Method for improving concentration of fetal free DNA in maternal peripheral blood

Abstract

The invention relates to a method for increasing the concentration of fetal free DNA in maternal peripheral blood. The method comprises the following steps: S1, extracting free DNA in maternal peripheral blood; s2, performing terminal repair on the free DNA to obtain a terminal repair product; s3, carrying out fragment screening on the terminal repair product to obtain enriched fetal free DNA (Deoxyribose Nucleic Acid); s4, performing linker connection on the enriched fetal free DNA to obtain a linker connection product; s5, carrying out PCR (Polymerase Chain Reaction) amplification on the linker connection product to obtain an amplification product of the fetal free DNA; and S6, purifying and recovering the amplification product of the fetal free DNA to obtain the constructed fetal free DNA library. The invention also relates to the method. According to the method disclosed by the invention, two magnetic bead screening steps are added, so that the fragments less than 143bp in the cfDNA are enriched, and the fetal concentration is increased to 1.5-5 times of that of the non-enriched fragments, so that the detection failure rate, the false positive rate and the false negative rate of NIPT detection of a sample are remarkably reduced, the accuracy of NIPT detection is improved, and a scientific decision basis is favorably provided for pregnant women.

Description

Technical Field

[0001] The present invention relates to the field of prenatal screening, and in particular to a method for increasing the concentration of fetal free DNA in maternal peripheral blood. Background Art

[0002] Birth defects are a major factor affecting the quality of my country's population, with a total incidence of approximately 5.6%. Among them, chromosomal aneuploidy is one of the most common genetic factors leading to birth defects. Clinically, Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), and Patau syndrome (trisomy 13) are the main types of fetal chromosomal aneuploidy, with incidence rates of 1 / 700, 1 / 6000, and 1 / 10000, respectively. Children with these diseases generally suffer from severe intellectual disability, developmental delay, and multiple organ malformations, and most are unable to take care of themselves. This not only threatens children's health and quality of life, but also imposes a long-term burden on families and sustainable socioeconomic development. In addition to abnormal chromosome number, copy number variations (such as chromosome microdeletion / microduplication syndrome) are also important genetic pathogenic factors.

[0003] Currently, there is no effective treatment for birth defects such as Down syndrome, Edwards syndrome, and Patau syndrome. Patients require lifelong care, placing significant emotional and financial strain on their families. Therefore, prenatal screening and diagnosis during pregnancy have become crucial for preventing birth defects. Traditional prenatal screening and diagnosis typically utilize invasive methods such as amniocentesis, chorionic villus sampling, and umbilical vein puncture. While highly accurate, these methods carry risks such as fetal loss, maternal bleeding, and infection, limiting their widespread use.

[0004] In 1997, it was first discovered that fetal DNA exists stably in the form of cell-free fetal DNA (cffDNA) and enters the maternal peripheral blood circulation. Since then, non-invasive prenatal genetic screening (NIPT) has been pioneered. It can screen for chromosomal aneuploidy and chromosomal microdeletion / microduplication syndrome, help reduce the risk of adverse pregnancy outcomes, and provide pregnant women with a scientific basis for decision-making.

[0005] Before using NIPT to perform high-throughput sequencing analysis of fetal free DNA in maternal peripheral blood, a library of fetal free DNA needs to be constructed, including the following steps: (1) extracting free DNA from maternal peripheral blood; (2) performing end-repair on the free DNA to obtain end-repair products; (3) performing adapter ligation on the end-repair products to obtain adapter ligation products; (4) performing PCR amplification on the adapter ligation products to obtain amplified products; (5) purifying and recovering the amplified products to obtain the constructed library. However, the fetal concentration of some libraries obtained by this method is relatively low (<4%), which can easily lead to subsequent NIPT test failures and false positive or false negative results in NIPT, and then blood needs to be re-drawn for secondary testing, which increases the cycle and cost of NIPT. In addition, for samples with low fetal concentrations, even if blood is re-drawn, it is still difficult to increase the fetal concentration to the minimum detection limit of NIPT. Therefore, increasing the concentration of fetal free DNA in maternal peripheral blood is crucial to reducing the detection failure rate of NIPT and improving the accuracy of NIPT detection. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to overcome the defects or shortcomings of the prior art and provide a method for increasing the concentration of fetal free DNA in maternal peripheral blood.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A method for increasing the concentration of fetal free DNA in maternal peripheral blood comprises the steps of:

[0009] S1. Extract free DNA from peripheral blood of pregnant women;

[0010] S2, performing end repair on the free DNA to obtain end repair products;

[0011] S3. Screen the end-repair products for fragments to obtain enriched fetal free DNA;

[0012] S4, performing adapter ligation on the enriched fetal free DNA to obtain an adapter ligation product;

[0013] S5, performing PCR amplification on the adapter ligation product to obtain an amplified product of fetal cell-free DNA;

[0014] S6. Fragment sorting is performed on the amplified products of fetal free DNA to obtain a constructed fetal free DNA library.

[0015] Compared to existing technologies, this method adds two magnetic bead screening steps (fragment screening of end-repair products and fragment screening of amplified products) to enrich fragments <143bp in cfDNA, increasing the fetal concentration to 1.5-5 times that of the unenriched version. When used in NIPT, the library constructed using this method can significantly reduce the failure rate, false positive rate, and false negative rate of NIPT testing, thereby improving the accuracy of NIPT testing and providing scientific decision-making for pregnant women.

[0016] In one embodiment, step S3 includes: S31, adding 1.0-2.0 times the volume of purified magnetic beads to the end repair product of step S2, mixing thoroughly and letting it stand at room temperature until it is clarified; S32, aspirating the supernatant into 1.0-2.0 times the volume of purified magnetic beads, mixing thoroughly and letting it stand at room temperature until it is clarified; S33, aspirating and discarding the supernatant, washing with 80% ethanol, and then eluting with enzyme-free nucleic acid water to obtain enriched fetal free DNA.

[0017] In one embodiment, step S6 includes: S61, adding 0.8-1.2 times the volume of purified magnetic beads to the amplified product of step S5, mixing thoroughly, and letting it stand at room temperature until it is clear; S62, aspirating the supernatant into 0.8-1.2 times the volume of purified magnetic beads, mixing thoroughly, and letting it stand at room temperature until it is clear; S63, aspirating and discarding the supernatant, washing with 80% ethanol, and then eluting with enzyme-free nucleic acid water to obtain a screened library.

[0018] In one embodiment, step S4 includes: using 0.8-1.5 times the volume of purification magnetic beads to purify and recover the adapter ligation product.

[0019] In one embodiment, in step S5, the reaction procedure of the PCR amplification is: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 65°C for 30s, extension at 72°C for 30s, 16-19 cycles; incubation at 72°C for 5min, and storage at 4°C.

[0020] In one embodiment, in step S2, the end repair reaction system includes free DNA, end repair buffer and end repair enzyme; the end repair reaction procedure is 12°C for 15 minutes, 37°C for 15 minutes, 72°C for 20 minutes, and finally stored at 4°C.

[0021] In one embodiment, the volumes of the purification magnetic beads in step S31 and step S32 are 1.2-1.8 times and 1.2-1.8 times, respectively.

[0022] In one embodiment, the volumes of the purification magnetic beads in step S61 and step S62 are 0.8-1.0 times and 1.0-1.2 times, respectively.

[0023] In one embodiment, the volume of the purification magnetic beads in step S4 is 1.0-1.5 times.

[0024] In one embodiment, step S1 uses a two-step method to centrifuge the maternal peripheral blood sample to obtain plasma, and uses a magnetic bead method to extract free DNA in the plasma.

[0025] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a library fragment distribution diagram of library S4201 and library S4301 in Example 1;

[0027] Figure 2 This is a fragment distribution diagram of the library S4201-1 in Example 1;

[0028] Figure 3 This is a fragment distribution diagram of library S4201-2 in Example 1;

[0029] Figure 4 This is a fragment distribution diagram of the library S4301-1 in Example 1;

[0030] Figure 5 This is a fragment distribution diagram of library S4301-2 in Example 1;

[0031] Figure 6 This is the library fragment distribution diagram of library S4301-3 in Example 1. DETAILED DESCRIPTION

[0032] In response to the problem that the concentration of fetal free DNA in the library of the existing library construction method is low (<4%), which leads to an increase in the failure rate of NIPT detection and a decrease in accuracy of the library, the present invention considers enriching the fetal free DNA in the library in a targeted manner and removing the maternal free DNA in the library to increase the concentration of fetal free DNA in the library. According to research, there is a significant difference in length between DNA fragments of fetal origin (cffDNA fragments) and DNA fragments of maternal origin. The DNA fragments of fetal origin (cffDNA fragments) are smaller, with a length mainly concentrated in the range of 75-205bp, and mostly concentrated in 143bp, while the length of DNA fragments of maternal origin is mostly concentrated in 166bp. This length difference provides a theoretical basis for enriching fetal free DNA through fragment screening.

[0033] Based on these length differences, the present invention conducted fragment screening experiments on different products during library construction, specifically end-repair products, adapter-ligated products, and amplified products. The study found that fragment screening of end-repair products alone increased the relative proportion of cell-free fetal DNA, but fragment screening of adapter-ligated products alone or amplified products alone did not significantly change the relative proportion of cell-free fetal DNA in the library.

[0034] Furthermore, the present invention attempts to combine different fragment screening operations. After extensive experimental exploration, it was discovered that fragment screening of end-repair products and fragment screening of amplification products have a synergistic effect. When these two screening operations are used in combination, the relative proportion of fetal free DNA in the library can be significantly increased (by more than 1.5 times), providing an effective technical means for enriching fetal free DNA.

[0035] Based on this, the present invention provides a method for increasing the concentration of fetal free DNA in maternal peripheral blood, comprising the following steps:

[0036] S1. Extract cell-free DNA (cfDNA) from maternal peripheral blood;

[0037] S2, performing end repair on the free DNA to obtain end repair products;

[0038] S3. Screen the end-repair products for fragments to obtain enriched fetal free DNA;

[0039] S4, performing adapter ligation on the enriched fetal free DNA to obtain an adapter ligation product;

[0040] S5. performing PCR amplification on the adapter-ligated product to obtain an amplified product of fetal cell-free DNA (cffDNA);

[0041] S6. Fragment sorting is performed on the amplified products of fetal free DNA to obtain a constructed fetal free DNA library.

[0042] Based on this method, the present invention further conducted experimental operations and verifications on various parameters, which are described in detail below in conjunction with the examples.

[0043] In the following examples, cfDNA was extracted using the magnetic bead-based cell-free DNA extraction kit from Meiji Biotechnology, and the fetal cell-free DNA library was constructed using the NGS Fast DNA Library Prep set for Illumina library construction kit from Kangwei Century. The magnetic beads involved in each example were all PCR purification magnetic beads from Kesheng, and the short linkers and index primers involved were NGS Multiplex OligoS0 for Illumina (Index Primers set Ⅰ).

[0044] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional in the art. For experimental methods in the examples where specific experimental conditions are not specified, conventional experimental conditions or the experimental conditions recommended by the manufacturer are generally used. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0045] Example 1

[0046] This embodiment 1 provides a method for increasing the concentration of fetal free DNA in maternal peripheral blood, comprising the following steps:

[0047] S1. Extract free DNA from the peripheral blood of pregnant women: Use a two-step method to centrifuge the maternal peripheral blood sample to obtain plasma, and aspirate 600 μL of plasma for free DNA extraction; wherein, the free DNA is extracted using the magnetic bead free DNA extraction kit of Meiji Bio.

[0048] S2. Perform end repair on the free DNA obtained in step S1 to obtain end repair products; wherein the reaction system and reaction procedure of the end repair are shown in Tables 1 and 2.

[0049] Table 1 End repair reaction system

[0050] Reagent name volume Extracted free DNA 43μL End repair buffer 6.5 μL End repair enzymes 2μL Overall system 51.5μL

[0051] Table 2 End repair reaction procedure

[0052] Reaction temperature Reaction time 12℃ 15min 37℃ 15min 72℃ 20min 4℃ hold

[0053] S3, screening the end-repair products obtained in step S2 to obtain enriched fetal free DNA, specifically including steps S31-S33:

[0054] S31. Add nuclease-free water to the end-repair product of step S2 to 60 μL, then add 60-120 μL (1.0-2.0 times the volume) of purified magnetic beads, vortex to mix thoroughly, and let stand at room temperature for 5 minutes;

[0055] S32. Place the sample obtained in step S31 on a magnetic rack until the solution is clear, pipette the supernatant into 60-120 μL (1.0-2.0 times the volume) of purified magnetic beads, vortex to mix thoroughly, and let stand at room temperature for 5 minutes;

[0056] S33. Place the sample obtained in step S32 on a magnetic rack until the solution is clear. After aspirating the supernatant, add 200 μL of 80% ethanol and wash twice. Fully aspirate the ethanol, then let it stand at room temperature to allow the ethanol to fully evaporate. Then add 32 μL of nuclease-free water, vortex and mix. After standing at room temperature for 5 minutes, place it on a magnetic rack until the solution is clear. Aspirate 30 μL of supernatant (end repair sorting product), that is, the enriched fetal free DNA, and transfer it to a new 200 μL PCR tube for use.

[0057] S4. Perform adapter ligation on the enriched fetal free DNA to obtain an adapter ligation product: wherein the adapter ligation reaction system is shown in Table 3.

[0058] Table 3 Reaction system for linker ligation

[0059] Reagent name volume End-repair sorting products 30μL Nuclease-free water 21.5μL Ligation buffer 14 μL DNA ligase 3μL Short connector 4μL Overall system 72.5μL

[0060] The reaction system was incubated at 20°C for 15 min, and then 58-108.8 μL (0.8-1.5 times the volume) of purified magnetic beads were used to recover the ligation product. 24 μL of nuclease-free water was added for elution to obtain 21 μL of eluted product, i.e., the adapter ligation product, which was transferred to a new 200 μL PCR tube for the next experiment.

[0061] S5. Perform PCR amplification on the linker-ligated product to obtain an amplified product of fetal cell-free DNA; wherein the reaction system and reaction procedure of the PCR amplification are shown in Tables 4 and 5.

[0062] The PCR primer sequence is as follows: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'.

[0063] Table 4 PCR amplification reaction system

[0064] Reagent name volume Ligation of purified products 21 μL PCR amplification reagents 25 μL PCR primers 2μL Index Primers 2μL Overall system 50μL

[0065] Table 5 PCR amplification reaction program

[0066]

[0067] S6. Purify and recover the amplified product of the fetal cell-free DNA to obtain a constructed fetal cell-free DNA library, which specifically includes steps S61-S63:

[0068] S61. Add 40-60 μL (0.8-1.2 times the volume) of purified magnetic beads to the amplified product obtained in step S5, vortex and oscillate to mix thoroughly, and let stand at room temperature for 5 minutes;

[0069] S62. Perform fragment sorting on the sample obtained in step S61: Place the sample obtained in step S61 on a magnetic rack until the solution is clear, pipette the supernatant into 40-60 μL (0.8-1.2 times the volume) of purified magnetic beads, vortex to mix thoroughly, and let stand at room temperature for 5 min;

[0070] S63. Place the sample obtained in step S62 on a magnetic rack until the solution is clear, discard the supernatant, add 200 μL of 80% ethanol and wash twice, fully discard the ethanol, and then let it stand at room temperature to allow the ethanol to fully evaporate. Then add 32 μL of nuclease-free water, vortex and mix, let it stand at room temperature for 5 minutes, and then place it on a magnetic rack until the solution is clear. Pipette 30 μL of supernatant (eluted product) and transfer it to a new 1.5 mL centrifuge tube to obtain the screened library.

[0071] Example 2

[0072] The library construction method provided in Example 2 is similar to that in Example 1, except that in Example 2, only fragment screening of the end-repair products is performed, including the following steps:

[0073] S1. Extraction of free DNA from the peripheral blood of pregnant women: Use a two-step method to centrifuge the maternal peripheral blood sample to obtain plasma, and aspirate 600 μL of plasma for extraction of free DNA.

[0074] S2. Perform end repair on the free DNA obtained in step S1 to obtain end repair products; wherein the reaction system and reaction procedure of the end repair are shown in Tables 1 and 2.

[0075] S3, screening the end-repair products obtained in step S2 to obtain enriched fetal free DNA, specifically including steps S31-S33:

[0076] S31. Add nuclease-free water to the end-repair product of step S2 to a volume of 60 μL, then add 60-120 μL (i.e., 1.0-2.0 times the volume) of purified magnetic beads, vortex to mix thoroughly, and let stand at room temperature for 5 minutes;

[0077] S32. Place the sample obtained in step S31 on a magnetic rack until the solution is clear, pipette the supernatant into 60-120 μL (1.0-2.0 times the volume) of purified magnetic beads, vortex to mix thoroughly, and let stand at room temperature for 5 minutes;

[0078] S33. Place the sample obtained in step S32 on a magnetic rack until the solution is clear. After aspirating the supernatant, add 200 μL of 80% ethanol and wash twice. Fully aspirate the ethanol, then let it stand at room temperature to allow the ethanol to fully evaporate. Then add 32 μL of nuclease-free water, vortex and mix. After standing at room temperature for 5 minutes, place it on a magnetic rack until the solution is clear. Aspirate 30 μL of supernatant (end repair sorting product), that is, the enriched fetal free DNA, and transfer it to a new 200 μL PCR tube for use.

[0079] S4. Perform adapter ligation on the enriched fetal free DNA to obtain an adapter ligation product: wherein the adapter ligation reaction system is shown in Table 3.

[0080] The reaction system was incubated at 20°C for 15 min, and then 58-108.8 μL (0.8-1.5 times the volume) of purified magnetic beads were used to recover the ligation product. 24 μL of nuclease-free water was added for elution to obtain 21 μL of eluted product, i.e., the adapter ligation product, which was transferred to a new 200 μL PCR tube for the next experiment.

[0081] S5. Perform PCR amplification on the linker-ligated product to obtain an amplified product of fetal cell-free DNA; wherein the reaction system and reaction procedure of the PCR amplification are shown in Tables 4 and 5.

[0082] S6, purifying and recovering the amplified product of the fetal cell-free DNA to obtain a constructed fetal cell-free DNA library, specifically comprising steps S61 and S63:

[0083] S61. Add 40-60 μL (0.8-1.2 times the volume) of purified magnetic beads to the amplified product obtained in step S5, vortex and oscillate to mix thoroughly, and let stand at room temperature for 5 minutes;

[0084] S63. Place the sample obtained in step S61 on a magnetic rack until the solution is clear, discard the supernatant, add 200 μL of 80% ethanol and wash twice, fully discard the ethanol, and then let it stand at room temperature to allow the ethanol to fully evaporate. Then add 32 μL of nuclease-free water, vortex and mix, let it stand at room temperature for 5 minutes, and then place it on a magnetic rack until the solution is clear. Pipette 30 μL of supernatant (eluted product) and transfer it to a new 1.5 mL centrifuge tube to obtain the screened library.

[0085] Example 3

[0086] The library construction method provided in Example 3 is similar to that in Example 1, except that the library construction method in Example 3 only performs fragment sorting on the amplified products, and includes the following steps:

[0087] S1. Extraction of free DNA from the peripheral blood of pregnant women: Use a two-step method to centrifuge the maternal peripheral blood sample to obtain plasma, and aspirate 600 μL of plasma for extraction of free DNA.

[0088] S2. Perform end repair on the free DNA obtained in step S1 to obtain end repair products; wherein the reaction system and reaction procedure of the end repair are shown in Tables 1 and 2.

[0089] S4. Adapter ligation is performed on the end-repair product obtained in step S2 to obtain an adapter-ligated product. The adapter-ligated reaction system is shown in Table 3. The reaction system is incubated at 20°C for 15 min, and then the ligated product is recovered using 58-108.8 μL (0.8-1.5 volumes) of purified magnetic beads. 24 μL of nuclease-free water is then added for elution to obtain 21 μL of eluted product, i.e., the adapter-ligated product. This eluted product is transferred to a new 200 μL PCR tube for the next step.

[0090] S5. Perform PCR amplification on the linker-ligated product to obtain an amplified product of fetal cell-free DNA; wherein the reaction system and reaction procedure of the PCR amplification are shown in Tables 4 and 5.

[0091] S6. Sorting the amplified products of fetal cell-free DNA to obtain a constructed fetal cell-free DNA library, specifically including steps S61-S63:

[0092] S61. Add 40-60 μL (0.8-1.2 times the volume) of purified magnetic beads to the amplified product obtained in step S5, vortex and oscillate to mix thoroughly, and let stand at room temperature for 5 minutes;

[0093] S62. Place the sample obtained in step S61 on a magnetic rack until the solution is clear, pipette the supernatant into 40-60 μL (0.8x-1.2x) of purified magnetic beads, vortex to mix thoroughly, and let stand at room temperature for 5 min.

[0094] S63. Place the sample obtained in step S62 on a magnetic rack until the solution is clear, discard the supernatant, add 200 μL of 80% ethanol and wash twice, fully discard the ethanol, and then let it stand at room temperature to allow the ethanol to fully evaporate. Then add 32 μL of nuclease-free water, vortex and mix, let it stand at room temperature for 5 minutes, and then place it on a magnetic rack until the solution is clear. Pipette 30 μL of supernatant (eluted product) and transfer it to a new 1.5 mL centrifuge tube to obtain the screened library.

[0095] Comparative Example 1

[0096] Comparative Example 1 provides a library construction method, comprising the following steps:

[0097] (1) Extraction of free DNA from peripheral blood of pregnant women: The maternal peripheral blood sample was centrifuged using a two-step method to obtain plasma, and 600 μL was drawn for extraction of free DNA.

[0098] (2) Performing end repair on the free DNA to obtain end repair products; wherein the reaction system and reaction procedure of the end repair are shown in Tables 1 and 2.

[0099] (3) Performing connector ligation on the end-repair product to obtain a connector ligation product: wherein the reaction system of the connector ligation is shown in Table 6.

[0100] The adapter-ligated reaction system was incubated at 20°C for 15 min, and then 58 μL (0.8 times the volume) of purified magnetic beads was added to recover the ligated product. 24 μL of nuclease-free water was then added for elution to obtain 21 μL of eluted product, i.e., the adapter-ligated product. The adapter-ligated product was transferred to a new 200 μL PCR tube for the next experiment.

[0101] Table 6 Reaction system for linker ligation

[0102] Reagent name volume End repair products 51.5μL Ligation buffer 14 μL DNA ligase 3μL Short connector 4μL Overall system 72.5μL

[0103] (4) Performing PCR amplification on the adapter ligation product to obtain an amplified product; wherein the adapter ligation reaction system is shown in Table 4 and Table 7.

[0104] Table 7 PCR amplification reaction program

[0105]

[0106] (5) Purify and recover the amplified product to obtain the constructed library: add 50 μL (1.0 times the volume) of purified magnetic beads to the amplified product, vortex to mix it thoroughly, and let it stand at room temperature for 5 minutes; place it on a magnetic rack until the solution is clear, aspirate the supernatant, add 200 μL 80% ethanol to wash twice, and aspirate the ethanol thoroughly; let it stand at room temperature to allow the ethanol to evaporate completely; add 32 μL nuclease-free water, vortex to mix it thoroughly, and let it stand at room temperature for 5 minutes; place the centrifuge tube on a magnetic rack, aspirate 30 μL of the eluted product after the solution is clarified, and transfer it to a new 1.5 mL centrifuge tube. The obtained product is the constructed control group library.

[0107] Test Example 1

[0108] To examine the effect of end-repair product screening in step S3 on fetal concentration, libraries were constructed using the methods of Example 2 and Comparative Example 1, respectively, on two clinically collected maternal peripheral blood samples (sample numbers "S4201" and "S4301"). Referring to Table 8, each sample was divided into three aliquots; the first aliquot served as the control group, and a library was constructed using the method of Comparative Example 1 to obtain the control group library. The second and third aliquots were constructed using the method of Example 2 to obtain screened libraries, which were numbered +1 and +2, respectively, to distinguish them from the control group library.

[0109] The library quality test of the above samples was performed separately: 2 μL library and 198 μL Qubit reagent were mixed thoroughly, and the concentration and fragment size quality tests were performed. The results are shown in Table 8 and Figure 1-3 shown.

[0110] See Table 8 and Figure 1-5 Compared with the control libraries S4201 and S4301, libraries S4201-1, S4201-2, S4301-1, and S4301-2 performed fragment screening on the end-repair products, which increased the proportion of free DNA in the range of <240bp in the library fragment distribution map, and the fetal concentration increased by 1.16-1.65 times.

[0111] Test Example 2

[0112] The method of Example 1 was used to construct a library for the peripheral blood sample "S4301" of the pregnant woman in Test Example 1, and the screened library was obtained, and its number was added with "3". The sample was subjected to library quality inspection: 2 μL library and 198 μL Qubit reagent were mixed thoroughly, and the concentration and fragment size quality inspection were performed. The results are shown in Table 8 and Figure 6 shown.

[0113] See Figure 4-6 Compared with libraries S4301-1 and S4301-2, library S4301-3 performed fragment sorting on the amplified products (step S6), which resulted in a significant change in the fragment distribution of the library and a significant increase in the fetal concentration (2.02 times), which was much higher than that of the library without fragment sorting of the amplified products. That is, steps S3 and S6 may have a synergistic effect on improving the fetal concentration.

[0114] Table 8 Specific processing parameters and library quality inspection results of test example 1-2

[0115]

[0116] Test Example 3

[0117] In order to verify whether step S3 and step S6 have a synergistic effect, the methods of Example 1, Example 3 and Comparative Example 1 were used to construct libraries for peripheral blood samples collected from pregnant women (sample numbered "S4605"): Referring to Table 9, sample "S4605" was divided into three equal parts; the first sample was used as the control group, and the library was constructed using the method of Comparative Example 1 to obtain a control group library. The second sample was constructed using the method of Example 3 to obtain a screened library, which was numbered + "1". The third sample was constructed using the method of Example 1 to obtain a screened library, which was numbered + "2".

[0118] The library quality tests of the above samples were performed respectively, and the results are shown in Table 9.

[0119] Compared with S4605, S4605-1 only performed fragment sorting of the amplified products, and the fetal concentration of the obtained library (1.11 times) did not change significantly. Compared with S4605-1, S4605-2 performed fragment screening of the end-repair products (S3) and fragment sorting of the amplified products, which significantly increased the fetal concentration (2.32 times), and was significantly better than the effect of only performing fragment screening of the amplified products on the fetal concentration in Example 3. Therefore, step S3 for fragment screening of the end-repair products and step S62 for fragment sorting of the amplified products have a synergistic effect on improving the fetal concentration.

[0120] As shown in Test Examples 1-2, fragment screening of end-repair products helps increase fetal concentration, and the best effect is achieved when the purification magnetic bead volumes in steps S31 / S32 are 1.5x / 1.5x. Therefore, the purification magnetic bead volumes in steps S31 and S32 were selected as 90 μL (1.5x) and 90 μL (1.5x) for subsequent experiments.

[0121] Table 9 Specific processing parameters and library quality inspection results of Test Example 3

[0122]

[0123] Test Example 4

[0124] In order to explore the effect of the purification and recovery conditions in step S6 on the fetal concentration, the methods of Example 1 and Example 2 were used to construct libraries for three clinically collected pregnant women's peripheral blood samples (sample numbers were "S5801", "S5804", and "S5507" respectively):

[0125] Referring to Table 10, samples "S5801" and "S5804" were each divided into four equal parts. The first part was constructed according to the method of Example 2 to obtain a screened library, which was numbered +1. The second, third, and fourth parts were constructed according to the method of Example 1 to obtain screened libraries, which were numbered +2, +3, and +4, respectively.

[0126] Sample "S5507" was divided into three equal parts; the first part was prepared according to the method of Example 2 to construct library 6, resulting in a screened library numbered + "1". The second and third parts were prepared according to the method of Example 1 to obtain screened libraries, numbered + "2" and + "3", respectively.

[0127] The library quality tests of the above samples were performed respectively, and the results are shown in Table 10.

[0128] Table 10 Specific processing parameters and library quality inspection results of Test Example 4

[0129]

[0130] Compared with S5801-1 and S5804-1, fragment sorting of PCR products (step S62) helps to increase the fetal concentration. Among them, the effect is better when the volume of purification magnetic beads in steps S61 / S62 is 1.0 times / 1.0 times and 0.9 times / 1.0 times. Comparing S5801-2 to S5801-4 and S5804-2 to S5804-4, it can be seen that increasing the proportion of purification magnetic beads in steps S61 and S62 has no obvious effect on fetal concentration, and may even lead to a decrease in fetal concentration. Compared with S5801 and S5804, S5507 reduces the proportion of purification magnetic beads in S61 and S62, which has a poor effect on improving fetal concentration. Based on the above results, a fragment screening ratio of 0.9 times / 1.0 times is selected for steps S61 / S62 for subsequent experiments.

[0131] Test Example 5

[0132] In order to explore the effect of the proportion of magnetic beads recovered from the linker ligation product on the fetal concentration, the methods of Example 1 and Example 2 were used to construct libraries for two clinically collected pregnant women's peripheral blood samples (sample numbers "S5601" and "S5401" respectively):

[0133] Referring to Table 11, the sample numbered "S5601" was divided into four equal parts. The first part was constructed according to the method of Example 2 to obtain a screened library, which was numbered +1. The second, third, and fourth parts were constructed according to the method of Example 1 to obtain screened libraries, which were numbered +2, +3, and +4, respectively.

[0134] The sample numbered "S5401" was divided into three equal parts; the first part was prepared according to the method of Example 2 to obtain a screened library, which was numbered + "1". The second and third parts were prepared according to the method of Example 1 to obtain screened libraries, which were numbered + "2" and + "3", respectively.

[0135] The library quality tests of the above samples were performed respectively, and the results are shown in Table 11.

[0136] Table 11 Specific processing parameters and library quality inspection results of test case 5

[0137]

[0138] Compared with samples S5601-1 and S5401-1, fragment sorting of the amplified products significantly increased the fetal concentration (1.98-2.32 times). Compared with sample S5601-2, reducing the proportion of magnetic beads used to recover the ligation products (samples S5601-1, S5601-3, and S5601-4) significantly decreased the fetal concentration. However, compared with S5401-2, increasing the proportion of magnetic beads used to recover the ligation products to 1.5 times (sample S5401-3) did not significantly increase the library concentration or fetal concentration. Taking all factors into consideration, 1.2 times the number of magnetic beads was selected for the recovery of the ligation products.

[0139] Test Example 6

[0140] The results of test examples 1-4 showed that magnetic bead screening of the end repair products resulted in a low overall concentration of the library (2.5-16 ng / μL), and the low overall concentration of the library would also affect the detection efficiency of NIPT. Based on this, the method of Example 1 was used to construct a library of 4 clinically collected pregnant women's peripheral blood samples (sample numbers were "S6092", "S6402", "S6905", and "S6906") to explore the effect of PCR amplification on the library concentration:

[0141] Referring to Table 12, in this Test Example 6, the sample was divided into two equal parts; both samples were constructed according to the method of Example 1, except that the number of PCR amplification cycles for the first sample was 11, and the number of PCR amplification cycles for the second sample was 16-19. The library number obtained from the second sample was added with "1".

[0142] The library quality tests of the above samples were performed respectively, and the results are shown in Table 11.

[0143] Table 12 Specific processing parameters and library quality inspection results of each sample in Test Example 6

[0144]

[0145] After increasing the number of cycles, the concentration of the library ranged from 10 to 14 ng / μL, and the proportion of the library with a concentration range of 2.5 to 10 ng / μL decreased. The concentration of the library at cycle 20 was not significantly different from that at cycle 16.

[0146] Example 4

[0147] Based on Test Examples 1-6, this Example 4 provides a method for increasing the concentration of fetal free DNA in maternal peripheral blood, comprising the following steps:

[0148] S1. Extraction of free DNA from the peripheral blood of pregnant women: Use a two-step method to centrifuge the maternal peripheral blood sample to obtain plasma, and aspirate 600 μL of plasma for extraction of free DNA; among them, use Meiji Bio's magnetic bead-based free DNA extraction kit for extraction of free DNA.

[0149] S2. Perform end repair on the free DNA obtained in step S1 to obtain end repair products; wherein the reaction system and reaction procedure of the end repair are shown in Tables 1 and 2.

[0150] S3, performing fragment screening on the end-repair products obtained in step S2 to obtain enriched fetal free DNA, specifically including steps S31-S36:

[0151] S31. Add nuclease-free water to the volume of the end-repair product from step S2 to 60 μL, then add 90 μL (1.5 times the volume) of purified magnetic beads, vortex to mix thoroughly, and let stand at room temperature for 5 minutes;

[0152] S32. Place the sample obtained in step S31 on a magnetic rack until the solution is clear, pipette the supernatant into 90 μL (1.5 times the volume) of purified magnetic beads, vortex to mix thoroughly, and let stand at room temperature for 5 min;

[0153] S33. Place the sample obtained in step S32 on a magnetic rack until the solution is clear. After aspirating the supernatant, add 200 μL of 80% ethanol and wash twice. Fully aspirate the ethanol, then let it stand at room temperature to allow the ethanol to fully evaporate. Then add 32 μL of nuclease-free water, vortex and mix. After standing at room temperature for 5 minutes, place it on a magnetic rack until the solution is clear. Aspirate 30 μL of supernatant (end repair sorting product), that is, the enriched fetal free DNA, and transfer it to a new 200 μL PCR tube for use.

[0154] S4. Adapter ligation was performed on the enriched cell-free fetal DNA to obtain an adapter-ligated product. The adapter-ligated reaction system is shown in Table 3. This reaction system was incubated at 20°C for 15 minutes. The ligated product was then recovered using 87 μL (1.2 volumes) of purified magnetic beads. Elution was then performed with 24 μL of nuclease-free water to obtain 21 μL of eluted product, i.e., the adapter-ligated product. This eluted product was transferred to a new 200 μL PCR tube for the next step.

[0155] S5. Perform PCR amplification on the linker ligation product to obtain an amplified product of fetal free DNA; wherein the reaction system and reaction procedure of PCR amplification are shown in Table 4 and Table 13.

[0156] Table 13 PCR amplification reaction program

[0157]

[0158] S6. Sorting the amplified products of fetal cell-free DNA to obtain a constructed fetal cell-free DNA library, specifically including steps S61-S63:

[0159] S61. Add 45 μL (0.9 times the volume) of purified magnetic beads to the amplified product obtained in step S5, vortex and oscillate to mix thoroughly, and let stand at room temperature for 5 minutes;

[0160] S62. Perform fragment sorting on the sample obtained in step S61: Place the sample obtained in step S61 on a magnetic rack until the solution is clear, pipette the supernatant into 50 μL (1.0 times the volume) of purified magnetic beads, vortex to mix thoroughly, and let stand at room temperature for 5 min;

[0161] S63. Place the sample obtained in step S62 on a magnetic rack until the solution is clear, discard the supernatant, add 200 μL of 80% ethanol and wash twice, fully discard the ethanol, and then let it stand at room temperature to allow the ethanol to fully evaporate. Then add 32 μL of nuclease-free water, vortex and mix, let it stand at room temperature for 5 minutes, and then place it on a magnetic rack until the solution is clear. Pipette 30 μL of supernatant (eluted product) and transfer it to a new 1.5 mL centrifuge tube to obtain the screened library.

[0162] Among the above embodiments, embodiment 4 is the best embodiment.

[0163] Verification Example 1

[0164] The methods of Example 4 and Comparative Example 1 were respectively used to build a library for 5 interstitial samples (sample numbers were "202411", "202412", "202413", "202414", and "202415". The interstitial samples were provided by the National Health Commission Clinical Center for the 2024 National Peripheral Blood Fetal Chromosome Aneuploidy (T21, T18, and T13) High-Throughput Sequencing Detection Inter-Laboratory Quality Evaluation (Proficiency Verification) Activities): The 5 interstitial samples were plasma simulation samples in a lyophilized powder state. 1200 μL of nuclease-free water was added to each sample, allowed to stand at room temperature for 5-10 minutes, and inverted to mix until the dry powder was completely dissolved. Each dissolved sample was divided into two portions, 600 μL each. The first portion was built using the method of Comparative Example 1 to obtain a control group library; the second portion was built using the method of Example 4 to obtain a screened library and numbered it + "1" to distinguish it from the control group library.

[0165] The library constructed for each sample was quality checked, and the results are shown in Table 14. The library concentration of the screened library was between 8.5-11 ng / μL, which is within a reasonable range. Sequencing was then completed using a BGI high-throughput sequencer. The sequencing data was analyzed and aligned with the human genome reference sequence (GRch37 / hg19). The proportion of fetal free DNA in the mother was calculated, and the Z-score value of each chromosome was calculated. A fetal free DNA concentration (FF) of <4% was considered a test failure. When FF>4%, a Z-score value of chr13, chr18, and chr21>3 was considered positive.

[0166] Table 14 Library quality inspection results and sequencing results of Verification Example 1

[0167]

[0168] Referring to Table 14, compared to Comparative Example 1, the concentration of fetal free DNA after enrichment using the method of Example 4 was significantly increased by 3-5 times. As the concentration of enriched fetal free DNA increased, the Z-score value of sample 202412chr18 increased from the critical value of 3.7841 to 27.9715, indicating the presence of chr18 trisomy. Simultaneously, the Z-score values ​​of sample 202414chr21 and sample 202415chr13 increased to 24.92 and 28.0536, respectively, representing 4.15-fold and 5.56-fold increases, indicating the presence of chr21 trisomy and chr13 trisomy, respectively, making the results more accurate and reliable.

[0169] Verification Example 2

[0170] The peripheral blood libraries of 8 pregnant women aged 10-20 weeks (all of whom were confirmed to have normal fetuses by clinical follow-up) were constructed using the methods of Example 4 and Comparative Example 1, respectively: each sample was divided into two parts, the first part was constructed using the method of Comparative Example 1 to obtain a control group library; the second part was constructed using the method of Example 4 to obtain a screened library and numbered "1".

[0171] The library constructed for each sample was quality checked, and the library concentration of the screened library was between 5-11.5 ng / μL, slightly lower than the inter-laboratory quality control, but still within a reasonable range. Sequencing was then completed using a Zhenmai high-throughput sequencer. The sequencing data was analyzed and aligned with the human genome reference sequence (GRch37 / hg19). The proportion of fetal free DNA in the mother was calculated, and the Z-score value of each chromosome was calculated. A fetal free DNA concentration (FF) of <4% was considered a test failure. When FF>4%, a Z-score value of chr13, chr18, and chr21>3 was considered positive.

[0172] Referring to Table 15, compared to Comparative Example 1, the enriched fetal free DNA concentration in Example 4 increased by 1.5-5 times, and the fetal concentration in five samples increased from the original critical value (3%-4%) to at least 7%. The chr21 Z-score value of the control library for sample S0905 was close to 3, indicating a possible false positive. However, the chr21 Z-score value of the library constructed using the method of Example 4 was significantly lower, demonstrating that the method of the present invention can improve the detection accuracy of NIPT.

[0173] Table 15 Library quality inspection results and sequencing results of Verification Example 2

[0174]

[0175] Verification Example 3

[0176] To verify the enrichment effect of the method of the present invention on fetal free DNA in samples with low fetal concentration, peripheral blood libraries were constructed from 23 pregnant women aged 7-9 weeks (all fetuses were confirmed to be normal by clinical follow-up) using the methods of Example 4 and Comparative Example 1, respectively. Refer to Table 6. Each sample was divided into two parts. The first part was constructed using the method of Comparative Example 1 to obtain a control group library; the second part was constructed using the method of Example 4 to obtain a screened library and number it + "1". The library constructed for each sample was quality-checked. The library concentration of the screened library was between 2.5-11.5 ng / μL, which was lower than the inter-laboratory quality sample but still within a reasonable range. Sequencing was then completed using a Zhenmai high-throughput sequencer.

[0177] Compared with the peripheral blood samples of pregnant women aged 10-20 weeks in Validation Example 2, the concentration of fetal free DNA in the peripheral blood of pregnant women aged 7-9 weeks was generally low, among which the fetal free DNA concentration of about 44% of the control group libraries was below the critical value (4%). Compared with the control group library, only 3 samples (S1104, S1118, S1121) of the library constructed using Example 4 had no significant change in fetal free DNA concentration (1.3-1.33 times), which may be caused by the low gestational age. The fetal free DNA concentration of the remaining 20 samples increased to 1.5-2.5 times the original value. Moreover, most of the samples with fetal free DNA concentration at or below the critical value were enriched by the method of Example 4, and the fetal concentration was significantly improved, thereby reducing the detection failure rate of NIPT.

[0178] Table 16 Library quality inspection results and sequencing results of Verification Example 3

[0179]

[0180] In addition, in the control group library constructed using the method of Comparative Example 1, the Z-score values ​​of chr21 of sample S1901, sample S1903, and sample S1112, and the Z-score value of chr13 of sample S1112 showed false positives. In the library constructed using Example 4, the Z-score value of chr21 in sample S1903 decreased from 3.5083 to 1.6437, excluding the presence of chr21 trisomy; the Z-score values ​​of chr13 and chr21 in sample S1112 decreased to 0.2907 and 0.0727, respectively, excluding chr13 trisomy and chr21 trisomy, which is consistent with the clinical follow-up of the sample.

[0181] Therefore, constructing a library using the method of Example 4 of the present invention before NIPT testing can reduce the false positive rate of NIPT testing and improve the accuracy of NIPT testing.

[0182] Verification Example 4

[0183] In order to verify the detection effect of the library constructed by the present invention on different sequencing platforms, the peripheral blood of 12 pregnant women aged 7-25 weeks (clinical follow-up confirmed that the fetuses were all normal) was constructed using the methods of Example 4 and Comparative Example 1 respectively: each sample was divided into two parts, and the first part of each part was constructed using the method of Comparative Example 1 to obtain a control group library; the second part was constructed using the method of Example 4 to obtain a screened library and numbered + "1" to distinguish it from the control group library.

[0184] The library constructed for each sample was quality checked, and the library concentration of the screened library was between 4.5-16 ng / μL, slightly lower than the inter-laboratory quality product, but still within a reasonable range. The library was then sequenced using a BGI high-throughput sequencer.

[0185] Table 17 Library quality inspection results and sequencing results of Verification Example 4

[0186]

[0187] Referring to Table 17, after constructing libraries on different sequencing platforms using the same method (methods of Example 4 and Comparative Example 1), it was found that the concentration of fetal free DNA increased to 1.5-2.5 times the original value, indicating that the method provided by the present invention has good platform compatibility.

[0188] In addition, the same results were achieved in peripheral blood samples of pregnant women aged 7-25 weeks, indicating that the method provided by the present invention is suitable for low fetal concentration samples with a smaller gestational age, and can detect adverse pregnancies in pregnant women earlier, providing a basis for scientific decision-making for pregnant women.

[0189] Verification Example 5

[0190] In order to verify the chromosome microdeletion / microduplication abnormality detection effect of the library constructed by the present invention, the method of Example 4 was used to build a library for 3 reference samples from Jingliang (sample numbers were "GWS012", "GWS016", and "GWS023", wherein "GWS012" and "GWS016" were two microdeletion samples with deletion lengths of 29.4M and 5.65M, respectively; "GWS023" samples were 13 trisomy, 18 trisomy, and 21 trisomy) to obtain the screened library. The library constructed for each reference product was quality inspected and then sequenced using a Zhenmai high-throughput sequencer. The fetal concentration and Z-score results of each reference product were analyzed, and CNV analysis was performed on each reference product to obtain CNV results.

[0191] Table 18 Library quality inspection results and sequencing results of Verification Example 5

[0192]

[0193] Table 19 CNV results of verification example 5

[0194]

[0195] Referring to Table 18, the Z-score values ​​of chr13, chr18, and chr21 of sample GWS023 were all greater than 3.0, indicating the presence of chr13 trisomy, chr18 trisomy, and chr21 trisomy, which are consistent with the results of the reference product.

[0196] Referring to Table 19, the CNV type of samples GWS012 and GWS016 is Del, and the test results are consistent with the chromosomal microdeletion / microduplication abnormality status of the reference, and the length of the deletion is consistent with the reference.

[0197] Therefore, the library obtained by the method of the present invention can be used to detect copy number variation (chromosomal microdeletion / microduplication syndrome) with high accuracy.

[0198] In summary, the method provided by the present invention for increasing the concentration of fetal free DNA in maternal blood has the following advantages:

[0199] (1) The present invention enriches fragments <143 bp in cfDNA by adding two steps of magnetic bead screening (i.e., fragment screening of end-repair products and fragment screening of amplification products), thereby increasing the fetal concentration to 1.5-5 times that of the unenriched one.

[0200] (2) NIPT testing was performed on the library constructed by the present invention. It was found that the present invention can solve the problem of NIPT test failure caused by low cffDNA. In particular, for pregnant women with a shorter gestational age (7-9 weeks), the concentration of fetal free DNA in samples originally at the critical value (4%) can be significantly increased, thereby reducing the failure rate of NIPT testing. Therefore, the method of the present invention is conducive to the application of early NIPT and helps to detect adverse pregnancies earlier.

[0201] (3) The library constructed by the method provided by the present invention can reduce the false positive rate and false negative rate of NIPT and improve the accuracy of chromosomal aneuploidy detection, such as Down syndrome, Edwards syndrome and Patau syndrome. In addition, the method of the present invention can also improve the accuracy of copy number variation detection (such as chromosomal microdeletion / microduplication syndrome). Therefore, the method of the present invention combined with NIPT can significantly reduce the risk of adverse pregnancy outcomes and provide a scientific basis for decision-making for pregnant women.

[0202] (4) The method provided by the present invention is suitable for increasing the concentration of fetal free DNA in the plasma of pregnant women aged 7-25 weeks and has wide applicability.

[0203] (5) The library construction method provided by the present invention can significantly increase the concentration of fetal free DNA in samples on different sequencing platforms, showing good platform compatibility.

[0204] (6) Compared with the prior art methods that use paired-end sequencing and bioinformatics analysis of short fragments, the library constructed by the method of the present invention has lower sequencing costs and shorter analysis time for NIPT testing. Compared with the prior art methods that use gel excision to recover DNA fragments, the present invention can accurately enrich fetal cfDNA of the desired length, avoid large amounts of adapter dimer contamination, and improve the reproducibility of the method.

[0205] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A method for increasing the concentration of fetal free DNA in maternal peripheral blood, characterized in that: Including steps: S1. Extract free DNA from peripheral blood of pregnant women; S2, performing end repair on the free DNA to obtain end repair products; S3. Screen the end-repair products for fragments to obtain enriched fetal free DNA; S4, performing adapter ligation on the enriched fetal free DNA to obtain an adapter ligation product; S5, performing PCR amplification on the adapter ligation product to obtain an amplified product of fetal cell-free DNA; S6. Fragment sorting is performed on the amplified products of fetal free DNA to obtain a constructed fetal free DNA library.

2. The method according to claim 1, characterized in that Step S3 includes: S31, adding 1.0-2.0 times the volume of purified magnetic beads to the end repair product of step S2, mixing thoroughly and letting it stand at room temperature until it is clear; S32, aspirating the supernatant into 1.0-2.0 times the volume of purified magnetic beads, mixing thoroughly and letting it stand at room temperature until it is clear; S33, aspirating and discarding the supernatant, washing with 80% ethanol, and then eluting with enzyme-free nucleic acid water to obtain enriched fetal free DNA.

3. The method according to claim 2, characterized in that Step S6 includes: S61, adding 0.8-1.2 times the volume of purified magnetic beads to the amplified product of step S5, mixing thoroughly and letting it stand at room temperature until it is clear; S62, aspirating the supernatant into 0.8-1.2 times the volume of purified magnetic beads, mixing thoroughly and letting it stand at room temperature until it is clear; S63, aspirating and discarding the supernatant, washing with 80% ethanol, and then eluting with enzyme-free nucleic acid water to obtain the screened library.

4. The method according to claim 3, characterized in that Step S4 includes: using 0.8-1.5 times the volume of purification magnetic beads to purify and recover the connector ligation product.

5. The method according to claim 4, characterized in that In step S5, the reaction procedure of the PCR amplification is: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 65°C for 30s, extension at 72°C for 30s, 16-19 cycles; incubation at 72°C for 5min, and storage at 4°C.

6. The method according to claim 4, characterized in that In step S2, the end repair reaction system includes free DNA, end repair buffer and end repair enzyme; the end repair reaction procedure is 12°C for 15 minutes, 37°C for 15 minutes, 72°C for 20 minutes, and finally stored at 4°C.

7. The method according to claim 4, characterized in that The volumes of the purification magnetic beads in step S31 and step S32 are 1.2-1.8 times and 1.2-1.8 times, respectively.

8. The method according to claim 4, characterized in that The volumes of the purified magnetic beads in step S61 and step S62 are 0.8-1.0 times and 1.0-1.2 times, respectively.

9. The method according to claim 4, characterized in that The volume of the purification magnetic beads in step S4 is 1.0-1.5 times.

10. The method according to any one of claims 1 to 9, characterized in that: In step S1, a maternal peripheral blood sample is centrifuged in two steps to obtain plasma, and free DNA in the plasma is extracted using a magnetic bead method.

Images