Method for detecting embryo chromosome abnormality by using blastocyst culture solution
阅读说明：本技术 一种利用囊胚培养液检测胚胎染色体异常的方法 (Method for detecting embryo chromosome abnormality by using blastocyst culture solution ) 是由 陆思嘉 蔡立义 姚兵 于 2015-11-05 设计创作，主要内容包括：本发明涉及一种利用囊胚培养液检测胚胎染色体异常的方法,通过从胚胎早期体外培养液即囊胚培养液中检测胚胎来源的游离DNA,对微量DNA进行均匀的全基因组扩增,再利用二代测序等方法对扩增后的DNA产物进行分析,从而判断胚胎的染色体情况,即是否出现染色体整体或局部的非整倍体。本发明选择囊胚培养液这种体外受精操作过程中胚胎体外培养阶段的废弃物作为检测样本,这种非侵入性无创的方法既避免了常规卵裂球活检或囊胚滋养层细胞活检取样时对胚胎造成的细胞损伤,又不给临床增添额外的麻烦,同时简化了样本获取时的操作,安全可靠性及简便性更高。(The invention relates to a method for detecting embryo chromosome abnormality by using a blastocyst culture solution, which detects free DNA (deoxyribonucleic acid) of an embryo source from an embryo early-stage in-vitro culture solution, namely the blastocyst culture solution, performs uniform whole genome amplification on trace DNA, and analyzes the amplified DNA product by using methods such as second-generation sequencing and the like so as to judge whether the chromosome condition of the embryo, namely whether integral or local aneuploidy of chromosomes occurs or not. The invention selects the blastula culture solution which is the waste in the extra-embryonic-body culture stage in the in-vitro fertilization operation process as the detection sample, and the noninvasive method avoids the cell damage to the embryo caused by the conventional blastomere biopsy or the blastula trophoblast cell biopsy during sampling, does not add extra trouble to clinic, simplifies the operation during sample acquisition, and has higher safety reliability and simplicity.)
1. A method for detecting embryo chromosome abnormality by utilizing blastocyst culture solution is characterized by comprising the following steps:
(1) obtaining a blastocyst culture solution: obtaining fertilized eggs by a single sperm injection method, culturing the fertilized eggs to the blastomere stage of day 3, transferring the fertilized eggs to a newly prepared blastocyst culture microdroplet for blastocyst culture, sucking out embryos forming the blastocysts, transferring the embryos to a new blastocyst culture solution or entering a vitrification cryopreservation process, wherein the remaining protoblastocyst culture solution is a sample required to be collected during detection;
(2) collecting a blastocyst culture solution: transferring the blastula proembryogenesis culture solution obtained in the step (1) into a lysate, centrifuging, and then carrying out the next whole genome amplification step on a sample;
(3) whole genome amplification of trace DNA in blastocyst culture solution: adding lyase into the mixture of the blastocyst culture solution and the lysate obtained in the step (2), uniformly mixing, incubating, inactivating the lyase, taking out a lysate, and adding the lysate into a PCR reaction tube for genome amplification reaction;
(4) analyzing the DNA product after the whole genome amplification to identify whether the chromosome state of the embryo is normal: the analysis adopts second-generation sequencing, nucleic acid chip or immunofluorescence detection.
2. The method according to claim 1, wherein the composition of the lysate in step (2) is Tris-Cl 25-45mM with pH 7.0-8.0, EDTA 0.5-3mM, KCl 10-25mM, and 0.05% -5% detergent selected from one or more of Triton X-100, Triton X-114, Tween 20, NP40 and SDS.
3. The method of claim 2, wherein the lysate is preferably composed of Tris-Cl 40mM, EDTA1mM, KCl 15mM, and Triton X-100% at pH 7.2.
4. The method according to claim 1, wherein the lyase in step (3) is selected from one or more of proteinase K, Qiagen Protease, pepsin, papain, trypsin and lysozyme, and the concentration of the lyase is 1-25 μ g/ml.
5. The method according to claim 4, wherein the concentration of the lytic enzyme is preferably 20 μ g/ml.
6. The method according to claim 1, wherein the incubation temperature in step (3) is 30-60 ℃, the incubation time is 1min to 12h, the inactivation temperature is 75-95 ℃, and the inactivation time is 1-15 min.
7. The method according to claim 6, wherein in step (3), preferably, the incubation temperature is 40 ℃ and the incubation time is 3h, the inactivation temperature is 90 ℃ and the inactivation time is 5 min.
8. The method of claim 1, wherein the PCR reaction in step (3) is carried out in a PCR reaction tube containing the amplification mixture, 0.5-20% of a PCR inhibitor antagonist selected from one or more of DMSO, betaine, formamide, glycerol and albumin, 5-100. mu.M NG and NT primers, 50-200. mu.M amplification primers, and 0.5-10 units of a nucleic acid polymerase selected from Phi29DNA polymerase, Bst DNA polymerase, Vent polymerase, Deep Vent polymerase, KlenFragment DNA polymerase, AMV reverse transcriptase, HIV reverse transcriptase, I, MMLV reverse transcriptase, DNA polymerase, DNA polymerase, DNA, polymerase, DNA polymerase, DNA polymerase, DNA polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA, polymerase, DNA,one or more of ultra-fidelity DNA polymerase, Taq polymerase, e.coli DNA polymerase, LongAmp Taq DNA polymerase, and OneTaq DNA polymerase.
9. The method of claim 8, wherein the amplification mixture comprises 10-25mM Tris-HCl, 5-25mM (NH)4)2SO4，5-30mM KCl，0.5-5mM MgSO40.1% -20% DMSO and 0.05-5% Triton X-100.
10. The method of claim 9, wherein the amplification mixture preferably comprises 15mM Tris-HCl, 15mM (NH)4)2SO4，20mM KCl，1mM MgSO45% DMSO and 2% Triton X-100.
11. The method of claim 8, wherein said NG and NT primers comprise, from 5 'to 3', a universal sequence and a variable sequence, wherein said universal sequence consists of three or two of the four bases G, A, C and T, provided that said universal sequence does not include both G and C; the amplification primers comprise the universal sequence and do not comprise the variable sequence.
12. The method of claim 11, wherein the variable sequence is selected from the group consisting of: (N) nGGG, (N) nTTT, (N) mTNTNG, (N) xGTGG (N) y, wherein N is any nucleotide capable of base pairing with a natural nucleic acid, N is a positive integer selected from 3 to 17, m is a positive integer selected from 3 to 15, and x and y are each a positive integer selected from 3 to 13.
13. The method of claim 12, wherein said NG and NT primers comprise the sequences of SEQ ID NO 1[ GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNN ], SEQ ID NO 2[ GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG ], SEQ ID NO 3[ GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT ], SEQ ID NO 4[ GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG ] or SEQ ID NO 5[ GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN ], wherein N is any nucleotide that can base pair with a native nucleic acid; the amplification primers have the sequence of SEQ ID NO 6[ GTGAGTGATGGTTGAGGTAGTGTGGAG ] from 5 'to 3'.
14. The method of claim 1, wherein the thermocycling procedure for whole genome amplification in step (3) is as follows:
(1) reacting at a first denaturation temperature of between 90 and 98 ℃ for 5 to 20 s;
(2) reacting at a first annealing temperature between 5-15 ℃ for 5-60s, at a second annealing temperature between 15-25 ℃ for 5-60s, at a third annealing temperature between 25-35 ℃ for 30-80s, at a fourth annealing temperature between 35-45 ℃ for 5-60s, and at a fifth annealing temperature between 45-55 ℃ for 5-60 s;
(3) reacting at a first extension temperature between 55 and 80 ℃ for 10 to 150 min;
(4) reacting at a second denaturation temperature of between 90 and 98 ℃ for 5 to 30 s;
(5) reacting at a sixth annealing temperature between 45-70 ℃ for 10-30 s;
(6) reacting at a second extension temperature between 60 and 80 ℃ for 1 to 10 min;
(7) repeating steps (4) to (6) for 5 to 50 cycles;
(8) continuing the extension reaction at 60-80 deg.C for 1-10 min;
(9) and (4) refrigerating and storing the amplified product at 0-5 ℃.
15. The method of claim 14, wherein the thermocycling procedure for whole genome amplification in step (3) is preferably as follows:
(1) reacting at the first denaturation temperature of 95 ℃ for 10 s;
(2) reacting at a first annealing temperature of 10 ℃ for 45s, at a second annealing temperature of 20 ℃ for 45s, at a third annealing temperature of 30 ℃ for 60s, at a fourth annealing temperature of 40 ℃ for 45s, and at a fifth annealing temperature of 50 ℃ for 45 s;
(3) reacting at a first extension temperature of 62 ℃ for 90 min;
(4) reacting at the second denaturation temperature of 95 ℃ for 20 s;
(5) reacting at the sixth annealing temperature of 59 ℃ for 20 s;
(6) reacting at a second extension temperature of 72 ℃ for 3 min;
(7) repeating the steps (4) to (6) for 10 to 30 cycles;
(8) continuing the extension reaction at 72 ℃ for 5 min;
(9) the amplified product was stored at 4 ℃ under refrigeration.
Technical Field
The invention relates to the fields of biomedicine and molecular cell biology, in particular to a method for detecting and analyzing the state of an embryo chromosome by using a blastocyst culture solution.
Background
The technology of test tube babies is a powerful technical means for resisting infertility, and the technical process is that a plurality of eggs (usually 8-15) are obtained from mothers, then the eggs are fertilized in vitro by sperms of fathers, when fertilized eggs grow in vitro culture solution for 5 days, embryos are cysts which are vesicular structures consisting of about 80-100 cells, namely blastula, and after 2-3 blastula are placed into the uteri of mothers, 1-3 blastula placed into the uteri can successfully develop according to the normal pregnancy period under the ideal condition until birth. However, for various reasons, the success rate from the insertion of the blastocyst into the uterus to the birth of the fetus is not high, usually about 40%, and besides the health reasons of the mother, the quality of the fertilized egg is one of the important reasons for the failure of the blastocyst development.
The chromosome of the fertilized egg is derived from the sperm of the mother source, the sperm of the father source, and the chromosomal abnormality of the fertilized egg is caused by the chromosomal abnormality of either one of them. There are 44 normal human zygotes, 22 pairs of autosomes (called diploids) and two sex chromosomes (XY in males and XX in females) in each of the embryonic cells and the cells of fetuses, infants and adults. In abnormal situations, more or less than diploid events may occur for all or part of any chromosome, called aneuploidy, which is the most common form of chromosomal abnormality that results in embryonic failure. In conventional tube baby technology, 2-3 relatively "normal" embryos are picked from a plurality (usually 8-15) and placed into the mother's uterus, relying solely on morphological observation under a microscope. The normal morphology under the microscope cannot reflect whether the chromosome is normal, and the embryo with normal morphology and abnormal chromosome is mistakenly selected to be placed into the uterus of a mother, so that many test-tube infants fail to be pregnant.
In recent years, several techniques, collectively called Preimplantation Genetic Screening (PGS), have been established to detect the chromosomal status of embryos cultured in vitro to Screen for embryos with normal chromosomes to be placed into the mother's uterus, thereby improving the success rate of conception. Research shows that the success rate of the test-tube infant placed in the uterus after PGS is operated can be improved to more than 60 percent, and various PGS methods comprise immunofluorescence assay (FISH), chip assay, second-generation sequencing assay and the like. The biological samples required by the various tests are one to a plurality of cells collected from in vitro cultured embryos, and the detection of the few cells reflects whether chromosomes of the whole embryos are normal.
Specifically, embryonic trophoblast cells (trophoblast) are extracted when fertilized eggs cultured in vitro for 5 days develop to the blastocyst stage. Generally, the method is carried out under a microscope, one or more trophoblast cells are sucked by a capillary glass tube to lyse the cells, a trace amount of DNA in the cells is released, and after the trace amount of DNA is subjected to whole genome amplification, the chromosome state of the cells can be detected by a method such as a nucleic acid chip or second-generation sequencing and the like (see patent application CN104711362A, published as 2015, 6, months and 17 days). Theoretically, the chromosome state of the extracted cells is consistent with that of other cells in the embryo, and the chromosome state of the embryo can be known to be normal or not by detecting the cells. It is believed that the aspiration of several trophoblast cells at this time does not adversely affect the development of the embryo, and that this procedure does not actually affect the health of the born infant. However, the time of appearance of this technology is still short (only a few years), and its long-term impact on the life-long health of humans remains to be observed.
In addition, methods have been developed for embryo quality detection using blastocoel fluid by first obtaining free DNA from the blastocoel fluid by means of a micropipetting technique using sterile needle aspiration under a micromanipulator (see patent application CN104450923A, published as 2015 3-25 days; and journal articles Luca Gianariol, M. Cristana Magli, Alessandra Pomanate, et al. blast center: a source of DNA for prediction genetic testing. solutions from a pilot. fetiliity and Sterility,2014,102(6):1692 and 1698.). However, the blastocoel fluid is the fluid in the blastocoel cavity, and holes or punctures are still needed to obtain the blastocoel fluid, and the intervention causes inevitable damage to the embryo.
In summary, the main disadvantages of the prior art are:
1. the requirement on embryo operation technology is high during cell sampling, if the operation is wrong and rough, the embryo can be seriously damaged, and the embryo development can be stopped due to the serious damage.
2. Even with good handling, cell loss and slight damage to the embryo are inevitable when sampling cells. Although there is no evidence that cell loss and mild injury adversely affect embryonic development and postnatal health, the time of appearance of this technique is short (only a few years), and its long-term impact on the life-long health of humans remains to be observed.
3. In a few cases, there are cases where the chromosome state of several cells obtained by sampling differs from the chromosome state of other cells in the embryo, resulting in erroneous detection results.
Therefore, a non-invasive technical means which can detect the chromosome condition of the embryo without damaging the embryo itself is a real need to eliminate the hidden danger of health influence and ensure the safety of embryo detection.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a method for detecting chromosome abnormality of an embryo by using a blastocyst culture solution, which does not cause any damage to the embryo, and has the advantages of simple operation, higher safety and higher reliability.
In order to achieve the above object, the present invention provides a method for detecting chromosomal abnormality of an embryo using a blastocyst culture solution, comprising the steps of:
(1) obtaining a blastocyst culture solution: obtaining fertilized eggs by a single sperm injection method, culturing the fertilized eggs to the blastomere stage of day 3, and then transferring the fertilized eggs to a newly prepared blastocyst culture microdroplet for blastocyst culture, wherein the liquid change is necessary at the day 3 so as to remove the pollution of the exfoliated granulocytes and unfertilized sperms;
sucking out the embryo with the formed blastocyst, transferring the embryo into a new blastocyst culture solution or entering a vitrification cryopreservation process, wherein the rest of the original blastocyst culture solution is about 1 microliter to 500 microliters, preferably 10 microliters to 200 microliters, and the embryo is a sample which needs to be collected for genetic screening (PGS) before embryo implantation;
(2) collecting a blastocyst culture solution: transferring the blastula proembryogenesis culture solution obtained in the step (1) into a lysate, centrifuging, and then carrying out the next whole genome amplification step on a sample;
(3) whole genome amplification of trace DNA in blastocyst culture solution: adding lyase into the mixture of the blastocyst culture solution and the lysate obtained in the step (2), uniformly mixing, incubating, inactivating the lyase, taking out a lysate, and adding the lysate into a PCR reaction tube for PCR reaction;
(4) analyzing the DNA product after the whole genome amplification to identify whether the chromosome state of the embryo is normal: the analysis adopts second-generation sequencing, nucleic acid chip or immunofluorescence detection.
The components of the cracking liquid in the step (2) are Tris-Cl 25-45mM with the pH value of 7.0-8.0, EDTA 0.5-3mM, KCl 10-25mM and a detergent with the concentration of 0.05% -5%, wherein the detergent is one or more of Triton X-100, Triton X-114, Tween 20, NP40 and SDS. Preferably, the lysate composition is Tris-Cl 40mM at pH 7.2, EDTA1mM, KCl 15mM, and Triton X-100 at 3%.
The lyase in the step (3) is selected from one or more of proteinase K, Qiagen Protease, pepsin, papain, trypsin and lysozyme, and the concentration of the lyase is 1-25 mu g/ml, preferably 20 mu g/ml; in the step (3), the incubation temperature is 30-60 ℃, the incubation time is 1 min-12 h, the inactivation temperature is 75-95 ℃, and the inactivation time is 1-15 min; preferably, the incubation temperature is 40 ℃ and the incubation time is 3h, the inactivation temperature is 90 ℃ and the inactivation time is 5 min.
The PCR reaction tube for carrying out the PCR reaction in the step (3) contains an amplification mixed solution, 0.5-20% of a PCR inhibitor antagonist, 5-20mM dNTP, 5-100 mu M NG and NT primers, 50-200 mu M of an amplification primer and 0.5-10 units of nucleic acid polymerase, wherein the PCR inhibitor antagonist is selected from one or more of DMSO, betaine, formamide, glycerol and albumin, and the nucleic acid polymerase is selected from Phi29DNA polymerase, Bst DNA polymerase and Vent polySynthase, Deep Vent polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase,One or more of ultra-fidelity DNA polymerase, Taq polymerase, e.coli DNA polymerase, LongAmp Taq DNA polymerase, and OneTaq DNA polymerase.
The amplification mixture comprises 10-25mM Tris-HCl, 5-25mM (NH)4)2SO4，5-30mM KCl，0.5-5mM MgSO40.1% -20% DMSO and 0.05-5% Triton X-100. Preferably, the amplification mixture has a composition of 15mM Tris-HCl, 15mM (NH)4)2SO4，20mM KCl，1mM MgSO45% DMSO and 2% Triton X-100.
The NG and NT primers comprise, from 5 'to 3', a universal sequence and a variable sequence, wherein the universal sequence consists of three or two of the four bases G, A, C and T, provided that the universal sequence does not include G and C at the same time; the amplification primers comprise the universal sequence and do not comprise the variable sequence. The variable sequence is selected from the group consisting of: (N) nGGG, (N) nTTT, (N) mTNTNG, (N) xGTGG (N) y, wherein N is any nucleotide capable of base pairing with a natural nucleic acid, N is a positive integer selected from 3 to 17, m is a positive integer selected from 3 to 15, and x and y are each a positive integer selected from 3 to 13.
Preferably, the NG and NT primers comprise the sequences of SEQ ID NO 1[ GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNN ], SEQ ID NO 2[ GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG ], SEQ ID NO 3[ GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT ], SEQ ID NO 4[ GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG ] or SEQ ID NO 5[ GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN ], wherein N is any nucleotide that can base pair with a natural nucleic acid; the amplification primers have the sequence of SEQ ID NO 6[ GTGAGTGATGGTTGAGGTAGTGTGGAG ] from 5 'to 3'.
The thermocycling procedure for whole genome amplification in step (3) is as follows:
(1) reacting at a first denaturation temperature of between 90 and 98 ℃ for 5 to 20 s;
(2) reacting at a first annealing temperature between 5-15 ℃ for 5-60s, at a second annealing temperature between 15-25 ℃ for 5-60s, at a third annealing temperature between 25-35 ℃ for 30-80s, at a fourth annealing temperature between 35-45 ℃ for 5-60s, and at a fifth annealing temperature between 45-55 ℃ for 5-60 s;
(3) reacting at a first extension temperature between 55 and 80 ℃ for 10 to 150 min;
(4) reacting at a second denaturation temperature of between 90 and 98 ℃ for 5 to 30 s;
(5) reacting at a sixth annealing temperature between 45-70 ℃ for 10-30 s;
(6) reacting at a second extension temperature between 60 and 80 ℃ for 1 to 10 min;
(7) repeating steps (4) to (6) for 5 to 50 cycles;
(8) continuing the extension reaction at 60-80 deg.C for 1-10 min;
(9) and (4) refrigerating and storing the amplified product at 0-5 ℃.
Preferably, the thermocycling procedure for whole genome amplification in step (3) is as follows:
(1) reacting at the first denaturation temperature of 95 ℃ for 10 s;
(2) reacting at a first annealing temperature of 10 ℃ for 45s, at a second annealing temperature of 20 ℃ for 45s, at a third annealing temperature of 30 ℃ for 60s, at a fourth annealing temperature of 40 ℃ for 45s, and at a fifth annealing temperature of 50 ℃ for 45 s;
(3) reacting at a first extension temperature of 62 ℃ for 90 min;
(4) reacting at the second denaturation temperature of 95 ℃ for 20 s;
(5) reacting at the sixth annealing temperature of 59 ℃ for 20 s;
(6) reacting at a second extension temperature of 72 ℃ for 3 min;
(7) repeating the steps (4) to (6) for 10 to 30 cycles;
(8) continuing the extension reaction at 72 ℃ for 5 min;
(9) the amplified product was stored at 4 ℃ under refrigeration.
The present invention detects free DNA derived from an embryo from an early embryo in vitro culture solution (blastocyst culture solution) to determine the chromosome condition of the embryo (whether or not chromosome aneuploidy occurs wholly or locally). Since the embryo releases a very small amount (about several tens picograms) of DNA into the blastocyst culture solution during early in vitro culture and development, in order to detect chromosomal aneuploidy using such a small amount of DNA, it is necessary to amplify the DNA uniformly and largely. The volume of the blastocyst culture solution is about 30 microliters, so that the embryo-derived DNA in the culture solution is highly diluted, and the components of the embryo culture solution are complex, wherein certain components can inhibit the amplification of the DNA. The technical scheme of the invention overcomes various technical problems and successfully establishes a technical method for detecting the embryo chromosome aneuploidy from the blastocyst culture solution.
Therefore, compared with the prior art, the method avoids the cell loss and damage to the embryo caused by the conventional PGS detection sampling method, and simplifies the operation of obtaining the PGS sample; in addition, because the blastocyst culture solution is originally waste in the in vitro culture stage of the embryo in the operation process of the test-tube infant, the technology detects the waste, hardly adds extra trouble to clinic, and realizes the evaluation of the chromosome state of the corresponding embryo.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a result analysis of chromosome detection of a specimen A using a blastocyst culture solution and blastocytes, respectively, in example 1 of the present invention;
FIG. 2 is a graph showing the analysis of the results of chromosome detection of a B sample using blastocyst culture fluid and blastocytes, respectively, in example 1 of the present invention.
Detailed Description
The following description will be made in further detail with reference to the accompanying drawings and examples of the present invention.
Example 1
A, B two embryo samples fertilized in vitro are selected, and the chromosome state of the embryo samples is evaluated by adopting a blastula cell detection method and a blastula culture solution detection method respectively, and the specific steps are as follows:
1. obtaining of blastocyst Medium
1) After the fertilized eggs obtained by the single sperm injection method are cultured to the day 3 blastomere stage, the embryos are transferred to newly prepared blastocyst culture microdroplets for blastocyst culture.
2) Sucking out the embryo with the formed blastocyst, transferring the embryo into a new blastocyst culture solution or entering a vitrification cryopreservation process, wherein the rest of the blastcyst culture solution (about 30ul) is the sample A and the sample B which need to be collected for PGS.
2. Collection of blastocyst Medium
1) Placing a collection tube filled with 10 microliters of lysate (Tris-Cl 40mM with pH of 7.2, EDTA1mM, KCl 15mM and Triton X-100 with the concentration of 3%) for 2min at room temperature, placing the sample collection tube in a mini centrifuge after the lysate is thawed, and centrifuging for 30s to ensure that the lysate is completely accumulated at the bottom of the tube.
2) The blastocyst medium from step one 1.2) was transferred to the lysate in its entirety using a mouth pipette.
3) Marking the name of the sample on the collection tube by using a marker pen, centrifuging the sample by using a micro centrifuge for 30s, and immediately carrying out the next whole genome amplification step or putting the sample into a freezing storage at-20 ℃ or-80 ℃.
3. Whole genome amplification of trace DNA in blastocyst culture solution
1) The mixture of blastocyst medium and lysate was thawed at room temperature.
2) Adding protease into the tube, blowing up and down, and mixing uniformly.
3) The tube was incubated at 40 ℃ for 3 h.
4) The tube was left at 90 ℃ for 5min to inactivate the lyase.
5) The lysate was removed from the tube and added to a PCR reaction tube.
6) The amplification mix (15mM Tris-HCl, 15mM (NH) was added to the PCR reaction tube4)2SO4，20mM KCl，1mM MgSO45% DMSO and 2% Triton X-100), 5% DMSO, 10mM dNTP, 50. mu.M NG (5 ' -GT GAG TGA TGG TTG AGG TAG TGT GGA GNNNNNGGG-3 ') and NT (5 ' -GT G)AG TGA TGG TTG AGG TAG TGT GGA GNNNNNTTT-3 '), 100. mu.M amplification primer (5'-GT GAG TGA TGG TTG AGG TAG TGT GGA G-3'), 1 unit of BstDNA polymerase, 1 unit of DeepVentR.
7) Placing the PCR reaction tube in a PCR instrument for whole genome amplification, wherein the thermal cycle program comprises the following steps:
4. and performing second-generation sequencing on the amplified DNA product according to a conventional method to identify whether the chromosome state of the embryo is normal.
The results of the second generation sequencing data show that in the sample A, a blastocyst culture solution detection method (figure A1) and a blastocyst cell detection method (figure A2) can detect a plurality of chromosome abnormalities; in the B sample, both the blastocyst culture solution detection method (FIG. B1) and the blastocyst cell detection method (FIG. B2) were determined to be chromosome-normal. The results show that the two methods of blastocyst culture solution detection and blastocyst cell detection are adopted to obtain the same result for identifying the chromosome state of the embryo, and the accuracy and reliability of the non-invasive detection method are further verified.
While the foregoing is directed to embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.
<110> Zhikang medical science and technology (Suzhou) Co., Ltd
<120> method for detecting embryo chromosome abnormality by using blastocyst culture solution
<160> 6
<210> 1
<211> 35
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 1
GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNN
<210> 2
<211> 35
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 2
GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG
<210> 3
<211> 35
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 3
GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT
<210> 4
<211> 35
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 4
GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG
<210> 5
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 5
GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN
<210> 6
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 6
GTGAGTGATGGTTGAGGTAGTGTGGAG