CN113278688A - Primer combination and kit for detecting HPV integration gene locus - Google Patents
Primer combination and kit for detecting HPV integration gene locus Download PDFInfo
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Abstract
The invention provides a primer combination and a kit for detecting HPV integration gene locus, belonging to the technical field of biological detection, wherein the kit has the advantages of rapidness, convenience, accuracy, low false positive rate and the like; HPV integration sites ARAP2, CASC21, CDH13, CNTN5 and CNTNAP2 related to the occurrence of the cervical cancer are obtained by a high-throughput sequencing method and a fluorescent PCR method, and can be used as molecular markers for evaluating the cervical cancer occurrence risk of HPV positive patients according to the integration of HPV in cervical exfoliated cell DNA to the gene sites; the kit assembled by HPV integration is applied to the evaluation of the risk of cervical cancer of HPV infection positive patients, has good innovation and is helpful for clinical diagnosis.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a primer combination and a kit for detecting HPV integration gene loci.
Background
Cervical cancer has 570,000 new cases and 311,000 deaths each year worldwide, with over 130,000 new cases per year in our country. Persistent infection with high-risk Human Papillomaviruses (HPV) is considered to be the leading cause of cervical cancer. With the continuous development of high-throughput sequencing technology, scientific research finds that the occurrence of cervical cancer is a long-term process, the integration of HPV genome into host genome is considered as a key step of the occurrence of cervical cancer, and the HPV DNA is integrated to continuously express oncoprotein and increase the instability of the host genome, so that the normal cervical epithelium is induced to develop into precancerous lesion and finally become carcinogenic. At present, HPV genomic integration is an important mark of malignant transformation of cervical intraepithelial neoplasia to cervical cancer, and is well accepted, in benign lesions caused by HPV, intracellular viral DNA is usually located outside a host genome, in high-level intratumoral lesions and cervical cancer, HPV and DNA are usually present in the host genome in an integrated form, and the integration rate is remarkably increased along with the severity of cervical lesions; meanwhile, multiple high-throughput sequencing studies find that although HPV integration events occur in the samples of precancerous lesions and cervical carcinoma, the statistic result shows that the HPV integration event occurrence rate (81%) of the cervical carcinoma samples is obviously higher than that (48%) of the CINs samples, which indicates that the process of HPV integration event occurrence from precancerous lesions to cancerous lesions is an increasing process. Therefore, the detection of HPV integration event can be used as an important assessment factor for the occurrence risk of cervical lesion, and the integration site of HPV and human genome can be used as a molecular marker for assessing the occurrence risk of cervical cancer of HPV positive patients.
The risk assessment of cervical cancer can be predicted based on HPV integration sites, virus integration detection is carried out by adopting a high-throughput sequencing method, an HPV frequent integration site map is calculated and drawn, and LSIL, HSIL in HPV positive patients and HPV integration sites in cervical cancer are determined. The invention aims to find a primer combination and a kit for detecting HPV integration gene sites, which are used for evaluating the cervical cancer occurrence risk of HPV positive patients.
Disclosure of Invention
Aiming at the problems in the prior art, the invention discloses five new HPV integration sites (ARAP2, CASC21, CDH13, CNTN5 and CNTNAP2) related to the occurrence of cervical cancer by utilizing a high-throughput sequencing method and a fluorescent PCR method, and prepares a kit for detecting the occurrence risk of the cervical cancer according to the integration of the HPV into the sites, and the kit has the advantages of rapidness, convenience, accuracy and the like.
The technical scheme for solving the technical problems is as follows:
a primer combination for detecting an HPV integration gene site, the primer combination comprising SEQ ID NO: 6 to SEQ ID NO: 15 complete sequence.
Preferably, the HPV integration gene sites are ARAP2, CASC21, CDH13, CNTN5, CNTNAP2, respectively.
The 5 integration sites are the gene sites for integrating the human genome sequence inserted by partial or all of HPV16, 18, 56 and 66.
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 HPV56 is https:// www.ncbi.nlm.nih.gov/nuccore/EF 177176.1;
the website address of HPV66 is https:// www.ncbi.nlm.nih.gov/nuccore/LC 511686.1.
The website corresponding to the human chromosome is as follows:
the website address of Chr4 is http: // www.ncbi.nlm.nih.gov/nuccore/NC _ 000004.11;
the website address of Chr7 is http:// www.ncbi.nlm.nih.gov/nuccore/NC _ 000007.13;
the website address of Chr8 is http:// www.ncbi.nlm.nih.gov/nuccore/NC _ 000008.10;
the website address of Chr11 is http:// www.ncbi.nlm.nih.gov/nuccore/NC _ 000011.9;
the website address of Chr16 is http:// www.ncbi.nlm.nih.gov/nuccore/NC _ 000016.9.
Table 1 shows the integration sites of the genomic sequences of HPV on the human genome, wherein the "HPV breakpoints" shown in the third column are inserted into the "breakpoints on the human genome" shown in the second column of the "genes" shown in the first column, respectively, and the specific "genes", "breakpoints on the human genome", "HPV breakpoints" are shown in table 1:
TABLE 1 site of integration of the HPV genomic sequences on the human genome
| Gene | Breakpoint on human genome | HPV breakpoints |
| ARAP2 | Chr4：35694937 | HPV56：5500 |
| CASC21 | Chr8：127229304 | HPV18：2498 |
| CDH13 | Chr16：83320304 | HPV66：678 |
| CNTN5 | Chr11：100013476 | HPV16：4345 |
| CNTNAP2 | Chr7：147910076 | HPV56：5298 |
In the above table, "Chr 4: 35694937 "for example, indicating HPV integration at human chromosome 4, position 35694937; with "HPV 56: 5500 "as an example means that the breakpoint integrated in the human genome HPV is base 5500.
The sequences of the upstream primer and the downstream primer of the integration site ARAP2 are shown as SEQ ID NO: 6 and SEQ ID NO: 7 is shown in the specification;
the sequences of the upstream primer and the downstream primer of the integration site CASC21 are shown as SEQ ID NO: 8 and SEQ ID NO: 9 is shown in the figure;
the sequences of the upstream primer and the downstream primer of the integration site CDH13 are shown as SEQ ID NO: 10 and SEQ ID NO: 11 is shown in the figure;
the sequences of the upstream primer and the downstream primer of the integration site CNTN5 are shown as SEQ ID NO: 12 and SEQ ID NO: 13 is shown in the figure;
the sequences of the upstream primer and the downstream primer of the integration site CNTNAP2 are shown as SEQ ID NO: 14 and SEQ ID NO: shown at 15.
PCR amplification 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
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 upstream and downstream primers in Table 2, reducing the length of the primer sequences in Table 2 (deleting part of bases, retaining only part or all of the 3' sequence), making changes to individual bases; the above operation is within the scope of the present invention as long as it does not affect PCR amplification.
Preferably, the sequence of the integration site ARAP2 is as set forth in SEQ ID NO: 1 is shown in the specification;
the sequence of the integration site CASC21 is shown as SEQ ID NO: 2 is shown in the specification;
the sequence of the integration site CDH13 is shown in SEQ ID NO: 3 is shown in the specification;
the sequence of the integration site CNTN5 is shown as SEQ ID NO: 4 is shown in the specification;
the sequence of the integration site CNTNAP2 is shown in SEQ ID NO: 5, respectively.
The sequences of the integration sites formed after insertion of the HPV genome 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
The integration sequence in the above table refers to a sequence obtained on a human chromosome after fusion of a part or all of the genomic sequence of HPV, i.e., a part of the sequence of HPV and a part of the sequence of human genome. Those skilled in the art can make appropriate additions, reductions or changes according to the integrated sequences in table 3, such as adding different restriction enzyme site sequences, protecting bases, etc., reducing the integrated sequence length (e.g., deleting part of bases) in table 3, making changes to individual bases; such manipulations are within the scope of the present invention, as long as a substantial portion of the bases of the integrated sequences in Table 3 are retained.
The HPV integration sites related to the generation of the cervical cancer as well as 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.
A kit for detecting HPV integration gene locus comprises the primer combination, and the sequence is shown as SEQ ID NO: 6 to SEQ ID NO: shown at 15.
Preferably, the kit further comprises a DNA extraction reagent and a reagent for PCR amplification.
Preferably, in the kit, the DNA extraction reagent is: buffer ACL, RNaseA, protease K, Buffer ACL, Buffer WA, Buffer WB, and Elution Buffer, available from Napkinson Zan Biotech, Inc., having a product number of DC 112. More specifically, the DNA extraction reagent may be purchased as a commercial DNA extraction kit, or may be used for extracting human cervical exfoliated cell DNA by referring to the DNA extraction reagent described in the fifth edition of molecular biology laboratory Manual (F.M. Osber et al) or by appropriately adjusting the DNA extraction reagent.
Preferably, in the kit, the PCR amplification reagent is: DNA polymerase, buffer solution matched with the DNA polymerase, solution containing magnesium ions, fluorescent dye and double distilled water. More specifically, the fluorescent dye is SYBR Green.
Preferably, in the kit, the PCR amplification reagent is 2 XTaq Pro Universal SYBR qPCR Master Mix purchased from NyVon Nuo Zan Biotech Co.
The use method of the kit comprises the following steps:
1) extracting cervical exfoliated cell DNA, collecting human cervical exfoliated cells as a sample, and extracting the exfoliated cell DNA by using a DNA extraction reagent; the method for extracting the DNA of the human cervical exfoliated cells is not particularly limited, and only needs to ensure that the concentration of the DNA is more than 15 ng/mu L, the purity A260/A280 is not less than 1.5, and the purity A260/A230 is not less than 1.
2) And (3) detecting virus integration by a high-throughput sequencing method:
virus integration detection: 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) are designed. 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 16-24 hours at 65 ℃, and target products are captured to elute non-hybridized DNA fragments. The obtained fragments are subjected to 16-round PCR amplification, purification and secondary hybridization capture, 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;
analyzing the virus integration detection result: the splice, low quality and repeat sequences were filtered and the quality of the sequences before and after filtering was evaluated. Ensuring that the sequence Q30 after filtration is more than 80 percent and the sequence length is more than 100 bp. And (3) rapidly aligning the sequences of the preprocessed sequencing data to HPV virus and human genome reference sequences by adopting sequence alignment software. Based on the alignment, the sequence of the chimera was identified (one aligned partially with the HPV genome and partially with the human genome). For these chimeric sequences, local clustering is performed according to the position on the human genome, and the clustered sequences are locally aligned with the reference genome, and it is determined whether the sample incorporates a partial sequence of HPV and whether the HPV partial sequence is incorporated in the genes ARAP2, CASC21, CDH13, CNTN5, CNTNAP2, based on the alignment result.
3) Detection of HPV integration sites by fluorescent PCR: using SEQ ID N0: 6 to SEQ ID NO: 15) the integrated sequence obtained in step 2) was subjected to fluorescent PCR detection.
4) And (4) analyzing results: step 3), amplification is positive, namely the result is positive when the integration site is detected, namely the cervical cancer high risk group is detected; 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 select proper amplification conditions according to the PCR amplification reagent as long as the amplification is positive, the PCR amplification method is preferably 95 ℃ 30s, 95 ℃ 7s, 60 ℃ 20s, 95 ℃ 15s, 60 ℃ 60s, 95 ℃ 15s and 40 cycles, and the parameters can be properly adjusted according to actual conditions.
Compared with the prior art, the invention has the beneficial effects that: the kit for detecting the risk of cervical cancer has the advantages of rapidness, convenience, accuracy, low false positive rate and the like; HPV integration sites ARAP2, CASC21, CDH13, CNTN5 and CNTNAP2 related to the occurrence of the cervical cancer are obtained by a high-throughput sequencing method and a fluorescent PCR method, and can be used as molecular markers for evaluating the cervical cancer occurrence risk of HPV positive patients according to the integration of HPV in cervical exfoliated cell DNA to the gene sites; the kit assembled by HPV integration is applied to the evaluation of the risk of cervical cancer of HPV infection positive patients, has good innovation and is helpful for clinical diagnosis.
Drawings
FIG. 1 is a flow chart of the method of using the kit for detecting a high risk group of cervical cancer according to the present invention;
FIG. 2 is a diagram showing the HPV integration positivity distribution of all integration gene sites in LSIL, HSIL and cervical cancer samples;
FIG. 3 is a graph showing the detection positivity distribution among all samples for 5 integrated gene loci, wherein A represents the detection positivity of 5 gene loci.
Detailed Description
The technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to 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 of the present invention without any inventive step, are within the 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) Taking a cervical exfoliated cell sample, centrifuging for 5min, collecting cells, and removing supernatant. Add 200. mu.l PBS and 20. mu.l protease K in sequence, shake and mix well.
2) Adding 200 μ l Buffer BCL, shaking, mixing, adding 56 deg.C water bath for 10min, mixing several times while turning upside down, and purifying with column. Add 150. mu.l of absolute ethanol, shake and mix well, centrifuge briefly to collect the liquid on the inner wall of the tube cap.
3) The FastPurg DNA Mini Columns II adsorption column was placed in a 2ml Collection tube, and the mixture (including flocculent precipitate) was transferred to the adsorption column. Centrifuge at 12,000rpm (13,400 Xg) for 1 min. The filtrate was discarded and the adsorption column was placed in the collection tube. Mu.l of Buffer WA WAs added to the adsorption column along the tube wall and centrifuged at 12,000rpm (13,400 Xg) for 1 min.
4) The filtrate was discarded and the adsorption column was placed in the collection tube. 600. mu.l of Buffer WB was added along the tube wall, centrifuged at 12,000rpm (13,400 Xg) for 1min, and the filtrate was discarded.
5) And (4) repeating the step.
6) The adsorption column was placed in a collection tube. The column was centrifuged at 12,000rpm (13,400 Xg) for 2 min.
7) The column was transferred to a new 1.5ml centrifuge tube. 50-200 μ l of Elution Buffer is dripped into the center of the adsorption column membrane, and the adsorption column membrane is placed for 2-5min at room temperature. Centrifuge 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
And detecting the HPV integration sites in the cervical tissue genome DNA by using a high-throughput sequencing method.
1) Viral integration detection of genomic DNA samples of cervical exfoliated cells: designing HPV full-length probes (HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82) containing 18 subtypes, wherein the detailed sequence of the probes is shown in patent CN202011260519.5 already filed by the applicant, a probe set for HPV typing and integration detection of human papilloma virus 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, then carrying out end repair and A treatment on the small fragments, ensuring that the total amount of the constructed DNA library is not less than 1500ng and the main peak of the length of the library fragment is about 350-550 bp, applying HPV probes containing 18 subtypes to hybridize with the genome DNA at 65 ℃ for 16-24 h by a liquid phase probe hybridization capture technology, capturing the eluted 16-550 DNA fragments of target products, amplifying the DNA fragments by PCR, purifying and performing secondary hybridization capture, wherein 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 fragment is about 350-550 bp. Adding the sequencing library into a gene sequencer Nextseq CN500 for sequencing;
2) analyzing the virus integration detection result: first, the sequences with low quality, repeats and linker primer contamination were removed, and the remaining sequences were aligned to both 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, 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. From the alignment, it can be determined whether the sample has integrated and integrated in the ARAP2, CASC21, CDH13, CNTN5, CNTNAP2 gene locus.
Example 3
HPV integration sites were detected by fluorescence PCR.
1. Fluorescent PCR amplification
Upstream and downstream primers were designed to amplify the HPV integration sites, as shown in Table 2, formulated according to the system of Table 4:
TABLE 4 primer systems
| Reagent | Concentration of | Volume of |
| 2×Taq Pro Universal SYBR qPCR Master Mix | 10 | |
| Primer 1 | 10μM | 0.4 |
| Primer 2 | 10μM | 0.4 |
| Template DNA | X | |
| Pure water | 9.2-X | |
| Total volume | 20 |
Each tube was mixed well separately, amplified in a standard PCR instrument and amplified using the program in table 5:
TABLE 5 PCR amplification procedure
2. And (4) analyzing results: if the amplification is positive, the integration site is detected, namely the HPV is integrated into any gene site of ARAP2, CASC21, CDH13, CNTN5 and CNTNAP2, the high risk group of cervical cancer can be judged; otherwise, the result is negative, i.e. not belonging to the high risk group of cervical cancer.
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, HPV integration sites in cervical tissue genome DNA are detected by a high-throughput sequencing method according to the method described in the embodiment 2, all integrated gene sites are obtained after result analysis, and the integration sites are obtained through statistics.
Analysis of Experimental results
FIG. 1 is a flow chart of the method of using the kit for detecting a high risk group of cervical cancer according to the present invention. The application method of the kit for detecting the high risk group of cervical cancer comprises the following steps of extracting cervical exfoliated cell genome DNA of a subject, detecting HPV integration sites through high-throughput sequencing virus integration detection and fluorescence PCR, and making risk assessment for judging the occurrence of the cervical cancer of an HPV positive patient by the subject according to a detection result.
FIG. 2 analysis of HPV integration positivity 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 integration sites of ARAP2, CASC21, CDH13, CNTN5 and CNTNAP2 have higher 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, so that HPV integration can be used as an index for evaluating the malignancy degree of cervical lesions and predicting the cervical cancer risk.
FIG. 3 is a diagram showing HPV integration positive rate of gene loci in all samples detected by fluorescence PCR method, five integration gene loci of the invention, ARAP2, CASC21, CDH13, CNTN5 and CNTNAP 2. In 1472 samples, the detection positive rate of 5 gene loci is 6.13%, the results show that the detection positive rate of the gene loci 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 an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Sequence listing
<110> Wuhan Kaideweis Biotech Co., Ltd
<120> primer combination and kit for detecting HPV integration gene locus
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 166
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtggaaggct ttataggtaa gtgtgcagaa ggtgtagcaa ctgactggct agacaaacca 60
ggtgcttcat catctatatt tgcataaata tcatataggc catcaaatgc cttgagaaca 120
tcttggcaag tgaaaagagc cactcttttt tttttttttt ttttga 166
<210> 2
<211> 340
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cctgcctcgg ccttccaaag tgctaggatt ataggcatga gccaccatgc cctgcctgac 60
aaatcttgag tcagatatta gtaaatgcaa acattacaca tacatacagt catacaacac 120
agacaacaaa tacacacata cagtacacac aataaataaa tagtacataa tacaaccata 180
catatacaat acagacacac aacatactat ttgcgtttgc gcttggagga gggggctgag 240
gtagatgtag taggagcaga gcgtttctta gaggatacag gaggtttagt acggacccca 300
acctgcatta aaaacttcct gccaagaggg aactgatcca 340
<210> 3
<211> 150
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gttttcgtaa catgtaataa ctaggatgta aataaaagtc acctgcatca gcaataattg 60
tatattgtgg agaccctgga actataggaa gtaatgaagg agcttggtca gttatattaa 120
tgggtatatc aggacctgat actaaaggaa 150
<210> 4
<211> 899
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tgggactaca ggcgcccgcc accgcgcccg gctaattttt tgtattttta gtagagacgg 60
ggtttcacct tgttagccag gatggtctcg atctcctgac ctcatgatcc acccgcctcg 120
gcctcccaga gtgctgggat tacaggcgtg agccaccgcg cccggccctc gccaactttt 180
aatttttaaa agttatttaa aacaaaaaga cccagctagt atcactattc taaaagagaa 240
agtttacaga aatacacaca cacacataca cgcgtgcgca catgctaaaa agagcaggcg 300
ctgagctctg agttaaggtt aactttatga tatcattaca ttgtcctgct tactcttcca 360
tttcaccaag gcccagtgaa aactttctca ctaaccagca ccaatccaca gctacagata 420
cacttgctcc tgtaagaccc cctttaacag tagatcctgt gggcccttct gatccttcta 480
tagtttcttt agtggaagaa actagtttta ttgatgctgg tgcaccaaca tctgtacctt 540
ccattccccc agatgtatca ggatttagta ttactacttc aactgatacc acacctgcta 600
tattagatat taataatact gttactactg ttactacaca taataatccc actttcactg 660
acccatctgt attgcagcct ccaacacctg cagaaactgg agggcatttt acactttcat 720
catccactat tagtacacat aattatgaag aaattcctat ggatacattt attgttagca 780
caaaccctaa cacagtaact agtagcacac ccataccagg gtctcgccca gtggcacgcc 840
taggattata tagtcgcaca acacaacaag ttaaagttgt agaccctgct tttgtaacc 899
<210> 5
<211> 150
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ataacgatta tttttaaaaa tattcagaat taggtgttgg atagtaagct gacccaacca 60
aataaatatg tttctggctt tgaaatttgg tttgtctagc cagtcagttg ctacaccttc 120
tgcacactta cctataaagc cttccacatt 150
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
agtgtgcaga aggtgtagca a 21
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tgccaagatg ttctcaaggc 20
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tatttgcgtt tgcgcttgga g 21
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gcaggttggg gtccgtacta 20
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aaagtcacct gcatcagcaa t 21
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
accaagctcc ttcattactt cc 22
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gtgggccctt ctgatccttc 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ccctggtatg ggtgtgctac 20
<210> 14
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ggatagtaag ctgacccaac ca 22
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aggtgtagca actgactggc 20
Claims (8)
1. A primer combination for detecting HPV integration gene sites, wherein the primer combination comprises SEQ ID NO: 6 to SEQ ID NO: 15 complete sequence.
2. The primer combination of claim 1, wherein the HPV integration gene sites are ARAP2, CASC21, CDH13, CNTN5, CNTNAP 2;
the sequences of the upstream primer and the downstream primer of the integration site ARAP2 are shown as SEQ ID NO: 6 and SEQ ID NO: 7 is shown in the specification;
the sequences of the upstream primer and the downstream primer of the integration site CASC21 are shown as SEQ ID NO: 8 and SEQ ID NO: 9 is shown in the figure;
the sequences of the upstream primer and the downstream primer of the integration site CDH13 are shown as SEQ ID NO: 10 and SEQ ID NO: 11 is shown in the figure;
the sequences of the upstream primer and the downstream primer of the integration site CNTN5 are shown as SEQ ID NO: 12 and SEQ ID NO: 13 is shown in the figure;
the sequences of the upstream primer and the downstream primer of the integration site CNTNAP2 are shown as SEQ ID NO: 14 and SEQ ID NO: shown at 15.
3. The primer combination of claim 2, wherein the sequence of the integration site ARAP2 is as set forth in SEQ ID NO: 1 is shown in the specification;
the sequence of the integration site CASC21 is shown as SEQ ID NO: 2 is shown in the specification;
the sequence of the integration site CDH13 is shown in SEQ ID NO: 3 is shown in the specification;
the sequence of the integration site CNTN5 is shown as SEQ ID NO: 4 is shown in the specification;
the sequence of the integration site CNTNAP2 is shown in SEQ ID NO: 5, respectively.
4. A kit for detecting an HPV integration gene site comprising the primer combination of claim 1.
5. The kit of claim 4, further comprising DNA extraction reagents and PCR amplification reagents.
6. The kit of claim 5, wherein the DNA extraction reagent is: buffer ACL, RNaseA, protease K, Buffer ACL, Buffer WA, Buffer WB, and Elutionbuffer.
7. The kit of claim 5, wherein the PCR amplification reagents are: DNA polymerase, buffer solution matched with the DNA polymerase, solution containing magnesium ions, fluorescent dye and double distilled water.
8. The kit of claim 5 or 7, wherein the PCR amplification reagent is 2 XTaq Pro Universal SYBR qPCR Master Mix.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114196755A (en) * | 2021-12-23 | 2022-03-18 | 首都医科大学附属北京佑安医院 | Composition for diagnosing cervical lesions in a subject and use thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005049861A2 (en) * | 2003-11-10 | 2005-06-02 | Epigenomics Ag | Method for the analysis of gynaecological cell proliferative disorders |
| JP2012044924A (en) * | 2010-08-26 | 2012-03-08 | Japan Health Science Foundation | Marker and method for cervix uteri cancer screening |
| 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 |
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2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005049861A2 (en) * | 2003-11-10 | 2005-06-02 | Epigenomics Ag | Method for the analysis of gynaecological cell proliferative disorders |
| JP2012044924A (en) * | 2010-08-26 | 2012-03-08 | Japan Health Science Foundation | Marker and method for cervix uteri cancer screening |
| 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114196755A (en) * | 2021-12-23 | 2022-03-18 | 首都医科大学附属北京佑安医院 | Composition for diagnosing cervical lesions in a subject and use thereof |
| CN114196755B (en) * | 2021-12-23 | 2022-07-15 | 首都医科大学附属北京佑安医院 | Composition for diagnosing cervical lesions in a subject and use thereof |
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Application publication date: 20210820 |