Quality control product for noninvasive prenatal detection of fetal chromosomal trisomy syndrome and preparation method thereof

Abstract

The invention discloses a quality control product for noninvasive prenatal detection of fetal chromosome trisomy syndrome and a preparation method thereof, the quality control product comprises a positive quality control product and a negative quality control product, the positive quality control product is obtained by adding nucleic acid fragments of trisomy syndrome genome DNA into background plasma; the negative quality control product is obtained by adding nucleic acid fragments of normal karyotype genome DNA into background plasma; the length of the nucleic acid fragment is 130 to 200 bp; the background plasma is peripheral blood plasma of normal karyotype and pregnant fetus women. According to the invention, peripheral blood plasma of a pregnant woman with normal karyotype and pregnant fetus is used as background plasma, a DNA extraction correction coefficient is introduced, and nucleic acid fragments of trisomatic negative and positive genomes 130-200bp with sex chromosome XY are added to prepare the quality control product, so that the quality control product is more accurate and reliable, and is more suitable for pregnant woman plasma; and whether each index of the noninvasive antenatal detection kit meets the technical requirements of a product or not can be reflected more completely and truly.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology. More specifically, the present invention relates to a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome and a preparation method thereof. Background Art

[0002] With the continuous development of Next Generation Sequencing (NGS) technology, non-invasive prenatal testing (NIPT) has gradually been widely used in clinical testing. Non-invasive prenatal testing technology is mainly used to detect whether the fetus has chromosomal abnormalities, among which trisomy 21, 18, and 13 are more common. The incidence of trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome) in live births is 1 / 700, 1 / 6000, and 1 / 10000, respectively. There is currently no specific drug or treatment for affected individuals. The widespread use of non-invasive prenatal testing technology and its kits will help reduce the incidence of birth defects, while also avoiding irreversible effects on the fetus caused by invasive testing methods.

[0003] There are many types of kits currently used in the market for non-invasive prenatal testing, which usually cover supporting reagents such as nucleic acid extraction, library construction, and high-throughput sequencing. The quality control products included in the kits or sold independently on the market are generally composed of DNA nucleic acid, artificial plasma, normal non-pregnant female plasma, etc. The invention patent CN104531842A "A positive quality control product for non-invasive prenatal detection of fetal trisomy 21, 18 and 13 and its preparation method" describes the preparation method of the positive quality control product, which has the following defects:

[0004] 1. The positive nucleic acid fragments are 160 to 200 bp, which is different from the approximately 140 bp cell-free fetal DNA (cffDNA) fragment size found by Lo YM et al., and cannot highly simulate the peripheral blood plasma of real pregnant women;

[0005] 2. The quality control products only contain the preparation method of positive quality control products, but lack the preparation method of negative quality control products, and the quality control reagents are incomplete;

[0006] 3. Positive quality control products are stored in the form of DNA nucleic acid, and in actual clinical testing, it is impossible to monitor the nucleic acid extraction process in the non-invasive prenatal testing process.

[0007] 4. The preparation method only proves the positive-negative consistency rate, lacks an effective method to evaluate the fetal free DNA fraction (fetal fraction, FF), and there is no sufficient evidence to show the proportion and content of positive or negative nucleic acids.

[0008] Therefore, it is very meaningful to provide a more comprehensive and accurate quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome. Summary of the Invention

[0009] Based on this, the purpose of the present invention is to provide a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome. Using this quality control product, fetal chromosomal trisomy syndrome can be detected more comprehensively and accurately.

[0010] The technical solutions for achieving the above-mentioned invention objectives include the following.

[0011] The first aspect of the present invention provides a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome, including a positive quality control product and a negative quality control product. The positive quality control product is obtained by adding nucleic acid fragments of genomic DNA of chromosomes 21, 18 and 13 trisomy syndrome to background plasma; the negative quality control product is obtained by adding nucleic acid fragments of genomic DNA with normal karyotype to background plasma; the nucleic acid fragments are 130 to 200 bp; and the background plasma is peripheral blood plasma of a woman with a normal karyotype and pregnant with a female fetus.

[0012] A second aspect of the present invention provides a method for preparing a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome, comprising the following steps:

[0013] (1) The genomic DNA of chromosome 21, 18, and 13 trisomy syndrome and the normal karyotype genomic DNA were digested with enzymes and screened with magnetic beads to recover nucleic acid fragments of 130 to 200 bp;

[0014] (2) DNA was extracted from peripheral blood plasma of pregnant women with normal karyotypes and female fetuses, and the concentration of nucleic acid fragments ranging from 140 bp to 320 bp was read. The total amount of nucleic acid fragments ranging from 140 bp to 320 bp in each milliliter of peripheral blood plasma was calculated using a DNA extraction correction factor of 0.6 to 0.8;

[0015] (3) According to the final concentration of the positive control product and the formula: Calculate the input amount of 130-200 bp nucleic acid fragments of chromosome 21, 18, and 13 trisomy syndrome in each milliliter of peripheral blood plasma; based on the final concentration of the negative quality control and the formula: Calculate the input amount of 130-200 bp nucleic acid fragments of normal karyotype genome in each milliliter of peripheral blood plasma;

[0016] (4) Take peripheral blood plasma, add 130-200 bp nucleic acid fragments of chromosome trisomy syndrome 21, 18, and 13 respectively according to the amount of input in step (3), and mix thoroughly to obtain a positive quality control product; take peripheral blood plasma, add 130-200 bp nucleic acid fragments of normal karyotype genome according to the amount of input in step (3), and mix thoroughly to obtain a negative quality control product.

[0017] The quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome of the present invention is obtained by adding fragments of trisomy negative and positive genomic DNA (chromosomes 21, 18, and 13 trisomy syndrome) with a main peak length of 130bp to 200bp to the peripheral blood plasma of women with normal karyotype and pregnant with female fetuses; the fragment size of 130bp to 200bp is consistent with the size of real cffDNA fragments, so that the quality control product is more in line with the peripheral blood plasma of pregnant women with normal karyotype and pregnant with female fetuses, and highly simulates the real state of cffDNA. The properties, state, and composition of the peripheral blood plasma of pregnant women with normal karyotype and pregnant with female fetuses are highly consistent with those of the peripheral blood plasma of the population to be tested non-invasively before birth. Therefore, the quality control product of the present invention can more truly reflect the detection performance of various non-invasive prenatal detection kits on the market, and can perform quality control on the entire process of nucleic acid extraction, library construction, high-throughput sequencing, bioinformatics statistics and analysis. The simultaneous use of negative and positive quality control products can more completely and truly reflect whether the various indicators of the non-invasive prenatal detection kit meet the product technical requirements.

[0018] Before the quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome of the present invention is prepared, an extraction correction coefficient is introduced into the calculation formula for calculating the total amount of nucleic acid contained in each milliliter of plasma in the extraction process of background plasma, which can effectively reduce the DNA loss of the target background plasma during the extraction process and increase the accuracy of the quality control product; in addition, during preparation, the negative and positive quality control products are calculated and prepared by the ratio of the content within the negative and positive DNA nucleic acid fragments of the trisomy with sex chromosomes XY to the content within the DNA nucleic acid fragment range in the background plasma. Therefore, in addition to evaluating the positive and negative conformity rate of the quality control product, FF can also be evaluated by the chrY% proportion, and whether the concentration of the prepared quality control product reaches the expected effect can be evaluated, thereby maximizing the accuracy of the prepared quality control product, increasing the credibility of the detection, and making the quality control product more accurate, reliable, and more consistent with the plasma of pregnant women. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram of the enzyme-cut nucleic acid fragments of the genomic DNA NG09394 (47 / XY / +21) of chromosome 21 trisomy syndrome in Example 1 of the present invention.

[0020] Figure 2This is a diagram of the enzyme-cut nucleic acid fragments of the genomic DNA NA03769 (47 / XY / +18) of chromosome 18 trisomy syndrome in Example 1 of the present invention.

[0021] Figure 3 This is a diagram of the enzyme-cut nucleic acid fragments of the genomic DNA NA02948 (47 / XY / +13) of chromosome 13 trisomy syndrome in Example 1 of the present invention.

[0022] Figure 4 This is a diagram of the enzyme-cut nucleic acid fragments of the normal karyotype genomic DNA NA24385 (46 / XY) in Example 1 of the present invention.

[0023] Figure 5 This is a diagram of free DNA fragments in background plasma in Example 1 of the present invention.

[0024] Figure 6 This is a diagram showing the sequencing results of the negative and positive quality controls in Example 2 of the present invention. DETAILED DESCRIPTION

[0025] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual (2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0028] In some embodiments of the present invention, a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome is disclosed, including a positive quality control product and a negative quality control product. The positive quality control product is obtained by adding nucleic acid fragments of genomic DNA of chromosome 21, 18, and 13 trisomy syndrome to background plasma; the negative quality control product is obtained by adding nucleic acid fragments of genomic DNA with normal karyotype to background plasma; the nucleic acid fragments are 130 to 200 bp; and the background plasma is peripheral blood plasma of a woman with a normal karyotype and pregnant with a female fetus.

[0029] In existing relevant literature, plasma from non-pregnant women with normal karyotype is usually used as a solvent (background plasma). When preparing positive quality control products and negative quality control products in the present invention, peripheral blood plasma from pregnant women with normal karyotype and female fetuses is used as background plasma. The peripheral blood plasma from pregnant women with normal karyotype and female fetuses (i.e., plasma from pregnant women who have completed non-invasive prenatal testing and whose fetuses are female) is highly consistent with the properties, state, and composition of the peripheral blood plasma of the population to be tested non-invasively (pregnant women to be tested), and can more truly reflect the detection performance of various non-invasive prenatal testing kits on the market, and can perform quality control on the entire process of nucleic acid extraction, library construction, high-throughput sequencing, bioinformatics statistics and analysis.

[0030] The nucleic acid fragments of chromosome 21, 18, and 13 trisomy syndrome genomic DNA are selected with a size of 130bp to 200bp. The fragment size is consistent with the real cffDNA fragment size, making the quality control product more in line with the peripheral blood plasma of pregnant women and highly simulating the real state of cffDNA.

[0031] In addition, in the present invention, negative quality control products are added. The simultaneous use of negative and positive quality control products can more completely and truly reflect whether the various indicators of the non-invasive prenatal detection kit meet the product technical requirements.

[0032] In some embodiments, the nucleic acid fragment is 140 to 170 bp.

[0033] In some embodiments, the trisomy 21, 18, and 13 genomic DNAs are NG09394, NA03769, and NA02948, respectively, and the normal karyotype genomic DNA is NA24385.

[0034] In other embodiments of the present invention, a method for preparing a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome is disclosed, comprising the following steps:

[0035] (1) The genomic DNA of chromosome 21, 18, and 13 trisomy syndrome and the normal karyotype genomic DNA were digested with enzymes and screened with magnetic beads to recover nucleic acid fragments of 130 to 200 bp;

[0036] (2) DNA was extracted from peripheral blood plasma of pregnant women with normal karyotypes and female fetuses, and the concentration of nucleic acid fragments ranging from 140 bp to 320 bp was read. The total amount of nucleic acid fragments ranging from 140 bp to 320 bp in each milliliter of peripheral blood plasma was calculated using a DNA extraction correction factor of 0.6 to 0.8;

[0037] (3) According to the final concentration of the positive control product and the formula: Calculate the input amount of 130-200 bp nucleic acid fragments of chromosome 21, 18, and 13 trisomy syndrome in each milliliter of peripheral blood plasma; based on the final concentration of the negative quality control and the formula: Calculate the input amount of 130-200 bp nucleic acid fragments of normal karyotype genome in each milliliter of peripheral blood plasma;

[0038] (4) Take peripheral blood plasma, add 130-200 bp nucleic acid fragments of chromosome trisomy syndrome 21, 18, and 13 respectively according to the amount of input in step (3), and mix thoroughly to obtain a positive quality control product; take peripheral blood plasma, add 130-200 bp nucleic acid fragments of normal karyotype genome according to the amount of input in step (3), and mix thoroughly to obtain a negative quality control product.

[0039] In the present invention, the negative and positive quality control products are calculated and prepared by calculating the ratio of the content of the DNA nucleic acid fragments within the range of the negative (46 / XY) and positive (47 / XY / +21, 47 / XY / +18, 47 / XY / +13) trisomy with XY as the sex chromosome to the content of the DNA nucleic acid fragments within the range of the background plasma. In addition to evaluating the positive and negative conformity rate of the quality control products, the ch FF is evaluated by the percentage of Y chromosome (the quality control product is obtained by mixing negative or positive nucleic acid with the plasma of pregnant women and fetuses. The percentage of Y chromosome can be used to calculate the percentage of added nucleic acid in plasma, and the percentage of nucleic acid in plasma can be considered as the percentage of fetal DNA distributed in maternal plasma. The percentage of Y chromosome can be used to infer FF). This is to evaluate whether the concentration of the prepared quality control product has achieved the expected effect, to maximize the accuracy of the prepared quality control product, to increase the credibility of the test, and to make the quality control product more accurate, reliable, and more consistent with the plasma of pregnant women.

[0040] The present invention introduces an extraction correction factor into the formula for calculating the total nucleic acid content per milliliter of plasma during the background plasma extraction process before quality control product preparation. The correction factor can be set to any value between 0.6 and 0.8, with a preferred value of 0.65. This approach effectively reduces DNA loss during the extraction process of the target background plasma and increases the accuracy of the quality control product.

[0041] In some embodiments, the final concentration of the positive control in step (3) is 4-6%, and the final concentration of the negative control is 8-12%.

[0042] In some embodiments, a nucleic acid fragment of 140 to 170 bp is recovered in step (1).

[0043] In some embodiments, the method for calculating the total amount of nucleic acid fragments of 140 bp to 320 bp per milliliter of peripheral blood plasma in step (2) includes: using the concentration of the nucleic acid fragments of 140 bp to 320 bp to estimate the amount of nucleic acid fragments of 140 bp to 320 bp per milliliter of peripheral blood plasma, and then dividing it by the DNA extraction correction factor to obtain.

[0044] In some embodiments, the DNA extraction correction factor in step (2) is 0.6 to 0.7, more preferably 0.65.

[0045] In some embodiments, the peripheral blood plasma in step (2) is mixed peripheral blood plasma from 3 to 4 pregnant women with normal karyotypes.

[0046] In some embodiments, the genomic DNA of trisomy 21, 18, and 13 in step (1) are NG09394, NA03769, and NA02948, respectively, and the normal karyotype genomic DNA is NA24385.

[0047] In some embodiments, a method for preparing a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome is disclosed, comprising the following steps:

[0048] (1) The trisomy genomic DNA NG09394, NA03769, NA02948 and the normal karyotype genomic DNA NA24385 were digested with enzymes and screened with magnetic beads to recover nucleic acid fragments of 140 to 170 bp;

[0049] (2) DNA was extracted from peripheral blood plasma of pregnant women with normal karyotypes and female fetuses, and the concentration of nucleic acid fragments ranging from 140 bp to 320 bp was read. The total amount of nucleic acid fragments ranging from 140 bp to 320 bp in each milliliter of peripheral blood plasma was calculated using a DNA extraction correction factor of 0.6 to 0.7;

[0050] (3) According to the calculation formula: Calculate the input amount of 130-200 bp nucleic acid fragment of trisomy syndrome genomic DNA per milliliter of peripheral blood plasma; according to the calculation formula: Calculate the input amount of 130-200 bp nucleic acid fragment of normal karyotype genomic DNA per ml of peripheral blood plasma;

[0051] (4) Take peripheral blood plasma, add 130-200 bp nucleic acid fragments of trisomy syndrome genomic DNA according to the amount of input in step (3), mix thoroughly, and obtain a positive quality control product; take peripheral blood plasma, add 130-200 bp nucleic acid fragments of normal karyotype genomic DNA according to the amount of input in step (3), mix thoroughly, and obtain a negative quality control product.

[0052] In the present invention, genomic DNA of trisomy 21, 18, and 13 and genomic DNA with normal karyotype can be obtained from commercial human genomic DNA, such as NG09394 (karyotype 47 / XY / +21), NA03769 (karyotype 47 / XY / +18), NA02948 (karyotype 47 / XY / +13), and NA24385 (karyotype 46 / XY) from the Coriell Institute; or from genomic DNA extracted from aborted tissue, or from human peripheral blood with corresponding karyotype characteristics. Background plasma is plasma from a pregnant woman with a normal karyotype and a female fetus, which can be commercial plasma from a pregnant woman with a female fetus, or from donations, or from scientific research projects of cooperative institutions, or plasma from multiple donors can be mixed.

[0053] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0054] Example 1 Preparation Method of Quality Control Material for Non-invasive Prenatal Detection of Fetal Chromosomal Trisomy

[0055] The following steps are involved:

[0056] 1. Enzyme fragmentation of genomic DNA of chromosomal trisomy syndrome and normal karyotype genomic DNA

[0057] In this embodiment, the genomic DNA of chromosome 21, 18, and 13 trisomy syndromes uses NG09394 (karyotype 47 / XY / +21), NA03769 (karyotype 47 / XY / +18), and NA02948 (karyotype 47 / XY / +13), and the normal karyotype genomic DNA uses NA24385 (karyotype 46 / XY). The genomic DNA of chromosome trisomy syndrome and the normal karyotype genomic DNA are enzymatically fragmented using the VAHTS Universal Plus Fragmentas Kit (Nanjing Novozymes Biotechnology Co., Ltd.). The enzyme fragmentation reaction system is shown in Table 1. The reaction program is set on a PCR amplifier (miniAMP): 37°C, 40 minutes (can be 15 to 60 minutes); 4°C, storage; hot cover 85°C.

[0058] Table 1

[0059]

[0060]

[0061] 2. Recover 140bp~170bp enzyme-digested nucleic acid fragments

[0062] AMPure XP magnetic beads (Beckman Coulter) were used to screen 50 μL of enzyme digestion reaction products. The first round of magnetic beads was 50 μL, and the second round of magnetic beads was 50 μL. Then 100 μL of nuclease-free water was used for elution. The fragment size analysis of the enzyme digestion nucleic acid fragment screening products of NG09394, NA03769, NA02948, and NA24385 was performed using Agilent 4200 TapeStation. The results were as follows: Figures 1 to 4 As shown, it indicates that the screening obtained a fragment with a main peak of 140 bp to 170 bp.

[0063] The concentrations of nucleic acid fragments within the range of 70 bp to 300 bp of the enzyme-digested nucleic acid were read from the Agilent 4200 TapeStation analysis software, as shown in Table 2. 70 bp to 300 bp represents the distribution region of all nucleic acids in the product; 130 bp to 200 bp represents the main distribution region of the substance concentration; and 140 bp to 170 bp represents the region where the main peak is located.

[0064] Table 2

[0065]

[0066] 3. Extract DNA from background plasma

[0067] The background plasma was obtained from peripheral blood plasma of three women with normal karyotypes and pregnant with female fetuses. The obtained plasma was mixed in equal proportions to form 50 mL of mixed plasma. DNA was extracted using a magnetic bead free DNA extraction kit (Guangzhou Meiji Biotechnology Co., Ltd.) with three replicates.

[0068] The extraction steps are as follows:

[0069] a. Sample Lysis: Add 1500 μL of Lysis Buffer, 60 μL of Magnetic Bead Solution, 900 μL of plasma sample, and 60 μL of Proteinase K to a centrifuge tube, vortex to mix, and incubate at room temperature for 10 minutes. Then, place the tube on a magnetic rack until the solution is clear and discard the supernatant.

[0070] b. Wash with Wash Buffer 1: Add 800 μL of Wash Buffer 1 to resuspend the magnetic beads. Place the beads on a magnetic rack and let them stand until the solution is clear. Discard the supernatant.

[0071] c. Wash with Wash Buffer 2: (New reagents must be added with anhydrous ethanol before use) Add 800 μL of Wash Buffer 2 to resuspend the magnetic beads, place on a magnetic rack and let stand until the solution is clear, discard the supernatant; and repeat the washing step with Wash Buffer 2 once.

[0072] d. Drying the magnetic beads: Centrifuge the centrifuge tube for 5 seconds and place it on a magnetic rack. Discard any remaining liquid and dry the magnetic beads at room temperature or no higher than 37°C.

[0073] e. DNA elution and recovery: Add 60 μL of elution buffer to the dried magnetic beads, resuspend the beads, incubate at room temperature for 5 minutes, place on a magnetic stand and let stand until the solution is clear, and transfer the supernatant to a new centrifuge tube for storage.

[0074] The extracted nucleic acid was analyzed by Agilent 4200 TapeStation for fragment size analysis. Figure 5 As shown, the shortest fragment is approximately 150 bp, the longest fragment is approximately 310 bp, and the main peak is 216 bp. This is somewhat delayed compared to the approximately 167 bp found by Lo YM et al. for cell-free DNA fragments in pregnant women. Considering the low concentration of cfDNA and the presence of cohesive ends and varying lengths at both ends of cfDNA, which can affect electrophoresis speed, the detected values ​​are higher than the theoretical values. Therefore, the test results are considered to be in line with expectations.

[0075] The concentration of background plasma nucleic acids in the range of 140 bp to 320 bp was read from the Agilent 4200 TapeStation analysis software (since the distribution of nucleic acid sizes in plasma is basically stable in pregnant women, and mixing of maternal plasma from three sources can effectively reduce individual differences, this range can be fixed), as shown in Table 3.

[0076] Table 3

[0077] Analyze samples Concentration (pmol / L) Background plasma-1 495 Background plasma-2 533 Background plasma-3 517

[0078] According to Table 3, the average background plasma nucleic acid concentration of the three cases was calculated to be 515 pmol / L. The total amount of nucleic acid per milliliter of plasma in this batch of mixed plasma was calculated to be approximately 52820.5 pmol. The 0.65 in the formula is the extraction correction factor.

[0079] 4. Calculate the amount of enzyme-digested nucleic acid in the positive and negative quality controls

[0080] The positive control samples include T21 (T represents triploid, the same below), T18, and T13. The input amount of the three positive enzyme-cleaved nucleic acid fragments (140-170 bp) is calculated separately. The calculation formula for the input amount of each enzyme-cleaved nucleic acid fragment per milliliter of plasma is (assuming the final concentration of the positive control samples is 5%):

[0081]

[0082] The formula for calculating the amount of 46XY enzyme-digested nucleic acid fragment (140-170 bp) in the negative control per milliliter of plasma (assuming the final concentration of the negative control is 10%) is as follows:

[0083]

[0084] According to the calculation formula of the input amount of enzyme-cut nucleic acid fragments of positive quality control products and negative quality control products, the input volume of each enzyme-cut nucleic acid fragment in each milliliter of plasma can be obtained, as shown in Table 4.

[0085] Table 4

[0086] sample Volume (μL) NG09394(T21) enzyme digested nucleic acid fragment 0.67 NA03769(T18) enzyme digested nucleic acid fragments 0.55 NA02948(T13) enzyme digested nucleic acid fragments 0.63 NA24385(46XY) enzyme digested nucleic acid fragment 0.70

[0087] 5. Prepare positive and negative quality control products

[0088] Prepare 10 mL each of positive and negative quality control products according to the calculation results in Table 4.

[0089] Preparation of positive quality control: Take 10 mL of mixed plasma, add 6.7 μL of T21-digested nucleic acid fragment, 5.5 μL of T18-digested nucleic acid fragment, and 6.3 μL of T13-digested nucleic acid fragment, respectively, and mix thoroughly by inversion to obtain a positive quality control with a T21, T18, and T13 concentration of 5% each.

[0090] Preparation of negative quality control: Take 10 mL of mixed plasma, add 7.0 μL of 46XY enzyme-digested nucleic acid fragment, and mix thoroughly by inversion to obtain a negative quality control with a concentration of 10%.

[0091] Example 2 Accuracy Assessment of the Quality Control Prepared in Example 1

[0092] The accuracy of the positive quality control product and the negative quality control product prepared in Example 1 was evaluated. The process included nucleic acid extraction, library preparation, sequencing, bioinformatics analysis and other links. Each quality control product was tested with 9 replicates.

[0093] Nucleic acids from positive and negative controls were extracted using a cfDNA universal library construction kit (Guangdong KingMed Future Biotechnology Co., Ltd., which includes modules such as end repair and A addition, adapter ligation, and fragment screening or purification). The nucleic acids were then prepared into libraries suitable for high-throughput sequencing using a PCR-free library construction method (the PCR-free library construction method can greatly restore the nucleic acid information of the sample itself, such as nucleic acid length and mutual ratio; if the PCR library construction method is used, competitive enrichment of shorter fragments is very likely to occur, resulting in changes in the mutual ratio of nucleic acids). Sequencing was then performed using the Illumina Nextseq 550Dx, and the sequencing data were subjected to bioinformatics analysis and statistics.

[0094] The sequencing results of negative and positive quality control products are as follows Figure 6 As shown in Tables 5 and 6.

[0095] Table 5

[0096]

[0097]

[0098] Table 6

[0099] Negative control UniqueReads Z-21 Z-18 Z-13 chrY% 1 4735262 1.474 0.142 0.602 9.241 2 4193253 -0.948 0.624 -1.306 11.715 3 4025590 0.790 0.612 0.882 11.983 4 3615569 1.586 0.125 1.228 10.211 5 3615963 1.806 -0.088 0.676 9.207 6 3899761 -0.045 0.457 -0.551 10.871 7 3403306 0.506 0.354 1.815 11.422 8 4382129 -0.117 0.152 -0.084 8.267 9 4249962 0.434 1.078 -0.058 11.539 AVG 4013422 0.610 0.384 0.356 10.495 CV 10.6％ 148.9％ 92.0％ 267.9％ 12.6％

[0100] Figure 6 The results showed that the sequencing results of the negative and positive quality control samples prepared by the present invention were normal and qualified. The samples used paired-end sequencing (read1, read4) with paired-end molecular tags (read2, read3). The total sequencing cycle number was 88 cycles, with a total output of 45.89G and a sequencing Q30 of 93.00%. The sequencing results can be used for subsequent analysis.

[0101] In bioinformatics analysis of high-throughput sequencing results, UniqueReads represents unique DNA sequences aligned to the human genome. Z-21 is the Z-score for chromosome 21, Z-18 is the Z-score for chromosome 18, and Z-13 is the Z-score for chromosome 13. The Z-score is a reference value used to determine whether a particular chromosome is aneuploid. It is calculated as: (chromosome content in the sample - average chromosome content in the model) ÷ standard deviation of the reference sample for that chromosome. A Z-score within the range of [-3.00, 3.00] indicates that the chromosome is normal. chrY% is the percentage of uniquely aligned sequences on the Y chromosome compared to the total number of autosomes.

[0102] As shown in Table 5, all positive quality control products indicated trisomy 21, trisomy 18, and trisomy 13, and their Z values ​​were all above 5; and the chrY% was in the range of 12% to 18%. The actual detection values ​​were consistent with the expected theoretical values ​​of the preparation (the theoretical concentration of each positive chromosome was 5%, and the total theoretical concentration was 15%). When analogized to FF, it can be inferred that the FF of the positive quality control products was between 12% and 18%, which met the requirement of ±20% of the theoretical concentration of general quality control products.

[0103] As can be seen from Table 6, all negative quality control products did not indicate trisomy 21, trisomy 18, and trisomy 13, and their Z values ​​were all between -3.00 and 3.00; and the chrY% was all in the range of 8% to 12%. The actual detection value was consistent with the expected preparation theoretical value (theoretical value is 10%). When analogized to FF, it can be inferred that the FF of the negative quality control products was between 8% and 12%, which met the requirement of ±20% of the theoretical concentration of general quality control products.

[0104] Example 3: Verification of the quality control product of the present invention using a commercial non-invasive prenatal detection kit

[0105] In this example, the negative quality control product and the positive quality control product prepared in Example 1 of the present invention were detected using the fetal chromosome aneuploidy (T13 / T18 / T21) detection kit (reversible terminal termination sequencing method) (National Medical Device Registration No. 20153400461), the fetal chromosome aneuploidy data analysis software (Zhejiang Medical Device Registration No. 20182210231), and the gene sequencer (National Medical Device Registration No. 20153220460) of Hangzhou Berry Genomics Diagnostic Technology Co., Ltd.

[0106] The test results of the positive quality control products are shown in Table 7.

[0107] Table 7

[0108]

[0109]

[0110] The test results of negative control products are shown in Table 8.

[0111] Table 8

[0112] Negative control UniqueReads Z-21 Z-18 Z-13 chrY% 1-1 4169328 1.426 1.245 -0.725 11.258 2-1 4390667 1.522 -0.457 0.394 10.242 3-1 4464978 0.803 1.63 1.337 9.609 4-1 5061593 -0.169 0.672 0.96 8.872 5-1 4044361 1.69 0.975 0.716 9.934 6-1 3837434 0.321 0.871 1.234 8.683 7-1 5262754 1.376 -0.103 -0.425 10.529 8-1 3545078 0.458 0.209 1.012 9.778 9-1 4778392 -0.379 0.825 -0.298 10.988 AVG 4394954 0.783 0.652 0.467 9.988 CV 12.9％ 98.0％ 101.3％ 165.0％ 8.7％

[0113] As can be seen from the results in Tables 7 and 8, all positive quality control products indicated trisomy 21, trisomy 18, and trisomy 13, their Z values ​​were all above 5, and chrY% was in the range of 12% to 18%. The test results were consistent with the results in Example 2; all negative quality control products did not indicate trisomy 21, trisomy 18, and trisomy 13, their Z values ​​were between -3.00 and 3.00, and chrY% was in the range of 8% to 12%. The test results were consistent with the results in Example 2. Therefore, the performance of the quality control products of the present invention in commercial non-invasive prenatal detection kits is consistent with the performance demonstrated in Example 2.

[0114] Example 4: Verification of Extraction Correction Coefficient

[0115] This example verifies the extraction correction coefficient of step 3 of Example 1, and the process is as follows:

[0116] 1. A negative quality control was prepared using NA24385 (karyotype 46XY). Based on the principle analysis of commercially available cell-free nucleic acid extraction reagents, the extraction and recovery efficiency of cfDNA can generally reach between 0.5 and 0.8. Therefore, in this example, the extraction correction factors were set to 0.4, 0.5, 0.65, 0.85, and 1, respectively, with three replicates for each factor. The preparation process was consistent with the steps in Example 1. The total nucleic acid content per milliliter of mixed plasma and the amount of NA24385 (46XY) enzyme-digested nucleic acid fragments input under different extraction correction factors are shown in Table 9.

[0117] Table 9

[0118]

[0119] As shown in Table 9, 3.42 μL of NA24385-cleaved nucleic acid fragments were added to 3 mL of mixed plasma with an extraction correction factor of 0.4; 2.74 μL of NA24385-cleaved nucleic acid fragments were added to 3 mL of mixed plasma with an extraction correction factor of 0.5; 2.11 μL of NA24385-cleaved nucleic acid fragments were added to 3 mL of mixed plasma with an extraction correction factor of 0.65; 1.71 μL of NA24385-cleaved nucleic acid fragments were added to 3 mL of mixed plasma with an extraction correction factor of 0.8; and 1.37 μL of NA24385-cleaved nucleic acid fragments were added to 3 mL of mixed plasma with an extraction correction factor of 1.

[0120] The negative quality control products prepared with different extraction correction coefficients were subjected to nucleic acid extraction, library preparation, sequencing, and bioinformatics analysis, and the process was consistent with that in Example 2.

[0121] The chrY% values ​​of the sequencing results of negative quality control products with different extraction correction coefficients are shown in Table 10.

[0122] Table 10

[0123] Extraction correction factor 0.4 0.5 0.65 0.8 1 chrY%-1 13.174 14.512 10.815 10.168 8.481 chrY%-2 13.232 12.356 9.673 8.237 8.270 chrY%-3 12.076 12.058 10.121 9.062 7.877 AVG 12.828 12.975 10.203 9.156 8.209 CV 5.1％ 10.3％ 5.6％ 10.6％ 3.7％

[0124] From the comparative test results of negative quality control products with different extraction correction coefficients, it can be seen that when the extraction correction coefficient is 0.65, the chrY% of the negative quality control product is closest to the theoretical value of 10%. After the correction coefficient is adjusted, the lower the coefficient, the greater the chrY% of the negative quality control product is than the theoretical value of 10%, and the higher the coefficient, the smaller the chrY% of the negative quality control product is than the theoretical value of 10%. Considering the need to meet the requirement of ±20% of the theoretical concentration of general quality control products, in this process, coefficients of 0.4 and 0.5 cannot meet the conditions, and a coefficient of 1 is close to the critical level. Taking into account the degree of closeness to the theoretical value and the fault tolerance, the extraction correction coefficient in the present invention is preferably 0.65.

[0125] Example 5 Selection of background plasma

[0126] In this example, 10 mL of positive control and 10 mL of negative control were prepared using plasma from non-pregnant women with normal karyotype (donated or from research projects of cooperative institutions) according to the preparation process in Example 1, and the test results were compared with those in Example 2. The following steps were included:

[0127] 1. Referring to Example 1, nucleic acid was extracted from plasma of non-pregnant women with normal karyotype, and fragment analysis was performed using an Agilent 4200 TapeStation to obtain a concentration within the range of 140 bp to 320 bp, as shown in Table 11.

[0128] Table 11

[0129] Analyze samples Concentration (pmol / L) Non-pregnant female plasma-1 379 Non-pregnant female plasma-2 364 Non-pregnant female plasma-3 418

[0130] 2. The average nucleic acid concentration in non-pregnant female plasma is 387 pmol / L, which means the total nucleic acid content per milliliter of plasma is approximately 39,692.3 pmol. The volume of enzyme-cleaved nucleic acid fragments per milliliter of plasma can then be calculated, as shown in Table 12.

[0131] Table 12

[0132] sample Volume (μL) NG09394(T21) enzyme digested nucleic acid fragment 0.50 NA03769(T18) enzyme digested nucleic acid fragments 0.41 NA02948(T13) enzyme digested nucleic acid fragments 0.47 NA24385(46XY) enzyme digested nucleic acid fragment 0.53

[0133] 3. Prepare 10 mL each of positive quality control product and negative quality control product according to the calculation results.

[0134] Preparation of positive quality control: Take 10 mL of mixed plasma from non-pregnant women, add 5.0 μL of nucleic acid digested with T21, 4.1 μL of nucleic acid digested with T18, and 4.7 μL of nucleic acid digested with T13, respectively, and mix thoroughly by inversion to obtain the positive quality control.

[0135] Preparation of negative quality control: Take 10 mL of non-pregnant female mixed plasma, add 5.3 μL of 46XY enzyme-cut nucleic acid, and mix thoroughly by inversion to obtain negative quality control.

[0136] 4. The obtained negative and positive quality control products were subjected to nucleic acid extraction, library preparation, sequencing, and bioinformatics analysis, and the process was consistent with that in Example 2.

[0137] The comparison of chrY% of negative and positive quality control products prepared from plasma of non-pregnant women and plasma of pregnant women with fetal fetuses is shown in Table 13.

[0138] Table 13

[0139]

[0140]

[0141] From the comparison results in Table 13, it can be seen that the average chrY% of the positive quality control prepared from non-pregnant female plasma is 11.187, and the average chrY% of the negative quality control is 6.855. The average chrY% of the positive quality control prepared from pregnant female fetal plasma is 16.077, and the average chrY% of the negative quality control is 10.495. The concentrations of the negative and positive quality controls prepared using non-pregnant female plasma are lower than the expected target values, while the concentrations of the negative and positive quality controls prepared using pregnant female fetal plasma are more in line with the expected target values. Therefore, when preparing negative and positive quality controls in the present invention, pregnant female fetal plasma is used as background plasma.

[0142] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0143] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. 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, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome, characterized in that: The test comprises a positive quality control product and a negative quality control product. The positive quality control product is obtained by adding nucleic acid fragments of genomic DNA of chromosome 21, 18 and 13 trisomy syndrome to background plasma; the negative quality control product is obtained by adding nucleic acid fragments of genomic DNA of normal karyotype to background plasma; the nucleic acid fragments are 130 to 200 bp; and the background plasma is peripheral blood plasma of a woman with a normal karyotype and a pregnant woman with a female fetus.

2. The quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome according to claim 1, characterized in that: The nucleic acid fragment is 140 to 170 bp.

3. The quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome according to claim 1 or 2, characterized in that: The genomic DNA of the trisomy syndrome of chromosomes 21, 18 and 13 are NG09394, NA03769 and NA02948 respectively, and the genomic DNA of the normal karyotype is NA24385.

4. A method for preparing a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy syndrome, characterized in that: The following steps are involved: (1) The genomic DNA of chromosome 21, 18, and 13 trisomy syndrome and the normal karyotype genomic DNA were digested with enzymes and screened with magnetic beads to recover nucleic acid fragments of 130 to 200 bp; (2) DNA was extracted from peripheral blood plasma of pregnant women with normal karyotypes and female fetuses, and the concentration of nucleic acid fragments ranging from 140 bp to 320 bp was read. The total amount of nucleic acid fragments ranging from 140 bp to 320 bp in each milliliter of peripheral blood plasma was calculated using a DNA extraction correction factor of 0.6 to 0.8; (3) According to the final concentration of the positive control product and the formula: Calculate the input amount of 130-200 bp nucleic acid fragments of chromosome 21, 18, and 13 trisomy syndrome in each milliliter of peripheral blood plasma; based on the final concentration of the negative quality control and the formula: Calculate the input amount of 130-200 bp nucleic acid fragments of normal karyotype genome in each milliliter of peripheral blood plasma; (4) Take peripheral blood plasma, add 130-200 bp nucleic acid fragments of chromosome trisomy syndrome 21, 18, and 13 respectively according to the amount of input in step (3), and mix thoroughly to obtain a positive quality control product; take peripheral blood plasma, add 130-200 bp nucleic acid fragments of normal karyotype genome according to the amount of input in step (3), and mix thoroughly to obtain a negative quality control product.

5. The method for preparing a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy according to claim 4, characterized in that: The final concentration of the positive control product in step (3) is 4-6%, and the final concentration of the negative control product is 8-12%.

6. The method for preparing a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy according to claim 4, characterized in that: In step (1), a nucleic acid fragment of 140 to 170 bp is recovered.

7. The method for preparing a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy according to claim 4, characterized in that: The method for calculating the total amount of nucleic acid fragments of 140 bp to 320 bp per milliliter of peripheral blood plasma in step (2) comprises: using the concentration of the nucleic acid fragments of 140 bp to 320 bp to estimate the amount of nucleic acid fragments of 140 bp to 320 bp per milliliter of peripheral blood plasma, and then dividing it by the DNA extraction correction factor to obtain.

8. The method for preparing a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy according to claim 4, characterized in that: The DNA extraction correction factor in step (2) is 0.6 to 0.7, more preferably 0.

65.

9. The method for preparing a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy according to claim 4, characterized in that: The peripheral blood plasma in step (2) is a mixed peripheral blood plasma of 3 to 4 women with normal karyotypes and pregnant with female fetuses; and / or, the genomic DNA of chromosome 21, 18, and 13 trisomy syndrome in step (1) are NG09394, NA03769, and NA02948, respectively, and the normal karyotype genomic DNA is NA24385.

10. The method for preparing a quality control product for non-invasive prenatal detection of fetal chromosomal trisomy according to claim 4, characterized in that: The following steps are involved: (1) The trisomy genomic DNA NG09394, NA03769, NA02948 and the normal karyotype genomic DNA NA24385 were digested with enzymes and screened with magnetic beads to recover nucleic acid fragments of 140 to 170 bp; (2) DNA was extracted from peripheral blood plasma of pregnant women with normal karyotypes and female fetuses, and the concentration of nucleic acid fragments ranging from 140 bp to 320 bp was read. The total amount of nucleic acid fragments ranging from 140 bp to 320 bp in each milliliter of peripheral blood plasma was calculated using a DNA extraction correction factor of 0.6 to 0.7; (3) According to the calculation formula: Calculate the input amount of 130-200 bp nucleic acid fragment of trisomy syndrome genomic DNA per milliliter of peripheral blood plasma; according to the calculation formula: Calculate the input amount of 130-200 bp nucleic acid fragment of normal karyotype genomic DNA per ml of peripheral blood plasma; (4) Take peripheral blood plasma, add 130-200 bp nucleic acid fragments of trisomy syndrome genomic DNA according to the amount of input in step (3), mix thoroughly, and obtain a positive quality control product; take peripheral blood plasma, add 130-200 bp nucleic acid fragments of normal karyotype genomic DNA according to the amount of input in step (3), mix thoroughly, and obtain a negative quality control product.

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