CN111918965A - Enrichment method of fetal free nucleic acid and application thereof - Google Patents
Enrichment method of fetal free nucleic acid and application thereof Download PDFInfo
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Abstract
A fetal free nucleic acid enrichment method comprises the steps of carrying out PCR amplification on a sample containing fetal free nucleic acid to obtain a fetal free nucleic acid enrichment product; the PCR amplification comprises the steps of firstly carrying out first linear amplification on a sample by adopting a forward primer or a reverse primer of a primer pair, and then adding another primer into a first amplification product to carry out second exponential amplification, thus obtaining a fetal free nucleic acid enrichment product.
Description
The application relates to the field of nucleic acid enrichment, in particular to a fetal free nucleic acid enrichment method and application thereof.
In 1997, Lo et al extracted plasma free DNA (abbreviated cfDNA) from pregnant woman plasma and serum, used Y chromosome specificity for fetal sex diagnosis, and first confirmed the presence of fetal free DNA in maternal plasma, which is an important finding that new eosin is brought to the field of noninvasive prenatal diagnosis. The mechanism of the origin of fetal free DNA is not well understood, but it is believed that placental trophoblast cells release free fetal DNA following apoptosis. Fetal free DNA completely disappeared from the mother within 2 hours after delivery. The fetal free DNA can be used for fetal sex determination, chromosome abnormality detection, RHD blood type judgment and the like. With the development of a new generation of sequencing technology, cfDNA is successfully applied to prenatal noninvasive fetal chromosome aneuploidy screening, and fetal chromosome abnormality is detected by the separation of pregnant woman plasma free DNA and high-throughput sequencing.
The current methods for prenatal noninvasive detection by high-throughput sequencing can be divided into three categories, namely methods based on whole genomes, based on SNP loci and based on target region enrichment. The method based on the whole genome can detect all chromosome abnormalities, so the method is widely applied, such as NIFTY product adopting whole genome sequencing for Huada gene, but the concentration of fetal free DNA in a plasma sample is required to be higher than 3.5%, and the fetal free DNA with the concentration lower than 3.5% can cause false negative, thus the result detection is inaccurate. In large sample statistics, the fetal free DNA concentration in 1% of plasma samples is less than 3.5%, so this sample cannot be detected by the whole genome method, and it is necessary to re-draw blood and then re-perform non-invasive prenatal detection. The method brings a challenge to noninvasive prenatal detection, the concentration of free fetal DNA rises along with the increase of the gestational period, so that the problem of low concentration of free fetal DNA can be solved to a certain extent by delaying blood drawing, but the detection cost is undoubtedly increased, the anxiety of puerperae is caused, and therefore, the improvement of the concentration of free fetal DNA without changing the existing conditions has important significance to noninvasive prenatal detection. In addition, in some detection of the dominant monogenic diseases, false negative results occur due to low fetal free DNA concentration, so that the improvement of the fetal free DNA concentration is also significant for accurately detecting the diseases.
Disclosure of Invention
The purpose of the application is to provide a novel enrichment method of fetal free nucleic acid and application thereof.
The following technical scheme is adopted in the application:
one aspect of the present application discloses a method for enriching fetal free nucleic acid, which comprises performing PCR amplification on a sample containing fetal free nucleic acid to obtain a product enriched in fetal free nucleic acid; wherein, the PCR amplification comprises the steps of firstly adopting a forward primer or a reverse primer in a primer pair to carry out primary amplification on a sample containing the fetal free nucleic acid, then adding the other primer in the primer pair into a primary amplification product to carry out secondary amplification, and obtaining a product of the secondary amplification, namely a fetal free nucleic acid enrichment product; wherein the first amplification is linear amplification and the second amplification is exponential amplification.
Preferably, the fetal-free nucleic acid is fetal-free DNA.
It should be noted that the present application was completed based on the following findings of the inventors: through a large amount of experimental researches, the inventor of the application finds that PCR linear amplification has obvious preference on short-fragment nucleic acid. Because the fetal free DNA fragments in the plasma free DNA of the pregnant women are generally shorter than the fetal free DNA fragments, the inventor provides a method for enriching the fetal free nucleic acid, and creatively adopts two times of PCR amplification to realize the enrichment of the fetal free nucleic acid. Wherein, the first amplification only adds a forward primer or a reverse primer in an amplification system, only linear amplification can be carried out because of only the forward primer or the reverse primer, and the step has obvious preference on shorter fetal free nucleic acid and can obtain more fetal free nucleic acid fragments; and then carrying out second amplification, wherein during the second amplification, the other primer in the primer pair is added into the product obtained by the first amplification, and the other primer and the primer remained in the product obtained by the first amplification form a complete primer pair, so that the exponential amplification can be carried out.
Preferably, the number of PCR cycles for the first amplification is 10 to 50.
Preferably, the number of PCR cycles for the first amplification is 10 to 20.
Preferably, the PCR reaction conditions for the first amplification include pre-denaturation at 98 ℃ for 2min, followed by 10-20 or 10-50 cycles: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 5min after circulation is finished.
Preferably, the number of PCR cycles for the second amplification is 10 to 20.
Preferably, the number of PCR cycles for the second amplification is 10 to 15.
Preferably, the PCR reaction conditions for the second amplification include pre-denaturation at 98 ℃ for 2min, followed by 10-15 or 10-20 cycles: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 5min after circulation is finished.
Preferably, in the fetal free nucleic acid enrichment method of the present application, the sample containing fetal free nucleic acids is peripheral blood of a pregnant woman.
More preferably, the sample containing fetal free nucleic acids is maternal plasma.
More preferably, the sample containing fetal free nucleic acids is maternal plasma free nucleic acids.
Preferably, the fetal free nucleic acid enrichment method of the present application further comprises, prior to the PCR amplification, sequentially performing end repair, a-addition, and a-adapter on the sample.
It should be noted that the end repair, the addition of a, and the addition of a linker are performed on the sample, and the purpose is to construct a library for the subsequent purposes, so that the constructed library has enriched fetal free nucleic acids, which facilitates the detection of the fetal free nucleic acids. Also, after the adapter is added, for PCR amplification, only one forward primer or reverse primer of the universal primer may be used for the first amplification, and another primer may be added for the second amplification. If the adapter is not added, the sample is directly subjected to PCR amplification, and the forward primer or reverse primer added for the first amplification may be more than one primer, for example, multiple forward primers or multiple reverse primers may be added, and is not particularly limited herein.
Preferably, the fetal free nucleic acid enrichment method of the present application further comprises, after the ligation, purifying the ligated product and performing PCR amplification on the purified ligated product.
Preferably, the adaptor product is purified using magnetic beads.
The application also discloses application of the enrichment method in detection of the fetal free nucleic acid, preparation of a kit for detecting the fetal free nucleic acid or a device for detecting the fetal free nucleic acid.
Preferably, in the application of the fetal episomal nucleic acid enrichment method of the present application, each detection comprises detection of fetal episomal nucleic acid concentration and/or detection of fetal chromosomal genetic abnormality; wherein the detection of the genetic abnormality of the fetal chromosome comprises at least one of detection of aneuploidy of the fetal chromosome, detection of copy number variation of the fetal gene and detection of fetal monogenic diseases.
The fetal free nucleic acid enrichment method 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, so that the subsequent detection of the fetal free nucleic acid is facilitated. Such as detection of fetal free nucleic acid concentration, detection of genetic abnormality of fetal chromosomes, etc., wherein the detection of genetic abnormality of fetal chromosomes includes detection of fetal chromosomal aneuploidy, detection of fetal gene copy number variation, detection of fetal monogenopathy, etc. Of course, according to the guidance of the fetal free nucleic acid enrichment method of the present application, related reagents can also be combined into a corresponding fetal free nucleic acid detection kit; or according to the invention idea of the fetal free nucleic acid enrichment method, a fetal free nucleic acid detection device is specially developed; and is not particularly limited herein.
The application further discloses a method for constructing the free nucleic acid sequencing library, which comprises the steps of carrying out PCR amplification on a free nucleic acid sample by adopting the fetal free nucleic acid enrichment method, and then carrying out cyclization on a PCR amplification product to obtain the free nucleic acid sequencing library. Wherein, the free nucleic acid sample refers to a sample containing fetal free nucleic acid.
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 free nucleic acid sequencing library construction method of the present application is particularly suitable for sequencing detection of fetal free nucleic acid, such as analysis of fetal free nucleic acid concentration, fetal chromosomal aneuploidy variation, fetal gene copy number variation, fetal single-gene-related disease variation, etc., because the fetal free nucleic acid enrichment method of the present application is used, and a large amount of fetal free nucleic acid is enriched in the library finally constructed.
Preferably, in the method for constructing an isolated nucleic acid sequencing library, before circularizing the PCR amplification product, the method further comprises purifying the PCR amplification product and circularizing the purified PCR amplification product.
Preferably, the PCR amplification product is purified by using magnetic beads.
The application also discloses an application of the method for constructing the free nucleic acid sequencing library in detection of free nucleic acid in fetus, preparation of a kit for detecting free nucleic acid in fetus or a device for detecting free nucleic acid in fetus.
Preferably, in the application of the method for constructing the episomal nucleic acid sequencing library, each detection comprises detection of fetal episomal nucleic acid concentration and/or detection of fetal chromosomal genetic abnormality; wherein the detection of the genetic abnormality of the fetal chromosome comprises at least one of detection of aneuploidy of the fetal chromosome, detection of copy number variation of the fetal gene and detection of fetal monogenic diseases.
It can be understood that, the method for constructing the free nucleic acid sequencing library of the present application adopts the method for enriching fetal free nucleic acid of the present application, and a large amount of fetal free nucleic acid is enriched in the library finally constructed, so that the method can be applied to detection of fetal free nucleic acid concentration, detection of fetal chromosomal aneuploidy, detection of fetal gene copy number variation, detection of fetal single-gene diseases, and the like. Of course, according to the guidance of the method for constructing the free nucleic acid sequencing library, related reagents can be combined into a corresponding fetal free nucleic acid detection kit; or according to the invention idea of the method for constructing the free nucleic acid sequencing library, a fetal free nucleic acid detection device is specially developed; and is not particularly limited herein.
In yet another aspect, the present application discloses a method for detecting fetal free nucleic acid, which comprises, using the method for enriching fetal free nucleic acid of the present application, performing fetal free nucleic acid enrichment on a sample containing fetal free nucleic acid, and then detecting the enriched product.
Preferably, the enriched product is detected, including but not limited to high throughput sequencing.
The key point of the fetal free nucleic acid detection method is that the fetal free nucleic acid enrichment method is adopted to enrich the fetal free nucleic acid in the free nucleic acid, so that the detection sensitivity and accuracy of the fetal free nucleic acid are improved, and a general nucleic acid detection means can be adopted for a specific detection method, wherein the most accurate and comprehensive nucleic acid sequencing is adopted; of course, in addition to nucleic acid sequencing, in the case of fetal free nucleic acid being effectively enriched, other conventional nucleic acid detection methods can be used for detection, and are not specifically limited herein.
Preferably, the fetal free nucleic acid detection method further comprises the steps of cyclizing the enriched product, preparing a DNA nanosphere by using the cyclized product, and sequencing the DNA nanosphere.
It should be noted that, circularizing the enriched product and preparing DNA nanospheres is a nucleic acid sequencing library construction method in an implementation manner of the present application, and it is understood that circularizing and DNA nanosphere preparation are not essential steps for different sequencing platforms or sequencing methods, and are not limited herein.
Preferably, the fetal free nucleic acid detection method of the present application further comprises, before circularizing the enriched product, purifying the enriched product, and circularizing the purified enriched product; preferably, the enriched product is purified by magnetic beads.
The beneficial effect of this application lies in:
the fetal free nucleic acid enrichment method creatively adopts twice PCR amplification to perform fetal free nucleic acid enrichment, makes full use of the amplification preference of PCR linear amplification to short-segment nucleic acid in the first amplification, enables the fetal free nucleic acid of a short segment to be better amplified, and then amplifies the first amplification preference by using the second amplification index, thereby realizing the fetal free nucleic acid enrichment and improving the proportion of the fetal free nucleic acid to the total free nucleic acid. The fetal free nucleic acid enrichment method has important significance for improving the accuracy of fetal free nucleic acid detection, reducing noninvasive prenatal detection false negative and improving the detection success rate.
FIG. 1 is a schematic diagram of a library construction process based on a fetal episomal nucleic acid enrichment method in an embodiment of the present application;
FIG. 2 is a graph of the comparison of the fetal free DNA concentration of the present method, NIFTY post-pooling sequencing analysis in the examples of the present application with the fetal free DNA concentration of the pooling sequencing analysis using the fetal free nucleic acid enrichment method of the examples;
FIG. 3 is a graph showing the effect of the original concentration of fetal free DNA on the enrichment increase ratio when the fetal free DNA is enriched by the fetal free nucleic acid enrichment method of the example in the example of the present application;
FIG. 4 is a graph showing the comparative analysis of the size distribution of the library inserts obtained by the fetal free nucleic acid enrichment method of the example and the size distribution curve of the library inserts obtained by the conventional method, i.e., NIFTY pooling in the examples of the present application;
FIG. 5 is a graph of the comparison of the fetal free DNA concentration of the present method, i.e., NIFTY post-pooling sequencing analysis, to that of a comparative test method for pooling sequencing analysis in the examples of the present application;
FIG. 6 is a graph showing the results of comparing the concentration of fetal free DNA in the present method, i.e., NIFTY post-pooling sequencing analysis, the concentration of fetal free DNA in the pooling sequencing analysis performed by the comparative test method, and the concentration of fetal free DNA in the pooling sequencing analysis performed by the fetal free nucleic acid enrichment method of the example.
The low fetal free DNA concentration in plasma samples is an important factor causing the failure of noninvasive prenatal detection and is also a direct reason for false negative of many fetal monogenic disease detections. Although the concentration of free fetal DNA increases with the increase of the gestational week, on the one hand, it cannot be completely guaranteed that the concentration of free fetal DNA can meet the detection requirement; on the other hand, the non-invasive obstetrical examination or the late discovery of fetal monogenic diseases is not beneficial to subsequent diagnosis and treatment along with the increase of the gestational period, and the anxiety of the puerpera is increased. Therefore, how to increase the concentration of fetal free DNA in a sample without changing the gestational period of the sample or changing the sample per se is important for noninvasive birth detection and fetal genetic disease detection, and the enrichment of fetal free DNA is realized to facilitate subsequent detection.
In this application, "fetal free DNA concentration" means the ratio of the amount of free DNA derived from a fetus to the total amount of free DNA. "fetal free DNA concentration" is sometimes also referred to as "fetal concentration", "fetal free DNA ratio". Similarly, the term "fetal free nucleic acid concentration" as used herein refers to the ratio of the amount of fetal free nucleic acid to the amount of total free nucleic acid. The "fetal free nucleic acid concentration" is sometimes also referred to as "fetal free nucleic acid ratio".
Through a great deal of research, the application finds that the PCR linear amplification has obvious preference on short-fragment nucleic acid. In pregnant plasma free DNA, fetal free DNA fragments are generally shorter than free maternal DNA fragments. Based on the above research and understanding, the present application creatively provides that when a sample containing fetal free nucleic acid is subjected to PCR amplification, a forward primer or a reverse primer in a primer pair is firstly used for first amplification, the preference of linear PCR amplification of a one-way primer to short-segment amplification is utilized, so that the fetal free nucleic acid of a short segment is better amplified, then another primer in the primer pair is added into a product obtained by the first amplification for second amplification, the second amplification is conventional PCR exponential amplification, and the content of the fetal free nucleic acid is further increased, so that the enrichment of the fetal free nucleic acid is realized.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Examples
The enrichment effect of free DNA of fetus was compared by comparing the enrichment method of free nucleic acid of fetus with the existing method using 24 plasma samples of pregnant women carrying male fetus. The pregnant woman plasma free DNA is extracted by a QIAGEN free DNA extraction kit, and the obtained cfDNA is dissolved in 40 mu L of AE solution for subsequent tests.
The existing method directly adopts NIFTY library construction kit (cargo number BOX3) of combined probe anchoring polymerization sequencing method of Shenzhen Hua Dagen GmbH to prepare free nucleic acid library for 24 cases of pregnant woman plasma samples carrying male fetus. The steps comprise repairing the tail end, adding A, adding a joint and carrying out conventional PCR amplification on a product added with the joint, namely simultaneously adding a forward primer and a reverse primer in a PCR reaction for carrying out 12 rounds of exponential amplification, and the specific method refers to the NIFTY library building kit specification and is not described herein in a repeated way. The library obtained was then sequenced on a sequencer BGISEQ-500. Finally, the concentration of fetal free DNA, chromosomal abnormality, etc. in each sample are analyzed based on the sequencing results, and the specific analysis procedure is carried out by referring to the method disclosed in the patent "noninvasive detection of fetal genetic abnormality" (No. CN103403183B), which is not described herein in detail. The results of the sequencing analysis of the obtained fetal free DNA concentration and chromosomal abnormalities are shown in Table 1.
TABLE 1 fetal free DNA concentration and chromosomal status obtained by sequencing analysis by Current methods
| Sample numbering | Chromosomal abnormalities | Fetal free DNA concentration | Sample numbering | Chromosomal abnormalities | Fetal |
| 1 | No abnormality | 4.3％ | 13 | No abnormality | 14.2％ |
| 2 | No abnormality | 5.9％ | 14 | No abnormality | 15.7％ |
| 3 | No abnormality | 6.8％ | 15 | No abnormality | 18.4％ |
| 4 | No abnormality | 9.6％ | 16 | No abnormality | 16.7％ |
| 5 | No abnormality | 9.6％ | 17 | No abnormality | 16.9％ |
| 6 | No abnormality | 9.8％ | 18 | No abnormality | 19.5％ |
| 7 | No abnormality | 10.0％ | 19 | T21 | 19.5％ |
| 8 | No abnormality | 10.5％ | 20 | No abnormality | 20.3％ |
| 9 | No abnormality | 10.6％ | 21 | T18 | 21.1％ |
| 10 | No abnormality | 12.4％ | 22 | No abnormality | 22.7％ |
| 11 | No abnormality | 12.5％ | 23 | No abnormality | 25.6％ |
| 12 | No abnormality | 13.2％ | 24 | No abnormality | 32.4％ |
The fetal free nucleic acid enrichment method of the embodiment also adopts NIFTY library construction kit (cargo number BOX3) of joint probe anchoring polymerization sequencing method of Shenzhen Hua Dagen GmbH for library preparation. Unlike the method described in the kit product specification, as shown in fig. 1, after the end of 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 this example, namely, the first amplification (also called "first round PCR") and the second amplification (also called "second round PCR"), and then the subsequent banking and sequencing are performed, wherein the steps before and after the two PCR amplifications are the same as the existing method, and referring to the NIFTY banking kit specification, the analysis of the final sequencing off-machine data is also the same as the existing method, and referring to the patent "noninvasive detection of fetal genetic abnormality" (No. CN 103403183B).
The specific steps of the fetal free nucleic acid enrichment method and the related library construction and sequencing are as follows:
(1) end repair and addition of A
The end repair and A reaction system is as follows: 40. mu.L of cfDNA, 9. mu.L of the end-repair reaction solution, and 1. mu.L of the end-repair mix enzyme, and a total amount of 50. mu.L. The used reagent is derived from NIFTY library construction kit (cargo number BOX3) of Shenzhen Hua Dagen Gen York Co.
After uniformly mixing, carrying out reaction under the following reaction conditions: 15min at 37 ℃ and 15min at 65 ℃.
(2) Add the piecing
The joint connection reaction system is as follows: the end-repair-and-A reaction product (50. mu.L), the ligation reaction solution (24. mu.L), 1. mu.M sequencing linker (5. mu.L), and ligase (1. mu.L) were added, and the total amount was 80. mu.L. The used reagent is derived from NIFTY library construction kit (cargo number BOX3) of Shenzhen Hua Dagen Gen York Co.
Reaction conditions are as follows: 30min at 23 ℃.
After completion of the reaction, 60. mu.L of AXYGEN purified magnetic beads was added to purify the DNA, and the obtained DNA was dissolved in 20. mu.L of distilled water. AXYGEN purified magnetic beads were purchased from MAG-FRAG-I-50, Corning incorporated, USA, and specific purification procedures were referred to product specifications and will not be described herein.
(3) Two PCR amplifications
Performing first PCR amplification, namely linear amplification, wherein a reaction system of the linear amplification comprises: PCR reaction mixture 25. mu.L, 10. mu.M Universal primer 1 2.5. mu. L, AXYGEN purified product 20. mu.L, totaling 47.5. mu.L. Wherein, the universal primer 1 is the forward primer. The used reagent is derived from NIFTY library construction kit (cargo number BOX3) of Shenzhen Hua Dagen Gen York Co.
And (3) PCR reaction conditions: pre-denaturation at 98 ℃ for 2 min; then 20 cycles were performed: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30 s; after the circulation was completed, the extension was carried out at 72 ℃ for 5 min. Thus obtaining the linear amplification product.
And performing second PCR amplification, namely performing exponential amplification, wherein the reaction system of the exponential amplification comprises: the linear amplification product was 47.5. mu.L, the amount of the universal primer 2 used was 2.5. mu.L at 10. mu.M, and the total amount was 50. mu.L. Wherein, the universal primer 2 is a reverse primer. The used reagent is derived from NIFTY library construction kit (cargo number BOX3) of Shenzhen Hua Dagen Gen York Co.
And (3) PCR reaction conditions: pre-denaturation at 98 ℃ for 2 min; then 10 cycles were performed: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30 s; after the circulation was completed, the extension was carried out at 72 ℃ for 5 min. Thus obtaining exponential amplification products.
After the reaction is finished, 60 μ L of AXYGEN purified magnetic beads are added to the exponential amplification product for purification, and the obtained DNA is dissolved in 20 μ L of distilled water, thus obtaining the enrichment product of fetal free DNA.
(4) Cyclization of
160ng of the enriched product of the fetal free DNA is taken, water is supplemented to 48 mu L, denaturation is carried out for 5 minutes at 98 ℃, ice is immediately inserted for cooling for 5 minutes, 13 mu L of cyclization buffer solution is added, 1 mu L of ligase is then added, reaction is carried out for 30 minutes at 37 ℃, and a cyclization product is obtained and is used in a refrigerator at-20 ℃. The used reagent is derived from NIFTY library construction kit (cargo number BOX3) of Shenzhen Hua Dagen Gen York Co.
(5) DNA Nanosphere (DNB) preparation
DNA nanospheres, namely DNB for sequencing, 10. mu.L of the circularized product was taken, 2. mu.L of DNB preparation buffer and 1. mu.L of enzyme were added, and reaction was carried out at 37 ℃ for 30 minutes to obtain DNB. The used reagent is derived from NIFTY library construction kit (cargo number BOX3) of Shenzhen Hua Dagen Gen York Co.
(6) Sequencing on machine
The obtained DNB is sequenced by using a high-throughput sequencer BGISEQ-500, and the sequencing type double-ended sequencing is 50bp and the tag is 10 bp.
(7) Data analysis
And (3) filtering the sequencing data obtained in the step (6), comparing the filtered sequencing data with bwa, and performing whole genome analysis on the obtained data, wherein the whole genome analysis comprises chromosome abnormality, fetal gender, fetal free DNA concentration and the like, and the specific steps refer to a method disclosed in a patent 'noninvasive detection of fetal genetic abnormality' with an authorization publication No. CN 103403183B. The results of off-line analysis of the library construction and sequencing using the fetal free DNA enrichment method of this example are shown in Table 2, and Table 2 shows the statistics of the concentration of fetal free DNA analyzed by sequencing and the chromosomal abnormality. The results of the fetal sex tests show that 24 samples are male and correspond to the actual conditions.
TABLE 2 fetal free DNA concentration based on fetal free DNA enrichment method library-building sequencing analysis
| Sample numbering | Chromosomal abnormalities | Fetal free DNA concentration | Sample numbering | Chromosomal abnormalities | Fetal |
| 1 | No abnormality | 6.6％ | 13 | No abnormality | 18.7％ |
| 2 | No abnormality | 8.6％ | 14 | No abnormality | 21.1％ |
| 3 | No abnormality | 9.8％ | 15 | No abnormality | 23.7％ |
| 4 | No abnormality | 14.3％ | 16 | No abnormality | 21.0％ |
| 5 | No abnormality | 13.7％ | 17 | No abnormality | 22.1％ |
| 6 | No abnormality | 13.8％ | 18 | No abnormality | 25.3％ |
| 7 | No abnormality | 14.6％ | 19 | T21 | 23.3％ |
| 8 | No abnormality | 13.7％ | 20 | No abnormality | 22.2％ |
| 9 | No abnormality | 14.6％ | 21 | T18 | 22.5％ |
| 10 | No abnormality | 16.5％ | 22 | No abnormality | 25.0％ |
| 11 | No abnormality | 16.5％ | 23 | No abnormality | 27.6％ |
| 12 | No abnormality | 16.0％ | 24 | No abnormality | 36.0％ |
And (3) comparison test:
in order to exclude the reasons that the fetal concentration is not increased due to the increase of amplification cycles, this example further performs a comparative experiment based on the fetal free DNA enrichment method, i.e. after obtaining the purified products of the magnetic beads after adding the linker, the magnetic bead purified products are directly subjected to 30 cycles of conventional PCR amplification, and the universal primer 1 and the universal primer 2 are directly added into the PCR reaction system at the same time, and the amount added is the same as that of the "(3) two PCR amplifications" in the fetal free DNA enrichment method of this example, i.e. the reaction system comprises: PCR reaction mixture 25. mu.L, universal primer 1 at 10. mu.M 2.5. mu.L, universal primer 2 at 10. mu.M 2.5. mu. L, AXYGEN purified product 20. mu.L, totaling 50. mu.L.
And (3) PCR reaction conditions: pre-denaturation at 98 ℃ for 2 min; then 30 cycles were performed: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30 s; after the circulation was completed, the extension was carried out at 72 ℃ for 5 min. PCR amplification products for comparative experiments were obtained.
After the reaction, 60. mu.L of AXYGEN purified magnetic beads were added to the PCR amplification product of the comparative experiment for purification, and the obtained DNA was dissolved in 20. mu.L of distilled water, i.e., the purified PCR amplification product of the comparative experiment.
The 30 cycles of PCR amplification were performed so that the number of amplification cycles was equal to the total number of cycles of two PCR amplifications in the fetal isolated nucleic acid enrichment method of this example, i.e., (3) 20 cycles of the first amplification and 10 cycles of the second amplification in the two PCR amplifications ".
The subsequent steps, including circularization, DNA nanosphere preparation, on-machine sequencing and data analysis, were all the same as the pooling, sequencing and data analysis of the fetal free DNA enrichment method of this example, i.e. performed with reference to "(4) circularization" to "(7) data analysis". The used reagent is derived from NIFTY library construction kit (cargo number BOX3) of Shenzhen Hua Dagen Gen York Co.
The results of the comparative off-line data analysis are shown in Table 3, and Table 3 shows the statistics of the concentration of fetal free DNA analyzed by sequencing and the chromosomal abnormality. The results of the fetal sex tests show that 24 samples are male and correspond to the actual conditions.
TABLE 3 fetal free DNA concentration and chromosomal status obtained by sequencing analysis of the comparative experiments
| Sample numbering | Chromosomal abnormalities | Fetal free DNA concentration | Sample numbering | Chromosomal abnormalities | Fetal |
| 1 | No abnormality | 4.2％ | 13 | No abnormality | 14.2％ |
| 2 | No abnormality | 5.7％ | 14 | No abnormality | 15.4％ |
| 3 | No abnormality | 6.9％ | 15 | No abnormality | 18.4％ |
| 4 | No abnormality | 9.5％ | 16 | No abnormality | 16.4％ |
| 5 | No abnormality | 9.6％ | 17 | No abnormality | 16.7％ |
| 6 | No abnormality | 9.8％ | 18 | No abnormality | 19.5％ |
| 7 | No abnormality | 10.1％ | 19 | T21 | 19.7％ |
| 8 | No abnormality | 10.5％ | 20 | No abnormality | 20.2％ |
| 9 | No abnormality | 10.4％ | 21 | T18 | 21.2％ |
| 10 | No abnormality | 12.5％ | 22 | No abnormality | 22.7％ |
| 11 | No abnormality | 12.5％ | 23 | No abnormality | 25.5％ |
| 12 | No abnormality | 13.1％ | 24 | No abnormality | 32.4％ |
In the fetal free nucleic acid enrichment method of this example, i.e., (3) two PCR amplifications ", the universal primer 1, i.e., the forward primer, and the universal primer 2, i.e., the reverse primer, are primers used in the NIFTY library construction kit. All tests of this example, including the fetal free nucleic acid enrichment method of this example, the current method, i.e., the NIFTY banking kit, and the comparative test, used the same primers and reagents, the only difference being the PCR amplification step. The fetal free nucleic acid enrichment method of this example was performed by PCR amplification in two cycles, and the conventional method performed only 12 cycles of exponential amplification, while the comparative experiment performed 30 cycles of exponential amplification. The general primer 1 of the embodiment is a sequence shown in SEQ ID NO.1, and the general primer 2 is a sequence shown in SEQ ID NO. 2; the 5' end of the universal primer 1 has a phosphorylation modification.
SEQ ID NO.1：5’-GAACGACATGGCTACGA-3’
SEQ ID NO.2：5’-TGTGAGCCAAGGAGTTG-3’
Comparing the results in tables 1 and 2 shows that the concentration of fetal free DNA obtained by the method of fetal free DNA enrichment using linear amplification followed by exponential amplification, i.e., the method of fetal free nucleic acid enrichment of this example, is improved to various degrees, and the statistical analysis chart is shown in FIG. 2. FIG. 2 is a graph showing the comparison between the concentration of free fetal DNA obtained by the conventional method, i.e., the library construction sequencing analysis of the NIFTY library construction kit, and the concentration of free fetal DNA obtained by the library construction sequencing analysis of the NIFTY library construction kit of the present embodiment, wherein the abscissa is the concentration of free fetal DNA obtained by the library construction sequencing analysis of the fetal free nucleic acid enrichment method of the present embodiment, the ordinate is the concentration of free fetal DNA obtained by the library construction sequencing analysis of the fetal free nucleic acid enrichment method of the present embodiment, the dotted line is a fitting curve showing the concentration of free fetal DNA obtained by the fetal free nucleic acid enrichment method of the present embodiment, and each point is the concentration of free fetal DNA of each sample; the results in FIG. 2 show that the concentration of fetal free DNA obtained by the method for enrichment of fetal free nucleic acid of this example is improved by about 1.224 times on average, i.e., y is 1.224x, R is higher than that obtained by the conventional method2＝0.885。
The results of Table 2 were statistically analyzed using the fetal free DNA concentrations of Table 1 as initial concentrations, and the results are shown in FIG. 3, in which the relationship between the initial concentrations and the increase ratios of the fetal free DNA concentrations was analyzed. FIG. 3 is a graph of the effect of the original concentration of fetal free DNA on the enrichment increase ratio when the fetal free DNA enrichment method of the present application is used for enriching fetal free DNA, wherein the abscissa is the initial concentration of fetal free DNA based on the statistics of Table 1, the ordinate is the ratio of fetal free DNA enrichment relative to the initial concentration based on the statistics of Table 2, the dotted line is a curve fitted to the fetal free DNA enrichment ratios of 24 samples, and each point is the ratio of fetal free DNA enrichment of 24 samples; FIG. 3 shows that the fetal free DNA enrichment method of the present application has a stronger effect on the samples with lower fetal free DNA concentration, the fetal free DNA concentration increases more proportionally, and the fetal free DNA concentration increases with the increase of the fetal free DNA concentration, the fetal free DNA increase proportion of the fetal free DNA increases with the enrichment method of the present application decreases; as can be seen, the fetal free DNA enrichment method of this example has a better enrichment effect on low concentrations of fetal free DNA.
The products of the prior art method and the fetal-free nucleic acid enrichment method of this example were analyzed to compare the size distributions of the library inserts obtained by the different methods, and the results are shown in FIG. 4. FIG. 4 is a graph comparing the size distribution of fragments obtained by the method for enrichment of fetal free nucleic acid of this example with the size distribution of fragments obtained by the conventional method, namely, NIFTY pooling kit pooling, wherein the abscissa is the size of the library insert and the ordinate is the fragment ratio, the solid line is the size distribution curve of fragments obtained by the conventional method, namely, NIFTY pooling kit pooling, and the dotted line is the size distribution curve of fragments obtained by the method for enrichment of fetal free nucleic acid of this example, and the results of FIG. 4 show that the product of the method for enrichment of fetal free nucleic acid of this example has a major fragment ratio significantly greater than that of the conventional method, indicating that the method for enrichment of fetal free nucleic acid of this example is indeed capable of enriching smaller DNA fragments, in accordance with expectations.
Comparing the results in tables 1 and 3 shows that the concentration of free fetal DNA analyzed by the prior art method, i.e., NIFTY library kit library sequencing, is equivalent to the concentration of free fetal DNA analyzed by the comparative test method, and the greatest difference between the comparative test method and the prior art method is only that the number of cycles of PCR amplification is increased, thus it can be seen that the concentration of free fetal DNA cannot be increased by exponential amplification with high cycles, and the statistical analysis graphs in tables 1 and 3 are shown in fig. 5. FIG. 5 is a comparison analysis chart of the concentration of free DNA in fetus obtained by sequencing analysis after NIFTY library construction according to the prior art and the concentration of free DNA in fetus obtained by library construction sequencing analysis according to a comparison test method, wherein the abscissa is the concentration of free DNA in fetus obtained by library construction sequencing analysis according to the comparison test method, i.e., the number of PCR amplification cycles is 30 cycles, the ordinate is the concentration of free DNA in fetus obtained by library construction sequencing analysis of the NIFTY library construction kit, the dotted line is a fitting curve of the multiple relation of the two, and each point is the concentration of free DNA in fetus in each sample; the results in FIG. 5 show that there is no statistical difference between the concentration of free fetal DNA obtained by amplification using the prior art method and the comparative test method, y is 0.99x, R2＝0.98。
Comparing the results in tables 2 and 3 shows that, under otherwise identical conditions, the fetal free nucleic acid enrichment method of the present example employs two-step PCR amplification, and the comparison test employs one-step PCR amplification, although the total PCR amplification cycles are the same, and the reagents and primers are the same, the fetal free nucleic acid enrichment method of the present example has significantly better fetal free DNA enrichment effect. It is demonstrated that in the fetal free nucleic acid enrichment method of this example, the linear amplification of the first amplification can indeed preferentially enrich for small pieces of DNA, thereby increasing the fetal free DNA concentration.
Comparing and analyzing the data in the tables 1, 2 and 3, the result is shown in fig. 6, in the figure, the abscissa is 24 samples in sequence, the ordinate is the fetal free DNA concentration, and each group of column charts is the fetal free DNA concentration of the prior method, namely, NIFTY library kit library building sequencing analysis, the fetal free DNA concentration of the comparative test method sequencing analysis, and the fetal free DNA concentration of the fetal free nucleic acid enrichment method library building sequencing analysis in the example, in sequence from left to right; the results of fig. 6 show that the concentrations of the fetal free DNA obtained by the conventional method and the comparative test method are equivalent, which indicates that the concentrations of the fetal free DNA obtained by the conventional method and the comparative test method are equivalent, and that the concentrations of the fetal free DNA obtained by the fetal free nucleic acid enrichment method in the present example are significantly higher than those of the conventional method and the comparative test method in 24 samples, which indicates that the fetal free nucleic acid enrichment method of the present example has significant enrichment effect on the fetal free DNA, can improve the proportion of the fetal free DNA, and has important significance in improving the detection accuracy of the fetal free nucleic acid, reducing the non-invasive prenatal detection false negative, and improving the detection success rate.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
Claims (19)
- A method for enriching fetal free nucleic acid, comprising: comprises carrying out PCR amplification on a sample containing fetal free nucleic acid to obtain a fetal free nucleic acid enriched product;the PCR amplification comprises the steps of firstly carrying out primary amplification on a sample containing fetal free nucleic acid by adopting a forward primer or a reverse primer in a primer pair, then adding the other primer in the primer pair into a primary amplification product to carry out secondary amplification, wherein a product obtained by the secondary amplification is a fetal free nucleic acid enrichment product;the first amplification is linear amplification and the second amplification is exponential amplification.
- The enrichment method according to claim 1, wherein: the fetal-free nucleic acid is fetal-free DNA.
- The enrichment method according to claim 1 or 2, characterized in that: the number of PCR cycles of the first amplification is 10-50; preferably, the number of PCR cycles of the first amplification is 10 to 20.
- The enrichment method according to claim 3, wherein: the PCR reaction conditions for the first amplification include pre-denaturation at 98 ℃ for 2min, and then 10-20 or 10-50 cycles: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 5min after circulation is finished.
- The enrichment method according to any one of claims 1 to 3, wherein: the PCR cycle number of the second amplification is 10-20; preferably, the number of PCR cycles for the second amplification is 10 to 15.
- The enrichment method according to claim 5, wherein: the PCR reaction conditions for the second amplification include pre-denaturation at 98 ℃ for 2min, and then 10-15 or 10-20 cycles: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 5min after circulation is finished.
- The enrichment method according to any one of claims 1 to 6, wherein: the sample is peripheral blood of a pregnant woman; preferably, the sample is maternal plasma; preferably, the sample is maternal plasma free nucleic acid.
- The enrichment method according to any one of claims 1 to 6, wherein: before the PCR amplification, the method also comprises the steps of sequentially carrying out end repair, A addition and joint addition on the sample.
- The enrichment method according to claim 8, wherein: after the adding of the joint, purifying a joint product, and performing the PCR amplification on the purified joint product; preferably, the purification is magnetic bead purification.
- Use of the enrichment method according to any one of claims 1-9 for detection of fetal free nucleic acid, for the preparation of a kit for detection of fetal free nucleic acid or a device for detection of fetal free nucleic acid.
- Use according to claim 10, characterized in that: in the detection of the fetal free nucleic acid, the detection kit of the fetal free nucleic acid and the detection device of the fetal free nucleic acid, the detection comprises the detection of the concentration of the fetal free nucleic acid and/or the detection of the genetic abnormality of the fetal chromosome; preferably, the detection of fetal chromosomal genetic abnormality comprises at least one of detection of fetal chromosomal aneuploidy, detection of fetal gene copy number variation, and detection of fetal monogenic disorder.
- A method for constructing an isolated nucleic acid sequencing library, which is characterized by comprising the following steps: comprising performing PCR amplification on an isolated nucleic acid sample by the enrichment method according to any one of claims 1 to 9, and then circularizing the PCR amplification product to obtain the isolated nucleic acid sequencing library.
- The construction method according to claim 12, wherein: before circularizing the PCR amplification product, purifying the PCR amplification product, and circularizing the purified PCR amplification product; preferably, the purification is magnetic bead purification.
- Use of the method according to claim 12 or 13 for detecting free fetal nucleic acid, preparing a kit for detecting free fetal nucleic acid, or detecting a device for detecting free fetal nucleic acid.
- Use according to claim 14, characterized in that: in the detection of the fetal free nucleic acid, the detection kit of the fetal free nucleic acid and the detection device of the fetal free nucleic acid, the detection comprises the detection of the concentration of the fetal free nucleic acid and/or the detection of the genetic abnormality of the fetal chromosome; preferably, the detection of fetal chromosomal genetic abnormality comprises at least one of detection of fetal chromosomal aneuploidy, detection of fetal gene copy number variation, and detection of fetal monogenic disorder.
- A method for detecting fetal free nucleic acid, comprising: comprising subjecting a sample containing fetal free nucleic acid to fetal free nucleic acid enrichment using the enrichment method of any one of claims 1 to 9 and then detecting the enriched product.
- The detection method according to claim 16, characterized in that: the detection of the enriched product includes, but is not limited to, high-throughput sequencing detection.
- The detection method according to claim 16, characterized in that: and the method also comprises the steps of carrying out cyclization on the enriched product, preparing the DNA nanospheres by adopting the cyclization product, and carrying out sequencing detection on the DNA nanospheres.
- The detection method according to claim 18, characterized in that: before the enriched product is cyclized, purifying the enriched product, and cyclizing the purified enriched product; preferably, the purification is magnetic bead purification.
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| CN103403183B (en) * | 2011-06-29 | 2014-10-15 | 深圳华大基因医学有限公司 | Noninvasive detection of fetal genetic abnormality |
| WO2016095736A1 (en) * | 2014-12-18 | 2016-06-23 | 深圳华大基因研究院 | Target region enrichment method based on multiplex pcr, and reagent |
| CN107083440A (en) * | 2017-06-13 | 2017-08-22 | 深圳华大智造科技有限公司 | Kit for detecting chromosome aneuploidy and preparation method and application thereof |
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| GB201410646D0 (en) * | 2014-06-14 | 2014-07-30 | Illumina Cambridge Ltd | Methods of increasing sequencing accuracy |
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| CN103403183B (en) * | 2011-06-29 | 2014-10-15 | 深圳华大基因医学有限公司 | Noninvasive detection of fetal genetic abnormality |
| WO2016095736A1 (en) * | 2014-12-18 | 2016-06-23 | 深圳华大基因研究院 | Target region enrichment method based on multiplex pcr, and reagent |
| CN107083440A (en) * | 2017-06-13 | 2017-08-22 | 深圳华大智造科技有限公司 | Kit for detecting chromosome aneuploidy and preparation method and application thereof |
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