WO2017076359A1 - 一种利用囊胚培养液检测胚胎染色体异常的方法 - Google Patents

一种利用囊胚培养液检测胚胎染色体异常的方法 Download PDF

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WO2017076359A1
WO2017076359A1 PCT/CN2016/104753 CN2016104753W WO2017076359A1 WO 2017076359 A1 WO2017076359 A1 WO 2017076359A1 CN 2016104753 W CN2016104753 W CN 2016104753W WO 2017076359 A1 WO2017076359 A1 WO 2017076359A1
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primer
reacting
amplification
temperature
sequence
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PCT/CN2016/104753
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English (en)
French (fr)
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陆思嘉
蔡立义
姚兵
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序康医疗科技（苏州）有限公司
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Priority to AU2016351034A priority Critical patent/AU2016351034B2/en
Priority to JP2018543417A priority patent/JP2019506873A/ja
Priority to EP16861627.4A priority patent/EP3372690A4/en
Priority to BR112018009105A priority patent/BR112018009105A8/pt
Priority to KR1020207028157A priority patent/KR20200118221A/ko
Priority to US15/774,186 priority patent/US20180327821A1/en
Priority to KR1020187016022A priority patent/KR20180088830A/ko
Publication of WO2017076359A1 publication Critical patent/WO2017076359A1/zh
Priority to AU2020286291A priority patent/AU2020286291A1/en

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Definitions

  • the invention relates to the fields of biomedicine and molecular cell biology, and particularly relates to a method for detecting and analyzing the state of an embryo chromosome by using a blastocyst culture solution.
  • IVF technology is a powerful technical means to fight infertility.
  • the technical process is to first obtain multiple eggs from the mother (usually 8-15), and then in vitro fertilize the eggs with the father's sperm.
  • the embryo When grown in vitro for 5 days, the embryo is a saccular structure consisting of about 80-100 cells, ie, blastocysts. After placing 2-3 blastocysts into the mother's uterus, ideally, the blastocyst placed in the uterus can be One to three have developed successfully during normal pregnancy until birth.
  • the success rate of blastocyst implantation into the uterus to the birth of the fetus is usually not high, usually only about 40%.
  • the quality of the fertilized egg is one of the important reasons for the failure of blastocyst development.
  • the chromosome of the fertilized egg comes from the mother's egg and the parent's sperm, and any one of the chromosome abnormalities causes the chromosomal abnormality of the fertilized egg.
  • all or part of any chromosome may be more or less diploid, called aneuploidy abnormality, and aneuploidy abnormality is the most common chromosomal abnormal form that causes embryonic development failure. .
  • PGS Preimplantation Genetic Screen
  • FISH immunofluorescence detection
  • chip detection chip detection
  • second-generation sequencing detection The biological samples required for the above various tests are one to several cells collected from the in vitro cultured embryos, and the detection of the small number of cells reflects whether the chromosomes of the entire embryo are normal.
  • embryonic trophoblast cells can be extracted when the fertilized egg cultured in vitro for 5 days develops to the blastocyst stage (trophoblast).
  • the general operation is to use a capillary glass tube to absorb one to several trophoblast cells to lyse the cells, release the trace DNA, and then use the nucleic acid chip or the second generation sequencing method to detect the trace DNA.
  • the chromosomal state of the cells see invention patent application CN104711362A, published on June 17, 2015). In theory, the chromosomal state in several cells taken up is consistent with other cells in the embryo. By detecting these cells, it is possible to know whether the chromosomal state of the embryo is normal.
  • the free DNA is obtained in the blastocyst fluid, that is, using a micro-puncture technique under a micromanipulator, using a sterile needle suction.
  • Blastocyst fluid free DNA see invention patent application CN104450923A, published on March 25, 2015; and journal literature Luca Gianaroli, M. Chris Magli, Alessandra Pomante, et al. Blastocentesis: a source of DNA for preimplantation genetic testing .Results from a pilot study.Fertility and Sterility, 2014, 102(6): 1692-1698.).
  • the blastocyst fluid is a liquid in the blastocyst cavity. To obtain a blastocyst fluid, it is still necessary to punch or pierce the blastocyst, and its interventional effect will still cause inevitable damage to the embryo.
  • a non-invasive technical means that does not damage the embryo itself, but can also check the chromosomal condition of the embryo is a realistic need to eliminate the hidden dangers of health and ensure the safety of embryo detection.
  • the object of the present invention is to provide a method for detecting chromosomal abnormalities of an embryo by using a blastocyst culture solution, which does not cause any damage to the embryo, and is simple in operation and safety. And higher reliability.
  • the present invention provides a method for detecting an chromosomal abnormality of an embryo using a blastocyst culture solution, which comprises the following steps:
  • blastocyst culture solution The fertilized egg is obtained by single sperm injection method, and cultured until the blastomere stage on the 3rd day, and then transferred to the newly prepared blastocyst culture droplet for blastocyst culture, at this time It is necessary to change the liquid on the third day, in order to remove the contamination of the detached granulosa cells and unfertilized sperm;
  • the embryos forming the blastocysts are aspirated, transferred to a new blastocyst culture solution or enter a vitrification cryopreservation process, and the remaining protoplast culture medium is about 1 microliter to 500 microliters, preferably 10 microliters to 200 microliters, Samples to be collected for preimplantation genetic screening (PGS);
  • the whole genome-amplified DNA product is analyzed to identify whether the chromosomal state of the embryo is normal: the second generation sequencing, nucleic acid chip or immunofluorescence detection is used for the analysis.
  • the embryos forming the blastocyst are aspirated, transferred to a new blastocyst culture solution or enter a vitrification cryopreservation process, and the remaining primary blastocyst culture solution is about 1 microliter to 500 microliters.
  • L preferably 10 microliters to 200 microliters, is the sample that needs to be collected for preimplantation genetic screening (PGS).
  • the component of the lysate in the step (2) is Tris-Cl 25-45 mM having a pH of 7.0 to 8.0, EDTA 0.5-3 mM, KCl 10-25 mM, and a concentration of 0.05% to 5% of the detergent.
  • the stain is one or more of Triton X-100, Triton X-114, Tween 20, NP40, and SDS.
  • the components of the lysate are Tris-Cl 40 mM having a pH of 7.2, EDTA 1 mM, KCl 15 mM, and 3% Triton X-100.
  • the primer used includes an NG primer, an NT primer, and an amplification primer.
  • the NG primer and the NT primer comprise a universal sequence and a variable sequence from the 5' end to the 3' end, wherein the universal sequence consists of three or two of four bases of G, A, C and T Composition, provided that the universal sequence does not include G and C at the same time;
  • variable sequence of the NG primer is selected from the group consisting of: (N) nGGG, (N) xGTGG(N)y, or a combination thereof;
  • variable sequence of the NT primer is selected from the group consisting of: (N) nTTT, (N) mTNTNG, or a combination thereof; wherein N is any nucleotide that can base pair with a natural nucleic acid, each n independently Is a positive integer selected from 3-17, each m is independently a positive integer selected from 3-15, and x and y are each a positive integer selected from 3-13;
  • the amplification primer comprises the universal sequence and does not comprise the variable sequence.
  • 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 to 25 ⁇ g/ml, preferably 20 ⁇ g/ml; incubation temperature in step (3) is 30-60 ° C, incubation time is 1 min to 12 h, inactivation temperature is 75-95 ° C, inactivation time is 1-15 min; preferably, incubation temperature is 40 ° C, The incubation time was 3 h, the inactivation temperature was 90 ° C, and the inactivation time was 5 min.
  • the PCR reaction tube in the PCR reaction in the step (3) contains the amplification mixture, 0.5%-20% of the PCR inhibitor antagonist, 5-20 mM dNTP, 5-100 ⁇ M NG and NT primer, 50-200 ⁇ M amplification.
  • a primer 0.5-10 units of nucleic acid polymerase
  • the PCR inhibitor antagonist is selected from one or more of DMSO, betaine, formamide, glycerol and albumin
  • the nucleic acid polymerase being selected from the group consisting of Phi29 DNA polymerase, Bst DNA polymerase, Vent polymerase, 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. col DNA polymerase, LongAmp Taq DNA polymerase, and OneTaq DNA polymerase.
  • the components of the amplification mixture are 10-25 mM Tris-HCl, 5-25 mM (NH 4 ) 2 SO 4 , 5-30 mM KCl, 0.5-5 mM MgSO 4 , 0.1%-20% DMSO and 0.05-5% Triton. X-100.
  • the components of the amplification mixture are 15 mM Tris-HCl, 15 mM (NH 4 ) 2 SO 4 , 20 mM KCl, 1 mM MgSO 4 , 5% DMSO and 2% Triton X-100.
  • the NG and NT primers comprise a universal sequence and a variable sequence from the 5' end to the 3' end, wherein the universal sequence consists of three or two of the four bases G, A, C and T, provided that The universal sequences do not include G and C at the same time; the amplification primers comprise the universal sequence and do not comprise the variable sequence.
  • variable sequence is selected from the group consisting of: (N) nGGG, (N) nTTT, (N) mTNTNG, (N) xGTGG(N)y, wherein N is any nucleoside capable of base pairing with a natural nucleic acid
  • the acid n is a positive integer selected from 3-17, m is a positive integer selected from 3-15, and x and y are each a positive integer selected from 3-13.
  • the NG and NT primers comprise SEQ ID NO: 1 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNNNNNN], SEQ ID NO: 2 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG], SEQ ID NO: 3 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT], SEQ ID NO: 4 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG] or the sequence of SEQ ID NO: 5 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN], wherein N is any nucleotide that can base pair with a natural nucleic acid; the amplification primer has SEQ ID NO: 6 from 5' to 3' Sequence of [GTGAGTGATGGTTGAGGTAGTGTGGAG].
  • step (3) The thermal cycling procedure for whole genome amplification in step (3) is as follows:
  • the thermal cycling procedure for whole genome amplification in step (3) is as follows:
  • the amplification product obtained from the above step (9), according to technical requirements including, but not limited to, Illumina Hiseq, Miseq, Life Technology PGM, Proton sequencer, conventional database construction, sequencing, Data analysis and other steps detect the copy number of each chromosome and chromosome part in the sample genome.
  • the copy number of the normal chromosome and the chromosome part is 2.
  • the copy number is greater than 2 (such as ⁇ 2.5) or less than 2 (such as ⁇ 1.8)
  • it is a copy number anomaly that is, the chromosome is abnormal.
  • This normal or abnormal test result represents the normal or abnormal chromosome of the culture-derived embryo. If the chromosomal abnormal embryo is implanted into the mother, it will lead to embryo failure, abortion and other adverse consequences. Only when the embryo with normal chromosomes is implanted into the mother has a higher chance of successful conception.
  • the present invention detects embryonic-derived free DNA from an embryonic early in vitro culture medium (blastocyst culture solution) to determine the chromosomal condition of the embryo (whether or not a whole or partial aneuploidy of the chromosome appears). Since embryos release a very small amount (about several tens of picograms) of DNA into blastocyst culture fluid during early in vitro culture and development, in order to use such a small amount of DNA for chromosome aneuploidy detection, DNA must be first large. Uniform amplification of amplitude.
  • the volume of the blastocyst culture solution is about 30 ⁇ L, so the embryo-derived DNA in the culture solution is highly diluted, and the composition of the embryo culture solution is complicated, and some of the components may inhibit the amplification of DNA.
  • the technical solution in the present invention overcomes the above various technical problems, and successfully establishes a technical method for detecting embryonic chromosome aneuploidy from blastocyst culture fluid.
  • the present invention avoids the cell loss and damage caused by the conventional PGS detection sampling method to the embryo, and simplifies the operation of the PGS sample acquisition; in addition, since the blastocyst culture fluid is originally performed for the test tube baby The waste in the in vitro culture stage of the embryo during the operation, the technology of the present invention detects the waste, and almost no additional trouble is added to the clinical condition to evaluate the chromosomal state of the corresponding embryo.
  • the present invention also provides a test kit for detecting chromosomal abnormalities of an embryo using a blastocyst culture solution, the kit comprising the following components:
  • a primer for PCR comprising an NG primer, an NT primer, and an amplification primer
  • the NG primer and the NT primer comprise a universal sequence and a variable sequence from the 5' end to the 3' end, wherein the universal sequence consists of three or two of four bases of G, A, C and T Composition, provided that the universal sequence does not include G and C at the same time;
  • variable sequence of the NG primer is selected from the group consisting of: (N) nGGG, (N) xGTGG(N)y, or a combination thereof; and the variable sequence of the NT primer is selected from the group consisting of: (N) nTTT, (N) mTNTNG, or a combination thereof; wherein N is any nucleotide that can base pair with a natural nucleic acid, each n being independently a positive integer selected from 3-17, each m being independently selected from 3- a positive integer of 15, x and y are positive integers selected from 3-13, respectively;
  • the amplification primer comprises the universal sequence and does not comprise the variable sequence
  • the NG primer, the NT primer, and the amplification primer have the same general purpose sequence.
  • the universal sequence has a length of from 20 to 35 nt, preferably from 25 to 30 nt.
  • sequence of the NG primer and the NT primer is selected from the group consisting of SEQ ID NO.: 1-5; and the sequence of the amplification primer is shown in SEQ ID NO.: 6.
  • the kit further comprises one or more additional detection-related reagents selected from the group consisting of reagents for sequencing, nucleic acid chips, immunofluorescence detection reagents, or combination.
  • the kit further comprises a lysate or a lytic enzyme.
  • the kit further comprises a label or a label indicating that the amount of the blastocyst culture fluid collected by the kit is 10-100 ⁇ l, preferably 15-80 ⁇ l, more Good place, 20-60 ⁇ l.
  • the present invention also provides a use of the test kit of the present invention for preparing a product for detecting chromosomal abnormalities of an embryo using a blastocyst culture solution.
  • Example 1 is a result of analyzing chromosomes of A sample by blastocyst culture solution and blastocyst cells in Example 1 of the present invention
  • Fig. 2 is a graph showing the results of chromosome detection of B samples by blastocyst culture medium and blastocyst cells in Example 1 of the present invention.
  • the present inventors discovered for the first time through extensive screening and testing that the embryos were cultured in a small amount of culture solution, and a very small amount of the culture solution was taken out for detection, and the obtained chromosomal abnormality was found. The test results are extremely accurate. On the basis of this, the inventors completed the present invention.
  • the blastocyst culture fluid used in the detection technique of the present invention is a cell-free blastocyst culture fluid.
  • the present invention provides a method for genetically detecting a depleted medium (i.e., a culture solution separated from the culture system) after culturing a blastocyst, thereby identifying whether the embryonic chromosome is abnormal.
  • a depleted medium i.e., a culture solution separated from the culture system
  • the method of performing genetic detection on the "lean" culture solution (that is, the culture solution separated from the culture system) after culturing the blastocyst is not particularly limited, and can be detected by a conventional method, such as two. Generation sequencing, nucleic acid chips, immunofluorescence detection, fluorescent PCR detection, first generation sequencing, third generation sequencing, mass spectrometry detection, or a combination thereof.
  • the detecting method comprises the following steps:
  • blastocyst culture solution The fertilized egg is obtained by single sperm injection method, and cultured until the blastomere stage on the 3rd day, and then transferred to the newly prepared blastocyst culture droplet for blastocyst culture, at this time It is necessary to change the liquid on the third day, in order to remove the contamination of the detached granulosa cells and unfertilized sperm;
  • the embryos forming the blastocysts are aspirated, transferred to a new blastocyst culture solution or enter a vitrification cryopreservation process, and the remaining protoplast culture medium is about 1 microliter to 500 microliters, preferably 10 microliters to 200 microliters, Samples to be collected for preimplantation genetic screening (PGS);
  • the whole genome-amplified DNA product is analyzed to identify whether the chromosomal state of the embryo is normal: the second generation sequencing, nucleic acid chip or immunofluorescence detection is used for the analysis.
  • the embryos forming the blastocyst are aspirated, transferred to a new blastocyst culture solution or enter a vitrification cryopreservation process, and the remaining primary blastocyst culture solution is about 1 microliter to 500 microliters.
  • L preferably 10 microliters to 200 microliters, is the sample that needs to be collected for preimplantation genetic screening (PGS).
  • the embryo is cultured in a very small amount of the culture solution, and a very small amount of the culture solution is detected, and the detection result of the obtained chromosomal abnormality is extremely accurate.
  • the present invention adopts a single embryo culture system, that is, only one embryo is cultured in one culture liquid droplet.
  • the detection results of chromosomal abnormalities obtained by this system are more accurate.
  • the embryos forming the blastocysts are aspirated, transferred to a new blastocyst culture solution, or enter the vitrification cryopreservation process, and the remaining primary blastocyst culture solution (about 30 ul) is the sample A and sample B to be collected for PGS.
  • the embryos forming the blastocyst are aspirated.
  • lysate Tris-Cl 40 mM with pH 7.2, EDTA 1 mM, KCl 15 mM, and 3% Triton X-100
  • step 2 1) Transfer the original blastocyst culture solution in the second step of step 1 to the lysate with a mouth pipette.
  • the thermal cycle program is:
  • the amplified DNA product is subjected to second generation sequencing according to a conventional method to identify whether the chromosomal state of the embryo is normal.
  • the results of the second-generation sequencing data showed that in the A sample, the blastocyst culture solution detection method (Fig. A1) and the blastocyst cell detection method (Fig. A2) could detect multiple chromosomal abnormalities; in the B sample, the blastocyst culture Both the liquid detection method (Fig. B1) and the blastocyst detection method (Fig. B2) were judged to be normal chromosomes.
  • the above results indicate that the two methods of blastocyst culture and blastocyst detection have the same results for identifying the embryonic chromosome state, which further confirms that the non-invasive detection method is accurate and reliable.
  • the cell test showed abnormality, and the test result of the culture solution also showed abnormality.
  • the second type of cell test showed normal, and the test results of the culture solution also showed normal.
  • the third type The cell test showed normal, and the test result of the culture solution showed abnormality.
  • the fourth type The cell test showed abnormality, and the test result of the culture solution also showed normal.
  • the results showed that when the cell test was used as the gold standard, the sensitivity of the calculated culture solution was 88.2%, the specificity was 84.0%, the positive predictive value was 78.9, and the negative predictive value was 91.3%. Although the indicators are not 100%, the non-invasive method of providing a test result sufficiently close to the gold standard is sufficient to confirm the beneficial value of the present invention.
  • the embryos of 8 patients with fertility difficulties caused by different reasons were tested for embryo culture solution according to the method of Example 1 of the present invention, and the embryos with normal chromosomes were selected for implantation into the mother uterus according to the detection results.
  • results of the present invention showed that patients with No. 3 and No. 8 did not have a high-quality fertilized egg, and the embryo was chromosomal abnormal, and thus no embryo transfer was performed.
  • results of the remaining 6 patients fully showed that the embryos with normal chromosomes were selected by the method of the present invention to be successfully implanted once after the mother was implanted, and the success rate of embryo transfer and survival was 5/6 (ie, , 83.3%), that is, only one patient did not succeed.

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Abstract

提供了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。

Description

一种利用囊胚培养液检测胚胎染色体异常的方法 技术领域
本发明涉及生物医学和分子细胞生物学领域，具体涉及一种利用囊胚培养液对胚胎染色体的状态进行检测和分析的方法。
背景技术
试管婴儿技术是一项对抗不孕不育的强大技术手段，其技术过程为，首先从母亲获得多个卵子(通常8-15个)，再以父亲的精子对卵子进行体外受精，受精卵在体外培养液中生长5天时，胚胎是由约80～100个细胞组成的囊状结构即囊胚，将2-3个囊胚放置入母亲子宫后，理想情况下，置入子宫的囊胚可有1个至3个按正常孕期成功发育，直至出生。但由于各种原因，从囊胚置入子宫到胎儿出生的成功率不高，通常只有40％左右，除母亲自身的健康原因以外，受精卵质量是导致囊胚发育失败的重要原因之一。
受精卵的染色体来自于母源的卵子和父源的精子，任何一方的染色体异常均导致受精卵的染色体异常。正常人的受精卵、每个胚胎细胞及胎儿、婴幼儿直至成人的每个细胞内均有44条即22对常染色体(称为二倍体)和两条性染色体(男性为XY，女性为XX)。在异常情况下，任何染色体的全部或局部均可能出现多于或少于二倍体的情况，称为非整倍体异常，非整倍体异常是最常见的导致胚胎发育失败的染色体异常形式。在常规试管婴儿技术中，仅依靠显微镜下的形态观察，从多个(通常8-15个)胚胎中挑选2-3个相对“正常”者置入母亲子宫。而显微镜下的形态正常并无法反映染色体是否正常，错误地挑选形态正常而染色体异常的胚胎置入母亲子宫导致了很多试管婴儿受孕失败。
近年以来，人们已建立了一些技术，统称为胚胎植入前遗传学筛查(Preimplantation Genetic Screen，PGS)对体外培养胚胎的染色体状态进行检测，以筛选染色体正常的胚胎置入母亲子宫,从而提高受孕成功率。有研究表明，经过PGS再置入子宫的试管婴儿操作可将成功率提高到60％以上，各种PGS方法包括免疫荧光检测(FISH)，芯片检测，二代测序检测等。上述各种检测所需的生物样本都是从体外培养胚胎中采集的一个至数个细胞，通过对这少量细胞的检测反映整个胚胎的染色体是否正常。
具体而言，当体外培养5天的受精卵发育至囊胚期时可提取胚胎滋养层细胞 (trophoblast)。一般操作为在显微镜下，用毛细玻璃管吸取一个至数个滋养层细胞将细胞裂解，释放其中的微量DNA，将微量DNA进行全基因组扩增后，可采用核酸芯片或二代测序等方法检测细胞的染色体状态(参见发明专利申请CN104711362A，公开日为2015年6月17日)。理论上讲，吸取出来的几个细胞中的染色体状态与胚胎中其它细胞一致，检测这几个细胞即可了解该胚胎的染色体状态是否正常。一般认为，此时吸取几个滋养层细胞不会对胚胎发育造成不良影响，从已出生婴儿的健康状况来看，这一操作也确实没有健康影响。但是，该技术出现的时间尚短(仅数年)，其对人的终生健康是否存在远期影响仍然有待观察。
此外，人们也已研发出利用囊胚腔液进行胚胎质量检测的方法，首先在囊胚腔液中获取游离的DNA，即在显微操作仪下使用微穿刺技术，利用无菌的针头吸取得到囊胚腔液游离DNA(参见发明专利申请CN104450923A，公开日为2015年3月25日；以及期刊文献Luca Gianaroli,M.Cristina Magli,Alessandra Pomante,et al.Blastocentesis:a source of DNA for preimplantation genetic testing.Results from a pilot study.Fertility and Sterility,2014,102(6):1692-1698.)。然而，囊胚腔液是囊胚腔中的液体，欲获取囊胚腔液仍需要在囊胚上打洞或刺穿，其介入性仍会对胚胎造成不可避免的损伤。
综上所述，现有技术所存在的主要缺点为：
1.细胞取样时对胚胎操作技术要求较高，如果操作失误、粗暴操作会导致胚胎严重损伤，损伤过于严重可造成胚胎发育终止。
2.即使良好操作，细胞取样时不可避免地对胚胎造成细胞损失和轻微损伤。尽管现在尚无证据表明细胞损失和轻微损伤会对胚胎发育和出生后健康发生不良影响，但该技术出现的时间尚短(仅数年)，其对人的终生健康是否存在远期影响仍然有待观察。
3.在少数情况下，存在取样获得的几个细胞的染色体状态与胚胎中其它细胞的染色体状态不同的情况，导致检测结果失误。
因此，一种不损伤胚胎本身，而又可以对胚胎染色体状况进行检查的非侵入性的技术手段，是消除健康影响隐患、确保胚胎检测安全性的现实需求。
发明内容
本发明的目的就是针对上述现有技术中的不足，提供一种利用囊胚培养液检测胚胎染色体异常的方法，其不会对胚胎带来任何损伤，操作简单，安全性 和可靠性更高。
为了实现上述目的，本发明提供了一种利用囊胚培养液检测胚胎染色体异常的方法，其包括如下步骤：
(1)囊胚培养液的获得：通过单精子注射方法获得受精卵，将其培养至第3天卵裂球期之后转移至新制备的囊胚培养微滴中进行囊胚培养，此时在第3天进行换液是必须的，目的是去除脱落的颗粒细胞及未受精精子的污染；
将形成囊胚的胚胎吸出，转移至新的囊胚培养液中或者进入玻璃化冻存流程，剩余的原囊胚培养液约1微升至500微升，优选10微升至200微升，即为进行胚胎植入前遗传学筛查(PGS)需要收集的样本；
(2)囊胚培养液的采集：将步骤(1)中获取的原囊胚培养液转移至裂解液中，离心后，样本进入下一步的全基因组扩增步骤；
(3)囊胚培养液中微量DNA的全基因组扩增：向步骤(2)中获得的囊胚培养液与裂解液的混合物中加入裂解酶，混匀后孵育，而后使裂解酶失活，取出裂解产物加入PCR反应管中进行PCR反应；和
(4)将全基因组扩增后的DNA产物进行分析，以鉴定胚胎的染色体状态是否正常：分析时采用二代测序、核酸芯片或者免疫荧光检测。
在一优选例中，换液2-3天后将形成囊胚的胚胎吸出，转移至新的囊胚培养液中或者进入玻璃化冻存流程，剩余的原囊胚培养液约1微升至500微升，优选10微升至200微升，即为进行胚胎植入前遗传学筛查(PGS)需要收集的样本。
其中，步骤(2)中裂解液的成分是PH为7.0～8.0的Tris-Cl 25-45mM，EDTA 0.5-3mM，KCl 10-25mM以及浓度为0.05％-5％的去污剂，所述去污剂为Triton X-100、Triton X-114、吐温20、NP40和SDS中的一种或多种。优选地，裂解液的成分为PH为7.2的Tris-Cl 40mM，EDTA 1mM，KCl 15mM以及3％的Triton X-100。
在一优选实施方式中，在步骤(3)中，使用的引物包括NG引物、NT引物以及扩增引物，
其中，所述的NG引物和NT引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C；
所述NG引物的可变序列选自下组：(N)nGGG、(N)xGTGG(N)y、或其组合； 而所述NT引物的可变序列选自下组：(N)nTTT、(N)mTNTNG、或其组合；其中N为任意的可与天然核酸进行碱基配对的核苷酸，各n独立地是选自3-17的正整数，各m独立地是选自3-15的正整数，x和y分别是选自3-13的正整数；
而所述扩增引物包含所述通用序列且不包含所述可变序列。
步骤(3)中的裂解酶选自蛋白酶K、Qiagen Protease、胃蛋白酶、木瓜蛋白酶、胰蛋白酶和溶菌酶中的一种或多种，所述裂解酶的浓度为1-25μg/ml，优选为20μg/ml；步骤(3)中孵育温度为30-60℃，孵育时间为1min至12h，失活温度为75-95℃，失活时间为1-15min；优选地，孵育温度为40℃，孵育时间为3h，失活温度为90℃，失活时间为5min。
步骤(3)中进行PCR反应时的PCR反应管中含有扩增混合液、0.5％-20％的PCR抑制物对抗剂、5-20mM dNTP、5-100μM NG和NT引物、50-200μM扩增引物、0.5-10单位核酸聚合酶，所述PCR抑制物对抗剂选自DMSO、甜菜碱、甲酰胺、甘油和白蛋白中的一种或多种，所述核酸聚合酶选自Phi29DNA聚合酶、Bst DNA聚合酶、Vent聚合酶、Deep Vent聚合酶、Klenow Fragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、
Figure PCTCN2016104753-appb-000001
超保真DNA聚合酶、Taq聚合酶、E.col i DNA聚合酶、LongAmp Taq DNA聚合酶和OneTaq DNA聚合酶中的一种或多种。
所述扩增混合液的成分为10-25mM Tris-HCl，5-25mM(NH4)2SO4，5-30mM KCl，0.5-5mM MgSO4，0.1％-20％DMSO和0.05-5％Triton X-100。优选地，所述扩增混合液的成分为15mM Tris-HCl，15mM(NH4)2SO4，20mM KCl，1mM MgSO4，5％DMSO和2％Triton X-100。
所述NG和NT引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C；所述扩增引物包含所述通用序列且不包含所述可变序列。所述可变序列选自下组：(N)nGGG、(N)nTTT，(N)mTNTNG，(N)xGTGG(N)y，其中N为任意的可与天然核酸进行碱基配对的核苷酸，n是选自3-17的正整数，m是选自3-15的正整数，x和y分别是选自3-13的正整数。
优选地，所述NG和NT引物包括SEQ ID NO:1[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNN]、SEQ ID NO:2[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG]、SEQ ID NO:3[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT]、SEQ ID NO:4 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG]或SEQ ID NO:5[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN]的序列，其中N为任意的可与天然核酸进行碱基配对的核苷酸；所述扩增引物从5’到3’具有SEQ ID NO:6[GTGAGTGATGGTTGAGGTAGTGTGGAG]的序列。
步骤(3)中全基因组扩增的热循环程序如下所述：
(1)在介于90-98℃之间的第一变性温度反应5-20s；
(2)在介于5-15℃之间的第一退火温度反应5-60s，在介于15-25℃之间的第二退火温度反应5-60s，在介于25-35℃之间的第三退火温度反应30-80s，在介于35-45℃之间的第四退火温度反应5-60s，在介于45-55℃之间的第五退火温度反应5-60s；
(3)在介于55-80℃之间的第一延伸温度反应10-150min；
(4)在介于90-98℃之间的第二变性温度反应5-30s；
(5)在介于45-70℃之间的第六退火温度反应10-30s；
(6)在介于60-80℃之间的第二延伸温度反应1-10min；
(7)重复步骤(4)到(6)5至50个循环；
(8)在介于60-80℃之间的温度下继续延伸反应1-10min；
(9)将扩增后的产物在0-5℃下冷藏保存。
优选地，步骤(3)中全基因组扩增的热循环程序如下所述：
(1)在第一变性温度95℃下反应10s；
(2)在第一退火温度10℃下反应45s，在第二退火温度20℃下反应45s，在第三退火温度30℃下反应60s，在第四退火温度40℃下反应45s，在第五退火温度50℃下反应45s；
(3)在第一延伸温度62℃下反应90min；
(4)在第二变性温度95℃下反应20s；
(5)在第六退火温度59℃下反应20s；
(6)在第二延伸温度72℃下反应3min；
(7)重复步骤(4)到(6)10至30个循环；
(8)在72℃下继续延伸反应5min；
(9)将扩增后的产物在4℃下冷藏保存。
将从上述步骤(9)得到的扩增产物，按照包括但不限于Illumina Hiseq、Miseq，Life Technology PGM、Proton测序仪的技术要求、经常规建库、测序、 数据分析等步骤对样本基因组中各染色体和染色体局部的拷贝数进行检测。正常染色体及染色体局部的拷贝数为2。当拷贝数大于2(如≥2.5)或小于2(如≤1.8)时均属于拷贝数异常，亦即染色体出现异常。这一正常或异常的检测结果即代表了培养液来源胚胎的染色体正常或异常。如果将染色体异常的胚胎植入母体会导致胚胎着床失败、流产等不良后果。只有将染色体正常的胚胎植入母体才有较高机会成功受孕。
本发明从胚胎早期体外培养液(囊胚培养液)中检测胚胎来源的游离DNA，从而判断该胚胎的染色体情况(是否出现染色体整体或局部的非整倍体)。由于胚胎在早期体外培养发育过程中会向囊胚培养液中释放极少量(约几十皮克)的DNA，要想利用如此微量的DNA进行染色体非整倍体检测，必须先对DNA进行大幅度的均一扩增。而囊胚培养液的体积约为30微升，因此培养液中的胚胎来源DNA是高度稀释的，同时胚胎培养液成份复杂，其中的某些成份会对DNA的扩增产生抑制作用。本发明中的技术方案克服了上述各种技术难题，成功地建立了从囊胚培养液中检测胚胎染色体非整倍体的技术方法。
因此，与现有技术相比，本发明避免了常规PGS检测取样方法对胚胎造成的细胞损失和损伤，并且简化了PGS样本获取时的操作；此外，由于囊胚培养液本来是进行试管婴儿的操作过程中胚胎体外培养阶段的废弃物，本发明技术对这一废弃物进行检测，几乎不给临床增添额外麻烦而实现了对相应胚胎染色体状态进行评估。
在另一方面，本发明还提供了一种利用囊胚培养液检测胚胎染色体异常的检测试剂盒，所述试剂盒含有如下组分：
(i)用于PCR的引物,所述引物包括NG引物、NT引物以及扩增引物，
其中，所述的NG引物和NT引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C；
所述NG引物的可变序列选自下组：(N)nGGG、(N)xGTGG(N)y、或其组合；而所述NT引物的可变序列选自下组：(N)nTTT、(N)mTNTNG、或其组合；其中N为任意的可与天然核酸进行碱基配对的核苷酸，各n独立地是选自3-17的正整数，各m独立地是选自3-15的正整数，x和y分别是选自3-13的正整数；
而所述扩增引物包含所述通用序列且不包含所述可变序列；
以及(ii)任选的囊胚培养液。
在另一优选例中，所述的NG引物、NT引物以及扩增引物具有相同的通用 序列。
在另一优选例中，所述的通用序列的长度为20-35nt，较佳地25-30nt。
在另一优选例中，所述NG引物和NT引物的序列选自SEQ ID NO.:1-5；而所述扩增引物的序列如SEQ ID NO.:6所示。
在另一优选例中，所述试剂盒还包括一种或多种额外的检测相关试剂，所述检测相关试剂选自下组：用于测序的试剂、核酸芯片、免疫荧光检测试剂、或其组合。
在另一优选例中，所述试剂盒还包括裂解液或裂解酶。
在另一优选例中，所述试剂盒还包括标签或说明书，所述标签或说明书注明所述试剂盒采集的囊胚培养液的量为10-100μl，较佳地，15-80μl，更佳地，20-60μl。
在另一方面，本发明还提供了一种本发明所述检测试剂盒的用途，用于制备利用囊胚培养液检测胚胎染色体异常的产品。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1为本发明的实施例1中分别利用囊胚培养液与囊胚细胞对A样本进行染色体检测的结果分析；
图2为本发明的实施例1中分别利用囊胚培养液与囊胚细胞对B样本进行染色体检测的结果分析。
具体实施方式
本发明人经过长期广泛而深入的研究，通过大量筛选和测试，首次意外地发现，在少量的培养液中对胚胎进行培养，取出极微量的培养液进行检测后发现，所得到的染色体异常的检测结果具有极高的精确性。在此基础上，本发明人完成了本发明。
术语
用于本发明检测技术的囊胚培养液为无细胞囊胚培养液。
检测方法
本发明提供了一种对囊胚进行培养后的乏培养液(depleted medium)(即从所述培养体系中分离出的培养液)进行基因检测，从而鉴定胚胎染色体是否异常的方法。
在本发明中，对囊胚进行培养后的“乏”培养液(即从所述培养体系中分离出的培养液)进行基因检测的方法不受特别的限制，可用常规方法进行检测，如二代测序、核酸芯片、免疫荧光检测、荧光PCR检测、一代测序、三代测序、质谱检测、或其组合。
在一种实施方式中，所述检测方法包括如下步骤：
(1)囊胚培养液的获得：通过单精子注射方法获得受精卵，将其培养至第3天卵裂球期之后转移至新制备的囊胚培养微滴中进行囊胚培养，此时在第3天进行换液是必须的，目的是去除脱落的颗粒细胞及未受精精子的污染；
将形成囊胚的胚胎吸出，转移至新的囊胚培养液中或者进入玻璃化冻存流程，剩余的原囊胚培养液约1微升至500微升，优选10微升至200微升，即为进行胚胎植入前遗传学筛查(PGS)需要收集的样本；
(2)囊胚培养液的采集：将步骤(1)中获取的原囊胚培养液转移至裂解液中，离心后，样本进入下一步的全基因组扩增步骤；
(3)囊胚培养液中微量DNA的全基因组扩增：向步骤(2)中获得的囊胚培养液与裂解液的混合物中加入裂解酶，混匀后孵育，而后使裂解酶失活，取出裂解产物加入PCR反应管中进行PCR反应；和
(4)将全基因组扩增后的DNA产物进行分析，以鉴定胚胎的染色体状态是否正常：分析时采用二代测序、核酸芯片或者免疫荧光检测。
在一优选例中，换液2-3天后将形成囊胚的胚胎吸出，转移至新的囊胚培养液中或者进入玻璃化冻存流程，剩余的原囊胚培养液约1微升至500微升，优选10微升至200微升，即为进行胚胎植入前遗传学筛查(PGS)需要收集的样本。
本发明的主要优点包括：
(1)在本发明中，将胚胎在很少量的培养液中培养，并且对极微量的培养液进行检测，所得到的染色体异常的检测结果居然具有极高的精确性。
(2)本发明采用单胚胎培养体系，即一个培养液微滴中只培养一个胚胎， 该体系所得到的染色体异常的检测结果更准确。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，本发明实施例中所用的材料和试剂均为市售产品。
实施例1
选择A、B两个体外受精的胚胎样本，分别采用囊胚细胞检测的方法和囊胚培养液检测的方法评估其染色体状态，具体步骤如下所述：
1.囊胚培养液的获得
1)通过单精子注射方法获得的受精卵培养至第3天卵裂球期后，将胚胎转移至新制备的囊胚培养微滴中进行囊胚培养。
2)将形成囊胚的胚胎吸出，转移至新的囊胚培养液中/或者进入玻璃化冻存流程，剩余的原囊胚培养液(约30ul)即为进行PGS需要收集的样本A及样本B，优选换液2-3天后，将形成囊胚的胚胎吸出。
2.囊胚培养液的采集
1)将装有10微升裂解液(PH为7.2的Tris-Cl 40mM，EDTA 1mM，KCl 15mM以及3％的Triton X-100)的采集管室温放置2min，待裂解液解冻后，将样本采集管置于迷你离心机中，离心30s，保证裂解液全部聚集在管底。
2)将步骤1的第2)步中的原囊胚培养液用口吸管全部转移至裂解液中。
3)用记号笔在采集管上标记样本名称，微型离心机离心30s，样本可立即进入下一步全基因组扩增步骤或放入-20℃或-80℃冷冻保存。
3.囊胚培养液中微量DNA的全基因组扩增
1)将囊胚培养液与裂解液的混合物于室温下融解。
2)向管中加入蛋白酶，上下吹打混匀。
3)将管子置于40℃孵育3h。
4)将管子置于90℃5min以失活裂解酶。
5)从管中取出裂解产物加入一个PCR反应管中。
6)向PCR反应管中加入扩增混合液(15mM Tris-HCl，15mM(NH4)2SO4，20mM KCl，1mM MgSO4，5％DMSO和2％Triton X-100)，5％DMSO，10mM dNTP，50μM NG(5′-GT GAG TGA TGG TTG AGG TAG TGT GGA GNNNNNGGG-3′)和NT(5′-GT GAG TGA TGG TTG AGG TAG TGT GGA GNNNNNTTT-3′)引物，100μM扩增引物(5′-GT GAG TGA TGG TTG AGG TAG TGT GGA G-3′)，1单位BstDNA聚合酶，1单位DeepVentR。
7)将PCR反应管置于PCR仪中进行全基因组扩增，热循环程序为：
Figure PCTCN2016104753-appb-000002
4.将扩增后的DNA产物按常规方法进行二代测序以鉴定胚胎的染色体状态是否正常。
二代测序数据结果表明，在A样本中，囊胚培养液检测方法(图A1)与囊胚细胞检测方法(图A2)均能检出多条染色体异常；而在B样本中，囊胚培养液检测方法(图B1)与囊胚细胞检测方法(图B2)均判定为染色体正常。上述结果表明，采用囊胚培养液检测与囊胚细胞检测这两种方法对鉴定胚胎染色体状态取得相同的结果，进一步证实了该非侵入性的检测方法准确、可靠。
实施例2
随机选取42个体外培养胚胎，分别按照实施例1中的方法在囊胚培养液检测与囊胚细胞检测这两种方法之间进行对比。检测结果的对应关系，逻辑上可出现4种组合形式；
第一类.细胞检测显示异常，培养液检测结果亦显示异常。
第二类.细胞检测显示正常，培养液检测结果亦显示正常。
第三类.细胞检测显示正常，培养液检测结果显示异常。
第四类.细胞检测显示异常，培养液检测结果亦显示正常。
42个对比结果中，这四种对应关系的例数分布如表1所示：
表1
Figure PCTCN2016104753-appb-000003
结果显示，当以细胞检测为金标准时，计算所得培养液检测的敏感度为88.2％，特异性为84.0％，阳性预示值为78.9，阴性预示值为91.3％。尽管各项指标均不为100％，但以无创方法提供充分接近于金标准的检测结果，足以证实本发明的有益价值。
实施例3
对8例因不同原因导致生育困难患者的胚胎按照本发明实施例1的方法实施胚胎培养液检测，并依据检测结果挑选染色体正常的胚胎植入母亲子宫。
结果如表2所示。
表2
Figure PCTCN2016104753-appb-000004
Figure PCTCN2016104753-appb-000005
通常，平衡易位患者的配子(即精子或卵子)中的80％存在染色体异倍体，自然受孕成功率很低。常规试管婴儿方法亦无法鉴别存在染色体异倍体的胚胎，成功率非常低。
本发明的结果显示，3号和8号患者由于未检测到高质量的受精卵，胚胎染色体异常，因此未作胚胎移植。除此之外，其余6例患者的结果充分显示，用本发明的方法挑选染色体正常的胚胎植入患者母体后一次即可成功受孕，并且胚胎移植且保持存活的成功率为5/6(即，83.3％)，也就是说，只有一例患者没有获得成功。
因此，结果表明，用本发明的方法可获得非常高的受孕率和胚胎存活率。
本发明所公开的实施例的上述说明，使本领域技术人员能够实现或使用本发明，同时，以上仅为本发明的较佳实施例而已，并不用以限制本发明实施例，凡在本发明实施例的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明实施例的保护范围之内。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (20)

  1. 一种利用囊胚培养液检测胚胎染色体异常的方法，其特征在于，包括如下步骤：
    (1)囊胚培养液的获得：通过单精子注射方法获得受精卵，将其培养至第3天卵裂球期之后转移至新制备的囊胚培养微滴中进行囊胚培养，将形成囊胚的胚胎吸出，转移至新的囊胚培养液中或者进入玻璃化冻存流程，剩余的原囊胚培养液即检测时所需要收集的样本；
    (2)囊胚培养液的采集：将步骤(1)中获取的原囊胚培养液转移至裂解液中，离心后，样本进入下一步的全基因组扩增步骤；
    (3)囊胚培养液中微量DNA的全基因组扩增：向步骤(2)中获得的囊胚培养液与裂解液的混合物中加入裂解酶，混匀后孵育，而后使裂解酶失活，取出裂解产物加入PCR反应管中进行基因组扩增反应；和
    (4)将全基因组扩增后的DNA产物进行分析，以鉴定胚胎的染色体状态是否正常：分析时采用二代测序、核酸芯片或者免疫荧光检测。
  2. 根据权利要求1所述的方法，其特征在于，步骤(2)中裂解液的成分是PH为7.0～8.0的Tris-Cl 25-45mM，EDTA 0.5-3mM，KCl 10-25mM以及浓度为0.05％-5％的去污剂，所述去污剂为Triton X-100、Triton X-114、吐温20、NP40和SDS中的一种或多种。
  3. 根据权利要求2所述的方法，其特征在于，裂解液的成分优选为PH为7.2的Tris-Cl 40mM，EDTA 1mM，KCl 15mM以及3％的Triton X-100。
  4. 根据权利要求1所述的方法，其特征在于，步骤(3)中的裂解酶选自蛋白酶K、Qiagen Protease、胃蛋白酶、木瓜蛋白酶、胰蛋白酶和溶菌酶中的一种或多种，所述裂解酶的浓度为1-25μg/ml。
  5. 根据权利要求4所述的方法，其特征在于，所述裂解酶的浓度优选为20μg/ml。
  6. 根据权利要求1所述的方法，其特征在于，步骤(3)中孵育温度为30-60℃，孵育时间为1min至12h，失活温度为75-95℃，失活时间为1-15min。
  7. 根据权利要求6所述的方法，其特征在于，步骤(3)中优选的，孵育温度为40℃，孵育时间为3h，失活温度为90℃，失活时间为5min。
  8. 根据权利要求1所述的方法，其特征在于，步骤(3)中进行PCR反应时 的PCR反应管中含有扩增混合液、0.5％-20％的PCR抑制物对抗剂、5-20mM dNTP、5-100μM NG和NT引物、50-200μM扩增引物、0.5-10单位核酸聚合酶，所述PCR抑制物对抗剂选自DMSO、甜菜碱、甲酰胺、甘油和白蛋白中的一种或多种，所述核酸聚合酶选自Phi29 DNA聚合酶、Bst DNA聚合酶、Vent聚合酶、Deep Vent聚合酶、Klenow Fragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、
    Figure PCTCN2016104753-appb-100001
    超保真DNA聚合酶、Taq聚合酶、E.coli DNA聚合酶、LongAmp Taq DNA聚合酶和OneTaq DNA聚合酶中的一种或多种。
  9. 根据权利要求8所述的方法，其特征在于，所述扩增混合液的成分为10-25mM Tris-HCl，5-25mM(NH4)2SO4，5-30mM KCl，0.5-5mM MgSO4，0.1％-20％DMSO和0.05-5％Triton X-100。
  10. 根据权利要求9所述的方法，其特征在于，所述扩增混合液的成分优选为15mM Tris-HCl，15mM(NH4)2SO4，20mM KCl，1mM MgSO4，5％DMSO和2％Triton X-100。
  11. 根据权利要求8所述的方法，其特征在于，所述NG和NT引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C；所述扩增引物包含所述通用序列且不包含所述可变序列。
  12. 根据权利要求11所述的方法，其特征在于，所述可变序列选自下组：(N)nGGG、(N)nTTT，(N)mTNTNG，(N)xGTGG(N)y，其中N为任意的可与天然核酸进行碱基配对的核苷酸，n是选自3-17的正整数，m是选自3-15的正整数，x和y分别是选自3-13的正整数。
  13. 根据权利要求12所述的方法，其特征在于，所述NG和NT引物包括SEQ ID NO:1[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNN]、SEQ ID NO:2[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG]、SEQ ID NO:3[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT]、SEQ ID NO:4[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG]或SEQ ID NO:5[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN]的序列，其中N为任意的可与天然核酸进行碱基配对的核苷酸；所述扩增引物从5’到3’具有SEQ ID NO:6[GTGAGTGATGGTTGAGGTAGTGTGGAG]的序列。
  14. 根据权利要求1所述的方法，其特征在于，步骤(3)中全基因组扩增的热循环程序如下所述：
    (1)在介于90-98℃之间的第一变性温度反应5-20s；
    (2)在介于5-15℃之间的第一退火温度反应5-60s，在介于15-25℃之间的第二退火温度反应5-60s，在介于25-35℃之间的第三退火温度反应30-80s，在介于35-45℃之间的第四退火温度反应5-60s，在介于45-55℃之间的第五退火温度反应5-60s；
    (3)在介于55-80℃之间的第一延伸温度反应10-150min；
    (4)在介于90-98℃之间的第二变性温度反应5-30s；
    (5)在介于45-70℃之间的第六退火温度反应10-30s；
    (6)在介于60-80℃之间的第二延伸温度反应1-10min；
    (7)重复步骤(4)到(6)5至50个循环；
    (8)在介于60-80℃之间的温度下继续延伸反应1-10min；
    (9)将扩增后的产物在0-5℃下冷藏保存。
  15. 根据权利要求14所述的方法，其特征在于，步骤(3)中全基因组扩增的热循环程序如下所述：
    (1)在第一变性温度95℃下反应10s；
    (2)在第一退火温度10℃下反应45s，在第二退火温度20℃下反应45s，在第三退火温度30℃下反应60s，在第四退火温度40℃下反应45s，在第五退火温度50℃下反应45s；
    (3)在第一延伸温度62℃下反应90min；
    (4)在第二变性温度95℃下反应20s；
    (5)在第六退火温度59℃下反应20s；
    (6)在第二延伸温度72℃下反应3min；
    (7)重复步骤(4)到(6)10至30个循环；
    (8)在72℃下继续延伸反应5min；
    (9)将扩增后的产物在4℃下冷藏保存。
  16. 如权利要求1所述的方法，其特征在于，在步骤(3)中，进行PCR反应时使用的引物包括NG引物、NT引物以及扩增引物，
    其中，所述的NG引物和NT引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C；
    所述NG引物的可变序列选自下组：(N)nGGG、(N)xGTGG(N)y、或其组合； 而所述NT引物的可变序列选自下组：(N)nTTT、(N)mTNTNG、或其组合；其中N为任意的可与天然核酸进行碱基配对的核苷酸，各n独立地是选自3-17的正整数，各m独立地是选自3-15的正整数，x和y分别是选自3-13的正整数；
    而所述扩增引物包含所述通用序列且不包含所述可变序列。
  17. 一种利用囊胚培养液检测胚胎染色体异常的检测试剂盒，其特征在于，所述试剂盒含有如下组分：
    (i)用于PCR扩增的引物,所述引物包括NG引物、NT引物以及扩增引物，
    其中，所述的NG引物和NT引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C；
    所述NG引物的可变序列选自下组：(N)nGGG、(N)xGTGG(N)y、或其组合；而所述NT引物的可变序列选自下组：(N)nTTT、(N)mTNTNG、或其组合；其中N为任意的可与天然核酸进行碱基配对的核苷酸，各n独立地是选自3-17的正整数，各m独立地是选自3-15的正整数，x和y分别是选自3-13的正整数；
    而所述扩增引物包含所述通用序列且不包含所述可变序列；以及
    (ii)任选的囊胚培养液。
  18. 如权利要求17所述的试剂盒，其特征在于，所述的NG引物、NT引物以及扩增引物具有相同的通用序列。
  19. 如权利要求17所述的试剂盒，其特征在于，所述的通用序列的长度为20-35nt，较佳地25-30nt。
  20. 一种权利要求17所述检测试剂盒的用途，其特征在于，用于制备利用囊胚培养液检测胚胎染色体异常的产品。
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