CN113584141A - Sex identification method for mammalian embryo - Google Patents
Sex identification method for mammalian embryo Download PDFInfo
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- CN113584141A CN113584141A CN202110854857.XA CN202110854857A CN113584141A CN 113584141 A CN113584141 A CN 113584141A CN 202110854857 A CN202110854857 A CN 202110854857A CN 113584141 A CN113584141 A CN 113584141A
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- 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
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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention relates to a sex identification method of a mammal embryo. A method of sexing a mammalian embryo comprising the steps of: (1) obtaining an embryo; (2) obtaining genome DNA of the embryonic cells; (3) designing and synthesizing a primer; (4) QPCR reaction: adding fluorescent dye into a PCR reaction system; (5) after the curve was amplified and analyzed, the embryo sex was identified. The sex determination method of the mammal embryo, disclosed by the invention, has the advantages of simple design, high sensitivity, short time consumption, low initial cost, good universality, high automation degree and the like, and only needs one pair of primers for analyzing and detecting the specificity of an amplification reaction through a melting point curve in order to apply a real-time fluorescent quantitative PCR method.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a sex identification method of a mammalian embryo.
Background
The research on sex determination and control of mammals is a great subject of molecular embryology, and has important theoretical significance and practical application value. The mastering and control of livestock sex determination is a biological technology capable of obviously improving the breeding efficiency of livestock. Methods for sex control include X, Y sperm isolation and embryo sex determination and controlling the environment of insemination. X, Y sperm separation can damage sperm during separation, especially for fragile sheep sperm; moreover, the price of the instrument is expensive, and the instrument is difficult to configure in a common laboratory. Methods for regulating the internal environment of the uterus of female animals, i.e., the environment for insemination, are still under investigation and perfection. Embryo sexing, as part of sex control methods, is currently the main approach taken, progressing from individual, cellular to molecular levels.
The cytological method is mainly used for judging the sex of the embryo by analyzing the chromosome composition (female is XX, male is XY) of partial embryo cells. The immunological methods (i.e., cytotoxicity assays, indirect immunofluorescence and blastogenesis inhibition) are based on the presence of specific H-Y antigens on male embryos and are detected using H-Y antisera or H-Y monoclonal antibodies to determine the sex of the embryo. The sex identification is carried out by a molecular biology method (a Fluorescence In Situ Hybridization (FISH) method, a loop-enzyme isothermal amplification (LAMP) method and a PCR amplification method) according to specific genes between male and female. The PCR method is simple, rapid, accurate and sensitive, and becomes the most common and widely applied identification method at present. The PCR method essentially synthesizes primers according to specific gene segments on the chromosome, detects the existence of X/Y chromosome specific segments and realizes the purpose of sex identification. However, the detection rate of sex identification using chromosomes is low, and the identification time is long, so that embryos are often damaged inevitably, and the conception rate after displacement is reduced.
At present, the amplification of male specific DNA fragments, ZFX/ZFY genes, SRY genes, ALME genes and the like by taking trace embryonic cell DNA as a template is the mainstream trend of a PCR identification method. Animal sex identification and control technology has achieved certain achievements at present, and has certain influence on animal husbandry. But the method needs to explore a technical method which is rapid, simple, convenient, high in accuracy, low in cost and convenient for large-scale production.
However, when the sex of an embryo is determined, a part of embryo cells is cut out as a template, which may cause some damage to the embryo. In order to ensure the survival rate of the embryo, the identification effect is not ideal while the number of embryo cells is cut as little as possible. Therefore, the sensitivity and the identification efficiency of sex identification and a more stable identification effect are improved, and meanwhile, the method needs to explore a technology which is rapid, simple, convenient, high in accuracy, low in cost and convenient for large-scale production, and has great significance.
In view of the above, the present invention provides a novel method for identifying the sex of a mammalian embryo, which is a Real-time fluorescent Real-time PCR (Quantitative Real-time PCR) fluorescent dye method.
Disclosure of Invention
The invention aims to provide a sex determination method of mammalian embryos, which is a real-time fluorescence quantitative PCR method, has simple design, high sensitivity, short time consumption and good universality, can meet the requirement of ultra-micro templates for sex determination of embryos (namely higher sensitivity), can complete sex determination of embryos according to an amplification curve, and can perform sex determination of a plurality of embryos in the whole process.
In order to realize the purpose, the adopted technical scheme is as follows:
a method of sexing a mammalian embryo comprising the steps of:
(1) obtaining an embryo;
(2) obtaining genome DNA of the embryonic cells;
(3) designing and synthesizing a primer;
(4) QPCR reaction: adding a proper amount of fluorescent dye into a PCR reaction system;
(5) after the curve was amplified and analyzed, the embryo sex was identified.
Further, in the step (5), after the amplification curve, the embryo with the male-specific gene amplification curve is male, otherwise, the embryo is female.
Further, the amplification curve is at least one of an embryo DNA amplification curve and an embryo DNA dissolution curve.
Further, in the step (4), the amplification conditions of the QPCR reaction are: after pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s and annealing extension at 65 ℃ for 20s, the process was cycled 40 times.
Still further, in the step (4), every 10 μ L of PCR reaction system is: 2 XS 6Universal SYBR qPCR Mix 5. mu.L, upstream and downstream primers 0.25. mu.L each, DNA template 1. mu.L, dd H2O 3.5μL。
Further, the embryo is a live embryo, or an embryo produced by in vitro fertilization, or a frozen embryo.
Further, the sex determination method is used for determining sheep embryos.
Compared with the prior art, the invention has the beneficial effects that:
1. the technical scheme of the invention is a Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR) method, wherein a proper amount of fluorescent dye is added into a PCR reaction system, the fluorescent dye nonspecifically dopes into a DNA double strand and then emits a fluorescent signal, and SYBR dye molecules which are not doped into the strand cannot emit any fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous. SYBR binds only to double-stranded DNA and thus it can be determined whether the PCR reaction is specific by a lysis curve. Compared with common PCR and nested PCR, the method has higher sensitivity and shorter time, and can directly carry out judgment and analysis according to an amplification curve; compared with the FISH method, the method is more convenient and easier to operate, has low cost, and can also achieve higher identification efficiency and accuracy.
2. The technical scheme of the invention is used for identifying the sex of the sheep early embryo, has higher sensitivity, simpler and more convenient operation and economic cost, greatly shortens the reaction time, improves the identification efficiency, ensures the reliability of the identification result and has higher operability.
Drawings
FIG. 1 shows the results of blood genome amplification;
FIG. 2 is a blood genome amplification lysis curve;
FIG. 3 shows the result of sex determination and amplification of 10 embryos;
FIG. 4 is a 10 embryo sexing lysis curve.
Detailed Description
In order to further illustrate the method for sex determination of a mammalian embryo according to the present invention and achieve the intended purpose, the following embodiments are provided to describe the method for sex determination of a mammalian embryo according to the present invention, and the detailed description, structure, features and effects thereof are given below. In the following description, different "one embodiment" or "an embodiment" refers to not necessarily the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
Before describing in detail a method for sexing a mammalian embryo according to the present invention, it is necessary to further describe the related methods mentioned in the present invention in order to achieve better results.
At present, common molecular biology identification methods include DNA probe methods, Fluorescence In Situ Hybridization (FISH) methods, PCR amplification methods, and other major methods. But each has advantages and disadvantages. Specifically, the method comprises the following steps:
the DNA probe method designs and synthesizes a DNA probe according to the gene sequence of the Y chromosome, and hybridizes and marks cells in the embryo or DNA target genes of the embryo cells, wherein the positive one is a male embryo, and the negative one is a female embryo. The method has high identification accuracy, but the synthesis of the probe needs certain cost.
The FISH method mainly comprises 3 parts of preparation of a fluorescein probe, denaturation and hybridization of the probe and target DNA, and observation and identification. The probe is firstly combined with a reporter molecule, and is connected with a fluorescent dye through an immunocytochemistry process after hybridization to generate fluorescence, the method can be used for hybridization identification in the interphase and metaphase of cells, and the method has the advantages of strong specificity, high efficiency and low error rate, but needs longer time, has strict requirements on reagents and high identification cost, and is difficult to apply in production practice.
The PCR method is to add proper amount of buffer solution, template DNA, 4 kinds of deoxymononucleotide (dNTP) solution, heat-resistant DNA polymerase, Mg2+ and 1 pair of synthetic primers into a micro centrifugal tube according to the characteristics of DNA. The method comprises the steps of utilizing DNA to decompose helix denatured double strand at 90-95 ℃ in vitro to be cracked into single strand, combining a primer and the single strand according to the principle of base complementary pairing at low temperature (about 60 ℃), adjusting the temperature to the optimal reaction temperature (about 72 ℃) of DNA polymerase, taking d NTPs as a raw material, synthesizing a complementary strand along the direction from phosphoric acid to pentose (5'-3') under the action of the DNA polymerase, and repeatedly using a newly formed DNA fragment as a next reaction template, wherein the amplification product of the target DNA is increased at the speed of 2, so that the specific gene is amplified.
The conventional PCR is used for carrying out electrophoresis on an amplification product, the amplification product is stained by ethidium bromide, and the amplification result is detected under the irradiation of an ultraviolet lamp, so that the amplified specific segment DNA band is male, otherwise, the amplified specific segment DNA band is female, but the sex cannot be effectively identified under the condition of lower template concentration. The nested PCR utilizes a pair of sex control primers and a pair of inner primers to enrich a PCR template in advance, and then carries out the amplification of the second inner primer PCR to detect the amplification result under the irradiation of an ultraviolet lamp to identify the sex of the embryo. The method can amplify a large amount of trace DNA templates, improves the identification efficiency to a certain extent, but also increases the workload, prolongs the identification time and increases the pollution risk.
The Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR) is characterized in that the Ct value of a template product and the initial copy number of the template have a linear relation in the PCR amplification reaction process, the increase of a fluorescent signal is completely synchronous with the increase of a PCR product, and the fluorescent signal of a fluorescent chemical substance is used for carrying out Real-time detection on the PCR process, so that the existence of a certain gene can be judged, and the Quantitative analysis can be carried out on the certain gene. The purpose of quantitative analysis of the specific DNA sequence in the sample to be detected is achieved by measuring the total amount of products after each Polymerase Chain Reaction (PCR) cycle. High sensitivity: a single copy gene can be detected; the dynamic range is wide; high repeatability; high resolution; high speed: 40 PCR cycles were completed in 60 min; and an advanced optical detection system is matched, so that the edge effect is thoroughly eliminated, and a stable and excellent detection result is kept.
With the understanding of the related methods mentioned in the present invention, the method for sexing a mammalian embryo according to the present invention will be described in further detail with reference to specific examples:
the technical scheme of the invention is as follows:
a method of sexing a mammalian embryo comprising the steps of:
(1) obtaining an embryo;
(2) obtaining genome DNA of the embryonic cells;
(3) designing and synthesizing a primer;
(4) QPCR reaction: adding a proper amount of fluorescent dye into a PCR reaction system;
(5) after the curve was amplified and analyzed, the embryo sex was identified.
Preferably, in the step (5), after the amplification curve, the embryo with the SRY gene amplification curve is male, otherwise, the embryo is female.
Preferably, the amplification curve is at least one of an embryonic DNA amplification curve and an embryonic DNA lysis curve.
Preferably, in the step (4), the amplification conditions of the QPCR reaction are: after pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s and annealing extension at 65 ℃ for 20s, the process was cycled 40 times.
Further preferably, in the step (4), every 10 μ L of PCR reaction system is: 2 XS 6Universal SYBR qPCR Mix 5. mu.L, upstream and downstream primers 0.25. mu.L each, DNA template 1. mu.L, dd H2O 3.5μL。
Preferably, the embryo is a live embryo, or an embryo produced by in vitro fertilization, or a frozen embryo.
Preferably, the sex determination method is used for identifying sheep embryos.
Example 1.
The technical scheme of the embodiment is as follows:
(1) obtaining an embryo: including in vivo embryo collection or in vitro fertilization and frozen embryo can be used for sex determination.
(2) And (3) obtaining the genome DNA of the embryonic cell.
(3) Design and synthesis of primers.
Male specific gene primer sequence: upstream 5 '-TTGCACCCCTTCACATACAG-3'; downstream 5' -AGCCAATGTTACCCTATCGTG-3
Internal reference gene primer sequence: upstream 5 '-GGTCCACATGGCCTCCAAG-3';
downstream 5' -TCCCTTTCCTCAGGGCCTT-3
(4) QPCR reaction: adding excessive SYBR fluorescent dye into a PCR reaction system;
10. mu.L per PCR reaction: 2 XS 6Universal SYBR qPCR Mix 5. mu.L, upstream and downstream primers 0.25. mu.L each, DNA template 1. mu.L, dd H2O 3.5μL。
Q-PCR amplification conditions: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, annealing and extension at 65 ℃ for 20s, and 40 cycles.
(5) And (4) analyzing an amplification curve and identifying the sex.
Wherein the parts not described in detail are all conventional methods in the field. The partial amplification curve results were:
(1) blood DNA amplification curves
The male and female sheep blood DNA with known concentration is diluted to (male 5.4pg/uL, 5.6pg/uL, 5.2pg/uL and female 5.7pg/uL) and amplified according to the above-mentioned method system. The ". cndot." -labeled curve was used as the reference gene amplification curve, and unlabeled one was used as the male-specific gene amplification curve (three replicates each), and the ". DELTA." -was used as the blank. Consistent with the expected results, only ram genomes were amplified to obtain male specific gene amplification curves (amplification curves were obtained repeatedly) except that the male and female reference genes were obtained amplification curves, as shown in fig. 1 (in the figure, the amplification curve represents a fluorescence signal, the earlier the peak is, the earlier the fluorescence is received, the higher the curve is, the stronger the fluorescence signal is, and vice versa).
(2) Blood DNA amplification lysis curve
Corresponding to FIG. 1, the "B" peak curve is an internal reference gene lysis curve, the "A" peak curve is a male specific gene lysis curve (three replicates for each sample), the blank group has no lysis curve, and only the ram genome obtains a male specific gene lysis curve (the replicates obtain lysis curves) except for the internal reference gene male and female, as shown in FIG. 2 (in the figure, each curve has a peak in a normal range of about 85 ℃, and is a single peak, without dimer effect and non-specific amplification).
Sex determination of 10 embryos of unknown sex:
(1) embryonic DNA amplification curves
The marked curves are used as reference gene amplification curves of each embryo, the unmarked curves are male specific gene amplification curves (three repeats per embryo), and the delta is used as blank group. The 10 embryos are all amplified to obtain an internal reference gene curve, wherein the male specific gene curve obtained by amplifying 6 embryos (the amplification curve is obtained by three repetitions) is a male embryo, the other 4 embryos have no male specific gene amplification curve and are female embryos, as shown in fig. 3 (in the figure, the amplification curve represents a fluorescence signal, the earlier the starting peak is, the earlier the fluorescence is received, the higher the curve is, the stronger the fluorescence signal is, and vice versa).
(2) Embryo DNA melting Curve
Corresponding to FIG. 3, the "B" peak curve is the reference gene lysis curve, the "A" peak curve is the male specific gene lysis curve (three replicates per sample), and the blank group has no lysis curve. In addition to the normal lysis curves of the 10 embryo reference genes, the corresponding lysis curves of 6 embryos (including three repeats) amplified to obtain the male specific gene amplification curve were normal. As shown in fig. 4 (in the figure, each curve has a peak in the normal range around 85 ℃, is a single peak, has no dimer effect, and has no non-specific amplification).
The technical scheme of the invention is a Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR) SYBR dye method, and SYBR only combines with double-stranded DNA, so that whether PCR reaction is specific can be determined through a dissolution curve, the method is more convenient and easy to operate, the cost is low, the sensitivity is higher, the time is shorter, judgment and analysis can be directly carried out according to an amplification curve, the identification efficiency and the accuracy are higher, and the method has the advantages of Real-time and quantification.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
Claims (7)
1. A sex determination method of a mammalian embryo, characterized in that the sex determination method comprises the following steps:
(1) obtaining an embryo;
(2) obtaining genome DNA of the embryonic cells;
(3) designing and synthesizing a primer;
(4) QPCR reaction: adding fluorescent dye into a PCR reaction system;
(5) after the curve was amplified and analyzed, the embryo sex was identified.
2. The method of sex determination according to claim 1,
in the step (5), after the amplification curve, the embryo with the male specific gene amplification curve is male, otherwise, the embryo is female.
3. The method of sex determination according to claim 1,
the amplification curve is at least one of an embryo DNA amplification curve and an embryo DNA dissolution curve.
4. The method of sex determination according to claim 1,
in the step (4), the amplification conditions of the QPCR reaction are as follows: after pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s and annealing extension at 65 ℃ for 20s, the process was cycled 40 times.
5. The method of sex determination according to claim 4,
in the step (4), each 10 μ L of PCR reaction system is: 2 XS 6Universal SYBR qPCR Mix 5. mu.L, upstream and downstream primers 0.25. mu.L each, DNA template 1. mu.L, dd H2O 3.5μL。
6. The method of sex determination according to claim 1,
the embryo is a living embryo, or an embryo produced by in vitro fertilization, or a frozen embryo.
7. The method of sex determination according to claim 1,
the sex identification method is used for identifying sheep embryos.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115851967A (en) * | 2022-09-09 | 2023-03-28 | 海南省农业科学院畜牧兽医研究所 | Substance for detecting SRY gene and PCR detection method for sex identification of pig, goat and cattle |
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| CN115851967A (en) * | 2022-09-09 | 2023-03-28 | 海南省农业科学院畜牧兽医研究所 | Substance for detecting SRY gene and PCR detection method for sex identification of pig, goat and cattle |
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