CN114250302B - Composition and kit for detecting cervical intraepithelial neoplasia and application - Google Patents
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Abstract
The invention discloses a composition, a kit and an application for detecting cervical intraepithelial neoplasia, wherein the composition comprises a reagent capable of indicating the ACTR3B gene state in a biological sample, and when the existence of HPV gene insertion in or near the ACTR3B gene is detected, the cervical state is diagnosed as the cervical intraepithelial neoplasia. The invention determines the cervical intraepithelial neoplasia specific gene based on nanopore sequencing, and further performs reliability verification through Sanger sequencing. Therefore, the invention provides an effective means for diagnosing or identifying cervical lesions, colposcopic shunt, whether CIN I level intervenes or not and the like, particularly distinguishing cervicitis and cervical CIN.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a composition for detecting cervical intraepithelial neoplasia, a kit and application.
Background
Cervical Intraepithelial Neoplasia (CIN) belongs to cervical epithelial dysplasia, which is a dynamically changing process classified into three levels, i.e., low, medium and high, i.e., level I, II and III. High CIN refers to atypical lesions that involve the entire epithelial layer but do not infiltrate the basement membrane, also known as carcinoma in situ of the cervix. The continuous development can infiltrate into the basement membrane, and can form cervical infiltration cancer. Low grade CIN lesions refer to cervical epithelial dysplasia that only affects the upper 1/3 of the entire epithelial layer. CIN is a dynamically changing process, but timely treatment can reverse and return to normal. Therefore, the diagnosis of CIN is of great significance for preventing or early intervening in cervical cancer.
Currently, CIN is mainly a cytometric examination. Specifically, exfoliated cells of the cervical orifice are collected through a liquid-based thin-layer cell kit, a full-automatic thin-layer cell pelleter is used for flaking, and cytological classification diagnosis is carried out according to the form of cell nuclei to judge whether cancerous lesions exist. The examination means is poor in accuracy and specificity for CIN, missing examination often occurs, and since CIN is similar to a lesion of cervicitis, the two cannot be well distinguished by cytology.
In recent years, with the research on the relationship between Human Papilloma Virus (HPV) infection and the lower genital tract, it has been found that HPV infection is associated with the occurrence of CIN. HPV infection, a specific type of sexually transmitted disease, is the etiological agent for CIN and cervical cancer development. Molecular biology and epidemiological studies have shown that human papillomaviruses are oncogenic. HPV can be divided into different types according to their oncogenicity: the HPV16, 18, 45 and 56 are high-risk types, 11 types of HPV31, 33, 35 and the like are medium-risk types, and 8 types of HPV6, 11, 26 and the like are low-risk types. Therefore, the search for specific biomarkers of CIN is of great significance.
CN104870653A discloses molecular diagnostic markers PRDM14 and FAM19a4 for HPV-induced invasive cancers and high-grade precursor lesions thereof, which comprise detecting hypermethylation in PRDM14 and/or FAM19a4 genes in cells, whereas such hypermethylation indicates the presence of HPV-induced precursor lesions and HPV-induced invasive cancers with invasive potential.
However, there are currently no molecular markers for effectively distinguishing CIN from early lesions.
The information in this background is only for the purpose of illustrating the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.
Disclosure of Invention
On the basis of collecting a TCT sample without intraepithelial neoplasia due to cervical high-risk HPV infection, TCT or fresh tissue samples of cervical lesions CIN I, CIN II and CIN III and TCT or fresh tissue samples of cervical cancer, the invention utilizes the three-generation nanopore technology and detects HPV DNA integration positions based on the probe hybridization capture principle to obtain HPV integration information in different stage samples, and further analyzes to obtain a specific marker which can well distinguish cervicitis and CIN and is used for assisting clinical early diagnosis and treatment. Specifically, the present invention includes the following.
In a first aspect of the invention, there is provided a composition for cervical intraepithelial neoplasia detection comprising an agent capable of indicating the status of the ACTR3B gene in a biological sample.
According to the composition for detecting cervical intraepithelial neoplasia of the present invention, preferably, the biological sample is derived from cervical tissue of an HPV infected subject.
The composition for detecting cervical intraepithelial neoplasia according to the present invention preferably further comprises an agent capable of indicating the status of DOCK3 gene in a biological sample.
According to the composition for cervical intraepithelial neoplasia detection of the present invention, preferably, the reagent comprises a barcode linker and a barcode primer, and the status of the DOCK3 gene or ACTR3B gene is indicated by a method comprising the steps of:
a. collecting a biological sample from a subject, and separating DNA to obtain a DNA fragment with the length of 1-3 Kb;
b. separating a target sequence from the DNA fragment, constructing a library, connecting a bar code joint at the tail end, and enriching by adopting a bar code primer to enable the target sequence or part of the target sequence to pass through a nanopore located in a chip, wherein the chip is arranged near an electrode, the electrode can detect current passing through the nanopore, and information of the target sequence is detected through current change.
According to the composition for cervical intraepithelial neoplasia detection of the present invention, preferably, the biological sample is exfoliated cells, surgical or punctured tissue, and step a comprises adjusting the ultrasound frequency and energy and/or optimizing the disruption time and system to disrupt DNA in the biological sample to a length of 1-3 Kb.
The composition for detecting cervical intraepithelial neoplasia according to the invention preferably further comprises a capture probe for enriching the target sequence.
In a second aspect of the present invention, there is provided use of an agent in the preparation of a composition for cervical intraepithelial neoplasia detection by a method comprising the steps of, wherein the agent comprises an agent capable of indicating the status of the ACTR3B gene in a biological sample, the method comprising:
a. collecting a biological sample from a subject, and separating DNA to obtain a DNA fragment with the length of 1-3 Kb;
b. separating a target sequence from the DNA fragment, constructing a library, connecting a bar code joint at the tail end, and enriching by adopting a bar code primer to enable the target sequence or part of the target sequence to pass through a nanopore located in a chip, wherein the chip is arranged near an electrode, the electrode can detect current passing through the nanopore, and information of the target sequence is detected through current change.
According to the application, preferably, the enrichment comprises the steps of mixing a DNA fragment with the length of 1-3Kb with human cot-1DNA, evaporating to dryness, redissolving in an optimized hybridization solution, incubating at room temperature, keeping at 93-97 ℃ for 5-15 minutes, adding a probe set consisting of a plurality of probes, slowly cooling to the hybridization temperature by reducing the temperature by 0.1 ℃ per minute to improve the specificity of a captured target sequence and the binding stability of the long fragment, hybridizing at 63-67 ℃ for 4-16 hours, mixing a product with streptavidin magnetic beads, incubating for 45 minutes, washing the magnetic beads with a washing solution, and synthesizing and enriching by using a random primer, dNTP and an enzyme with single-stranded DNA as a guide strand synthesis activity.
According to the use of the present invention, preferably, the cervical state is diagnosed as cervical intraepithelial neoplasia when the presence of HPV gene insertion is detected inside or in the vicinity of ACTR3B gene.
According to the use of the present invention, preferably, the cervical state is diagnosed as cervicitis when the presence of HPV gene insertion is detected within or near the DOCK3 gene.
In a third aspect of the invention, there is provided a kit comprising a composition according to the first aspect and instructions for using the composition for detection.
The invention provides an effective means for diagnosing or identifying cervical lesions, colposcopic shunt, interference of CIN I grade and the like, particularly distinguishing cervicitis and cervical CIN.
Drawings
Figure 1 is a histogram of qPCR results in different tissues for the DOCK3 gene.
Figure 2 is a histogram of qPCR results in different tissues for the ACTR3B gene.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
The composition, kit and use for detecting cervical intraepithelial neoplasia of the present invention are described below.
Composition comprising a fatty acid ester and a fatty acid ester
The compositions of the present invention include agents capable of indicating the status of the DOCK3 gene in a biological sample, examples of which include, but are not limited to, barcode linkers and barcode primers. The specific sequences of the barcode linker and the barcode primer are not particularly limited as long as the genes can be ligated and enrichment of the DOCK3 gene is achieved. The composition of the present invention further comprises a reagent capable of indicating the status of ACTR3B gene in a biological sample, examples of which include, but are not limited to, additional barcode linkers and barcode primers, the specific sequences of which are not particularly limited as long as the gene can be ligated and the enrichment of ACTR3B gene is achieved. Preferably, the composition of the present invention comprises a reagent capable of simultaneously indicating the status of the DOCK3 and ACTR3B genes in a biological sample, and also preferably, the composition of the present invention consists of a reagent capable of simultaneously indicating the status of the DOCK3 and ACTR3B genes in a biological sample.
The status of the gene includes, but is not limited to, an increase or decrease in the expression level of the target gene (DOCK3 gene, ACTR3B), the presence or absence of insertion of a foreign gene into the target gene. A foreign gene refers to a gene derived from outside the organism of interest or study. In particular embodiments, the exogenous gene refers to an HPV gene, and examples of HPV genotyping include, but are not limited to: HPV genes related to common warts, flat warts, plantar warts, etc. such as HPV1, 2, 3, 4, 7, 10, 12, 15, etc.; HPV5, 8, 14, 17, 20, 36, 38 and other HPV genes related to epidermodysplasia verruciformis, vulvar cancer, penile cancer, anal cancer, prostatic cancer and bladder cancer; HPV-6, 11, 13, 32, 34, 40, 42, 43, 44, 54 and other HPV genes related to genital, anal, oropharyngeal and esophageal mucosa infection; HPV-16, 18, 30, 31, 33, 35, 53, 39 and the like related to cervical cancer, rectal cancer, oral cancer, tonsil cancer. HPV genes further include HPV32, 52, 56, 58, 68.
The gene insertion site is not particularly limited, and may be a site present within or near the gene of interest, and may be any site of any component/element thereof within or near the gene of interest, for example, any site including, but not limited to, upstream or downstream of a promoter, coding sequence, terminator, enhancer thereof.
Detection method
Step 1. sequencing a subject sample
First, a subject sample genomic DNA is sequenced in order to obtain a sequencing result. The type of the sample to be tested is not particularly limited, and includes, but is not limited to, TCT sample of cervical inflammation or hyperplasia, TCT sample of cervical lesion CIN or TCT sample of cervical cancer, or blood or tissue sample of the above-mentioned patient. Wherein, the TCT sample refers to a sample obtained according to a standard liquid-based thin-layer cell detection method. According to an embodiment of the present invention, before sequencing the genomic DNA of the sample infected with HPV in the subject, the method may further comprise: extracting genomic DNA from the subject sample. The extraction method is not particularly limited, and extraction may be performed by using a genomic DNA extraction method or an extraction kit known in the art.
According to an embodiment of the present invention, the subject genomic DNA is subjected to a sequencing step using nanopore sequencing technology (Nanorpore). Therefore, real-time monitoring of the sequencing process can be realized, and the target sequence is enriched, so that the sequencing time is obviously reduced. In addition, based on the technology, longer sequence reads (reads) can be obtained, the result of structural variation of a longer fragment is more reliable than that of millions of reads measured by an NGS platform, and meanwhile, the problem of sequence splicing caused by short sequences is avoided. In sequencing single DNA or RNA molecules, PCR amplification or chemical labeling of the sample is not required. Thus, a relatively low cost sequencing method for genotyping, high test mobility and rapid sample handling is provided. The instrument or sequencing platform for sequencing is not particularly limited, and examples of the instrument or platform for Nanopore sequencing include, but are not limited to, MinION sequencer, Gridios X5, Promethion, Flongle, by Oxford Nanopore Technologies. According to the library building and sequencing requirements of the nanopore sequencing technology platform, a sequencing library of the genome DNA of the sample of the subject is built, and then the sequencing library is sequenced so as to obtain a sequencing result.
Step 2. determining DNA fragment containing both HPV integration sequence and human genome sequence
Based on the above sequencing results, a DNA fragment containing both HPV sequences and human genome sequences was determined. Wherein, determining the DNA fragment comprising both HPV sequences and human genome sequences further comprises: filtering the sequencing result to obtain a filtered sequencing result; the filtered sequencing results are aligned with reference sequences to determine DNA fragments comprising both HPV integration sequences and human genome sequences. Wherein the reference sequence is human genome and HPV genome sequence.
It should be noted that, in the detection step of the present invention, the library creation includes a step of performing hybridization capture using a probe or a probe set, and the specific sequence of the probe or probe set used for the hybridization capture is not particularly limited as long as enrichment of HPV genome can be achieved. One skilled in the art can design such probes or probe sets based on the entire genome of the virus.
As used herein, unless otherwise indicated, the reference sequence is the human genomic data (hg18) downloaded (http:// genome. UCSC. edu /) from the UCSC database, including genes and duplicate annotations. COSMIC v58 and a series of oncogenes were downloaded from http:// www.sanger.ac.uk/genetics/CGP/Census. HPV genomic sequences were downloaded from http:// HPV-web. The dbSNP132 and thousand human genome project data that may be used herein are downloaded from the NCBI FTP website (http:// www.ncbi.nlm.nih.gov/Ftp /). Human disease-associated viral integration site data were obtained from http:// www.scbit.org/dbmi/drvis.
3. Authentication
The invention further includes validation of the sequencing data, which validated the reliability of HPV integration using sanger sequencing.
In the present invention, specific genes for diagnosis are obtained based on machine learning using HPV insertion data as characteristic data and disease progression as a label. Preferably, the machine learning is a random forest algorithm. Machine learning based analysis steps are known in the art.
Reagent kit
The invention further provides a kit comprising a composition as described herein. In addition to the above components, the kits of the present invention may include precautions related to the regulatory manufacture, use or sale of the diagnostic kit in a form prescribed by a governmental agency. In addition, the kits of the invention may be provided with detailed instructions for use, storage, and troubleshooting. The kit may optionally also be provided in a suitable device, preferably for robotic handling in a high throughput setting.
In certain embodiments, the components (e.g., oligonucleotides) of the kits of the invention may be provided as dry powders. When the reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be disposed in another container. The container will typically comprise at least one vial, test tube, flask, bottle, syringe, and/or other container means, optionally in which the solvent is placed in equal portions. The kit may further comprise means for a second container comprising a sterile, pharmaceutically acceptable buffer and/or other solvent.
In certain embodiments, the components of the kits of the invention may be provided in the form of a solution, e.g., an aqueous solution. The concentrations or amounts of these ingredients, when present in aqueous solution, are readily determinable by those skilled in the art according to the various requirements. For example, for storage purposes, for example, the concentration of the oligonucleotide may be present in a higher form, and when in the working state or in use, the concentration may be reduced to the working concentration, for example, by diluting the higher concentration solution.
Where more than one component is present in a kit, the kit will also typically comprise a second, third or other additional container into which additional components may be separately placed. In addition, combinations of various components may be included in the container. Any of the compositions or reagents described herein can be a component of a kit.
The scheme of the invention will be explained with reference to the following examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or apparatus used are not indicated by the manufacturer, but are conventional products available commercially, for example from Illumina.
Example 1
Firstly, sample information:
1. the clinical sample information of the present invention is as follows: 16 TCT samples of HPV high-risk infection of early cervical inflammation or hyperplasia, 20 TCT or fresh tissue samples of grade CIN I, CIN II and CIN III cervical lesions, and 10 fresh tissue samples of cervical cancer.
And performing quality inspection on the sample after genome DNA extraction, and grouping the sample after the quality inspection is qualified.
2. Group entry criteria
The study may be selected for entry when the patient meets all of the following criteria:
1) patients who were examined for cervical cancer in my hospital;
2) HPV detection of high risk patients;
3) patients with abnormal colposcopy, TCT examination, cervical biopsy results.
4) Follow-up can be performed regularly.
5) The patient or family member was able to understand the study protocol and was willing to participate in the study, providing written informed consent.
3. Exclusion criteria
Patients cannot be selected for clinical study when any of the following conditions exist:
1) all examinations of cervix are normal
2) HPV detection negative
3) Pathologically confirmed as cervical neuroendocrine cancer;
4) combined with other malignant tumors
5) The follow-up visit is not standard or the clinical data is not complete.
6) Patients or family members were unable to coordinate the study or provide written informed consent.
Secondly, sequencing by taking tissue DNA as a sample
The gDNA of the TCT samples used in the test was cleaved at a length of 1kb or more. And (3) carrying out fragment sorting of more than 1kb on the broken DNA sample, and constructing a library on the broken product by using a three-generation nanopore library construction method.
Carrying out hybridization capture on HPV related probes of the third generation library, enriching HPV sequence information and carrying out nanopore sequencing. And carrying out QC quality control on the third-generation off-line data, and filtering out qualified sequences. Performing Blast comparison on the qualified sequences to find HPV integrated sequences containing human genome sequences and virus sequences. And (4) performing gene position annotation on the integrated sequence, sorting high-frequency genes and annotating genes related to the cervical cancer. And carrying out sanger sequencing verification on the detected high-frequency integration genes.
The method specifically comprises the following steps:
1. enrichment of target region
1) 1 mu g of tissue gDNA is taken, 1K to 3K is broken by Covarism220, the tissue gDNA is mixed with 5 mu g of human cot-1DNA, the mixture is dried by distillation at 60 ℃ by using a vacuum suction filter pump, then re-dissolved in optimized hybridization solution (containing 10 XSSPE, 10 XDenhart solution and 0.3% SDS), the mixture is incubated for 10min at room temperature and then put into a PCR instrument, a mixed 120nt ssDNA probe is added after 10min at 95 ℃, the temperature is slowly reduced to the hybridization temperature by reducing 0.1 ℃ per minute, and then the mixture is placed at 65 ℃ for hybridization for 4 to 16 hours.
2) Mixing the product obtained in the step 1) with streptavidin magnetic beads, incubating for 45min on a PCR instrument, and subsequently cleaning the magnetic beads with a cleaning solution.
3) And (3) enriching the product obtained in the step 2) by using a random primer, dNTP, Klenow exo-and the like, and performing quality control by using the Qubit4.0 and Agilent2100 capillary electrophoresis after purifying the product by using Agencour AMPure XP magnetic beads.
2. Building warehouse
1) Taking 100ng of each sample DNA and the enriched DNA library obtained in the step 1), constructing the library by using a PCR coding Kit of Oxford nanopore technologies, and repairing the End by using NEB Ultra II End-prep reaction buffer and Ultra II End-prep enzyme mix;
2) ligating Barcode Adapters to the product of step 1) using Blunt/TALigase Master Mix;
3) and (3) enriching the product obtained in the step 2) by using Long Amp Taq 2x Master Mix and Barcode Primers, purifying the product by using Agencour AMpure XP magnetic beads, and performing quality control by using Qubit4.0 and Agilent2100 capillary electrophoresis.
4) Libraries of different barcodes were pooled together for on-machine sequencing.
Third, the results
The third generation nanopore technology has the advantages of long reading time, rapidness, convenience and the like, and can be well adapted to clinical development and application. In order to search for CIN specific HPV integration sites, 15 HPV high-risk type probes are designed based on the whole virus genome, and based on a three-generation nanopore sequencing platform, the cervical exfoliated cell samples of 20-position entry subjects are captured and detected by probe hybridization. Wherein 1351 genes detected in the cervicitis group are related to HPV gene insertion, 4948 genes detected in the CIN I group are related to HPV gene insertion, 5562 genes detected in the CIN II group are related to HPV gene insertion, and 4041 genes detected in the CIN III group are related to HPV gene insertion. The results of the gene detection and clinical information are shown in Table 1.
TABLE 1
Compared with the NGS pre-sequencing result, 322 new integration sites are obtained based on the gene data obtained by the three generations of nanopore sequencing platforms. The reliability of the newly found 322 integration positions was verified by sanger sequencing.
The database is established by taking the insertion data of the 15902 genes in total as characteristics and taking different lesions of the cervix as labels. And (3) carrying out classification training on the database by using a random forest algorithm, and selecting the genes with the highest weight from the database, wherein the genes are DOCK3 genes and ACTR3B genes, and the two genes are new integration sites. Next, the DOCK3 genes were used as inflammation group-specific genes, respectively, and ACTR3B was classified as a specific gene of CIN group (including I, II and III).
Example 2
This example was used to analyze the expression of the DOCK3 gene and ACTR3B gene in different cervical tissues.
First, experiment method
Specific primers for the DOCK3 gene and ACTR3B gene were designed based on known public information, and primers were artificially synthesized by primer synthesis companies.
1. Total RNA of cervical secretions was extracted and tested for concentration, purity and integrity.
2. Carrying out a reverse transcription reaction:
1. mu.g of the extracted total RNA was used as a template, and the following reaction system was constructed and placed on ice to carry out a reaction.
The reverse transcription reaction system was then constructed:
the reverse transcription reaction system was reacted at 37 ℃ for 15 minutes.
And carrying out SYBRGreen qPCR detection, and drawing a melting curve. And detecting the expression conditions of the DOCK3 gene and the ACTR3B gene in the tissues.
Second, experimental results
In cervical secretions of healthy people, active expression of DOCK3 and ACTR3B was detected, and expression of the corresponding mRNA was present. However, in the inflammation group, the mRNA amount of DOCK3 was significantly reduced, but the mRNA amount in the CIN group sample was comparable to that of the healthy control population (fig. 1). In CIN group, the expression of ACTR3B gene was significantly reduced, but the amount of DOCK3 mRNA was not significantly reduced (FIG. 2).
The results showed that the insertion of HPV into the DOCK3 gene and the ACTR3B gene blocked the expression of the corresponding genes, and that the insertion of HPV gene into and around the DOCK3 gene is a phenomenon peculiar to the inflammatory group. The insertion of the HPV gene into and around the ACTR3B gene is a phenomenon specific to patients in the CIN group.
The research of the invention provides an effective means for further diagnosing or identifying cervical lesions, particularly distinguishing cervical inflammation from cervical CIN.
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Claims (2)
1. Use of an agent in the preparation of a composition for use in differentiating cervicitis from cervical intraepithelial neoplasia detection by a method comprising the steps of, wherein the agent comprises an agent capable of indicating the status of ACTR3B and DOCK3 genes in a biological sample, the method comprising:
a. collecting a biological sample from a subject, and separating DNA to obtain a DNA fragment with the length of 1-3 Kb;
b. separating a target sequence from the DNA fragment, constructing a library, connecting a bar code joint at the tail end, and enriching by adopting a bar code primer to enable the target sequence or part of the target sequence to pass through a nanopore located in a chip, wherein the chip is arranged near an electrode, the electrode can detect current passing through the nanopore, and information of the target sequence is detected through current change;
diagnosing the subject as having or having a high risk of having cervical intraepithelial neoplasia when the presence of HPV gene insertion is detected within or near the ACTR3B gene; when the presence of HPV gene insertion is detected within or near the DOCK3 gene, the subject is diagnosed as a cervicitis patient.
2. The use of claim 1, wherein the enrichment comprises mixing a DNA fragment with a length of 1-3Kb with human cot-1DNA, evaporating to dryness, redissolving in an optimized hybridization solution, incubating at room temperature, maintaining at 93-97 ℃ for 5-15 minutes, adding a probe set consisting of a plurality of probes, slowly cooling to the hybridization temperature by lowering the temperature by 0.1 ℃ per minute to improve the specificity of the captured target sequence and the binding stability of the long fragment, hybridizing at 63-67 ℃ for 4-16 hours, mixing the product with streptavidin magnetic beads, incubating for 45 minutes, washing the magnetic beads with a washing solution, and synthesizing and enriching using a random primer, dNTP, an enzyme having a single-stranded DNA as a guide for duplex synthesis activity.
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