CN113337644A - Screening method and application of Linc HPV integration gene sites - Google Patents

Screening method and application of Linc HPV integration gene sites Download PDF

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CN113337644A
CN113337644A CN202110769725.7A CN202110769725A CN113337644A CN 113337644 A CN113337644 A CN 113337644A CN 202110769725 A CN202110769725 A CN 202110769725A CN 113337644 A CN113337644 A CN 113337644A
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陈世民
杨帆
黄晓园
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Wuhan Kaideweisi Biotechnology Co ltd
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Abstract

The invention discloses a screening method and application of an HPV integration gene locus of Linc type, belonging to the field of biological diagnosis, wherein corresponding PCR amplification upstream and downstream primers are designed aiming at the integration loci Linc02301, Linc01435, Linc00430 and Linc00564 of HPV, and are assembled to obtain a corresponding kit for detecting high risk group of cervical cancer.

Description

Screening method and application of Linc HPV integration gene sites
Technical Field
The invention belongs to the field of biological diagnosis, and particularly relates to a screening method and application of HPV integration gene loci related to cervical cancer.
Background
Cervical cancer refers to malignant tumors occurring in the cervix, vagina and cervix uteri, and is one of the most common gynecological malignancies, with the incidence second only to breast cancer. The cervical cancer of China ranks second in the incidence rate and third in the mortality rate of various cancers of Chinese women aged 15-44. Research shows that the persistent infection of high-risk HPV is a necessary condition for the generation of cervical cancer. The progress of molecular biology and cytogenetics proves that the integration of HPV and host chromosome is an important step in the cervical cell immortalization process, and is an important mark for causing the malignant progress of cervical intraepithelial neoplasia to cervical cancer. Integration of HPV with the host cell chromosome is found in more than 90% of cervical cancer tissues, and integration of HPV with the host cell genome is the most important risk factor for development of cervical cancer. Meanwhile, multiple research groups at home and abroad detect the HPV integration state in the population with large samples of different disease states (CIN1-3 and cervical cancer), and the result shows that the virus integration incidence rate is in positive correlation with the cervical cancer incidence rate and the virus integration state is in positive correlation with the cervical cancer disease degree. Therefore, the HPV integration is used as a marker of cervical lesion degree, and has very important significance for early auxiliary diagnosis of cervical precancerous lesion and cervical cancer.
Studies have shown that integration of HPV into the human genome is random. The HPV integration sites reported in the literature are quite many at present, and a plurality of high-frequency integration sites, such as POU5F1B, FHIT, KLF12 and the like, are found through sequencing of a large number of samples, and have certain promotion effects on carcinogenesis. However, studies show that long-chain non-coding RNAs (lincRNAs) play a key role in biomolecule epigenetic regulation, participate in the processes of generation and development of cancers, and play a relevant role in regulating and controlling the generation process of cervical cancer when HPV is integrated into Linc gene families, so that the formation of the cancers is promoted. Therefore, the method has important significance for the research on the Linc gene family of the HPV integration site.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for detecting Linc HPV integration gene loci in high risk population of cervical cancer, and a liquid phase hybridization capture combined NGS sequencing method is utilized to detect virus integration and PCR Sanger sequencing method is utilized to research and reveal a new type of Linc gene families (Linc02301, Linc01435, Linc00430 and Linc00564) of the HPV integration gene loci related to the occurrence of cervical cancer; according to the HPV integration to the locus, a kit for detecting the high risk group of cervical cancer is prepared. The kit has the advantages of rapidness, convenience, accuracy and the like, and is realized by the following technology.
The screening method of the Linc HPV integration gene locus comprises the following steps:
s1, extracting human cervical exfoliated cell DNA; generally, cervical exfoliated cells of a plurality of LSIL patients, cervical exfoliated cells of HSIL patients and cervical exfoliated cells of cervical cancer patients can be selected as samples; LSIL refers to low-grade squamous intraepithelial lesions, HSIL refers to high-grade squamous intraepithelial lesions, and is a distinct pathological stage of cervical intraepithelial neoplasia;
s2, designing HPV full-length probes containing 18 subtypes of HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV 82;
constructing a DNA sequencing library according to the requirement of Illumina, carrying out enzyme digestion treatment on sample genome DNA into a plurality of DNA fragments, then carrying out terminal repair and A addition treatment on the DNA fragments, wherein the total amount of the constructed DNA library is more than or equal to 1500ng, and the main peak of the fragment length of the DNA library is 350-550 bp;
hybridizing the HPV full-length probes of 18 subtypes with a genomic DNA library (according to a liquid phase probe hybridization capture technology), capturing target products, and eluting non-hybridized DNA fragments; the obtained DNA fragment is amplified through 16 PCR cycles, secondary hybridization capture is carried out after purification, the obtained purified amplification product is a sequencing library, the concentration of the sequencing library is more than or equal to 1 ng/mu L, and the main peak of the length of the sequencing library fragment is 350-550 bp; sequencing the sequencing library;
s3, analyzing the sequencing result of the step S2; filtering, removing the adaptor sequence, the low-quality sequence and the repetitive sequence, and evaluating the quality of the sequence before and after filtering to ensure that the filtered sequence Q30 is more than 80 percent and the sequence length is more than 100 bp; comparing the pre-processed sequencing data with HPV virus (HPV 16: NC-001526.2, HPV 18: AY262282.1, HPV 26: NC-001583.1, HPV 31: HQ537666.1, HPV 33: HQ537688.1, HPV 35: HQ537708.1, HPV 39: M62849.1 HPV 45: EF202156, HPV 51: KF436882.1, HPV 52: GQ472848.1, HPV 53: NC-001593.1, HPV 56: EF177176.1, HPV 58: HQ537777.1, HPV 59: EU918767.1, HPV 66: LR862085.1, 68: EU918769.1, HPV 73: LR862011.1, HPV 82: LR862056.1) and human genome (NCBI build 37, HG19) reference sequences, identifying several chimeras therefrom comparing the HPV genomes in part and comparing in part with human genome sequence; locally clustering the chimeric sequences according to positions on a human genome (assembling the chimeric sequences through end pairing to determine accurate integration sites), locally aligning the locally clustered sequences with a reference genome (generally, BWA v0.7.17, Samtools v1.9 and Picard v2.20.6 software is adopted for alignment), and determining whether the sample genome integrates HPV partial sequences in genes Linc02301, Linc01435, Linc00430 and Linc00564 according to local alignment results;
s4, and verifying HPV integration gene sites Linc02301, Linc01435, Linc00430 and Linc00564 related to the occurrence of cervical cancer by using conventional PCR and Sanger sequencing.
Preferably, in step S1, the concentration of human cervical exfoliated cell DNA is > 15 ng/. mu.L, the purity A260/A280 is greater than or equal to 1.5, and the purity A260/A230 is greater than or equal to 1.
Preferably, in step S2, the HPV full-length probes of 18 subtypes hybridize to the genomic DNA library in a manner of: hybridization was carried out at 65 ℃ for 16-24 h.
The invention also provides a primer combination for PCR amplification of the Linc HPV integration gene loci, wherein the Linc HPV integration gene loci are Linc02301, Linc01435, Linc00430 and Linc 00564; the four integration gene sites are formed by inserting partial or all sequences of HPV16, 18, 33 and 58 into human genome sequence.
The address of the sequence of HPV is as follows:
the website address of HPV16 is https:// www.ncbi.nlm.nih.gov/nuccore/NC _ 001526.2;
the website address of HPV18 is https:// www.ncbi.nlm.nih.gov/nuccore/AY 262282.1;
the website address of HPV33 is https:// www.ncbi.nlm.nih.gov/nuccore/HQ 537688.1;
the website address of HPV58 is http:// www.ncbi.nlm.nih.gov/nuccore/HQ 537777.1;
the insertion sites of the human genomic sequence include the following sites: chr 13: 87408594, Chr 13: 81127041, Chr 10: 107321167, Chr 14: 83702575. of the above sites, the amino acid sequence represented by "Chr 13: 87408594 "for example, indicates that HPV is integrated at position 87408594 on human chromosome 13.
The corresponding websites of human chromosomes are as follows:
the website address of Chr10 is http:// www.ncbi.nlm.nih.gov/nuccore/NC _ 000010.10;
the website address of Chr13 is http:// www.ncbi.nlm.nih.gov/nuccore/NC _ 000013.10;
the website address of Chr14 is http:// www.ncbi.nlm.nih.gov/nuccore/NC _ 000014.9;
the HPV site (or breakpoint) points include the following sites (or breakpoints): HPV 16: 2647. HPV 33: 1552. HPV 18: 6008. HPV 58: 3380. among the above breakpoints, the breakpoint expressed by "HPV 16: 2647 "for example, means that the site of integration into the human genome HPV (breakpoint) is base 2647.
Table 1 below is the site of integration of the genomic sequences of HPV16, 18, 33, 58 on the human genome. The "HPV sites" shown in the third column of Table 1 are inserted into the "sites on the human genome" shown in the second column of the "genes" shown in the first column, respectively.
TABLE 1 site of integration of the HPV genomic sequences on the human genome
Gene Breakpoint on human genome HPV breakpoints
Linc00430 Chr13：87408594 HPV16：2647
Linc00564 Chr13：81127041 HPV33：1552
Linc01435 Chr10：107321167 HPV18：6008
Linc02301 Chr14：83702575 HPV58：3380
The sequences of upstream and downstream primers of the HPV integration gene locus Linc02301 amplified by PCR are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6;
the sequences of upstream and downstream primers of the HPV integration gene locus Linc01435 amplified by PCR are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8;
the sequences of upstream and downstream primers of an HPV integration gene locus Linc00430 amplified by PCR are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10;
the sequences of the upstream primer and the downstream primer of the HPV integration gene locus Linc00564 obtained by PCR amplification are respectively shown as SEQ ID NO.11 and SEQ ID NO. 12.
The sequences of the PCR amplified upstream and downstream primer combinations for detecting HPV integration sites are shown in Table 2 below.
TABLE 2 PCR amplification of upstream and downstream primer combinations for detection of HPV integration sites
Figure BDA0003152511960000041
Those skilled in the art can appropriately add, reduce or change the length of the primer sequence shown in Table 2 above (delete part of the bases, retain only part or all of the 3' sequence) by adding different restriction enzyme site sequences, protecting bases, etc., and change the individual bases, based on the primer sequence shown in Table 2 above. The above operation is within the scope of the present invention as long as it does not affect PCR amplification.
Preferably, in the primer combination for amplifying the Linc HPV integration gene locus by PCR, the integration sequence of the HPV integration gene locus Linc02301 is shown as SEQ ID No.1, the integration sequence of the HPV integration gene locus Linc01435 is shown as SEQ ID No.2, the integration sequence of the HPV integration gene locus Linc00430 is shown as SEQ ID No.3, and the integration sequence of the HPV integration gene locus Linc00564 is shown as SEQ ID No. 4.
The sequences of integration sites formed after insertion of the above HPV genomes into the human genome are shown in Table 3 below.
TABLE 3 integration sequences formed after insertion of the HPV genome into the human genome
Figure BDA0003152511960000042
Figure BDA0003152511960000051
Those skilled in the art can make appropriate additions, reductions or changes, for example, adding different restriction enzyme site sequences, protecting bases, etc., according to the sequences of the integration sites described in the above table 3, reducing the sequence length (e.g., deleting part of bases) of the integration sites described in the above table 3, and making changes for individual bases; such manipulations are within the scope of the present invention, as long as most of the bases of the sequence of the integration site described in Table 2 of the present invention are still retained.
The HPV integrated gene locus related to the occurrence of the cervical cancer, and the primers and reagents used in the detection process can be assembled into a kit for detecting the high risk group of the cervical cancer, and the kit is used for detecting the high risk group of the cervical cancer.
The invention also provides a kit for detecting the high risk group of cervical cancer, which comprises the primer combination of the PCR amplification Linc HPV integration gene locus, as shown in SEQ ID NO.6-SEQ ID NO. 15.
The method for extracting the DNA of the human cervical exfoliated cells is not particularly limited, and the method can be used as long as the integrity of the extracted DNA sample is ensured, a commercial DNA extraction kit can be purchased, and the DNA extraction reagent described in the fifth edition (F.M. Oseber et al) of the well-compiled molecular biology experimental guidelines can be referred to or can be properly adjusted to be used for extracting the DNA of the human cervical exfoliated cells.
Preferably, the kit for detecting the high risk group of cervical cancer further comprises a DNA extraction reagent, a PCR reagent and a plurality of sample storage tubes.
The invention has no special strict limitation on the manufacturers, concentrations and systems of the reagents used for PCR amplification, and can be premixed reagents for PCR amplification or singly packaged reagents for PCR amplification as long as the target bands can be amplified.
Preferably, the DNA extraction reagent is Buffer ACL, RNaseA, protease K, Buffer ACL, Buffer WA, Buffer WB, and Elution Buffer;
the PCR reagent comprises DNA polymerase, an amplification buffer solution and double distilled water. When the kit is adopted, healthy normal cervical exfoliated cells (namely human cervical exfoliated cells which are not infected by HPV virus) can be selected as negative control, or liquid-based thin-layer cell preservation solution containing trace cervical cells can be selected as raw materials, DNA in the liquid-based thin-layer cell preservation solution is extracted, and a cell sample containing human cervical cell DNA is prepared by PCR amplification or clone transformation.
Preferably, the sample holding tubes are 1.5ml EP tubes, and each of the sample holding tubes contains 1 ml cell preservation solution.
Preferably, the PCR reagent is
Figure BDA0003152511960000061
Master Mix, available from Nanjing Novowed Biotech, Inc., cat # P511-01.
The use method of the kit for detecting the high risk group of cervical cancer comprises the following steps:
(1) collecting human cervical exfoliated cells as a sample and extracting exfoliated cell DNA by using a DNA extraction reagent;
(2) and detecting and extracting a cervical exfoliated cell DNA sequence by using a reversible terminal termination sequencing method, analyzing, filtering the joint sequence, the low-quality sequence and the repeated sequence, and evaluating the sequence quality before and after filtering. Ensuring that the filtered sequence Q30 is more than 80 percent and the sequence length is more than 100 bp;
and (3) rapidly comparing the sequences of the preprocessed sequencing data to HPV virus and human genome reference sequences by adopting sequence comparison software. Identifying a chimeric sequence (i.e., a sequence aligned partially with the HPV genome and partially with the human genome) from the alignment based on the results of the alignment; for the chimeric sequences, local clustering is carried out according to positions on a human genome, the clustered sequences are locally aligned with a reference genome, and whether a sample integrates a partial sequence of HPV or not and whether the HPV partial sequence is integrated in the genes (Linc02301, Linc01435, Linc00430 and Linc00564) are determined according to the alignment result;
(3) using SEQ ID N0: 5 to SEQ ID NO: 12, performing PCR amplification on the integrated sequence obtained in the step (2) by using upstream and downstream primer sequences shown in the specification;
(4) and (4) analyzing results: sequencing the PCR product amplified in the step (3), comparing the PCR product with a human genome and an HPV genome, and when a part of the human genome and a part of the HPV genome are compared at the same time, indicating that the genes are integrated by the HPV genome, wherein the joint of the compared human genome and the HPV genome is an integration site of HPV; the detection of the integration site indicates that the result is positive, namely the cervical cancer high risk group is obtained; otherwise, the result is negative, i.e. not belonging to the high risk group of cervical cancer.
The PCR amplification method is not particularly limited, and the reagent operation can be performed by selecting appropriate amplification conditions according to the PCR amplification reagent as long as the amplification target band can be ensured, and the preferred PCR amplification method comprises 25 cycles of 95 ℃ for 30s, 95 ℃ for 5s, 60 ℃ for 20s, 72 ℃ for 15s, and 72 ℃ for 5min, and the parameters can be properly adjusted according to actual conditions.
The detection method of the HPV integrated gene locus related to the occurrence of cervical cancer, the HPV integrated gene locus related to the occurrence of cervical cancer and the primer for detecting the HPV integrated gene locus can be assembled into a kit for detecting the high risk group of cervical cancer, and can also be used for manufacturing DNA chips or other products, and the products can be used for detecting the high risk group of cervical cancer.
Compared with the prior art, the invention has the advantages that: the invention discloses a new HPV integration gene locus Linc gene family (Linc02301, Linc01435, Linc00430 and Linc00564) related to the generation of cervical cancer by utilizing a liquid phase hybridization capture combined NGS sequencing method to detect virus integration and PCR Sanger sequencing method research, and prepares a kit for detecting high risk group of cervical cancer according to the integration of HPV in cervical exfoliated cell DNA to the gene locus. The kit assembled by HPV integration is applied to high risk groups for judging cervical cancer, has good innovation and is helpful for clinical diagnosis.
Drawings
FIG. 1 is a diagram showing the HPV integration positive rate distribution of all integration gene sites in LSIL, HSIL and cervical cancer samples in examples;
FIG. 2 is a distribution diagram of the detection positivity of the Linc gene family integration sites in all samples.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The cervical exfoliated cells of 988 LSIL patients, 358 HSIL patients and 126 cervical cancer patients were selected as samples.
The LSIL of the invention refers to low-grade squamous intraepithelial lesion, and the HSIL refers to high-grade squamous intraepithelial lesion, which is different pathological stages of cervical intraepithelial neoplasia.
1. Centrifuging the cervical exfoliated cell sample for 5min to collect cells, and removing supernatant; sequentially adding 200 mul PBS and 20 mul protease K, and uniformly mixing by oscillation;
2. adding 200 μ l Buffer BCL, shaking, mixing, adding 56 deg.C water bath for 10min, reversing the above process, mixing for several times, and purifying with column; adding 150 μ l of anhydrous ethanol, shaking, mixing, and centrifuging for a short time to collect liquid on the inner wall of the tube cover;
3. placing FastPuggDNA Mini Columns II adsorption column in 2ml Collection tube, transferring the above mixed solution (including flocculent precipitate) to adsorption column; centrifuging at 12,000rpm (13,400 Xg) for 1 min; discarding the filtrate, and placing the adsorption column in a collection tube; add 500. mu.l Buffer WA to the adsorption column along the tube wall, centrifuge for 1min at 12,000rpm (13,400 Xg);
4. discarding the filtrate, and placing the adsorption column in a collection tube; add 600. mu.l Buffer WB along the tube wall, centrifuge at 12,000rpm (13,400 Xg) for 1min, discard the filtrate;
5. repeating the step 4;
6. placing the adsorption column in a collection tube; centrifuging the column at 12,000rpm (13,400 Xg) for 2 min;
7. transferring the adsorption column to a new 1.5ml centrifuge tube; dripping 50-200 μ l of Elution Buffer into the center of the adsorption column membrane, and standing at room temperature for 2-5 min; centrifuging at 12,000rpm (13,400 Xg) for 1 min;
8. the adsorption column was discarded and the DNA product was stored at-20 ℃ until use.
Example 2: the integration site of HPV in cervical tissue genome DNA is detected by reversible end-termination sequencing.
1. Viral integration detection of genomic DNA samples of cervical exfoliated cells: designing HPV full-length probes containing 18 subtypes (HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82), wherein the sequence details of the probes are shown in the patent application CN112195287A of the applicant at 11, 12/2020 of 2020-A probe set for HPV typing and integration detection of human papillomavirus and a kit thereof;
constructing a DNA sequencing library according to the requirement of Illumina, carrying out enzyme digestion on sample genome DNA into small fragments by adopting an enzyme digestion mode, and then repairing and adding A to treat the tail ends of the small fragments; ensuring that the total amount of the constructed DNA library is more than or equal to 1500ng, and the main peak of the length of the library fragment is about 350-550 bp; according to a liquid phase probe hybridization capture technology, HPV probes containing 18 subtypes are applied to hybridize with a genomic DNA library for 16-24h at 65 ℃, and target products are captured to elute non-hybridized DNA fragments; the obtained fragments are amplified through 16 PCR cycles, purified and then captured by secondary hybridization, the obtained purified amplification product is a sequencing library, the concentration of the library is ensured to be more than or equal to 1 ng/mu l, and the main peak of the length of the library fragments is about 350-550 bp; adding the sequencing library into a gene sequencer Nextseq CN500 for sequencing;
2. analyzing the virus integration detection result:
firstly, removing sequences with low quality, repeated and polluted by adaptor primer, and simultaneously comparing the remaining sequences with human genome (NCBI build 37, HG) and HPV genome (HPV: NC _, HPV: AY, HPV: NC _, HPV: HQ, HPV: HQ, HPV: HQ, HPV: M HPV: EF202156, HPV: KF, HPV: GQ, HPV: NC _, HPV: EF, HPV: HQ, HPV EU: LR, HPV: EU, HPV: LR and HPV: LR);
second, sequences that are aligned completely to the human genome or the HPV viral genome are removed, leaving only chimeric sequences that are aligned partially to human and partially to the HPV genome at the same time. The assembly of the chimeric sequences by end pairing determines the exact integration site. The sequences assembled by end pairing are aligned again by using BWA v0.7.17, Samtools v1.9 and Picard v2.20.6 software, and the junction of the human genomic DNA and the HPV genomic DNA sequences is the integration site of HPV. According to the alignment result, whether the sample is integrated or not and whether the integration in the loci of Linc02301, Linc01435, Linc00430 and Linc00564 exists or not can be determined.
Example 3: the HPV integration sites were verified using PCR amplification and Sanger sequencing.
1. PCR amplification
Designing upstream and downstream primers capable of amplifying the HPV integration sites, wherein the primers are shown in Table 2 and are prepared according to the system shown in the following Table 4:
TABLE 4 primer systems
Figure BDA0003152511960000091
Each tube was mixed well and amplified in a standard PCR instrument according to the procedure shown in table 5 below:
TABLE 5 PCR amplification procedure
Figure BDA0003152511960000092
2. And sending the PCR product obtained after amplification to Wuhan Pongziaceae biotechnology limited company for Sanger sequencing.
(1) PCR products (the content is more than 200ng, the volume is more than 20ul) obtained after amplification are sent to Wuhan Pongziaceae biotechnology limited company for Sanger sequencing;
(2) PCR samples were purified and the sequence of the PCR fragments was determined by adding ABI3730XL sequencer.
3. And (4) analyzing and detecting the integration sites of the HPV according to the sequencing result, and detecting a new integration site.
(1) Opening a sequencing result by applying the text document;
(2) performing sequencing result comparison by using NCBI BLAST;
open BLAST page: http: // www.ncbi.nlm.nih.gov/BLAST/o click on the nucleotide BLAST section's nucleotide BLAST link to a new page; inputting the Sequence in the Enter Query Sequence part or pasting the Sequence; the Job Title component may also call a name for this Job; select "others" in the Choose Search Set section, select NCBI chromosome in the drop-down dialog; selecting the accuracy of the comparison in the Program Selection part, and generally selecting high similarity sequences to click a BLAST button to obtain a comparison result;
(3) judging the comparison result;
the Description part indicates that one part of the sequence is from human genome DNA, and the other part of the sequence is from HPV genome DNA; wherein the higher the Max score, Total score, Max identity, the higher the degree of similarity; the lower the E value, the higher the degree of similarity; the alignment of the most similar human DNA and HPV viral DNA is selected.
(4) Counting results;
according to the alignment result, whether the sample is integrated or not and whether any one or more sites of Linc02301, Linc01435, Linc00430 and Linc00564 gene sites are integrated or not can be determined;
example 4
LSIL, HSIL and cervical cancer samples from HPV infection are extracted into genome DNA according to the method described in the embodiment 1, integration sites of HPV in cervical tissue genome DNA are detected by a reversible end termination sequencing method according to the method described in the embodiment 2, and all integrated gene sites are obtained after result analysis.
FIG. 1 shows the result of HPV integration positive rate analysis of all integration gene sites of different pathological stage samples. The "all integration gene sites" described in the present invention refer to all HPV integration gene sites found in the process of detecting cervical exfoliated cell DNA by the inventors using the methods described in examples 1 and 2, and most of the HPV integration gene sites have low occurrence frequency. The Linc02301, Linc01435, Linc00430 and Linc00564 integration sites have high frequency. The sample is divided into LSIL, HSIL and cervical cancer samples, and the integration is positive, namely, part of the sample contains human genome sequence and part of the sample contains HPV genome sequence. According to the method for calculating the integration positive rate, the denominator is the total number of patients detected in a certain pathological stage, and the numerator is the number of the patients detected to be integration positive in the pathological stage. In 896 statistical LSIL samples, 339 HSIL samples and 114 cervical cancer samples, the detected HPV integration positive rate shows that the HPV integration positive rate is from 61.27% of LSIL to 68.14% of HSIL and then 92.98% of cervical cancer, and shows an increasing trend, and the result shows that the HPV integration positive rate is closely related to the malignancy degree of cervical lesions, and the higher the malignancy degree of cervical lesions, the higher the HPV integration positive rate is, so that HPV integration can be used as an index for evaluating the malignancy degree of cervical lesions and predicting the risk of cervical cancer;
FIG. 2 is a distribution diagram of the detection positive rate of Linc gene family integration sites in all samples, wherein the Linc gene families Linc02301, Linc01435, Linc00430 and Linc00564 disclosed by the invention are included. In 1472 samples, the detection rate of high-frequency gene loci (Linc02301, Linc01435, Linc00430 and Linc00564 is 5.15%, the above results show that the detection positive rate of Linc gene families is high, and the integration of the gene loci can be used as an index for evaluating the malignancy degree of cervical lesions and predicting the risk of cervical cancer;
the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Wuhan Kaideweis Biotech Co., Ltd
Screening method and application of <120> Linc HPV integration gene locus
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Claims (10)

  1. A screening method of Linc HPV integration gene sites is characterized by comprising the following steps:
    s1, extracting human cervical exfoliated cell DNA;
    s2, designing HPV full-length probes containing 18 subtypes of HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV 82;
    constructing a DNA sequencing library according to the requirement of Illumina, carrying out enzyme digestion treatment on sample genome DNA into a plurality of DNA fragments, then carrying out terminal repair and A addition treatment on the DNA fragments, wherein the total amount of the constructed DNA library is more than or equal to 1500ng, and the main peak of the fragment length of the DNA library is 350-550 bp;
    hybridizing the HPV full-length probes of 18 subtypes with a genome DNA library, capturing a target product, and eluting non-hybridized DNA fragments; the obtained DNA fragment is amplified through 16 PCR cycles, secondary hybridization capture is carried out after purification, the obtained purified amplification product is a sequencing library, the concentration of the sequencing library is more than or equal to 1 ng/mu L, and the main peak of the length of the sequencing library fragment is 350-550 bp; sequencing the sequencing library;
    s3, analyzing the sequencing result of the step S2; filtering, removing the adaptor sequence, the low-quality sequence and the repetitive sequence, and evaluating the quality of the sequence before and after filtering to ensure that the filtered sequence Q30 is more than 80 percent and the sequence length is more than 100 bp; comparing the preprocessed sequencing data with HPV virus and human genome reference sequences, and identifying a plurality of chimeric sequences of which parts are compared with HPV genomes and parts are compared with human genomes; aiming at the chimeric sequences, local clustering is carried out according to positions on a human genome, the locally clustered sequences are locally compared with a reference genome, and whether the sample genome integrates HPV partial sequences in genes Linc02301, Linc01435, Linc00430 and Linc00564 is determined according to local comparison results;
    s4, and verifying HPV integration gene sites Linc02301, Linc01435, Linc00430 and Linc00564 related to the occurrence of cervical cancer by using conventional PCR and Sanger sequencing.
  2. 2. The method for screening the integration gene loci of Linc HPV according to claim 1, wherein in step S1, the concentration of human cervical exfoliated cell DNA is > 15ng/μ L, the purity A260/A280 is not less than 1.5, and the purity A260/A230 is not less than 1.
  3. 3. The method for screening the Linc-type HPV integration gene locus according to claim 1, wherein the HPV full-length probes of 18 subtypes hybridize to the genomic DNA library in the step S2 in a manner that: hybridization was carried out at 65 ℃ for 16-24 h.
  4. 4, primer combination for PCR amplification of Linc HPV integration gene loci, which is characterized in that the Linc HPV integration gene loci are Linc02301, Linc01435, Linc00430 and Linc 00564;
    the sequences of upstream and downstream primers of the HPV integration gene locus Linc02301 amplified by PCR are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6;
    the sequences of upstream and downstream primers of the HPV integration gene locus Linc01435 amplified by PCR are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8;
    the sequences of upstream and downstream primers of an HPV integration gene locus Linc00430 amplified by PCR are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10;
    the sequences of the upstream primer and the downstream primer of the HPV integration gene locus Linc00564 obtained by PCR amplification are respectively shown as SEQ ID NO.11 and SEQ ID NO. 12.
  5. 5. The primer combination for PCR amplification of the Linc HPV integration gene locus according to claim 4, wherein the integration sequence of the HPV integration gene locus Linc02301 is shown as SEQ ID No.1, the integration sequence of the HPV integration gene locus Linc01435 is shown as SEQ ID No.2, the integration sequence of the HPV integration gene locus Linc00430 is shown as SEQ ID No.3, and the integration sequence of the HPV integration gene locus Linc00564 is shown as SEQ ID No. 4.
  6. 6. A kit for detecting the high risk group of cervical cancer, which comprises the primer combination for PCR amplification of the Linc HPV integration gene locus according to claim 4.
  7. 7. The kit for detecting the high risk group of cervical cancer according to claim 6, further comprising a DNA extraction reagent, a PCR reagent and a plurality of sample storage tubes.
  8. 8. The kit for detecting the cervical cancer high risk group according to claim 7, wherein the DNA extraction reagent is Buffer ACL, RNaseA, protease K, Buffer ACL, Buffer WA, Buffer WB, Elution Buffer;
    the PCR reagent comprises DNA polymerase, an amplification buffer solution and double distilled water.
  9. 9. The kit for detecting the high risk group of cervical cancer according to claim 7 or 8, wherein the sample-holding tubes are 1.5ml EP tubes, and each of the sample-holding tubes contains 1 ml cell-preserving solution.
  10. 10. The kit for detecting the high risk group of cervical cancer according to claim 7 or 8, wherein the PCR reagent is 2 prepared
    Figure FDA0003152511950000021
    Master Mix。
CN202110769725.7A 2021-07-07 2021-07-07 Screening method and application of Linc HPV integration gene sites Withdrawn CN113337644A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104830869A (en) * 2015-04-30 2015-08-12 珠海雅马生物工程有限公司 HPV-integrated gene sites related to occurrence of cervical carcinoma and application thereof
CN107739761A (en) * 2016-08-12 2018-02-27 嘉兴允英医学检验有限公司 It is a kind of to be used for HPV partings and the high-flux sequence detection method of integration
CN110724765A (en) * 2019-10-30 2020-01-24 元码基因科技（北京）股份有限公司 Gene containing HPV integration site and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104830869A (en) * 2015-04-30 2015-08-12 珠海雅马生物工程有限公司 HPV-integrated gene sites related to occurrence of cervical carcinoma and application thereof
CN107739761A (en) * 2016-08-12 2018-02-27 嘉兴允英医学检验有限公司 It is a kind of to be used for HPV partings and the high-flux sequence detection method of integration
CN110724765A (en) * 2019-10-30 2020-01-24 元码基因科技（北京）股份有限公司 Gene containing HPV integration site and application thereof

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