Blastocyst solution and embryo culture solution sample preservation solution as well as preparation method and application of blastocyst solution and embryo culture solution sample preservation solution

Abstract

The invention provides a blastocyst solution and embryo culture solution sample preservation solution and a preparation method and application thereof.The blastocyst solution and embryo culture solution cfDNA sample preservation solution is composed of a Tris-HCl solution, an EDTA solution, a KCl solution, TritonX-100 and Primer N. The preservation solution can stably preserve cfDNA in the blastocyst solution and the embryo culture solution for a long time, the preservation condition is relatively loose, the requirement for equipment conditions is low, the preservation time is short, and the preservation time is short. In the transportation process, ultralow-temperature cold chain transportation is not needed, the transportation requirement can be met only under the condition of 4 DEG C, samples can be stably transported and stored for 14 days under the condition of 4 DEG C, cfDNA degradation of blastocyst liquid and embryo culture liquid is effectively prevented, the samples can be stored for 6 months for a long time under the condition of-20 DEG C, and meanwhile, the preservation liquid is completely compatible with a downstream detection process and can be directly used for subsequent detection. And the method can be directly used for subsequent detection.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic gene detection technology, and more specifically, to a blastocyst fluid and embryo culture fluid sample preservation solution and a preparation method and application thereof. Background Art

[0002] Preimplantation genetic testing (PGT) is widely used in assisted reproductive technology (ART). PGT can detect single-gene genetic diseases, chromosome structure and number variations in embryos, and can improve embryo implantation effects, clinical pregnancy outcomes and live birth rates. However, the biopsy process involved in this technology is somewhat invasive to the embryo, and has high requirements for equipment and personnel.

[0003] With the continuous development of second-generation sequencing technology, noninvasive prenatal testing (NiPT) can be achieved by testing for aneuploidy and single-gene genetic diseases in the free DNA released by the fetus into the maternal peripheral blood. These free DNAs mainly come from fetal apoptotic cells. Some metabolomics experiments have shown that cells cultured in vitro can also release nucleic acids into the culture medium. In the study of Xu et al., it was shown that free DNA was also released when the embryo was cultured in vitro.

[0004] With the discovery of free DNA that can be used for genetic analysis in blastocyst fluid and spent embryo culture media (SCM), noninvasive PGT (NiPGT) has become a potential detection method with broad application prospects in the field of assisted reproduction.

[0005] As an important biological sample, blastocyst fluid and embryo culture fluid are easy to obtain and convenient to operate compared with traditional embryo biopsy. They are non-invasive, truly non-invasive and highly safe. At the same time, free DNA (cell-free DNA, cfDNA) can come from both euploid and aneuploid cells of the embryo, which is more conducive to the detection of chimeras. In recent years, there have been many reports on the use of blastocyst fluid and embryo culture fluid in clinical research.

[0006] It can be seen that the research and application of cfDNA in blastocyst fluid and embryo culture medium are becoming more and more extensive. By detecting the status of cfDNA in blastocyst fluid and embryo culture medium, the genetic information of the embryo can be effectively evaluated. However, the composition of blastocyst fluid and embryo culture medium is complex, and it contains various metabolites and high levels of nucleases, and the cfDNA in them is easily degraded.

[0007] In order to ensure that the small amount of cfDNA contained in the blastocyst fluid and embryo culture fluid samples meets the accuracy requirements of clinical testing, the blastocyst fluid and embryo culture fluid are required to be stored and transported at -80°C after collection, so as to maximize the original state of the cfDNA in the blastocyst fluid and embryo culture fluid. Cold chain storage and transportation Even so, relying solely on low temperature cannot effectively prevent the degradation of cfDNA in blastocyst fluid and embryo culture fluid samples within a certain period of time.

[0008] The volume of blastocyst fluid and embryo culture itself is small, generally less than 20 microliters, and the cfDNA content is low. Therefore, the total amount of cfDNA in a single sample is extremely limited. It cannot be extracted for nucleic acid and then subsequently tested like other biological samples. The original sample needs to be used for direct testing.

[0009] Therefore, it is necessary to develop a preservative that can stably preserve cfDNA in blastocyst fluid and embryo culture fluid samples at 4°C, effectively prevent the degradation of cfDNA in blastocyst fluid and embryo culture fluid, and have no effect on subsequent direct biological detection. At the same time, it can solve the problems that current samples rely on cold chain and cannot stably preserve cfDNA in blastocyst fluid and embryo culture fluid. Summary of the invention

[0010] In view of this, in order to solve the above problems, the present invention proposes a blastocyst fluid and embryo culture fluid sample preservation solution and a preparation method and application thereof, wherein the blastocyst fluid and embryo culture fluid cfDNA sample preservation solution is composed of Tris-HCl solution, EDTA solution, KCl solution, TritonX-100 and Primer N. The preservation solution can stably preserve the cfDNA in the blastocyst fluid and embryo culture fluid for a long time, and the storage conditions are relatively loose and have low requirements on equipment conditions. During transportation, there is no need to rely on ultra-low temperature cold chain transportation, and the transportation requirements can be met only at 4°C. The samples can be stably transported and preserved at 4°C for up to 14 days, effectively preventing the degradation of blastocyst fluid and embryo culture fluid cfDNA, and can be stored for up to 6 months at -20°C. At the same time, the preservation solution is fully compatible with downstream detection processes and can be directly used for subsequent detection without affecting subsequent detection.

[0011] A blastocyst fluid and embryo culture fluid sample preservation solution, characterized in that the working concentrations of the components in the blastocyst fluid and embryo culture fluid cfDNA sample preservation solution are: Tris-HCl: 13mM-33mM; EDTA: 7mM-15mM; KCl: 13mM-33mM; TritonX-100: 0.1%-0.5%; Primer N: 0.05μM-0.5μM.

[0012] The components in the sample preservation solution described in the present invention are reasonably selected. Compared with other preservation solutions at inappropriate concentrations, the preservation solution provided by the present invention can better maintain the stability of cfDNA in the sample, so that the data obtained are rich, accurate and reliable.

[0013] A method for preparing the sample preservation solution comprises: sequentially adding Tris HCl solution, EDTA solution, KCl solution, TritonX-100 and Primer N into a centrifuge tube, mixing well, and preparing the sample preservation solution.

[0014] In some embodiments, the volume ratio of the blastocyst fluid and embryo culture fluid to be stored to the storage fluid is: (2-4):1.

[0015] In some embodiments, the working concentrations of the components in the sample preservation solution are: Tris-HCl: 15 mM; EDTA: 1 mM; KCl: 10 mM; TritonX-100: 0.1%; Primer N: 0.05 μM.

[0016] In some embodiments, the working concentrations of the components in the sample preservation solution are: Tris-HCl: 33 mM; EDTA:

[0017] 3.5mM; KCl: 30mM; TritonX-100: 0.5%; Primer N: 0.5μM.

[0018] In some embodiments, the working concentrations of the components in the sample preservation solution are: Tris-HCl: 23 mM; EDTA: 10 mM; KCl: 23 mM; TritonX-100: 0.3%; Primer N: 0.2 μM.

[0019] The present invention also provides a reagent combination for detecting cfDNA in blastocyst fluid and embryo culture fluid, which includes the sample preservation solution, DNA library construction reagent, and cfDNA amplification and library construction kit as described above.

[0020] In the present invention, the DNA library construction reagents and cfDNA amplification and library construction reagents are reagents well known in the art, and the present invention is not limited thereto. They can all be used in conjunction with the sample preservation solution described in the present invention to construct a library of good quality.

[0021] Furthermore, the blastocyst fluid and embryo culture medium are placed in the sample preservation solution of the present invention, and the samples are stably transported and preserved at 4° C. for up to 14 days.

[0022] Furthermore, the blastocyst fluid and embryo culture medium are placed in the sample preservation solution of the present invention and can be stored for up to 6 months at -20°C.

[0023] Beneficial effects of the present invention: The present invention proposes a blastocyst fluid and embryo culture fluid sample preservation solution and a preparation method and application thereof, wherein the blastocyst fluid and embryo culture fluid cfDNA sample preservation solution is composed of Tris-HCl solution, EDTA solution, KCl solution, TritonX-100 and Primer N, and the preservation solution can stably preserve the cfDNA in the blastocyst fluid and embryo culture fluid for a long time, and the preservation conditions are relatively loose and have low requirements on equipment conditions. During transportation, there is no need to rely on ultra-low temperature cold chain transportation, and the transportation requirements can be met only at 4°C. The samples can be stably transported and preserved at 4°C for up to 14 days, and the degradation of blastocyst fluid and embryo culture fluid cfDNA can be effectively prevented. The samples can be preserved for a long time at -20°C for up to 6 months. At the same time, the preservation solution is fully compatible with downstream detection processes and can be directly used for subsequent detection without affecting subsequent detection. DETAILED DESCRIPTION

[0024] The present invention provides a sample preservation solution and its application in blastocyst fluid and embryo culture fluid cfDNA detection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted in particular that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0025] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0026] The present invention will be further described below in conjunction with embodiments:

[0027] Storage of Example Samples

[0028] Prepare the preservation solution according to the following formula:

[0029]

[0030]

[0031] A method for preparing the sample preservation solution comprises: sequentially adding Tris HCl solution, EDTA solution, KCl solution, TritonX-100 and Primer N into a centrifuge tube, mixing well, and preparing the sample preservation solution.

[0032] Usage of preservation solution: The ratio of the blastocyst fluid and embryo culture solution to be preserved to the preservation solution is (2-4):1, calculated by volume.

[0033] Storage of comparative samples

[0034] Prepare the preservation solution of each comparison ratio according to the formula in the following table:

[0035] Raw material name Final concentration range of storage solution components Tris-HCl 20mM-50mM EDTA 1mM-3.5mM NaCl 1mM-4mM Triton X-100 0.1％-0.5％ DTT 5mM-30mM

[0036] A method for preparing the sample preservation solution comprises: sequentially adding Tris HCl solution, EDTA solution, KCl solution, TritonX-100 and Primer N into a centrifuge tube, mixing well, and preparing the sample preservation solution.

[0037] Usage of preservation solution: The ratio of the blastocyst fluid and embryo culture solution to be preserved to the preservation solution is (2-4):1, calculated by volume.

[0038] Example 1: Comparative storage fluid test

[0039] Test plan: Preservation solution formulas 1, 2, 3 and the control preservation solution were used for embryo culture medium preservation (3 samples in each group). The samples were tested immediately after adding the preservation solution. The remaining samples were stored at 4°C for 15 days and then tested again. The detection performance of the samples before and after storage at 4°C in different preservation solutions was evaluated by evaluating the consistency of product concentration and CNV copy number variation coefficient.

[0040] 1. Sample pretreatment is required before testing

[0041] 1.1 Sample processing: Take out the culture fluid samples to be tested, centrifuge them in a microcentrifuge for 30-60 seconds, and place them neatly on the PCR plate in order;

[0042] 1.2 Take 10 μL of each sample and place it at the bottom of a 200 μL PCR tube. Then add 1 μL of lytic enzyme to each PCR tube and centrifuge instantly.

[0043] 1.3 Set the lysis program according to the table below, select "On 105℃" for "Hot Cover", and place the prepared PC, NC and samples to be lysed in the PCR instrument to complete the lysis reaction.

[0045] 2 Pre-amplification: 2.1 Take out buffer 2 and pre-amplification enzyme from the -20℃ refrigerator, thaw at room temperature, vortex buffer 2 to mix, centrifuge briefly (microcentrifuge, 2000 rpm, 3-5 sec), and set aside.

[0046] 2.2 Prepare the pre-amplification mixture according to the following table based on the reaction system and the number of reactions (N).

[0047]

[0048]

[0049] 2.3 Add 60 μL of pre-amplification mixture to the lysate of step 1.3, vortex and centrifuge briefly (microcentrifuge, 2000 rpm, 10-15 sec).

[0050] 2.4 Set the pre-amplification program according to the table below, select "On 105℃" for "Hot Cover", and place the sample in step 2.3 in the PCR instrument to complete the pre-amplification reaction.

[0051]

[0052] 3 Exponential expansion

[0053] 3.1 Take out buffer 3 and amplification enzyme from -20℃ refrigerator, thaw at room temperature, vortex buffer 3 to mix, and centrifuge briefly (microcentrifuge, 2000 rpm, 3-5 sec). According to the reaction system and the number of reactions (N), prepare the amplification mixture according to the table below.

[0054] Reagent name Volume (N = number of reaction samples) Buffer 3 60μL×(N+1) Amplification enzyme 1.6μL×(N+1) Total volume 61.6μL×(N+1)

[0055] 3.2 Add 60 μL of amplification mixture to the pre-amplification product in step 2.4 (the total volume of the solution is about 131

[0056] μL) and 2 μL of label primer, vortex to mix, and centrifuge briefly (microcentrifuge, 2000 rpm, 10-15 sec).

[0057] 3.3 Set the pre-amplification program according to the table below, select "On 105℃" for "Hot Cover", and place the sample in step 3.2 in the PCR instrument to complete the exponential amplification reaction.

[0058]

[0059] 4 Library purification

[0060] 4.1 Take out the magnetic beads from the 4℃ refrigerator in advance and equilibrate them to room temperature. Vortex the magnetic beads to mix them thoroughly.

[0061] 4.2 Add 1× magnetic beads to each tube of library (for example, if the initial volume of the library is 100 μL, add 100

[0062] μL magnetic beads), vortex to mix, centrifuge briefly (microcentrifuge, 2000 rpm, 3-5 sec), and let stand at room temperature for 5 min.

[0063] 4.3 Place the centrifuge tube on a magnetic rack to separate the magnetic beads from the supernatant. After about 5 minutes, the solution becomes clear. Carefully aspirate the supernatant and discard it. Do not blow the magnetic beads containing the target fragment. Note: Do not aspirate the magnetic beads.

[0064] 4.4 Keep the centrifuge tube fixed on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to the centrifuge tube, place it at room temperature for 30 seconds, and carefully discard the supernatant.

[0065] 4.5 Repeat step 4.4 once.

[0066] 4.6 Keep the centrifuge tube open on the magnetic rack and place it at room temperature for 5-10 minutes to allow the ethanol to evaporate completely and ensure it is fully dried.

[0067] 4.7 Add 30 μL of nuclease-free water, remove the centrifuge tube from the magnetic rack, vortex to completely resuspend the magnetic beads (15-30 seconds), centrifuge briefly (microcentrifuge, 2000 rpm, 3-5 seconds), leave at room temperature for 5 minutes, place the centrifuge tube in the magnetic rack to separate the magnetic beads and liquid. After about 5 minutes, the solution is clear, and carefully pipette 28 μL of supernatant into a new 1.5 ml centrifuge tube.

[0068] 5. Library Quantification

[0069] Use of purified pooled libraries dsDNA HS Assay Kit (Cat. No.: Q32851, Manufacturer: ThermoFisher Scientific) was used for quantification.

[0070] 6 Library on the machine

[0071] Next-generation sequencing.

[0072] 7 Data Analysis

[0073] Bioinformatics CNV data analysis.

[0074] The test data was evaluated by evaluating the library concentration by sequencing after amplification and library construction, and by testing the data on the machine.

[0075] Amplification product concentration:

[0076]

[0077] CNV test results:

[0078]

[0079]

[0080] The results show:

[0081] After samples were stored at 4°C for 15 days, the concentration of amplified products and the coefficient of variation CV of the samples stored in the preservation solutions of formula P1, P2 and P3 of the present invention were basically consistent with those before storage, and the results were in line with expectations; the preservation solution of the present invention can effectively protect cfDNA in blastocyst fluid and embryo culture medium at 4°C for 15 days, meeting the design requirements;

[0082] However, after the samples in the control group were stored in the C group preservation solution at 4°C for 15 days, the concentration of the amplified product decreased significantly, and the coefficient of variation CV and other QC data deteriorated significantly, indicating that the preservation performance was poor and the samples could not be stably preserved.

[0083] Embodiment 2:

[0084] Test plan: 10 embryo culture fluids (A001-A010) and 5 blastocyst fluids (A011-A015) were preserved using formula 3. The samples were tested immediately after adding the preservation fluid. The remaining samples were stored at -20°C for 6 months and then tested again.

[0085] 1. Sample pretreatment is required before testing

[0086] 1.1 Sample processing: Take out the culture fluid samples to be tested, centrifuge them in a microcentrifuge for 30-60 seconds, and place them neatly on the PCR plate in order;

[0087] 1.2 Take 10 μL of each sample and place it at the bottom of a 200 μL PCR tube. Then add 1 μL of lytic enzyme to each PCR tube and centrifuge instantly.

[0088] 1.3 Set the lysis program according to the table below, select "On 105℃" for "Hot Cover", and place the prepared PC, NC and samples to be lysed in the PCR instrument to complete the lysis reaction.

[0090] 2 Pre-amplification: 2.1 Take out buffer 2 and pre-amplification enzyme from the -20℃ refrigerator, thaw at room temperature, vortex buffer 2 to mix, centrifuge briefly (microcentrifuge, 2000 rpm, 3-5 sec), and set aside.

[0091] 2.2 Prepare the pre-amplification mixture according to the following table based on the reaction system and the number of reactions (N).

[0092]

[0093] 2.3 Add 60 μL of pre-amplification mixture to the lysate of step 1.3, vortex and centrifuge briefly (microcentrifuge, 2000 rpm, 10-15 sec).

[0094] 2.4 Set the pre-amplification program according to the table below, select "On 105℃" for "Hot Cover", and place the sample in step 2.3 in the PCR instrument to complete the pre-amplification reaction.

[0095]

[0096]

[0097] 3 Exponential expansion

[0098] 3.1 Take out buffer 3 and amplification enzyme from -20℃ refrigerator, thaw at room temperature, vortex buffer 3 to mix, and centrifuge briefly (microcentrifuge, 2000 rpm, 3-5 sec). According to the reaction system and the number of reactions (N), prepare the amplification mixture according to the table below.

[0099] Reagent name Volume (N = number of reaction samples) Buffer 3 60μL×(N+1) Amplification enzyme 1.6μL×(N+1) Total volume 61.6μL×(N+1)

[0100] 3.2 Add 60 μL of amplification mixture to the pre-amplification product in step 2.4 (the total volume of the solution is about 131

[0101] μL) and 2 μL of label primer, vortex to mix, and centrifuge briefly (microcentrifuge, 2000 rpm, 10-15 sec).

[0102] 3.3 Set the pre-amplification program according to the table below, select "On 105℃" for "Hot Cover", and place the sample in step 3.2 in the PCR instrument to complete the exponential amplification reaction.

[0103]

[0104] 4 Library purification

[0105] 4.1 Take out the magnetic beads from the 4℃ refrigerator in advance and equilibrate them to room temperature. Vortex the magnetic beads to mix them thoroughly.

[0106] 4.2 Add 1× magnetic beads to each tube of library (for example, if the initial volume of the library is 100 μL, add 100

[0107] μL magnetic beads), vortex to mix, centrifuge briefly (microcentrifuge, 2000 rpm, 3-5 sec), and let stand at room temperature for 5 min.

[0108] 4.3 Place the centrifuge tube on a magnetic rack to separate the magnetic beads from the supernatant. After about 5 minutes, the solution becomes clear. Carefully aspirate the supernatant and discard it. Do not blow the magnetic beads containing the target fragment. Note: Do not aspirate the magnetic beads.

[0109] 4.4 Keep the centrifuge tube fixed on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to the centrifuge tube, place it at room temperature for 30 seconds, and carefully discard the supernatant.

[0110] 4.5 Repeat step 4.4 once.

[0111] 4.6 Keep the centrifuge tube open on the magnetic rack and place it at room temperature for 5-10 minutes to allow the ethanol to evaporate completely and ensure it is fully dried.

[0112] 4.7 Add 30 μL of nuclease-free water, remove the centrifuge tube from the magnetic rack, vortex to completely resuspend the magnetic beads (15-30 seconds), centrifuge briefly (microcentrifuge, 2000 rpm, 3-5 seconds), leave at room temperature for 5 minutes, place the centrifuge tube in the magnetic rack to separate the magnetic beads and liquid. After about 5 minutes, the solution is clear, and carefully pipette 28 μL of supernatant into a new 1.5 ml centrifuge tube.

[0113] 5. Library Quantification

[0114] Use of purified pooled libraries dsDNA HS Assay Kit (Cat. No.: Q32851, Manufacturer: ThermoFisher Scientific) was used for quantification.

[0115] 6 Library on the machine

[0116] Next-generation sequencing.

[0117] 7 Data Analysis

[0118] Bioinformatics CNV data analysis.

[0119] The preservation performance of the sample preservation solution was evaluated by assessing the consistency of product concentration and CNV copy number variation coefficient; amplification product concentration:

[0120]

[0121] CNV test results:

[0122]

[0123] The above data show that after the blastocyst fluid and embryo culture fluid samples were preserved in the preservation solution of the present invention and stored at -20°C for 6 months, the concentration of the amplified product and the coefficient of variation CV were basically consistent with those before preservation, and the results were in line with expectations; the preservation solution of the present invention can effectively protect the blastocyst fluid and embryo culture fluid cfDNA for 6 months under -20°C conditions, meeting the design requirements.

[0124] Therefore, the preservation solution can preserve the cfDNA in the blastocyst fluid and embryo culture fluid stably for a long time, and the preservation conditions are relatively loose and have low requirements on equipment conditions. During transportation, there is no need to rely on ultra-low temperature cold chain transportation, and the transportation needs can be met at 4°C. The samples can be stably transported and preserved at 4°C for up to 14 days, effectively preventing the degradation of cfDNA in the blastocyst fluid and embryo culture fluid, and can be stored for up to 6 months at -20°C. At the same time, the preservation solution is fully compatible with downstream detection processes and can be directly used for subsequent detection without affecting subsequent detection and can be directly used for subsequent detection.

[0125] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A blastocyst fluid and embryo culture fluid sample storage solution, characterized in that: The working concentrations of the components in the blastocyst fluid and embryo culture fluid cfDNA sample preservation solution are: Tris-HCl: 13mM-33mM; EDTA: 7mM- 15mM; KCl: 13mM-33mM; TritonX-100: 0.1%-0.5%; Primer N: 0.05μM- 0.5μM.

2. The blastocyst fluid and embryo culture fluid sample preservation solution according to claim 1, characterized in that: The volume ratio of the blastocyst fluid and embryo culture fluid to be stored to the storage fluid is: (2-4):

1.

3. The blastocyst fluid and embryo culture fluid sample preservation solution according to claim 1, characterized in that: The working concentrations of the components in the sample preservation solution are: Tris-HCl: 15 mM; EDTA: 1 mM; KCl: 10 mM; TritonX-100: 0.1%; Primer N: 0.05μM.

4. The blastocyst fluid and embryo culture fluid sample preservation solution according to claim 1, characterized in that: The working concentrations of the components in the sample preservation solution are: Tris-HCl: 33 mM; EDTA: 3.5 mM; KCl: 30 mM; TritonX-100: 0.5%; Primer N: 0.5μM.

5. The blastocyst fluid and embryo culture fluid sample preservation solution according to claim 1, characterized in that: The working concentrations of the components in the sample preservation solution are: Tris-HCl: 23 mM; EDTA: 10 mM; KCl: 23 mM; TritonX-100: 0.3%; Primer N: 0.2 μM.

6. A method for preserving the sample using the preservative solution of any one of claims 1 to 5 above, wherein the blastocyst fluid and embryo culture solution are placed in the sample preservative solution of the present invention, and the sample is stably transported and stored at 4°C for up to 14 days.

7. A method for preserving the sample using the preservative solution of any one of claims 1 to 5 above, wherein the blastocyst fluid and embryo culture solution are placed in the sample preservative solution of the present invention, and can be stored for up to 6 months at -20°C.

8. A method for preparing the sample preservation solution according to claim 1, the method comprising: Tris HCl solution, EDTA solution, KCl solution, TritonX-100 and Primer N were added to the centrifuge tube in sequence, mixed evenly, and the sample preservation solution was prepared.

9. A reagent combination for detecting cfDNA in blastocyst fluid and embryo culture fluid, comprising the sample preservation solution described in any one of claims 1 to 5 above, a DNA library construction reagent, and a cfDNA amplification and library construction kit, wherein the DNA library construction reagent and the cfDNA amplification and library construction reagent are reagents well known in the art.

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