CN112996926A

CN112996926A - Construction method, detection device and application of target gene library

Info

Publication number: CN112996926A
Application number: CN201880098877.5A
Authority: CN
Inventors: 杨林; 张海萍; 高雅; 陈芳
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd; Shenzhen BGI Life Science Research Institute
Priority date: 2018-12-07
Filing date: 2018-12-07
Publication date: 2021-06-18
Also published as: WO2020113577A1

Abstract

A target gene library construction method, a detection device and application are provided. The target gene library construction method comprises the steps of extracting free RNA from a biological sample of a pregnant woman, wherein the free RNA comprises fetal free RNA; reverse transcribing free RNA into first-strand cDNA; amplifying the first strand cDNA by using a specific primer to obtain a specific amplification product, namely a fetus and maternal differential expression gene, namely a target gene; and carrying out second round PCR amplification on the amplification product by using the universal primer to obtain a target gene library.

Description

Construction method, detection device and application of target gene library

Technical Field

The application relates to the field of gene detection, in particular to a construction method of a target gene library, a detection device and application thereof.

Background

Prenatal diagnosis is an important means for preventing birth defects caused by genetic diseases. However, invasive sampling methods like chorionic villus sampling, amniotic fluid lancing, and cord blood lancing, which are used to obtain fetal genetic information, still pose certain risks. Therefore, the development of a non-invasive prenatal detection method has been an important research direction. During the early years, researchers have focused on how to obtain fetal nucleated red blood cells from the peripheral blood of pregnant women for genetic analysis. However, how to separate and enrich fetal nucleated red blood cells efficiently has been a big bottleneck in the development of the technology. In 1997, Luyuming published a scientific paper on "lancet" that confirmed the presence of free DNA from fetuses in the peripheral plasma of pregnant women by PCR amplification of Y chromosome-specific genes; this finding offers new possibilities for the detection of fetal genotypes by the peripheral blood of pregnant women.

The detection technology based on whole genome sequencing widely applied at present provides an important reference basis for screening of the infants suffering from Down's disease, and also avoids the birth of countless infants suffering from Down's disease. However, the conventional techniques can detect only chromosomal abnormalities, and cannot detect other single-gene mutations. However, the proportion of fetal defects caused by chromosome abnormality is less than 4% of the total number of congenital foolishing defects, and a considerable number of defective fetuses are not caused by chromosome number abnormality but by single gene mutation. Diseases caused by single gene mutation are important diseases affecting the health condition and the life quality of children, and most of the diseases cannot be found in early pregnancy. Although abnormality can be found in B-mode ultrasonography, the abnormality can be found only in the middle or late pregnancy, which brings heavy impact to pregnant women and their families. The most of the fetuses after birth have physiological abnormality, the life is difficult to take care of oneself, and great pain is brought to families.

In recent years, the development of high-throughput sequencing technology provides a new technical platform for noninvasive prenatal detection of monogenic diseases. The detection of the dominant single gene can be carried out by directly detecting the de novo mutation which may be present in the plasma; however, for recessive monogenes, it is difficult to detect whether the fetus carries the mutation information of both parents from the plasma with maternal background, since the mother generally carries the mutation information. Dennis Lo et al 2010 published an article based on maternal plasma free DNA whole genome sequencing combined with parental haplotype analysis to infer fetal genotype, which exemplifies beta thalassemia, and confirmed the application prospect of the method in genetic disease detection. Subsequently, it also published a report of noninvasive fetal beta poor mutation carrier detection based on target region capture, which is similar to the analytical method adopted in the previous article, except that by using the target region capture technique, the sequencing cost is greatly reduced, making it further close to clinical application. The haplotype-based analysis method can not only determine whether the fetus obtains pathogenic mutation from the father, but also realize the judgment of the condition of the inherited allele of the fetus from the mother. However, the method needs to obtain the gene information of parents and probands through experiments, and adopts a complex algorithm to guess whether the fetus is sick, so that the operation is complex, the cost is high, and the actual clinical use requirements cannot be met. Moreover, it is difficult to detect some gene structure variations by short plasma-free DNA fragments.

Disclosure of Invention

The application aims to provide a novel target gene library construction method, a fetal gene detection method based on the library construction method, a fetal gene detection device, a fetal gene detection reagent and application thereof.

The application specifically adopts the following technical scheme:

the first aspect of the present application discloses a method for constructing a target gene library, comprising the following steps: extracting free RNA from a biological sample derived from a pregnant woman, the free RNA comprising fetal free RNA; reverse transcribing the extracted free RNA to produce first strand cDNA; amplifying the first strand cDNA by using a specific primer to obtain a specific amplification product; wherein, the specific primer can specifically amplify a target gene, the target gene is a differential expression gene of a fetus and a mother body, and the 5' end sequence of the upstream primer and/or the downstream primer of the specific primer is the same as at least a part of the sequence of the universal primer; and carrying out second round PCR amplification on the specific amplification product by adopting the universal primer to obtain a target gene library.

Preferably, the differentially expressed gene is a gene that is expressed fetal-specifically, but rarely expressed maternal.

Preferably, the differentially expressed gene is at least one of the genes shown in table 1.

In the present application, the differentially expressed gene refers to a gene corresponding to an RNA which is derived from a mother and a fetus and is released into the peripheral blood of a pregnant woman with a significant difference in expression level. For example, a differentially expressed gene may be a gene that is expressed fetal-specifically, but is rarely expressed or expressed in very low amounts by the mother. Specifically, the differentially expressed genes may be any one or more of the genes shown in table 1. The method and the device construct the fetal target gene library by utilizing the differential expression of the fetus and the mother body, and further realize the subsequent detection of the fetal gene information. In addition, the sequence of the 5' end of the upstream primer and/or the downstream primer of the specific primer is the same as at least a part of the sequence of the universal primer, so that the subsequent universal primer can be combined with the end of the specific amplification product, thereby realizing the second round of PCR amplification on the specific amplification product. It can be understood that, in the actual application process, there may be multiple sets of specific primers, which amplify multiple target genes respectively, thereby obtaining multiple specific amplification products; the universal primer can amplify a plurality of different specific amplification products by adopting a group of primers; therefore, it is necessary to design a sequence identical to at least a part of the sequence of the universal primer at the 5' end of the forward primer and/or the reverse primer of each specific primer.

It should be noted that the biological sample of the pregnant woman contains free RNA of fetus, especially free mRNA (abbreviated as cfmRNA), so the present application creatively performs the construction of the target gene library of fetus directly on the free RNA extracted from the pregnant woman sample. The target gene library of the fetus constructed by the method can be used for directly detecting the specifically expressed cfmRNA of the fetus in the plasma or urine of a pregnant woman, and then deducing the genotype of the fetus, thereby solving the problem that mutation detection cannot be judged due to the fact that free DNA carries a large amount of maternal background. cfmRNA is a complete transcript sequence obtained after DNA translation, removes a large amount of intron redundancy, and can reflect structural variations of DNA occurring on exons, which are difficult to detect at the DNA level, while at the mRNA level, they are relatively easy to detect. Therefore, the target gene library construction of the application adopts cfmRNA to replace cfDNA for subsequent noninvasive detection of genetic disease related mutation, can detect some recessive genetic disease related mutation of a fetus, and can also detect structural variation of some genes, which are difficult to detect on the cfDNA level.

Preferably, in the target gene library constructing method of the present application, the biological sample is peripheral blood or urine.

It should be noted that the target gene library construction method of the present application is applicable to all samples of pregnant women containing fetal free RNA in principle, including but not limited to urine and plasma of peripheral blood.

Preferably, in the target gene library construction method of the present application, the reverse transcription of the extracted free RNA to generate the first strand cDNA specifically includes the reverse transcription of the free RNA using a random primer to obtain the first strand cDNA.

More preferably, the random primer is an N6 random primer.

It should be noted that the generation of the first strand cDNA using the N6 random primer is only a specific scheme used in one implementation manner of the present application, and does not exclude that other methods for generating the first strand cDNA may also be used, and is not limited herein.

Preferably, in the target gene library constructing method of the present application, the specific primer pair is at least one of the first to sixth primer pairs; the upstream and downstream primers of the first primer pair are respectively sequences shown in SEQ ID NO.1 and SEQ ID NO.2, the upstream and downstream primers of the second primer pair are respectively sequences shown in SEQ ID NO.3 and SEQ ID NO.4, the upstream and downstream primers of the third primer pair are respectively sequences shown in SEQ ID NO.5 and SEQ ID NO.6, the upstream and downstream primers of the fourth primer pair are respectively sequences shown in SEQ ID NO.7 and SEQ ID NO.8, the upstream and downstream primers of the fifth primer pair are respectively sequences shown in SEQ ID NO.9 and SEQ ID NO.10, and the upstream and downstream primers of the sixth primer pair are respectively sequences shown in SEQ ID NO.11 and SEQ ID NO. 12.

It should be noted that, the six primer pairs of the sequences shown in SEQ ID No.1 to SEQ ID No.12 are only primers for amplifying the common mutation site of the FGA gene in one implementation manner of the present application, on one hand, the primers can be properly adjusted without affecting the amplification effect or covering the mutation site, for example, increasing or decreasing a number of bases at the 5 'end or the 3' end, or adding a special purpose nucleic acid sequence at the 5 'end, for example, the six primer pairs of the present application have a partial linker sequence at the 5' end to facilitate the subsequent amplification by using a general primer; on the other hand, in the specific using process, one or more primer pairs can be selected and used according to the specific mutation sites to be detected, even in some special mutation sites which are not covered by the six primer pairs in the application, the primer pairs can be additionally designed, and the primer pairs are not specifically limited herein.

Preferably, in the target gene library construction method of the present application, the specific primers include at least one of a first primer pair to a seventh primer pair, the upstream and downstream primers of the first primer pair are respectively sequences shown by SEQ ID No.17 and SEQ ID No.18, the upstream and downstream primers of the second primer pair are respectively sequences shown by SEQ ID No.19 and SEQ ID No.20, the upstream and downstream primers of the third primer pair are respectively sequences shown by SEQ ID No.21 and SEQ ID No.22, the upstream and downstream primers of the fourth primer pair are respectively sequences shown by SEQ ID No.23 and SEQ ID No.24, the upstream and downstream primers of the fifth primer pair are respectively sequences shown by SEQ ID No.25 and SEQ ID No.26, the upstream and downstream primers of the sixth primer pair are respectively sequences shown by SEQ ID No.27 and SEQ ID No.28, and the upstream and downstream primers of the seventh primer pair are respectively sequences shown by SEQ ID No.29 and SEQ ID No. 30.

Likewise, seven primer pairs of the sequences shown in SEQ ID No.17 to SEQ ID No.30 are only primers for amplifying a common mutation site of the HESX1 gene in one embodiment of the present application; on one hand, the primer can be properly adjusted under the condition of not influencing the amplification effect or covering the mutation site; on the other hand, in a specific using process, one or more primer pairs can be selected and used according to a specific mutation site to be detected, even, in some specific mutation sites which are not covered by seven primer pairs in the application, the primer pairs can be additionally designed, and the specific mutation sites are not limited in the specification.

Preferably, in the target gene library constructing method of the present application, the first strand cDNA is amplified using a reference primer capable of specifically amplifying the housekeeping gene, while the first strand cDNA is amplified using a specific primer.

It should be noted that the target region of the housekeeping gene is amplified for the purpose of serving as a reference for the success or failure of the experiment; on the other hand, the method can be used as a reference for sample quantification.

Preferably, the housekeeping gene is the GAPDH gene.

Preferably, the upstream and downstream primers of the primer for amplifying the housekeeping gene GAPDH are the sequences shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.

It should be noted that the primers of the sequences shown in SEQ ID NO.13 and SEQ ID NO.14 are only primers for amplifying the housekeeping gene GAPDH used in one implementation manner of the present application, and it is not excluded that other primers can be designed or used as long as the amplification of the target gene is not affected.

Preferably, in the target gene library construction method of the present application, the universal primer includes a first primer and a second primer, the first primer is a sequence shown by SEQ ID No.15, and the second primer is a sequence shown by SEQ ID No. 16. Also, preferably, the 5' end of the first primer has a phosphorylation modification.

The universal primers are used for further amplifying and enriching PCR amplification products, so that a sequencing library which can be used for sequencing is obtained; wherein, the 5' end of the first primer has phosphorylation modification, and the function is to facilitate subsequent circularization of single-stranded DNA. It will be appreciated that the specific sequence of the universal primers can be designed according to different assay or sequencing platforms.

Preferably, the target gene library construction method further comprises denaturing the double-stranded nucleic acid amplification product into single-stranded nucleic acid after obtaining the double-stranded nucleic acid amplification product through the second round of PCR amplification, and connecting the single-stranded nucleic acid into circular single-stranded nucleic acid by using a nucleic acid mediated fragment, thereby obtaining the circular single-stranded target gene library, wherein the nucleic acid mediated fragment can be combined with two ends of the single-stranded nucleic acid by the base complementary pairing principle.

It should be noted that, depending on the sequencing platform selected, the target gene library can be adaptively processed. For example, the Illumina sequencing platform is used for sequencing, the library on the computer is required to be a double-stranded nucleic acid library, that is, a nucleic acid double-stranded amplification product obtained after the second round of PCR amplification is the library used on the computer; for another example, when sequencing is performed using a BGI or MGI sequencing platform, if the library on the computer is a circular single-stranded nucleic acid library, the double-stranded nucleic acid amplification product obtained after the second round of PCR amplification needs to be denatured into single-stranded nucleic acids, and the single-stranded nucleic acids are connected into circular single-stranded nucleic acids through a mediating segment, and the sequencing can be performed on the computer only after the circular single-stranded nucleic acid library is obtained.

In a second aspect of the present application, a method for detecting a fetal gene is disclosed, comprising the steps of:

constructing a target gene library by using the method of the first aspect of the present application to obtain a target gene library; sequencing the target gene library to obtain a sequencing result consisting of a plurality of sequencing data; analyzing the sequencing result to obtain the gene information of the fetus.

It should be noted that, the fetal gene detection method of the present application is actually based on the target gene library construction method of the present application, and the constructed target gene library is further sequenced, thereby realizing fetal gene detection. In an implementation manner of the present application, a high-throughput sequencing method is used to sequence the constructed target gene library, so that fetal genetic information including mutation information of recessive genetic monogenic disorder, fetal genotype information, genetic structure variation information, and the like can be obtained.

It is understood that the fetal genetic testing method of the present application may be a non-diagnostic testing method, for example, by which the fetal genotype information is obtained to predict the possible phenotype of the fetus, and when the genotype information does not directly correspond to the disease, the predicted phenotype information does not directly relate to the disease.

Preferably, the sequencing result is analyzed, and the method specifically comprises the following steps of filtering sequencing data; comparing the filtered sequencing data to a reference genome, and reserving the sequencing data of the reference genome on the unique comparison; and counting the base distribution condition of the target gene based on the sequencing data of the reference genome on the unique comparison to obtain the mutation information of the target gene and further obtain the gene information of the fetus.

Preferably, the sequencing result is analyzed, and further comprising comparing the target gene mutation information with a disease database to obtain the mutation information of the recessive genetic monogenic disorder of the fetus.

Other parts of the fetal gene detection method of the present application, such as biological sample type, first strand cDNA generation, etc., can be referred to the target gene library construction method of the first aspect of the present application, and will not be described herein in detail.

In the target gene library construction method or the fetal gene detection method, all or part of the steps can be realized by a special mechanical device, so that the automatic operation is realized.

A third aspect of the present application discloses a fetal gene detection apparatus, which comprises a free RNA extraction module, a reverse transcription module, a target gene amplification module, a target gene library generation module, a sequencing module, and an analysis module; a free RNA extraction module for extracting free RNA from a biological sample derived from a pregnant woman, the free RNA including fetal free RNA; the reverse transcription module is used for carrying out reverse transcription on the extracted free RNA to generate first-strand cDNA; the target gene amplification module is used for amplifying the first strand cDNA by adopting a specific primer to obtain a specific amplification product, the specific primer can specifically amplify a target gene, the target gene is a differential expression gene of a fetus and a mother body, and the 5' end sequence of an upstream primer and/or a downstream primer of the specific primer is the same as at least one part of sequence of a universal primer; the target gene library generating module is used for carrying out second round PCR amplification on the specific amplification product by adopting a universal primer to obtain a target gene library; the sequencing module is used for sequencing the target gene library to obtain a sequencing result consisting of a plurality of sequencing data; and the analysis module is used for analyzing the sequencing result to obtain the gene information of the fetus.

Preferably, in the fetal gene testing device of the present application, the biological sample related to the free RNA extraction module is peripheral blood or urine.

Preferably, in the fetal gene detection apparatus of the present application, the reverse transcription module performs reverse transcription on the extracted free RNA to generate first strand cDNA, specifically including performing reverse transcription on the free RNA by using a random primer to obtain the first strand cDNA; preferably, the random primer is an N6 random primer.

Preferably, in the fetal gene detection apparatus of the present application, the differentially expressed genes involved in the target gene amplification module are genes expressed specifically in the fetus, but hardly expressed in the mother. Preferably, the differentially expressed gene is at least one of the genes shown in table 1.

Preferably, in the fetal gene testing apparatus of the present application, the genetic information of the fetus in the analysis module includes at least one of mutation information of recessive genetic monogenic disorder, fetal genotype information, and genetic structural variation information.

Preferably, in the fetal gene detection apparatus of the present application, the analysis module further comprises a filtering unit, a reference genome alignment unit and a statistical unit; the filtering unit is used for filtering the sequencing data; the reference genome comparison unit is used for comparing the filtered sequencing data to a reference genome and reserving the sequencing data of the reference genome which is only compared; and the counting unit is used for counting the base distribution condition of the target gene based on the sequencing data of the reference genome on the unique comparison, obtaining the mutation information of the target gene and further obtaining the gene information of the fetus.

Preferably, in the fetal gene detection apparatus of the present application, the analysis module further includes a disease database comparison unit, and the disease database comparison unit is configured to compare the target gene mutation information with a disease database to obtain the mutation information of the recessive genetic monogenic disorder of the fetus.

The fourth aspect of the present application discloses the use of the target gene library construction method of the first aspect of the present application, the fetal gene detection method of the second aspect of the present application, or the fetal gene detection device of the third aspect of the present application in the detection of a single genetic disease of recessive inheritance or structural variation.

The fifth aspect of the application discloses a reagent for fetal FGA gene detection, which comprises specific primers for amplifying FGA genes, wherein the specific primers comprise at least one group from a first primer pair to a sixth primer pair, the upstream and downstream primers of the first primer pair are respectively sequences shown in SEQ ID NO.1 and SEQ ID NO.2, the upstream and downstream primers of the second primer pair are respectively sequences shown in SEQ ID NO.3 and SEQ ID NO.4, the upstream and downstream primers of the third primer pair are respectively sequences shown in SEQ ID NO.5 and SEQ ID NO.6, the upstream and downstream primers of the fourth primer pair are respectively sequences shown in SEQ ID NO.7 and SEQ ID NO.8, the upstream and downstream primers of the fifth primer pair are respectively sequences shown in SEQ ID NO.9 and SEQ ID NO.10, and the upstream and downstream primers of the sixth primer pair are respectively sequences shown in SEQ ID NO.11 and SEQ ID NO. 12.

It should be noted that, the reagent of the present application, its six primer pairs can amplify and cover the common mutation site of FGA gene; therefore, the reagent of the application can be directly used for FGA gene mutation detection on one hand, and can also be used for amplifying and enriching corresponding target fragments on the other hand. It is understood that the reagent of the present application can be used in other methods for detecting FGA gene based on PCR amplification target fragment besides the fetal FGA gene detection method of the present application, and is not specifically limited herein.

Preferably, the reagent of the present application further comprises a reference primer for amplifying the housekeeping gene GAPDH, wherein the upstream primer and the downstream primer of the reference primer are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14.

It should be noted that, in the reagent of the present application, six specific primer pairs and a primer pair for amplifying housekeeping gene GAPDH are provided, and these primer pairs may be mixed in proportion in advance according to the use requirement and the optimized multiplex PCR amplification scheme, or each primer pair may be packaged separately, and the primer mixture is configured according to the specific use condition, which is not limited herein. It will be appreciated that if amplification of the housekeeping gene is not required, the corresponding reference primer pair may not be used.

Preferably, the reagent of the application also comprises a universal primer for constructing a sequencing library, wherein the universal primer comprises a first primer and a second primer, the first primer is a sequence shown in SEQ ID NO.15, and the second primer is a sequence shown in SEQ ID NO. 16; also, preferably, the 5' end of the first primer has a phosphorylation modification.

The sixth aspect of the application discloses a kit for fetal FGA gene detection, which comprises the reagent for fetal FGA gene detection of the fifth aspect of the application.

Preferably, the kit further comprises at least one of a free RNA extraction reagent, a reverse transcription reagent, a PCR amplification reagent, and a nucleic acid purification reagent.

It should be noted that, for convenience of use, a universal primer can be completely constructed for the specific primer pair of the FGA gene, the housekeeping gene GAPDH amplification primer pair and/or the library, even various reagents involved in the fetal FGA gene detection method of the present application, to assemble a kit, so as to facilitate detection of the fetal FGA gene; of course, the free RNA extraction reagent, the reverse transcription reagent, the PCR amplification reagent, and the nucleic acid purification reagent may be used in a reagent that is conventional in laboratories, and are not particularly limited.

The seventh aspect of the application discloses a reagent for detecting a fetal HESX1 gene, which comprises specific primers for amplifying a HESX1 gene, wherein the specific primers comprise at least one group of a first primer pair to a seventh primer pair, the upstream and downstream primers of the first primer pair are respectively sequences shown in SEQ ID NO.17 and SEQ ID NO.18, the upstream and downstream primers of the second primer pair are respectively sequences shown in SEQ ID NO.19 and SEQ ID NO.20, the upstream and downstream primers of the third primer pair are respectively sequences shown in SEQ ID NO.21 and SEQ ID NO.22, the upstream and downstream primers of the fourth primer pair are respectively sequences shown in SEQ ID NO.23 and SEQ ID NO.24, the upstream and downstream primers of the fifth primer pair are respectively sequences shown in SEQ ID NO.25 and SEQ ID NO.26, the upstream and downstream primers of the sixth primer pair are respectively sequences shown in SEQ ID NO.27 and SEQ ID NO.28, and the upstream and downstream primers of the seventh primer pair are respectively sequences shown in SEQ ID NO.29 and SEQ ID NO. 30.

Likewise, the reagents of the present application, with seven primer pairs, were able to amplify and cover the common mutation site of the HESX1 gene; therefore, the reagent can be directly used for detecting the mutation of the HESX1 gene on one hand, and can also be used for amplifying and enriching the corresponding target fragment on the other hand. It is understood that the reagent of the present application can be used in other HESX1 gene detection methods based on PCR amplification of target fragment besides the fetal HESX1 gene detection method of the present application, and is not limited herein.

Likewise, the packaged form of the primers and the use or non-use of the reference primers as reagents for fetal FGA gene detection according to the fifth aspect of the present application are not described herein in detail.

Similarly, for convenience of use, universal primers can be constructed for the specific primer pair of the HESX1 gene, the housekeeping gene GAPDH amplification primer pair and/or the library, and even various reagents involved in the fetal HESX1 gene detection method can be assembled into a kit to facilitate detection of the fetal HESX1 gene; of course, the free RNA extraction reagent, the reverse transcription reagent, the PCR amplification reagent, and the nucleic acid purification reagent may be used in a reagent that is conventional in laboratories, and are not particularly limited.

An eighth aspect of the present application discloses a kit for detecting a fetal HESX1 gene, which comprises the reagent for detecting a fetal HESX1 gene of the seventh aspect of the present application.

Similarly, the packaged form of each reagent in the kit, such as the kit for fetal FGA gene detection of the sixth aspect of the present application, will not be described herein again.

The beneficial effect of this application lies in:

the target gene library construction method utilizes the gene expression difference of a fetus and a mother body to construct the target gene library of genes which are expressed by the fetus and hardly expressed by the mother body on the basis of free RNA extracted from a pregnant woman. According to the target gene library construction method and the fetal gene detection method based on the constructed target gene library, firstly, the interference of a maternal background can be eliminated by detecting the expression difference genes of a fetus and a maternal body, particularly the genes which are specifically expressed by the fetus and hardly expressed by the maternal body, and the problem that mutation detection cannot be judged due to the fact that a large amount of maternal background is carried in free DNA detection is solved; secondly, the target genes in the free RNA of the plasma of the pregnant woman are specifically amplified, so that the fetal genes with low expression level in the plasma can be well detected, and the problem that mRNA with low expression level is lost based on the whole transcriptome detection is solved; thirdly, cfmRNA is a complete transcript sequence obtained after DNA translation, a large amount of intron redundant information is removed, and structural variation of DNA on exons can be reflected, so that the application can detect the genetic structural variation, and the problem that the existing detection based on free DNA is difficult to detect the genetic structural variation is solved; fourthly, the target gene library constructed by the method is adopted for fetal gene detection, so that the information of parents and probands does not need to be acquired when detecting the recessive monogenic disease, the fetal haploid information does not need to be constructed, the operation is simpler, and the cost is lower. The target gene library construction method and the fetal gene detection method based on the constructed target gene library are simple to operate and low in cost, can meet the clinical use requirements, and lay a foundation for further popularization and use of noninvasive detection of pregnant women.

Drawings

FIG. 1 is a schematic technical flow chart of a method for constructing a target gene library in the example of the present application;

FIG. 2 is a graph of the coverage depth profile of each amplicon in accordance with one embodiment of the present application;

FIG. 3 is a graph of the reproducibility statistics of 2 duplicate sequenced samples of a pregnant plasma sample of a normal fetus in accordance with one embodiment of the present application;

FIG. 4 is a graph of the repetitive statistics of 2 duplicate sequencing samples of a maternal plasma sample from a fetus with congenital non-fibrinogenemia according to one embodiment of the present application;

FIG. 5 is a graph showing the statistics of the base coverage of four sequenced samples on the target pathogenic site in the first embodiment of the present application;

FIG. 6 is a graph showing the statistics of the base coverage of four sequenced samples on the target pathogenic site in example two of the present application.

Detailed Description

The existing fetal gene detection method is mainly carried out aiming at the fetal free DNA in the blood of a pregnant woman, and the detection method based on the fetal free DNA provided by Dennis Lo and the like can not only determine whether a fetus obtains pathogenic mutation from a father, but also judge the condition of the inherited allele of the fetus from a mother. However, the detection method of Dennis Lo et al is complex in operation and high in cost, and is difficult to meet the clinical use requirement; furthermore, more importantly, the detection of structural variation of genes is difficult with the detection method based on fetal free DNA.

The studies of the inventors of the present application show that the transcript profile of a pregnant woman and that of a fetus are not the same throughout pregnancy; there are differences in the temporal and spatial expression of different genes during the development of the fetus from a fertilized egg into an individual, e.g., for certain genes, the mother does not express, while the fetus is expressed at some time during or throughout pregnancy. The present inventors found that diseases caused by mutations in these genes whose fetal and maternal expression are different can be detected by directly detecting cfmRNA specifically expressed in plasma from a fetus. Fetus-specific expressed RNA is released into blood circulation through a placenta, and the mutation of the DNA level of the fetus can be deduced through detecting cfmRNA in the blood circulation.

Based on the above research and recognition, the present application creatively proposes a new target gene library construction method, which comprises extracting free RNA from a biological sample derived from a pregnant woman, the free RNA comprising fetal free RNA; reverse transcribing the extracted free RNA to produce first strand cDNA; amplifying the first strand cDNA by using a specific primer to obtain a specific amplification product, wherein the specific primer can specifically amplify a target gene, the target gene is a differential expression gene of a fetus and a mother body, and the 5' end sequence of an upstream primer and/or a downstream primer of the specific primer is the same as at least part of the sequence of a universal primer; and carrying out second round PCR amplification on the specific amplification product by adopting the universal primer to obtain the target gene library of the application.

On the basis of the target gene library construction method, the application further provides a fetal gene detection method based on the target gene library construction method, a fetal gene detection device and the like. Further, the target gene library construction method and the fetal gene detection method based on the application are specifically applied; the application provides a reagent and a kit for detecting fetal FGA genes; a reagent and a kit for detecting a fetal HESX1 gene.

The fetal gene detection method is based on plasma free RNA of a pregnant woman for detection, and can easily detect the variation types of the fetus, including gene structure variation, through cfmRNA specifically expressed in the plasma, so that whether the fetus carries parent mutation or not is analyzed. Specifically, some genes of the fetus are specifically expressed during the pregnancy of the pregnant woman and released to the blood circulation system of the mother through the placenta, and if the genes are related to certain diseases, the plasma cfRNA can be directly detected to detect the DNA mutation of the fetus, as shown in figure 1.

In the fetal gene detection method of the present application, for example, in the case of recessive pseudopathy, the genotype of the mother is AA or AA, and the decision logic is:

if only AA is detected in the plasma cfRNA, the genotype of the fetus is AA;

if only Aa is detected in plasma cfRNA, there are two cases: 1) a: 1, then the fetal genotype is Aa; 2) a < < a, i.e., A detected is much less than a, then the genotype of the fetus is aa;

if only aa is detected in plasma cfRNA, then the fetal genotype must be aa.

The target gene library construction method and the fetal gene detection method of the application utilize genes with fetal and maternal expression difference for detection, are particularly suitable for genes with fetal specific expression but maternal non-expression or maternal low expression, and the genes are shown in table 1.

TABLE 1 detectable Gene List

KRT7	CSH1	PSG8	GBP1P1	SEMA3B
TMEM54	KISS1	ENDOU	CLDN4	GPC3
PRKCZ	STAT1	EGFR	C2orf72	PLAC2
SDC1	CGA	DUSP4	PAPPA2	PSG9
LGALS13	CSH2	PHYHIPL	HIST2H3A	FN1
EFHD1	TFPI2	CTSF	HIST2H3C	NOS3
CAPN6	GBP1	TRIM29	TCL6	LOC100506655
XAGE3	PLAC4	RCN3	MFSD2A	PSG11
EBI3	HSD17B1	SPIRE2	ZFAT-AS1	SPTLC3
GH2	CSHL1	LOC100216001	INSL4	EXPH5
PAGE4	KRT8	FAM176A	DHRS2	HSPA2
ALPP	KRT18	SCIN	HES2	PSG6
INHBA	HPGD	ZNF500	WLS	PLAC1
LOC100505659	GADD45G	PRR16	PLCXD3	TACC2
FBLN1	LGALS14	LOC100128054	LOC100505483	PRPF40B
LOC388948	ADAM12	GRHL2	SVEP1	CRH
SERPINB2	PSG2	PPP1R32	CRYAB	EFS
HSD3B1	OLR1	C1orf130	HSPB8	TIMD4
KRT19	SLC30A2	FOSB	PKIB	ALDH3B2
SERPINE1	LOC285972	GCM1	PGF	KRT81
GDF15	HESX1	TRPV6	CYP11A1	MUC15
CYP19A1	TMEM139	TFAP2A	PSG5	PRSS8
PSG4	ZNF727	MMP11	PSG1	SH2D5
PSG3	TM4SF19	TUSC3	PAPPA	LOC728175
HMGCR	GLDN	C8orf39	VGLL3	CORO6
CCK	PGAP3	LOC100129935	MSX2P1	GH1
PABPN1L	FGA

Table 1 shows genes expressed specifically in the fetus but not expressed or expressed in a very small amount in the mother, and these genes can be detected by the fetal gene detection method of the present application for recessive genetic monogenic mutation, fetal genotype, genetic structural variation, and the like.

The target gene library construction method and the fetal gene detection method based on the constructed target gene library can be used for detecting the fetal genotype and easily realizing the detection of the gene structure variation; the method is simple to operate, has relatively low detection cost, can well meet the clinical detection use, and lays a foundation for further popularization and application of noninvasive obstetrical examination.

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.

Example one

Congenital afibrinogenemia is a bleeding symptom caused by the loss of a coagulation process, is mostly related to mutation on FGA genes, and is an autosomal recessive genetic disease. During pregnancy of a pregnant woman, the gene is expressed little or not in the mother, but is expressed specifically in the fetus. Therefore, the present example performed the detection of fetal FGA gene to verify the target gene library construction method and the fetal gene detection method of the present application.

Six primer pairs, namely specific primer pairs, are designed in the embodiment and cover common FGA mutation sites; the primer pairs designed in this example are shown in Table 2, and the common FGA mutation sites covered are shown in Table 3. In addition, this example also designed a pair of primer amplified GAPDH as a sample reference for successful experiments and sample quantification, and the specific primer sequences are shown in table 2. In Table 2, FGA-F01 and FGA-R01 are the first primer pair, FGA-F02 and FGA-R02 are the second primer pair, FGA-F03 and FGA-R03 are the third primer pair, FGA-F04 and FGA-R04 are the fourth primer pair, FGA-F05 and FGA-R05 are the fifth primer pair, FGA-F06 and FGA-R06 are the sixth primer pair, and GAPDH-F and GAPDH-R are the primer pairs for amplifying GAPDH.

TABLE 2 specific primer pairs and GAPDH amplification primers

Primer numbering	Sequence (5 '→ 3')	SEQ ID NO.
FGA-F01	GACCGCTTGGCCTCCGACTTTGTTTGCTGTAACTTGAAGATTTACC	1
FGA-R01	GACATGGCTACGATCCGACTTCTTCTCACCTATGTTAGGAGAG	2
FGA-F02	GACCGCTTGGCCTCCGACTTATTGCCTCGGGACAGTCAGAACCA	3
FGA-R02	GACATGGCTACGATCCGACTTCAGGACTGGTAAAGAGAAGGTCACC	4
FGA-F03	GACCGCTTGGCCTCCGACTTGCCAGGATTCCAGGTTCCGGTAC	5
FGA-R03	GACATGGCTACGATCCGACTTGGACTGGAGGGACTGCAACCTGG	6
FGA-F04	GACCGCTTGGCCTCCGACTTCGAGTAATCTCATTTCCACCAGGTCTC	7

FGA-R04	GACATGGCTACGATCCGACTTTGAACAGGTCATTGCCAAAGACTT	8
FGA-F05	GACCGCTTGGCCTCCGACTTCACTTTTCTCTGCATATAGTAGAC	9
FGA-R05	GACATGGCTACGATCCGACTTAATTGAAGTCCTGAAGCGCAAAG		10
FGA-F06	GACCGCTTGGCCTCCGACTTTGACTGCTTACCCAGTCTTCAT	11
FGA-R06	GACATGGCTACGATCCGACTTTATGTGAATGAATCTTTAAAGACTGC	12
GAPDH-F	GACCGCTTGGCCTCCGACTTTCAACGACCACTTTGTCAAGC	13
GAPDH-R	GACATGGCTACGATCCGACTTGCCAGACCCTGCACTTTTTAAG	14

In Table 2, the sequences of the underlined parts, i.e., the 20bp sequence of the 5 'end of the upstream primer and the 21bp sequence of the 5' end of the downstream primer, are linker sequences. In this example, partial linker sequences were introduced by primers during the first round of PCR on cDNA; in the second round of PCR, i.e., the universal primers amplify the specific PCR amplification products, the universal primers introduce the complete linker sequences containing the tag sequences, and these linker sequences can be adjusted according to different sequencing platforms.

TABLE 3 FGA mutation sites covered by specific primer pairs

Numbering	Mutations	Position at HG38
1	c.510+1G>T	chr4:154587511
2	c.1622delT(p.Val541Alafs)	chr4:154585807
3	c.1634A>T(p.Glu545Val)	chr4:154585795
4	c.104G>A(p.Arg35His)	chr4:154589513
5	c.103C>T(p.Arg35Cys)	chr4:154589514
6	c.571G>A(p.Gly191Arg)	chr4:154609725
7	c.1718G>T(p.Arg573Leu)	chr4:154585711
8	c.1629delG(p.Thr544Leufs)	chr4:154585800
9	c.711dupT(p.Lys238Terfs)	chr4:154586718
10	c.502C>T(p.Arg168Ter)	chr4:154587520

In the embodiment, free RNA in the blood of the pregnant woman is extracted, cfRNA is reversely transcribed into cDNA, then multiple PCR is combined to complete preparation of a targeted library, and mutation information is obtained through high-throughput sequencing. The details are as follows:

experimental samples: a pregnant woman who had a normal fetus had 1mL of a plasma sample, and the sample was divided into 2 portions on average as a replicate. A pregnant woman with a fetus with congenital non-fibrinogen blood disease is known to have a clinically diagnosed genetic mutation: c.103c > T (p.arg35cys), which is 1mL of the maternal plasma sample, was also divided equally into 2 samples as replicates.

cfRNA extraction

Extraction of cfRNA from plasma samples Using Qiagen

The Circulating Nucleic Acid Kit is extracted according to a standard operation flow, and finally the obtained total RNA is dissolved in 20 mu L of water.

First Strand cDNA Synthesis

SuperScript was synthesized using the first strand cDNA Synthesis kit from Thermofisher^TMIV First-Strand Synthesis System 18091200, as follows:

mu.L of 10mM dNTPs, 1. mu.L of 50 ng/. mu.L random primer and 1. mu.L of extracted RNA are added with 10. mu.L of RNase free H₂O, mixing, standing at 65 ℃ for 5 minutes, and standing on ice for 1 minute to obtain an RNA mixture. Wherein the random primer is N₆And (4) random primers.

Then 20. mu.L of a reverse transcription system was prepared, comprising: 5 XSSIV BUFFER 4. mu.L, 100mM DTT 1. mu.L, Ribonucleae Inhibitor 1. mu.L, SuperScript TM IV Reverse Transcriptase Transcriptase 1. mu.L at 200U/. mu.L, and RNA mixture 13. mu.L of the previous step.

The reverse transcription system was mixed rapidly and mixed at 23 ℃ for 10 minutes and at 55 ℃ for 10 minutes. After the reaction is finished, 30 mu L of Agencour AMPure XP magnetic beads are added to obtain a 50 mu L magnetic bead purification system, the purification is carried out according to the instruction, and finally 20 mu L of distilled water is used for dissolving DNA. Among them, Agencour AMPure XP magnetic beads are available from Beckmann Coulter, Inc., USA.

3. Specific PCR amplification

The PCR amplification enzyme was KAPA2G Fast Multiplex PCR Kit product K.K. KK5801 of Kapa company, USA.

The PCR reaction system comprises: the 2 Xkapa polymerase mixture (25. mu.L), a 10. mu.M primer pool (5. mu.L), and the obtained DNA (20. mu.L) were purified, and the total amount was 50. mu.L.

Wherein the primer pool comprises specific primer pairs shown in Table 1 and primer pairs for amplifying GAPDH, and the concentration of the upstream primer and the downstream primer of each primer pair in the primer pool is 10 μ M.

The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 2min, then 15 cycles were entered: 10s at 98 ℃, 2min at 62 ℃ and 30s at 72 ℃, and extending for 5min at 72 ℃ after the circulation is finished, thus finishing the PCR amplification.

After PCR, 1 volume of Agencour AMPure XP magnetic beads are added into PCR amplification products, namely 50 mu L of magnetic beads are added, purification is carried out according to the instruction, and DNA is dissolved by 20 mu L of distilled water after purification.

4. Universal PCR amplification

The PCR amplification enzyme was KAPA2G Fast Multiplex PCR Kit product, cat # KK5801, manufactured by Kapa, USA.

The reaction system comprises: the 2 Xkapa polymerase mixture was 25. mu.L, 2.5. mu.L of 10. mu.M first primer, and 2.5. mu.L of 10. mu.M second primer, and 20. mu.L of DNA obtained as a purified product of the specific PCR amplification was added, and the total amount was 50. mu.L.

The first primer is a sequence shown as SEQ ID NO.15, and the second primer is a sequence shown as SEQ ID NO. 16; and, the 5' end of the first primer has a phosphorylation modification.

SEQ ID NO.15：5’-GAACGACATGGCTACGATCCGACTT-3’

SEQ ID NO.16：

5’-TGTGAGCCAAGGAGTTGCTGCGTACATTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3’。

The reaction conditions are as follows: pre-denaturation at 98 ℃ for 2min, then 15 cycles were entered: 10s at 98 ℃, 2min at 62 ℃ and 30s at 72 ℃, and the extension is carried out for 5min at 72 ℃ after the circulation is finished.

After the reaction was completed, 1 volume of Agencour AMPure XP magnetic beads were added, 50. mu.L of magnetic beads were added, purification was performed according to the instructions, and after purification, 20. mu.L of distilled water was used to dissolve the DNA.

5. Sequencing on machine

And after the quality of the library is qualified, preparing the DNA nanospheres according to the requirements of the Huada gene BGISEQ-500 platform, and then carrying out machine sequencing on the DNA nanospheres, wherein the sequencing type has two ends of 50 bp. The preparation of the DNA nanosphere comprises the following steps: the double-stranded nucleic acid amplification product is denatured into single-stranded nucleic acid at high temperature, the single-stranded nucleic acid is connected into annular single-stranded nucleic acid by adopting a nucleic acid mediated fragment, and the annular single-stranded nucleic acid is subjected to rolling circle amplification, so that the DNA nanosphere is obtained. The detailed steps refer to the operational instructions of the Huada gene BGISEQ-500 platform.

6. Data analysis

Removing joints after the data is downloaded, and performing joint removal and low-quality filtering (see https:// gitubb.com/marcell/cutdata /) on the fastq file by adopting cutdata 1.18 software to obtain filtered reads (clean reads); comparing the fastq data with a reference genome by using BWA (BWA) or other comparison software, and counting the comparison rate, wherein the comparison rate is the reads/clean reads of the reference genome; removing reads of multiple comparison, counting the reads with the only comparison, counting the reads at all target amplification positions, counting the total number of the reads of each amplification sub-region and the coverage depth of the amplicons, counting the base distribution of target sites, and comparing site mutation information obtained according to the base distribution with a disease database to obtain a mutation detection result.

The statistics of the original reads, the truncated reads, the alignment reads and the target region reads for the off-line data of the four sequenced samples are shown in table 4. Of these, four sequencing samples, 2 duplicate sequencing samples of a maternal plasma sample of a normal fetus, and 2 duplicate sequencing samples of a maternal plasma sample of a fetus with congenital nonfibrinogenemia.

TABLE 4 statistical results of the data of the offline

Sample numbering	Original reads	Splices removal	Comparing reads	Target area reads
Sample 1 repeat 1	67394	65987	64991	63981
Sample 1 repeat 2	76542	74541	73923	73012
Sample 2 repeat 1	63495	62596	61463	60982
Sample No.2 repeat 2	79482	78210	77129	76365

In table 4, sample No.1 replicate 1 and sample No.1 replicate 2, i.e., 2 replicate sequencing samples of a pregnant woman plasma sample of a normal fetus; sample No.2 replicate sample No.1 and sample No.2 replicate 2, i.e., 2 replicate sequencing samples of maternal plasma samples of a fetus with congenital nonfibrinogenemia. The results in table 4 show that the data utilization rate of the off-line data of the fetal FGA gene detection method of this example can reach 97%, the comparison rate can reach 98%, and the target region proportion can reach 98%.

Counting the coverage rate of each specific primer pair and GAPDH amplification primer in four sequencing samples, wherein the result is shown in FIG. 2; in fig. 2, the abscissa FGA01, FGA02, FGA03, FGA04, FGA05, FGA06 is the coverage of the first to sixth primer pairs in four sequencing samples, 1-repeat 1, 1-repeat 2 are 2 duplicate sequencing samples of maternal plasma samples of normal fetuses, 2-repeat 1, 2-repeat 2 are 2 duplicate sequencing samples of maternal plasma samples of fetuses with congenital non-fibrinogenemia. The results in FIG. 2 show that the depth distribution obtained after sequencing of each amplicon (i.e., primer pair) is within 5-fold of the depth difference between the different amplicons in the four samples.

The results of counting the reproducibility of 2 duplicate sequenced samples of pregnant plasma samples of normal fetuses are shown in fig. 3, where the ordinate of fig. 3 is the coverage of repeat 1 and the abscissa is the coverage of repeat 2. The reproducibility of 2 replicate sequencing samples of a maternal plasma sample from a fetus with congenital non-fibrinogenemia was counted and the results are shown in fig. 4, with the coverage of replicate 1 on the ordinate and replicate 2 on the abscissa of fig. 4. The results in FIGS. 3 and 4 show that the depth stability of each amplicon obtained from different experiments on the same sample is very good, as shown by the two repeated R in FIG. 3²0.9666, two repetitions of R as shown in FIG. 4²0.9797, the repeatability of each sample is very good.

The base coverage condition of the target pathogenic site is counted through sequencing data, and the result is shown in fig. 5, the abscissa of fig. 5 is four sequencing samples respectively, the ordinate is the coverage depth ratio, █ represents the pathogenic base, □ represents the normal base, and fig. 5 shows the coverage degree of the target site c.103c > T of the four sequencing samples. The results in fig. 5 show that the maternal plasma samples of two normal fetuses can cover the pathogenic bases well, while the maternal plasma samples of two fetuses with congenital non-fibrinogenemia can also cover the normal bases well, indicating that the sequencing data of this example has good coverage of the target pathogenic sites.

The results of mutation analysis on the four sequenced samples are shown in table 5.

TABLE 5 sample test results

Sample numbering	Clinical test results	The detection result of this example
Sample 1 repeat 1	No mutation	No mutation was detected
Sample 1 repeat 2	No mutation	No mutation was detected
Sample 2 repeat 1	c.103C>T	c.103C>T
Sample No.2 repeat 2	c.103C>T	c.103C>T

The results in table 5 show that no mutations were detected for 2 replicate sequencing samples of a pregnant woman plasma sample of a normal fetus; and the results of 2 repeated sequencing samples of the plasma samples of pregnant women with congenital non-fibrinogenemia of the pregnant women detect C.103C > T, and are consistent with the expectation, which shows that the fetal FGA gene detection method of the embodiment can accurately detect the single-base mutation of the FGA gene.

Example two

Visual compartment dysplasia is a rare anterior malformation of the midline structure, also known as De-Morsier syndrome, involving a hyaline compartment defect and visual transmission to pathway dysplasia, which may be associated with other intracerebral dysplasias. Part of the pathogenic reasons are caused by mutation on the HESX1 gene. During pregnancy of a pregnant woman, the gene is expressed little or not in the mother, but is expressed specifically in the fetus. Therefore, the present example performed the detection of fetal HESX1 gene to verify the target gene library construction method and fetal gene detection method of the present application.

In the embodiment, 7 pairs of specific primers are designed to cover the common HESX1 mutation site; the primer pairs designed in this example are shown in table 6, and the covered common HESX1 mutation sites are shown in table 7. In addition, this example also uses a pair of primers to amplify GAPDH as a sample reference for successful experiments and sample quantification, and the specific primer sequences are shown in Table 1. In Table 6, HESX-F01 and HESX-R01 are the first primer pair, HESX F02 and HESX-R02 are the second primer pair, HESX-F03 and HESX-R03 are the third primer pair, HESX-F04 and HESX-R04 are the fourth primer pair, HESX F05 and HESX-R05 are the fifth primer pair, HESX-F06 and HESX-R06 are the sixth primer pair, and HESX-F07 and HESX-R07 are the seventh primer pair.

TABLE 6 specific primer pairs

Primer numbering	Sequence (5 '→ 3')	SEQ ID NO.
HESX1-01F	GACCGCTTGGCCTCCGACTTCACTTCTTTAGAGAAAGTTAAGTC	17
HESX1-01R	GACATGGCTACGATCCGACTTTCCTGTCTTAGAAAGTTTTAGC	18
HESX1-02F	GACCGCTTGGCCTCCGACTTGCTGGGCAAGTGTTCATTGAC	19
HESX1-02R	GACATGGCTACGATCCGACTTGGAGACATCCTCTCGTGGTCTGCA		20
HESX1-03F	GACCGCTTGGCCTCCGACTTCAGAGGCCAGAGCTGTTGCTC		21
HESX1-03R	GACATGGCTACGATCCGACTTGACATAAGTTACCATCTTTCC	22
HESX1-04F	GACCGCTTGGCCTCCGACTTGGGCAGACACCTGCAGCTCATC	23
HESX1-04R	GACATGGCTACGATCCGACTTTCTTCGGCCTCTATACCAACTC	24
HESX1-05F	GACCGCTTGGCCTCCGACTTAAGACTGTCTTTGAAAAGAGAG	25
HESX1-05R	GACATGGCTACGATCCGACTTCTTTTCAGTTTTGCACGCCGA	26
HESX1-06F	GACCGCTTGGCCTCCGACTTACAGAATCCAGATTTGGTTTCAA	27
HESX1-06R	GACATGGCTACGATCCGACTTTTTAACACTTAATATTTCCACTG	28

HESX1-07F	GACCGCTTGGCCTCCGACTTCTTCTAATTGCAGAGCATGAAGA	29
HESX1-07R	GACATGGCTACGATCCGACTTTCTTTACTATAACTAAAAGTGCCC		30

TABLE 7 HESX1 Gene mutation sites covered by specific primer pairs

Mutation numbering	Mutations	Position at HG38
18	c.18G>C(p.Gln6His)	chr3:57199901
19	c.313T>G(p.Trp105Gly)	chr3:57198797
20	c.357+2T>C	chr3:57198751
21	c.445G>A(p.Glu149Lys)	chr3:57198405
22	c.450_451delCA(p.Asp150Glufs)	chr3:57198399-57198400
23	c.478C>T(p.Arg160Cys)	chr3:57198277
24	c.509C>T(p.Ser170Leu)	chr3:57198246
25	c.511_512delCA(p.Gln171Valfs)	chr3:57198243-57198244
26	c.77T>C(p.Ile26Thr)	chr3:57199842

Experimental samples of this example: a pregnant woman who had a normal fetus had 1mL of a plasma sample numbered No.3, and the sample was divided into 2 portions on average as a replicate. The pregnant woman who is found to have visual compartment dysplasia and positive needle biopsy by MRI detection, and the gene mutation condition of the fetus through clinical diagnosis is as follows: ESX1, c.509c > T, p.ser170leu; 1mL of the pregnant plasma sample, numbered sample No.4, was also divided equally into 2 portions for repeat test.

In this example, the primers in Table 6 were used to prepare a sequencing library by the same library construction method as in example one, and the general primers used in the library preparation process were the same as in example one. Then, the same sequencing platform as that of the first embodiment is used for on-machine sequencing, and the same method as that of the first embodiment is used for data analysis.

In this example, the base coverage of the target pathogenic site is counted by the sequencing data, and the result is shown in fig. 6, where the abscissa in fig. 6 is four sequencing samples, sample No.3 repeat 1 and sample No.3 repeat 2, respectively, that is, 2 repeated sequencing samples of the plasma sample of the pregnant woman of the normal fetus; sample No.4 replicate 1 and sample No.4 replicate 2, i.e., 2 replicate sequencing samples of a pregnant woman's plasma sample with a fetus with dysplasia of the visual compartment; the ordinate is the coverage depth ratio, █ indicates the pathogenic base, □ indicates the normal base, and FIG. 6 shows the coverage of the target site c.509C > T by four sequencing samples. The results in fig. 6 show that the maternal plasma samples of two normal fetuses can cover the pathogenic bases well, while the maternal plasma samples of two fetuses with dystrophia can cover the normal bases well, indicating that the sequencing data of this example has good coverage of the target pathogenic sites.

The results of mutation analysis on the four sequenced samples are shown in table 8.

TABLE 8 sample test results

Sample numbering

Clinical test results

The detection result of this example

Sample No.3 repeat 1	No mutation	No mutation was detected
Sample No.3 repeat 2	No mutation	Not detectedTo mutation
Sample No.4 repeat 1	c.509C>T	c.509C>T
Sample No.4 repeat 2	c.509C>T	c.509C>T

The results in table 8 show that no mutations were detected for 2 replicate sequencing samples of a pregnant woman plasma sample of a normal fetus; the c.509C > T is detected in 2 repeated sequencing samples of the plasma sample of the pregnant woman of the fetus with dysplasia, and the result is consistent with the expectation, which shows that the fetal HESX1 gene detection method can accurately detect the single-base mutation of the HESX1 gene.

The target gene library construction method and the fetal gene detection method based on the constructed target gene library can detect not only the single base mutation of the FGA gene of the first embodiment and the single base mutation of the HESX1 gene of the second embodiment, but also genes which are similar to the FGA gene and the HESX1 gene and have fetal-specific expression but no expression or very little expression of mothers, such as the genes shown in table 1, and can also detect the genes, so long as the first round of PCR amplification is performed on cDNA by designing corresponding specific primer pairs covering mutation sites of each gene according to the inventive concept of the present application; the FGA gene detection method of the first embodiment of the present application can be referred to for extraction of cfRNA before specific amplification, first strand synthesis of cDNA, and subsequent general PCR amplification, on-machine sequencing, and data analysis.

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. For those skilled in the art to which the present application pertains, several simple deductions or substitutions may be made without departing from the concept of the present application, and all should be considered as belonging to the protection scope of the present application.

Claims

A method for constructing a library of target genes, comprising: comprises the following steps of (a) carrying out,

extracting free RNA from a biological sample derived from a pregnant woman, the free RNA comprising fetal free RNA;

reverse transcribing the extracted free RNA to produce first strand cDNA;

amplifying the first strand cDNA by using a specific primer to obtain a specific amplification product, wherein the specific primer can specifically amplify the target gene, the target gene is a differential expression gene of a fetus and a mother body, and the 5' end sequence of the upstream primer and/or the downstream primer of the specific primer is the same as at least a part of the sequence of the universal primer;

and carrying out second round PCR amplification on the specific amplification product by adopting a universal primer to obtain the target gene library.
The method of claim 1, wherein: the differentially expressed genes are genes that are expressed fetal-specifically, while being barely expressed maternal.
The method of claim 2, wherein: the differential expression gene is at least one of the genes shown in Table 1.
The method of claim 1, wherein: the biological sample is peripheral blood or urine.
The method of claim 1, wherein: performing reverse transcription on the extracted free RNA to generate first-strand cDNA, specifically performing reverse transcription on the free RNA by using a random primer to obtain first-strand cDNA; preferably, the random primer is an N6 random primer.
The method of claim 1, wherein: the specific primer is at least one of a first primer pair to a sixth primer pair;

the upstream and downstream primers of the first primer pair are respectively sequences shown in SEQ ID NO.1 and SEQ ID NO.2, the upstream and downstream primers of the second primer pair are respectively sequences shown in SEQ ID NO.3 and SEQ ID NO.4, the upstream and downstream primers of the third primer pair are respectively sequences shown in SEQ ID NO.5 and SEQ ID NO.6, the upstream and downstream primers of the fourth primer pair are respectively sequences shown in SEQ ID NO.7 and SEQ ID NO.8, the upstream and downstream primers of the fifth primer pair are respectively sequences shown in SEQ ID NO.9 and SEQ ID NO.10, and the upstream and downstream primers of the sixth primer pair are respectively sequences shown in SEQ ID NO.11 and SEQ ID NO. 12.
The method of claim 1, wherein: the specific primers are at least one group of primer pairs I to seven;

the upstream and downstream primers of the first primer pair are respectively sequences shown as SEQ ID NO.17 and SEQ ID NO.18, the upstream and downstream primers of the second primer pair are respectively sequences shown as SEQ ID NO.19 and SEQ ID NO.20, the upstream and downstream primers of the third primer pair are respectively sequences shown as SEQ ID NO.21 and SEQ ID NO.22, the upstream and downstream primers of the fourth primer pair are respectively sequences shown as SEQ ID NO.23 and SEQ ID NO.24, the upstream and downstream primers of the fifth primer pair are respectively sequences shown as SEQ ID NO.25 and SEQ ID NO.26, the upstream and downstream primers of the sixth primer pair are respectively sequences shown as SEQ ID NO.27 and SEQ ID NO.28, and the upstream and downstream primers of the seventh primer pair are respectively sequences shown as SEQ ID NO.29 and SEQ ID NO. 30.
The method according to any one of claims 1 to 7, wherein: further comprising amplifying the first strand cDNA with a reference primer capable of specifically amplifying the housekeeping gene while amplifying the first strand cDNA with a specific primer.
The method of claim 8, wherein: the housekeeping gene is the GAPDH gene.
The method of claim 9, wherein: the upstream and downstream primers of the reference primer are respectively sequences shown in SEQ ID NO.13 and SEQ ID NO. 14.
The method according to any one of claims 1 to 7, wherein: the universal primer comprises a first primer and a second primer, wherein the first primer is a sequence shown by SEQ ID NO.15, and the second primer is a sequence shown by SEQ ID NO. 16; also, preferably, the 5' end of the first primer has a phosphorylation modification.
The method according to any one of claims 1 to 7, wherein: the method further comprises the steps of denaturing the second round PCR amplification product into single-stranded nucleic acid after the second round PCR amplification, and connecting the single-stranded nucleic acid into circular nucleic acid by adopting a nucleic acid mediated fragment so as to obtain a circular target gene library, wherein the nucleic acid mediated fragment can be combined with two tail ends of the single-stranded nucleic acid by the base complementary pairing principle.
A method for detecting a fetal gene, comprising: comprises the following steps of (a) carrying out,

performing a target gene library construction using the method of any one of claims 1-12 to obtain the target gene library;

sequencing the target gene library to obtain a sequencing result consisting of a plurality of sequencing data;

and analyzing the sequencing result to obtain the gene information of the fetus.
The detection method according to claim 13, characterized in that: the genetic information of the fetus comprises at least one of mutation information of recessive genetic monogenic diseases, fetal genotype information and genetic structure variation information.
The detection method according to claim 13 or 14, characterized in that: analyzing the sequencing result, specifically comprising the following steps,

filtering the sequencing data;

comparing the filtered sequencing data to a reference genome, and reserving the sequencing data of the reference genome on the unique comparison;

and counting the base distribution condition of the target gene based on the sequencing data of the reference genome on the unique comparison to obtain the mutation information of the target gene and further obtain the gene information of the fetus.
The detection method according to claim 15, characterized in that: and analyzing the sequencing result, and further comprising comparing the target gene mutation information with a disease database to obtain the mutation information of the recessive genetic monogenic disease of the fetus.
A fetal gene detection device, comprising: comprises a free RNA extraction module, a reverse transcription module, a target gene amplification module, a target gene library generation module, a sequencing module and an analysis module;

the free RNA extraction module is used for extracting free RNA from a biological sample derived from a pregnant woman, wherein the free RNA comprises fetal free RNA;

the reverse transcription module is used for carrying out reverse transcription on the extracted free RNA to generate first-strand cDNA;

the target gene amplification module is used for amplifying the first strand cDNA by adopting a specific primer to obtain a specific amplification product, the specific primer can specifically amplify the target gene, the target gene is a differential expression gene of a fetus and a mother body, and the 5' end sequence of an upstream primer and/or a downstream primer of the specific primer is the same as at least one part of sequence of a universal primer;

the target gene library generating module is used for carrying out second round PCR amplification on the specific amplification product by adopting a universal primer to obtain the target gene library;

the sequencing module is used for sequencing the target gene library to obtain a sequencing result consisting of a plurality of sequencing data;

and the analysis module is used for analyzing the sequencing result to obtain the gene information of the fetus.
The detection device according to claim 17, wherein: in the free RNA extraction module, the biological sample is peripheral blood or urine.
The detection device according to claim 17, wherein: in the reverse transcription module, reverse transcription is carried out on the extracted free RNA to generate first-strand cDNA, and specifically, reverse transcription is carried out on the free RNA by adopting a random primer to obtain the first-strand cDNA; preferably, the random primer is an N6 random primer.
The detection device according to claim 17, wherein: in the target gene amplification module, the differentially expressed genes are genes expressed specifically in the fetus, but hardly expressed in the mother.
The detection device according to claim 20, wherein: the differential expression gene is at least one of the genes shown in Table 1.
The detection device according to claim 17, wherein: in the analysis module, the genetic information of the fetus comprises at least one of mutation information of recessive genetic monogenic diseases, fetal genotype information and genetic structure variation information.
The detection device according to any one of claims 17 to 22, wherein: the analysis module further comprises a filtering unit, a reference genome alignment unit and a statistical unit;

the filtering unit is used for filtering the sequencing data;

the reference genome comparison unit is used for comparing the filtered sequencing data to a reference genome and reserving the sequencing data of the reference genome which is only compared;

the statistic unit is used for counting the base distribution condition of the target gene based on the sequencing data of the unique comparative reference genome, obtaining the mutation information of the target gene and further obtaining the gene information of the fetus.
The detection device of claim 23, wherein: the analysis module further comprises a disease database comparison unit, and the disease database comparison unit is used for comparing the target gene mutation information with a disease database to obtain the mutation information of the recessive genetic monogenic disease of the fetus.
Use of the method according to any one of claims 1 to 12, the detection method according to any one of claims 13 to 16, or the detection device according to any one of claims 17 to 24 for the detection of monogenic disorders of recessive inheritance, or the detection of structural variations.
A reagent for fetal FGA gene detection, comprising: the kit comprises specific primers for amplifying FGA genes, wherein the specific primers comprise at least one group from a first primer pair to a sixth primer pair, the upstream and downstream primers of the first primer pair are respectively sequences shown as SEQ ID NO.1 and SEQ ID NO.2, the upstream and downstream primers of the second primer pair are respectively sequences shown as SEQ ID NO.3 and SEQ ID NO.4, the upstream and downstream primers of the third primer pair are respectively sequences shown as SEQ ID NO.5 and SEQ ID NO.6, the upstream and downstream primers of the fourth primer pair are respectively sequences shown as SEQ ID NO.7 and SEQ ID NO.8, the upstream and downstream primers of the fifth primer pair are respectively sequences shown as SEQ ID NO.9 and SEQ ID NO.10, and the upstream and downstream primers of the sixth primer pair are respectively sequences shown as SEQ ID NO.11 and SEQ ID NO. 12.
The reagent of claim 26, wherein: the kit also comprises a reference primer for amplifying the housekeeping gene GAPDH, wherein the upstream primer and the downstream primer of the reference primer are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14.
The reagent according to claim 26 or 27, characterized in that: the kit also comprises a universal primer for constructing a sequencing library, wherein the universal primer comprises a first primer and a second primer, the first primer is a sequence shown by SEQ ID NO.15, and the second primer is a sequence shown by SEQ ID NO. 16; also, preferably, the 5' end of the first primer has a phosphorylation modification.
A kit for fetal FGA gene detection, characterized in that: the kit comprising the reagent of any one of claims 26-28.
The kit of claim 29, wherein: the kit also comprises at least one of a free RNA extraction reagent, a reverse transcription reagent, a PCR amplification reagent and a nucleic acid purification reagent.
A reagent for detecting a fetal HESX1 gene, which is characterized in that: the primers comprise specific primers for amplifying the HESX1 gene, the specific primers comprise at least one group of primer pair I to primer pair VII, the upstream and downstream primers of the primer pair I are respectively sequences shown in SEQ ID NO.17 and SEQ ID NO.18, the upstream and downstream primers of the primer pair II are respectively sequences shown in SEQ ID NO.19 and SEQ ID NO.20, the upstream and downstream primers of the primer pair III are respectively sequences shown in SEQ ID NO.21 and SEQ ID NO.22, the upstream and downstream primers of the primer pair IV are respectively sequences shown in SEQ ID NO.23 and SEQ ID NO.24, the upstream and downstream primers of the primer pair V are respectively sequences shown in SEQ ID NO.25 and SEQ ID NO.26, the upstream and downstream primers of the primer pair VI are respectively sequences shown in SEQ ID NO.27 and SEQ ID NO.28, and the upstream and downstream primers of the primer pair VII are respectively sequences shown in SEQ ID NO.29 and SEQ ID NO. 30.
The reagent of claim 31, wherein: the kit also comprises a reference primer for amplifying the housekeeping gene GAPDH, wherein the upstream primer and the downstream primer of the reference primer are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14.
The reagent according to claim 31 or 32, characterized in that: the kit also comprises a universal primer for constructing a sequencing library, wherein the universal primer comprises a first primer and a second primer, the first primer is a sequence shown by SEQ ID NO.15, and the second primer is a sequence shown by SEQ ID NO. 16; also, preferably, the 5' end of the first primer has a phosphorylation modification.
A kit for detecting a fetal HESX1 gene is characterized in that: the kit comprising the reagent of any one of claims 31-33.
The kit of claim 34, wherein: the kit also comprises at least one of a free RNA extraction reagent, a reverse transcription reagent, a PCR amplification reagent and a nucleic acid purification reagent.