WO2019205132A1 - Method for enriching fetal free nucleic acids and application thereof - Google Patents

Method for enriching fetal free nucleic acids and application thereof Download PDF

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WO2019205132A1
WO2019205132A1 PCT/CN2018/085080 CN2018085080W WO2019205132A1 WO 2019205132 A1 WO2019205132 A1 WO 2019205132A1 CN 2018085080 W CN2018085080 W CN 2018085080W WO 2019205132 A1 WO2019205132 A1 WO 2019205132A1
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nucleic acid
fetal
free nucleic
detection
amplification
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PCT/CN2018/085080
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French (fr)
Chinese (zh)
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杨林
王逸丛
高雅
陈芳
张翔涵
于靖
张海萍
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深圳华大生命科学研究院
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Priority to PCT/CN2018/085080 priority Critical patent/WO2019205132A1/en
Priority to CN201880091896.5A priority patent/CN111918965A/en
Publication of WO2019205132A1 publication Critical patent/WO2019205132A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present application relates to the field of nucleic acid enrichment, and in particular to a method for enriching fetal free nucleic acid and its application.
  • cfDNA has been successfully applied to the screening of prenatal non-invasive fetal chromosome aneuploidy, and fetal chromosomal abnormalities are detected by separation of plasma free DNA from pregnant women combined with high-throughput sequencing.
  • Non-invasive prenatal testing This poses a challenge to non-invasive prenatal testing.
  • the concentration of fetal free DNA increases with the increase of gestational age. Therefore, delayed blood sampling can solve the problem of low fetal free DNA concentration to a certain extent, but this will undoubtedly increase the cost of detection and cause Maternal anxiety, so increasing fetal free DNA concentration without changing existing conditions is important for non-invasive prenatal testing.
  • increasing the concentration of fetal free DNA is also important for the accurate detection of such diseases.
  • the purpose of the present application is to provide a novel method for enriching fetal free nucleic acid and its use.
  • An aspect of the present application discloses a method for enriching fetal free nucleic acid, comprising performing PCR amplification on a sample containing fetal free nucleic acid to obtain a product of fetal free nucleic acid enrichment; wherein PCR amplification comprises first adopting primer pairing The forward or reverse primer is used to perform the first amplification of the sample containing the fetal free nucleic acid, and then the second amplification of the other primer in the primer pair is added to the first amplification product, the second time.
  • the amplified product is the fetal free nucleic acid enrichment product; wherein the first amplification is linear amplification and the second amplification is exponential amplification.
  • the fetal free nucleic acid is fetal free DNA.
  • the present application is completed based on the following findings of the inventors:
  • the inventors of the present application have found through extensive experimental research that linear amplification of PCR has a clear preference for short-fragment nucleic acids. Since the fetal free DNA fragment in the plasma free DNA of the pregnant woman is generally shorter than the free DNA fragment of the pregnant woman, the inventors of the present application have proposed a method for enriching the fetal free nucleic acid, and creatively adopting two PCR amplifications to realize the fetal free nucleic acid rich. set. Among them, the first amplification only adds a forward primer or a reverse primer to the amplification system. Since there are only forward primers or reverse primers, only linear amplification can be performed.
  • This step is for short fetal free nucleic acids. Hence, more fetal free nucleic acid fragments can be obtained; then a second amplification is performed, and in the second amplification, another primer in the primer pair is added to the first amplified product, The primers remaining in the first amplification product constitute a complete primer pair, and therefore, it is possible to perform exponential amplification, which is the same as normal PCR amplification, and can achieve exponential growth of the target fragment, so that the first amplification The preference for fetal free nucleic acid fragments is exponentially multiplied to achieve enrichment of fetal free nucleic acids.
  • the number of PCR cycles for the first amplification is 10-50.
  • the number of PCR cycles for the first amplification is 10-20.
  • the PCR reaction conditions for the first amplification include pre-denaturation at 98 ° C for 2 min, and then enter 10-20 or 10-50 cycles: denaturation at 98 ° C for 15 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 30 s, after the end of the cycle , extending at 72 ° C for 5 min.
  • the number of PCR cycles for the second amplification is 10-20.
  • the number of PCR cycles for the second amplification is 10-15.
  • the PCR conditions of the second amplification include pre-denaturation at 98 ° C for 2 min, and then enter 10-15 or 10-20 cycles: denaturation at 98 ° C for 15 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 30 s, after the end of the cycle , extending at 72 ° C for 5 min.
  • the sample containing the fetal free nucleic acid is pregnant women's peripheral blood.
  • the sample containing fetal free nucleic acid is maternal plasma.
  • the sample containing the fetal free nucleic acid is pregnant plasma free nucleic acid.
  • the fetal free nucleic acid enrichment method of the present application further comprises, before PCR amplification, sequentially repairing the sample, adding A, and adding a linker.
  • the end of the sample is repaired, A is added, and the adaptor is added for the purpose of subsequent library construction, so that the constructed library has enriched fetal free nucleic acid to facilitate detection of fetal free nucleic acid.
  • the first amplification may use only one forward primer or reverse primer of the universal primer, and the second amplification may add another primer.
  • the adapter is not added, the sample is directly amplified by PCR.
  • the first primer or the reverse primer added to the first amplification may have more than one primer, for example, multiple forward primers or multiple reverse primers may be added. This is not specifically limited.
  • the fetal free nucleic acid enrichment method of the present application further comprises, after adding the linker, purifying the addition product, and performing PCR amplification on the purified addition product.
  • the addition of the linker product is purified by magnetic beads.
  • the other side of the application discloses the application of the enrichment method of the present application in fetal free nucleic acid detection, preparation of fetal free nucleic acid detection kit or fetal free nucleic acid detection device.
  • each test comprises fetal free nucleic acid concentration detection and/or fetal chromosome genetic abnormality detection; wherein fetal chromosomal genetic abnormality detection includes fetal chromosome aneuploidy detection, fetal At least one of gene copy number variation detection and fetal monogenic disease detection.
  • the fetal free nucleic acid enrichment method of the present invention can effectively enrich the fetal free nucleic acid in the plasma free nucleic acid of the pregnant woman, and improve the proportion of the fetal free nucleic acid in the total free nucleic acid, thereby facilitating the subsequent release of the fetus.
  • the detection of nucleic acids therefore, the fetal free nucleic acid enrichment method of the present application can fully assist in the detection of fetal free nucleic acid to improve its detection accuracy.
  • fetal free nucleic acid concentration detection fetal chromosome genetic abnormality detection
  • fetal chromosome genetic abnormality detection includes fetal chromosome aneuploidy detection, fetal gene copy number variation detection, fetal single gene disease detection and the like.
  • the related reagents may also be combined into a corresponding fetal free nucleic acid detection kit; or the fetal free nucleic acid is specially developed according to the inventive idea of the fetal free nucleic acid enrichment method of the present application.
  • the detecting device is not specifically limited herein.
  • a further aspect of the present application discloses a method for constructing a free nucleic acid sequencing library, comprising performing PCR amplification of a free nucleic acid sample by using the fetal free nucleic acid enrichment method of the present application, and then cyclizing the PCR amplification product to obtain a free nucleic acid.
  • Sequencing library specifically refers to a sample containing fetal free nucleic acid.
  • the PCR amplification product in the library construction method of the present application is actually the fetal free nucleic acid enrichment product in the fetal free nucleic acid enrichment method of the present application, and the method for constructing the free nucleic acid sequencing library of the present application, since the application is adopted
  • the fetal free nucleic acid enrichment method, the final constructed library enriches the fetal free nucleic acid in a large amount, and therefore, is particularly suitable for sequencing and detecting fetal free nucleic acid, for example, analyzing fetal free nucleic acid concentration and fetal chromosome aneuploidy by sequencing.
  • the PCR amplification product is further purified, and the purified PCR amplification product is cyclized.
  • the PCR amplification product is purified using magnetic beads.
  • a further aspect of the present application discloses the use of the free nucleic acid sequencing library construction method of the present application in fetal free nucleic acid detection, preparation of a fetal free nucleic acid detection kit or a fetal free nucleic acid detection device.
  • each test comprises fetal free nucleic acid concentration detection and/or fetal chromosome genetic abnormality detection; wherein fetal chromosome genetic abnormality detection includes fetal chromosome aneuploidy detection, fetal At least one of gene copy number variation detection and fetal monogenic disease detection.
  • the method for constructing a free nucleic acid sequencing library of the present application because the fetal free nucleic acid enrichment method of the present application is adopted, the final constructed library is enriched in fetal free nucleic acid, and thus can be applied to the detection of fetal free nucleic acid concentration. , fetal chromosome aneuploidy detection, fetal gene copy number variation detection, fetal monogenic disease detection.
  • the related reagents may also be combined into a corresponding fetal free nucleic acid detection kit; or in accordance with the inventive idea of the free nucleic acid sequencing library construction method of the present application, the fetal free nucleic acid is specially developed.
  • the detecting device is not specifically limited herein.
  • a further aspect of the present application discloses a method for detecting fetal free nucleic acid, which comprises performing fetal free nucleic acid enrichment on a sample containing fetal free nucleic acid using the fetal free nucleic acid enrichment method of the present application, and then enriching The product was tested.
  • the enriched product is detected, including but not limited to high throughput sequencing assays.
  • the key of the fetal free nucleic acid detection method of the present application is to enrich the fetal free nucleic acid in the free nucleic acid by using the fetal free nucleic acid enrichment method of the present application, thereby improving the detection sensitivity and accuracy of the fetal free nucleic acid.
  • the specific detection method can adopt general nucleic acid detection means, among which the most accurate and comprehensive is nucleic acid sequencing; of course, in addition to nucleic acid sequencing, other conventional nucleic acid detection can also be used in the case where fetal free nucleic acid is effectively enriched. The method is tested and is not specifically limited herein.
  • the method for detecting fetal free nucleic acid of the present application further comprises: cyclizing the enriched product, preparing the DNA nanosphere by using the cyclized product, and performing sequencing detection on the DNA nanosphere.
  • cyclization of the enriched product and preparation of DNA nanospheres is a nucleic acid sequencing library construction method in an implementation manner of the present application, and it can be understood that for different sequencing platforms or sequencing methods, cyclization and DNA
  • the preparation of the nanospheres is not an essential step and is not specifically limited herein.
  • the method further comprises purifying the enriched product to cyclize the purified enriched product; preferably, the enriched product is purified. Magnetic beads were purified.
  • the fetal free nucleic acid enrichment method of the present application creatively uses two PCR amplifications to perform fetal free nucleic acid enrichment, and fully utilizes the amplification preference of short linear nucleic acid amplification by PCR linear amplification in the first amplification, so that the short fragment
  • the fetal free nucleic acid is better amplified, and then the second amplification exponential amplification is used to amplify the preference of the first amplification, thereby realizing fetal free nucleic acid enrichment and increasing fetal free nucleic acid to total free nucleic acid.
  • the fetal free nucleic acid enrichment method of the present application is important for improving the accuracy of fetal free nucleic acid detection, reducing false negatives in non-invasive prenatal testing, and improving the success rate of detection.
  • FIG. 1 is a schematic diagram of a library construction process based on a fetal free nucleic acid enrichment method in an embodiment of the present application
  • 2 is a comparative analysis of the fetal free DNA concentration of the existing method in the embodiment of the present application, that is, the NIFTY after the database is constructed, and the fetal free DNA concentration is analyzed by using the fetal free nucleic acid enrichment method of the embodiment;
  • FIG. 3 is a graph showing the effect of the fetal free DNA original concentration on the enrichment ratio when the fetus free DNA is enriched by the fetal free nucleic acid enrichment method of the embodiment;
  • FIG. 4 is a comparative analysis diagram of a library insert size distribution obtained by using the fetal free nucleic acid enrichment method of the embodiment and a library insert size distribution curve obtained by the existing method, that is, NIFTY database;
  • 5 is a comparative analysis of the fetal free DNA concentration of the existing method in the present embodiment, that is, the NIFTY post-sequence analysis of the fetal free DNA concentration and the comparative test method for the analysis of the fetal free DNA concentration;
  • fetal free DNA concentration of a conventional method in the present embodiment that is, a NIFTY post-sequence analysis, a fetal free DNA concentration by a comparative test method, and a fetal free nucleic acid enrichment method according to the embodiment.
  • the free DNA concentration of the fetus was analyzed by sequencing, and the comparison results of the three were plotted.
  • the low concentration of fetal free DNA in plasma samples is an important factor leading to the failure of non-invasive prenatal testing, and is also the direct cause of many false negatives in fetal monogenic diseases.
  • concentration of fetal free DNA will increase with the increase of gestational age, on the one hand, this can not fully guarantee the concentration of fetal free DNA can meet the detection needs; on the other hand, with the increase of gestational age, non-invasive or fetal monogenic disease The later it is discovered, it is not conducive to subsequent diagnosis and treatment, and it will also increase maternal anxiety. Therefore, how to increase the concentration of fetal free DNA in the sample without changing the sampling gestational age, or to change the sample itself, to achieve fetal free DNA enrichment, to facilitate subsequent detection, for non-invasive detection and fetal genetic disease detection are very important.
  • fetal free DNA concentration in the present application means the ratio of the amount of free DNA derived from the fetus to the total amount of free DNA.
  • Fetal free DNA concentration is sometimes referred to as “fetal concentration”, “fetal free DNA ratio”.
  • fetal free nucleic acid concentration in this application means the ratio of the amount of fetal free nucleic acid to the total amount of free nucleic acid.
  • Fetal free nucleic acid concentration is sometimes referred to as "fetal free nucleic acid ratio”.
  • PCR linear amplification has a clear preference for short fragment nucleic acids.
  • fetal free DNA fragments are generally shorter than free maternal DNA fragments.
  • the present application creatively proposes that when a sample containing fetal free nucleic acid is subjected to PCR amplification, the first primer or the reverse primer in the primer pair is used for the first amplification, and the unidirectional primer is used.
  • the preference of linear PCR amplification for short fragment amplification is such that the short fragment of the fetal free nucleic acid is better amplified, and then the other primer in the primer pair is added to the first amplified product for a second Secondary amplification, the second amplification is the conventional PCR index amplification, further increasing the content of fetal free nucleic acid, thereby achieving fetal free nucleic acid enrichment.
  • the plasma samples of pregnant women with male fetuses were used to compare the fetal free DNA enrichment method with the existing methods to compare the enrichment effects of fetal free DNA.
  • the plasma free DNA of pregnant women was extracted using QIAGEN free DNA extraction kit, and the obtained cfDNA was dissolved in 40 ⁇ L of AE solution for subsequent experiments.
  • the existing method that is, the NIFTY database kit (No. BOX3) of the joint probe anchor polymerization sequencing method of Shenzhen Huada Gene Co., Ltd. is used to carry out free nucleic acid library of plasma samples of 24 pregnant women with male fetuses. preparation.
  • the steps include end-repairing, adding A, adding a linker, and performing conventional PCR amplification on the product of the linker, that is, adding a forward primer and a reverse primer simultaneously in the PCR reaction for 12 rounds of exponential amplification, and the specific method is built with reference to NIFTY. Library kit instructions are not described here.
  • the obtained library was then sequenced on a sequencer BGISEQ-500.
  • the library preparation was also carried out using the NIFTY library kit (Cat. No. BOX3) of the joint probe anchor polymerization sequencing method of Shenzhen Huada Gene Co., Ltd. Different from the method of operation in the kit product specification, as shown in FIG. 1 , after the end of the plasma free DNA fragment is repaired, A is added, and the linker is added, two PCR amplifications are performed according to the fetal free nucleic acid enrichment method of the present example.
  • the first amplification also called “first round PCR”
  • the second amplification also called “second round PCR”
  • subsequent database construction and sequencing two PCR amplifications before and The subsequent steps are the same as the existing methods.
  • the final sequencing data is also the same as the existing method, and the patent "non-invasive detection of fetal genetic abnormality" (authorization bulletin number CN103403183B) is referred to.
  • the end-repair plus A reaction system was: 40 ⁇ L of cfDNA, 9 ⁇ L of the terminal repair reaction solution, and 1 ⁇ L of the terminal repair mixed enzyme, totaling 50 ⁇ L.
  • the reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
  • reaction was carried out under the conditions of 37 ° C for 15 min and 65 ° C for 15 min.
  • the ligation reaction system was: end-repair plus 50 ⁇ L of the A reaction product, 24 ⁇ L of the reaction mixture, 5 ⁇ L of the sequencing linker of 1 ⁇ M, and 1 ⁇ L of the ligase, totaling 80 ⁇ L.
  • the reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
  • AXYGEN purified magnetic beads were added for purification, and the obtained DNA was dissolved in 20 ⁇ L of distilled water.
  • the AXYGEN purified magnetic beads were purchased from Corning Incorporated MAG-FRAG-I-50. The specific purification steps are referred to the product specifications and are not described herein.
  • the first PCR amplification that is, linear amplification, linear amplification reaction system includes: 25 ⁇ L of PCR reaction mixture, 2.5 ⁇ L of 10 ⁇ M universal primer 1 and 20 ⁇ L of AXYGEN purified product, totaling 47.5 ⁇ L.
  • the universal primer 1 is a forward primer.
  • the reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
  • PCR reaction conditions pre-denaturation at 98 ° C for 2 min; then 20 cycles: denaturation at 98 ° C for 15 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 30 s; after the end of the cycle, extension at 72 ° C for 5 min. That is, a linear amplification product is obtained.
  • the second PCR amplification ie, the exponential amplification reaction system, includes: linearly amplified product 47.5 ⁇ L, 10 ⁇ M universal primer 2 dosage 2.5 ⁇ L, totaling 50 ⁇ L.
  • the universal primer 2 is a reverse primer.
  • the reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
  • PCR reaction conditions pre-denaturation at 98 ° C for 2 min; then 10 cycles: denaturation at 98 ° C for 15 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 30 s; after the end of the cycle, extension at 72 ° C for 5 min. That is, an exponential amplification product is obtained.
  • DNA nanospheres were used for sequencing of DNB, 10 ⁇ L of cyclized product was taken, 2 ⁇ L of DNB preparation buffer and 1 ⁇ L of enzyme were added, and reacted at 37 ° C for 30 minutes to obtain DNB.
  • the reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
  • the obtained DNB was sequenced using a high-throughput sequencer BGISEQ-500, sequencing type double-end sequencing 50 bp, label 10 bp.
  • the sequencing data obtained in step (6) is filtered and then compared using bwa, and the obtained data is subjected to genome-wide analysis, including chromosomal abnormality, fetal sex, fetal free DNA concentration, etc., and the specific steps refer to the patent “non-invasiveness of fetal genetic abnormality”. Detection”, authorization notice number CN103403183B, the method disclosed.
  • Table 2 summarizes the concentration of fetal free DNA and chromosomal abnormalities by sequencing analysis. Fetal gender test results showed that 24 samples were male, which was consistent with the actual situation.
  • this example further carried out a comparative test on the basis of the method of fetal free DNA enrichment, that is, after obtaining the purified product of the magnetic beads after the addition of the linker, directly to the magnetic
  • the bead-purified addition product was subjected to 30 cycles of conventional PCR amplification, and the universal primer 1 and the universal primer 2 were directly added to the PCR reaction system, and the amount of addition was the same as the fetal free DNA enrichment method of this example "(3)
  • the sub-PCR amplification was the same, that is, the reaction system included 25 ⁇ L of the PCR reaction mixture, 2.5 ⁇ L of 10 ⁇ M universal primer 1 , 2.5 ⁇ L of 10 ⁇ M universal primer 2, and 20 ⁇ L of AXYGEN purified product, totaling 50 ⁇ L.
  • PCR reaction conditions pre-denaturation at 98 ° C for 2 min; then 30 cycles: denaturation at 98 ° C for 15 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 30 s; after the end of the cycle, extension at 72 ° C for 5 min.
  • a PCR amplification product of the comparative test was obtained.
  • 30 cycles of PCR amplification are to make the number of amplification cycles equal to the total number of cycles of two PCR amplifications of the fetal free nucleic acid enrichment method of this example, that is, "(3) two PCR amplifications" 20 cycles of the first amplification plus 10 cycles of the second amplification.
  • Table 3 summarizes the concentration of fetal free DNA and chromosomal abnormalities by sequencing analysis. Fetal gender test results showed that 24 samples were male, which was consistent with the actual situation.
  • the universal primer 1 is a forward primer
  • the universal primer 2 is a reverse primer, which is a NIFTY library reagent.
  • Primers used in the cassette In all the tests of this example, including the fetal free nucleic acid enrichment method of the present example, the existing method, the NIFTY library kit, and the comparative test, the same primers and reagents were used, the only difference being the PCR amplification step.
  • the fetal free nucleic acid enrichment method of this example is performed by PCR amplification in two steps.
  • the existing method only performs exponential amplification of 12 cycles, and the comparative experiment performs exponential amplification of 30 cycles.
  • the universal primer 1 of this example is the sequence shown in SEQ ID NO. 1
  • the universal primer 2 is the sequence shown in SEQ ID NO. 2; the universal primer 1 has a phosphorylation modification at the 5' end.
  • SEQ ID NO. 1 5'-GAACGACATGGCTACGA-3'
  • the fetal free DNA enrichment method using the linear amplification and then exponential amplification that is, the fetal free nucleic acid enrichment method of this example
  • the final fetal free DNA concentration is improved to different degrees.
  • the statistical analysis chart is shown in Figure 2.
  • 2 is a comparative analysis of fetal free DNA concentration and the analysis of fetal free DNA concentration by using the fetal free nucleic acid enrichment method for sequencing analysis of the existing method, that is, the NIFTY library construction library sequencing analysis, in the figure, the abscissa
  • the fetal free DNA concentration was analyzed by sequencing.
  • the ordinate was the fetal free DNA concentration method for the fetal free nucleic acid enrichment method.
  • the dotted line was used as the fetal free nucleic acid enrichment method.
  • the fetal free DNA concentration is a fitting curve of the multiple of the existing method, and each point is the fetal free DNA concentration of each sample; the results of FIG. 2 show the fetal free DNA concentration ratio obtained by the fetal free nucleic acid enrichment method of this example.
  • FIG. 3 is a statistical diagram of the effect of the original concentration of fetal free DNA on the increase ratio of enrichment when the fetal free DNA is enriched by the fetal free DNA enrichment method of the present application.
  • the abscissa is the fetal release based on the data of Table 1.
  • the initial concentration of DNA the ordinate is the ratio of fetal free DNA enrichment relative to the initial concentration based on the data in Table 2, and the dotted line is the fitted curve of the fetal free DNA enrichment ratio of 24 samples, 24 samples at each point.
  • Proportion of fetal free DNA enrichment the results of Figure 3 show that the fetal free DNA enrichment method of the present application has a stronger effect on the increase of fetal free DNA concentration, and the fetal free DNA concentration increases more, with the fetus
  • the increase of free DNA concentration, the enrichment method of the present application has a decreased proportion of fetal free DNA; it can be seen that the fetal free DNA enrichment method of this example has a better enrichment effect on low concentration of fetal free DNA.
  • Figure 4 is a comparison of the size distribution of the fragment obtained by the fetal free nucleic acid enrichment method of the present example and the size distribution of the fragment obtained by the existing method, that is, the NIFTY library kit.
  • the abscissa is the size of the library insert, and the ordinate.
  • the solid line is the fragment size distribution curve obtained by the existing method, that is, the NIFTY library building kit
  • the dotted line is the fragment size distribution curve obtained by the fetal free nucleic acid enrichment method of the present example
  • the abscissa is the comparative test method, that is, the number of PCR amplification cycles.
  • the fetal free DNA concentration was analyzed by 30-cycle sequencing analysis.
  • the ordinate was the fetal free DNA concentration analyzed by the NIFTY library.
  • the dotted line is the fitting curve of the multiple relationship. The points are for each sample.
  • the fetal free nucleic acid enrichment method of this example was amplified by two-step PCR under the same conditions, and the comparison test was performed by one-step PCR amplification, although the total PCR of the two was performed. The number of amplification cycles is the same, and the reagents and primers used are also the same.
  • the fetal free nucleic acid enrichment method of this example has a better fetal free DNA enrichment effect.
  • the linear amplification of the first amplification can indeed preferentially enrich the small fragment DNA, thereby increasing the fetal free DNA concentration.
  • the fetal free DNA concentration of the existing method that is, the NIFTY library construction library sequencing analysis, the fetal free DNA concentration of the comparative test method, and the fetus free DNA enrichment method for the fetal free DNA analysis Concentration; the results of Figure 6 show that the concentration of fetal free DNA obtained by the existing method and the comparative test method is equivalent, indicating that the enrichment effect of fetal free DNA is equivalent, and in 24 samples, the fetal free nucleic acid enrichment method
  • the obtained fetal free DNA concentration is significantly higher than the existing methods and comparative test methods, indicating that the fetal free nucleic acid enrichment method of this example has a significant enrichment effect on fetal free DNA, and can increase the ratio of fetal free DNA, which improves The accuracy of fetal free nucleic acid detection, reducing false negatives in non-invasive prenatal testing, and improving the success rate of detection is of great significance.

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Abstract

A method for enriching fetal free nucleic acids, comprising performing PCR amplification on a sample containing fetal free nucleic acids to obtain a fetal free nucleic acid-enriched product; PCR amplification comprises first performing first linear amplification on the sample by using a forward or reverse primer of a primer pair, and then adding the other primer to a product of first amplification for second exponential amplification so as to obtain the fetal free nucleic acid-enriched product.

Description

一种胎儿游离核酸的富集方法及其应用Enrichment method of fetal free nucleic acid and application thereof 技术领域Technical field
本申请涉及核酸富集领域，特别是涉及一种胎儿游离核酸的富集方法及其应用。The present application relates to the field of nucleic acid enrichment, and in particular to a method for enriching fetal free nucleic acid and its application.
背景技术Background technique
1997年，Lo等人从孕妇血浆和血清中抽提血浆游离DNA(缩写cfDNA)，应用Y染色体特异性进行胎儿性别诊断，首次证实母体血浆中存在胎儿游离DNA，这一重要发现给无创性产前诊断领域带来了新的曙光。胎儿游离DNA的来源途径机制还不是很清楚，但主要认为胎盘滋养层细胞凋亡后释放游离胎儿DNA。胎儿游离DNA在分娩后于2小时内完全从母体中消失。胎儿游离DNA可用于胎儿性别鉴定、染色体异常检测、RHD血型判断等。随着新一代测序技术的发展，cfDNA成功应用于产前无创性胎儿染色体非整倍性的筛查，通过对孕妇血浆游离DNA的分离结合高通量测序来检测胎儿染色体异常。In 1997, Lo et al. extracted plasma free DNA (abbreviated as cfDNA) from pregnant women's plasma and serum, and used Y chromosome specificity for fetal sex diagnosis. It was first confirmed that fetal free DNA was present in maternal plasma. This important finding is for non-invasive production. The field of pre-diagnosis has brought a new dawn. The mechanism of the source pathway of fetal free DNA is not well understood, but it is mainly believed that the placental trophoblast cells release free fetal DNA after apoptosis. Fetal free DNA completely disappeared from the mother within 2 hours after delivery. Fetal free DNA can be used for fetal sex identification, chromosomal abnormality detection, RHD blood type judgment, and the like. With the development of next-generation sequencing technology, cfDNA has been successfully applied to the screening of prenatal non-invasive fetal chromosome aneuploidy, and fetal chromosomal abnormalities are detected by separation of plasma free DNA from pregnant women combined with high-throughput sequencing.
目前通过高通量测序进行产前无创性检测的方法可以分为三类，基于全基因组、基于SNP位点和基于目标区域富集的方法。其中基于全基因组的方法可以检测所有染色体异常情况，因此应用比较广泛，如华大基因采用全基因组测序的NIFTY产品，但其要求血浆样本中胎儿游离DNA的浓度必须高于3.5％，低于3.5％浓度的胎儿游离DNA会引起假阴性的产生，导致结果检测不准确。而在大样本的统计中，大概有1％血浆样本中的胎儿游离DNA浓度是小于3.5％的，因此这部分样本无法通过全基因组的方法进行检测，对于这部分样本需要重新抽血然后再重新进行无创产前检测。这对无创产前检测带来了挑战，胎儿游离DNA的浓度随着孕周的增加而上升，因此延迟抽血可以一定程度解决胎儿游离DNA浓度低的问题，但这无疑会增加检测成本并造成产妇焦虑情绪，因此在不改变现有条件的情况下提高胎儿游离DNA浓度对于无创产前检测具有重要意义。此外，在一些显性单基因病的检测中，由于胎儿游离DNA浓度过低导致假阴性结果时有发生，因此提高胎儿游离DNA浓度对于准确检测这类疾病也具有重要意义。Current methods for prenatal non-invasive detection by high-throughput sequencing can be divided into three categories, based on genome-wide, SNP-based and target-based region enrichment methods. Among them, the whole genome-based method can detect all chromosomal abnormalities, so it is widely used. For example, the NGFTY product of the whole genome sequencing using Huada gene requires that the concentration of fetal free DNA in plasma samples must be higher than 3.5%, lower than 3.5. % concentration of fetal free DNA can cause false negatives, resulting in inaccurate detection of results. In the large sample statistics, the fetal free DNA concentration in about 1% of the plasma samples is less than 3.5%, so this part of the sample cannot be detected by the genome-wide method. For this part of the sample, it is necessary to re-bleed and then re-sample. Non-invasive prenatal testing. This poses a challenge to non-invasive prenatal testing. The concentration of fetal free DNA increases with the increase of gestational age. Therefore, delayed blood sampling can solve the problem of low fetal free DNA concentration to a certain extent, but this will undoubtedly increase the cost of detection and cause Maternal anxiety, so increasing fetal free DNA concentration without changing existing conditions is important for non-invasive prenatal testing. In addition, in the detection of some dominant monogenic diseases, false negative results occur due to the low concentration of fetal free DNA. Therefore, increasing the concentration of fetal free DNA is also important for the accurate detection of such diseases.
发明内容Summary of the invention
本申请的目的是提供一种新的胎儿游离核酸的富集方法及其应用。The purpose of the present application is to provide a novel method for enriching fetal free nucleic acid and its use.
本申请采用了以下技术方案：This application uses the following technical solutions:
本申请的一方面公开了一种胎儿游离核酸的富集方法，包括对含有胎儿游离核酸的样品进行PCR扩增，获得胎儿游离核酸富集的产物；其中，PCR扩增包括先采用引物对中的正向引物或反向引物对含有胎儿游离核酸的样品进行第一次扩增，然后再向第一次扩增产物中加入引物对中的另一引物进行第二次扩增，第二次扩增的产物即胎儿游离核酸富集产物；其中，第一次扩增为线性扩增，第二次扩增为指数扩增。An aspect of the present application discloses a method for enriching fetal free nucleic acid, comprising performing PCR amplification on a sample containing fetal free nucleic acid to obtain a product of fetal free nucleic acid enrichment; wherein PCR amplification comprises first adopting primer pairing The forward or reverse primer is used to perform the first amplification of the sample containing the fetal free nucleic acid, and then the second amplification of the other primer in the primer pair is added to the first amplification product, the second time. The amplified product is the fetal free nucleic acid enrichment product; wherein the first amplification is linear amplification and the second amplification is exponential amplification.
优选的，胎儿游离核酸为胎儿游离DNA。Preferably, the fetal free nucleic acid is fetal free DNA.
需要说明的是，本申请基于发明人的以下发现而完成：本申请发明人经过大量实验研究发现，PCR线性扩增对短片段核酸具有明显的偏好性。由于孕妇血浆游离DNA中，胎儿游离DNA片段一般比孕妇游离DNA片段要短，因此本申请发明人提出了一种胎儿游离核酸的富集方法，创造性地采用两次PCR扩增实现胎儿游离核酸富集。其中，第一次扩增只在扩增体系中添加了正向引物或反向引物，由于只有正向引物或反向引物，所以只能进行线性扩增，该步骤对较短的胎儿游离核酸具有明显偏好性，能够获得更多的胎儿游离核酸片段；然后再进行第二次扩增，第二次扩增时，向第一次扩增的产物中加入引物对中的另一引物，与第一次扩增产物中残留的引物构成完整的引物对，因此，能够进行指数扩增，该步骤与正常的PCR扩增相同，能够实现靶标片段的指数倍增长，使得第一次扩增的胎儿游离核酸片段的偏好性被指数倍放大，从而实现胎儿游离核酸的富集。It should be noted that the present application is completed based on the following findings of the inventors: The inventors of the present application have found through extensive experimental research that linear amplification of PCR has a clear preference for short-fragment nucleic acids. Since the fetal free DNA fragment in the plasma free DNA of the pregnant woman is generally shorter than the free DNA fragment of the pregnant woman, the inventors of the present application have proposed a method for enriching the fetal free nucleic acid, and creatively adopting two PCR amplifications to realize the fetal free nucleic acid rich. set. Among them, the first amplification only adds a forward primer or a reverse primer to the amplification system. Since there are only forward primers or reverse primers, only linear amplification can be performed. This step is for short fetal free nucleic acids. Apparently, more fetal free nucleic acid fragments can be obtained; then a second amplification is performed, and in the second amplification, another primer in the primer pair is added to the first amplified product, The primers remaining in the first amplification product constitute a complete primer pair, and therefore, it is possible to perform exponential amplification, which is the same as normal PCR amplification, and can achieve exponential growth of the target fragment, so that the first amplification The preference for fetal free nucleic acid fragments is exponentially multiplied to achieve enrichment of fetal free nucleic acids.
优选的，第一次扩增的PCR循环数为10-50。Preferably, the number of PCR cycles for the first amplification is 10-50.
优选的，第一次扩增的PCR循环数为10-20。Preferably, the number of PCR cycles for the first amplification is 10-20.
优选的，第一次扩增的PCR反应条件包括，98℃预变性2min，然后进入10-20或10-50个循环：98℃变性15s、58℃退火30s、72℃延伸30s，循环结束后，72℃延伸5min。Preferably, the PCR reaction conditions for the first amplification include pre-denaturation at 98 ° C for 2 min, and then enter 10-20 or 10-50 cycles: denaturation at 98 ° C for 15 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 30 s, after the end of the cycle , extending at 72 ° C for 5 min.
优选的，第二次扩增的PCR循环数为10-20。Preferably, the number of PCR cycles for the second amplification is 10-20.
优选的，第二次扩增的PCR循环数为10-15。Preferably, the number of PCR cycles for the second amplification is 10-15.
优选的，第二次扩增的PCR反应条件包括，98℃预变性2min，然后进入10-15或10-20个循环：98℃变性15s、58℃退火30s、72℃延伸30s，循环结束后，72℃延伸5min。Preferably, the PCR conditions of the second amplification include pre-denaturation at 98 ° C for 2 min, and then enter 10-15 or 10-20 cycles: denaturation at 98 ° C for 15 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 30 s, after the end of the cycle , extending at 72 ° C for 5 min.
优选的，本申请的胎儿游离核酸富集方法中，含有胎儿游离核酸的样品为孕妇外周血。Preferably, in the fetal free nucleic acid enrichment method of the present application, the sample containing the fetal free nucleic acid is pregnant women's peripheral blood.
更优选的，含有胎儿游离核酸的样品为孕妇血浆。More preferably, the sample containing fetal free nucleic acid is maternal plasma.
更优选的，含有胎儿游离核酸的样品为孕妇血浆游离核酸。More preferably, the sample containing the fetal free nucleic acid is pregnant plasma free nucleic acid.
优选的，本申请的胎儿游离核酸富集方法，在PCR扩增之前，还包括对样品依序进行末端修复、加A、加接头。Preferably, the fetal free nucleic acid enrichment method of the present application further comprises, before PCR amplification, sequentially repairing the sample, adding A, and adding a linker.
需要说明的是，对样品进行末端修复、加A、加接头，其目的是为了后续的文库构建，使得构建的文库中具有富集的胎儿游离核酸，以便于胎儿游离核酸的检测。并且，在加接头以后，对于PCR扩增，第一次扩增可以只采用通用引物的一条正向引物或反向引物，第二次扩增再加入另一条引物。如果没有加接头，直接对样品进行PCR扩增，此时第一次扩增所加入的正向引物或反向引物可能不止一条引物，例如会加入多条正向引物或多条反向引物，在此不做具体限定。It should be noted that the end of the sample is repaired, A is added, and the adaptor is added for the purpose of subsequent library construction, so that the constructed library has enriched fetal free nucleic acid to facilitate detection of fetal free nucleic acid. Moreover, after the addition of the linker, for PCR amplification, the first amplification may use only one forward primer or reverse primer of the universal primer, and the second amplification may add another primer. If the adapter is not added, the sample is directly amplified by PCR. At this time, the first primer or the reverse primer added to the first amplification may have more than one primer, for example, multiple forward primers or multiple reverse primers may be added. This is not specifically limited.
优选的，本申请的胎儿游离核酸富集方法，在加接头之后，还包括对加接头产物进行纯化，对纯化的加接头产物进行PCR扩增。Preferably, the fetal free nucleic acid enrichment method of the present application further comprises, after adding the linker, purifying the addition product, and performing PCR amplification on the purified addition product.
优选的，对加接头产物进行纯化采用磁珠纯化。Preferably, the addition of the linker product is purified by magnetic beads.
本申请的另一面公开了本申请的富集方法在胎儿游离核酸检测、制备胎儿游离核酸检测试剂盒或胎儿游离核酸检测装置中的应用。The other side of the application discloses the application of the enrichment method of the present application in fetal free nucleic acid detection, preparation of fetal free nucleic acid detection kit or fetal free nucleic acid detection device.
优选的，本申请的胎儿游离核酸富集方法的应用中，各检测包括胎儿游离核酸浓度检测和/或胎儿染色体遗传异常检测；其中，胎儿染色体遗传异常检测包括胎儿染色体非整倍性检测、胎儿基因拷贝数变异检测和胎儿单基因病检测中的至少一种。Preferably, in the application of the fetal free nucleic acid enrichment method of the present application, each test comprises fetal free nucleic acid concentration detection and/or fetal chromosome genetic abnormality detection; wherein fetal chromosomal genetic abnormality detection includes fetal chromosome aneuploidy detection, fetal At least one of gene copy number variation detection and fetal monogenic disease detection.
需要说明的是，本申请的胎儿游离核酸富集方法，能够有效的对孕妇血浆游离核酸中的胎儿游离核酸进行富集，提高胎儿游离核酸在总游离核酸中的比例，从而方便后续对胎儿游离核酸的检测，因此，本申请的胎儿游离核酸富集方法完全可以辅助用于胎儿游离核酸检测，以提高其检测准确性。这些检测，例如胎儿游离核酸浓度检测、胎儿染色体遗传异常检测等，其中胎儿染色体遗传异常检测包括胎儿染色体非整倍性检测、胎儿基因拷贝数变异检测、胎儿单基因病检测等。当然，根据本申请胎儿游离核酸富集方法的指导，也可以将相关试剂组合成相应的胎儿游离核酸检测试剂盒；又或者按照本申请胎儿游离核酸富集方法的发明思路，特别研制胎儿游离核酸检测装置；在此不做具体限定。It should be noted that the fetal free nucleic acid enrichment method of the present invention can effectively enrich the fetal free nucleic acid in the plasma free nucleic acid of the pregnant woman, and improve the proportion of the fetal free nucleic acid in the total free nucleic acid, thereby facilitating the subsequent release of the fetus. The detection of nucleic acids, therefore, the fetal free nucleic acid enrichment method of the present application can fully assist in the detection of fetal free nucleic acid to improve its detection accuracy. These tests, such as fetal free nucleic acid concentration detection, fetal chromosome genetic abnormality detection, etc., wherein fetal chromosome genetic abnormality detection includes fetal chromosome aneuploidy detection, fetal gene copy number variation detection, fetal single gene disease detection and the like. Of course, according to the guidance of the fetal free nucleic acid enrichment method of the present application, the related reagents may also be combined into a corresponding fetal free nucleic acid detection kit; or the fetal free nucleic acid is specially developed according to the inventive idea of the fetal free nucleic acid enrichment method of the present application. The detecting device is not specifically limited herein.
本申请的再一面公开了一种游离核酸测序文库的构建方法，包括采用本申请的胎儿游离核酸富集方法对游离核酸样品进行PCR扩增，然后对PCR扩增产物进行环化，获得游离核酸测序文库。其中，游离核酸样品特指含有胎儿游离核酸的样品。A further aspect of the present application discloses a method for constructing a free nucleic acid sequencing library, comprising performing PCR amplification of a free nucleic acid sample by using the fetal free nucleic acid enrichment method of the present application, and then cyclizing the PCR amplification product to obtain a free nucleic acid. Sequencing library. Among them, the free nucleic acid sample specifically refers to a sample containing fetal free nucleic acid.
需要说明的是，本申请文库构建方法中PCR扩增产物实际上就是本申请胎儿游离核酸富集方法中的胎儿游离核酸富集产物，本申请的游离核酸测序文库 构建方法，由于采用了本申请的胎儿游离核酸富集方法，其最终构建的文库中，大量富集了胎儿游离核酸，因此，特别适用于对胎儿游离核酸的测序检测，例如通过测序分析胎儿游离核酸浓度、胎儿染色体非整倍性变异、胎儿基因拷贝数变异、胎儿单基因病变异等。It should be noted that the PCR amplification product in the library construction method of the present application is actually the fetal free nucleic acid enrichment product in the fetal free nucleic acid enrichment method of the present application, and the method for constructing the free nucleic acid sequencing library of the present application, since the application is adopted The fetal free nucleic acid enrichment method, the final constructed library, enriches the fetal free nucleic acid in a large amount, and therefore, is particularly suitable for sequencing and detecting fetal free nucleic acid, for example, analyzing fetal free nucleic acid concentration and fetal chromosome aneuploidy by sequencing. Sexual variation, fetal gene copy number variation, fetal monogenic disease variation, etc.
优选的，本申请的游离核酸测序文库构建方法中，在对PCR扩增产物进行环化之前，还包括对PCR扩增产物进行纯化，对纯化的PCR扩增产物进行环化。Preferably, in the method for constructing a free nucleic acid sequencing library of the present application, before the cyclization of the PCR amplification product, the PCR amplification product is further purified, and the purified PCR amplification product is cyclized.
优选的，对PCR扩增产物进行纯化采用磁珠纯化。Preferably, the PCR amplification product is purified using magnetic beads.
本申请的再一面公开了本申请的游离核酸测序文库构建方法在胎儿游离核酸检测、制备胎儿游离核酸检测试剂盒或胎儿游离核酸检测装置中的应用。A further aspect of the present application discloses the use of the free nucleic acid sequencing library construction method of the present application in fetal free nucleic acid detection, preparation of a fetal free nucleic acid detection kit or a fetal free nucleic acid detection device.
优选的，本申请的游离核酸测序文库构建方法的应用中，各检测包括胎儿游离核酸浓度检测和/或胎儿染色体遗传异常检测；其中，胎儿染色体遗传异常检测包括胎儿染色体非整倍性检测、胎儿基因拷贝数变异检测和胎儿单基因病检测中的至少一种。Preferably, in the application of the free nucleic acid sequencing library construction method of the present application, each test comprises fetal free nucleic acid concentration detection and/or fetal chromosome genetic abnormality detection; wherein fetal chromosome genetic abnormality detection includes fetal chromosome aneuploidy detection, fetal At least one of gene copy number variation detection and fetal monogenic disease detection.
可以理解，本申请的游离核酸测序文库构建方法，由于采用了本申请的胎儿游离核酸富集方法，其最终构建的文库中，大量富集了胎儿游离核酸，因此可以应用于胎儿游离核酸浓度检测、胎儿染色体非整倍性检测、胎儿基因拷贝数变异检测、胎儿单基因病检测等。当然，根据本申请游离核酸测序文库构建方法的指导，也可以将相关试剂组合成相应的胎儿游离核酸检测试剂盒；又或者按照本申请游离核酸测序文库构建方法的发明思路，特别研制胎儿游离核酸检测装置；在此不做具体限定。It can be understood that the method for constructing a free nucleic acid sequencing library of the present application, because the fetal free nucleic acid enrichment method of the present application is adopted, the final constructed library is enriched in fetal free nucleic acid, and thus can be applied to the detection of fetal free nucleic acid concentration. , fetal chromosome aneuploidy detection, fetal gene copy number variation detection, fetal monogenic disease detection. Of course, according to the guidance of the method for constructing a free nucleic acid sequencing library of the present application, the related reagents may also be combined into a corresponding fetal free nucleic acid detection kit; or in accordance with the inventive idea of the free nucleic acid sequencing library construction method of the present application, the fetal free nucleic acid is specially developed. The detecting device is not specifically limited herein.
本申请的再一面公开了一种胎儿游离核酸的检测方法，该方法包括，采用本申请的胎儿游离核酸富集方法，对含有胎儿游离核酸的样品进行胎儿游离核酸富集，然后对富集的产物进行检测。A further aspect of the present application discloses a method for detecting fetal free nucleic acid, which comprises performing fetal free nucleic acid enrichment on a sample containing fetal free nucleic acid using the fetal free nucleic acid enrichment method of the present application, and then enriching The product was tested.
优选的，对富集的产物进行检测，包括但不仅限于高通量测序检测。Preferably, the enriched product is detected, including but not limited to high throughput sequencing assays.
可以理解，本申请的胎儿游离核酸检测方法，其关键就是采用本申请的胎儿游离核酸富集方法对游离核酸中的胎儿游离核酸进行富集，从而提高胎儿游离核酸的检测灵敏度和准确性，至于具体的检测方法可以采用一般的核酸检测手段，其中，最为准确且全面的就是核酸测序；当然，除了核酸测序以外，在胎儿游离核酸被有效富集的情况下，也可以采用其它常规的核酸检测方法进行检测，在此不做具体限定。It can be understood that the key of the fetal free nucleic acid detection method of the present application is to enrich the fetal free nucleic acid in the free nucleic acid by using the fetal free nucleic acid enrichment method of the present application, thereby improving the detection sensitivity and accuracy of the fetal free nucleic acid. The specific detection method can adopt general nucleic acid detection means, among which the most accurate and comprehensive is nucleic acid sequencing; of course, in addition to nucleic acid sequencing, other conventional nucleic acid detection can also be used in the case where fetal free nucleic acid is effectively enriched. The method is tested and is not specifically limited herein.
优选的，本申请的胎儿游离核酸检测方法还包括，对富集产物进行环化，采用环化产物制备DNA纳米球，对DNA纳米球进行测序检测。Preferably, the method for detecting fetal free nucleic acid of the present application further comprises: cyclizing the enriched product, preparing the DNA nanosphere by using the cyclized product, and performing sequencing detection on the DNA nanosphere.
需要说明的是，对富集产物进行环化，并制备DNA纳米球，是本申请一种 实现方式中的核酸测序文库构建方式，可以理解，对于不同的测序平台或者测序方式，环化和DNA纳米球制备并非必须步骤，在此不做具体限定。It should be noted that cyclization of the enriched product and preparation of DNA nanospheres is a nucleic acid sequencing library construction method in an implementation manner of the present application, and it can be understood that for different sequencing platforms or sequencing methods, cyclization and DNA The preparation of the nanospheres is not an essential step and is not specifically limited herein.
优选的，本申请的胎儿游离核酸检测方法中，在对富集产物进行环化之前，还包括对富集产物进行纯化，对纯化的富集产物进行环化；优选的，富集产物纯化采用磁珠纯化。Preferably, in the method for detecting fetal free nucleic acid of the present application, before enriching the enriched product, the method further comprises purifying the enriched product to cyclize the purified enriched product; preferably, the enriched product is purified. Magnetic beads were purified.
本申请的有益效果在于：The beneficial effects of the present application are:
本申请的胎儿游离核酸富集方法，创造性地采用两次PCR扩增进行胎儿游离核酸富集，充分利用第一次扩增中PCR线性扩增对短片段核酸的扩增偏好性，使得短片段的胎儿游离核酸得到更好的扩增，然后再利用第二次扩增的指数扩增放大第一次扩增的偏好性，从而实现胎儿游离核酸富集，提高胎儿游离核酸占总游离核酸的比例。本申请的胎儿游离核酸富集方法对提高胎儿游离核酸检测准确性，降低无创产前检测假阴性，提高检测成功率具有重要意义。The fetal free nucleic acid enrichment method of the present application creatively uses two PCR amplifications to perform fetal free nucleic acid enrichment, and fully utilizes the amplification preference of short linear nucleic acid amplification by PCR linear amplification in the first amplification, so that the short fragment The fetal free nucleic acid is better amplified, and then the second amplification exponential amplification is used to amplify the preference of the first amplification, thereby realizing fetal free nucleic acid enrichment and increasing fetal free nucleic acid to total free nucleic acid. proportion. The fetal free nucleic acid enrichment method of the present application is important for improving the accuracy of fetal free nucleic acid detection, reducing false negatives in non-invasive prenatal testing, and improving the success rate of detection.
附图说明DRAWINGS
图1是本申请实施例中基于胎儿游离核酸富集方法的文库构建流程示意图；1 is a schematic diagram of a library construction process based on a fetal free nucleic acid enrichment method in an embodiment of the present application;
图2是本申请实施例中现有方法即NIFTY建库后测序分析的胎儿游离DNA浓度与采用实施例的胎儿游离核酸富集方法进行建库测序分析的胎儿游离DNA浓度对比分析图；2 is a comparative analysis of the fetal free DNA concentration of the existing method in the embodiment of the present application, that is, the NIFTY after the database is constructed, and the fetal free DNA concentration is analyzed by using the fetal free nucleic acid enrichment method of the embodiment;
图3是本申请实施例中采用实施例的胎儿游离核酸富集方法对胎儿游离DNA进行富集时，胎儿游离DNA原始浓度对富集增加比例的影响分析图；FIG. 3 is a graph showing the effect of the fetal free DNA original concentration on the enrichment ratio when the fetus free DNA is enriched by the fetal free nucleic acid enrichment method of the embodiment;
图4是本申请实施例中采用实施例的胎儿游离核酸富集方法获得的文库***片段大小分布与现有方法即NIFTY建库获得的文库***片段大小分布曲线对比分析图；4 is a comparative analysis diagram of a library insert size distribution obtained by using the fetal free nucleic acid enrichment method of the embodiment and a library insert size distribution curve obtained by the existing method, that is, NIFTY database;
图5是本申请实施例中现有方法即NIFTY建库后测序分析的胎儿游离DNA浓度与对比试验方法进行建库测序分析的胎儿游离DNA浓度对比分析图；5 is a comparative analysis of the fetal free DNA concentration of the existing method in the present embodiment, that is, the NIFTY post-sequence analysis of the fetal free DNA concentration and the comparative test method for the analysis of the fetal free DNA concentration;
图6是本申请实施例中现有方法即NIFTY建库后测序分析的胎儿游离DNA浓度、对比试验方法进行建库测序分析的胎儿游离DNA浓度，以及采用实施例的胎儿游离核酸富集方法进行建库测序分析的胎儿游离DNA浓度，三者的对比结果图。6 is a fetal free DNA concentration of a conventional method in the present embodiment, that is, a NIFTY post-sequence analysis, a fetal free DNA concentration by a comparative test method, and a fetal free nucleic acid enrichment method according to the embodiment. The free DNA concentration of the fetus was analyzed by sequencing, and the comparison results of the three were plotted.
具体实施方式detailed description
血浆样本中胎儿游离DNA浓度低是导致无创产前检测失败的重要因素，同 时也是很多胎儿单基因病检测假阴性的直接原因。虽然随着孕周的增加，胎儿游离DNA的浓度会提高，但是，一方面这不能完全保障胎儿游离DNA的浓度能够达到检测需求；另一方面，随着孕周增加无创产检或胎儿单基因病发现得越晚，不利于后续的诊治，同时也会增加产妇焦虑情绪。因此，如何在不改变取样孕周，或者不改变样本自身情况下，增加样本中胎儿游离DNA的浓度，实现胎儿游离DNA的富集，以方便后续检测，对于无创产检和胎儿基因病检测都很重要。The low concentration of fetal free DNA in plasma samples is an important factor leading to the failure of non-invasive prenatal testing, and is also the direct cause of many false negatives in fetal monogenic diseases. Although the concentration of fetal free DNA will increase with the increase of gestational age, on the one hand, this can not fully guarantee the concentration of fetal free DNA can meet the detection needs; on the other hand, with the increase of gestational age, non-invasive or fetal monogenic disease The later it is discovered, it is not conducive to subsequent diagnosis and treatment, and it will also increase maternal anxiety. Therefore, how to increase the concentration of fetal free DNA in the sample without changing the sampling gestational age, or to change the sample itself, to achieve fetal free DNA enrichment, to facilitate subsequent detection, for non-invasive detection and fetal genetic disease detection are very important.
需要说明的是，本申请中“胎儿游离DNA浓度”表示，胎儿来源的游离DNA数量占总游离DNA数量的比值。“胎儿游离DNA浓度”有时也称“胎儿浓度”，“胎儿游离DNA比例”。同样的，本申请中“胎儿游离核酸浓度”表示，胎儿游离核酸数量占总游离核酸数量的比值。“胎儿游离核酸浓度”有时也称“胎儿游离核酸比例”。It should be noted that the "fetal free DNA concentration" in the present application means the ratio of the amount of free DNA derived from the fetus to the total amount of free DNA. "Fetal free DNA concentration" is sometimes referred to as "fetal concentration", "fetal free DNA ratio". Similarly, "fetal free nucleic acid concentration" in this application means the ratio of the amount of fetal free nucleic acid to the total amount of free nucleic acid. "Fetal free nucleic acid concentration" is sometimes referred to as "fetal free nucleic acid ratio".
本申请经过大量的研究发现，PCR线性扩增对短片段核酸具有明显的偏好性。而孕妇血浆游离DNA中，胎儿游离DNA片段一般比游离母体DNA片段要短。基于以上研究和认识，本申请创造性的提出，对含有胎儿游离核酸的样品进行PCR扩增时，先采用引物对中的正向引物或反向引物进行第一次扩增，利用单向引物的线性PCR扩增对短片段扩增的偏好性，使得短片段的胎儿游离核酸得到更好的扩增，然后再向第一次扩增的产物中加入引物对中的另一引物，进行第二次扩增，第二次扩增就是常规的PCR指数扩增，进一步增加胎儿游离核酸的含量，从而实现胎儿游离核酸的富集。A large number of studies have found that PCR linear amplification has a clear preference for short fragment nucleic acids. In pregnant women's plasma free DNA, fetal free DNA fragments are generally shorter than free maternal DNA fragments. Based on the above research and knowledge, the present application creatively proposes that when a sample containing fetal free nucleic acid is subjected to PCR amplification, the first primer or the reverse primer in the primer pair is used for the first amplification, and the unidirectional primer is used. The preference of linear PCR amplification for short fragment amplification is such that the short fragment of the fetal free nucleic acid is better amplified, and then the other primer in the primer pair is added to the first amplified product for a second Secondary amplification, the second amplification is the conventional PCR index amplification, further increasing the content of fetal free nucleic acid, thereby achieving fetal free nucleic acid enrichment.
下面通过具体实施例对本申请作进一步详细说明。以下实施例仅对本申请进行进一步说明，不应理解为对本申请的限制。The present application is further described in detail below by way of specific embodiments. The following examples are only intended to further illustrate the present application and are not to be construed as limiting the invention.
实施例Example
采用24例怀有男胎的孕妇血浆样本，使用本例胎儿游离核酸富集方法和现有方法进行对比，比较胎儿游离DNA的富集效果。孕妇血浆游离DNA采用QIAGEN游离DNA提取试剂盒提取，得到的cfDNA溶于40μL的AE溶液中，用于后续试验。The plasma samples of pregnant women with male fetuses were used to compare the fetal free DNA enrichment method with the existing methods to compare the enrichment effects of fetal free DNA. The plasma free DNA of pregnant women was extracted using QIAGEN free DNA extraction kit, and the obtained cfDNA was dissolved in 40 μL of AE solution for subsequent experiments.
其中，现有方法，即直接采用深圳华大基因股份有限公司的联合探针锚定聚合测序法的NIFTY建库试剂盒(货号BOX3)对24例怀有男胎的孕妇血浆样本进行游离核酸文库制备。步骤包括对末端修复、加A、加接头，以及对加接头的产物进行常规PCR扩增，即在PCR反应中同时添加正向引物和反向引物进 行12轮指数扩增，具体方法参考NIFTY建库试剂盒说明书，在此不累述。然后将获得的文库在测序仪BGISEQ-500上进行测序。最后根据测序结果分析各样本中胎儿游离DNA的浓度、染色体异常情况等，具体分析步骤参照专利“胎儿遗传异常的无创性检测”(授权公告号CN103403183B)所公开的方法进行，在此不累述。测序分析获得的胎儿游离DNA浓度及染色体异常情况结果如表1所示。Among them, the existing method, that is, the NIFTY database kit (No. BOX3) of the joint probe anchor polymerization sequencing method of Shenzhen Huada Gene Co., Ltd. is used to carry out free nucleic acid library of plasma samples of 24 pregnant women with male fetuses. preparation. The steps include end-repairing, adding A, adding a linker, and performing conventional PCR amplification on the product of the linker, that is, adding a forward primer and a reverse primer simultaneously in the PCR reaction for 12 rounds of exponential amplification, and the specific method is built with reference to NIFTY. Library kit instructions are not described here. The obtained library was then sequenced on a sequencer BGISEQ-500. Finally, according to the sequencing results, the concentration of fetal free DNA and chromosomal abnormalities in each sample were analyzed. The specific analysis steps were carried out according to the method disclosed in the patent “non-invasive detection of fetal genetic abnormality” (authorization announcement number CN103403183B), which is not described here. . The results of fetal free DNA concentration and chromosomal abnormalities obtained by sequencing analysis are shown in Table 1.
表1 现有方法测序分析获得的胎儿游离DNA浓度及染色体情况Table 1 Fetal free DNA concentration and chromosomal status obtained by sequencing of existing methods
样本编号Sample number 染色体异常Chromosomal abnormality 胎儿游离DNA浓度Fetal free DNA concentration 样本编号Sample number 染色体异常Chromosomal abnormality 胎儿游离DNA浓度Fetal free DNA concentration
11 无异常No abnormality 4.3％4.3% 1313 无异常No abnormality 14.2％14.2%
22 无异常No abnormality 5.9％5.9% 1414 无异常No abnormality 15.7％15.7%
33 无异常No abnormality 6.8％6.8% 1515 无异常No abnormality 18.4％18.4%
44 无异常No abnormality 9.6％9.6% 1616 无异常No abnormality 16.7％16.7%
55 无异常No abnormality 9.6％9.6% 1717 无异常No abnormality 16.9％16.9%
66 无异常No abnormality 9.8％9.8% 1818 无异常No abnormality 19.5％19.5%
77 无异常No abnormality 10.0％10.0% 1919 T21T21 19.5％19.5%
88 无异常No abnormality 10.5％10.5% 2020 无异常No abnormality 20.3％20.3%
99 无异常No abnormality 10.6％10.6% 21twenty one T18T18 21.1％21.1%
1010 无异常No abnormality 12.4％12.4% 22twenty two 无异常No abnormality 22.7％22.7%
1111 无异常No abnormality 12.5％12.5% 23twenty three 无异常No abnormality 25.6％25.6%
1212 无异常No abnormality 13.2％13.2% 24twenty four 无异常No abnormality 32.4％32.4%
本例的胎儿游离核酸富集方法，同样采用深圳华大基因股份有限公司的联合探针锚定聚合测序法的NIFTY建库试剂盒(货号BOX3)进行文库制备。与试剂盒产品说明书中操作方法不同的是，如图1所示，在血浆游离DNA片段末端修复、加A以及加接头之后，按照本例的胎儿游离核酸富集方法进行两次PCR扩增，即第一次扩增(也称“第一轮PCR”)和第二次扩增(也称“第二轮PCR”)，然后再进行后续的建库和测序，两次PCR扩增之前和之后的步骤都与现有方法相同，参考NIFTY建库试剂盒说明书，最终测序下机数据的分析也与现有方法相同，参考专利“胎儿遗传异常的无创性检测”(授权公告号CN103403183B)。In the fetal free nucleic acid enrichment method of this example, the library preparation was also carried out using the NIFTY library kit (Cat. No. BOX3) of the joint probe anchor polymerization sequencing method of Shenzhen Huada Gene Co., Ltd. Different from the method of operation in the kit product specification, as shown in FIG. 1 , after the end of the plasma free DNA fragment is repaired, A is added, and the linker is added, two PCR amplifications are performed according to the fetal free nucleic acid enrichment method of the present example. That is, the first amplification (also called "first round PCR") and the second amplification (also called "second round PCR"), followed by subsequent database construction and sequencing, two PCR amplifications before and The subsequent steps are the same as the existing methods. Referring to the NIFTY library kit specification, the final sequencing data is also the same as the existing method, and the patent "non-invasive detection of fetal genetic abnormality" (authorization bulletin number CN103403183B) is referred to.
本例的胎儿游离核酸富集方法及前后相关的建库和测序的具体步骤如下：The specific steps of the fetal free nucleic acid enrichment method and the related database construction and sequencing in this example are as follows:
(1)末端修复和加A(1) End repair and add A
末端修复加A反应体系为：cfDNA 40μL、末端修复反应液9μL、末端修复混合酶1μL，总计50μL。所用试剂来源于深圳华大基因股份有限公司的NIFTY建库试剂盒(货号BOX3)。The end-repair plus A reaction system was: 40 μL of cfDNA, 9 μL of the terminal repair reaction solution, and 1 μL of the terminal repair mixed enzyme, totaling 50 μL. The reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
混匀后进行反应，反应条件为：37℃15min，65℃15min。After mixing, the reaction was carried out under the conditions of 37 ° C for 15 min and 65 ° C for 15 min.
(2)加接头(2) Add connector
接头连接反应体系为：末端修复加A反应产物50μL、连接反应液24μL、1μM的测序接头5μL、连接酶1μL，总计80μL。所用试剂来源于深圳华大基因股份有限公司的NIFTY建库试剂盒(货号BOX3)。The ligation reaction system was: end-repair plus 50 μL of the A reaction product, 24 μL of the reaction mixture, 5 μL of the sequencing linker of 1 μM, and 1 μL of the ligase, totaling 80 μL. The reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
反应条件：23℃30min。Reaction conditions: 23 ° C for 30 min.
反应完成后，加入60μL的AXYGEN纯化磁珠进行纯化，得到的DNA溶解于20μL蒸馏水中。AXYGEN纯化磁珠购自美国康宁公司MAG-FRAG-I-50，具体纯化步骤参考产品说明书，在此不累述。After completion of the reaction, 60 μL of AXYGEN purified magnetic beads were added for purification, and the obtained DNA was dissolved in 20 μL of distilled water. The AXYGEN purified magnetic beads were purchased from Corning Incorporated MAG-FRAG-I-50. The specific purification steps are referred to the product specifications and are not described herein.
(3)两次PCR扩增(3) Two PCR amplifications
第一次PCR扩增，即线性扩增，线性扩增的反应体系包括：PCR反应混合物25μL、10μM的通用引物1用量2.5μL、AXYGEN纯化产物20μL，总计47.5μL。其中，通用引物1即正向引物。所用试剂来源于深圳华大基因股份有限公司的NIFTY建库试剂盒(货号BOX3)。The first PCR amplification, that is, linear amplification, linear amplification reaction system includes: 25 μL of PCR reaction mixture, 2.5 μL of 10 μM universal primer 1 and 20 μL of AXYGEN purified product, totaling 47.5 μL. Among them, the universal primer 1 is a forward primer. The reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
PCR反应条件：98℃预变性2min；然后进行20个循环：98℃变性15s、58℃退火30s、72℃延伸30s；循环结束后，72℃延伸5min。即获得线性扩增产物。PCR reaction conditions: pre-denaturation at 98 ° C for 2 min; then 20 cycles: denaturation at 98 ° C for 15 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 30 s; after the end of the cycle, extension at 72 ° C for 5 min. That is, a linear amplification product is obtained.
第二次PCR扩增，即指数扩增，指数扩增的反应体系包括：线性扩增的产物47.5μL、10μM的通用引物2用量2.5μL，总计50μL。其中，通用引物2即反向引物。所用试剂来源于深圳华大基因股份有限公司的NIFTY建库试剂盒(货号BOX3)。The second PCR amplification, ie, the exponential amplification reaction system, includes: linearly amplified product 47.5 μL, 10 μM universal primer 2 dosage 2.5 μL, totaling 50 μL. Among them, the universal primer 2 is a reverse primer. The reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
PCR反应条件：98℃预变性2min；然后进行10个循环：98℃变性15s、58℃退火30s、72℃延伸30s；循环结束后，72℃延伸5min。即获得指数扩增产物。PCR reaction conditions: pre-denaturation at 98 ° C for 2 min; then 10 cycles: denaturation at 98 ° C for 15 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 30 s; after the end of the cycle, extension at 72 ° C for 5 min. That is, an exponential amplification product is obtained.
反应完成后，向指数扩增产物中加入60μL的AXYGEN纯化磁珠进行纯化，得到的DNA溶解于20μL蒸馏水中，即获得胎儿游离DNA的富集产物。After completion of the reaction, 60 μL of AXYGEN purified magnetic beads were added to the exponential amplification product for purification, and the obtained DNA was dissolved in 20 μL of distilled water to obtain an enriched product of fetal free DNA.
(4)环化(4) Cyclization
取160ng胎儿游离DNA的富集产物，补水至48μL，98℃变性5分钟，立即插冰上冷却5min，加入13μL环化缓冲液，然后加入1μL连接酶，37℃反应30min，获得环化产物，于-20℃冰箱待用。所用试剂来源于深圳华大基因股份有限公司的NIFTY建库试剂盒(货号BOX3)。Take 160 ng fetal free DNA enrichment product, hydrate to 48 μL, denaturation at 98 ° C for 5 minutes, immediately freeze on ice for 5 min, add 13 μL of cyclization buffer, then add 1 μL of ligase, react at 37 ° C for 30 min to obtain cyclized product. Wait at -20 ° C in the refrigerator. The reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
(5)DNA纳米球(DNB)制备(5) Preparation of DNA nanospheres (DNB)
DNA纳米球即用于测序的DNB，取10μL环化产物，加入2μL DNB制备缓冲液和1μL酶，37℃反应30分钟，获得DNB。所用试剂来源于深圳华大基因股份有限公司的NIFTY建库试剂盒(货号BOX3)。DNA nanospheres were used for sequencing of DNB, 10 μL of cyclized product was taken, 2 μL of DNB preparation buffer and 1 μL of enzyme were added, and reacted at 37 ° C for 30 minutes to obtain DNB. The reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
(6)上机测序(6) Sequencing on the machine
将获得的DNB使用高通量测序仪BGISEQ-500进行测序，测序类型双端测序50bp，标签10bp。The obtained DNB was sequenced using a high-throughput sequencer BGISEQ-500, sequencing type double-end sequencing 50 bp, label 10 bp.
(7)数据分析(7) Data analysis
将步骤(6)获得的测序数据通过过滤后使用bwa进行比对，得到的数据进行全基因组分析，包括染色体异常、胎儿性别、胎儿游离DNA浓度等，具体步骤参照专利“胎儿遗传异常的无创性检测”，授权公告号CN103403183B，公开的方法进行。采用本例的胎儿游离DNA富集方法进行文库构建、测序的下机数据分析结果如表2所示，表2统计了测序分析的胎儿游离DNA的浓度以及染色体异常情况。胎儿性别检测结果显示，24个样本都是男胎，与实际情况相符。The sequencing data obtained in step (6) is filtered and then compared using bwa, and the obtained data is subjected to genome-wide analysis, including chromosomal abnormality, fetal sex, fetal free DNA concentration, etc., and the specific steps refer to the patent “non-invasiveness of fetal genetic abnormality”. Detection", authorization notice number CN103403183B, the method disclosed. The results of the data analysis of the library construction and sequencing using the fetal free DNA enrichment method of this example are shown in Table 2. Table 2 summarizes the concentration of fetal free DNA and chromosomal abnormalities by sequencing analysis. Fetal gender test results showed that 24 samples were male, which was consistent with the actual situation.
表2 基于胎儿游离DNA富集方法建库测序分析获得的胎儿游离DNA浓度Table 2 Fetal free DNA concentration obtained by sequencing analysis based on fetal free DNA enrichment method
样本编号Sample number 染色体异常Chromosomal abnormality 胎儿游离DNA浓度Fetal free DNA concentration 样本编号Sample number 染色体异常Chromosomal abnormality 胎儿游离DNA浓度Fetal free DNA concentration
11 无异常No abnormality 6.6％6.6% 1313 无异常No abnormality 18.7％18.7%
22 无异常No abnormality 8.6％8.6% 1414 无异常No abnormality 21.1％21.1%
33 无异常No abnormality 9.8％9.8% 1515 无异常No abnormality 23.7％23.7%
44 无异常No abnormality 14.3％14.3% 1616 无异常No abnormality 21.0％21.0%
55 无异常No abnormality 13.7％13.7% 1717 无异常No abnormality 22.1％22.1%
66 无异常No abnormality 13.8％13.8% 1818 无异常No abnormality 25.3％25.3%
77 无异常No abnormality 14.6％14.6% 1919 T21T21 23.3％23.3%
88 无异常No abnormality 13.7％13.7% 2020 无异常No abnormality 22.2％22.2%
99 无异常No abnormality 14.6％14.6% 21twenty one T18T18 22.5％22.5%
1010 无异常No abnormality 16.5％16.5% 22twenty two 无异常No abnormality 25.0％25.0%
1111 无异常No abnormality 16.5％16.5% 23twenty three 无异常No abnormality 27.6％27.6%
1212 无异常No abnormality 16.0％16.0% 24twenty four 无异常No abnormality 36.0％36.0%
对比试验：Comparative Test:
为了排除不是扩增循环数增加带来的胎儿浓度提高的原因，本例进一步在胎儿游离DNA富集方法的基础上进行了对比试验，即获得加接头后的磁珠纯化产物后，直接对磁珠纯化的加接头产物进行30个循环的常规PCR扩增，PCR反应体系中直接同时加入通用引物1和通用引物2，加入的量与本例的胎儿游离DNA富集方法中“(3)两次PCR扩增”相同，即反应体系包括：PCR反应混合物25μL、10μM的通用引物1用量2.5μL、10μM的通用引物2用量2.5μL、AXYGEN纯化产物20μL，总计50μL。In order to eliminate the cause of the increase in fetal concentration caused by the increase in the number of amplification cycles, this example further carried out a comparative test on the basis of the method of fetal free DNA enrichment, that is, after obtaining the purified product of the magnetic beads after the addition of the linker, directly to the magnetic The bead-purified addition product was subjected to 30 cycles of conventional PCR amplification, and the universal primer 1 and the universal primer 2 were directly added to the PCR reaction system, and the amount of addition was the same as the fetal free DNA enrichment method of this example "(3) The sub-PCR amplification was the same, that is, the reaction system included 25 μL of the PCR reaction mixture, 2.5 μL of 10 μM universal primer 1 , 2.5 μL of 10 μM universal primer 2, and 20 μL of AXYGEN purified product, totaling 50 μL.
PCR反应条件：98℃预变性2min；然后进行30个循环：98℃变性15s、58℃退火30s、72℃延伸30s；循环结束后，72℃延伸5min。获得对比试验的PCR 扩增产物。PCR reaction conditions: pre-denaturation at 98 ° C for 2 min; then 30 cycles: denaturation at 98 ° C for 15 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 30 s; after the end of the cycle, extension at 72 ° C for 5 min. A PCR amplification product of the comparative test was obtained.
反应完成后，向对比试验的PCR扩增产物中加入60μL的AXYGEN纯化磁珠进行纯化，得到的DNA溶解于20μL蒸馏水中，即纯化的对比试验PCR扩增产物。After the completion of the reaction, 60 μL of AXYGEN purified magnetic beads were added to the PCR amplification product of the comparative test for purification, and the obtained DNA was dissolved in 20 μL of distilled water, that is, a purified comparative test PCR amplification product.
其中，30个循环的PCR扩增是为了使其扩增循环数与本例的胎儿游离核酸富集方法两次PCR扩增的总循环数相等，即“(3)两次PCR扩增”中第一次扩增的20个循环加上第二次扩增的10个循环。Among them, 30 cycles of PCR amplification are to make the number of amplification cycles equal to the total number of cycles of two PCR amplifications of the fetal free nucleic acid enrichment method of this example, that is, "(3) two PCR amplifications" 20 cycles of the first amplification plus 10 cycles of the second amplification.
后续步骤，包括环化、DNA纳米球制备、上机测序和数据分析都与本例的胎儿游离DNA富集方法的建库、测序和数据分析相同，即参照“(4)环化”至“(7)数据分析”进行。所用试剂来源于深圳华大基因股份有限公司的NIFTY建库试剂盒(货号BOX3)。Subsequent steps, including cyclization, DNA nanosphere preparation, sequencing on the machine, and data analysis are the same as the database construction, sequencing, and data analysis of the fetal free DNA enrichment method of this example, that is, refer to "(4) cyclization" to " (7) Data analysis is performed. The reagent used was obtained from NIFTY Library Kit (No. BOX3) of Shenzhen Huada Gene Co., Ltd.
对比试验的下机数据分析结果如表3所示，表3统计了测序分析的胎儿游离DNA的浓度以及染色体异常情况。胎儿性别检测结果显示，24个样本都是男胎，与实际情况相符。The results of the comparative data analysis of the comparative test are shown in Table 3. Table 3 summarizes the concentration of fetal free DNA and chromosomal abnormalities by sequencing analysis. Fetal gender test results showed that 24 samples were male, which was consistent with the actual situation.
表3 对比试验测序分析获得的胎儿游离DNA浓度及染色体情况Table 3 Comparison of fetal free DNA concentration and chromosomal status obtained by sequencing analysis
样本编号Sample number 染色体异常Chromosomal abnormality 胎儿游离DNA浓度Fetal free DNA concentration 样本编号Sample number 染色体异常Chromosomal abnormality 胎儿游离DNA浓度Fetal free DNA concentration
11 无异常No abnormality 4.2％4.2% 1313 无异常No abnormality 14.2％14.2%
22 无异常No abnormality 5.7％5.7% 1414 无异常No abnormality 15.4％15.4%
33 无异常No abnormality 6.9％6.9% 1515 无异常No abnormality 18.4％18.4%
44 无异常No abnormality 9.5％9.5% 1616 无异常No abnormality 16.4％16.4%
55 无异常No abnormality 9.6％9.6% 1717 无异常No abnormality 16.7％16.7%
66 无异常No abnormality 9.8％9.8% 1818 无异常No abnormality 19.5％19.5%
77 无异常No abnormality 10.1％10.1% 1919 T21T21 19.7％19.7%
88 无异常No abnormality 10.5％10.5% 2020 无异常No abnormality 20.2％20.2%
99 无异常No abnormality 10.4％10.4% 21twenty one T18T18 21.2％21.2%
1010 无异常No abnormality 12.5％12.5% 22twenty two 无异常No abnormality 22.7％22.7%
1111 无异常No abnormality 12.5％12.5% 23twenty three 无异常No abnormality 25.5％25.5%
1212 无异常No abnormality 13.1％13.1% 24twenty four 无异常No abnormality 32.4％32.4%
需要说明的是，本例的胎儿游离核酸富集方法中，即“(3)两次PCR扩增”中，通用引物1即正向引物，通用引物2即反向引物，是NIFTY建库试剂盒中采用的引物。本例的所有试验中，包括本例的胎儿游离核酸富集方法、现有方法即NIFTY建库试剂盒、以及对比试验，都采用相同的引物和试剂，唯一的不同在于PCR扩增步骤。本例的胎儿游离核酸富集方法是分两次进行PCR扩增， 现有方法只进行12个循环的指数扩增，对比试验进行30个循环的指数扩增。本例的通用引物1为SEQ ID NO.1所示序列，通用引物2为SEQ ID NO.2所示序列；通用引物1的5’端具有磷酸化修饰。It should be noted that in the fetal free nucleic acid enrichment method of the present example, that is, "(3) two-time PCR amplification", the universal primer 1 is a forward primer, and the universal primer 2 is a reverse primer, which is a NIFTY library reagent. Primers used in the cassette. In all the tests of this example, including the fetal free nucleic acid enrichment method of the present example, the existing method, the NIFTY library kit, and the comparative test, the same primers and reagents were used, the only difference being the PCR amplification step. The fetal free nucleic acid enrichment method of this example is performed by PCR amplification in two steps. The existing method only performs exponential amplification of 12 cycles, and the comparative experiment performs exponential amplification of 30 cycles. The universal primer 1 of this example is the sequence shown in SEQ ID NO. 1, and the universal primer 2 is the sequence shown in SEQ ID NO. 2; the universal primer 1 has a phosphorylation modification at the 5' end.
SEQ ID NO.1：5’-GAACGACATGGCTACGA-3’SEQ ID NO. 1: 5'-GAACGACATGGCTACGA-3'
SEQ ID NO.2：5’-TGTGAGCCAAGGAGTTG-3’SEQ ID NO. 2: 5'-TGTGAGCCAAGGAGTTG-3'
对比表1和表2的结果显示，采用先线性扩增再指数扩增的胎儿游离DNA富集方法，即本例的胎儿游离核酸富集方法，最终获得的胎儿游离DNA浓度有不同程度的提高，统计分析图如图2所示。图2是现有方法即NIFTY建库试剂盒建库测序分析的胎儿游离DNA浓度与采用本例胎儿游离核酸富集方法进行建库测序分析的胎儿游离DNA浓度对比分析图，图中，横坐标为NIFTY建库试剂盒建库测序分析的胎儿游离DNA浓度，纵坐标为本例胎儿游离核酸富集方法进行建库测序分析的胎儿游离DNA浓度，虚线为本例的胎儿游离核酸富集方法获得的胎儿游离DNA浓度比现有方法提高倍数的拟合曲线，各点为各样本的胎儿游离DNA浓度；图2的结果显示，本例的胎儿游离核酸富集方法，获得的胎儿游离DNA浓度比现有方法平均提高了约1.224倍，即y＝1.224x，R 2＝0.885。 Comparing the results of Table 1 and Table 2, the fetal free DNA enrichment method using the linear amplification and then exponential amplification, that is, the fetal free nucleic acid enrichment method of this example, the final fetal free DNA concentration is improved to different degrees. The statistical analysis chart is shown in Figure 2. 2 is a comparative analysis of fetal free DNA concentration and the analysis of fetal free DNA concentration by using the fetal free nucleic acid enrichment method for sequencing analysis of the existing method, that is, the NIFTY library construction library sequencing analysis, in the figure, the abscissa For the NIFTY database, the fetal free DNA concentration was analyzed by sequencing. The ordinate was the fetal free DNA concentration method for the fetal free nucleic acid enrichment method. The dotted line was used as the fetal free nucleic acid enrichment method. The fetal free DNA concentration is a fitting curve of the multiple of the existing method, and each point is the fetal free DNA concentration of each sample; the results of FIG. 2 show the fetal free DNA concentration ratio obtained by the fetal free nucleic acid enrichment method of this example. The existing method is improved by an average of about 1.224 times, that is, y=1.224x and R 2 =0.885.
以表1的胎儿游离DNA浓度为初始浓度，对表2的结果进行统计分析，分析不同初始浓度与胎儿游离DNA浓度增加比例的关系，结果如图3所示。图3采用本申请胎儿游离DNA富集方法对胎儿游离DNA进行富集时，胎儿游离DNA原始浓度对富集增加比例的影响统计图，图中，横坐标为基于表1的数据统计的胎儿游离DNA的初始浓度，纵坐标为基于表2的数据统计的相对于初始浓度的胎儿游离DNA富集的比例，虚线为24个样本胎儿游离DNA富集比例的拟合曲线，各点为24个样本胎儿游离DNA富集的比例；图3的结果显示，本申请的胎儿游离DNA富集方法对胎儿游离DNA浓度较低的样本的增加效果更强，胎儿游离DNA浓度增加比例更大，随着胎儿游离DNA浓度的增加，本申请的富集方法对胎儿游离DNA的增加比例有所下降；可见，本例的胎儿游离DNA富集方法对低浓度的胎儿游离DNA具有更好的富集效果。The fetal free DNA concentration in Table 1 was taken as the initial concentration, and the results of Table 2 were statistically analyzed to analyze the relationship between the different initial concentrations and the increase ratio of the fetal free DNA concentration. The results are shown in Fig. 3. Figure 3 is a statistical diagram of the effect of the original concentration of fetal free DNA on the increase ratio of enrichment when the fetal free DNA is enriched by the fetal free DNA enrichment method of the present application. In the figure, the abscissa is the fetal release based on the data of Table 1. The initial concentration of DNA, the ordinate is the ratio of fetal free DNA enrichment relative to the initial concentration based on the data in Table 2, and the dotted line is the fitted curve of the fetal free DNA enrichment ratio of 24 samples, 24 samples at each point. Proportion of fetal free DNA enrichment; the results of Figure 3 show that the fetal free DNA enrichment method of the present application has a stronger effect on the increase of fetal free DNA concentration, and the fetal free DNA concentration increases more, with the fetus The increase of free DNA concentration, the enrichment method of the present application has a decreased proportion of fetal free DNA; it can be seen that the fetal free DNA enrichment method of this example has a better enrichment effect on low concentration of fetal free DNA.
对现有方法和本例的胎儿游离核酸富集方法的产物进行分析，比较不同方法得到的文库***片段的大小分布，结果如图4所示。图4是本例的胎儿游离核酸富集方法获得的片段大小分布与现有方法即NIFTY建库试剂盒建库获得的片段大小分布对比图，图中，横坐标是文库***片段大小，纵坐标是片段比例，实线为现有方法即NIFTY建库试剂盒建库获得的片段大小分布曲线，虚线为本例的胎儿游离核酸富集方法获得的片段大小分布曲线，图4的结果显示，本例 的胎儿游离核酸富集方法的产物其主要片段比例明显大于现有方法，说明本例的胎儿游离核酸富集方法的确能够富集较小的DNA片段，与预期相符。The products of the existing method and the fetal free nucleic acid enrichment method of this example were analyzed, and the size distribution of the library insert obtained by the different methods was compared. The results are shown in Fig. 4. Figure 4 is a comparison of the size distribution of the fragment obtained by the fetal free nucleic acid enrichment method of the present example and the size distribution of the fragment obtained by the existing method, that is, the NIFTY library kit. In the figure, the abscissa is the size of the library insert, and the ordinate. Is the fragment ratio, the solid line is the fragment size distribution curve obtained by the existing method, that is, the NIFTY library building kit, and the dotted line is the fragment size distribution curve obtained by the fetal free nucleic acid enrichment method of the present example, and the result of FIG. 4 shows that The ratio of the main fragment of the fetal free nucleic acid enrichment method is significantly greater than that of the existing method, indicating that the fetal free nucleic acid enrichment method of this example can indeed enrich a small DNA fragment, which is consistent with expectations.
对比表1和表3的结果显示，现有方法即NIFTY建库试剂盒建库测序分析的胎儿游离DNA浓度与对比试验方法测序分析的胎儿游离DNA浓度，两组数据相当，对比试验方法和现有方法相比，最大的区别仅在于提高了PCR扩增的循环数，可见，采用高循环的指数扩增不能够提高胎儿游离DNA的浓度，表1和表3的统计分析图如图5所示。图5是现有方法即NIFTY建库后测序分析的胎儿游离DNA浓度与对比试验方法建库测序分析的胎儿游离DNA浓度对比分析图，图中，横坐标为对比试验方法即PCR扩增循环数为30个循环进行建库测序分析的胎儿游离DNA浓度，纵坐标为NIFTY建库试剂盒建库测序分析的胎儿游离DNA浓度，虚线为两者倍数关系的拟合曲线，各点为各样本的胎儿游离DNA浓度；图5的结果显示，现有方法和对比试验方法扩增获得的胎儿游离DNA浓度无统计学差异y＝0.99x，R 2＝0.98。 Comparing the results of Tables 1 and 3, the existing methods, namely the NIFTY library kit, the analysis of the fetal free DNA concentration and the comparative analysis of the fetal free DNA concentration, the two groups of data are comparable, the comparison test method and present Compared with the method, the biggest difference is only to increase the number of cycles of PCR amplification. It can be seen that the high-cycle exponential amplification can not increase the concentration of fetal free DNA. The statistical analysis of Table 1 and Table 3 is shown in Figure 5. Show. Fig. 5 is a comparative analysis of the fetal free DNA concentration of the existing method, that is, the NIFTY post-sequencing analysis, and the comparison of the fetal free DNA concentration by the comparison test method. In the figure, the abscissa is the comparative test method, that is, the number of PCR amplification cycles. The fetal free DNA concentration was analyzed by 30-cycle sequencing analysis. The ordinate was the fetal free DNA concentration analyzed by the NIFTY library. The dotted line is the fitting curve of the multiple relationship. The points are for each sample. Fetal free DNA concentration; the results in Figure 5 show that there is no statistical difference in the fetal free DNA concentration obtained by the existing method and the comparative test method y = 0.99x, R 2 = 0.98.
对比表2和表3的结果显示，在其它条件都相同的条件下，本例的胎儿游离核酸富集方法采用两步PCR扩增，对比试验采用一步PCR扩增，虽然两者的总的PCR扩增循环数相同，所采用的试剂和引物也都相同，但是，本例的胎儿游离核酸富集方法明显具有更好的胎儿游离DNA富集效果。说明本例的胎儿游离核酸富集方法中，第一次扩增的线性扩增的确能够偏好性地富集小片段DNA，从而提高胎儿游离DNA浓度。Comparing the results of Tables 2 and 3, the fetal free nucleic acid enrichment method of this example was amplified by two-step PCR under the same conditions, and the comparison test was performed by one-step PCR amplification, although the total PCR of the two was performed. The number of amplification cycles is the same, and the reagents and primers used are also the same. However, the fetal free nucleic acid enrichment method of this example has a better fetal free DNA enrichment effect. In the fetal free nucleic acid enrichment method of this example, the linear amplification of the first amplification can indeed preferentially enrich the small fragment DNA, thereby increasing the fetal free DNA concentration.
对比分析表1、表2和表3的数据，结果如图6所示，图中，横坐标依序为24个样本，纵坐标为胎儿游离DNA浓度，每组柱形图中由左至右依序为现有方法即NIFTY建库试剂盒建库测序分析的胎儿游离DNA浓度、对比试验方法测序分析的胎儿游离DNA浓度、本例胎儿游离核酸富集方法进行建库测序分析的胎儿游离DNA浓度；图6的结果显示，现有方法和对比试验方法获得的胎儿游离DNA浓度相当，说明两者对胎儿游离DNA的富集效果相当，而24个样本中，本例胎儿游离核酸富集方法获得的胎儿游离DNA浓度都明显要高于现有方法和对比试验方法，说明本例的胎儿游离核酸富集方法对胎儿游离DNA的富集效果明显，能够提高胎儿游离DNA的比例，这对提高胎儿游离核酸检测准确性，降低无创产前检测假阴性，提高检测成功率具有重要意义。Comparing and analyzing the data of Table 1, Table 2 and Table 3, the results are shown in Fig. 6. In the figure, the abscissa is 24 samples in sequence, and the ordinate is the fetal free DNA concentration, from left to right in each group of histograms. The fetal free DNA concentration of the existing method, that is, the NIFTY library construction library sequencing analysis, the fetal free DNA concentration of the comparative test method, and the fetus free DNA enrichment method for the fetal free DNA analysis Concentration; the results of Figure 6 show that the concentration of fetal free DNA obtained by the existing method and the comparative test method is equivalent, indicating that the enrichment effect of fetal free DNA is equivalent, and in 24 samples, the fetal free nucleic acid enrichment method The obtained fetal free DNA concentration is significantly higher than the existing methods and comparative test methods, indicating that the fetal free nucleic acid enrichment method of this example has a significant enrichment effect on fetal free DNA, and can increase the ratio of fetal free DNA, which improves The accuracy of fetal free nucleic acid detection, reducing false negatives in non-invasive prenatal testing, and improving the success rate of detection is of great significance.
以上内容是结合具体的实施方式对本申请所作的进一步详细说明，不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干简单推演或替换。The above content is a further detailed description of the present application in conjunction with the specific embodiments, and the specific implementation of the present application is not limited to the description. For the ordinary person skilled in the art to which the present invention pertains, a number of simple deductions or substitutions may be made without departing from the spirit of the present application.

Claims (19)

  1. 一种胎儿游离核酸的富集方法，其特征在于：包括对含有胎儿游离核酸的样品进行PCR扩增，获得胎儿游离核酸富集的产物；A method for enriching fetal free nucleic acid, comprising: performing PCR amplification on a sample containing fetal free nucleic acid to obtain a product of fetal free nucleic acid enrichment;
    所述PCR扩增包括先采用引物对中的正向引物或反向引物对含有胎儿游离核酸的样品进行第一次扩增，然后再向第一次扩增产物中加入引物对中的另一引物进行第二次扩增，第二次扩增的产物即胎儿游离核酸富集产物；The PCR amplification comprises first performing a first amplification of a sample containing fetal free nucleic acid using a forward primer or a reverse primer in a primer pair, and then adding another primer pair to the first amplification product. The primer is subjected to a second amplification, and the product of the second amplification is a fetal free nucleic acid-enriched product;
    所述第一次扩增为线性扩增，所述第二次扩增为指数扩增。The first amplification is linear amplification and the second amplification is exponential amplification.
  2. 根据权利要求1所述的富集方法，其特征在于：所述胎儿游离核酸为胎儿游离DNA。The enrichment method according to claim 1, wherein the fetal free nucleic acid is fetal free DNA.
  3. 根据权利要求1或2所述的富集方法，其特征在于：所述第一次扩增的PCR循环数为10-50；优选地，所述第一次扩增的PCR循环数为10-20。The enrichment method according to claim 1 or 2, wherein the number of PCR cycles of the first amplification is 10-50; preferably, the number of PCR cycles of the first amplification is 10- 20.
  4. 根据权利要求3所述的富集方法，其特征在于：所述第一次扩增的PCR反应条件包括，98℃预变性2min，然后进入10-20或10-50个循环：98℃变性15s、58℃退火30s、72℃延伸30s，循环结束后，72℃延伸5min。The enrichment method according to claim 3, wherein the PCR reaction conditions of the first amplification comprise pre-denaturation at 98 ° C for 2 min, and then enter 10-20 or 10-50 cycles: denaturation at 98 ° C for 15 s. Annealing at 58 ° C for 30 s, 72 ° C for 30 s, after the end of the cycle, extending at 72 ° C for 5 min.
  5. 根据权利要求1-3任一项所述的富集方法，其特征在于：所述第二次扩增的PCR循环数为10-20；优选地，所述第二次扩增的PCR循环数为10-15。The enrichment method according to any one of claims 1 to 3, wherein the number of PCR cycles of the second amplification is 10-20; preferably, the number of PCR cycles of the second amplification It is 10-15.
  6. 根据权利要求5所述的富集方法，其特征在于：所述第二次扩增的PCR反应条件包括，98℃预变性2min，然后进入10-15或10-20个循环：98℃变性15s、58℃退火30s、72℃延伸30s，循环结束后，72℃延伸5min。The enrichment method according to claim 5, wherein the PCR reaction conditions of the second amplification comprise pre-denaturation at 98 ° C for 2 min, and then enter 10-15 or 10-20 cycles: denaturation at 98 ° C for 15 s. Annealing at 58 ° C for 30 s, 72 ° C for 30 s, after the end of the cycle, extending at 72 ° C for 5 min.
  7. 根据权利要求1-6任一项所述的富集方法，其特征在于：所述样品为孕妇外周血；优选的，所述样品为孕妇血浆；优选的，所述样品为孕妇血浆游离核酸。The enrichment method according to any one of claims 1 to 6, wherein the sample is maternal peripheral blood; preferably, the sample is maternal plasma; preferably, the sample is maternal plasma free nucleic acid.
  8. 根据权利要求1-6任一项所述的富集方法，其特征在于：所述PCR扩增之前，还包括对样品依序进行末端修复、加A、加接头。The enrichment method according to any one of claims 1 to 6, wherein before the PCR amplification, the method further comprises sequentially repairing the sample, adding A, and adding a joint.
  9. 根据权利要求8所述的富集方法，其特征在于：所述加接头之后，还包括对加接头产物进行纯化，对纯化的加接头产物进行所述PCR扩增；优选的，所述纯化为磁珠纯化。The enrichment method according to claim 8, wherein after the addition of the linker, the method further comprises purifying the addition product, and performing the PCR amplification on the purified addition product; preferably, the purification is Magnetic beads were purified.
  10. 根据权利要求1-9任一项所述的富集方法在胎儿游离核酸检测、制备胎儿游离核酸检测试剂盒或胎儿游离核酸检测装置中的应用。The use of the enrichment method according to any one of claims 1 to 9 in fetal free nucleic acid detection, preparation of fetal free nucleic acid detection kit or fetal free nucleic acid detection device.
  11. 根据权利要求10所述的应用，其特征在于：所述胎儿游离核酸检测、胎儿游离核酸检测试剂盒和胎儿游离核酸检测装置中，所述检测包括胎儿游离核酸浓度检测和/或胎儿染色体遗传异常检测；优选的，所述胎儿染色体遗传异常检测包括胎儿染色体非整倍性检测、胎儿基因拷贝数变异检测和胎儿单基因病 检测中的至少一种。The use according to claim 10, wherein in said fetal free nucleic acid detection, fetal free nucleic acid detection kit and fetal free nucleic acid detection device, said detection comprises fetal free nucleic acid concentration detection and/or fetal chromosomal genetic abnormality Preferably, the fetal chromosomal genetic abnormality detection comprises at least one of fetal chromosome aneuploidy detection, fetal gene copy number variation detection, and fetal monogenic disease detection.
  12. 一种游离核酸测序文库的构建方法，其特征在于：包括采用权利要求1-9任一项所述的富集方法对游离核酸样品进行PCR扩增，然后对PCR扩增产物进行环化，获得所述游离核酸测序文库。A method for constructing a free nucleic acid sequencing library, comprising: performing PCR amplification on a free nucleic acid sample by the enrichment method according to any one of claims 1-9, and then cyclizing the PCR amplification product to obtain The free nucleic acid sequencing library.
  13. 根据权利要求12所述的构建方法，其特征在于：在对PCR扩增产物进行环化之前，还包括对PCR扩增产物进行纯化，对纯化的PCR扩增产物进行环化；优选的，所述纯化为磁珠纯化。The method according to claim 12, characterized in that before the cyclization of the PCR amplification product, the method further comprises purifying the PCR amplification product, and cyclizing the purified PCR amplification product; preferably, Purification is magnetic bead purification.
  14. 根据权利要求12或13所述的构建方法在胎儿游离核酸检测、制备胎儿游离核酸检测试剂盒或胎儿游离核酸检测装置中的应用。The use of the construction method according to claim 12 or 13 in fetal free nucleic acid detection, preparation of fetal free nucleic acid detection kit or fetal free nucleic acid detection device.
  15. 根据权利要求14所述的应用，其特征在于：所述胎儿游离核酸检测、胎儿游离核酸检测试剂盒和胎儿游离核酸检测装置中，所述检测包括胎儿游离核酸浓度检测和/或胎儿染色体遗传异常检测；优选的，所述胎儿染色体遗传异常检测包括胎儿染色体非整倍性检测、胎儿基因拷贝数变异检测和胎儿单基因病检测中的至少一种。The use according to claim 14, wherein said fetal free nucleic acid detection, fetal free nucleic acid detection kit and fetal free nucleic acid detection device, said detection comprises fetal free nucleic acid concentration detection and / or fetal chromosome genetic abnormality Preferably, the fetal chromosomal genetic abnormality detection comprises at least one of fetal chromosome aneuploidy detection, fetal gene copy number variation detection, and fetal monogenic disease detection.
  16. 一种胎儿游离核酸的检测方法，其特征在于：包括采用权利要求1-9任一项所述的富集方法，对含有胎儿游离核酸的样品进行胎儿游离核酸富集，然后对富集产物进行检测。A method for detecting fetal free nucleic acid, comprising: enriching fetal free nucleic acid for a sample containing fetal free nucleic acid by using the enrichment method according to any one of claims 1-9, and then performing enrichment product on the sample Detection.
  17. 根据权利要求16所述的检测方法，其特征在于：所述对富集产物进行检测，包括但不仅限于高通量测序检测。The method of detecting according to claim 16, wherein said detecting the enriched product comprises, but is not limited to, high throughput sequencing detection.
  18. 根据权利要求16所述的检测方法，其特征在于：还包括，对富集产物进行环化，采用环化产物制备DNA纳米球，对DNA纳米球进行测序检测。The detection method according to claim 16, further comprising: cyclizing the enriched product, preparing the DNA nanosphere by using the cyclized product, and performing sequencing detection on the DNA nanosphere.
  19. 根据权利要求18所述的检测方法，其特征在于：在对富集产物进行环化之前，还包括对富集产物进行纯化，对纯化的富集产物进行环化；优选的，所述纯化为磁珠纯化。The detection method according to claim 18, further comprising: prior to cyclizing the enriched product, purifying the enriched product, and cyclizing the purified enriched product; preferably, the purifying is Magnetic beads were purified.
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