the fluorescence signal of the fluorescence quantitative PCR when the embryo does not contain Y chromosome is as follows: only 1 amplification curve corresponding to SEQ ID NO. 12 was detected in the VIC channel as shown in FIG. 3, and none of the remaining channels and probes had amplification curves as shown in FIG. 4, i.e., the embryo was female in sex.
The results of the examination of the culture solutions of the 8 cases of embryonic cells were compared by the above-described assessment method, wherein 5 cases of embryos containing the Y chromosome and 3 cases of embryos containing no Y chromosome were used. And then, performing gene detection on the blastula cells of the 8 embryos by using a high-throughput gene sequencing technology, wherein the detection result completely accords with the detection result of the culture solution of the embryonic cells.
The primers and the probes have the same accuracy rate when used for carrying out fluorescence quantitative PCR detection and high-throughput gene sequencing technology detection on the embryo culture solution, the detection result of each sample is effective, no detection failure sample exists, and the detection method is stable and reliable.
Example 3
A kit for identifying the sex of embryo chromosome before implantation comprises the primer and probe SEQ ID NO 1-12, PCR buffer solution and DNA polymerase.
The method for detecting the sex of the embryo chromosome before the transplantation by using the kit is the same as the example 1.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
SEQUENCE LISTING
<110> Hospital for reproduction in Hebei province
<120> primer and probe for identifying embryo chromosome sex and application thereof
<130> 2021
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 1
tgtgcctcct ggaagaatga 20
<210> 2
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 2
ccgaagcggg atctgcgta 19
<210> 3
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 3
gatgactgta cgaaagccag 20
<210> 4
<211> 21
<212> DNA
<213> Artificial Synthesis
<400> 4
cacgccgaat cctgtaactc t 21
<210> 5
<211> 21
<212> DNA
<213> Artificial Synthesis
<400> 5
atcctcctcc accacagttt c 21
<210> 6
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 6
atgacgagca cataacttta 20
<210> 7
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 7
acctaacaag caattctaaa cct 23
<210> 8
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 8
tttgacaaag tggctgctg 19
<210> 9
<211> 21
<212> DNA
<213> Artificial Synthesis
<400> 9
attcttgtag tacctatggg a 21
<210> 10
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 10
tctgcatcct atcggcgat 19
<210> 11
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 11
tgaagatcaa gatgactgct 20
<210> 12
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 12
tgctgggcgc cagggtggc 19
Claims (7)
1. A primer and probe for identifying the sex of an embryo chromosome, characterized in that: the method comprises the following steps:
an upstream primer SEQ ID NO 1, a downstream primer SEQ ID NO 2 and a probe SEQ ID NO 3 which are designed according to the gene SRY;
an upstream primer SEQ ID NO. 4, a downstream primer SEQ ID NO. 5 and a probe SEQ ID NO. 6 which are designed according to the specific region of the Y chromosome; an upstream primer SEQ ID NO. 7, a downstream primer SEQ ID NO. 8 and a probe SEQ ID NO. 9;
specific primers and probes of an internal standard gene beta-actin: an upstream primer SEQ ID NO 10, a downstream primer SEQ ID NO 11 and a probe SEQ ID NO 12;
the fluorescent groups carried by the probe SEQ ID NO. 3 and the probe SEQ ID NO. 9 are different from the fluorescent groups carried by the probe SEQ ID NO. 6 and the probe SEQ ID NO. 12.
2. Primers and probes for the sex determination of chromosomes in embryos according to claim 1, characterized in that: the probe SEQ ID NO. 3 and the probe SEQ ID NO. 9 carry FAM fluorophore; the probe SEQ ID NO. 6 and the probe SEQ ID NO. 12 carry a VIC fluorophore.
3. A method for sex determination of embryo chromosomes using the primers and probes according to claim 1 or 2, characterized in that: the method comprises the following steps:
a. extracting free DNA in the embryo culture solution;
b. in a fluorescent quantitative PCR reaction system A, the free DNA is taken as a template, the primer and the probe SEQ ID NO 1-6 are added, and fluorescent quantitative multiple PCR reaction is carried out; in a fluorescent quantitative PCR reaction system B, the free DNA is taken as a template, the primer and the probe SEQ ID NO. 7-12 are added, and fluorescent quantitative multiplex PCR reaction is carried out;
c. detecting fluorescent quantitative multiplex PCR reaction by using fluorescent detection channels corresponding to fluorescent groups carried by the probes SEQ ID NO. 3, 6, 9 and 12 respectively; if the probes SEQ ID NO. 3 and 12 detect fluorescence signals and at least one of the probes SEQ ID NO. 6 and 9 detects fluorescence signals, the embryo contains Y chromosome; if the probes SEQ ID NO 3, 6 and 9 have NO fluorescence signal detection and the probes SEQ ID NO 12 have NO fluorescence signal detection, the embryo does not contain Y chromosome; if NO fluorescent signal is detected in any of the probes SEQ ID NO. 3, 6, 9, 12, the detection fails.
4. A method of performing sex determination of chromosomes in embryos according to claim 3, wherein: in the step b, the fluorescent quantitative PCR reaction system A is 23ul and comprises:
the fluorescent quantitative PCR reaction system B is 23ul and comprises:
the concentrations of the primers and the probes in the fluorescent quantitative PCR reaction system A and the fluorescent quantitative PCR reaction system B are both 10 uM; the concentration of the template is more than or equal to 10 ng/ul.
5. A method of performing sex determination of chromosomes in embryos according to claim 3, wherein: the conditions of the fluorescent quantitative multiplex PCR reaction are as follows: pre-denaturation: 95 deg.C for 5 min; pre-amplification: 94 ℃, 20s, 57 ℃, 20s, 72 ℃, 60s, said pre-amplification for a total of 4 cycles; detection and amplification: the detection amplification was carried out for 35 cycles at 94 ℃ for 15s, 56 ℃ for 60 s.
6. A kit for identifying the sex of a chromosome of a preimplantation embryo, which is characterized in that: comprising the primer and probe of claim 1 or 2.
7. The kit for sex identification of chromosomes in a preimplantation embryo according to claim 6, wherein: also included are PCR buffers and DNA polymerases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111341135.0A CN113881761A (en) | 2021-11-12 | 2021-11-12 | Primer and probe for identifying sex of embryo chromosome and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111341135.0A CN113881761A (en) | 2021-11-12 | 2021-11-12 | Primer and probe for identifying sex of embryo chromosome and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113881761A true CN113881761A (en) | 2022-01-04 |
Family
ID=79017444
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111341135.0A Pending CN113881761A (en) | 2021-11-12 | 2021-11-12 | Primer and probe for identifying sex of embryo chromosome and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113881761A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148484A (en) * | 2015-03-27 | 2016-11-23 | 上海透景生命科技股份有限公司 | A kind of diagnose the test kit that Y chromosome is micro-deleted |
| CN106811512A (en) * | 2015-12-01 | 2017-06-09 | 北京爱普益生物科技有限公司 | A kind of micro-deleted method in quick detection people Y chromosome AZF areas, kit and its preparation |
| CN110607358A (en) * | 2019-09-18 | 2019-12-24 | 河北医科大学第二医院 | Primer and probe for identifying chromosome sex of fetus in early pregnancy, identification method and kit |
| CN113584142A (en) * | 2021-08-04 | 2021-11-02 | 塔里木大学 | Sex identification method for mammalian embryo |
-
2021
- 2021-11-12 CN CN202111341135.0A patent/CN113881761A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148484A (en) * | 2015-03-27 | 2016-11-23 | 上海透景生命科技股份有限公司 | A kind of diagnose the test kit that Y chromosome is micro-deleted |
| CN106811512A (en) * | 2015-12-01 | 2017-06-09 | 北京爱普益生物科技有限公司 | A kind of micro-deleted method in quick detection people Y chromosome AZF areas, kit and its preparation |
| CN110607358A (en) * | 2019-09-18 | 2019-12-24 | 河北医科大学第二医院 | Primer and probe for identifying chromosome sex of fetus in early pregnancy, identification method and kit |
| CN113584142A (en) * | 2021-08-04 | 2021-11-02 | 塔里木大学 | Sex identification method for mammalian embryo |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2021007403A (en) | Method of detecting chromosome abnormality in embryo by using blastocyst culture solution | |
| KR101781147B1 (en) | Methods and genotyping panels for detecting alleles, genomes, and transcriptomes | |
| EP3406736A1 (en) | Methods for the diagnosis of fetal abnormalities | |
| CN113286892B (en) | Method and product for detecting embryo health condition by using blastula culture solution | |
| EP3099818B1 (en) | Preimplantation assessment of embryos through detection of free embryonic dna | |
| CN109504784B (en) | MiRNA molecular marker for predicting early embryo quality in human assisted reproduction technology and application thereof | |
| CN112251518B (en) | Molecular marker associated with lambing number and growth traits in goat RSAD2 gene and application thereof | |
| CN109920479B (en) | Method for identifying chromosome inversion carrying state of embryo | |
| CN114480610A (en) | Method for detecting translocation fragment monomer or trisomy in latent equilibrium translocation carrier embryo | |
| CN109182493B (en) | Primer and kit for detecting human 16p11.2 microdeletion syndrome and detection method thereof | |
| CN111440857B (en) | Method for non-invasive genetic detection of embryo before implantation | |
| Ren et al. | Preimplantation genetic diagnosis for Duchenne muscular dystrophy by multiple displacement amplification | |
| CN112029850A (en) | Primer pair, probe and kit for detecting thalassemia gene and using method | |
| CN109371122B (en) | Reference gene for panda milk miRNA detection and application thereof | |
| KR101686446B1 (en) | SNP markers for discriminating Yeonsan Ogye breed of chicken and use thereof | |
| CN113881761A (en) | Primer and probe for identifying sex of embryo chromosome and application thereof | |
| CN113684282B (en) | Method for detecting pig hairless character based on EDAR gene expression quantity and application thereof | |
| CN114480609A (en) | Method for identifying chromosome insertion translocation carrying embryo and normal embryo | |
| CN104388587A (en) | One-step detection kit for influenza B virus and influenza B virus detection method | |
| Song et al. | Genome-wide screening of severe male factor infertile patients using BAC-array comparative genomic hybridization (CGH) | |
| CN108998545B (en) | Application of cynoglossus semilaevis exosome piR-mmu-31018127 | |
| CN109112194B (en) | Application of cynoglossus semilaevis sex label piR-mmu-72274 | |
| CN111378724A (en) | RNA amplification detection method and detection kit | |
| CN114752664B (en) | SNP (Single nucleotide polymorphism) marker for identifying genetic sex of coilia ectenes as well as primer and application of SNP marker | |
| CN109971863B (en) | Application of cynoglossus semilaevis sex tag piR-mmu-29271668 |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220104 |
|
| RJ01 | Rejection of invention patent application after publication |