CN112195281A - Probe for detecting human papilloma virus HPV56 and kit thereof - Google Patents

Abstract

The invention discloses a probe for detecting human papillomavirus HPV56 and a kit thereof, wherein the probe group comprises a probe capable of specifically capturing HPV56 genome; the invention has the advantages of comprehensive detection range, greatly improved detection efficiency and accurate result.

Description

Probe for detecting human papilloma virus HPV56 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 HPV56 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 HPV56, and the probe information is shown in Table 1

The invention also provides a kit for detecting typing and integration of human papillomavirus HPV 56. The kit comprises the probe for detecting typing and integration of human papillomavirus HPV 56; 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 HPV56 and a kit thereof, which mainly comprises the following steps:

(1) design of capture probes for HPV56

(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 HPV56 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 HPV56 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 specific 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 HPV56 and a kit thereof

<130> 0

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 33541

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe type HPV56

<400> 1

gaaagtttca atcatacttt tatatattgg gagtgaccga aaagggttta agaccgaaaa 60

cggtacatat aaaaggcagc ttattctgtg tggacatatc aaacggtaca tataaaaggc 120

agcttattct gtgtggacat atccatggag ccacaattca acaatccaca ggaacgtcca 180

cgaagcctgc accacttgag gtggacatat ccatggagcc acaattcaac aatccacagg 240

aacgtccacg aagcctgcac cacttgagtg aggtattaga aataccttta attgatctta 300

atatccatgg agccacaatt caacaatcca caggaacgtc cacgaagcct gcaccacttg 360

agtgaggtat tagaaatacc tttaattgat cttagattac cacaggaacg tccacgaagc 420

ctgcaccact tgagtgaggt attagaaata cctttaattg atcttagatt atcatgtgta 480

tattgcaaaa aagaactaag gaacgtccac gaagcctgca ccacttgagt gaggtattag 540

aaataccttt aattgatctt agattatcat gtgtatattg caaaaaagaa ctagtgaggt 600

attagaaata cctttaattg atcttagatt atcatgtgta tattgcaaaa aagaactaac 660

acgtgctgag gtatataatt ttgccgtaac tgagattatc atgtgtatat tgcaaaaaag 720

aactaacacg tgctgaggta tataattttg ccgtaactga attaaaatta gtgtataggg 780

atgattttcc ttcacgtgct gaggtatata attttgccgt aactgaatta aaattagtgt 840

atagggatga ttttccttcg taagtgtgca gagtatgttt attgttttat aggaattaaa 900

attagtgtat agggatgatt ttccttcgta agtgtgcaga gtatgtttat tgttttatag 960

taaagttaga aaatataggt attatgacta tttcgtaagt gtgcagagta tgtttattgt 1020

tttatagtaa agttagaaaa tataggtatt atgactattc agtgtatgga gctacactag 1080

aaagtataac tagtatgttt attgttttat agtaaagtta gaaaatatag gtattatgac 1140

tattcagtgt atggagctac actagaaagt ataactaaaa aacagtaaag ttagaaaata 1200

taggtattat gactattcag tgtatggagc tacactagaa agtataacta aaaaacagtt 1260

atgtgattta ttaataaggt gcttcagtgt atggagctac actagaaagt ataactaaaa 1320

aacagttatg tgatttatta ataaggtgct acagatgtca aagtccgtta actccggagg 1380

aaaaaaaaac agttatgtga tttattaata aggtgctaca gatgtcaaag tccgttaact 1440

ccggaggaaa agcaattgca ttgtgacaga aaaagacgat ttcgctacag atgtcaaagt 1500

ccgttaactc cggaggaaaa gcaattgcat tgtgacagaa aaagacgatt tcatctaata 1560

gcacatggtt ggaccgggtc atgagcaatt gcattgtgac agaaaaagac gatttcatct 1620

aatagcacat ggttggaccg ggtcatgttt ggggtgctgg agacaaacat ctagagaacc 1680

tagcatctaa tagcacatgg ttggaccggg tcatgtttgg ggtgctggag acaaacatct 1740

agagaaccta gagaatctac agtataatcc gtaatggtaa agtttggggt gctggagaca 1800

aacatctaga gaacctagag aatctacagt ataatccgta atggtaaagt accaacgctg 1860

caagacgttg tattagaact aacgagaatc tacagtataa tccgtaatgg taaagtacca 1920

acgctgcaag acgttgtatt agaactaaca cctcaaacag aaattgacct acagtgcaat 1980

gagctacagt ataatccgta atggtaaagt accaacgctg caagacgttg tattagaact 2040

aacacctcaa acagaaattg acctacagtg caatgagcaa ttggtaccaa cgctgcaaga 2100

cgttgtatta gaactaacac ctcaaacaga aattgaccta cagtgcaatg agcaattgga 2160

cagctcagag gatgaggatg aggcgctgca agacgttgta ttagaactaa cacctcaaac 2220

agaaattgac ctacagtgca atgagcaatt ggacagctca gaggatgagg atgaggatga 2280

agtcacctca aacagaaatt gacctacagt gcaatgagca attggacagc tcagaggatg 2340

aggatgagga tgaagtagac catttgcagg agcggccaca gcaaaacaga aattgaccta 2400

cagtgcaatg agcaattgga cagctcagag gatgaggatg aggatgaagt agaccatttg 2460

caggagcggc cacagcaagc tagattgacc tacagtgcaa tgagcaattg gacagctcag 2520

aggatgagga tgaggatgaa gtagaccatt tgcaggagcg gccacagcaa gctagacgag 2580

caattggaca gctcagagga tgaggatgag gatgaagtag accatttgca ggagcggcca 2640

cagcaagcta gacaagctaa acaacatacg tgttaccgga cagctcagag gatgaggatg 2700

aggatgaagt agaccatttg caggagcggc cacagcaagc tagacaagct aaacaacata 2760

cgtgttacct aatacaccag ctcagaggat gaggatgagg atgaagtaga ccatttgcag 2820

gagcggccac agcaagctag acaagctaaa caacatacgt gttacctaat gaagtagacc 2880

atttgcagga gcggccacag caagctagac aagctaaaca acatacgtgt tacctaatac 2940

acgtaccttg ttgtgagtgt aagtttgtgg agaccatttg caggagcggc cacagcaagc 3000

tagacaagct aaacaacata cgtgttacct aatacacgta ccttgttgtg agtgtaagtt 3060

tgtggtgcag gctagacaag ctaaacaaca tacgtgttac ctaatacacg taccttgttg 3120

tgagtgtaag tttgtggtgc agttggacat tcagagtacc aaagaggacc gacaagctaa 3180

acaacatacg tgttacctaa tacacgtacc ttgttgtgag tgtaagtttg tggtgcagtt 3240

ggacattcag agtaccaaag aggaaataca cgtaccttgt tgtgagtgta agtttgtggt 3300

gcagttggac attcagagta ccaaagagga cctgcgtgtt gtacaacagc tgcttatggg 3360

tgcgacgtac cttgttgtga gtgtaagttt gtggtgcagt tggacattca gagtaccaaa 3420

gaggacctgc gtgttgtaca acagctgctt atgggtgcgt taacgcagtt ggacattcag 3480

agtaccaaag aggacctgcg tgttgtacaa cagctgctta tgggtgcgtt aacagtaacg 3540

tgcccactct gcgcatcaac ctgcgtgttg tacaacagct gcttatgggt gcgttaacag 3600

taacgtgccc actctgcgca tcaagtaact aactgcaatg gcgtcacctg aaggtacagt 3660

gttgtacaac agctgcttat gggtgcgtta acagtaacgt gcccactctg cgcatcaagt 3720

aactaactgc aatggcgtca cctgaaggta cagatgggcg ttaacagtaa cgtgcccact 3780

ctgcgcatca agtaactaac tgcaatggcg tcacctgaag gtacagatgg ggaggggaag 3840

ggatgttgtg gatggtttca gtaacgtgcc cactctgcgc atcaagtaac taactgcaat 3900

ggcgtcacct gaaggtacag atggggaggg gaagggatgt tgtggatggt ttgtgcccac 3960

tctgcgcatc aagtaactaa ctgcaatggc gtcacctgaa ggtacagatg gggaggggaa 4020

gggatgttgt ggatggtttg aagtagaggc aagtaactaa ctgcaatggc gtcacctgaa 4080

ggtacagatg gggaggggaa gggatgttgt ggatggtttg aagtagaggc aattgtagaa 4140

aaaaaaacag gaaactgcaa tggcgtcacc tgaaggtaca gatggggagg ggaagggatg 4200

ttgtggatgg tttgaagtag aggcaattgt agaaaaaaaa acgatgggga ggggaaggga 4260

tgttgtggat ggtttgaagt agaggcaatt gtagaaaaaa aaacaggaga taaaatatca 4320

gatgatgaaa gtgacgagga gggaagtaga ggcaattgta gaaaaaaaaa caggagataa 4380

aatatcagat gatgaaagtg acgaggagga tgaaatagat acagatttag atggatttat 4440

aggataaaat atcagatgat gaaagtgacg aggaggatga aatagataca gatttagatg 4500

gatttataga cgattcatat atacaaaata tacaggcaga cgggatgaaa tagatacaga 4560

tttagatgga tttatagacg attcatatat acaaaatata caggcagacg cagaaacagc 4620

tcaacaattg ttgcaagtac aaatagatac agatttagat ggatttatag acgattcata 4680

tatacaaaat atacaggcag acgcagaaac agctcaacaa ttgttgcaag tacaaacagc 4740

acgacgattc atatatacaa aatatacagg cagacgcaga aacagctcaa caattgttgc 4800

aagtacaaac agcaccgtaa gataaacaga cgttgcaaaa accgcagaaa cagctcaaca 4860

attgttgcaa gtacaaacag caccgtaaga taaacagacg ttgcaaaaac taaaacgaaa 4920

gtatatagct agtccattaa ggacagctca acaattgttg caagtacaaa cagcaccgta 4980

agataaacag acgttgcaaa aactaaaacg aaagtatata gctagtccat taagggatat 5040

tcagcaccgt aagataaaca gacgttgcaa aaactaaaac gaaagtatat agctagtcca 5100

ttaagggata ttagtaatca gcaaactgtg tgccgggaag gctaaaacga aagtatatag 5160

ctagtccatt aagggatatt agtaatcagc aaactgtgtg ccgggaagga gtaaaacgga 5220

ggcttatttt atcagaccta cacgaaagta tatagctagt ccattaaggg atattagtaa 5280

tcagcaaact gtgtgccggg aaggagtaaa acggaggctt attttatcag acctacaaga 5340

cagcgggagt ccattaaggg atattagtaa tcagcaaact gtgtgccggg aaggagtaaa 5400

acggaggctt attttatcag acctacaaga cagcgggtat ggcgggatat tagtaatcag 5460

caaactgtgt gccgggaagg agtaaaacgg aggcttattt tatcagacct acaagacagc 5520

gggtatggca atacattgga aacagtaatc agcaaactgt gtgccgggaa ggagtaaaac 5580

ggaggcttat tttatcagac ctacaagaca gcgggtatgg caatacattg gaaactctgg 5640

aaacaggagt aaaacggagg cttattttat cagacctaca agacagcggg tatggcaata 5700

cattggaaac tctggaaaca ccagaacagg tagatgaaga ggtaccaaga cagcgggtat 5760

ggcaatacat tggaaactct ggaaacacca gaacaggtag atgaagaggt acagggacgt 5820

gggtgcggga atacacaaaa tggagctctg gaaacaccag aacaggtaga tgaagaggta 5880

cagggacgtg ggtgcgggaa tacacaaaat ggaggctcac aaaacagtac ctatagtaac 5940

aatagacagg gacgtgggtg cgggaataca caaaatggag gctcacaaaa cagtacctat 6000

agtaacaata gtgaggactc tgtaatacat atggatattg atagaaggct cacaaaacag 6060

tacctatagt aacaatagtg aggactctgt aatacatatg gatattgata gaaacaatga 6120

aacgccaaca caacaattgc aggacgtgag gactctgtaa tacatatgga tattgataga 6180

aacaatgaaa cgccaacaca acaattgcag gacttgttta aaagtagcaa tttacaaggt 6240

aaattgaaac aatgaaacgc caacacaaca attgcaggac ttgtttaaaa gtagcaattt 6300

acaaggtaaa ttatattata aatttaaaga agtgtatggt attcccttgt ttaaaagtag 6360

caatttacaa ggtaaattat attataaatt taaagaagtg tatggtattc cattttcaga 6420

attggtgcgt acgtttaaaa gtgatatatt ataaatttaa agaagtgtat ggtattccat 6480

tttcagaatt ggtgcgtacg tttaaaagtg atagtacatg ttgcaatgat tggatatgtg 6540

ctataccatt ttcagaattg gtgcgtacgt ttaaaagtga tagtacatgt tgcaatgatt 6600

ggatatgtgc tatatttggt gttaatgaaa cattagccga ggcacagtac atgttgcaat 6660

gattggatat gtgctatatt tggtgttaat gaaacattag ccgaggcact aaaaactata 6720

ataaaaccac actgtatgta ttatcatttg gtgttaatga aacattagcc gaggcactaa 6780

aaactataat aaaaccacac tgtatgtatt atcatcgtaa atgtttaaca tgtacatggg 6840

gggtttaaaa actataataa aaccacactg tatgtattat catcgtaaat gtttaacatg 6900

tacatggggg gttatagtaa tgcgtataat tagataaaaa ctataataaa accacactgt 6960

atgtattatc atcgtaaatg tttaacatgt acatgggggg ttatagtaat gcgtataatt 7020

agatatacat gtggcgtatg tattatcatc gtaaatgttt aacatgtaca tggggggtta 7080

tagtaatgcg tataattaga tatacatgtg gcaaaaacag aaaaacaatt gcaaatcatc 7140

gtaaatgttt aacatgtaca tggggggtta tagtaatgcg tataattaga tatacatgtg 7200

gcaaaaacag aaaaacaatt gcaaaagcat taagctatag taatgcgtat aattagatat 7260

acatgtggca aaaacagaaa aacaattgca aaagcattaa gctcaatatt aaatgtacca 7320

caggagcaaa tgttaatgtg gcaaaaacag aaaaacaatt gcaaaagcat taagctcaat 7380

attaaatgta ccacaggagc aaatgttaat tcaaccacca aaaatacgaa gtccggcaaa 7440

aacagaaaaa caattgcaaa agcattaagc tcaatattaa atgtaccaca ggagcaaatg 7500

ttaattcaac caccaaaaat acgaagtcct gctgtaagct caatattaaa tgtaccacag 7560

gagcaaatgt taattcaacc accaaaaata cgaagtcctg ctgtagcttt atatttttat 7620

aaaacagcag ctcaatatta aatgtaccac aggagcaaat gttaattcaa ccaccaaaaa 7680

tacgaagtcc tgctgtagct ttatattttt ataaaacagc aatgtcaaag caaatgttaa 7740

ttcaaccacc aaaaatacga agtcctgctg tagctttata tttttataaa acagcaatgt 7800

caaatattag tgatgtgtat ggaattcaac caccaaaaat acgaagtcct gctgtagctt 7860

tatattttta taaaacagca atgtcaaata ttagtgatgt gtatggagac acaccagaat 7920

ggccaccaaa aatacgaagt cctgctgtag ctttatattt ttataaaaca gcaatgtcaa 7980

atattagtga tgtgtatgga gacacaccag aatggcctgc tgtagcttta tatttttata 8040

aaacagcaat gtcaaatatt agtgatgtgt atggagacac accagaatgg atacaaagac 8100

aaacacaatt gcaacgcttt atatttttat aaaacagcaa tgtcaaatat tagtgatgtg 8160

tatggagaca caccagaatg gatacaaaga caaacacaat tgcaacacag tttaccaatg 8220

tcaaatatta gtgatgtgta tggagacaca ccagaatgga tacaaagaca aacacaattg 8280

caacacagtt tacaggatag tcaatttgaa ttatcattag tgatgtgtat ggagacacac 8340

cagaatggat acaaagacaa acacaattgc aacacagttt acaggatagt caatttgaat 8400

tatctaaaat ggtgcgtgta tggagacaca ccagaatgga tacaaagaca aacacaattg 8460

caacacagtt tacaggatag tcaatttgaa ttatctaaaa tggtgcagtg ggcacaccag 8520

aatggataca aagacaaaca caattgcaac acagtttaca ggatagtcaa tttgaattat 8580

ctaaaatggt gcagtgggca tttgataatg aagtggatac aaagacaaac acaattgcaa 8640

cacagtttac aggatagtca atttgaatta tctaaaatgg tgcagtgggc atttgataat 8700

gaagtaacag atgacacagt ttacaggata gtcaatttga attatctaaa atggtgcagt 8760

gggcatttga taatgaagta acagatgata gccaaattgc gtttcaatcg taaccaggat 8820

agtcaatttg aattatctaa aatggtgcag tgggcatttg ataatgaagt aacagatgat 8880

agccaaattg cgtttcaatc gtaacaatta gcagcaattt gaattatcta aaatggtgca 8940

gtgggcattt gataatgaag taacagatga tagccaaatt gcgtttcaat cgtaacaatt 9000

agcagatgta gacactaaaa tggtgcagtg ggcatttgat aatgaagtaa cagatgatag 9060

ccaaattgcg tttcaatcgt aacaattagc agatgtagac agcacgtaac aagcgcagtg 9120

ggcatttgat aatgaagtaa cagatgatag ccaaattgcg tttcaatcgt aacaattagc 9180

agatgtagac agcacgtaac aagccttttt aaaaaacaga tgatagccaa attgcgtttc 9240

aatcgtaaca attagcagat gtagacagca cgtaacaagc ctttttaaaa agcaatcgta 9300

aggcaaaata tgtagccaaa ttgcgtttca atcgtaacaa ttagcagatg tagacagcac 9360

gtaacaagcc tttttaaaaa gcaatcgtaa ggcaaaatat gtaaaggatt gtgggcgttt 9420

caatcgtaac aattagcaga tgtagacagc acgtaacaag cctttttaaa aagcaatcgt 9480

aaggcaaaat atgtaaagga ttgtggaata atgtgcacaa ttagcagatg tagacagcac 9540

gtaacaagcc tttttaaaaa gcaatcgtaa ggcaaaatat gtaaaggatt gtggaataat 9600

gtgtagacat tatattttta aaaagcaatc gtaaggcaaa atatgtaaag gattgtggaa 9660

taatgtgtag acattataaa agggcacaac agcaacaaat gaatatgtgc cagagcaatc 9720

gtaaggcaaa atatgtaaag gattgtggaa taatgtgtag acattataaa agggcacaac 9780

agcaacaaat gaatatgtgc cagtggataa agcggattgt ggaataatgt gtagacatta 9840

taaaagggca caacagcaac aaatgaatat gtgccagtgg ataaagcaca tatgtagtaa 9900

aacagatgaa ggggtggaat aatgtgtaga cattataaaa gggcacaaca gcaacaaatg 9960

aatatgtgcc agtggataaa gcacatatgt agtaaaacag atgaaggggg tgaacagcaa 10020

caaatgaata tgtgccagtg gataaagcac atatgtagta aaacagatga agggggtgat 10080

tggaaaccca ttgtacaatt tttaagcagc aacaaatgaa tatgtgccag tggataaagc 10140

acatatgtag taaaacagat gaagggggtg attggaaacc cattgtacaa tttttaagat 10200

atcaaggcac atatgtagta aaacagatga agggggtgat tggaaaccca ttgtacaatt 10260

tttaagatat caaggggtcg atttcatttc atttctaagt tactttaggg ggtgattgga 10320

aacccattgt acaattttta agatatcaag gggtcgattt catttcattt ctaagttact 10380

ttaaattatt tctacaagga acacctttgg aaacccattg tacaattttt aagatatcaa 10440

ggggtcgatt tcatttcatt tctaagttac tttaaattat ttctacaagg aacacctaaa 10500

cataacaggg gtcgatttca tttcatttct aagttacttt aaattatttc tacaaggaac 10560

acctaaacat aactgtttgg tactttgtgg accgccaaat acaggttaaa ttatttctac 10620

aaggaacacc taaacataac tgtttggtac tttgtggacc gccaaataca ggtaaatccg 10680

tatttgctat gagtcttata aagtttctgt ttggtacttt gtggaccgcc aaatacaggt 10740

aaatccgtat ttgctatgag tcttataaag ttttttcaag ggtctgtcat ttcatttgtg 10800

aattcacaaa tacaggtaaa tccgtatttg ctatgagtct tataaagttt tttcaagggt 10860

ctgtcatttc atttgtgaat tcacaaagcc acttttggtt gcagccggta aatccgtatt 10920

tgctatgagt cttataaagt tttttcaagg gtctgtcatt tcatttgtga attcacaaag 10980

ccacttttgg ttgcagccat tagacatttt caagggtctg tcatttcatt tgtgaattca 11040

caaagccact tttggttgca gccattagac acgtataaac ttgggttgtt ggatgcgtaa 11100

acagaaacaa agccactttt ggttgcagcc attagacacg tataaacttg ggttgttgga 11160

tgcgtaaaca gaaatatgtt ggaaatatat agacgattat ttaaggtgct aaacttgggt 11220

tgttggatgc gtaaacagaa atatgttgga aatatataga cgattattta aggaatttgg 11280

tagatggaaa tcctataagt ttagattatg ttggaaatat atagacgatt atttaaggaa 11340

tttggtagat ggaaatccta taagtttaga tagaaaacat aaacaattag tacaaataaa 11400

atgtccaagt ttagatagaa aacataaaca attagtacaa ataaaatgtc caccattact 11460

aattacaacc aatataaatc ctcgtatagc gtataaatta cgatattaga aaacataaac 11520

aattagtaca aataaaatgt ccaccattac taattacaac caatataaat cctcgtatag 11580

cgtataaatt acgatattta cacagtccac cattactaat tacaaccaat ataaatcctc 11640

gtatagcgta taaattacga tatttacaca gtagaatgtt agtgtttcag tttcaaaatc 11700

catttctaaa tcctcgtata gcgtataaat tacgatattt acacagtaga atgttagtgt 11760

ttcagtttca aaatccattt ccattagata ataatggtaa tcctgtttac acagtagaat 11820

gttagtgttt cagtttcaaa atccatttcc attagataat aatggtaatc ctgtatatga 11880

attaagtaat gtaaactgga aatgtgtaga atgttagtgt ttcagtttca aaatccattt 11940

ccattagata ataatggtaa tcctgtatat gaattaagta atgtaaactg gaaatgtttc 12000

tttacaatcc atttccatta gataataatg gtaatcctgt atatgaatta agtaatgtaa 12060

actggaaatg tttctttaca aggacgtggt ccagattaaa tttggccatt agataataat 12120

ggtaatcctg tatatgaatt aagtaatgta aactggaaat gtttctttac aaggacgtgg 12180

tccagattaa atttggataa cgacgtgtat atgaattaag taatgtaaac tggaaatgtt 12240

tctttacaag gacgtggtcc agattaaatt tggataacga cgaggacaaa gaaaacaatg 12300

gagacattaa gtaatgtaaa ctggaaatgt ttctttacaa ggacgtggtc cagattaaat 12360

ttggataacg acgaggacaa agaaaacaat ggagacgctt tcccaaagga cgtggtccag 12420

attaaatttg gataacgacg aggacaaaga aaacaatgga gacgctttcc caacgtttaa 12480

cgtagtgcca gaacaaggac gtggtccaga ttaaatttgg ataacgacga ggacaaagaa 12540

aacaatggag acgctttccc aacgtttaac gtagtgccag aacaaaatac tagacggtcc 12600

agattaaatt tggataacga cgaggacaaa gaaaacaatg gagacgcttt cccaacgttt 12660

aacgtagtgc cagaacaaaa tactagactg ttttgggata acgacgagga caaagaaaac 12720

aatggagacg ctttcccaac gtttaacgta gtgccagaac aaaatactag actgttttga 12780

aaaagatagt agatgcgagg acaaagaaaa caatggagac gctttcccaa cgtttaacgt 12840

agtgccagaa caaaatacta gactgttttg aaaaagatag tagatgtatt gcagatccca 12900

acgtttaacg tagtgccaga acaaaatact agactgtttt gaaaaagata gtagatgtat 12960

tgcagatcat atagaatatt ggaaagctgt caacgtttaa cgtagtgcca gaacaaaata 13020

ctagactgtt ttgaaaaaga tagtagatgt attgcagatc atatagaata ttggaaagct 13080

gtgcgacatg aaatactaga ctgttttgaa aaagatagta gatgtattgc agatcatata 13140

gaatattgga aagctgtgcg acatgaaaat gtgctatact ataaagcaag gactgttttg 13200

aaaaagatag tagatgtatt gcagatcata tagaatattg gaaagctgtg cgacatgaaa 13260

atgtgctata ctataaagca agagaaaatg agatcatata gaatattgga aagctgtgcg 13320

acatgaaaat gtgctatact ataaagcaag agaaaatgac attactgtac taaaccacca 13380

gatatcatat agaatattgg aaagctgtgc gacatgaaaa tgtgctatac tataaagcaa 13440

gagaaaatga cattactgta ctaaaccacc agatggtgcc ttgtgcgaca tgaaaatgtg 13500

ctatactata aagcaagaga aaatgacatt actgtactaa accaccagat ggtgccttgt 13560

ttacaagtat gtaaagcaaa agcaaaatgt gctatactat aaagcaagag aaaatgacat 13620

tactgtacta aaccaccaga tggtgccttg tttacaagta tgtaaagcaa aagcatgtag 13680

tgcgagaaaa tgacattact gtactaaacc accagatggt gccttgttta caagtatgta 13740

aagcaaaagc atgtagtgca atagaagtgc aaatagcact ggaacattac tgtactaaac 13800

caccagatgg tgccttgttt acaagtatgt aaagcaaaag catgtagtgc aatagaagtg 13860

caaatagcac tggaatcatt aaggtttaca agtatgtaaa gcaaaagcat gtagtgcaat 13920

agaagtgcaa atagcactgg aatcattaag tacaacaata tataacaatg aagagtggac 13980

attgcaatag aagtgcaaat agcactggaa tcattaagta caacaatata taacaatgaa 14040

gagtggacat taagagacac cgtagaggaa ctatggctta ctggtacaac aatatataac 14100

aatgaagagt ggacattaag agacaccgta gaggaactat ggcttactga acctaaaaac 14160

gtatttaaaa aagaaggaca acagagtgga cattaagaga caccgtagag gaactatggc 14220

ttactgaacc taaaaacgta tttaaaaaag aaggacaaca tatagaagta tggtttgatg 14280

gtataagaga caccgtagag gaactatggc ttactgaacc taaaaacgta tttaaaaaag 14340

aaggacaaca tatagaagta tggtttgatg gtagtaaaaa caagaaccta aaaacgtatt 14400

taaaaaagaa ggacaacata tagaagtatg gtttgatggt agtaaaaaca attgtcgtaa 14460

atatgtagcc tggaaatata tatatataga agtatggttt gatggtagta aaaacaattg 14520

tcgtaaatat gtagcctgga aatatatata ttacaatgga gattgtgggt ggcaaaaagt 14580

gtgattgtcg taaatatgta gcctggaaat atatatatta caatggagat tgtgggtggc 14640

aaaaagtgtg ttctggggta gactatagag gtatatatta tgtattacaa tggagattgt 14700

gggtggcaaa aagtgtgttc tggggtagac tatagaggta tatattatgt acatgatggc 14760

cacaaaacat actacacaga cttgttctgg ggtagactat agaggtatat attatgtaca 14820

tgatggccac aaaacatact acacagactt tgaacaagag gccaaaaaat ttgggtgtaa 14880

aaatattatg tacatgatgg ccacaaaaca tactacacag actttgaaca agaggccaaa 14940

aaatttgggt gtaaaaacat atgggaagta catatggaaa gtacatgatg gccacaaaac 15000

atactacaca gactttgaac aagaggccaa aaaatttggg tgtaaaaaca tatgggaagt 15060

acatatggaa aatgagagta acacagactt tgaacaagag gccaaaaaat ttgggtgtaa 15120

aaacatatgg gaagtacata tggaaaatga gagtatttat tgtcctgact ctgtgtctag 15180

ttgaacaaga ggccaaaaaa tttgggtgta aaaacatatg ggaagtacat atggaaaatg 15240

agagtattta ttgtcctgac tctgtgtcta gtacctgtag ggtgtaaaaa catatgggaa 15300

gtacatatgg aaaatgagag tatttattgt cctgactctg tgtctagtac ctgtagatac 15360

aacgtatccc ctgttgaaac acatatggga agtacatatg gaaaatgaga gtatttattg 15420

tcctgactct gtgtctagta cctgtagata caacgtatcc cctgttgaaa ctgttaacga 15480

gagagtattt attgtcctga ctctgtgtct agtacctgta gatacaacgt atcccctgtt 15540

gaaactgtta acgaatacaa cacccacaag accaccactt attgtcctga ctctgtgtct 15600

agtacctgta gatacaacgt atcccctgtt gaaactgtta acgaatacaa cacccacaag 15660

accaccacca ccacctccag atacaacgta tcccctgttg aaactgttaa cgaatacaac 15720

acccacaaga ccaccaccac cacctccacg tccgtgggca accaagacgc cgcagtattg 15780

ttaacgaata caacacccac aagaccacca ccaccacctc cacgtccgtg ggcaaccaag 15840

acgccgcagt atcccacaga ccaggaaaac gacccagaaa tacaacaccc acaagaccac 15900

caccaccacc tccacgtccg tgggcaacca agacgccgca gtatcccaca gaccaggaaa 15960

acgacccaga ctacgggacc accacctcca cgtccgtggg caaccaagac gccgcagtat 16020

cccacagacc aggaaaacga cccagactac gggaatcaga atttgactcc tccagagaac 16080

gtccgtgggc aaccaagacg ccgcagtatc ccacagacca ggaaaacgac ccagactacg 16140

ggaatcagaa tttgactcct ccagagagtc ccacgcaacc gcagtatccc acagaccagg 16200

aaaacgaccc agactacggg aatcagaatt tgactcctcc agagagtccc acgcaaagtg 16260

tgtcacaaca cacacacacc cacagaccag gaaaacgacc cagactacgg gaatcagaat 16320

ttgactcctc cagagagtcc cacgcaaagt gtgtcacaac acacacacac atcagcgaca 16380

aagtgtgtca caacacacac acacatcagc gacacagaca ataccgacag tagaagtaga 16440

agtatcaaca acaacaacca ccctggtgat aagactacac acagacaata ccgacagtag 16500

aagtagaagt atcaacaaca acaaccaccc tggtgataag actacgcctg tagtacattt 16560

aaaaggtgaa cctaacagtc aacaacaaca accaccctgg tgataagact acgcctgtag 16620

tacatttaaa aggtgaacct aacagattaa aatgttgtag atatcgattt caaaaatact 16680

acgcctgtag tacatttaaa aggtgaacct aacagattaa aatgttgtag atatcgattt 16740

caaaaatata aaacattgtt tgtggatgta acatcaaccg cctgtagtac atttaaaagg 16800

tgaacctaac agattaaaat gttgtagata tcgatttcaa aaatataaaa cattgtttgt 16860

ggatgtaaca tcaacataac agattaaaat gttgtagata tcgatttcaa aaatataaaa 16920

cattgtttgt ggatgtaaca tcaacatatc attggacaag tacagacaat aaagattaaa 16980

atgttgtaga tatcgatttc aaaaatataa aacattgttt gtggatgtaa catcaacata 17040

tcattggaca agtacagaca ataaaaatta tagaatataa aacattgttt gtggatgtaa 17100

catcaacata tcattggaca agtacagaca ataaaaatta tagcataatt acaattatat 17160

ataaggatga aacataaaac attgtttgtg gatgtaacat caacatatca ttggacaagt 17220

acagacaata aaaattatag cataattaca attatatata aggatgaaac acacatatca 17280

ttggacaagt acagacaata aaaattatag cataattaca attatatata aggatgaaac 17340

acaacgaaac agctttttaa gtcatgtaaa aattcattgg acaagtacag acaataaaaa 17400

ttatagcata attacaatta tatataagga tgaaacacaa cgaaacagct ttttaagtca 17460

tgtaaaaatt cccgcataat tacaattata tataaggatg aaacacaacg aaacagcttt 17520

ttaagtcatg taaaaattcc cagtagtgta caggttagtt tgggacaaat gagcacaacg 17580

aaacagcttt ttaagtcatg taaaaattcc cagtagtgta caggttagtt tgggacaaat 17640

gagttttcca taaagtgctg tatatattgt ataaacgaaa cagcttttta agtcatgtaa 17700

aaattcccag tagtgtacag gttagtttgg gacaaatgag ttttccataa agtgctgtat 17760

atattgtata tacccagtag tgtacaggtt agtttgggac aaatgagttt tccataaagt 17820

gctgtatata ttgtatatac atttgtgtta ttgtaacaca caaatacgtg aagtgagttt 17880

tccataaagt gctgtatata ttgtatatac atttgtgtta ttgtaacaca caaatacgtg 17940

aagtgtacct gccatacatt gctgctacgc atagttttcc ataaagtgct gtatatattg 18000

tatatacatt tgtgttattg taacacacaa atacgtgaag tgtacctgcc atacattgct 18060

gctacgcata tatcatttgt gttattgtaa cacacaaata cgtgaagtgt acctgccata 18120

cattgctgct acgcatatat attgcaacca ttgatttttg tgttattggt gtggtgtacc 18180

tgccatacat tgctgctacg catatatatt gcaaccattg atttttgtgt tattggtgtg 18240

tttgcgcttt gcttttgtgt ttgtttgctt gtgattgcaa ccattgattt ttgtgttatt 18300

ggtgtgtttg cgctttgctt ttgtgtttgt ttgcttgtgt gtcatgttgt cccgcttttg 18360

ctatctgcct ctggtttgcg ctttgctttt gtgtttgttt gcttgtgtgt catgttgtcc 18420

cgcttttgct atctgcctct gtgttttcca gttgtatatt attaataata ttggtgtcat 18480

gttgtcccgc ttttgctatc tgcctctgtg ttttccagtt gtatattatt aataatattg 18540

ttttggtttg ttatagccac atcctttttt aatgtgtttt ccagttgtat attattaata 18600

atattgtttt ggtttgttat agccacatcc ttttttaata catttataat atttttgata 18660

tttttttact gtcgttttgg tttgttatag ccacatcctt ttttaataca tttataatat 18720

ttttgatatt tttttactgt cctgtgctgt gtatatattt accgtatttg tggtacattt 18780

ataatatttt tgatattttt ttactgtcct gtgctgtgta tatatttacc gtatttgtgg 18840

ataataaata atatgtaaat gtagtagtac tgtcctgtgc tgtgtatata tttaccgtat 18900

ttgtggataa taaataatat gtaaatgtag tagtactgtt actactatgg ttgcccaccg 18960

tgccacacga cgcgataata aataatatgt aaatgtagta gtactgttac tactatggtt 19020

gcccaccgtg ccacacgacg caaacgcgca tctgcaacac aactatataa aacgttacta 19080

ctatggttgc ccaccgtgcc acacgacgca aacgcgcatc tgcaacacaa ctatataaaa 19140

catgtaagtt gtctggtaca tgtccagagg atggccacac gacgcaaacg cgcatctgca 19200

acacaactat ataaaacatg taagttgtct ggtacatgtc cagaggatgt tgttaataaa 19260

atagagcaaa aaacgcaaac gcgcatctgc aacacaacta tataaaacat gtaagttgtc 19320

tggtacatgt ccagaggatg ttgttaataa aatagagcaa aaaacatggg ctgtatataa 19380

aacatgtaag ttgtctggta catgtccaga ggatgttgtt aataaaatag agcaaaaaac 19440

atgggctgat aaaatattgc aatggggaag ttcatgtaag ttgtctggta catgtccaga 19500

ggatgttgtt aataaaatag agcaaaaaac atgggctgat aaaatattgc aatggggaag 19560

tttatttaca taccagagga tgttgttaat aaaatagagc aaaaaacatg ggctgataaa 19620

atattgcaat ggggaagttt atttacatat tttggaggcc ttggcattgg tatgttaata 19680

aaatagagca aaaaacatgg gctgataaaa tattgcaatg gggaagttta tttacatatt 19740

ttggaggcct tggcattggt acaggaactg ggtaaaatat tgcaatgggg aagtttattt 19800

acatattttg gaggccttgg cattggtaca ggaactgggt ctgggggtcg tgcaggctat 19860

gttccattgg ggggtctggg ggtcgtgcag gctatgttcc attggggtct aggccttcca 19920

caatagttga tgtaactccg gcgcgaccac ctattgttgt ggaatccgta gggggtctag 19980

gccttccaca atagttgatg taactccggc gcgaccacct attgttgtgg aatccgtagg 20040

gcctacagac ccttccattg ttacattagt tgcggcgcga ccacctattg ttgtggaatc 20100

cgtagggcct acagaccctt ccattgttac attagttgag gagtccagtg ttatagaatc 20160

tggtgcaggg atgggcctac agacccttcc attgttacat tagttgagga gtccagtgtt 20220

atagaatctg gtgcagggat tcctaatttt actgggtctg ggggatttga aaggagtcca 20280

gtgttataga atctggtgca gggattccta attttactgg gtctggggga tttgaaatta 20340

catcctcatc aacaactaca cctgccgtgt tgtcctaatt ttactgggtc tgggggattt 20400

gaaattacat cctcatcaac aactacacct gccgtgttgg atattacacc aacctctagt 20460

actgtacatg tcggatttga aattacatcc tcatcaacaa ctacacctgc cgtgttggat 20520

attacaccaa cctctagtac tgtacatgtc agtagtaccc atataactta catcctcatc 20580

aacaactaca cctgccgtgt tggatattac accaacctct agtactgtac atgtcagtag 20640

tacccatata accaatccgt tatttatcct gccgtgttgg atattacacc aacctctagt 20700

actgtacatg tcagtagtac ccatataacc aatccgttat ttattgatcc ccctgttatt 20760

gaggcccgga tattacacca acctctagta ctgtacatgt cagtagtacc catataacca 20820

atccgttatt tattgatccc cctgttattg aggccccaca aacaggccag tagtacccat 20880

ataaccaatc cgttatttat tgatccccct gttattgagg ccccacaaac aggcgaggtg 20940

tctggcaata ttttaattag cacacccttg atccccctgt tattgaggcc ccacaaacag 21000

gcgaggtgtc tggcaatatt ttaattagca cacccacatc tggtatacat agctatgaag 21060

aaatacccca caaacaggcg aggtgtctgg caatatttta attagcacac ccacatctgg 21120

tatacatagc tatgaagaaa tacctcgtaa aacatttgct gcgaggtgtc tggcaatatt 21180

ttaattagca cacccacatc tggtatacat agctatgaag aaatacctcg taaaacattt 21240

gctgttcacg gttctggtac attagcacac ccacatctgg tatacatagc tatgaagaaa 21300

tacctcgtaa aacatttgct gttcacggtt ctggtacaga acctattagt agtactcctc 21360

ccacatctgg tatacatagc tatgaagaaa tacctcgtaa aacatttgct gttcacggtt 21420

ctggtacaga acctattagt agtactccta ttccaggctc ctcgtaaaac atttgctgtt 21480

cacggttctg gtacagaacc tattagtagt actcctattc caggctttag gcgtattgca 21540

gctcctagat tatatagaac agaacctatt agtagtactc ctattccagg ctttaggcgt 21600

attgcagctc ctagattata tagaaaagca tttcagcagg ttaaggtaac tgaccctgct 21660

taggcgtatt gcagctccta gattatatag aaaagcattt cagcaggtta aggtaactga 21720

ccctgcattt cttgatagac ctgcaacatt agtatctgca agcatttcag caggttaagg 21780

taactgaccc tgcatttctt gatagacctg caacattagt atctgctgat aatccacttt 21840

ttgaaggtac tgacacatcg catttcttga tagacctgca acattagtat ctgctgataa 21900

tccacttttt gaaggtactg acacatcttt agctttttct ccgtcgggtg tggctcctgc 21960

tgataatcca ctttttgaag gtactgacac atctttagct ttttctccgt cgggtgtggc 22020

tcctgaccct gattttatga atatagtagc attacatagc tgacacatct ttagcttttt 22080

ctccgtcggg tgtggctcct gaccctgatt ttatgaatat agtagcatta cataggcctg 22140

catttactac acgtagcttt agctttttct ccgtcgggtg tggctcctga ccctgatttt 22200

atgaatatag tagcattaca taggcctgca tttactacac gtaggggtgg tgtacggtgg 22260

ctcctgaccc tgattttatg aatatagtag cattacatag gcctgcattt actacacgta 22320

ggggtggtgt acgttttagt aggcttggca gaaagggacc ctgattttat gaatatagta 22380

gcattacata ggcctgcatt tactacacgt aggggtggtg tacgttttag taggcttggc 22440

agaaaggcta ctatacgcat tacataggcc tgcatttact acacgtaggg gtggtgtacg 22500

ttttagtagg cttggcagaa aggctactat acaaacacgt agaggcacac aaatagggcc 22560

tgcatttact acacgtaggg gtggtgtacg ttttagtagg cttggcagaa aggctactat 22620

acaaacacgt agaggcacac aaataggtgc ccgtgtggtg tacgttttag taggcttggc 22680

agaaaggcta ctatacaaac acgtagaggc acacaaatag gtgcccgtgt gcattattat 22740

tatgatataa gtcccgtttt agtaggcttg gcagaaaggc tactatacaa acacgtagag 22800

gcacacaaat aggtgcccgt gtgcattatt attatgatat aagtcctatt gcaccaaaca 22860

cgtagaggca cacaaatagg tgcccgtgtg cattattatt atgatataag tcctattgca 22920

caggctgagg aaattgaacg taagcgaggc acacaaatag gtgcccgtgt gcattattat 22980

tatgatataa gtcctattgc acaggctgag gaaattgaac gtaagccatt attgtctggt 23040

gcccgtgtgc attattatta tgatataagt cctattgcac aggctgagga aattgaacgt 23100

aagccattat tgtctgcaaa taattcattt gatggccgtg cattattatt atgatataag 23160

tcctattgca caggctgagg aaattgaacg taagccatta ttgtctgcaa ataattcatt 23220

tgatggccta tatgatatta tgatataagt cctattgcac aggctgagga aattgaacgt 23280

aagccattat tgtctgcaaa taattcattt gatggcctat atgatatttc gtaaaaccta 23340

ttgcacaggc tgaggaaatt gaacgtaagc cattattgtc tgcaaataat tcatttgatg 23400

gcctatatga tatttcgtaa aatatagatg atgaagcagg ctgaggaaat tgaacgtaag 23460

ccattattgt ctgcaaataa ttcatttgat ggcctatatg atatttcgta aaatatagat 23520

gatgaagcac ctggttctgc aaataattca tttgatggcc tatatgatat ttcgtaaaat 23580

atagatgatg aagcacctgg tttgtctagc cagtcagttg ctacaccttc tgcacatttc 23640

gtaaaatata gatgatgaag cacctggttt gtctagccag tcagttgcta caccttctgc 23700

acacttacct ataaagcctt ccacattgtc ttttgctgtc tagccagtca gttgctacac 23760

cttctgcaca cttacctata aagccttcca cattgtcttt tgctagtaac accactaatg 23820

taactgcccc tttaggcctt ctgcacactt acctataaag ccttccacat tgtcttttgc 23880

tagtaacacc actaatgtaa ctgccccttt aggtaatgtg tgggaaacac cattttactt 23940

acctataaag ccttccacat tgtcttttgc tagtaacacc actaatgtaa ctgccccttt 24000

aggtaatgtg tgggaaacac cattttattc aggtcctttg ctagtaacac cactaatgta 24060

actgcccctt taggtaatgt gtgggaaaca ccattttatt caggtcctga catagtgttg 24120

cctacaggcc ccctagtaac accactaatg taactgcccc tttaggtaat gtgtgggaaa 24180

caccatttta ttcaggtcct gacatagtgt tgcctacagg ccccagtacg tggccccttt 24240

aggtaatgtg tgggaaacac cattttattc aggtcctgac atagtgttgc ctacaggccc 24300

cagtacgtgg ccctttgttc ctcagtctcc ttgtaatgtg tgggaaacac cattttattc 24360

aggtcctgac atagtgttgc ctacaggccc cagtacgtgg ccctttgttc ctcagtctcc 24420

ttatgatgtt acattcaggt cctgacatag tgttgcctac aggccccagt acgtggccct 24480

ttgttcctca gtctccttat gatgttaccc atgatgtata tatacaggga tcctgacata 24540

gtgttgccta caggccccag tacgtggccc tttgttcctc agtctcctta tgatgttacc 24600

catgatgtat atatacaggg atcctccttt gcggcccttt gttcctcagt ctccttatga 24660

tgttacccat gatgtatata tacagggatc ctcctttgca ttatggcctg tgtatttttt 24720

tagacgtagg cgtatgatgt tacccatgat gtatatatac agggatcctc ctttgcatta 24780

tggcctgtgt atttttttag acgtaggcgc cgtaaacgta ttccctattt ttcccatgat 24840

gtatatatac agggatcctc ctttgcatta tggcctgtgt atttttttag acgtaggcgc 24900

cgtaaacgta ttccctattt ttttgcagat ggtatacagg gatcctcctt tgcattatgg 24960

cctgtgtatt tttttagacg taggcgccgt aaacgtattc cctatttttt tgcagatggc 25020

gacgtggcgg cctttgcatt atggcctgtg tattttttta gacgtaggcg ccgtaaacgt 25080

attccctatt tttttgcaga tggcgacgtg gcggcctagt gaaaatcatt atggcctgtg 25140

tattttttta gacgtaggcg ccgtaaacgt attccctatt tttttgcaga tggcgacgtg 25200

gcggcctagt gaaaataagg tgtatcacgt aggcgccgta aacgtattcc ctattttttt 25260

gcagatggcg acgtggcggc ctagtgaaaa taaggtgtat ctacctccaa cacctgtttc 25320

aaagggcgcc gtaaacgtat tccctatttt tttgcagatg gcgacgtggc ggcctagtga 25380

aaataaggtg tatctacctc caacacctgt ttcaaaggtt gtggctttgc agatggcgac 25440

gtggcggcct agtgaaaata aggtgtatct acctccaaca cctgtttcaa aggttgtggc 25500

aacggattcc tatgtaaaac gggcgacgtg gcggcctagt gaaaataagg tgtatctacc 25560

tccaacacct gtttcaaagg ttgtggcaac ggattcctat gtaaaacgca ctagtatatt 25620

tctacctcca acacctgttt caaaggttgt ggcaacggat tcctatgtaa aacgcactag 25680

tatattttat ccgtaaggca gttcacgatt gcttgccgta gcggattcct atgtaaaacg 25740

cactagtata ttttatccgt aaggcagttc acgattgctt gccgtaggac atccctatta 25800

ctctgtgact aaggacaata cctagtatat tttatccgta aggcagttca cgattgcttg 25860

ccgtaggaca tccctattac tctgtgacta aggacaatac caaaacaaac attatccgta 25920

aggcagttca cgattgcttg ccgtaggaca tccctattac tctgtgacta aggacaatac 25980

caaaacaaac attcccaaag ttagtgcata tcggacatcc ctattactct gtgactaagg 26040

acaataccaa aacaaacatt cccaaagtta gtgcatatca atatagggta tttagggtac 26100

ggttgcccga ccaccaaaac aaacattccc aaagttagtg catatcaata tagggtattt 26160

agggtacggt tgcccgaccc taataagttt gggcttccag atactaatat ttagtgcata 26220

tcaatatagg gtatttaggg tacggttgcc cgaccctaat aagtttgggc ttccagatac 26280

taatatttat aatccggacc aggaacgcaa tatagggtat ttagggtacg gttgcccgac 26340

cctaataagt ttgggcttcc agatactaat atttataatc cggaccagga acggttagtg 26400

tgggcatggt tgcccgaccc taataagttt gggcttccag atactaatat ttataatccg 26460

gaccaggaac ggttagtgtg ggcatgtgta ggtttggagg taggccgccc taataagttt 26520

gggcttccag atactaatat ttataatccg gaccaggaac ggttagtgtg ggcatgtgta 26580

ggtttggagg taggccgcgg acagccttat aatccggacc aggaacggtt agtgtgggca 26640

tgtgtaggtt tggaggtagg ccgcggacag cctttaggtg ctgggctaag tggccatcca 26700

ttgtttatgg gcatgtgtag gtttggaggt aggccgcgga cagcctttag gtgctgggct 26760

aagtggccat ccattgttta ataggctgga tgatactgag tgtaggtttg gaggtaggcc 26820

gcggacagcc tttaggtgct gggctaagtg gccatccatt gtttaatagg ctggatgata 26880

ctgaaagttc caatttagcc ctttaggtgc tgggctaagt ggccatccat tgtttaatag 26940

gctggatgat actgaaagtt ccaatttagc aaataataat gttatagaag ttaggtgctg 27000

ggctaagtgg ccatccattg tttaataggc tggatgatac tgaaagttcc aatttagcaa 27060

ataataatgt tatagaagat agtagggaca tccattgttt aataggctgg atgatactga 27120

aagttccaat ttagcaaata ataatgttat agaagatagt agggacaata tatcagttga 27180

tataggctgg atgatactga aagttccaat ttagcaaata ataatgttat agaagatagt 27240

agggacaata tatcagttga tggcaagcaa acacagttgt ggcaaataat aatgttatag 27300

aagatagtag ggacaatata tcagttgatg gcaagcaaac acagttgtgt attgttggat 27360

gtactcccgc tatgggtgaa caatatatca gttgatggca agcaaacaca gttgtgtatt 27420

gttggatgta ctcccgctat gggtgaacat tggactaaag gtgctgtgtg taagtccaca 27480

ctgtattgtt ggatgtactc ccgctatggg tgaacattgg actaaaggtg ctgtgtgtaa 27540

gtccacacaa gttaccacag gggactgccc gcctcttgca tcattggact aaaggtgctg 27600

tgtgtaagtc cacacaagtt accacagggg actgcccgcc tcttgcatta attaatacac 27660

ctatagagga tggggacatg acaagttacc acaggggact gcccgcctct tgcattaatt 27720

aatacaccta tagaggatgg ggacatgata gacacaggat ttggcgctat ggactttaag 27780

gttaattaat acacctatag aggatgggga catgatagac acaggatttg gcgctatgga 27840

ctttaaggtg ttgcaggaat ctaaggctga ggtaccttta gatgatagac acaggatttg 27900

gcgctatgga ctttaaggtg ttgcaggaat ctaaggctga ggtaccttta gacattgtac 27960

aatccacctg taaatatcct ggatagacac aggatttggc gctatggact ttaaggtgtt 28020

gcaggaatct aaggctgagg tacctttaga cattgtacaa tccacctgta aatatcctga 28080

cggactttaa ggtgttgcag gaatctaagg ctgaggtacc tttagacatt gtacaatcca 28140

cctgtaaata tcctgactat ttaaaaatgt ctgcagcgta cggtgttgca ggaatctaag 28200

gctgaggtac ctttagacat tgtacaatcc acctgtaaat atcctgacta tttaaaaatg 28260

tctgcagcgt actatggtga tgacattgta caatccacct gtaaatatcc tgactattta 28320

aaaatgtctg cagcgtacta tggtgattct atgtggtttt acttacgcag ggacattgta 28380

caatccacct gtaaatatcc tgactattta aaaatgtctg cagcgtacta tggtgattct 28440

atgtggtttt acttacgcag ggaacaatta tccacctgta aatatcctga ctatttaaaa 28500

atgtctgcag cgtactatgg tgattctatg tggttttact tacgcaggga acaattattt 28560

gccagacatt atcctgacta tttaaaaatg tctgcagcgt actatggtga ttctatgtgg 28620

ttttacttac gcagggaaca attatttgcc agacattatt ttaatagggc tatttaaaaa 28680

tgtctgcagc gtactatggt gattctatgt ggttttactt acgcagggaa caattatttg 28740

ccagacatta ttttaatagg gctggtaaag tctatgtggt tttacttacg cagggaacaa 28800

ttatttgcca gacattattt taatagggct ggtaaagttg gggaaacaat acctgcagag 28860

ttatatttaa ttgccagaca ttattttaat agggctggta aagttgggga aacaatacct 28920

gcagagttat atttaaaggg tagcaatggt agagaacccc ctccgagttc gttggggaaa 28980

caatacctgc agagttatat ttaaagggta gcaatggtag agaaccccct ccgagttctg 29040

tatatgttgc tacgcctagt gggtctatga aagggtagca atggtagaga accccctccg 29100

agttctgtat atgttgctac gcctagtggg tctatgatta cgtctgaggc acagttattt 29160

aataaacctt cctccgagtt ctgtatatgt tgctacgcct agtgggtcta tgattacgtc 29220

tgaggcacag ttatttaata aaccttattg gttgcaacgt gcctgtatat gttgctacgc 29280

ctagtgggtc tatgattacg tctgaggcac agttatttaa taaaccttat tggttgcaac 29340

gtgcccaagg ccataataat ggcctagtgg gtctatgatt acgtctgagg cacagttatt 29400

taataaacct tattggttgc aacgtgccca aggccataat aatggcattt gctggggtaa 29460

tcgtgggtct atgattacgt ctgaggcaca gttatttaat aaaccttatt ggttgcaacg 29520

tgcccaaggc cataataatg gcatttgctg gggtaatcaa ttacgtctga ggcacagtta 29580

tttaataaac cttattggtt gcaacgtgcc caaggccata ataatggcat ttgctggggt 29640

aatcaattat ttgttactgt atttaataaa ccttattggt tgcaacgtgc ccaaggccat 29700

aataatggca tttgctgggg taatcaatta tttgttactg tagtagatac tactttaata 29760

aaccttattg gttgcaacgt gcccaaggcc ataataatgg catttgctgg ggtaatcaat 29820

tatttgttac tgtagtagat actactagaa gtaataaacc ttattggttg caacgtgccc 29880

aaggccataa taatggcatt tgctggggta atcaattatt tgttactgta gtagatacta 29940

ctattggttg caacgtgccc aaggccataa taatggcatt tgctggggta atcaattatt 30000

tgttactgta gtagatacta ctagaagtac taacatgact aggcatttgc tggggtaatc 30060

aattatttgt tactgtagta gatactacta gaagtactaa catgactatt agtactgcta 30120

cagaacagtt aagtaaatat ggtagtagat actactagaa gtactaacat gactattagt 30180

actgctacag aacagttaag taaatatgcg taacgaaaaa ttaatcagta ccttagacat 30240

gattagtact gctacagaac agttaagtaa atatgcgtaa cgaaaaatta atcagtacct 30300

tagacatgtg gaggaatatg aattacaatt tgtttttcaa tgcgtaacga aaaattaatc 30360

agtaccttag acatgtggag gaatatgaat tacaatttgt ttttcaattc gtaaaaatta 30420

ctttgtctgc agaggttatg ggtggaggaa tatgaattac aatttgtttt tcaattcgta 30480

aaaattactt tgtctgcaga ggttatggca tatttacata atatgacgta taacctactg 30540

gcgtaaaaat tactttgtct gcagaggtta tggcatattt acataatatg acgtataacc 30600

tactggagga ctggaatatt gggttatccc cgccagtggc cgaggactgg aatattgggt 30660

tatccccgcc agtggccacc agcctagaag ataaatatag atatgttaga agcacagcta 30720

taacatgtca acgggaacag cggccaccag cctagaagat aaatatagat atgttagaag 30780

cacagctata acatgtcaac gggaacagcc accaacagaa aaacaggacc cattagctaa 30840

aagaagcaca gctataacat gtcaacggga acagccacca acagaaaaac aggacccatt 30900

agctaaatat aaattttggg atgttaactt acaggacagt tgccaccaac agaaaaacag 30960

gacccattag ctaaatataa attttgggat gttaacttac aggacagttt ttctacagac 31020

ctggatcaat ttccactggg ttataaattt tgggatgtta acttacagga cagtttttct 31080

acagacctgg atcaatttcc actgggtaga aaatttttac gtaaactggg cactaggtca 31140

atttctacag acctggatca atttccactg ggtagaaaat ttttacgtaa actgggcact 31200

aggtcaaagc ctgctgtagc tacctctaaa aagcgatctg cagaaaattt ttacgtaaac 31260

tgggcactag gtcaaagcct gctgtagcta cctctaaaaa gcgatctgct cctacctcca 31320

cctctacacc agcaaaacgt aaagcctgct gtagctacct ctaaaaagcg atctgctcct 31380

acctccacct ctacaccagc aaaacgtaaa aggcggtagt gtgttgttgt gtgtttgtgt 31440

agctcctacc tccacctcta caccagcaaa acgtaaaagg cggtagtgtg ttgttgtgtg 31500

tttgtgtaac tgtgtttgtg tgttgtatat atggtatgtt taaaaggcgg tagtgtgttg 31560

ttgtgtgttt gtgtaactgt gtttgtgtgt tgtatatatg gtatgtttgt gtatgtgctt 31620

tattttatac tttgtatgtg taactgtgtt tgtgtgttgt atatatggta tgtttgtgta 31680

tgtgctttat tttatacttt gtatgtgtat gttgtgtttg tgtaaatgtt tgtgtgaaat 31740

gtgtgtatgt gctttatttt atactttgta tgtgtatgtt gtgtttgtgt aaatgtttgt 31800

gtgaaatgtt tgtgtgtgta ttcattgtat gtatgactgt atatgttgtg tttgtgtaaa 31860

tgtttgtgtg aaatgtttgt gtgtgtattc attgtatgta tgactgtata tatgtgtaat 31920

gtttgtgtgt ctgtaataaa cgtttgtgtg tgtattcatt gtatgtatga ctgtatatat 31980

gtgtaatgtt tgtgtgtctg taataaacat gaatgagtgc ttttacgcgt ggttgcataa 32040

aatatatgtg taatgtttgt gtgtctgtaa taaacatgaa tgagtgcttt tacgcgtggt 32100

tgcataaact aaggtgtgtc attattgtgg cttttgtttt gcatgaatga gtgcttttac 32160

gcgtggttgc ataaactaag gtgtgtcatt attgtggctt ttgttttgta agttattgtg 32220

tacagtgtac tatgtgtatt gactaaggtg tgtcattatt gtggcttttg ttttgtaagt 32280

tattgtgtac agtgtactat gtgtattgtg catacatata tataccataa catactccat 32340

tgtaagttat tgtgtacagt gtactatgtg tattgtgcat acatatatat accataacat 32400

actccatttt gttgtttttc cgccattttg taccgtaaac cgtgcataca tatatatacc 32460

ataacatact ccattttgtt gtttttccgc cattttgtac cgtaaaccga attcggttgc 32520

atggcctagt gccattattt attgttgttt ttccgccatt ttgtaccgta aaccgaattc 32580

ggttgcatgg cctagtgcca ttatttaaac taaaaggaat tcggttgcat ggcctagtgc 32640

cccgaattcg gttgcatggc ctagtgccat tatttaaact aaaaggaatt cggttgcatg 32700

gcctagtgcc attatttaaa ccaaaaggcc cttttcagca gccattattt aaaccaaaag 32760

gcccttttca gcagaacagt taatcctttg gcatattgcc gtttcctgtg ttttatactt 32820

gaattatgta cagtaccgca caacagttaa tcctttggca tattgccgtt tcctgtgttt 32880

tatacttgaa ttatgtacag taccgcaccc tgtattactc acaggtacta tgactgccaa 32940

cgtgttttat acttgaatta tgtacagtac cgcaccctgt attactcaca ggtactatga 33000

ctgccaactc gtattttatc tgcatacttt agtgctgttg gccctgtatt actcacaggt 33060

actatgactg ccaactcgta ttttatctgc atactttagt gctgttgggc acacattttt 33120

atacatgtgt ctgcaacttt gctcgtattt tatctgcata ctttagtgct gttgggcaca 33180

catttttata catgtgtctg caactttggt gttttggctt gcagaataca ctatgtaggc 33240

cgggcacaca tttttataca tgtgtctgca actttggtgt tttggcttgc agaatacact 33300

atgtaggcca agtatctgtc agtatctgtt ttgcaaacat gggtgttttg gcttgcagaa 33360

tacactatgt aggccaagta tctgtcagta tctgttttgc aaacatgtaa catacaatta 33420

ctcatttttt aaaaccgttt agccaagtat ctgtcagtat ctgttttgca aacatgtaac 33480

atacaattac tcatttttta aaaccgttta cggtcgtgca aaaacaggtt tcttttaatt 33540

g 33541

<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 HPV56, characterized by comprising the following probes: SEQ ID NO. 1.

2. A kit for detecting human papillomavirus HPV56, 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 HPV56 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.

Images