CN112195286B - Probe for detecting human papilloma virus HPV26 and kit thereof - Google Patents

Abstract

The invention discloses a probe for detecting human papillomavirus HPV26 and a kit thereof, wherein the probe group comprises probes capable of specifically capturing HPV26 genome; the invention has comprehensive detection range, greatly improved detection efficiency and accurate result.

Description

Probe for detecting human papilloma virus HPV26 and kit thereof

Technical field:

The invention relates to a probe and a kit thereof, in particular to a probe and a kit for capturing and sequencing human papillomavirus HPV26 genes.

The background technology is as follows:

Cervical cancer refers to a malignancy occurring in the cervical vagina and cervix, one of the most common gynaecological malignancies, next to breast cancer. Cervical cancer is the second leading cancer incidence and the third leading mortality in women aged 15-44 in China. Numerous studies have demonstrated that cervical cancer is predominantly caused by persistent infection with high-risk human papillomaviruses (Human Papillomavirus, HPV) and that the presence of high-risk HPV DNA is detected in 99.7% of cervical cancer patients. Currently, more than 200 HPV genotypes have been identified, about 40 being associated with genital tract infections. HPV is classified into high-risk type and low-risk type according to the level of risk of cervical cancer, wherein 13 genotypes including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and the like are classified into high-risk type, and 5 genotypes including HPV 26, 53, 66, 73, 82 and the like are classified into medium-risk type. These 18 types of HPV are mainly associated with cervical intraepithelial neoplasia, cinii, iii grade and cervical cancer. The low risk is generally unrelated to malignant lesions, mainly causing condyloma acuminatum and low grade CIN (CINI). Therefore, by detecting whether high-risk HPV infection exists in cervical exfoliated cells and periodically detecting whether HPV virus in cervical exfoliated cells is continuously infected or not, the detection method is particularly important in early screening, auxiliary diagnosis, post-healing evaluation and other links of cervical cancer.

In general, HPV is present in a free state after infection of host cells, and its DNA is still free in the cytoplasm of the host cells, and as the host cells divide and replicate, HPV DNA is inserted into the human genome, a process also known as HPV integration. Once HPV DNA is accidentally integrated into the host cell chromosome, viral proteins will replicate in disorder, and host cell genetic material structure and associated gene expression changes, resulting in uncontrolled division of the host cell, thereby inducing canceration. Although HPV DNA is detected in 99.7% or more of cervical cancers, cervical cancer does not occur in every patient infected with high-risk HPV. With the progress of molecular biology and cytogenetics, the integration of HPV with host chromosomes is an important step in cervical cell immortalization, an important marker that leads to cervical intraepithelial neoplasia to cervical cancer malignancy. Integration of HPV with the host cell chromosome, which is the most important risk factor for cervical cancer development, is found in more than 90% of cervical cancer tissues. Low-risk HPV rarely causes cervical cancer, irrespective of viral load in the host, because it rarely undergoes viral integration. The integration rates of high-risk HPV in HSIL and squamous cell carcinoma were 76.2% and 88.2%, respectively, with only 37% in LSIL. HPV exists in a non-free state in invasive cervical cancer (about 60.9%) 20% higher than carcinoma in situ. Approximately 90% of HPV 16-positive cervical cancer cases are integrated, with only 16% of cases in pure free form. It is indicated that the incidence of viral integration is positively correlated with the incidence of cervical cancer, and the status of viral integration is positively correlated with the extent of cervical cancer. HPV DNA and human genome integration are used as markers of cervical lesion degree, and have very important significance for early diagnosis of cervical cancer.

The early symptoms of cervical cancer are not obvious, and the death rate caused by cervical cancer can be effectively reduced only by early screening and early warning of cervical cancer. Traditional pathological detection means, such as cervical smear detection, are easily affected by factors such as sampling, staining, subjective judgment of cytopathologists and the like, so that the application of cytopathologists to detect HPV has the defects of low sensitivity, poor specificity, high false negative rate and false positive rate, incapability of typing HPV and the like. The current real-time fluorescent Polymerase Chain Reaction (PCR) method commonly used in clinic has low detection flux, is difficult to meet the requirement of simultaneously detecting a plurality of types of HPV clinically when detecting different types of HPV respectively, and is not suitable for large-scale screening of cervical cancer. More importantly, the methods used at present can only judge whether HPV infection and HPV infection type exist or not, and cannot judge whether HPV DNA is integrated with human genome or not, and HPV integration event is an important factor for cervical cancer. The high-throughput sequencing technology (next-generation sequencing technology) has the advantages of accuracy, high sensitivity, rapidness and low cost, and can sequence a plurality of genes at the same time, so that the method can realize HPV typing detection and HPV integration detection. The liquid phase hybridization gene capturing technology can enrich relevant gene fragments of a target region, and can greatly reduce the cost of human gene sequencing, genetic research and gene diagnosis by combining a new generation gene sequencing technology, improve the sequencing depth and more accurately discover the mutation information of a specific region. The design of the capture probe is the core of the liquid phase hybridization capture technology. In summary, the probe set and the kit for detecting HPV typing and integration provided by the invention can accurately type 18 high-risk HPVs at one time and determine the integration condition of the HPVs in human genome, have the advantages of processing a plurality of samples at one time, being high in sensitivity and specificity, and have great clinical significance.

The invention comprises the following steps:

The present invention aims to solve the above-mentioned shortcomings of the prior art and provide a capture probe set for detecting HPV and a kit thereof; according to the 18 HPV whole genome sequences, the invention designs a synthetic probe sequence, and specifically captures, amplifies and sequences target fragments so as to achieve the purpose of detecting samples. The probe set comprises probes capable of specifically capturing all 18 HPV genomes. 18 HPV types and 5 HPV integration conditions can be comprehensively, rapidly and accurately detected, wherein the 18 HPV types are HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82 respectively; the 5 HPVs integrate as HPV16, 18, 33, 52, 58 respectively.

The invention adopts the following technical scheme:

the invention provides a probe for detecting HPV26 typing and integration of human papillomavirus, and the information of the probe is shown in Table 1

The invention also provides a kit for detecting HPV26 typing and integration of human papillomavirus. The kit comprises the probe for detecting human papillomavirus HPV26 typing and integration; preferably, the kit further comprises a library buffer 1, a library buffer 2, an enzyme 1, an enzyme 2, a linker, a PCR amplification primer 1, a PCR amplification primer 2, a PCR reaction mixture, a blocking sequence 1, a blocking sequence 2, a blocking reagent, a hybridization buffer, a PCR amplification primer 3, a PCR amplification reaction solution, a positive control, a negative control, a capture buffer, a cleaning buffer 1, a cleaning buffer 2 and a capture magnetic bead; preferably, the blocking sequence 1 is Cot-1 DNA and the blocking sequence 2 is a double-stranded oligonucleotide.

Further, the components of the kit are shown in Table 2.

The invention also discloses a method for using the probe for detecting human papillomavirus HPV26 typing and integration and a kit thereof, which mainly comprises the following steps:

(1) Design of Capture probes for HPV26

(2) Sample DNA extraction, fragmentation and construction of DNA library

(3) Library target region targeted capture and quantification

(4) High throughput sequencing

(5) Bioinformatic analysis of gene sequences

The invention provides a method for capturing HPV genes, which specifically comprises the following steps: extracting whole genome DNA of cervical exfoliated cells, randomly breaking the whole genome DNA into fragments with the size of 200-300bp, and constructing a DNA library by using partial fragments containing the sequence of HPV genes; the HPV probes (carrying biotin marks and capable of being combined by hybridization with HPVDNA sequences) related by the invention are hybridized with the constructed pre-library, and sequences containing HPV genes can be hybridized specifically by combining biotin on the probes with magnetic beads; washing and removing the sequence without HPV, thereby enriching HPV gene fragments, and capturing HPV sequences by the kit; HPV genotype of the sample to be tested and whether integration with human genome occurs can be obtained by sequencing through high throughput of a sequencer Nextseq CN500,500 and analyzing HPV information in the sequencing result.

The invention has the following beneficial effects:

(1) The invention designs the probe capable of rapidly and accurately capturing the complete sequence of HPV26 genome, and can effectively improve the detection specificity. The traditional methods such as fluorescent quantitative PCR only aim at a certain section of the HPV sequence, and the detection range of the probe can cover the whole length of the HPV genome.

(2) The invention uses HPV probes to sequence library samples captured with target areas by using a high-throughput sequencing technology. The HPV sequence is targeted, enriched and captured by using the HPV probe, and then high-throughput sequencing is carried out, so that the detection sensitivity is high.

(3) According to the invention, a plurality of samples can be processed at one time, different Barcode is added into a plurality of different samples to distinguish, and then the samples are mixed for on-machine sequencing, so that the detection flux can be effectively improved.

Description of the drawings:

FIG. 1 shows the parameters of a PCR instrument for end repair, 3' end addition "A" reaction;

FIG. 2 shows the parameters of the Pre-PCR procedure.

The specific embodiment is as follows:

The invention is further illustrated by the following examples. Unless otherwise indicated, all reagents commonly used in the art for use in the present invention are commercially available.

Example 1: design and preparation of the Probe set of the invention

According to the base complementary pairing principle, oligonucleotide probes complementary to the DNA sequence of HPV26 are designed. Because of the complex structure of the genome sequence, irregular regions such as high GC, high AT regions and the like exist, and meanwhile, because of the limitation of the second generation sequencing read length, the design of the probe becomes particularly critical, and a plurality of factors such as coverage rate, uniformity, capture efficiency and the like need to be comprehensively balanced. The specific principle of probe design is as follows:

(1) Length: the selectable interval is 80-150bp, and 100bp is generally selected.

(2) Number of probe layers: i.e. how many probes are covered on average over the target area, 3 layers are generally recommended and a shingled design is required.

(3) Specificity: i.e., the uniqueness of the probe in the genomic range, the higher the specificity, the higher the capture efficiency of the probe. Designing probes in low complexity areas should be avoided; but in special cases, such as where some hotspots are located exactly in low complexity areas, then it is necessary to comprehensively evaluate the sequence of that area as well as adjacent areas and carefully place the probes.

(4) Binding ability: the GC content is about 50%, and the probe capturing capacity is the strongest. The high GC region has strong probe binding capacity, but the DNA fragment has stronger self-binding capacity (longer length) and larger probe competition resistance; the DNA fragment of the high AT region itself is weak in binding ability but also weak in binding ability of the probe thereto, and therefore, it is necessary to appropriately increase the number of probes in the high GC and high AT regions to compensate for the disadvantages in competitive resistance and binding ability.

Preparing a probe: the designed probes are synthesized on a chip in a large quantity by a photo-chemical synthesis method, and the biotin-labeled probes are obtained through PCR or transcription reaction.

Example 2: the kit of the invention comprises, preparation and use

The kit reagent for HPV detection described in the example.

The kit comprises the following components: the invention relates to a capture probe for detecting HPV, a library buffer solution 1, a library buffer solution 2, an enzyme 1, an enzyme 2, a connector, a PCR amplification primer 1, a PCR amplification primer 2, a PCR reaction mixture, a blocking sequence 1, a blocking sequence 2, a blocking reagent, a hybridization buffer solution, a PCR amplification primer 3, a PCR amplification reaction solution, a positive control, a negative control, a capture buffer solution, a cleaning buffer solution 1, a cleaning buffer solution 2 and a capture magnetic bead. The information of the specific composition is shown in table 2,

The using method of the kit comprises the following steps:

1. Sample library preparation

(1) Fragmenting: taking out 1000 ng DNA, thawing, shaking, mixing, adding water to 100 μl, and breaking with Biorupter Pico ultrasonic breaker. The specific parameters are set as follows: setting parameters of ON 30 seconds and OFF 30 seconds as 1 cycle after the temperature of the cold circulation instrument is reduced to 4 ℃, wherein every 10 cycles are one round, and carrying out 3 rounds in total; 1 μl of the sample was used for fragment detection using Qsep a1, and the main peak of the sample detection after normal disruption was about 150: 150 bp-200: 200 bp.

(2) End repair plus a: and (3) carrying out end repair and adding ' A ' to the 3' end of the sample obtained in the last step. The specific reaction system is as follows: 35. mu.L of DNA fragment, 5. Mu.L of ERA buffer, 10. Mu.L of end repair enzyme. After mixing, a PCR program is run, and parameters of a PCR instrument are set as shown in FIG. 1 (50 mu L system, hot cover 85 ℃);

(3) And (3) adding a joint: and (3) performing joint connection on the sample obtained in the last step. The specific reaction system is as follows: 50. mu.L of the sample from which the reaction of the previous step was completed, 5. Mu.L of the linker, 15. Mu.L of enzyme-free water, 20. Mu.L of the ligation buffer, 10. Mu.L of ligase; after mixing, the PCR instrument program (no hot lid is required) was run and PCR instrument parameters were set: closing the heat cover, cooling to 4 ℃ for 30 minutes at 20 DEG C

(4) Purifying magnetic beads: immediately after the end of the procedure, the reaction product of the previous step was purified using purification magnetic beads. The purification system is as follows: 100. mu.L of the reaction product of the previous step, 100. Mu.L of purified magnetic beads.

(5) Pre-PCR amplification reaction: the purified product of the above step is used as a template for the Pre-PCR reaction. The specific reaction system is as follows: 20. mu.L of the purified product of the previous step, 25. Mu.L of the PCR reaction mixture, 2.5. Mu.L of the PCR amplification reaction primer 1 (20. Mu.M), 2.5. Mu.L of the PCR amplification reaction primer 2 (20. Mu.M). After mixing, the sample was placed on a PCR apparatus and the PCR procedure was started as shown in FIG. 2 (50. Mu.L system, hot lid temperature 105 ℃);

note that: TPE1.0index No. 1-8 in PCR amplification primer 1 (Table 3); TPE2.0index number in PCR amplification reaction primer 2 is 1# to 12#; the index number used is recorded (table 4).

(6) Purifying magnetic beads: immediately after the end of the procedure, the reaction product of the previous step was purified using 50. Mu.L of purification beads.

2. Sample liquid phase hybridization

(1) Standing and thawing the sealing sequence 1 and the sealing sequence 2 to prepare a mixed solution 1, wherein the mixed solution specifically comprises the following components: 750ng library, 5. Mu.L blocking sequence 1, 2. Mu.L blocking sequence 2.

(2) Taking out the blocking reagent and the probe in the kit, standing, thawing, shaking, mixing, and preparing a mixed solution 2 (ice operation) according to 5 mu L of the blocking reagent and 2 mu L of the probe.

(3) And taking out the hybridization buffer solution in the kit, thawing at room temperature, taking out 20 mu L of the hybridization buffer solution after thawing, and incubating the hybridization buffer solution in a constant-temperature mixing instrument at 65 ℃ for later use.

(4) 13 Mu L of hybridization buffer solution is added into the mixed solution 2, and after being blown and mixed uniformly, the liquid is collected by short centrifugation and is placed at room temperature for standby.

(5) Using a temperature-controllable vacuum drier, setting a drying temperature at 45-50 ℃, opening a pipe cover until the volume of the mixed solution 1 is less than 10 mu L, supplementing to 10 mu L by using enzyme-free water, gently sucking and beating, uniformly mixing, centrifuging for a short time, and placing on ice for standby. The concentration time should not be too long, so that the evaporation to dryness is prevented.

(6) The concentrated mixture 2 was placed in a PCR apparatus and incubated at 95℃for 5min and at 65℃for 2min (the temperature of the hot lid was 105 ℃) and no removal was required after the incubation.

(7) The hybridization buffer-added mixture 1 was placed in a PCR apparatus and incubated at 65℃for 2min (thermal cover temperature 105 ℃).

(8) And (3) adding 20 mu L of the mixed solution 1 into the mixed solution 2, fully and uniformly mixing, hybridizing for 16-24 hours at 65 ℃, keeping the hybridized product in a PCR instrument, and entering a capturing step.

3. Capturing

(1) Taking out the magnetic beads in the kit in advance, balancing for 30min at room temperature, and shaking and mixing uniformly.

(2) The 50. Mu.L of magnetic beads were removed and placed in a fresh reaction tube, and the tube was placed on a magnetic rack for 1min until the liquid was clear, taking care to discard the liquid (note not to attract the beads).

(3) 200 Mu L of capture buffer solution is added to the magnetic beads, after blowing and mixing evenly, a centrifuge tube is placed on a magnetic rack for standing for 1min until liquid is clear, and the liquid is carefully sucked and abandoned (note that the magnetic beads are not sucked).

(4) Repeating the step (3) for three times.

(5) Re-adding 200 mu L of capture buffer solution into the magnetic beads to re-suspend the magnetic beads, adding the re-suspended magnetic beads into a hybridization product, and placing a PCR tube on a rotary mixer to combine for 30min at room temperature.

(6) Taking out the cleaning buffer solution 1 and the cleaning buffer solution 2 in the kit, standing the cleaning buffer solution 1 at room temperature for later use, and preheating the cleaning buffer solution 2 at 65 ℃ for more than 5 minutes for later use.

(7) The captured product was placed on a magnetic rack for 1min and allowed to stand for 1min until the liquid was clear, taking care to discard the liquid (note not to attract magnetic beads).

(8) The centrifuge tube was removed from the magnetic rack, 200. Mu.L of washing buffer 1 was added, and after mixing by blowing, the tube was rotated on a mixer for 15min, placed on the magnetic rack for 1min until the liquid was clear, and the liquid was carefully removed (note not to be attached to the beads).

(9) The centrifuge tube was removed from the magnetic rack, 200. Mu.L of a pre-heated washing buffer 2 at 65℃was added, and after being mixed well by blowing, the mixture was placed on a constant temperature shaker (65℃at 800 rpm) for 10 minutes, and placed on the magnetic rack for 1 minute until the liquid was clear, and the liquid was carefully sucked off (note not to suck the beads).

(10) The step (9) is repeated three times.

(11) The centrifuge tube was removed from the magnetic rack, 200. Mu.L of freshly prepared 80% ethanol was added, and the mixture was allowed to stand on the magnetic rack for 30sec until the liquid was clear, and the liquid was carefully removed (note not to the beads)

(12) The reaction tube is left on the upper magnetic frame and dried for 3-5 min at room temperature to volatilize the residual ethanol, and the surface of the magnetic beads is matt (excessive drying can lead to the reduction of DNA yield).

(13) The centrifuge tube was removed from the magnetic rack and 30 μl of enzyme-free water was added to resuspend the beads for use.

(14) A mixed solution 3 is prepared, and a 0.2mL PCR tube is used, wherein the specific reaction system is as follows: 18. Mu.LPCR amplification buffer, 1. Mu.LPCR amplification primer, 1. Mu.LPCR amplification reaction solution.

(15) 20. Mu.L of the above reaction system was added to the washed product and thoroughly mixed.

(16) PCR amplification was performed in a PCR apparatus according to the reaction procedure of Table 5.

(17) Purification after amplification, purification of the amplified product was performed using commercial nucleic acid purification magnetic beads, and specific procedures were performed as directed by the kit instructions. All purified products were pipetted onto ice and steps 2-3 were repeated and either the sequencing phase was entered or stored at-20.+ -. 5 ℃.

4. Capture library quality control

(1) The concentration of the library is determined using nucleic acid quantification kit QubitdsDNA HS ASSAY KIT and the associated instrumentation, and the library concentration should be greater than 1 ng/. Mu.L. Otherwise, the library is not satisfactory and hybridization capture should be performed again.

(2) Taking a sample library or a reference substance library, and determining the size of library fragments by using STANDARD CARTRIDGE KIT (S2) and a matched instrument, wherein the size of the library fragments is concentrated between 250bp and 500bp, and no obvious small fragment and large fragment miscellaneous peaks exist. Otherwise, the library sample does not meet the requirements, and hybridization capture should be carried out again.

5. Library qPCR quantification

The library to be enriched on-boarding is quantified using KAPA Library Quantification Kit kit to adjust to the appropriate on-boarding concentration.

(1) Reagent preparation: 1) Preparing a proper amount of DNA dilution buffer: 1XIDTE buffer was diluted to 0.1X using enzyme free water, approximately 1.2mL dilution buffer per library. The buffer was allowed to warm to room temperature before use. 2) The components in the thawing kit on ice are fully and uniformly mixed before use, and are briefly centrifuged and placed on ice for standby.

(2) Sample preparation: 1) An appropriate library dilution (using enzyme-free water) was prepared. 1. Mu.L of enrichment library was taken for 1: 100. 1: 10000. 1:20000 dilution.

(3) To each well of the PCR reaction plate to be reacted, 8. Mu.L of a mixed solution containing the primer was added, and the mixed solution was composed of: 5 mu LMaster Mix,1 mu L primer, 1.8 mu L water, 0.2 mu LLow Rox.

(4) To each sample well was added the corresponding 2. Mu.L of diluted DNA library (volume ratio 1:1000 and 1:20000).

(5) To each of the wells of the standard, 2. Mu.L of DNA standard was added in order from low concentration to high concentration.

(6) To the negative control wells, 2 μl dilution buffer was added.

(7) Sealing the PCR reaction plate, and centrifuging in a microplate centrifuge for 1min.

(8) The reaction plate was loaded onto a qPCR instrument and the qPCR instrument was run according to the parameters of table 6 below.

(9) Concentration calculations of the library to be tested were performed according to KAPA Library Quantification Kit kit instructions. If the final total molar mass of the library is less than 0.02p molar, library hybridization enrichment or library construction is needed again, and if the library is unqualified again, detection is stopped.

6. Sequencing on machine

Sequencing was performed on a gene sequencer Nextseq CN with a sequencing reagent having a sequencing read length of 300 cycles (Paired-End Reads, 2X 150 cycles), suggesting a sequencing data volume of 800M per sample library. The number of sample libraries to be sequenced is not higher than 48, and NextSeq 500 Mid Output v2 Reagent kit (300 cycles) reagent is recommended for detection; the number of sample libraries to be sequenced is higher than 48 and not higher than 96, and NextSeq 500 High Output v2 Reagent kit (300 cycles) reagent is recommended for detection.

(1) Taking REAGENT CARTRIDGE, HT1 and flow cell out in advance, placing the flow cell in a room temperature balance 30min,Reagent Cartridge, placing the flow cell in a room temperature water bath for 1h, standing the HT1 at room temperature, thawing the HT1 at room temperature, and shaking and mixing the materials uniformly.

(2) Mixing all sample libraries to be sequenced according to the requirement of sequencing data, diluting the concentration of each library to 4nM by using a nucleic acid quantitative kit QubitdsDNA HS ASSAY KIT and a matched instrument, taking a new centrifuge tube with equal volume, taking each library for mixing, and keeping the total volume after mixing at least 5 mu L.

(3) Fresh 0.2N NaOH was prepared, and 1. Mu.L of 1N NaOH was added to 4. Mu.L of purified water and mixed well.

(4) Taking 5 mu L of 4 nM mixed library into a new 1.5 mL centrifuge tube, adding 5 mu L of freshly prepared 0.2N NaOH, blowing and mixing uniformly, standing for 5min at room temperature to denature the 4 nM mixed library, adding 990 mu LHT1 after denaturalization, and diluting the mixed library to 20 pM.

(5) A new 1.5 mL centrifuge tube was added to 1365. Mu.L HT1 after mixing.

(6) 135. Mu.L of the mixed library diluted to 20 pM. Mu.L was added to 1365. Mu.L of HT1 to give a total volume of 1500. Mu.L of the final on-press mixed library.

(7) Taking out REAGENT CARTRIDGE thawed in a room temperature water bath, drying by dust-free paper, turning over for 5 times, and uniformly mixing the reagents.

(8) The final on-machine mix library mixed with PhiX control was injected into well 10 using a clean 1 mL pipette tip to puncture the well 10 seal with Load Samples tag on REAGENT CARTRIDGE.

(9) And (3) carrying out reagent loading operation according to the prompt of the operation setting of the sequencer, and clicking a start button to sequence after the self-checking of the sequencer is finished, wherein the sequencing time is 26-29h.

7. HPV nucleic acid typing and integrated biological information analysis flow

(1) After sequencing was completed, BCL2fastq v2.20.0.422 software from illumina was used to convert the BCL files generated by sequencing into fastq files corresponding to the samples.

(2) And using the iltiming v0.6 software to read the record information of InterOp catalogues in the sequencing file, and carrying out quality analysis and evaluation on the sequencing. Fastp v0.20.0 for quality control, the Q30 mass value is required to be more than 80%.

(3) After the quality assessment is completed, the data is preprocessed, and the quality control is carried out on the original data according to the base and the quality of the fastq file (based on fastp v0.20.0 software).

(4) Sequence alignment: the base sequences in fastq files were aligned to human reference genome hg38 (GRCh 38) and HPV genomes to generate bam files (based on BWA v0.7.17, samtoolsv1.9 and picard v2.20.6 software: alignment).

(5) HPV type panel analysis: the numbers and proportions of reads for each HPV in the samples were analyzed by sequence alignment files using a typing detection module (based on HPVHPVKITTYPER V1.0.0 software).

(6) HPV integration analysis: using an integration detection module (based on hpvkitHPVFusionCallerv software), HPV integration sites in samples were analyzed by site comparison of sequences of HPV and human genome in alignment in sequence alignment files and annotated in conjunction with databases.

(7) And (3) result judgment: typing analysis, wherein if the single HPV type is MEAN DEPTH or more than 10, the type is judged to be positive in typing; and (3) carrying out integration analysis, wherein the HPV integration read number in the unit data volume is more than or equal to 3, and judging that the type is positive for integration.

Example 3: use effect verification of the kit

The present invention will be described in further detail with reference to the following embodiments.

1. High throughput sequencing detection of HPV genotyping integration of 20 reference samples and 16 clinical samples

Sample numbers S1-S36, wherein S1-S18 are 10-4 copies/mL of typing plasmid reference, HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82, S19 and S20 are SiHa cells (known HPV16 integration sample) and HeLa cell DNA (known HPV18 integration sample) with concentrations of 0.25 ng/. Mu.L, respectively, and S21-S36 are clinical samples. Detection result: the HPV type and the integration state corresponding to the reference sample are detected by using the method and the kit provided by the invention, so that the method and the kit can accurately and rapidly detect the actual sample, and have practical use value. The probe set and the kit for HPV typing and integration detection provided by the invention have the characteristics of simplicity and convenience in operation, high flux, good specificity, high sensitivity and the like. The results were as follows: table 7 shows the quality control results of the high-throughput sequencing of 36 samples, table 8 shows the typing results of the high-throughput sequencing of 36 samples, and Table 9 shows the integration results of the high-throughput sequencing of 18 samples.

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SEQUENCE LISTING

<110> Kadetweisi biotechnology Co., ltd

<120> A probe for detecting human papillomavirus HPV26 and kit therefor

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<160> 14

<170> PatentIn version 3.5

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<211> 24119

<212> DNA

<213> Artificial Sequence

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aaacagataa tcgtagtgac cagcaggcca gacaagctgg acaagaagtg tgttacagaa 1980

ttgaagcaca atgttgtatg gcgtgaccag caggccagac aagctggaca agaagtgtgt 2040

tacagaattg aagcacaatg ttgtatgtgt aatagtatag tgcagctagc tgtgcagagc 2100

agtgtgttac agaattgaag cacaatgttg tatgtgtaat agtatagtgc agctagctgt 2160

gcagagcagt cgacagaacg ttcgagtgct ggagcagatg gtgtaatagt atagtgcagc 2220

tagctgtgca gagcagtcga cagaacgttc gagtgctgga gcagatgtta atggaagacg 2280

tgtccttggt gtgccatcag agtcgacaga acgttcgagt gctggagcag atgttaatgg 2340

aagacgtgtc cttggtgtgc catcagtgtg ctgcacagta aacctgcaat ggactgtgaa 2400

agtgtgctgc acagtaaacc tgcaatggac tgtgaaggta caaatgagga ggggcggggg 2460

tgtacagggt ggttttcagt agaagctata gtggaaaaac gaaggtacaa atgaggaggg 2520

gcgggggtgt acagggtggt tttcagtaga agctatagtg gaaaaacata caggggacac 2580

aatatcagat gatgaaacag gggtggtttt cagtagaagc tatagtggaa aaacatacag 2640

gggacacaat atcagatgat gaaacagaca atagtagtga tacagggtcg gacctaatag 2700

catacagggg acacaatatc agatgatgaa acagacaata gtagtgatac agggtcggac 2760

ctaataggat ttatagatga tagtagtata agtgattatg gacaatagta gtgatacagg 2820

gtcggaccta ataggattta tagatgatag tagtataagt gattcgtaag aacaggaggt 2880

aacccaggca ttgtttcagg ggatttatag atgatagtag tataagtgat tcgtaagaac 2940

aggaggtaac ccaggcattg tttcaggcac aacaaaaaca ggcaaataca aaggcagtgc 3000

gcagaacagg aggtaaccca ggcattgttt caggcacaac aaaaacaggc aaatacaaag 3060

gcagtgcgca atttaaaacg aaagttacta ggtagtcaga ggcacaacaa aaacaggcaa 3120

atacaaaggc agtgcgcaat ttaaaacgaa agttactagg tagtcagaac agcccgttgc 3180

aagacataac aaatcaacac cgcaatttaa aacgaaagtt actaggtagt cagaacagcc 3240

cgttgcaaga cataacaaat caacacagac agcaaagcga cagtcagcag aatacacacc 3300

aacagcccgt tgcaagacat aacaaatcaa cacagacagc aaagcgacag tcagcagaat 3360

acacaccaag taaataattc acaggccaaa aggagagccg gacagcaaag cgacagtcag 3420

cagaatacac accaagtaaa taattcacag gccaaaagga gagccgtgga cagtgtaccg 3480

gacagcgggt atggctatac caagtaaata attcacaggc caaaaggaga gccgtggaca 3540

gtgtaccgga cagcgggtat ggctatactg aagtggaaac tcttacgccc gtacaggtag 3600

ccgtggacag tgtaccggac agcgggtatg gctatactga agtggaaact cttacgcccg 3660

tacaggtaga taaacaatat gaagaaaatg gcgggttgcc actgaagtgg aaactcttac 3720

gcccgtacag gtagataaac aatatgaaga aaatggcggg ttgcctagtg tgtgtagtca 3780

gggggggtca acgtactcag gataaacaat atgaagaaaa tggcgggttg cctagtgtgt 3840

gtagtcaggg ggggtcaacg tactcagtgg aagatatcga tgtagacaca catgtaaaca 3900

gtgtgtgtag tcaggggggg tcaacgtact cagtggaaga tatcgatgta gacacacatg 3960

taaacagtgt tacacaaata tgtgaattat taaaatgtag gtggaagata tcgatgtaga 4020

cacacatgta aacagtgtta cacaaatatg tgaattatta aaatgtagta atgtaaaagc 4080

agcattgtta agtaaattta agtgttacac aaatatgtga attattaaaa tgtagtaatg 4140

taaaagcagc attgttaagt aaatttaaaa cagtatatgg tgtaagtttt gcagaactag 4200

gtaatgtaaa agcagcattg ttaagtaaat ttaaaacagt atatggtgta agttttgcag 4260

aactagtacg ggtgtttaaa agtgacaaaa ccgtatgttc aacagtatat ggtgtaagtt 4320

ttgcagaact agtacgggtg tttaaaagtg acaaaaccgt atgttcagat tgggtgtgtg 4380

cagcattcgg tgtggcaggc gtacgggtgt ttaaaagtga caaaaccgta tgttcagatt 4440

gggtgtgtgc agcattcggt gtggcaggct ctgtagcaga aagtattaaa tcattaatac 4500

tcagattggg tgtgtgcagc attcggtgtg gcaggctctg tagcagaaag tattaaatca 4560

ttaatacaac aatattgttt atattatcat atacaatgtt ggctctgtag cagaaagtat 4620

taaatcatta atacaacaat attgtttata ttatcatata caatgtttaa catgtaattg 4680

gggagtaata gtactacgta caacaatatt gtttatatta tcatatacaa tgtttaacat 4740

gtaattgggg agtaatagta ctacgtatag tgcgctttac atgtgcaaaa aacagaacaa 4800

ttaacatgta attggggagt aatagtacta cgtatagtgc gctttacatg tgcaaaaaac 4860

agaacaacaa ttaaaaactg cctatgtatg ttattaaatg gctagtgcgc tttacatgtg 4920

caaaaaacag aacaacaatt aaaaactgcc tatgtatgtt attaaatgtg ccagaaacgc 4980

aattactaat tgaaccacca acaattaaaa actgcctatg tatgttatta aatgtgccag 5040

aaacgcaatt actaattgaa ccaccaaaat tgcgaagtac agcagtagca ttatattttt 5100

gtgccagaaa cgcaattact aattgaacca ccaaaattgc gaagtacagc agtagcatta 5160

tatttttata aaacagggtt gtccaatata agtgagacat aattgaacca ccaaaattgc 5220

gaagtacagc agtagcatta tatttttata aaacagggtt gtccaatata agtgagacat 5280

atggagatac accagaatgg aattgcgaag tacagcagta gcattatatt tttataaaac 5340

agggttgtcc aatataagtg agacatatgg agatacacca gaatggatag tacgacaaac 5400

tataaaacag ggttgtccaa tataagtgag acatatggag atacaccaga atggatagta 5460

cgacaaacac aattagaaca tagttttgat gcgtatacat tatggagata caccagaatg 5520

gatagtacga caaacacaat tagaacatag ttttgatgcg tatacatttg atttatcaaa 5580

aatggtgcaa tgggcgttcg acacaattag aacatagttt tgatgcgtat acatttgatt 5640

tatcaaaaat ggtgcaatgg gcgttcgatc atgacataac agatgatagt gaaattgcat 5700

tttgatttat caaaaatggt gcaatgggcg ttcgatcatg acataacaga tgatagtgaa 5760

attgcattta aatcgtaaca gttagctgac atagatagta gatcatgaca taacagatga 5820

tagtgaaatt gcatttaaat cgtaacagtt agctgacata gatagtacgt aagctgcctt 5880

tttaaaaagc aattgtcagg aacagatgat agtgaaattg catttaaatc gtaacagtta 5940

gctgacatag atagtacgta agctgccttt ttaaaaagca attgtcaggc aaaatatgta 6000

gcatttaaat cgtaacagtt agctgacata gatagtacgt aagctgcctt tttaaaaagc 6060

aattgtcagg caaaatatgt aaaagactgt gttaaatcgt aacagttagc tgacatagat 6120

agtacgtaag ctgccttttt aaaaagcaat tgtcaggcaa aatatgtaaa agactgtgca 6180

accatgacta gagtacgtaa gctgcctttt taaaaagcaa ttgtcaggca aaatatgtaa 6240

aagactgtgc aaccatgact agacattata aaagagcaca gacgtaagct gcctttttaa 6300

aaagcaattg tcaggcaaaa tatgtaaaag actgtgcaac catgactaga cattataaaa 6360

gagcacagaa acgatctatg tgggcaaaat atgtaaaaga ctgtgcaacc atgactagac 6420

attataaaag agcacagaaa cgatctatgt gtatgtcaca atggctacaa tatagatgtt 6480

ctgacattat aaaagagcac agaaacgatc tatgtgtatg tcacaatggc tacaatatag 6540

atgttctaaa atagaagagg gcgggtcgtg gaaggaaatt gcgtgtatgt cacaatggct 6600

acaatataga tgttctaaaa tagaagaggg cgggtcgtgg aaggaaattg ccaaattttt 6660

aaggtttcaa catgtaaact ttaaaataga agagggcggg tcgtggaagg aaattgccaa 6720

atttttaagg tttcaacatg taaactttat ttatttttta caagtgttaa aacagttttt 6780

aagccaaatt tttaaggttt caacatgtaa actttattta ttttttacaa gtgttaaaac 6840

agtttttaaa gggtacccca aagcacaatt gtatagtaat attttatttt ttacaagtgt 6900

taaaacagtt tttaaagggt accccaaagc acaattgtat agtaatatat ggaccgccaa 6960

atactggtaa gtcacagttt gcaagggtac cccaaagcac aattgtatag taatatatgg 7020

accgccaaat actggtaagt cacagtttgc aatgagtttt ataaaatttc gtaaagggtc 7080

agggaccgcc aaatactggt aagtcacagt ttgcaatgag ttttataaaa tttcgtaaag 7140

ggtcagtcat ttcatatgta aattcaaata gccatttttg gcatttcgta aagggtcagt 7200

catttcatat gtaaattcaa atagccattt ttggctgcag cctttagaag cgtaaaaagt 7260

tgcagtatta gatgcgtagt catttcatat gtaaattcaa atagccattt ttggctgcag 7320

cctttagaag cgtaaaaagt tgcagtatta gatgcgtata catatagctg ctggttatgg 7380

ctgcagcctt tagaagcgta aaaagttgca gtattagatg cgtatacata tagctgctgg 7440

ttatatattg ataaatattt acgtaacttt ttagatgggt attagatgcg tatacatata 7500

gctgctggtt atatattgat aaatatttac gtaacttttt agatggaaat ccctgttgta 7560

tagacagaaa acatagaata tattgataaa tatttacgta actttttaga tggaaatccc 7620

tgttgtatag acagaaaaca tagaagccta ctgcaagtta ccgtaccccc attaataagg 7680

aaatccctgt tgtatagaca gaaaacatag aagcctactg caagttaccg tacccccatt 7740

aataattacc tcaaatataa atcctcaaga agataactag cctactgcaa gttaccgtac 7800

ccccattaat aattacctca aatataaatc ctcaagaaga taactcactt ttgtatttac 7860

atagtagagt aacagtgaat tacctcaaat ataaatcctc aagaagataa ctcacttttg 7920

tatttacata gtagagtaac agtgatacca tttccaaata catttccatt tgacagcact 7980

cacttttgta tttacatagt agagtaacag tgataccatt tccaaataca tttccatttg 8040

acagcaatgg gaatcctgta tcgtaattga ctgatgtata catagtagag taacagtgat 8100

accatttcca aatacatttc catttgacag caatgggaat cctgtatcgt aattgactga 8160

tgtaaattgg aaaataccat ttccaaatac atttccattt gacagcaatg ggaatcctgt 8220

atcgtaattg actgatgtaa attggaaaag ctttttttcc accacctggt ccagcatttc 8280

catttgacag caatgggaat cctgtatcgt aattgactga tgtaaattgg aaaagctttt 8340

tttccaccac ctggtccaga ttagatttgg aggatcgtaa ttgactgatg taaattggaa 8400

aagctttttt tccaccacct ggtccagatt agatttggag gaggacgcgg acaaagaaaa 8460

tggagaattg gaaaagcttt ttttccacca cctggtccag attagatttg gaggaggacg 8520

cggacaaaga aaatggagaa cctttgccag cgtttaacgt agtgcctttt ttccaccacc 8580

tggtccagat tagatttgga ggaggacgcg gacaaagaaa atggagaacc tttgccagcg 8640

tttaacgtag tgccaggaga aaatacagat tagatttgga ggaggacgcg gacaaagaaa 8700

atggagaacc tttgccagcg tttaacgtag tgccaggaga aaatactaga ctattatgaa 8760

ctggacagga ggacgcggac aaagaaaatg gagaaccttt gccagcgttt aacgtagtgc 8820

caggagaaaa tactagacta ttatgaactg gacagtaata aattaaatgg agaacctttg 8880

ccagcgttta acgtagtgcc aggagaaaat actagactat tatgaactgg acagtaataa 8940

attaactgat caaattgatt attggcgttt aacgtagtgc caggagaaaa tactagacta 9000

ttatgaactg gacagtaata aattaactga tcaaattgat tattggaaac tggtacgatg 9060

ccaggagaaa atactagact attatgaact ggacagtaat aaattaactg atcaaattga 9120

ttattggaaa ctggtacgat atgaatgtgc aatattttac agtaataaat taactgatca 9180

aattgattat tggaaactgg tacgatatga atgtgcaata ttttataaag ctcgtgaagg 9240

aaaccgtaaa tgtataaacg gaaactggta cgatatgaat gtgcaatatt ttataaagct 9300

cgtgaaggaa accgtaaatg tataaaccac caggtggtgc cctctactgt tgtgtgtaaa 9360

agctcgtgaa ggaaaccgta aatgtataaa ccaccaggtg gtgccctcta ctgttgtgtg 9420

taaacaaaag gcatggcagg caattgaaat acatatagcc caccaggtgg tgccctctac 9480

tgttgtgtgt aaacaaaagg catggcaggc aattgaaata catatagcat tgcagtcgtt 9540

aataaacacg gactataatc aaaaggcatg gcaggcaatt gaaatacata tagcattgca 9600

gtcgttaata aacacggact ataatacaga agcttggaca cgtagagaca caagctatga 9660

gcattgcagt cgttaataaa cacggactat aatacagaag cttggacacg tagagacaca 9720

agctatgaaa tgtatatgac agaacctaaa cattgtttta cagaagcttg gacacgtaga 9780

gacacaagct atgaaatgta tatgacagaa cctaaacatt gttttaaaaa agaaggaaca 9840

acggtaacag tggtatttgt gaaatgtata tgacagaacc taaacattgt tttaaaaaag 9900

aaggaacaac ggtaacagtg gtatttgatt gtaataagga aaatacaatg gattatatta 9960

aaagaaggaa caacggtaac agtggtattt gattgtaata aggaaaatac aatggattat 10020

attaggtgga aatatgtgta ttataaaact gatataggga ggtggaaata tgtgtattat 10080

aaaactgata tagggtggtg taaaggtact ggagatgttg cgtaaaaagg gatatattat 10140

acacaagggg catataagca gggtggtgta aaggtactgg agatgttgcg taaaaaggga 10200

tatattatac acaaggggca tataagcagt attacgtgga ctttaaacaa gaggcggaag 10260

caaaagggat atattataca caaggggcat ataagcagta ttacgtggac tttaaacaag 10320

aggcggaaaa atatgggaca ggtgtgcaat gggctgtacg cagtattacg tggactttaa 10380

acaagaggcg gaaaaatatg ggacaggtgt gcaatgggct gtacatgtgt gtggtcaggt 10440

aatctgttgt cctgaattta aaatatggga caggtgtgca atgggctgta catgtgtgtg 10500

gtcaggtaat ctgttgtcct gaatttgtat ctagtacctg cagcagcaac caaatatcct 10560

gtgtgtggtc aggtaatctg ttgtcctgaa tttgtatcta gtacctgcag cagcaaccaa 10620

atatccactg ctaaaactgc tgagccagta tcaaacgcct tgtatctagt acctgcagca 10680

gcaaccaaat atccactgct aaaactgctg agccagtatc aaacgccacc acccagacca 10740

ccgaagccta cgtgcccgta ctgctaaaac tgctgagcca gtatcaaacg ccaccaccca 10800

gaccaccgaa gcctacgtgc ccgtgggcac caaggaaacc gaggcgccat acccaggaac 10860

accacccaga ccaccgaagc ctacgtgccc gtgggcacca aggaaaccga ggcgccatac 10920

ccaggaaagc gacgacgact cagtggacct gacaccacct gggcaccaag gaaaccgagg 10980

cgccataccc aggaaagcga cgacgactca gtggacctga caccaccgtc accacagtca 11040

ccactgtcac cacagctgca aagcgacgac gactcagtgg acctgacacc accgtcacca 11100

cagtcaccac tgtcaccaca gctgccacac agcccggaca gtcagtggac tatacaaacc 11160

gtcaccacag tcaccactgt caccacagct gccacacagc ccggacagtc agtggactat 11220

acaaacaaca acctacacag tacaagtgga ggccatcaca cacagcccgg acagtcagtg 11280

gactatacaa acaacaacct acacagtaca agtggaggcc atcacccggg aagggacacg 11340

agtagtgacc aaactgtgtc aacaacctac acagtacaag tggaggccat cacccgggaa 11400

gggacacgag tagtgaccaa actgtgttta tagtacacct aaaaggtgat acaaatagtc 11460

ccgggaaggg acacgagtag tgaccaaact gtgtttatag tacacctaaa aggtgataca 11520

aatagtttaa aatgtttaag atatagattt aaaaagcatg tttatagtac acctaaaagg 11580

tgatacaaat agtttaaaat gtttaagata tagatttaaa aagcataaag gattgtattg 11640

caatgtatca tctacctgga caaatagttt aaaatgttta agatatagat ttaaaaagca 11700

taaaggattg tattgcaatg tatcatctac ctggcattgg accagtaatt ttaaaatgtt 11760

taagatatag atttaaaaag cataaaggat tgtattgcaa tgtatcatct acctggcatt 11820

ggaccagtaa tgataccaat caacaaggca aggattgtat tgcaatgtat catctacctg 11880

gcattggacc agtaatgata ccaatcaaca aggcattgta acaattacct ttaacagtat 11940

aacacaacgg cattgtaaca attaccttta acagtataac acaacgtaat aattttttaa 12000

caactgttaa aataccacaa agtataactt caacattggg aataatgtcc gtaataattt 12060

tttaacaact gttaaaatac cacaaagtat aacttcaaca ttgggaataa tgtcattgta 12120

atatagtgta tattttacca acacacaagc aaagtataac ttcaacattg ggaataatgt 12180

cattgtaata tagtgtatat tttaccaaca cacaagccaa tatgtgctgc taacacacct 12240

atatacctgc attgtaatat agtgtatatt ttaccaacac acaagccaat atgtgctgct 12300

aacacaccta tatacctgta atcattattc cttttatagt ttatgtgtta gccaatatgt 12360

gctgctaaca cacctatata cctgtaatca ttattccttt tatagtttat gtgtttgtgc 12420

tttgcgtgtg tgtgtgtgtg ttgctgtgtt gtaatcatta ttccttttat agtttatgtg 12480

tttgtgcttt gcgtgtgtgt gtgtgtgttg ctgtgttgtt tgttgccact tttgctttcc 12540

atttatgtgt gtgctttgcg tgtgtgtgtg tgtgttgctg tgttgtttgt tgccactttt 12600

gctttccatt tatgtgtttg cagcctcgct attattagtg ttttgttttt tgtttgttgc 12660

cacttttgct ttccatttat gtgtttgcag cctcgctatt attagtgttt tgtttttggt 12720

ttgtggtatc tacatcatat ataactaccg tttgcagcct cgctattatt agtgttttgt 12780

ttttggtttg tggtatctac atcatatata actacctata ttgtgtatat ttgcttattt 12840

tttatacctt tggtttgtgg tatctacatc atatataact acctatattg tgtatatttg 12900

cttatttttt atacctgctt gttttttaca tttttatact gtaatggtac tatattgtgt 12960

atatttgctt attttttata cctgcttgtt ttttacattt ttatactgta atggtaattg 13020

ctacttagtc cctttaataa acgtgttact gcttgttttt tacattttta tactgtaatg 13080

gtaattgcta cttagtccct ttaataaacg tgttacaatg gtagctgtcc gtgcccctcg 13140

acgcaaacgg ctacttagtc cctttaataa acgtgttaca atggtagctg tccgtgcccc 13200

tcgacgcaaa cgagcatcag ctacagactt atacaaaaca tgtaaggccc gagcatcagc 13260

tacagactta tacaaaacat gtaaggccgc tggtacgtgc cctcctgatg ttattcctaa 13320

aattgaaggt tctacccttg ctgataagaa ggccgctggt acgtgccctc ctgatgttat 13380

tcctaaaatt gaaggttcta cccttgctga taagatatta caatggagtg gtttgggaat 13440

atttttaggc taaaattgaa ggttctaccc ttgctgataa gatattacaa tggagtggtt 13500

tgggaatatt tttaggtggt cttggtatag gtacaggaac tgggtctggt attacaatgg 13560

agtggtttgg gaatattttt aggtggtctt ggtataggta caggaactgg gtctggtggg 13620

cgtactggat acattcccct aggagggggg tggtcttggt ataggtacag gaactgggtc 13680

tggtgggcgt actggataca ttcccctagg agggggtggt agaccctctg ttgtggatat 13740

cggccctact gggcgtactg gatacattcc cctaggaggg ggtggtagac cctctgttgt 13800

ggatatcggc cctacccgtc cgcccattat tattgaacct gtgggtcctg tggtagaccc 13860

tctgttgtgg atatcggccc tacccgtccg cccattatta ttgaacctgt gggtcctaca 13920

gaaccttcta tagttacttt ggtggaggac ccgtccgccc attattattg aacctgtggg 13980

tcctacagaa ccttctatag ttactttggt ggaggaatct agtattatac aatctggagc 14040

ccctataccc tacagaacct tctatagtta ctttggtgga ggaatctagt attatacaat 14100

ctggagcccc tatacctaca tttagtggtg gcaatggctt tgaacttacg aatctagtat 14160

tatacaatct ggagccccta tacctacatt tagtggtggc aatggctttg aacttaccac 14220

atcctctgca acaacacctg ctgtgttggc tacatttagt ggtggcaatg gctttgaact 14280

taccacatcc tctgcaacaa cacctgctgt gttggacatc accccctctg ctggtactgt 14340

acatgtaacc cacatcctct gcaacaacac ctgctgtgtt ggacatcacc ccctctgctg 14400

gtactgtaca tgtaacaagt accaatatac aaaatccatt atatattgaa catcaccccc 14460

tctgctggta ctgtacatgt aacaagtacc aatatacaaa atccattata tattgaaccc 14520

cctatagata taccacaggc cggggaagcc aagtaccaat atacaaaatc cattatatat 14580

tgaaccccct atagatatac cacaggccgg ggaagcatca ggtcatatat ttactacaac 14640

gtccacagcg aaccccctat agatatacca caggccgggg aagcatcagg tcatatattt 14700

actacaacgt ccacagctgg cacacatagt tatgaagaaa ttccaatggg catcaggtca 14760

tatatttact acaacgtcca cagctggcac acatagttat gaagaaattc caatggaagt 14820

atttgcttct actaatggaa caggattagg ctggcacaca tagttatgaa gaaattccaa 14880

tggaagtatt tgcttctact aatggaacag gattagaacc tattagtagt acacctattc 14940

ctggtataca gtatttgctt ctactaatgg aacaggatta gaacctatta gtagtacacc 15000

tattcctggt atacaacgag tgtcagctcc tcgtttgtat agtaaggccg aacctattag 15060

tagtacacct attcctggta tacaacgagt gtcagctcct cgtttgtata gtaaggccta 15120

tcaacaggta aaggttacag atcccaattc aacgagtgtc agctcctcgt ttgtatagta 15180

aggcctatca acaggtaaag gttacagatc ccaattttat tggtaatccc tccacatttg 15240

ttacctttgg cctatcaaca ggtaaaggtt acagatccca attttattgg taatccctcc 15300

acatttgtta cctttgataa tcctgcatat gaacctatag atgaaacacg gtaatccctc 15360

cacatttgtt acctttgata atcctgcata tgaacctata gatgaaacac ttacatcgta 15420

ttccagtagt actgtagcac ctgaccccgt acatcgtatt ccagtagtac tgtagcacct 15480

gaccccgatt ttttggacat tattgcattg catcgtccgg cccttacatc tcgcaaaggt 15540

actgtacgcg accccgattt tttggacatt attgcattgc atcgtccggc ccttacatct 15600

cgcaaaggta ctgtacgcta tagtaggttg ggtcaaaagg ccactatgac gtccggccct 15660

tacatctcgc aaaggtactg tacgctatag taggttgggt caaaaggcca ctatgaaaac 15720

acgtagtgga aaacaaattg gagctacagg ctatagtagg ttgggtcaaa aggccactat 15780

gaaaacacgt agtggaaaac aaattggagc tacagtacat tattatcatg atattagtcc 15840

tatacagtca aacacgtagt ggaaaacaaa ttggagctac agtacattat tatcatgata 15900

ttagtcctat acagtctttt gctgaacacg aagaaattga attgcagccg tacattatta 15960

tcatgatatt agtcctatac agtcttttgc tgaacacgaa gaaattgaat tgcagccttt 16020

acatacatct acccattcat ctgcaccttc ttttgctgaa cacgaagaaa ttgaattgca 16080

gcctttacat acatctaccc attcatctgc acctttgttt gatatatcgt aagaccctga 16140

tacagttccc tttacataca tctacccatt catctgcacc tttgtttgat atatcgtaag 16200

accctgatac agttcctagc atacatacgc cgcgcatgtc atattcccct gtttgatata 16260

tcgtaagacc ctgatacagt tcctagcata catacgccgc gcatgtcata ttcccctaca 16320

acattaccag ttccaagatc gtactccaac ctagcataca tacgccgcgc atgtcatatt 16380

cccctacaac attaccagtt ccaagatcgt actccaatgt gttttcctct attaatactt 16440

ccactaccac ctacaacatt accagttcca agatcgtact ccaatgtgtt ttcctctatt 16500

aatacttcca ctaccaatgt tactgtgcct ttatccacct catttgaact gtgttttcct 16560

ctattaatac ttccactacc aatgttactg tgcctttatc cacctcattt gaactacctg 16620

tatatagtgg gtcagacatt tacacgcccg ttactgtgcc tttatccacc tcatttgaac 16680

tacctgtata tagtgggtca gacatttaca cgcccacatc ttccccgaca tggccatcat 16740

tgccccccct acctgtatat agtgggtcag acatttacac gcccacatct tccccgacat 16800

ggccatcatt gcccccccca cccaccacta acttacctgc aatagttgtg cccacatctt 16860

ccccgacatg gccatcattg ccccccccac ccaccactaa cttacctgca atagttgtgc 16920

atggggataa ttattattta tggccctatc cccacccacc actaacttac ctgcaatagt 16980

tgtgcatggg gataattatt atttatggcc ctatatttat ttaatccata aacgccgtaa 17040

acgtcgtacg catggggata attattattt atggccctat atttatttaa tccataaacg 17100

ccgtaaacgt cgtacttatt ttttttcaga tggctttgtg gcgtactagt attatttatg 17160

gccctatatt tatttaatcc ataaacgccg taaacgtcgt acttattttt tttcagatgg 17220

ctttgtggcg tactagtgac agtttattta atccataaac gccgtaaacg tcgtacttat 17280

tttttttcag atggctttgt ggcgtactag tgacagcaag gtatatcttc ctcccacccc 17340

tggccttatt ttttttcaga tggctttgtg gcgtactagt gacagcaagg tatatcttcc 17400

tcccacccct gtgtctcggg ttgtcaacac ggatgaatat gttttttcag atggctttgt 17460

ggcgtactag tgacagcaag gtatatcttc ctcccacccc tgtgtctcgg gttgtcaaca 17520

cggatgaata tgtgtgacag caaggtatat cttcctccca cccctgtgtc tcgggttgtc 17580

aacacggatg aatatgtaac tcgcaccggc atatattatt cgtagggcag ctcgtgtctc 17640

gggttgtcaa cacggatgaa tatgtaactc gcaccggcat atattattcg tagggcagct 17700

ctcgtttatt aacattagga catccatatt tttctcgcac cggcatatat tattcgtagg 17760

gcagctctcg tttattaaca ttaggacatc catatttttc catacctaaa actggccaaa 17820

aggccgaaat tcctccatac ctaaaactgg ccaaaaggcc gaaattccta aggtatctgc 17880

ctatcagtac agggtattta gagtgcacct acctgatcct aataaatttg gatcctaagg 17940

tatctgccta tcagtacagg gtatttagag tgcacctacc tgatcctaat aaatttggat 18000

tgcctgatcc acagttatat aatcctgaca cagagagtgc acctacctga tcctaataaa 18060

tttggattgc ctgatccaca gttatataat cctgacacag aacgcctggt gtgggcctgt 18120

gttggtgttg aagtgcctga tccacagtta tataatcctg acacagaacg cctggtgtgg 18180

gcctgtgttg gtgttgaagt tggtagagga cagccattag gcattggcct taggaacgcc 18240

tggtgtgggc ctgtgttggt gttgaagttg gtagaggaca gccattaggc attggcctta 18300

gtggacatcc tttgtttaat aagttggatg atagttggta gaggacagcc attaggcatt 18360

ggccttagtg gacatccttt gtttaataag ttggatgata ccgaaaactc tcatttggct 18420

actgtaacgt aagagtggac atcctttgtt taataagttg gatgataccg aaaactctca 18480

tttggctact gtaacgtaag acactgacaa cagggacaat gtttcagttg ataccgaaaa 18540

ctctcatttg gctactgtaa cgtaagacac tgacaacagg gacaatgttt cagttgataa 18600

taaacaaaca cagttatgta ttataggttg tacgacactg acaacaggga caatgtttca 18660

gttgataata aacaaacaca gttatgtatt ataggttgta caccgccctt gggagagcac 18720

tggggtattg gcaaataaac aaacacagtt atgtattata ggttgtacac cgcccttggg 18780

agagcactgg ggtattggca ctatatgtaa aaatacacag acacaacgtg gggacaccgc 18840

ccttgggaga gcactggggt attggcacta tatgtaaaaa tacacagaca caacgtgggg 18900

attgcccccc cttagaatta atttccagca ttactatatg taaaaataca cagacacaac 18960

gtggggattg ccccccctta gaattaattt ccagcattat tgaggatggc gatatgattg 19020

atacaggctt tggggattgc ccccccttag aattaatttc cagcattatt gaggatggcg 19080

atatgattga tacaggcttt ggagctatgg attttactgc cttacaggct accttgagga 19140

tggcgatatg attgatacag gctttggagc tatggatttt actgccttac aggctaccaa 19200

atcagatgtg cccattgata ttagtcaatc caccaggctt tggagctatg gattttactg 19260

ccttacaggc taccaaatca gatgtgccca ttgatattag tcaatccaca tgtaaatatc 19320

ctgattatct taaggagcta tggattttac tgccttacag gctaccaaat cagatgtgcc 19380

cattgatatt agtcaatcca catgtaaata tcctgattat cttaaaatgt ctgaccaaat 19440

cagatgtgcc cattgatatt agtcaatcca catgtaaata tcctgattat cttaaaatgt 19500

ctgcagatac atatggaaac agcatgttta aatcagatgt gcccattgat attagtcaat 19560

ccacatgtaa atatcctgat tatcttaaaa tgtctgcaga tacatatgga aacagcatgt 19620

ttttttttcc cacatgtaaa tatcctgatt atcttaaaat gtctgcagat acatatggaa 19680

acagcatgtt tttttttctt cgccgggaac aattatttgc cagacatttg cagatacata 19740

tggaaacagc atgttttttt ttcttcgccg ggaacaatta tttgccagac atttttataa 19800

taaggcgggg gctgttgggg cgtatatacc ttcgccggga acaattattt gccagacatt 19860

tttataataa ggcgggggct gttggggcgt atatacccac cactttgtat attaaaggtg 19920

ctgaatcaga taataaggcg ggggctgttg gggcgtatat acccaccact ttgtatatta 19980

aaggtgctga atcaggcagg gagcccccta catcttctat ttattctgcc ccaccacttt 20040

gtatattaaa ggtgctgaat caggcaggga gccccctaca tcttctattt attctgctac 20100

acctagtggc tctatggtta cttcggatgg gcagggagcc ccctacatct tctatttatt 20160

ctgctacacc tagtggctct atggttactt cggcgtaaca actatttaat aagccatact 20220

ggttacaacg ctacacctag tggctctatg gttacttcgg cgtaacaact atttaataag 20280

ccatactggt tacaacgtgc acagggtcat aataatggta tctgttgggg gttacttcgg 20340

cgtaacaact atttaataag ccatactggt tacaacgtgc acagggtcat aataatggta 20400

tctgttgggg caatcaattg tttgttacgc acaactattt aataagccat actggttaca 20460

acgtgcacag ggtcataata atggtatctg ttggggcaat caattgtttg ttacctgtgt 20520

tgataccacg tgcacagggt cataataatg gtatctgttg gggcaatcaa ttgtttgtta 20580

cctgtgttga taccacccgc agtactaacc ttaccattag tacattatgg ggcaatcaat 20640

tgtttgttac ctgtgttgat accacccgca gtactaacct taccattagt acattatctg 20700

cagcatctgc atccactcca tttaaaccac ccgcagtact aaccttacca ttagtacatt 20760

atctgcagca tctgcatcca ctccatttaa accatctgat tataaacaat ttataagaca 20820

tggcgaagtc tgcagcatct gcatccactc catttaaacc atctgattat aaacaattta 20880

taagacatgg cgaagaatat gaattacaat ttatatttca gttgtgtatc tgattataaa 20940

caatttataa gacatggcga agaatatgaa ttacaattta tatttcagtt gtgtaaaata 21000

acacttacaa cagatgttat ggcttacaga atatgaatta caatttatat ttcagttgtg 21060

taaaataaca cttacaacag atgttatggc ttacatacat ttaatgacgt actccatatt 21120

ggaggattta tttcagttgt gtaaaataac acttacaaca gatgttatgg cttacataca 21180

tttaatgacg tactccatat tggaggattg gaattttgga aataacactt acaacagatg 21240

ttatggctta catacattta atgacgtact ccatattgga ggattggaat tttggactaa 21300

ccttacctcc cactgctagt acatttaatg acgtactcca tattggagga ttggaatttt 21360

ggactaacct tacctcccac tgctagtttg gaagcgtact ataggtttat taaaaactct 21420

ggaattttgg actaacctta cctcccactg ctagtttgga agcgtactat aggtttatta 21480

aaaactctgc tactacctgt cagcgtaacg cccctcctga gtttggaagc gtactatagg 21540

tttattaaaa actctgctac tacctgtcag cgtaacgccc ctcctgtgcc aaaggaagat 21600

ccttttcaaa aatttaaatc tctgctacta cctgtcagcg taacgcccct cctgtgccaa 21660

aggaagatcc ttttcaaaaa tttaaatttt gggatgtaga tttaaaagaa aaattttctg 21720

tgccaaagga agatcctttt caaaaattta aattttggga tgtagattta aaagaaaaat 21780

tttctattga tttggatcaa tttccactag ggcgtaagtt tttgggatgt agatttaaaa 21840

gaaaaatttt ctattgattt ggatcaattt ccactagggc gtaagtttat gttacaggcc 21900

ggcatacaac ggcggccgat tgatttggat caatttccac tagggcgtaa gtttatgtta 21960

caggccggca tacaacggcg gccgaaacta ggcaccaaac gtcccttatc ttctacctcg 22020

tttatgttac aggccggcat acaacggcgg ccgaaactag gcaccaaacg tcccttatct 22080

tctacctctt cctctaccaa acgcaaaaaa cgtaaacttc ctcttcctct accaaacgca 22140

aaaaacgtaa acttactaaa taattccgca tgtttgtgtg tgtgtatatg tttgtggtat 22200

gttatgtaag gtgtgtttga ctaaataatt ccgcatgttt gtgtgtgtgt atatgtttgt 22260

ggtatgttat gtaaggtgtg tttgtatgtg tgtacaactg tatgtaatgt aaaggcgtga 22320

tgtttgtggt atgttatgta aggtgtgttt gtatgtgtgt acaactgtat gtaatgtaaa 22380

ggcgtgtttt gtatgtatgt attatatgtc gtaatggtta tgtgtgtaca actgtatgta 22440

atgtaaaggc gtgttttgta tgtatgtatt atatgtcgta atggttatgt gttttcctgt 22500

ttgtcgtaat acttgttatg ttttgtatgt atgtattata tgtcgtaatg gttatgtgtt 22560

ttcctgtttg tcgtaatact tgttatttaa taaagtatga atgtgtcttc cgtaatggtt 22620

atgtgttttc ctgtttgtcg taatacttgt tatttaataa agtatgaatg tgtcttccgt 22680

aatggttaca tgtctttact acactatttg tcatttgttt taataaagta tgaatgtgtc 22740

ttccgtaatg gttacatgtc tttactacac tatttgtcat ttgttttacc cctgaggtaa 22800

tgggaggaac cttaggtggt ttacccctga ggtaatggga ggaaccttag gtggtgtccc 22860

ttataattat tatattacac aagtttacct tttatatgta tttcactaaa cttttgtagg 22920

gtgtccctta taattattat attacacaag tttacctttt atatgtattt cactaaactt 22980

ttgtagtgtt atatttttac tttttatatt ttctattact accttttata tgtatttcac 23040

taaacttttg tagtgttata tttttacttt ttatattttc tattactatc tattgtccca 23100

accgttttcg gtcgttcctg tgttatattt ttacttttta tattttctat tactatctat 23160

tgtcccaacc gttttcggtc gttccttatt ttagttttta tccaactttc cgtatgtatc 23220

tatctattgt cccaaccgtt ttcggtcgtt ccttatttta gtttttatcc aactttccgt 23280

atgtatcctg caggaacagt taatcctttg gcagacaact tttagttttt atccaacttt 23340

ccgtatgtat cctgcaggaa cagttaatcc tttggcagac aacacatcct gcctcctacg 23400

cttggcttgc cattttggcc acatcctgcc tcctacgctt ggcttgccat tttggcacta 23460

taagtggcgc gcctgtatta gtcacatata tttaaacaat acttacataa gcactttttt 23520

ggcactataa gtggcgcgcc tgtattagtc acatatattt aaacaatact tacataagca 23580

ctttttctta cattataata aaactgctgt taggcacata tatatttaaa caatacttac 23640

ataagcactt tttcttacat tataataaaa ctgctgttag gcacatattt ttattttatt 23700

ttataggtct tttaagtgct cttacattat aataaaactg ctgttaggca catattttta 23760

ttttatttta taggtctttt aagtgcatag ttggctaaca tatacacttt tgtttgccat 23820

atttttattt tattttatag gtcttttaag tgcatagttg gctaacatat acacttttgt 23880

ttgccaacta tgtgtctgac acatactgtt gtaacccatg catagttggc taacatatac 23940

acttttgttt gccaactatg tgtctgacac atactgttgt aacccatagt taaacacagg 24000

tgtgtatgta accgaaatgg ccaactatgt gtctgacaca tactgttgta acccatagtt 24060

aaacacaggt gtgtatgtaa ccgaaatgtg ttttgttacg tacgtaaagt ttctttata 24119

<210> 2

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 2

caagcagaag acggcatacg agattgcatg acgtgactgg agttcagacg tgt 53

<210> 3

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 3

caagcagaag acggcatacg agattgctat cggtgactgg agttcagacg tgt 53

<210> 4

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 4

caagcagaag acggcatacg agatcacaag cagtgactgg agttcagacg tgt 53

<210> 5

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 5

caagcagaag acggcatacg agattcgctg atgtgactgg agttcagacg tgt 53

<210> 6

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 6

caagcagaag acggcatacg agatcgcttt gtgtgactgg agttcagacg tgt 53

<210> 7

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 7

caagcagaag acggcatacg agatccgatg aagtgactgg agttcagacg tgt 53

<210> 8

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 8

caagcagaag acggcatacg agattgacca cagtgactgg agttcagacg tgt 53

<210> 9

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 9

caagcagaag acggcatacg agatgaagcc atgtgactgg agttcagacg tgt 53

<210> 10

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 10

caagcagaag acggcatacg agatttctgg tggtgactgg agttcagacg tgt 53

<210> 11

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 11

caagcagaag acggcatacg agatccgaaa acgtgactgg agttcagacg tgt 53

<210> 12

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 12

caagcagaag acggcatacg agatcgaaaa gggtgactgg agttcagacg tgt 53

<210> 13

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 13

caagcagaag acggcatacg agataaccag ctgtgactgg agttcagacg tgt 53

<210> 14

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> Joint

<400> 14

ctacaataat tcatgtataa aactaagggc tagtataaaa gcagacattt tcgtaaccaa 60

Claims (3)

1. A kit for detecting human papillomavirus HPV26 comprising the probe: SEQ ID NO.1; the length interval of the probe is 80-150bp; the number of probe layers is 3 and a shingled design is required.

2. A method for using the kit for detecting human papillomavirus HPV26 of claim 1, for non-diagnostic and non-therapeutic purposes, comprising the steps of:

(1) Extracting sample DNA, fragmenting, constructing DNA library,

(2) Hybridization of the constructed library with the probe according to claim 1, targeted capture and quantification of the target region,

(3) High throughput sequencing, high throughput sequencing of the sample,

(4) Bioinformatic analysis of gene sequences.

3. The kit of claim 1 for non-diagnostic non-therapeutic human papillomavirus HPV detection.