CN112301166A - Probe for detecting human papilloma virus HPV51 and kit thereof - Google Patents

Abstract

The invention discloses a probe for detecting human papillomavirus HPV51 and a kit thereof, wherein the probe group comprises a probe capable of specifically capturing HPV51 genome; the invention has the advantages of comprehensive detection range, greatly improved detection efficiency and accurate result.

Description

Probe for detecting human papilloma virus HPV51 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 papilloma virus HPV51 genes.

Background：

Cervical cancer refers to a malignant tumor that occurs in the cervix, vagina and cervix, and is one of the most common gynecological malignancies, second only to breast cancer. The cervical cancer is the second most serious cancer incidence and the third most serious mortality of women aged 15-44 in China. A large number of researches prove that the cervical cancer is mainly caused by continuous infection of high-risk Human Papilloma Virus (HPV), and the existence of high-risk HPV DNA is detected in 99.7 percent of cervical cancer patients. Currently, over 200 HPV genotypes have been identified, about 40 being associated with genital tract infections. The HPV is classified into high-risk types and low-risk types according to the high and low risk of causing cervical cancer, wherein the high-risk types comprise 13 genotypes such as HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and the like which are classified as high-risk types, and 5 genotypes such as HPV 26, 53, 66, 73, 82 and the like which are classified as medium-risk types. These 18 types of HPV are primarily associated with the development of cervical intraepithelial neoplasia, CIN ii, iii and cervical cancer. The low risk forms are generally unrelated to malignant lesions, mainly causing condyloma acuminata and low grade cin (cini). Therefore, the detection of whether high-risk HPV infection exists in the exfoliated cervical cells and the periodic detection of whether persistent infection of HPV virus exists in the exfoliated cervical cells are particularly important in early screening, auxiliary diagnosis, post-cure evaluation and other links of cervical cancer.

Usually, HPV exists in a free state after infecting a host cell, and its DNA remains freely present in the cytoplasm of the host cell, and the HPV DNA is inserted into the human genome following replication by division of the host cell, a process also known as HPV integration. Once HPV DNA is accidentally integrated into the host cell chromosome, viral proteins will replicate disorderly, and the host cell genetic material structure and associated gene expression will change, resulting in uncontrolled division of the host cell, thereby inducing carcinogenesis. Although HPV DNA is detected in more than 99.7% of cervical cancers, not every patient infected with high-risk HPV develops cervical cancer. With the progress of molecular biology and cytogenetics, the integration of HPV with host chromosomes is an important step in the immortalization process of cervical cells, and is an important marker for the malignant progress of cervical intraepithelial neoplasia to cervical cancer. Integration of HPV with host cell chromosomes is found in more than 90% of cervical cancer tissues, and integration of HPV with host cell genomes is the most important risk factor for development of cervical cancer. Low risk HPV types rarely cause cervical cancer, independent of viral load in the host, because of their poor viral integration. The integration rates of high-risk HPV in HSIL and squamous cell carcinoma were 76.2% and 88.2%, respectively, and only 37% in LSIL. HPV exists in a non-free state in invasive cervical cancer (about 60.9%), which is 20% higher than that in situ cancer. HPV 16-positive cervical cancer cases are nearly 90% integrated, with only 16% of cases occurring in pure free form. The result shows that the incidence rate of virus integration is in positive correlation with the incidence rate of cervical cancer, and the virus integration state is in positive correlation with the disease degree of cervical cancer. The integration of HPV DNA and human genome can be used as a marker of cervical lesion degree, and has very important significance for early diagnosis of cervical cancer.

The early symptoms of the cervical cancer are not obvious, and the mortality rate caused by the cervical cancer can be effectively reduced only by early screening and early warning of the cervical cancer as soon as possible. Traditional pathological detection means, such as cervical smear detection, are easily influenced by factors such as material taking, staining and subjective judgment of cytopathologists, so that the application of cytopathology 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 fluorescence Polymerase Chain Reaction (PCR) method which is commonly used clinically has low detection flux, is difficult to meet the requirement of simultaneously detecting multiple types clinically for different types of HPV by respectively detecting different types of HPV, and is not suitable for large-scale screening of cervical cancer. More importantly, the methods used at present can only judge whether HPV infection exists or not and the type of HPV infection, and cannot judge whether HPV DNA is integrated with human genome, and an HPV integration event is an important factor for the generation of cervical cancer. The high-throughput sequencing technology (next-generation sequencing technology) has the advantages of accuracy, high sensitivity, rapidness and low cost, can simultaneously sequence a plurality of genes, and can realize HPV typing detection and HPV integration detection. The liquid phase hybridization gene capturing technology can enrich the relevant gene segments of the target area, and the cost of human gene sequencing, genetic research and gene diagnosis can be greatly reduced by combining a new generation gene sequencing technology, the sequencing depth is improved, and the variation information of the specific area is more accurately found. The design of the capture probe is the core of the liquid phase hybridization capture technology. In conclusion, the invention provides the probe set and the kit for detecting HPV typing and integration, which can accurately type 18 high-risk types of HPV at one time and determine the integration condition of the high-risk types of HPV in a human genome, have the advantages of high sensitivity and high specificity by processing a plurality of samples at one time, and have great clinical significance.

Disclosure of Invention：

The present invention is directed to solving the above-mentioned deficiencies of the prior art, and provides a capture probe set for detecting HPV and a kit thereof; the invention designs and synthesizes probe sequences according to the 18 HPV whole genome sequences, and carries out specific capture, amplification and sequencing on target fragments so as to achieve the purpose of detecting samples. The probe set comprises probes capable of specifically capturing all 18 HPV genomes. The method can comprehensively, quickly and accurately detect 18 HPV types and 5 HPV integration conditions, wherein the 18 HPV types are respectively HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82; the 5 HPV integrations were HPV16, 18, 33, 52, 58, respectively.

The invention adopts the following technical scheme:

the invention provides a probe for detecting typing and integration of human papillomavirus HPV51, and the probe information is shown in Table 1

The invention also provides a kit for detecting typing and integration of human papillomavirus HPV 51. The kit comprises the probe for detecting typing and integration of human papillomavirus HPV 51; preferably, the kit further comprises a library construction buffer solution 1, a library construction buffer solution 2, an enzyme 1, an enzyme 2, a joint, a PCR amplification primer 1, a PCR amplification primer 2, a PCR reaction mixed solution, 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; 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 typing and integration of human papillomavirus HPV51 and a kit thereof, which mainly comprises the following steps:

(1) design of capture probes for HPV51

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

(3) Library target region targeted capture and quantitation

(4) High throughput sequencing

(5) Bioinformatic analysis of gene sequences

The invention provides a method for capturing HPV genes, which comprises the following specific capturing processes: extracting cervical exfoliated cell whole genome DNA, randomly breaking the cervical exfoliated cell whole genome DNA into fragments with the size of 200-300bp, and constructing a DNA library, wherein partial fragments contain HPV gene sequences; the HPV probe (carrying biotin label and capable of specifically hybridizing with the HPV DNA sequence) related by the invention is hybridized with the constructed pre-library, and the sequence containing HPV gene is combined with magnetic beads through biotin on the probe and can be specifically hybridized; washing away the HPV-free sequence to enrich the HPV gene fragments, so that the HPV sequence is captured by the kit; and (3) obtaining the HPV genotype of the sample to be detected and whether the sample to be detected is integrated with the human genome or not by sequencing the HPV in the sequencing result in a high-throughput manner by a sequencer Nextseq CN500 and analyzing the HPV information in the sequencing result.

The invention has the following beneficial effects:

(1) the invention designs a probe which can rapidly and accurately capture the complete sequence of the HPV51 genome and can effectively improve the detection specificity. The existing methods such as fluorescent quantitative PCR are only directed at a certain region of an HPV sequence, and the detection range of the probe can cover the whole HPV genome length.

(2) The invention utilizes HPV probes to sequence a library sample of a captured target region by utilizing a high-throughput sequencing technology. The HPV probes are used for carrying out targeted enrichment capture on the HPV sequences, and then high-throughput sequencing is carried out, so that the detection sensitivity is high.

(3) The invention can process a plurality of samples at one time, distinguish a plurality of different samples by adding different Barcodes, and then mix on-machine sequencing, thereby effectively improving the detection flux.

Drawings：

FIG. 1 shows the parameters of a PCR apparatus with a repaired end and an "A" reaction at the 3' end;

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

The specific implementation mode is as follows:

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

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

Based on the base-complementary pairing principle, oligonucleotide probes complementary-paired with the HPV51 DNA sequence are designed. Due to the complex structure of genome sequences, irregular regions such as high GC and high AT regions exist, and due to the limitation of the read length of the second-generation sequencing, the design of probes becomes particularly critical, and various 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) The number of layers of the probes is as follows: i.e., how many probes are covered on average over the target area, 3 layers are generally suggested and a shingled design is required.

(3) Specificity: i.e., the uniqueness of the probe on the genome-wide scale, the higher the specificity, the higher the capture efficiency of the probe. Designing probes in low complexity regions should be avoided; however, in special cases, for example, where some hot spots happen to be located in a low complexity region, it is necessary to comprehensively evaluate the sequence of the region and adjacent regions, and carefully place probes.

(4) Binding capacity: the GC content is in a region of about 50%, and the probe capture capacity is strongest. The high GC area has strong probe binding capacity, but the DNA fragment has stronger self binding capacity (longer length) and larger probe competition resistance; since the high-AT region has a weak ability to bind to DNA fragments themselves, but also a weak ability to bind to probes, the number of probes needs to be increased appropriately in the high-GC and high-AT regions to compensate for the disadvantages in terms of competitive resistance and binding ability.

Preparing a probe: a large amount of designed probes are synthesized on a chip by a photochemical synthesis method, and then the biotin-labeled probes are obtained by PCR or transcription reaction.

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

The reagent kit for HPV detection described in this example.

The kit comprises the following components: the kit comprises a capture probe for detecting HPV, a library construction buffer solution 1, a library construction buffer solution 2, an enzyme 1, an enzyme 2, a joint, a PCR amplification primer 1, a PCR amplification primer 2, a PCR reaction mixed solution, 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 specific composition information is shown in table 2,

the using method of the kit comprises the following steps:

1. sample library preparation

(1) Fragmentation: 1000 ng of DNA is taken out, thawed, shaken and mixed evenly, water is added for supplementing to 100 mu L, and the mixture is interrupted by a Bioruptor Pico ultrasonic interrupting instrument. The specific parameters are set as follows: after the temperature of the cooling circulator is reduced to 4 ℃, setting parameters ON for 30 seconds, setting OFF for 30 seconds to be 1 cycle, setting one cycle for every 10 cycles, and performing 3 cycles in total; taking 1 mu L of sample to carry out fragment detection by using Qsep1, and detecting the main peak of the sample after normal fragmentation to be about 150 bp-200 bp.

(2) End repair plus a: and (3) performing 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, run the PCR program and set the PCR instrument parameters as in FIG. 1 (50 μ L system, hot lid 85 ℃);

(3) adding a joint: and (4) performing joint connection on the sample obtained in the last step. The specific reaction system is as follows: 50 μ L of the sample after the reaction in the previous step, 5 μ L of linker, 15 μ L of enzyme-free water, 20 μ L of linking buffer, and 10 μ L of ligase; after mixing, run the PCR instrument program (without heat lid required), set the PCR instrument parameters: closing the hot cover, cooling to 20 ℃ for 30 minutes, and finishing the process

(4) Magnetic bead purification: immediately after the procedure is finished, the reaction product of the previous step is purified by using purified magnetic beads. The purification system was as follows: 100. mu.L of the reaction product of the previous step and 100. mu.L of purified magnetic beads.

(5) Pre-PCR amplification reaction: the purified product of the previous step is used as a template to carry out 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 PCR amplification reaction primer 1 (20. mu.M), and 2.5. mu.L of PCR amplification reaction primer 2 (20. mu.M). After mixing, the samples were placed on a PCR instrument and the PCR program was started as shown in FIG. 2 (50. mu.L system, hot lid temperature 105 ℃);

note: TPE1.0index number in the PCR amplification reaction primer 1 is 1# -8 # (Table 3); TPE2.0index number in the PCR amplification reaction primer 2 is 1# to 12 #; the index number used was recorded (table 4).

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

2. Liquid phase hybridization of samples

(1) Standing and unfreezing the closed sequence 1 and the closed sequence 2 to prepare a mixed solution 1, which specifically comprises the following steps: 750ng library, 5. mu.L of blocking sequence 1, 2. mu.L of blocking sequence 2.

(2) And (3) taking out the blocking reagent and the probe in the kit, standing, thawing, shaking, uniformly mixing, and preparing a mixed solution 2 according to 5 mu L of the blocking reagent and 2 mu L of the probe (operation on ice).

(3) Taking out the hybridization buffer solution in the kit, placing the hybridization buffer solution at room temperature for thawing, taking out 20 mu L of the hybridization buffer solution after thawing, and placing the hybridization buffer solution in a constant temperature mixer at 65 ℃ for incubation for later use.

(4) Add 13. mu.L of hybridization buffer to mixture 2, blow and mix well, centrifuge briefly and collect the liquid, stand at room temperature for use.

(5) Using a temperature-controllable vacuum drier, setting the drying temperature at 45-50 ℃, opening a tube cover until the volume of the mixed solution 1 is concentrated to be less than 10 mu L, supplementing the mixed solution to 10 mu L with non-enzyme water, slightly sucking, uniformly mixing, centrifuging for a short time, and placing on ice for later use. The concentration time should not be too long to prevent evaporation.

(6) And placing the concentrated mixed solution 2 in a PCR instrument for incubation at 95 ℃ for 5min and at 65 ℃ for 2min (the temperature of a hot cover is 105 ℃), wherein the mixed solution does not need to be taken out after incubation is finished.

(7) The mixture 1 with the addition of hybridization buffer was incubated in a PCR apparatus at 65 ℃ for 2min (hot lid temperature 105 ℃).

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

3. Capture

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

(2) Remove 50. mu.L of magnetic beads into a new reaction tube, place the centrifuge tube on a magnetic rack, stand for 1min until the liquid is clear, and carefully aspirate the liquid (take care not to aspirate the magnetic beads).

(3) Adding 200 mu L of capture buffer solution into the magnetic beads, blowing and uniformly mixing, placing the centrifugal tube on a magnetic frame, standing for 1min until the liquid is clear, and carefully sucking away the liquid (not sucking the magnetic beads).

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

(5) And adding 200 mu L of capture buffer solution into the magnetic beads again to resuspend the magnetic beads, adding the resuspended magnetic beads into the hybridization products, and placing the PCR tube on a rotary vortex mixer to combine the magnetic beads at room temperature for 30 min.

(6) And taking out the washing buffer solution 1 and the washing buffer solution 2 in the kit, standing the washing buffer solution 1 at room temperature for later use, and preheating the washing buffer solution 2 at 65 ℃ for more than 5min for later use.

(7) The capture product was placed on a magnetic rack and allowed to stand for 1 minute for 1min until the liquid cleared, and the liquid was carefully aspirated (taking care not to attract the beads).

(8) The centrifuge tube was removed from the magnetic rack, 200. mu.L of washing buffer 1 was added, after pipetting and mixing, the tube was rotated on the mixer for 15min, placed on the magnetic rack and left to stand for 1min until the liquid was clear, and the liquid was carefully aspirated away (care was taken not to aspirate the beads).

(9) The centrifuge tube was removed from the magnetic rack, 200. mu.L of 65 ℃ preheated washing buffer 2 was added, after being blown and mixed well, placed on a constant temperature shaker (65 ℃, 800 rpm) for 10 minutes, placed on the magnetic rack and left for 1min until the liquid was clear, and the liquid was carefully discarded (care was taken not to attract the beads).

(10) Repeating the step (9) three times.

(11) Removing the centrifuge tube from the magnetic rack, adding 200 μ L of freshly prepared 80% ethanol, standing on the magnetic rack for 30sec until the liquid is clear, carefully aspirating the liquid (taking care not to aspirate the magnetic beads)

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

(13) The centrifuge tube was removed from the magnetic frame, 30. mu.L of enzyme-free water was added, and the beads were resuspended for use.

(14) Preparing a mixed solution 3, using a 0.2mL PCR tube, and adopting a specific reaction system as follows: 18 mu of LPCR amplification buffer solution, 1 mu of LPCR amplification primer and 1 mu of LPCR amplification reaction solution.

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

(16) The PCR product was placed in a PCR apparatus to perform PCR amplification according to the reaction procedure shown in Table 5.

(17) And (3) purifying after amplification, and purifying an amplification product by using commercial nucleic acid purification magnetic beads, wherein the specific operation steps are carried out according to the instruction of a kit. Sucking all purified products, placing on ice, repeating the steps 2-3, and entering a sequencing stage or storing at-20 +/-5 ℃.

4. Quality control of captured libraries

(1) The concentration of the library is determined by using a nucleic acid quantitative Kit QubitdsDNA HS Assay Kit and a matched instrument, and the concentration of the library is more than 1 ng/. mu.L. Otherwise, the hybrid capture should be performed again.

(2) Taking a sample library or a reference library, and determining the size of the library fragment by using a Standard card Kit (S2) and a matched instrument, wherein the size of the library fragment should be concentrated between 250bp and 500bp, and no obvious small fragment and large fragment hybrid peak exists. Otherwise, the library sample is not qualified, and the hybridization capture is carried out again.

5. Library qPCR quantification

The on-machine enrichment Library was quantitated using the KAPA Library Quantification Kit to adjust to the appropriate on-machine concentration.

(1) Preparation of reagents: 1) preparing appropriate amount of DNA dilution buffer: 1XIDTE buffer was diluted to 0.1X using enzyme-free water, and approximately 1.2mL of dilution buffer was required for each library. The buffer was allowed to come to room temperature before use. 2) The components in the kit are unfrozen on ice, fully and uniformly mixed before use, centrifuged for a short time, and placed on ice for later use.

(2) Sample preparation: 1) appropriate dilutions of the library (using enzyme-free water) were prepared. Take 1 μ L enrichment library for 1: 100. 1: 10000. 1:20000 to dilute.

(3) Adding 8 mu L of mixed solution mixed with primers into each hole of a PCR reaction plate to be reacted, wherein the mixed solution specifically comprises the following components: mu.L of LMaster Mix, 1. mu.L of primer, 1.8. mu.L of water, 0.2. mu.L of 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 well of the standard, 2. mu.L of each DNA standard was added in the order from low concentration to high concentration.

(6) To the negative control wells 2. mu.L of dilution buffer was added.

(7) The PCR reaction plate is sealed and put into a microplate centrifuge for centrifugation for 1 min.

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

(9) The concentration of the Library to be detected is calculated according to the KAPA Library Quantification Kit instruction. And if the final total molar mass of the library is less than 0.02p moles, performing hybrid enrichment on the library again or performing library building again, and if the final total molar mass of the library is unqualified again, terminating the detection.

6. Sequencing on machine

Sequencing was performed on a gene sequencer Nextseq CN500 using sequencing reagents with 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 the sample libraries to be sequenced is not more than 48, and the NextSeq 500 Mid Output v2 Reagent kit (300 cycles) is recommended to be used for detection; the number of libraries of samples to be sequenced is higher than 48 and not higher than 96, and it is recommended that the NextSeq 500 High Output v2 Reagent kit (300 cycles) reagents be tested.

(1) Taking out Reagent Cartridge, HT1 and Flow Cell in advance, placing the Flow Cell in room temperature for balancing for 30min, placing the Reagent Cartridge in room temperature water bath for 1h, and placing HT1 in room temperature for standing, thawing, shaking and mixing uniformly.

(2) Mixing all sample libraries to be sequenced according to the sequencing data volume requirement, diluting the concentration of each library to 4 nM by using a nucleic acid quantitative Kit QubitdsDNA HS Assay Kit and a matched instrument, taking a new centrifugal tube with the same volume, taking each library for mixing, and enabling the total volume after mixing to be not less than 5 mu L.

(3) Prepare fresh 0.2N NaOH, add 1 μ L1N NaOH into 4 μ L purified water and mix to get final product.

(4) And (3) taking 5 mu L of the 4 nM mixed library to a new 1.5 mL centrifuge tube, adding 5 mu L of freshly prepared 0.2N NaOH, uniformly blowing, uniformly mixing, standing for 5min at room temperature for denaturation of the 4 nM mixed library, adding 990 mu LHT1 after uniformly mixing after denaturation, and diluting the mixed library to 20 pM.

(5) A new 1.5 mL centrifuge tube was added to the well-mixed 1365. mu.L of HT 1.

(6) 135. mu.L of the pooled library diluted to 20 pM was added to 1365. mu.L of HT1 to make the final on-machine pooled library volume 1500. mu.L.

(7) Taking out the Reagent card thawed in the room-temperature water bath, drying by using the dust-free paper sassafras, turning for 5 times, and uniformly mixing the reagents.

(8) Using a clean 1 mL pipette tip to puncture the 10-well foiling port with the Load Samples tag on the Reagent Cartridge, 1300 μ L of the final on-machine mix library mixed with the PhiX control was injected into the 10-well.

(9) And (3) carrying out reagent loading operation according to the running setting prompt of the sequencer, clicking a 'start' button to carry out sequencing after the self-checking of the sequencer is completed, and carrying out sequencing for 26-29 h.

7. HPV nucleic acid typing and integration biological information analysis process

(1) After the sequencing is completed, BCL2fastq v2.20.0.422 software of the illumina company is used for converting a BCL file generated by the sequencing into a fastq file corresponding to the sample.

(2) Reading record information of InterOp catalogues in a sequencing file by using illuminate v0.6 software, and performing quality analysis and evaluation on the sequencing. And quality control is carried out on Fastp v0.20.0, and the quality value of Q30 is required to be more than 80%.

(3) And after the quality evaluation is finished, preprocessing the data, and performing quality control on the original data according to the base and the quality of the fastq file (based on fastp v0.20.0 software).

(4) And (3) sequence alignment: alignment of the base sequences in the fastq file onto the human reference genome hg38 (GRCh 38) and the HPV genome generates a bam file (based on BWA v0.7.17, samtools v1.9 and picard v2.20.6 software: alignment).

(5) HPV type differential analysis: the number of reads and the proportion of the various HPVs in the sample were analyzed by sequence alignment files using a typing detection module (based on hpvHPKitTyper v1.0 software).

(6) HPV integration analysis: the HPV integration sites in the samples were analyzed and annotated in conjunction with the database by site comparison of the aligned HPV and human genome sequences in the alignment file using an integration detection module (based on hpkithvfusioncallerrv 1 software).

(7) And (4) judging a result: performing typing analysis, and judging that the single HPV type differential Mean depth is more than or equal to 10 as positive type; and (4) integration analysis, and if the number of HPV integration reads in the unit data volume is more than or equal to 3, judging that the type is integrated positively.

Example 3: verification of Using Effect of the kit of the present invention

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

1. High throughput sequencing assay for HPV typing integration in 20 reference samples and 16 clinical samples

The sample numbers are S1-S36, wherein S1-S18 are typing plasmid reference substances of 10^4 copies/mL, HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82 respectively, S19 and S20 are SiHa cells (known HPV16 integrated sample) and HeLa cell DNA (known HPV18 integrated sample) with the concentration of 0.25 ng/muL respectively, and S21-S36 are clinical samples. And (3) detection results: the HPV type and the integration state corresponding to the reference sample are detected by using the method and the kit, which shows that the method and the kit can be used for accurately and rapidly detecting 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 simple and convenient operation, high flux, good specificity, high sensitivity and the like. The results are as follows: table 7 shows the high-throughput sequencing quality control results of 36 samples, table 8 shows the high-throughput sequencing typing results of 36 samples, and table 9 shows the high-throughput sequencing integration results of 18 samples.

SEQUENCE LISTING

<110> Wuhan Kaideweis Biotech Co., Ltd

<120> a probe for detecting human papillomavirus HPV51 and a kit thereof

<130> 0

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 28398

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe type HPV51

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cgaaaaaaaa aggttccatg aaatagcggg agaccacttg ggcctgaaga aaagcaaaaa 1800

ttggtggacg aaaaaaaaag gttccatgaa atagcgggac gttggacggg gcacgtagct 1860

aattgctggc ggtggacgaa aaaaaaaggt tccatgaaat agcgggacgt tggacggggc 1920

acgtagctaa ttgctggcaa cgtacacgac aacgtaacga aacccaagtg aatagcggga 1980

cgttggacgg ggcacgtagc taattgctgg caacgtacac gacaacgtaa cgaaacccaa 2040

gtgtaataaa gcccgtagtg gtaatgtacc cgggacgttg gacggggcac gtagctaatt 2100

gctggcaacg tacacgacaa cgtaacgaaa cccaagtgta ataaagcccg tagtggtaat 2160

gtaccacaat gcaacgtaca cgacaacgta acgaaaccca agtgtaataa agcccgtagt 2220

ggtaatgtac cacaattaaa agatgtagta ttgcatttaa caccacagac cccaagtgta 2280

ataaagcccg tagtggtaat gtaccacaat taaaagatgt agtattgcat ttaacaccac 2340

agactgaaat tgacttgcac gtatacgagc taataaagcc cgtagtggta atgtaccaca 2400

attaaaagat gtagtattgc atttaacacc acagactgaa attgacttgc acgtatacga 2460

gcaatttgac aagatgtagt attgcattta acaccacaga ctgaaattga cttgcacgta 2520

tacgagcaat ttgacagctc agaggaggag gatgaagtag ataatcgtag agctcagagg 2580

aggaggatga agtagataat cgtagtgacc agctaccaga aagacgggct ggacaggcta 2640

cgtgttacag aattgaagct ccgtgttgca cgtgaccagc taccagaaag acgggctgga 2700

caggctacgt gttacagaat tgaagctccg tgttgcaggt gttcaagtgt agtacaactg 2760

gcagtggaaa ggacaggcta cgtgttacag aattgaagct ccgtgttgca ggtgttcaag 2820

tgtagtacaa ctggcagtgg aaagcagtgg agacaccctt cgcgctacgt gttacagaat 2880

tgaagctccg tgttgcaggt gttcaagtgt agtacaactg gcagtggaaa gcagtggaga 2940

cacccttcgc gttgtacagc agaaggtgtt caagtgtagt acaactggca gtggaaagca 3000

gtggagacac ccttcgcgtt gtacagcaga tgttaatggg cgaactaagc ctggtttgcc 3060

cgtaagcagt ggagacaccc ttcgcgttgt acagcagatg ttaatgggcg aactaagcct 3120

ggtttgcccg tgttgtgcga acaactagca acggcgatgg actatgttaa tgggcgaact 3180

aagcctggtt tgcccgtgtt gtgcgaacaa ctagcaacgg cgatggactg tgaaggtaca 3240

gaggatgagg gggcggggtg taacgtgttg tgcgaacaac tagcaacggc gatggactgt 3300

gaaggtacag aggatgaggg ggcggggtgt aatgggtggt tttttgttga agcaatagta 3360

gaatgtgaag gtacagagga tgagggggcg gggtgtaatg ggtggttttt tgttgaagca 3420

atagtagaaa aaaaaacagg agataatgtt tcggatgatg aggaatgggt ggttttttgt 3480

tgaagcaata gtagaaaaaa aaacaggaga taatgtttcg gatgatgagg atgaaacgta 3540

agatgataca ggatctgatt taaaaaaaac aggagataat gtttcggatg atgaggatga 3600

aacgtaagat gatacaggat ctgatttaat aaactttata gatagtgaaa ctagtatttg 3660

cagggatgaa acgtaagatg atacaggatc tgatttaata aactttatag atagtgaaac 3720

tagtatttgc agtcaggcgg aacaggagac agcacgggcg ttgtaaactt tatagatagt 3780

gaaactagta tttgcagtca ggcggaacag gagacagcac gggcgttgtt tcaggcccaa 3840

gaattacagg caaacaaaga ggctttgcag tcaggcggaa caggagacag cacgggcgtt 3900

gtttcaggcc caagaattac aggcaaacaa agaggctgtg catcagttaa aacgaaagtt 3960

tctcagtcag gcggaacagg agacagcacg ggcgttgttt caggcccaag aattacaggc 4020

aaacaaagag gctgtgcatc agttaaaacg aaagtttcta gtcgcgttgt ttcaggccca 4080

agaattacag gcaaacaaag aggctgtgca tcagttaaaa cgaaagtttc tagtcagccc 4140

gcgaagcagc ccattagttt caggcccaag aattacaggc aaacaaagag gctgtgcatc 4200

agttaaaacg aaagtttcta gtcagcccgc gaagcagccc attaggagac attacaggct 4260

gtgcatcagt taaaacgaaa gtttctagtc agcccgcgaa gcagcccatt aggagacatt 4320

acaaatcaaa acaacacaca cagccatagt caggcagccc gcgaagcagc ccattaggag 4380

acattacaaa tcaaaacaac acacacagcc atagtcaggc aaacgagtca caagttaaaa 4440

ggagattact ggacagaatc aaaacaacac acacagccat agtcaggcaa acgagtcaca 4500

agttaaaagg agattactgg acagttatcc ggacagcgga tatggcaata cacaagcgag 4560

tcacaagtta aaaggagatt actggacagt tatccggaca gcggatatgg caatacacaa 4620

gtggaaactg tggaagcaac gttgcaggta gatggggtta tccggacagc ggatatggca 4680

atacacaagt ggaaactgtg gaagcaacgt tgcaggtaga tgggcaacat ggcggttcac 4740

agaacagtgt gtgtaggtgg aaactgtgga agcaacgttg caggtagatg ggcaacatgg 4800

cggttcacag aacagtgtgt gtagtagcgg ggggggcagt gttatggatg tggaaagggc 4860

aacatggcgg ttcacagaac agtgtgtgta gtagcggggg gggcagtgtt atggatgtgg 4920

aaacaacaga aagctgtgca aatgtagaac taaacaagta gcgggggggg cagtgttatg 4980

gatgtggaaa caacagaaag ctgtgcaaat gtagaactaa acagtatatg tgaagtatta 5040

aaaagcagta cgtaaaacaa cagaaagctg tgcaaatgta gaactaaaca gtatatgtga 5100

agtattaaaa agcagtacgt aaaaagcaac gttaatggca aaatttaaag agttgtcagt 5160

atatgtgaag tattaaaaag cagtacgtaa aaagcaacgt taatggcaaa atttaaagag 5220

ttgtatggta ttagttataa tgagttggta cgggtgaaag caacgttaat ggcaaaattt 5280

aaagagttgt atggtattag ttataatgag ttggtacggg tgtttaaaag tgataaaaca 5340

tgttgtatag attgggatgg tattagttat aatgagttgg tacgggtgtt taaaagtgat 5400

aaaacatgtt gtatagattg ggtttgtgca ttgtttggcg tttccccaat ggtagctaaa 5460

agtgataaaa catgttgtat agattgggtt tgtgcattgt ttggcgtttc cccaatggta 5520

gcagaaaatt taaaaacact aattaagcca ttttgcaagt gataaaacat gttgtataga 5580

ttgggtttgt gcattgtttg gcgtttcccc aatggtagca gaaaatttaa aaacactaat 5640

taagccattt tgcatggttt gtgcattgtt tggcgtttcc ccaatggtag cagaaaattt 5700

aaaaacacta attaagccat tttgcatgta ctaccatata caatgtttat catgtggcag 5760

aaaatttaaa aacactaatt aagccatttt gcatgtacta ccatatacaa tgtttatcat 5820

gtgattgggg caccattgta ttacgtataa ttaggttgca tgtactacca tatacaatgt 5880

ttatcatgtg attggggcac cattgtatta cgtataatta ggttttcatg tgcaaaaaac 5940

agaacaacaa ttgctagatt ggggcaccat tgtattacgt ataattaggt tttcatgtgc 6000

aaaaaacaga acaacaattg ctaagtgttt aagtacatta gtaaatatcc cacaatggtt 6060

ttcatgtgca aaaaacagaa caacaattgc taagtgttta agtacattag taaatatccc 6120

acaatcacaa atgtttatag aaccaccaaa attacggtgc aaaaaacaga acaacaattg 6180

ctaagtgttt aagtacatta gtaaatatcc cacaatcaca aatgtttata gaaccaccaa 6240

aattacgtag tacaccgtgt ttaagtacat tagtaaatat cccacaatca caaatgttta 6300

tagaaccacc aaaattacgt agtacacctg tggcattata tttttataga acaggcgttt 6360

aagtacatta gtaaatatcc cacaatcaca aatgtttata gaaccaccaa aattacgtag 6420

tacacctgtg gcattatatt tttatagaac aggcatcaca aatgtttata gaaccaccaa 6480

aattacgtag tacacctgtg gcattatatt tttatagaac aggcatatca aacattagca 6540

atacatatgg agagacaaat gtttatagaa ccaccaaaat tacgtagtac acctgtggca 6600

ttatattttt atagaacagg catatcaaac attagcaata catatggagt acacctgtgg 6660

cattatattt ttatagaaca ggcatatcaa acattagcaa tacatatgga gagacacctg 6720

aatggattac acgacaaacg caactacggc atatcaaaca ttagcaatac atatggagag 6780

acacctgaat ggattacacg acaaacgcaa ctacaacata gttttgagga tagtaccttt 6840

gaattataca cctgaatgga ttacacgaca aacgcaacta caacatagtt ttgaggatag 6900

tacctttgaa ttatcacaaa tggtgcaatg ggcatttgac catgaagcaa catagttttg 6960

aggatagtac ctttgaatta tcacaaatgg tgcaatgggc atttgaccat gaagtattag 7020

atgatagtga aatagcattt cattatgcat agttttgagg atagtacctt tgaattatca 7080

caaatggtgc aatgggcatt tgaccatgaa gtattagatg atagtgaaat agcatttcat 7140

tcgtaaccac aaatggtgca atgggcattt gaccatgaag tattagatga tagtgaaata 7200

gcatttcatt cgtaacaatt agcagatata gatagtacgt atgcagcatg ggcatttgac 7260

catgaagtat tagatgatag tgaaatagca tttcattcgt aacaattagc agatatagat 7320

agtacgtatg cagcgttttt aaagtattag atgatagtga aatagcattt cattcgtaac 7380

aattagcaga tatagatagt acgtatgcag cgtttttaaa gagtaattgc caagcaaaat 7440

atgtagatga tagtgaaata gcatttcatt cgtaacaatt agcagatata gatagtacgt 7500

atgcagcgtt tttaaagagt aattgccaag caaaatatgt aagtgaaata gcatttcatt 7560

cgtaacaatt agcagatata gatagtacgt atgcagcgtt tttaaagagt aattgccaag 7620

caaaatatgt aaaagattgt ggcatttcat tcgtaacaat tagcagatat agatagtacg 7680

tatgcagcgt ttttaaagag taattgccaa gcaaaatatg taaaagattg tgggcacaat 7740

tagcagatat agatagtacg tatgcagcgt ttttaaagag taattgccaa gcaaaatatg 7800

taaaagattg tgggaccatg gcacggcatt acagcgtttt taaagagtaa ttgccaagca 7860

aaatatgtaa aagattgtgg gaccatggca cggcattaca aacgagcaca aagaaaatca 7920

ttatctatgt cagtttaaag agtaattgcc aagcaaaata tgtaaaagat tgtgggacca 7980

tggcacggca ttacaaacga gcacaaagaa aatcattatc tatgtcagcc tgggtaaaag 8040

attgtgggac catggcacgg cattacaaac gagcacaaag aaaatcatta tctatgtcag 8100

cctggataag gtatagatgt gatagagcaa aggcaaacga gcacaaagaa aatcattatc 8160

tatgtcagcc tggataaggt atagatgtga tagagcaaag gatggaggca actggagaga 8220

aattgctaaa tttgcctgga taaggtatag atgtgataga gcaaaggatg gaggcaactg 8280

gagagaaatt gctaaatttt taagatatca aggtgtaaac tttatgtcct ttagatggag 8340

gcaactggag agaaattgct aaatttttaa gatatcaagg tgtaaacttt atgtccttta 8400

ttcaaatgtt taaacagttt ttaaaaggaa cacaactgga gagaaattgc taaattttta 8460

agatatcaag gtgtaaactt tatgtccttt attcaaatgt ttaaacagtt tttaaaagga 8520

acaccataag atatcaaggt gtaaacttta tgtcctttat tcaaatgttt aaacagtttt 8580

taaaaggaac accaaaacac aattgcatag tcatatatgg cccaccattc aaatgtttaa 8640

acagttttta aaaggaacac caaaacacaa ttgcatagtc atatatggcc caccaaacac 8700

aggcaagtca ttatttgcaa tgagccccaa aacacaattg catagtcata tatggcccac 8760

caaacacagg caagtcatta tttgcaatga gcctaatgaa gtttcgtaaa gggtccatta 8820

tttcatccaa acacaggcaa gtcattattt gcaatgagcc taatgaagtt tcgtaaaggg 8880

tccattattt catatgtaaa ctctggtagt catttttggt tacagcctaa tgaagtttcg 8940

taaagggtcc attatttcat atgtaaactc tggtagtcat ttttggttac agccactaga 9000

ggcgtataaa atagcattgt tagatgtgca agggtccatt atttcatatg taaactctgg 9060

tagtcatttt tggttacagc cactagaggc gtataaaata gcattgttag atgcgtatat 9120

gtaaactctg gtagtcattt ttggttacag ccactagagg cgtataaaat agcattgtta 9180

gatgcgtata cgtatgggtg ttggacatat attgatcgcc actagaggcg tataaaatag 9240

cattgttaga tgcgtatacg tatgggtgtt ggacatatat tgatcagtat ttaagaaact 9300

ttttagatgg taatccatag aggcgtataa aatagcattg ttagatgcgt atacgtatgg 9360

gtgttggaca tatattgatc agtatttaag aaacttttta gatggtaatc catgtaggcg 9420

tatacgtatg ggtgttggac atatattgat cagtatttaa gaaacttttt agatggtaat 9480

ccatgtagta tagatagaaa acataggagt ttaataccag tatttaagaa actttttaga 9540

tggtaatcca tgtagtatag atagaaaaca taggagttta atacaattag tatgtccacc 9600

attactaata acgtcaatgt agtatagata gaaaacatag gagtttaata caattagtat 9660

gtccaccatt actaataacg tcaaacataa atccacaaga ggcgtaaaac ctaatgtcaa 9720

ttagtatgtc caccattact aataacgtca aacataaatc cacaagaggc gtaaaaccta 9780

atgtatttac atacaagggt aacagtatta aagttttaca taaatccaca agaggcgtaa 9840

aacctaatgt atttacatac aagggtaaca gtattaaagt ttttaaatac atttccattt 9900

gataacaatg ggacgtagta tttacataca agggtaacag tattaaagtt tttaaataca 9960

tttccatttg ataacaatgg gacgtatgtg tatacattga atgatgaaaa ttggaaatta 10020

aatacatttc catttgataa caatgggacg tatgtgtata cattgaatga tgaaaattgg 10080

aaaaattttt tttccaccac atggtccaga ttagattaaa tacatttcca tttgataaca 10140

atgggacgta tgtgtataca ttgaatgatg aaaattggaa aaattttttt tccaccacat 10200

ggtccagcat ttccatttga taacaatggg acgtatgtgt atacattgaa tgatgaaaat 10260

tggaaaaatt ttttttccac cacatggtcc agattagatt tggaggccat ttgataacaa 10320

tgggacgtat gtgtatacat tgaatgatga aaattggaaa aatttttttt ccaccacatg 10380

gtccagatta gatttggagg aggaatgatg aaaattggaa aaattttttt tccaccacat 10440

ggtccagatt agatttggag gaggaagagg acaaagaaaa tggagaccct atgattggaa 10500

aaattttttt tccaccacat ggtccagatt agatttggag gaggaagagg acaaagaaaa 10560

tggagaccct cgtacaccgt ttaaatgtgt gccttttttc caccacatgg tccagattag 10620

atttggagga ggaagaggac aaagaaaatg gagaccctcg tacaccgttt aaatgtgtgc 10680

caggagaaaa tacagattag atttggagga ggaagaggac aaagaaaatg gagaccctcg 10740

tacaccgttt aaatgtgtgc caggagaaaa tactagactg ttatgaactg gacaggagga 10800

agaggacaaa gaaaatggag accctcgtac accgtttaaa tgtgtgccag gagaaaatac 10860

tagactgtta tgaactggac agtgataaat tagaatggag accctcgtac accgtttaaa 10920

tgtgtgccag gagaaaatac tagactgtta tgaactggac agtgataaat tagtagatca 10980

aattaactat tggatactag actgttatga actggacagt gataaattag tagatcaaat 11040

taactattgg acattgttac gatatgaagc tgctatgttt tcgtaagcac gggttagtag 11100

atcaaattaa ctattggaca ttgttacgat atgaagctgc tatgttttcg taagcacggg 11160

aaagaaactt acgaacaatc aatcaccagg tagacgatat gaagctgcta tgttttcgta 11220

agcacgggaa agaaacttac gaacaatcaa tcaccaggta gtaccagcaa caacagtatc 11280

aaaacaaaag gcccgggaaa gaaacttacg aacaatcaat caccaggtag taccagcaac 11340

aacagtatca aaacaaaagg cctgtcaagc aattgaacgt aacatggcct tacagtacca 11400

gcaacaacag tatcaaaaca aaaggcctgt caagcaattg aacgtaacat ggccttacaa 11460

tcgcttaaca aatcagacta taacatggaa ccacagtatc aaaacaaaag gcctgtcaag 11520

caattgaacg taacatggcc ttacaatcgc ttaacaaatc agactataac atggaaccat 11580

ggactgtcaa gcaattgaac gtaacatggc cttacaatcg cttaacaaat cagactataa 11640

catggaacca tggacacgta gggagacatg ttatgaacta tggcaagcaa ttgaacgtaa 11700

catggcctta caatcgctta acaaatcaga ctataacatg gaaccatgga cacgtaggga 11760

gacatgttat gaactatggc gcttaacaaa tcagactata acatggaacc atggacacgt 11820

agggagacat gttatgaact atggtgtgtg gctcccaagc aatgtttcaa aaaggggggg 11880

gtgtgtggct cccaagcaat gtttcaaaaa ggggggcata actgtaacag ttatatttga 11940

tggaaataag gacacgtaaa tggactatac aagctggaag ggggcataac tgtaacagtt 12000

atatttgatg gaaataagga cacgtaaatg gactatacaa gctggaaatt tatatatata 12060

tatgataatg ataagtgggt aaggacacgt aaatggacta tacaagctgg aaatttatat 12120

atatatatga taatgataag tgggtaaaga caaatggaaa tgtggactat acgggtatat 12180

ttatatatat atatgataat gataagtggg taaagacaaa tggaaatgtg gactatacgg 12240

gtatatatta cactgtaaat tcaaaaaaag aatattatga gacaaatgga aatgtggact 12300

atacgggtat atattacact gtaaattcaa aaaaagaata ttatgtacag tttaaagatg 12360

aagccaaaat atatggggct attacactgt aaattcaaaa aaagaatatt atgtacagtt 12420

taaagatgaa gccaaaatat atggggcaca acagtgggag gtctatatgt atggtactgt 12480

gtacagttta aagatgaagc caaaatatat ggggcacaac agtgggaggt ctatatgtat 12540

ggtactgtaa taacatgtcc tgaatatgta tctagtacct ttaaagatga agccaaaata 12600

tatggggcac aacagtggga ggtctatatg tatggtactg taataacatg tcctgaatat 12660

gtatctagta cctgcagggg gcacaacagt gggaggtcta tatgtatggt actgtaataa 12720

catgtcctga atatgtatct agtacctgca gcgacgcgtt atccactact acaactgcaa 12780

cagtgggagg tctatatgta tggtactgta ataacatgtc ctgaatatgt atctagtacc 12840

tgcagcgacg cgttatccac tactacaact gttgaacgta ataacatgtc ctgaatatgt 12900

atctagtacc tgcagcgacg cgttatccac tactacaact gttgaacaac tatcaaacac 12960

cccaacgacc aatcccctgc agcgacgcgt tatccactac tacaactgtt gaacaactat 13020

caaacacccc aacgaccaat ccccttacca cctgcgtggg cgccaaagaa gcccagattg 13080

aacaactatc aaacacccca acgaccaatc cccttaccac ctgcgtgggc gccaaagaag 13140

cccagacaca acagcgaaaa cgacagcgac ttactgacct taccacctgc gtgggcgcca 13200

aagaagccca gacacaacag cgaaaacgac agcgacttac tgagcccgac tcctccacaa 13260

tctccccact gtccgtggac acaacagcga aaacgacagc gacttactga gcccgactcc 13320

tccacaatct ccccactgtc cgtggacaat acaaacaacc aaatacactg tggaagtcac 13380

aacagcgaaa acgacagcga cttactgagc ccgactcctc cacaatctcc ccactgtccg 13440

tggacaatac aaacaaccaa atacactgtg gaagtcccga ctcctccaca atctccccac 13500

tgtccgtgga caatacaaac aaccaaatac actgtggaag tggaagcact aacactggag 13560

ggcaccaaag tgcaaggaca atacaaacaa ccaaatacac tgtggaagtg gaagcactaa 13620

cactggaggg caccaaagtg caactcagac tgcgtttata gtgcatttaa aaggtgtgga 13680

agcactaaca ctggagggca ccaaagtgca actcagactg cgtttatagt gcatttaaaa 13740

ggtgatacaa attgtttaaa atgttttaga tacagaacac tggagggcac caaagtgcaa 13800

ctcagactgc gtttatagtg catttaaaag gtgatacaaa ttgtttaaaa tgttttagat 13860

acagatgcaa ctcagactgc gtttatagtg catttaaaag gtgatacaaa ttgtttaaaa 13920

tgttttagat acagatttac aaaacacaaa gggttatata aaaacgggtg atacaaattg 13980

tttaaaatgt tttagataca gatttacaaa acacaaaggg ttatataaaa acgtatcctc 14040

aacctggcat tggaccagta atactaacaa attgtttaaa atgttttaga tacagattta 14100

caaaacacaa agggttatat aaaaacgtat cctcaacctg gcattggacc agtaatacta 14160

aaagatttac aaaacacaaa gggttatata aaaacgtatc ctcaacctgg cattggacca 14220

gtaatactaa aacaggcatt gttaccattg tgtttgacag tgctcctcaa cctggcattg 14280

gaccagtaat actaaaacag gcattgttac cattgtgttt gacagtgcac atcaacggga 14340

aacatttata aaaaccatta aagaacaggc attgttacca ttgtgtttga cagtgcacat 14400

caacgggaaa catttataaa aaccattaaa gtacccccaa gtgtaacact gtcattggga 14460

attcacatca acgggaaaca tttataaaaa ccattaaagt acccccaagt gtaacactgt 14520

cattgggaat tatgacactg taactagtgt aatatatgta ttgagtaccc ccaagtgtaa 14580

cactgtcatt gggaattatg acactgtaac tagtgtaata tatgtattgt acatatatac 14640

tgtcacaagc caatatgtgc tgccaagtgt aacactgtca ttgggaatta tgacactgta 14700

actagtgtaa tatatgtatt gtacatatat actgtcacaa gccaatatgt gctgctaatt 14760

gtattatgac actgtaacta gtgtaatata tgtattgtac atatatactg tcacaagcca 14820

atatgtgctg ctaattgtat agacatattg taaccattgc agtggtacat atatactgtc 14880

acaagccaat atgtgctgct aattgtatag acatattgta accattgcag tgtttattat 14940

tttgctattt gtgctttgct tgtgagccaa tatgtgctgc taattgtata gacatattgt 15000

aaccattgca gtgtttatta ttttgctatt tgtgctttgc ttgtgtgtgt gtcttgtgtt 15060

ggctaattgt atagacatat tgtaaccatt gcagtgttta ttattttgct atttgtgctt 15120

tgcttgtgtg tgtgtcttgt gttgtgttgt ttgttgccgc tgtttattat tttgctattt 15180

gtgctttgct tgtgtgtgtg tcttgtgttg tgttgtttgt tgccgctact gctgtcccaa 15240

tacgtgtttg cagctgcctt agtgtgtgtc ttgtgttgtg ttgtttgttg ccgctactgc 15300

tgtcccaata cgtgtttgca gctgccttat tattaatttt atgtttttgg tttgttgttg 15360

cttgtgttgt gttgtttgtt gccgctactg ctgtcccaat acgtgtttgc agctgcctta 15420

ttattaattt tatgtttttg gtttgttgtt gcaacatcac tgctgtccca atacgtgttt 15480

gcagctgcct tattattaat tttatgtttt tggtttgttg ttgcaacatc ccaattaact 15540

acattttttg tatatttgtt attaatttta tgtttttggt ttgttgttgc aacatcccaa 15600

ttaactacat tttttgtata tttgattttt ttttacttac cttgtttact tttacatcca 15660

acatcccaat taactacatt ttttgtatat ttgatttttt tttacttacc ttgtttactt 15720

ttacatctat atacattttt acttttgcaa taaacttgga ttttttttta cttaccttgt 15780

ttacttttac atctatatac atttttactt ttgcaataaa cttgttatat ttttgtgatt 15840

aaatatggtg gctacacgtc tatatacatt tttacttttg caataaactt gttatatttt 15900

tgtgattaaa tatggtggct acacgtgcac ggcgtcggaa gcgagcatct gtaacacatt 15960

atatttttgt gattaaatat ggtggctaca cgtgcacggc gtcggaagcg agcatctgta 16020

acacaattat attctaccgt aaaagctgct ggtacatggt gcacggcgtc ggaagcgagc 16080

atctgtaaca caattatatt ctaccgtaaa agctgctggt acatgtcctc ctgatgttgt 16140

gaataaggtt gaaggtacca caattatatt ctaccgtaaa agctgctggt acatgtcctc 16200

ctgatgttgt gaataaggtt gaaggtacta cattggccga taaaatatta cagtggagat 16260

tatattctac cgtaaaagct gctggtacat gtcctcctga tgttgtgaat aaggttgaag 16320

gtactacatt ggccgataaa atattacagt ggagtggggt cctcctgatg ttgtgaataa 16380

ggttgaaggt actacattgg ccgataaaat attacagtgg agtgggttgg gtatattttt 16440

gggtggccta ggtattggaa taaggttgaa ggtactacat tggccgataa aatattacag 16500

tggagtgggt tgggtatatt tttgggtggc ctaggtattg gtactgggtc tgaataaggt 16560

tgaaggtact acattggccg ataaaatatt acagtggagt gggttgggta tatttttggg 16620

tggcctaggt attggtactg ggtctggcta cattggccga taaaatatta cagtggagtg 16680

ggttgggtat atttttgggt ggcctaggta ttggtactgg gtctggatct ggggggcgta 16740

ctggataggt tgggtatatt tttgggtggc ctaggtattg gtactgggtc tggatctggg 16800

gggcgtactg gatatatccc tttaggtggt gggggtcgcc caggcgttac tgggtctgga 16860

tctggggggc gtactggata tatcccttta ggtggtgggg gtcgcccagg cgtggtggat 16920

attgctcctg caaggccacc tattataata tccctttagg tggtgggggt cgcccaggcg 16980

tggtggatat tgctcctgca aggccaccta ttataattga cctatggcac catactgaac 17040

cttctatggt ggatattgct cctgcaaggc cacctattat aattgaccta tggcaccata 17100

ctgaaccttc tatagtaaat ttggttgagg actctagtat tattcagatt gacctatggc 17160

accatactga accttctata gtaaatttgg ttgaggactc tagtattatt cagtctgggt 17220

ctcctatacc tacctttact ggtaccgtag taaatttggt tgaggactct agtattattc 17280

agtctgggtc tcctatacct acctttactg gtaccgatgg ctttgaaatt acttcatctt 17340

ccacaacagt ctgggtctcc tatacctacc tttactggta ccgatggctt tgaaattact 17400

tcatcttcca caacaacccc tgctgtgttg gacatcaccc catctgcccg atggctttga 17460

aattacttca tcttccacaa caacccctgc tgtgttggac atcaccccat ctgctggtac 17520

tgtacatgtt tctagtacta acattgacaa cccctgctgt gttggacatc accccatctg 17580

ctggtactgt acatgtttct agtactaaca ttgaaaatcc tttatatatt gaacctccat 17640

ccattgacct gctgtgttgg acatcacccc atctgctggt actgtacatg tttctagtac 17700

taacattgaa aatcctttat atattgaacc tccatccatt gaggctcgct ggtactgtac 17760

atgtttctag tactaacatt gaaaatcctt tatatattga acctccatcc attgaggctc 17820

cacaatctgg agaagtgtca gatatatgaa aatcctttat atattgaacc tccatccatt 17880

gaggctccac aatctggaga agtgtcagat atatatttac tagtacacta ctctggtact 17940

catgggtgag gctccacaat ctggagaagt gtcagatata tatttactag tacactactc 18000

tggtactcat gggtatgaag aaatacctat ggaagtgttt gcatccaatt tactagtaca 18060

ctactctggt actcatgggt atgaagaaat acctatggaa gtgtttgcat ccaatgtcag 18120

tactggtact gaacctatta gcagcacgaa gaaataccta tggaagtgtt tgcatccaat 18180

gtcagtactg gtactgaacc tattagcagc acacctactc caggggttag tcgcatagct 18240

gctccccatg tcagtactgg tactgaacct attagcagca cacctactcc aggggttagt 18300

cgcatagctg ctccccgctt gtatagtaag tcctacacac aggttaacac ctactccagg 18360

ggttagtcgc atagctgctc cccgcttgta tagtaagtcc tacacacagg ttaaagttac 18420

aaatcctgat tttattagta agccatcccc cgcttgtata gtaagtccta cacacaggtt 18480

aaagttacaa atcctgattt tattagtaag ccatccacat ttgttacatt taataatcct 18540

gcttttgaag ttacaaatcc tgattttatt agtaagccat ccacatttgt tacatttaat 18600

aatcctgctt ttgagcctat tgacacatcc ataacttttg aggaacccca catttgttac 18660

atttaataat cctgcttttg agcctattga cacatccata acttttgagg aacctgcgta 18720

tgttgcacct gatcctgatt ttctggaagc ctattgacac atccataact tttgaggaac 18780

ctgcgtatgt tgcacctgat cctgattttc tggatattat tacactgcac cgccctgccc 18840

ttacatcctg cgtatgttgc acctgatcct gattttctgg atattattac actgcaccgc 18900

cctgccctta catctcgtag aggcacagta cgctttagta ggttaggata ttattacact 18960

gcaccgccct gcccttacat ctcgtagagg cacagtacgc tttagtaggt taggtcaaaa 19020

ggccacccgt agcactcgta gtggcaactc gtagaggcac agtacgcttt agtaggttag 19080

gtcaaaaggc cacccgtagc actcgtagtg gcaaacaaat tggtgctcgt gtacattatt 19140

atcatgacgt agaggcacag tacgctttag taggttaggt caaaaggcca cccgtagcac 19200

tcgtagtggc aaacaaattg gtgctcgtgt acattattat ggtcaaaagg ccacccgtag 19260

cactcgtagt ggcaaacaaa ttggtgctcg tgtacattat tatcatgata ttagtagaat 19320

tgcaccagct gatgggtcaa aaggccaccc gtagcactcg tagtggcaaa caaattggtg 19380

ctcgtgtaca ttattatcat gatattagta gaattgcacc agctgatgaa cttggtggca 19440

aacaaattgg tgctcgtgta cattattatc atgatattag tagaattgca ccagctgatg 19500

aacttgaacg taagccttta ctttcacctt ctaaacaaat tggtgctcgt gtacattatt 19560

atcatgatat tagtagaatt gcaccagctg atgaacttga acgtaagcct ttactttcac 19620

cttctaataa ttatatcatg atattagtag aattgcacca gctgatgaac ttgaacgtaa 19680

gcctttactt tcaccttcta ataattatag ttatgacatt tcgtatgatt atattagtag 19740

aattgcacca gctgatgaac ttgaacgtaa gcctttactt tcaccttcta ataattatag 19800

ttatgacatt tcgtatgatt tagatgaagc gaacttgaac gtaagccttt actttcacct 19860

tctaataatt atagttatga catttcgtat gatttagatg aagctgaaac aggttttata 19920

cagccaacgt aagcctttac tttcaccttc taataattat agttatgaca tttcgtatga 19980

tttagatgaa gctgaaacag gttttataca gcccacacac accacgttat gacatttcgt 20040

atgatttaga tgaagctgaa acaggtttta tacagcccac acacaccaca cctatgtcac 20100

actcctcttt gtctaggcag ttgcccacct atgtcacact cctctttgtc taggcagttg 20160

ccctccttat cttcatctat gtcttcatct tcgtaaaatg ttactattcc attttcaact 20220

acatatgccc tccttatctt catctatgtc ttcatcttcg taaaatgtta ctattccatt 20280

ttcaactaca tattctgttc ctattcatac agggcctgat gtggtcgtaa aatgttacta 20340

ttccattttc aactacatat tctgttccta ttcatacagg gcctgatgtg gtattgccca 20400

catctcctac agtatggcct tatgtattct gttcctattc atacagggcc tgatgtggta 20460

ttgcccacat ctcctacagt atggccttat gttccccaca cttccattga caccaagcat 20520

tctatattgc ccacatctcc tacagtatgg ccttatgttc cccacacttc cattgacacc 20580

aagcattcta ttgttatact aggtggggat tactatttgt ggcccttccc cacacttcca 20640

ttgacaccaa gcattctatt gttatactag gtggggatta ctatttgtgg ccctatacac 20700

atttactacg caaacgccgt aaacgattga caccaagcat tctattgtta tactaggtgg 20760

ggattactat ttgtggccct atacacattt actacgcaaa cgccgtaaac gtatacccta 20820

tttttgttat actaggtggg gattactatt tgtggcccta tacacattta ctacgcaaac 20880

gccgtaaacg tataccctat ttttttacag atggcattgt ggcgcgggga ttactatttg 20940

tggccctata cacatttact acgcaaacgc cgtaaacgta taccctattt ttttacagat 21000

ggcattgtgg cgcactaatg acagccctat acacatttac tacgcaaacg ccgtaaacgt 21060

ataccctatt tttttacaga tggcattgtg gcgcactaat gacagcaagg tgtatttgcc 21120

acctgtacgc aaacgccgta aacgtatacc ctattttttt acagatggca ttgtggcgca 21180

ctaatgacag caaggtgtat ttgccacctg cacctgtgtc cgtataccct atttttttac 21240

agatggcatt gtggcgcact aatgacagca aggtgtattt gccacctgca cctgtgtctc 21300

gaattgtgaa tacagaagaa cgcactaatg acagcaaggt gtatttgcca cctgcacctg 21360

tgtctcgaat tgtgaataca gaagaatata tcacacgcac cggcatatat tactcgtaag 21420

gcacctgtgt ctcgaattgt gaatacagaa gaatatatca cacgcaccgg catatattac 21480

tcgtaaggca gttccagact aataacatta ggacatccct atatcacacg caccggcata 21540

tattactcgt aaggcagttc cagactaata acattaggac atccctattt tccaatacct 21600

aaaacctcaa cgcgtgctgc ggcagttcca gactaataac attaggacat ccctattttc 21660

caatacctaa aacctcaacg cgtgctgcta ttcctaaagt atctgcattt caatacaggg 21720

ctattttcca atacctaaaa cctcaacgcg tgctgctatt cctaaagtat ctgcatttca 21780

atacagggta tttagggtac agttaccaga tcctaacaag gctattccta aagtatctgc 21840

atttcaatac agggtattta gggtacagtt accagatcct aacaagtttg gactcccgga 21900

tccaaattta tataatccag gtatttaggg tacagttacc agatcctaac aagtttggac 21960

tcccggatcc aaatttatat aatccagaca cagataggtt ggtgtggggt tgtgtgggcg 22020

tttggactcc cggatccaaa tttatataat ccagacacag ataggttggt gtggggttgt 22080

gtgggcgttg aggtgggcag aggacagccc cttggtgttg agacacagat aggttggtgt 22140

ggggttgtgt gggcgttgag gtgggcagag gacagcccct tggtgttggc cttagtggtc 22200

atcccttatt taataaatat cgttgaggtg ggcagaggac agccccttgg tgttggcctt 22260

agtggtcatc ccttatttaa taaatatgat gacacagaaa attcacgcat agcaaatggc 22320

ggccttagtg gtcatccctt atttaataaa tatgatgaca cagaaaattc acgcatagca 22380

aatggcacgt aacaacaaga tgttagagat aacacatctg gatgacacag aaaattcacg 22440

catagcaaat ggcacgtaac aacaagatgt tagagataac acatctgttg acaacaaaca 22500

gactcagtta tgtataatag gcacgtaaca acaagatgtt agagataaca catctgttga 22560

caacaaacag actcagttat gtataatagg ctgtgctcca cctattgggg aacactgggg 22620

gttgacaaca aacagactca gttatgtata ataggctgtg ctccacctat tggggaacac 22680

tggggtattg gcactaccgt aaaaaacaca cctgtacctc ggctgtgctc cacctattgg 22740

ggaacactgg ggtattggca ctaccgtaaa aaacacacct gtacctccag gagactgccc 22800

ccccctggaa cttgtatcct ttggcactac cgtaaaaaac acacctgtac ctccaggaga 22860

ctgccccccc ctggaacttg tatcctctgt cattcaggat ggcgatatga ttgatacagg 22920

tccaggagac tgcccccccc tggaacttgt atcctctgtc attcaggatg gcgatatgat 22980

tgatacaggg tttggagcta tggatttcgc tgccctacag tctgtcattc aggatggcga 23040

tatgattgat acagggtttg gagctatgga tttcgctgcc ctacaggcca ccaaatcaga 23100

cgtccctttg gatatttcac cagggtttgg agctatggat ttcgctgccc tacaggccac 23160

caaatcagac gtccctttgg atatttcaca gtctgtttgt aaatatcctg attatttaag 23220

ggtttggagc tatggatttc gctgccctac aggccaccaa atcagacgtc cctttggata 23280

tttcacagtc tgtttgtaaa tatcctgatt atttaaaaag gccaccaaat cagacgtccc 23340

tttggatatt tcacagtctg tttgtaaata tcctgattat ttaaaaatgt ctgcagacac 23400

atatggtaat tccatgtttc agtctgtttg taaatatcct gattatttaa aaatgtctgc 23460

agacacatat ggtaattcca tgttttttca tttacgcagg gagcaaatct ttgctaggca 23520

tcctgattat ttaaaaatgt ctgcagacac atatggtaat tccatgtttt ttcatttacg 23580

cagggagcaa atctttgcta ggcactatta taataaatgt ctgcagacac atatggtaat 23640

tccatgtttt ttcatttacg cagggagcaa atctttgcta ggcactatta taataaactt 23700

gtaggtgttg gggaagttca tttacgcagg gagcaaatct ttgctaggca ctattataat 23760

aaacttgtag gtgttgggga agacattcct aacgattatt atattaaggg tagtgggaca 23820

ttcctaacga ttattatatt aagggtagtg gtaatggccg tgaccctata gaaagttata 23880

tatactctgc tactcccagt gggtctatga taacatggta atggccgtga ccctatagaa 23940

agttatatat actctgctac tcccagtggg tctatgataa catctgattc tcaaattttt 24000

aataagcctt attggcctct gctactccca gtgggtctat gataacatct gattctcaaa 24060

tttttaataa gccttattgg ctccaccgtg cgcagggtca caataatggc atttgccagt 24120

gggtctatga taacatctga ttctcaaatt tttaataagc cttattggct ccaccgtgcg 24180

cagggtcaca ataatggcat ttgctggaac aatcagtctg attctcaaat ttttaataag 24240

ccttattggc tccaccgtgc gcagggtcac aataatggca tttgctggaa caatcagctt 24300

tttattacct gtgttgtttt aataagcctt attggctcca ccgtgcgcag ggtcacaata 24360

atggcatttg ctggaacaat cagcttttta ttacctgtgt tgatactacg ctccaccgtg 24420

cgcagggtca caataatggc atttgctgga acaatcagct ttttattacc tgtgttgata 24480

ctaccagaag tacaaattta actattagcg ggtcacaata atggcatttg ctggaacaat 24540

cagcttttta ttacctgtgt tgatactacc agaagtacaa atttaactat tagcactgcg 24600

ctggaacaat cagcttttta ttacctgtgt tgatactacc agaagtacaa atttaactat 24660

tagcactgcc actgctgcgg tttccccaac atttactccg atactaccag aagtacaaat 24720

ttaactatta gcactgccac tgctgcggtt tccccaacat ttactccaag taactttaag 24780

caatatatta ggcatggggg cactgccact gctgcggttt ccccaacatt tactccaagt 24840

aactttaagc aatatattag gcatggggaa gagtatgaat tgcaatttat ttttcaattc 24900

caagtaactt taagcaatat attaggcatg gggaagagta tgaattgcaa tttatttttc 24960

aattatgtaa aattacttta actacagagg taatggcttg aagagtatga attgcaattt 25020

atttttcaat tatgtaaaat tactttaact acagaggtaa tggcttattt acacacaatg 25080

gatcctacca ttcttgaacg taaaattact ttaactacag aggtaatggc ttatttacac 25140

acaatggatc ctaccattct tgaacagtgg aattttggat taacattacc tccgtctgct 25200

atttacacac aatggatcct accattcttg aacagtggaa ttttggatta acattacctc 25260

cgtctgctag tttggaggcg taatataggt ttgttagaac agtggaattt tggattaaca 25320

ttacctccgt ctgctagttt ggaggcgtaa tataggtttg ttagaacgta agctactagc 25380

tgtcaaaagg acacccctcg gattaacatt acctccgtct gctagtttgg aggcgtaata 25440

taggtttgtt agaacgtaag ctactagctg tcaaaaggac acccctccac aggctaagct 25500

agtttggagg cgtaatatag gtttgttaga acgtaagcta ctagctgtca aaaggacacc 25560

cctccacagg ctaagccaga tcctttggcc aaatatacgt aagctactag ctgtcaaaag 25620

gacacccctc cacaggctaa gccagatcct ttggccaaat ataaattttg ggatgttgat 25680

ttaaaggaac gattttccca caggctaagc cagatccttt ggccaaatat aaattttggg 25740

atgttgattt aaaggaacga ttttctttag atttagacca atttgcattg ggtcgcaatt 25800

ttgggatgtt gatttaaagg aacgattttc tttagattta gaccaatttg cattgggtcg 25860

caagtttttg ttgcaggttg gcgtacaacg caagcccaag tttttgttgc aggttggcgt 25920

acaacgcaag cccagaccag gccttaaacg cccggcctca tcggcatcct cttcctcttc 25980

ctcttcagcc aaacgtaccc agaccaggcc ttaaacgccc ggcctcatcg gcatcctctt 26040

cctcttcctc ttcagccaaa cgtaaacgtg ttaaaaagta atgtatgtta gtttttgggc 26100

atcctcttcc tcttcctctt cagccaaacg taaacgtgtt aaaaagtaat gtatgttagt 26160

ttttgtcgta ttgtgcacac tgttgtcgta ctgtatgaac gtgttaaaaa gtaatgtatg 26220

ttagtttttg tcgtattgtg cacactgttg tcgtactgta tgtatatgtt tgtgtatgta 26280

ctgtatgtgt ttttgtggtc gtattgtgca cactgttgtc gtactgtatg tatatgtttg 26340

tgtatgtact gtatgtgttt ttgtgtgtgt gtgtgttgtt gttcctgtat gtatgagata 26400

tgtttgtgta tgtactgtat gtgtttttgt gtgtgtgtgt gttgttgttc ctgtatgtat 26460

gagttatgta tgtttattat taataaacta tgtggtgtgt gtgtgtgtgt tgttgttcct 26520

gtatgtatga gttatgtatg tttattatta ataaactatg tggtgtgtgt gtgtgtgttt 26580

ttgcatgact gcatttggtt atgtatgttt attattaata aactatgtgg tgtgtgtgtg 26640

tgtgtttttg catgactgca tttgtatgac atgtacgggt gtatgtgggt attacatgtg 26700

tgtgtgtgtg tttttgcatg actgcatttg tatgacatgt acgggtgtat gtgggtatta 26760

cattatcccc gtaggtcaag ggtggtgttt cggtggcgtg tgtgtgtttt tgcatgactg 26820

catttgtatg acatgtacgg gtgtatgtgg gtattacatt atccccgtag gtcaagggtg 26880

gtgtttcggt ggcgtcccgt atgacatgta cgggtgtatg tgggtattac attatccccg 26940

taggtcaagg gtggtgtttc ggtggcgtcc ctattgccct acccattttt tgcagcactt 27000

atccccgtag gtcaagggtg gtgtttcggt ggcgtcccta ttgccctacc cattttttgc 27060

agcacaacag tttatatttg tgctatttag ttatacttgg cgtccctatt gccctaccca 27120

ttttttgcag cacaacagtt tatatttgtg ctatttagtt atactttgta gcttccattt 27180

tgttacagct gcagccatca acagtttata tttgtgctat ttagttatac tttgtagctt 27240

ccattttgtt acagctgcag ccattttgag tgcaaccgat ttcggttcgt gtactttttt 27300

gtagcttcca ttttgttaca gctgcagcca ttttgagtgc aaccgatttc ggttcgtgta 27360

cttttagtat atttgccaag ttttaaacca caactgcctt gagtgcaacc gatttcggtt 27420

cgtgtacttt tagtatattt gccaagtttt aaaccacaac tgccagttgt ttttggcata 27480

aaccatcatt tttttatgag tatatttgcc aagttttaaa ccacaactgc cagttgtttt 27540

tggcataaac catcattttt ttatgacata gtgcatacat ccgcccgccc acgccttgca 27600

gttgtttttg gcataaacca tcattttttt atgacatagt gcatacatcc gcccgcccac 27660

gccttgtact tggcgcgcct taccggcgct agtcatacga catagtgcat acatccgccc 27720

gcccacgcct tgtacttggc gcgccttacc ggcgctagtc atacaaccta ttagtcattt 27780

gtactttaac aattgttgtg tacttggcgc gccttaccgg cgctagtcat acaacctatt 27840

agtcatttgt actttaacaa ttgttggcac actgttttcc gccctataat aatttaacca 27900

acctattagt catttgtact ttaacaattg ttggcacact gttttccgcc ctataataat 27960

ttaactgctt ataggcatgt attttttggc atattttatg gcacactgtt ttccgcccta 28020

taataattta actgcttata ggcatgtatt ttttggcata ttttatctta ctaattgcat 28080

agttggcagg tcaaatacct gcttataggc atgtattttt tggcatattt tatcttacta 28140

attgcatagt tggcaggtca aatactatgt ttttagtgcc aagtttctat cctacttatc 28200

ttactaattg catagttggc aggtcaaata ctatgttttt agtgccaagt ttctatccta 28260

cttataaacc atcttactca tcgtaaggtg tgctacacct atgtttttag tgccaagttt 28320

ctatcctact tataaaccat cttactcatc gtaaggtgtg ctacacaaat gtgttaccta 28380

accgatttgt gttctgcc 28398

<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 (7)

1. A probe for detecting human papillomavirus HPV51, characterized by comprising the following probes: SEQ ID NO. 1.

2. A kit for detecting human papillomavirus HPV51, characterized in that it comprises the probe of claim 1.

3. The kit of claim 2, wherein the kit further comprises a pooling buffer 1, a pooling 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, a positive control, a negative control, a capture buffer, a wash buffer 1, a wash buffer 2, and capture magnetic beads.

4. The kit according to claim 3, wherein the blocking sequence 1 is Cot-1 DNA.

5. The kit of claim 4, wherein the blocking sequence 2 is a double-stranded oligonucleotide.

6. A method for detecting human papillomavirus HPV51 using the probe of claim 1, characterized in that it comprises the following steps:

(1) extracting sample DNA, fragmenting, constructing a DNA library,

(2) hybridizing the probe of claim 1 with the library to perform target capture and quantification of the target region,

(3) and a high-throughput sequencing method,

(4) and bioinformatic analysis of gene sequences.

7. The use of the probe of claim 1 for human papillomavirus HPV detection of non-diagnostic non-therapeutic interest.

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