CN112266983A - Probe set for detecting human papilloma viruses HPV16 and HPV18 and kit thereof - Google Patents

Abstract

The invention discloses a probe set for detecting Human Papillomavirus (HPV) 16 and HPV18 and a kit thereof, wherein the probe set comprises genes capable of specifically capturing HPV16 and 18; the invention has the advantages of comprehensive detection range, greatly improved detection efficiency and accurate result.

Description

Probe set for detecting human papilloma viruses HPV16 and HPV18 and kit thereof

Technical Field：

The invention relates to a probe set and a kit thereof, in particular to a probe set and a kit for capturing and sequencing human papilloma virus HPV16 and 18 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 disadvantages of the prior art, and provides a capture probe set for detecting HPV16 and 18 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 set for detecting human papilloma viruses HPV16 and HPV18, which is believed to be shown in Table 1

The invention also provides a kit for HPV typing and integration detection of human papillomavirus. The kit comprises the probe set for HPV typing and integration detection of human papillomavirus; 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 set for detecting human papillomavirus HPV16 and HPV18 and a kit thereof, which mainly comprises the following steps:

(1) design of Capture probes for 2 types of HPV

(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 the probe group which can rapidly and accurately capture the complete sequence of the 2 types of HPV genomes, 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 uses HPV probe group to capture the library sample of the target area and uses high-throughput sequencing technology to sequence. 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.

(4) The kit can detect the integration condition of HPV in human genome, and the kits aiming at HPV in the market at present are all aiming at HPV typing, and no kit capable of detecting HPV integration exists.

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 DNA sequences of 2 types of HPV 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 kit comprises the following components: the invention relates to a capture probe group for detecting human papillomavirus HPV16 and HPV18, which comprises a database establishing buffer solution 1, a database establishing 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 set for detecting human papillomavirus HPV16 and HPV18 and a kit thereof

<130> 0

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 25221

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe type HPV16

<400> 1

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tgtatgtctt gttgcagatc atcaagaaca cgtagagaaa cccagctgta atccgtaatg 1680

gagatacacc tacacttgtt gcagatcatc aagaacacgt agagaaaccc agctgtaatc 1740

cgtaatggag atacacctac attgcatgaa tatatgttag atttgcaacc agagcccagc 1800

tgtaatccgt aatggagata cacctacatt gcatgaatat atgttagatt tgcaaccaga 1860

gacaactgat ctctactgtt atgagcaatt aaattgcatg aatatatgtt agatttgcaa 1920

ccagagacaa ctgatctcta ctgttatgag caattaaatg acagctcaga ggaggaggat 1980

gaaatagatg gtccgacaac tgatctctac tgttatgagc aattaaatga cagctcagag 2040

gaggaggatg aaatagatgg tccagctgga caagcagaac cggacagagc ccatactgtt 2100

atgagcaatt aaatgacagc tcagaggagg aggatgaaat agatggtcca gctggacaag 2160

cagaaccgga cagagcccat tacaatgaca gctcagagga ggaggatgaa atagatggtc 2220

cagctggaca agcagaaccg gacagagccc attacaatat tgtaaccttt tgttgcaagt 2280

gtgacccagc tggacaagca gaaccggaca gagcccatta caatattgta accttttgtt 2340

gcaagtgtga ctctacgctt cggttgtgcg tacaaagcac acacgcaata ttgtaacctt 2400

ttgttgcaag tgtgactcta cgcttcggtt gtgcgtacaa agcacacacg tagacattcg 2460

tactttggaa gacctgttaa tgggccgtag acattcgtac tttggaagac ctgttaatgg 2520

gcacactagg aattgtgtgc cccatctgtt ctcagaaacc ataatctacc atggctgatc 2580

ctgcagggca cactaggaat tgtgtgcccc atctgttctc agaaaccata atctaccatg 2640

gctgatcctg caggtaccaa tggggaagag ggtacgggat gtaatctcag aaaccataat 2700

ctaccatggc tgatcctgca ggtaccaatg gggaagaggg tacgggatgt aatggatggt 2760

tttatgtaga ggctgtagtg gaaaaggtac caatggggaa gagggtacgg gatgtaatgg 2820

atggttttat gtagaggctg tagtggaaaa aaaaacaggg gcgtatatat cagatgacga 2880

gaacgggatg gttttatgta gaggctgtag tggaaaaaaa aacaggggcg tatatatcag 2940

atgacgagaa cgaaaatgac agtgatacag gtgaagattt ggtagaaaac aggggcgtat 3000

atatcagatg acgagaacga aaatgacagt gatacaggtg aagatttggt agattttata 3060

gtaaatgata atgattattt aacaccgaaa atgacagtga tacaggtgaa gatttggtag 3120

attttatagt aaatgataat gattatttaa cacaggcaga aacagagaca gcaccgtagt 3180

tgtttgattt tatagtaaat gataatgatt atttaacaca ggcagaaaca gagacagcac 3240

cgtagttgtt tactgcacag gaagcaaaac aacatagagc gtaagcacag gcagaaacag 3300

agacagcacc gtagttgttt actgcacagg aagcaaaaca acatagagcg taagtacagg 3360

ttctaaaacg aaagtatttg ggtagtactg cacaggaagc aaaacaacat agagcgtaag 3420

tacaggttct aaaacgaaag tatttgggta gtccacttag tgatattagt ggatgtgtag 3480

acaatgcagt acaggttcta aaacgaaagt atttgggtag tccacttagt gatattagtg 3540

gatgtgtaga caataatatt agtcctagat taaaagctat atgtagtcca cttagtgata 3600

ttagtggatg tgtagacaat aatattagtc ctagattaaa agctatatgt atagaaaaac 3660

aaagtagagc tgcaaaaagg agattaatat tagtcctaga ttaaaagcta tatgtataga 3720

aaaacaaagt agagctgcaa aaaggagatt atttgaaagc gaagacagcg ggtatggcaa 3780

tactgttaaa agctatatgt atagaaaaac aaagtagagc tgcaaaaagg agattatttg 3840

aaagcgaaga cagcgggtat ggcaatactg aagtggagaa aaacaaagta gagctgcaaa 3900

aaggagatta tttgaaagcg aagacagcgg gtatggcaat actgaagtgg aaactcagca 3960

gatgttacag gtagaagatt tgaaagcgaa gacagcgggt atggcaatac tgaagtggaa 4020

actcagcaga tgttacaggt agaagggcgc catgagactg aaacaccatg tagtcagtga 4080

agtggaaact cagcagatgt tacaggtaga agggcgccat gagactgaaa caccatgtag 4140

tcagtatagt ggtggaagtg ggggtggttg cagtcagagg gcgccatgag actgaaacac 4200

catgtagtca gtatagtggt ggaagtgggg gtggttgcag tcagtacagt agtggaagtg 4260

ggggagaggg tgttagtgta tagtggtgga agtgggggtg gttgcagtca gtacagtagt 4320

ggaagtgggg gagagggtgt tagtgaaaga cacactatcg tacaaacacc acttacagta 4380

cagtagtgga agtgggggag agggtgttag tgaaagacac actatcgtac aaacaccact 4440

tacaaatatt ttaaatgtac taaaaactag tacgtaagtg aaagacacac tatcgtacaa 4500

acaccactta caaatatttt aaatgtacta aaaactagta cgtaaaaggc agcaatgtta 4560

gcaaaattta aagagttcaa atattttaaa tgtactaaaa actagtacgt aaaaggcagc 4620

aatgttagca aaatttaaag agttatacgg ggtgagtttt tcagaattag taagaccaaa 4680

ggcagcaatg ttagcaaaat ttaaagagtt atacggggtg agtttttcag aattagtaag 4740

accatttaaa agtaataaat caacgtgttg cgattggacg gggtgagttt ttcagaatta 4800

gtaagaccat ttaaaagtaa taaatcaacg tgttgcgatt ggtgtattgc tgcatttgga 4860

cttacaccca gtatagcggt gtattgctgc atttggactt acacccagta tagctgacag 4920

tataaaaaca ctattacaac aatattgttt atatttacac attcaaagtt tagcatgagc 4980

tgacagtata aaaacactat tacaacaata ttgtttatat ttacacattc aaagtttagc 5040

atgttcatgg ggaatggttg tgttactatt agtaagattg tttatattta cacattcaaa 5100

gtttagcatg ttcatgggga atggttgtgt tactattagt aagatataaa tgtggaaaaa 5160

atagagaaac aattgaagtt catggggaat ggttgtgtta ctattagtaa gatataaatg 5220

tggaaaaaat agagaaacaa ttgaaaaatt gctgtctaaa ctattatgtg tgtctccgat 5280

ataaatgtgg aaaaaataga gaaacaattg aaaaattgct gtctaaacta ttatgtgtgt 5340

ctccaatgtg tatgatgata gagcctccaa aattgcgaat tgctgtctaa actattatgt 5400

gtgtctccaa tgtgtatgat gatagagcct ccaaaattgc gtagtacagc agcagcatta 5460

tattggtata aaacaggaat gtgtatgatg atagagcctc caaaattgcg tagtacagca 5520

gcagcattat attggtataa aacaggtata tcaaatatta gtgaagtgta tggagaccca 5580

aaattgcgta gtacagcagc agcattatat tggtataaaa caggtatatc aaatattagt 5640

gaagtgtatg gagacacgcc agaatggata caaagacgca ttatattggt ataaaacagg 5700

tatatcaaat attagtgaag tgtatggaga cacgccagaa tggatacaaa gacaaacagt 5760

attacaacat agttatattg gtataaaaca ggtatatcaa atattagtga agtgtatgga 5820

gacacgccag aatggataca aagacaaaca gtattacaac atagcaggta tatcaaatat 5880

tagtgaagtg tatggagaca cgccagaatg gatacaaaga caaacagtat tacaacatag 5940

ttttaatgat tgtacatttg aattcacgcc agaatggata caaagacaaa cagtattaca 6000

acatagtttt aatgattgta catttgaatt atcacagatg gtacaatggg cctacgataa 6060

tgaccaacat agttttaatg attgtacatt tgaattatca cagatggtac aatgggccta 6120

cgataatgac atagtagacg atagtgaaat tgcatataaa tatgcacaga tggtacaatg 6180

ggcctacgat aatgacatag tagacgatag tgaaattgca tataaatcgt aacaattggc 6240

agacactaat agtacgtaaa gtgcggtaca atgggcctac gataatgaca tagtagacga 6300

tagtgaaatt gcatataaat cgtaacaatt ggcagacact aatagtacgt aaagtgccat 6360

agtagacgat agtgaaattg catataaatc gtaacaattg gcagacacta atagtacgta 6420

aagtgccttt ctaaaaagta attcacaggc aaaaattgat agtgaaattg catataaatc 6480

gtaacaattg gcagacacta atagtacgta aagtgccttt ctaaaaagta attcacaggc 6540

aaaaattgta aaggattgca caattggcag acactaatag tacgtaaagt gcctttctaa 6600

aaagtaattc acaggcaaaa attgtaaagg attgtgcaac aatgtgtaga cattatggca 6660

gacactaata gtacgtaaag tgcctttcta aaaagtaatt cacaggcaaa aattgtaaag 6720

gattgtgcaa caatgtgtag acattataaa cgagcgcctt tctaaaaagt aattcacagg 6780

caaaaattgt aaaggattgt gcaacaatgt gtagacatta taaacgagca gaaaaaaaac 6840

aaatgagtat gagtcgtaaa ggattgtgca acaatgtgta gacattataa acgagcagaa 6900

aaaaaacaaa tgagtatgag tcaatggata aaatatagat gtgatagggt agatgcaaca 6960

atgtgtagac attataaacg agcagaaaaa aaacaaatga gtatgagtca atggataaaa 7020

tatagatgtg atagggtaga tgatggtaaa cgagcagaaa aaaaacaaat gagtatgagt 7080

caatggataa aatatagatg tgatagggta gatgatggag gtgattggaa gcaaattgtt 7140

atgtttgtca atggataaaa tatagatgtg atagggtaga tgatggaggt gattggaagc 7200

aaattgttat gtttttaagg tatcaaggtg tagagtttat gtcattgatg gaggtgattg 7260

gaagcaaatt gttatgtttt taaggtatca aggtgtagag tttatgtcat ttttaactgc 7320

attaaaaaga tttttgcaag gcatactaag gtatcaaggt gtagagttta tgtcattttt 7380

aactgcatta aaaagatttt tgcaaggcat acctaaaaaa aattgcatat tactatatgg 7440

tgcagcttta actgcattaa aaagattttt gcaaggcata cctaaaaaaa attgcatatt 7500

actatatggt gcagctaaca caggtaaatc attatttggt atgagtacct aaaaaaaatt 7560

gcatattact atatggtgca gctaacacag gtaaatcatt atttggtatg agtttaatga 7620

aatttctgca agggtctgta atatgtgcag ctaacacagg taaatcatta tttggtatga 7680

gtttaatgaa atttctgcaa gggtctgtaa tatgttttgt aaattctaaa agccattttt 7740

ggttacgttt aatgaaattt ctgcaagggt ctgtaatatg ttttgtaaat tctaaaagcc 7800

atttttggtt acaaccatta gcagcgtaca aaataggtat gttagagttt tgtaaattct 7860

aaaagccatt tttggttaca accattagca gcgtacaaaa taggtatgtt agatgcgtat 7920

acagtgccct gttggaacta catagaccat tagcagcgta caaaataggt atgttagatg 7980

cgtatacagt gccctgttgg aactacatag atgacaattt aagaacgtaa ttggatggaa 8040

atttaggcgt atacagtgcc ctgttggaac tacatagatg acaatttaag aacgtaattg 8100

gatggaaatt tagtttctat ggatgtaaag catagaccat tggtactgac aatttaagaa 8160

cgtaattgga tggaaattta gtttctatgg atgtaaagca tagaccattg gtacaactaa 8220

acgtacctcc attattaatt acatctagtt tctatggatg taaagcatag accattggta 8280

caactaaacg tacctccatt attaattaca tctaacatta cgtatggtac agattctagg 8340

tggcctcaac taaacgtacc tccattatta attacatcta acattacgta tggtacagat 8400

tctaggtggc cttatttaca taatagattg gtggtgttta catttcacat tacgtatggt 8460

acagattcta ggtggcctta tttacataat agattggtgg tgtttacatt tcctaatgag 8520

tttccatttg acgaaaacgg aaatcctcct aatgagtttc catttgacga aaacggaaat 8580

ccagtgtatg agcttaatga taagaactgg aaatcctttt tctcaaggac gtggtccaga 8640

ttaagttttc catttgacga aaacggaaat ccagtgtatg agcttaatga taagaactgg 8700

aaatcctttt tctcaaggac gtggtccaga ttaagtttgc acgaggccag tgtatgagct 8760

taatgataag aactggaaat cctttttctc aaggacgtgg tccagattaa gtttgcacga 8820

ggacgaggac aaggaaaacg atggagtcct ttttctcaag gacgtggtcc agattaagtt 8880

tgcacgagga cgaggacaag gaaaacgatg gagactcttt gccaacgttt aaatgtgtgt 8940

caggactcct ttttctcaag gacgtggtcc agattaagtt tgcacgagga cgaggacaag 9000

gaaaacgatg gagactcttt gccaacgttt aaatgtgtgt caggacctca aggacgtggt 9060

ccagattaag tttgcacgag gacgaggaca aggaaaacga tggagactct ttgccaacgt 9120

ttaaatgtgt gtcaggacaa aatactagga cgtggtccag attaagtttg cacgaggacg 9180

aggacaagga aaacgatgga gactctttgc caacgtttaa atgtgtgtca ggacaaaata 9240

ctatttgcac gaggacgagg acaaggaaaa cgatggagac tctttgccaa cgtttaaatg 9300

tgtgtcagga caaaatacta acacattatg aaaatgatag tacgagactc tttgccaacg 9360

tttaaatgtg tgtcaggaca aaatactaac acattatgaa aatgatagta cagacctacg 9420

tgaccatata gactattgga aacgactctt tgccaacgtt taaatgtgtg tcaggacaaa 9480

atactaacac attatgaaaa tgatagtaca gacctacgtg accatataga ctattggaaa 9540

caccaaaata ctaacacatt atgaaaatga tagtacagac ctacgtgacc atatagacta 9600

ttggaaacac cgtagcctag aatgtgctat ttattacaag gcccagacct acgtgaccat 9660

atagactatt ggaaacaccg tagcctagaa tgtgctattt attacaaggc cagagaaatg 9720

ggatttaaac atattaacca ccacaccgta gcctagaatg tgctatttat tacaaggcca 9780

gagaaatggg atttaaacat attaaccacc aggtggtgcc aacactggct gtatcaaaga 9840

ataggccaga gaaatgggat ttaaacatat taaccaccag gtggtgccaa cactggctgt 9900

atcaaagaat aaagcattac aagcaattga actgcaacta acgcaggtgg tgccaacact 9960

ggctgtatca aagaataaag cattacaagc aattgaactg caactaacgt tagaaacaat 10020

atataactca caatatagta atgagcatta caagcaattg aactgcaact aacgttagaa 10080

acaatatata actcacaata tagtaatgaa aagtggacat tacaagacgt tagccttgaa 10140

gtgtagaaac aatatataac tcacaatata gtaatgaaaa gtggacatta caagacgtta 10200

gccttgaagt gtatttaact gcaccaacag gatgtataaa aaaaaaagtg gacattacaa 10260

gacgttagcc ttgaagtgta tttaactgca ccaacaggat gtataaaaaa acatggatat 10320

acagtggaag tgcagtttga tgggtattta actgcaccaa caggatgtat aaaaaaacat 10380

ggatatacag tggaagtgca gtttgatgga gacatcgtaa atacacgtaa ttatacaaac 10440

tggaacatgg atatacagtg gaagtgcagt ttgatggaga catcgtaaat acacgtaatt 10500

atacaaactg gacacatata tatatttgtg aagaagcatc agtgagacat cgtaaataca 10560

cgtaattata caaactggac acatatatat atttgtgaag aagcatcagt aactgtggta 10620

gagggtcaag ttgactatta tggggacaca tatatatatt tgtgaagaag catcagtaac 10680

tgtggtagag ggtcaagttg actattatgg tttatattat gttcatgaag gaatacgaac 10740

atagtaactg tggtagaggg tcaagttgac tattatggtt tatattatgt tcatgaagga 10800

atacgaacat attttgtgca gtttaaagat gcgtaagaaa aatgtttata ttatgttcat 10860

gaaggaatac gaacatattt tgtgcagttt aaagatgcgt aagaaaaata tagtaaaaat 10920

aaagtatggg aagttccgta gggattttgt gcagtttaaa gatgcgtaag aaaaatatag 10980

taaaaataaa gtatgggaag ttccgtaggg tggtcaggta atattatgtc ctacatctgt 11040

gttatagtaa aaataaagta tgggaagttc cgtagggtgg tcaggtaata ttatgtccta 11100

catctgtgtt tagcagcaac gaagtatcct ctcctgaaat tatgtggtca ggtaatatta 11160

gtcctacat ctgtgtttag cagcaacgaa gtatcctctc ctgaaattat taggcagcac 11220

ttggccaacc accccgccgc gacttagcag caacgaagta tcctctcctg aaattattag 11280

gcagcacttg gccaaccacc ccgccgcgac ccataccaaa gccgtcgcct tgggcaccga 11340

agattaggca gcacttggcc aaccaccccg ccgcgaccca taccaaagcc gtcgccttgg 11400

gcaccgaaga aacacagacg actatccagc gaccaagatc agacccatac caaagccgtc 11460

gccttgggca ccgaagaaac acagacgact atccagcgac caagatcaga gccagacacc 11520

ggaaacccct gccacaccac taaaaacaca gacgactatc cagcgaccaa gatcagagcc 11580

agacaccgga aacccctgcc acaccactaa gttgttgcac agagactcag tggacagtgc 11640

tccagccaga caccggaaac ccctgccaca ccactaagtt gttgcacaga gactcagtgg 11700

acagtgctcc aatcctcact gcatttaaca gctcacacaa aggagttgtt gcacagagac 11760

tcagtggaca gtgctccaat cctcactgca tttaacagct cacacaaagg acggattaac 11820

tgtaatagta acactacacc catccaatcc tcactgcatt taacagctca cacaaaggac 11880

ggattaactg taatagtaac actacaccca tagtacattt aaaaggtgcg tataatactt 11940

taagacggat taactgtaat agtaacacta cacccatagt acatttaaaa ggtgcgtata 12000

atactttaaa atgtttaaga tatagattta aaaagcattg tacagtacat ttaaaaggtg 12060

cgtataatac tttaaaatgt ttaagatata gatttaaaaa gcattgtaca ttgtatactg 12120

cagtgtcgtc tacatggcat tggaaggtgc gtataatact ttaaaatgtt taagatatag 12180

atttaaaaag cattgtacat tgtatactgc agtgtcgtct acatggcatt ggacaaatgt 12240

ttaagatata gatttaaaaa gcattgtaca ttgtatactg cagtgtcgtc tacatggcat 12300

tggacaggac ataatgtaaa acataaaagt gcaatcattg tatactgcag tgtcgtctac 12360

atggcattgg acaggacata atgtaaaaca taaaagtgca attgttacac ttacatatga 12420

tagtgaatgg caacgggaca ggacataatg taaaacataa aagtgcaatt gttacactta 12480

catatgatag tgaatggcaa cgtgaccaat ttttgtctca agttaaaata ccaaattgtt 12540

acacttacat atgatagtga atggcaacgt gaccaatttt tgtctcaagt taaaatacca 12600

aaaactatta cagtgtctac tggatttatg tctatcgtga ccaatttttg tctcaagtta 12660

aaataccaaa aactattaca gtgtctactg gatttatgtc tatatgacaa atcttgatac 12720

tgcatccaca acattaaact attacagtgt ctactggatt tatgtctata tgacaaatct 12780

tgatactgca tccacaacat tactggcgtg ctttttgctt tgcttttgtg tgctttatga 12840

caaatcttga tactgcatcc acaacattac tggcgtgctt tttgctttgc ttttgtgtgc 12900

ttttgtgtgt ctgcctatta atacgtccgc tgcttactgg cgtgcttttt gctttgcttt 12960

tgtgtgcttt tgtgtgtctg cctattaata cgtccgctgc ttttgtctgt gtctacatac 13020

acatcattaa taatagtgtg tctgcctatt aatacgtccg ctgcttttgt ctgtgtctac 13080

atacacatca ttaataatat tggtattact attgtggata acagcagcct ctgcgttgtc 13140

tgtgtctaca tacacatcat taataatatt ggtattacta ttgtggataa cagcagcctc 13200

tgcgtttagg tgttttattg tatatattat atttgtggta ttactattgt ggataacagc 13260

agcctctgcg tttaggtgtt ttattgtata tattatattt gtttatatac cattattttt 13320

aatacataca ccgtatgttt atataccatt atttttaata catacaccgt aacgcttttt 13380

aattacataa tgtatatgta cataatgtaa ttgttacata taattgttgt atacctgcac 13440

gctttttaat tacataatgt atatgtacat aatgtaattg ttacatataa ttgttgtata 13500

ccataactta ctattttttc ttttttattt tcataatttt ttctttttta ttttcatata 13560

taattttttt ttttgtttgt ttgtttgttt tttaataaac tgttattact taacacgtag 13620

acacaaacgt tctgctaata aactgttatt acttaacacg tagacacaaa cgttctgcaa 13680

aacgcacaaa acgtgcatcg gctacccaac tttataaaac cgtaaaacag gcaggcaaac 13740

gttctgcaaa acgcacaaaa cgtgcatcgg ctacccaact ttataaaacc gtaaaacagg 13800

caggtacatg tccacctgac attataccta aggttcggct acccaacttt ataaaaccgt 13860

aaaacaggca ggtacatgtc cacctgacat tatacctaag gttgaaggca aaactattgc 13920

tgatcaaata ttacaggcag gtacatgtcc acctgacatt atacctaagg ttgaaggcaa 13980

aactattgct gatcaaatat tacaatatgg aagtatgggt gtattttttg gtgggtgaag 14040

gcaaaactat tgctgatcaa atattacaat atggaagtat gggtgtattt tttggtgggt 14100

taggaattgg aacagggtcg ggtacaggcg gacgcatatg gaagtatggg tgtatttttt 14160

ggtgggttag gaattggaac agggtcgggt acaggcggac gcactgggta tattccattg 14220

ggaacaaggc ctcccttagg aattggaaca gggtcgggta caggcggacg cactgggtat 14280

attccattgg gaacaaggcc tcccacagct acagatacac ttgctcctgt aagaccactg 14340

ggtatattcc attgggaaca aggcctccca cagctacaga tacacttgct cctgtaagac 14400

cccctttaac agtagatcct gtgggccctt ctgatccaca gctacagata cacttgctcc 14460

tgtaagaccc cctttaacag tagatcctgt gggcccttct gatccttcta tagtttcttt 14520

agtggaagaa actagcccct ttaacagtag atcctgtggg cccttctgat ccttctatag 14580

tttctttagt ggaagaaact agttttattg cgtatggtgc accaacatct gtacctcctt 14640

ctatagtttc tttagtggaa gaaactagtt ttattgcgta tggtgcacca acatctgtac 14700

cttccattcc cccagatgta tcaggattta gtattgtttt attgcgtatg gtgcaccaac 14760

atctgtacct tccattcccc cagatgtatc aggatttagt attactactt caactgatac 14820

cacacctgct atatttccat tcccccagat gtatcaggat ttagtattac tacttcaact 14880

gataccacac ctgctatatt agatattaat aatactgtta ctactgttac tacactacta 14940

cttcaactga taccacacct gctatattag atattaataa tactgttact actgttacta 15000

cacataataa tcccactttc actgacccat ctgtagatat taataatact gttactactg 15060

ttactacaca taataatccc actttcactg acccatctgt attgcagcct ccaacacctg 15120

cagaaactgg agggccacat aataatccca ctttcactga cccatctgta ttgcagcctc 15180

caacacctgc agaaactgga gggcatttta cactttcatc atccactatt agtacattgc 15240

agcctccaac acctgcagaa actggagggc attttacact ttcatcatcc actattagta 15300

cacataatta tgaagaaatt cctatggata catttggcat tttacacttt catcatccac 15360

tattagtaca cataattatg aagaaattcc tatggataca tttattgtta gcacaaaccc 15420

taacacagta actagcacat aattatgaag aaattcctat ggatacattt attgttagca 15480

caaaccctaa cacagtaact agtagcacac ccataccagg gtctcgccca gtggcattgt 15540

tagcacaaac cctaacacag taactagtag cacacccata ccagggtctc gcccagtggc 15600

acgcctagga ttatatagtc gcacaacaca acaggagcac acccatacca gggtctcgcc 15660

cagtggcacg cctaggatta tatagtcgca caacacaaca ggttaaagtt gtagaccctg 15720

cttttgtaac cactcgtggc acgcctagga ttatatagtc gcacaacaca acaggttaaa 15780

gttgtagacc ctgcttttgt aaccactccc actaaactta ttacatatga taatccacgc 15840

ctaggattat atagtcgcac aacacaacag gttaaagttg tagaccctgc ttttgtaacc 15900

actcccacta aacttattac atatgataat cctgccacgc ctaggattat atagtcgcac 15960

aacacaacag gttaaagttg tagaccctgc ttttgtaacc actcccacta aacttattac 16020

atatgataat cctgcggtta aagttgtaga ccctgctttt gtaaccactc ccactaaact 16080

tattacatat gataatcctg catatgaagg tatagatgtg gataatacat tatatctccc 16140

actaaactta ttacatatga taatcctgca tatgaaggta tagatgtgga taatacatta 16200

tatttttcta gtaatgataa tagtattaat atagcgcata tgaaggtata gatgtggata 16260

atacattata tttttctagt aatgataata gtattaatat agctccagat cctgactttt 16320

tggatatagt tgcttttttt ctagtaatga taatagtatt aatatagctc cagatcctga 16380

ctttttggat atagttgctt tacataggcc agcattaacc tctaggcgta ctggcccaga 16440

tcctgacttt ttggatatag ttgctttaca taggccagca ttaacctcta ggcgtactgg 16500

cattaggtac agtagaattg gtaataaaca aacacacata ggccagcatt aacctctagg 16560

cgtactggca ttaggtacag tagaattggt aataaacaaa cactacgtac tcgtagtgga 16620

aaatctatag gtgctggcat taggtacagt agaattggta ataaacaaac actacgtact 16680

cgtagtggaa aatctatagg tgctaaggta cattattatt atgatttaag tactacacta 16740

cgtactcgta gtggaaaatc tataggtgct aaggtacatt attattatga tttaagtact 16800

attgatcctg cagaagaaat agaattacaa actatggtac attattatta tgatttaagt 16860

actattgatc ctgcagaaga aatagaatta caaactataa caccttctac atatactacc 16920

acttcaccgt aagccgatcc tgcagaagaa atagaattac aaactataac accttctaca 16980

tatactacca cttcaccgta agcctcacct acttctatta ataatggatt atatgaacac 17040

cttctacata tactaccact tcaccgtaag cctcacctac ttctattaat aatggattat 17100

atgatatttc gtaagatgac tttattacag atactcagcc tcacctactt ctattaataa 17160

tggattatat gatatttcgt aagatgactt tattacagat acttctacaa ccccggtacc 17220

atctgtaccc tctactgata tttcgtaaga tgactttatt acagatactt ctacaacccc 17280

ggtaccatct gtaccctcta catctttatc aggttatatt cctgcaaata caacatctac 17340

aaccccggta ccatctgtac cctctacatc tttatcaggt tatattcctg caaatacaac 17400

aattcctttt ggtggtgcat acaatattcc tttagcatct ttatcaggtt atattcctgc 17460

aaatacaaca attccttttg gtggtgcata caatattcct ttagtatcag gtcctgatat 17520

acccattaat ataactcctt ttggtggtgc atacaatatt cctttagtat caggtcctga 17580

tatacccatt aatataactg accaagctcc ttcattaatt cctatagttc caggggtatc 17640

aggtcctgat atacccatta atataactga ccaagctcct tcattaattc ctatagttcc 17700

agggtctcca caatatacaa ttattgctgc gtaagtgacc aagctccttc attaattcct 17760

atagttccag ggtctccaca atatacaatt attgctgcgt aaggtgactt ttatttacat 17820

cctagttatt acatgccagg gtctccacaa tatacaatta ttgctgcgta aggtgacttt 17880

tatttacatc ctagttatta catgttacga aaacgacgta aacgtttacc atattggtga 17940

cttttattta catcctagtt attacatgtt acgaaaacga cgtaaacgtt taccatattt 18000

tttttcagat gtctctttgg ctgcctagtg aggccgttac gaaaacgacg taaacgttta 18060

ccatattttt tttcagatgt ctctttggct gcctagtgag gccactgtct acttgcctcc 18120

tgtcccagta tctaaaccat attttttttc agatgtctct ttggctgcct agtgaggcca 18180

ctgtctactt gcctcctgtc ccagtatcta aggttgtaag cacggatgaa tatgtttttt 18240

cagatgtctc tttggctgcc tagtgaggcc actgtctact tgcctcctgt cccagtatct 18300

aaggttgtaa gcacggatga atatgttgca cgcacctagt gaggccactg tctacttgcc 18360

tcctgtccca gtatctaagg ttgtaagcac ggatgaatat gttgcacgca caaacatata 18420

ttatccgtaa gggccactgt ctacttgcct cctgtcccag tatctaaggt tgtaagcacg 18480

gatgaatatg ttgcacgcac aaacatatat tatccgtaag gaacatccag acaggttgta 18540

agcacggatg aatatgttgc acgcacaaac atatattatc cgtaaggaac atccagacta 18600

cttgcagttg gacatcccta ttttcctatt aacaaacata tattatccgt aaggaacatc 18660

cagactactt gcagttggac atccctattt tcctattaaa aaacctaaca ataacaaaat 18720

attagttcct aactacttgc agttggacat ccctattttc ctattaaaaa acctaacaat 18780

aacaaaatat tagttcctaa agtatcagga ttacaataca gggtatttag aaaaaaacct 18840

aacaataaca aaatattagt tcctaaagta tcaggattac aatacagggt atttagaata 18900

catttacctg accccaataa gtttggtttt ccgtatcagg attacaatac agggtattta 18960

gaatacattt acctgacccc aataagtttg gttttcctga cacctcattt tataatccag 19020

atacacagcg gctacattta cctgacccca ataagtttgg ttttcctgac acctcatttt 19080

ataatccaga tacacagcgg ctggtttggg cctgtgtagg tgttgaggta ggctgacacc 19140

tcattttata atccagatac acagcggctg gtttgggcct gtgtaggtgt tgaggtaggt 19200

cgtggtcagc cattaggtgt gggcattagt ggcggctggt ttgggcctgt gtaggtgttg 19260

aggtaggtcg tggtcagcca ttaggtgtgg gcattagtgg ccatccttta ttaaataaat 19320

tggatgacac agggtcgtgg tcagccatta ggtgtgggca ttagtggcca tcctttatta 19380

aataaattgg atgacacaga aacgtatagt gcttcgtaag caacgtaagg tgggccatcc 19440

tttattaaat aaattggatg acacagaaac gtatagtgct tcgtaagcaa cgtaaggtgt 19500

ggataataga gaatgtatat ctatggatta caaaacgtat agtgcttcgt aagcaacgta 19560

aggtgtggat aatagagaat gtatatctat ggattacaaa caaacacaat tgtgtttaat 19620

tggttgcaaa cctggataat agagaatgta tatctatgga ttacaaacaa acacaattgt 19680

gtttaattgg ttgcaaacca cctatagggg aacactgggg caaaggatcc ccaacaaaca 19740

caattgtgtt taattggttg caaaccacct ataggggaac actggggcaa aggatcccca 19800

tgtaccaatg ttgcagtaaa tccaggtgat tgccacctat aggggaacac tggggcaaag 19860

gatccccatg taccaatgtt gcagtaaatc caggtgattg tccaccatta gagttaataa 19920

acacagttat tccatgtacc aatgttgcag taaatccagg tgattgtcca ccattagagt 19980

taataaacac agttattcag gatggtgata tggttgatac tggctttggt gcgtccacca 20040

ttagagttaa taaacacagt tattcaggat ggtgatatgg ttgatactgg ctttggtgct 20100

atggacttta ctacattaca ggctaacaaa agggatggtg atatggttga tactggcttt 20160

ggtgctatgg actttactac attacaggct aacaaaagtg aagttccact ggatatttgt 20220

acatctattt gcgctatgga ctttactaca ttacaggcta acaaaagtga agttccactg 20280

gatatttgta catctatttg caaatatcca gattatatta aaatggtgtc aggtgaagtt 20340

ccactggata tttgtacatc tatttgcaaa tatccagatt atattaaaat ggtgtcagaa 20400

ccatatggcg acagcttatt tttttattta cggcaaatat ccagattata ttaaaatggt 20460

gtcagaacca tatggcgaca gcttattttt ttatttacga agggaacaaa tgtttgttag 20520

acatttattt aaccagatta tattaaaatg gtgtcagaac catatggcga cagcttattt 20580

ttttatttac gaagggaaca aatgtttgtt agacatttat ttaatagggc tgccatatgg 20640

cgacagctta tttttttatt tacgaaggga acaaatgttt gttagacatt tatttaatag 20700

ggctggtact gttggtgaaa atgtaccaga cggaagggaa caaatgtttg ttagacattt 20760

atttaatagg gctggtactg ttggtgaaaa tgtaccagac gatttataca ttaaaggctc 20820

tgggtctact gcatagggct ggtactgttg gtgaaaatgt accagacgat ttatacatta 20880

aaggctctgg gtctactgca aatttagcca gttcaaatta ttttcctaca ccgacgattt 20940

atacattaaa ggctctgggt ctactgcaaa tttagccagt tcaaattatt ttcctacacc 21000

tagtggttct atggttacct ctgcgtacca aagcaaattt agccagttca aattattttc 21060

ctacacctag tggttctatg gttacctctg cgtaccaaat attcaataaa ccttattggt 21120

tacaacgagc acttttccta cacctagtgg ttctatggtt acctctgcgt accaaatatt 21180

caataaacct tattggttac aacgagcaca gggccacaat aatggcctag tggttctatg 21240

gttacctctg cgtaccaaat attcaataaa ccttattggt tacaacgagc acagggccac 21300

aataatggca tttgttgggg taacctattc aataaacctt attggttaca acgagcacag 21360

ggccacaata atggcatttg ttggggtaac caactatttg ttactgttgt tgatactaca 21420

cgcagagggc cacaataatg gcatttgttg gggtaaccaa ctatttgtta ctgttgttga 21480

tactacacgc agtacaaata tgtcattatg tgctgccata tctacccaac tatttgttac 21540

tgttgttgat actacacgca gtacaaatat gtcattatgt gctgccatat ctacttcaga 21600

aactacatat aaaaatacta actttgtaca aatatgtcat tatgtgctgc catatctact 21660

tcagaaacta catataaaaa tactaacttt aaggagtacc tacgacatgg ggaggaatat 21720

gatttcttca gaaactacat ataaaaatac taactttaag gagtacctac gacatgggga 21780

ggaatatgat ttacagttta tttttcaact gtgcaaaata accttggagt acctacgaca 21840

tggggaggaa tatgatttac agtttatttt tcaactgtgc aaaataacct taactgcaga 21900

cgttatgaca tacatacatt ctatgtacag tttatttttc aactgtgcaa aataacctta 21960

actgcagacg ttatgacata catacattct atgaattcca ctattttgga ggactggaat 22020

tttggtaact gcagacgtta tgacatacat acattctatg aattccacta ttttggagga 22080

ctggaatttt ggtctacaac ctcccccagg aggcacacta gaagatgaat tccactattt 22140

tggaggactg gaattttggt ctacaacctc ccccaggagg cacactagaa gatacttata 22200

ggtttgtaac atcccaggca attgcggtct acaacctccc ccaggaggca cactagaaga 22260

tacttatagg tttgtaacat cccaggcaat tgcttgtcaa aaacatacac ctccagcacc 22320

taaaggatac ttataggttt gtaacatccc aggcaattgc ttgtcaaaaa catacacctc 22380

cagcacctaa agaagatccc cttaaaaaat acactttttg ggaaggcttg tcaaaaacat 22440

acacctccag cacctaaaga agatcccctt aaaaaataca ctttttggga agtaaattta 22500

aaggaaaagt tttctgcaga cctaggaaga tccccttaaa aaatacactt tttgggaagt 22560

aaatttaaag gaaaagtttt ctgcagacct agatcagttt cctttaggac gcaaattttt 22620

actacacttt ttgggaagta aatttaaagg aaaagttttc tgcagaccta gatcagtttc 22680

ctttaggacg caaattttta ctacaagcag gattgaaggc cgtaaattta aaggaaaagt 22740

tttctgcaga cctagatcag tttcctttag gacgcaaatt tttactacaa gcaggattga 22800

aggccaaacc aaaatttaca tatcagtttc ctttaggacg caaattttta ctacaagcag 22860

gattgaaggc caaaccaaaa tttacattag gaaaacgaaa agctacaccc accacctcat 22920

cgcaggattg aaggccaaac caaaatttac attaggaaaa cgaaaagcta cacccaccac 22980

ctcatctacc tctacaactg ctaaacgcaa aaaacgtaag ctaggaaaac gaaaagctac 23040

acccaccacc tcatctacct ctacaactgc taaacgcaaa aaacgtaagc tgtaagtatt 23100

gtatgtatgt tgaattagtg tctacctcta caactgctaa acgcaaaaaa cgtaagctgt 23160

aagtattgta tgtatgttga attagtgttg tttgttgtgt atatgtttgt atgtgcttgt 23220

agctgtaagt attgtatgta tgttgaatta gtgttgtttg ttgtgtatat gtttgtatgt 23280

gcttgtatgt gcttgtaaat attaagttgt atgtgtgttt gttgtttgtt gtgtatatgt 23340

ttgtatgtgc ttgtatgtgc ttgtaaatat taagttgtat gtgtgtttgt atgtatggta 23400

taataaacac gtgtgtatgt gatgtgcttg taaatattaa gttgtatgtg tgtttgtatg 23460

tatggtataa taaacacgtg tgtatgtgtt tttaacgtat tgtgtaacta ttgtgtccgt 23520

atgtatgtat ggtataataa acacgtgtgt atgtgttttt aacgtattgt gtaactattg 23580

tgtccgtaaa cataaataaa cttattgttt caacacctac tgtttttaac gtattgtgta 23640

actattgtgt ccgtaaacat aaataaactt attgtttcaa cacctactaa ttgtgttgtg 23700

gttattcatt gtatataaac tgcaacataa ataaacttat tgtttcaaca cctactaatt 23760

gtgttgtggt tattcattgt atataaacta tatttgctac atcctgtttt tgttttatat 23820

aattgtgttg tggttattca ttgtatataa actatatttg ctacatcctg tttttgtttt 23880

atatatacta tattttgtag cgccagcggc cattttgtag ctatatttgc tacatcctgt 23940

ttttgtttta tatatactat attttgtagc gccagcggcc attttgtagc ttcaaccgaa 24000

ttcggttgcc gtatttttgg cctatatttt gtagcgccag cggccatttt gtagcttcaa 24060

ccgaattcgg ttgccgtatt tttggcacaa aatgtgtttt tttaaatagt tctatgtcag 24120

cagcttcaac cgaattcggt tgccgtattt ttggcacaaa atgtgttttt ttaaatagtt 24180

ctatgtcagc aactatggtt taaacttgta cgtttcctgc tcacaaaatg tgttttttta 24240

aatagttcta tgtcagcaac tatggtttaa acttgtacgt ttcctgcttg cccgtagtgc 24300

caaatccctg ttttcctgac ccagcaacta tggtttaaac ttgtacgttt cctgcttgcc 24360

cgtagtgcca aatccctgtt ttcctgacct gcactgcttg ccaaccattc cattgttttt 24420

tttgcccgta gtgccaaatc cctgttttcc tgacctgcac tgcttgccaa ccattccatt 24480

gttttttaca ctgcactatg tgcaactact gaatcactat gctgcactgc ttgccaacca 24540

ttccattgtt ttttacactg cactatgtgc aactactgaa tcactatgta cattgtgtca 24600

tataaaataa atcactcgta gacactgcac tatgtgcaac tactgaatca ctatgtacat 24660

tgtgtcatat aaaataaatc actcgtagcc aacgccttac ataccgctgt taggcacata 24720

tgtacattgt gtcatataaa ataaatcact cgtagccaac gccttacata ccgctgttag 24780

gcacatattt ttggcttgtt ttaactaacc taattgcata tgcgccaacg ccttacatac 24840

cgctgttagg cacatatttt tggcttgttt taactaacct aattgcatat ttggcataag 24900

gtttaaactt ctaaggccaa ctttttggct tgttttaact aacctaattg catatttggc 24960

ataaggttta aacttctaag gccaactaaa tgtcacccta gttcatacat gaactgtgta 25020

attggcataa ggtttaaact tctaaggcca actaaatgtc accctagttc atacatgaac 25080

tgtgtaaagg ttagtcatac attgttcatt tgtaaaactg caatgtcacc ctagttcata 25140

catgaactgt gtaaaggtta gtcatacatt gttcatttgt aaaactgcac atgggtgtgt 25200

gcaaaccgtt ttgggttaca c 25221

<210> 2

<211> 27751

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe type HPV18

<400> 2

attaatactt ttaacaattg tagtatataa aaaagggagt aaccgaaaac ggtcgggacc 60

gaaaacggtg tatataaaag atgtgagaaa cacaccacaa agggagtaac cgaaaacggt 120

cgggaccgaa aacggtgtat ataaaagatg tgagaaacac accacaatac tatggcgcgc 180

tttgaggatc caacacggcg ccacaatact atggcgcgct ttgaggatcc aacacggcga 240

ccctacaagc tacctgatct gtgcacggaa ctgaacactt cactgcaaga catagaaata 300

cggcgaccct acaagctacc tgatctgtgc acggaactga acacttcact gcaagacata 360

gaaataacct gtgtatattg caagacagta ttggaactta cgaccctaca agctacctga 420

tctgtgcacg gaactgaaca cttcactgca agacatagaa ataacctgtg tatattgcaa 480

gacagtattg gaaccggaac tgaacacttc actgcaagac atagaaataa cctgtgtata 540

ttgcaagaca gtattggaac ttacagaggt atttgaattt gcatttaaag atttcctgtg 600

tatattgcaa gacagtattg gaacttacag aggtatttga atttgcattt aaagatttat 660

ttgtggtgta tagagacagt ataccgccgt atgccctgtg tatattgcaa gacagtattg 720

gaacttacag aggtatttga atttgcattt aaagatttat ttgtggtgta tagagacagt 780

ataccgccgt atgcacagag gtatttgaat ttgcatttaa agatttattt gtggtgtata 840

gagacagtat accgccgtat gccgtacata aatgtataga tttttattct agaatttgtg 900

gtgtatagag acagtatacc gccgtatgcc gtacataaat gtatagattt ttattctaga 960

attagagaat taagacatta ttcagactct gtgttggtgt atagagacag tataccgccg 1020

tatgccgtac ataaatgtat agatttttat tctagaatta gagaattaag acattattca 1080

gactctgtgt atgggccgta cataaatgta tagattttta ttctagaatt agagaattaa 1140

gacattattc agactctgtg tatggagaca cattggaaaa actaactaac actgtgccat 1200

aaatgtatag atttttattc tagaattaga gaattaagac attattcaga ctctgtgtat 1260

ggagacacat tggaaaaact aactaaagag aattaagaca ttattcagac tctgtgtatg 1320

gagacacatt ggaaaaacta actaacactg ggttatacaa tttattaata aggtgcctgc 1380

ggtgccagag aattaagaca ttattcagac tctgtgtatg gagacacatt ggaaaaacta 1440

actaacactg ggttatacaa tttattaata aggtgcctgc ggtgccgggt tatacaattt 1500

attaataagg tgcctgcggt gccagaaacc gttgaatcca gcagaaaaac ttagacacct 1560

taatgaaaaa cgacgatttc acaacaaatt tattaataag gtgcctgcgg tgccagaaac 1620

cgttgaatcc agcagaaaaa cttagacacc ttaatgaaaa acgacgattt cacaacatag 1680

cgtgccagaa accgttgaat ccagcagaaa aacttagaca ccttaatgaa aaacgacgat 1740

ttcacaacat agctgggcac tatagaggcc agtgccattc gagacacctt aatgaaaaac 1800

gacgatttca caacatagct gggcactata gaggccagtg ccattcgtgc tgcaaccgag 1860

cacgacagga acgactccaa cagctgggca ctatagaggc cagtgccatt cgtgctgcaa 1920

ccgagcacga caggaacgac tccaacgacg cagagaaaca caagtataat attaagtcgt 1980

acgacgcaga gaaacacaag tataatatta agtcgtaatg gacctaaggc aacattgcaa 2040

gacattgtat tgcatttaga gccccaaaat gaaattccgg ttgcatggac ctaaggcaac 2100

attgcaagac attgtattgc atttagagcc ccaaaatgaa attccggttg accttctatg 2160

tcacgagcaa ttaagcgact cgtattgcat ttagagcccc aaaatgaaat tccggttgac 2220

cttctatgtc acgagcaatt aagcgactca gaggaagaaa acgatgaaat agatggagtt 2280

attgaccttc tatgtcacga gcaattaagc gactcagagg aagaaaacga tgaaatagat 2340

ggagttaatc atcaacattt accagcccga cgagccgaac ccagaggaag aaaacgatga 2400

aatagatgga gttaatcatc aacatttacc agcccgacga gccgaaccac aacgtcacac 2460

aatgttgtgt atgtgttgta atcatcaaca tttaccagcc cgacgagccg aaccacaacg 2520

tcacacaatg ttgtgtatgt gttgtaagtg tgaagccaga attgagctag tagtagaaag 2580

cccacaacgt cacacaatgt tgtgtatgtg ttgtaagtgt gaagccagaa ttgagctagt 2640

agtagaaagc tcagcagacg accttcgagc attccagcag cagtgtgaag ccagaattga 2700

gctagtagta gaaagctcag cagacgacct tcgagcattc cagcagctgt ttctgaacac 2760

cctgtccttt gtgtgtccgt gctcagcaga cgaccttcga gcattccagc agctgtttct 2820

gaacaccctg tcctttgtgt gtccgtggtg tgcatcccag cagtaagcaa caatggctga 2880

ttgtttctga acaccctgtc ctttgtgtgt ccgtggtgtg catcccagca gtaagcaaca 2940

atggctgatc cagaaggtac agacggggag ggcacgggtt gcaccctgtc ctttgtgtgt 3000

ccgtggtgtg catcccagca gtaagcaaca atggctgatc cagaaggtac agacggggag 3060

ggcacgggtt gggtgtgcat cccagcagta agcaacaatg gctgatccag aaggtacaga 3120

cggggagggc acgggttgta acggctggtt ttatgtacaa gctattgtag accagaaggt 3180

acagacgggg agggcacggg ttgtaacggc tggttttatg tacaagctat tgtagacaaa 3240

aaaacaggag atgtaatatc agatgacgag gccagaaggt acagacgggg agggcacggg 3300

ttgtaacggc tggttttatg tacaagctat tgtagacaaa aaaacaggag atgtaatatc 3360

agatgacgag ggtaacggct ggttttatgt acaagctatt gtagacaaaa aaacaggaga 3420

tgtaatatca gatgacgagg acgaaacgta aacagacaca gggtcggata tacaaaaaaa 3480

caggagatgt aatatcagat gacgaggacg aaacgtaaac agacacaggg tcggatatgg 3540

tagattttat tgatacacaa ggaacatttt gcgaggacga aacgtaaaca gacacagggt 3600

cggatatggt agattttatt gatacacaag gaacattttg tgaacaggca gagctagaga 3660

cagcacaggc agaggacgaa acgtaaacag acacagggtc ggatatggta gattttattg 3720

atacacaagg aacattttgt gaacaggcag agctagagac agcacaggca ttggtagatt 3780

ttattgatac acaaggaaca ttttgtgaac aggcagagct agagacagca caggcattgt 3840

tcccgtagca ggaggtccac aatgcgtaac aggtagattt tattgataca caaggaacat 3900

tttgtgaaca ggcagagcta gagacagcac aggcattgtt cccgtagcag gaggtccaca 3960

atgcgtaaca gtgaacaggc agagctagag acagcacagg cattgttccc gtagcaggag 4020

gtccacaatg cgtaacaagt gttgcatgtt ttaaaacgaa agtttgcagg gtgaacaggc 4080

agagctagag acagcacagg cattgttccc gtagcaggag gtccacaatg cgtaacaagt 4140

gttgcatgtt ttaaaacgaa agtttgcagg gttcccgtag caggaggtcc acaatgcgta 4200

acaagtgttg catgttttaa aacgaaagtt tgcaggaggc agcacagaaa acagtccatt 4260

aggggagcgg gttcccgtag caggaggtcc acaatgcgta acaagtgttg catgttttaa 4320

aacgaaagtt tgcaggaggc agcacagaaa acagtccatt aggggagcgg caagtgttgc 4380

atgttttaaa acgaaagttt gcaggaggca gcacagaaaa cagtccatta ggggagcggc 4440

tggaggtgga tacagagtta agtccacggt caagtgttgc atgttttaaa acgaaagttt 4500

gcaggaggca gcacagaaaa cagtccatta ggggagcggc tggaggtgga tacagagtta 4560

agtccacggt ggaggcagca cagaaaacag tccattaggg gagcggctgg aggtggatac 4620

agagttaagt ccacggttac aagaaatatc tttaaatagt gggcagaaaa ggaggcagca 4680

cagaaaacag tccattaggg gagcggctgg aggtggatac agagttaagt ccacggttac 4740

aagaaatatc tttaaatagt gggcagaaaa ggctggaggt ggatacagag ttaagtccac 4800

ggttacaaga aatatcttta aatagtgggc agaaaaaggc aaaaaggcgg ctgtttacaa 4860

tatcagatag gtggatacag agttaagtcc acggttacaa gaaatatctt taaatagtgg 4920

gcagaaaaag gcaaaaaggc ggctgtttac aatatcagat agacaagaaa tatctttaaa 4980

tagtgggcag aaaaaggcaa aaaggcggct gtttacaata tcagatagtg gctatggctg 5040

ttctgaagtg gaagcacaag aaatatcttt aaatagtggg cagaaaaagg caaaaaggcg 5100

gctgtttaca atatcagata gtggctatgg ctgttctgaa gtggaagcaa cacagggcaa 5160

aaaggcggct gtttacaata tcagatagtg gctatggctg ttctgaagtg gaagcaacac 5220

agattcaggt aactacaaat ggcgaacatg gcggcgtggc tatggctgtt ctgaagtgga 5280

agcaacacag attcaggtaa ctacaaatgg cgaacatggc ggcaatgtat gtagtggcgg 5340

cagtacggag gctatagatt caggtaacta caaatggcga acatggcggc aatgtatgta 5400

gtggcggcag tacggaggct atagacaacg ggggcacaga gggcaacaac agcaggcaat 5460

gtatgtagtg gcggcagtac ggaggctata gacaacgggg gcacagaggg caacaacagc 5520

agtgtagacg gtacaagtga caatagcaat atagatagac aacgggggca cagagggcaa 5580

caacagcagt gtagacggta caagtgacaa tagcaatata gaaaatgtaa atccacaatg 5640

taccatagca caattgtgta gacggtacaa gtgacaatag caatatagaa aatgtaaatc 5700

cacaatgtac catagcacaa ttaaaagact tgttaaaagt aaacaataaa caaggaatgt 5760

aaatccacaa tgtaccatag cacaattaaa agacttgtta aaagtaaaca ataaacaagg 5820

agctatgtta gcagtattta aagacacata tgggctaaaa gacttgttaa aagtaaacaa 5880

taaacaagga gctatgttag cagtatttaa agacacatat gggctatcat ttacagattt 5940

agttagaaat tttaagagct atgttagcag tatttaaaga cacatatggg ctatcattta 6000

cagatttagt tagaaatttt aaaagtgata aaaccacgtg tacagattgg gttacgggct 6060

atcatttaca gatttagtta gaaattttaa aagtgataaa accacgtgta cagattgggt 6120

tacagctata tttggagtaa acccaacaat agcagttaca gatttagtta gaaattttaa 6180

aagtgataaa accacgtgta cagattgggt tacagctata tttggagtaa acccaacaat 6240

agcagaagga tttaaaaagt gataaaacca cgtgtacaga ttgggttaca gctatatttg 6300

gagtaaaccc aacaatagca gaaggattta aaacactaat acagccattt atattcagct 6360

atatttggag taaacccaac aatagcagaa ggatttaaaa cactaataca gccatttata 6420

ttatcgtacc atattcaatg tctagactgt aaatggaagg atttaaaaca ctaatacagc 6480

catttatatt atcgtaccat attcaatgtc tagactgtaa atggggagta ttaatattag 6540

ccctgttgcg ttacatatcg taccatattc aatgtctaga ctgtaaatgg ggagtattaa 6600

tattagccct gttgcgttac aaatgtggta agagtagact aacagttgct aaagggggga 6660

gtattaatat tagccctgtt gcgttacaaa tgtggtaaga gtagactaac agttgctaaa 6720

ggtttaagta cgttgttaca cgtacctgaa acttgaatgt ggtaagagta gactaacagt 6780

tgctaaaggt ttaagtacgt tgttacacgt acctgaaact tgtatgttaa ttcaaccacc 6840

aaaattgcga agtagggttt aagtacgttg ttacacgtac ctgaaacttg tatgttaatt 6900

caaccaccaa aattgcgaag tagtgttgca gcactatatt ggtatagaac aggaacgttg 6960

ttacacgtac ctgaaacttg tatgttaatt caaccaccaa aattgcgaag tagtgttgca 7020

gcactatatt ggtatagaac aggaatatca aatatgtatg ttaattcaac caccaaaatt 7080

gcgaagtagt gttgcagcac tatattggta tagaacagga atatcaaata ttagtgaagt 7140

aatgggagac acaccttcaa ccaccaaaat tgcgaagtag tgttgcagca ctatattggt 7200

atagaacagg aatatcaaat attagtgaag taatgggaga cacacctgag tggatccaaa 7260

attgcgaagt agtgttgcag cactatattg gtatagaaca ggaatatcaa atattagtga 7320

agtaatggga gacacacctg agtggataca aagacgtgtt gcagcactat attggtatag 7380

aacaggaata tcaaatatta gtgaagtaat gggagacaca cctgagtgga tacaaagact 7440

tactattata caacagcact atattggtat agaacaggaa tatcaaatat tagtgaagta 7500

atgggagaca cacctgagtg gatacaaaga cttactatta tacaacatgg gcactatatt 7560

ggtatagaac aggaatatca aatattagtg aagtaatggg agacacacct gagtggatac 7620

aaagacttac tattatacaa catggaatag tggtatagaa caggaatatc aaatattagt 7680

gaagtaatgg gagacacacc tgagtggata caaagactta ctattataca acatggaata 7740

gatgatagct atcaaatatt agtgaagtaa tgggagacac acctgagtgg atacaaagac 7800

ttactattat acaacatgga atagatgata gcaattttga tttgtcagat tagtgaagta 7860

atgggagaca cacctgagtg gatacaaaga cttactatta tacaacatgg aatagatgat 7920

agcaattttg atttgtcaga aatggtacac tgagtggata caaagactta ctattataca 7980

acatggaata gatgatagca attttgattt gtcagaaatg gtacaatggg catttgataa 8040

tgagctgact acaaagactt actattatac aacatggaat agatgatagc aattttgatt 8100

tgtcagaaat ggtacaatgg gcatttgata atgagctgac agatgaaagc atggaataga 8160

tgatagcaat tttgatttgt cagaaatggt acaatgggca tttgataatg agctgacaga 8220

tgaaagcgat atggcatttg aatcgtactg aaatggtaca atgggcattt gataatgagc 8280

tgacagatga aagcgatatg gcatttgaat cgtacttatt agcagacagc aacagcacgt 8340

aagctgcctc agatgaaagc gatatggcat ttgaatcgta cttattagca gacagcaaca 8400

gcacgtaagc tgccttttta aaaagcaatt gccaagctaa atatttaaag cgatatggca 8460

tttgaatcgt acttattagc agacagcaac agcacgtaag ctgccttttt aaaaagcaat 8520

tgccaagcta aatatttaaa agattgtgct attagcagac agcaacagca cgtaagctgc 8580

ctttttaaaa agcaattgcc aagctaaata tttaaaagat tgtgccacaa tgtgcaaaca 8640

ttataggcgt ttaaaaagca attgccaagc taaatattta aaagattgtg ccacaatgtg 8700

caaacattat aggcgagccc aaaaacgaca aatgaatatg tcacagtggg attgtgccac 8760

aatgtgcaaa cattataggc gagcccaaaa acgacaaatg aatatgtcac agtggatacg 8820

atttagatgt tcaaaaatag atgaaggggg agcccaaaaa cgacaaatga atatgtcaca 8880

gtggatacga tttagatgtt caaaaataga tgaaggggga gattggagac caatagtgca 8940

attcctgcgg gatacgattt agatgttcaa aaatagatga agggggagat tggagaccaa 9000

tagtgcaatt cctgcgatac caacaaatag agtttataac atttttaggg ggggagattg 9060

gagaccaata gtgcaattcc tgcgatacca acaaatagag tttataacat ttttaggagc 9120

cttaaaatca tttttaaaag gaacccccag cgataccaac aaatagagtt tataacattt 9180

ttaggagcct taaaatcatt tttaaaagga acccccaaaa aaaattgttt agtattttgt 9240

ggaccagcag gagccttaaa atcattttta aaaggaaccc ccaaaaaaaa ttgtttagta 9300

ttttgtggac cagcaaatac aggaaaatca tattttggaa tgagttttaa aaaaattgtt 9360

tagtattttg tggaccagca aatacaggaa aatcatattt tggaatgagt tttatacact 9420

ttatacaagg agcagtaata tcatttgtga tacaggaaaa tcatattttg gaatgagttt 9480

tatacacttt atacaaggag cagtaatatc atttgtgaat tccactagtc atttttggtt 9540

ggaaccgttt acactttata caaggagcag taatatcatt tgtgaattcc actagtcatt 9600

tttggttgga accgttaaca gatactaagg tggccatgtt agatgcgtag tgaattccac 9660

tagtcatttt tggttggaac cgttaacaga tactaaggtg gccatgttag atgcgtaaac 9720

gaccacgtgt tggacatact ttgatacctt aacagatact aaggtggcca tgttagatgc 9780

gtaaacgacc acgtgttgga catactttga tacctatatg agaacgtagt tagatggcaa 9840

tccaataagg caacgaccac gtgttggaca tactttgata cctatatgag aacgtagtta 9900

gatggcaatc caataagtat tgatagaaag cacaaaccat taatacaaca tatgagaacg 9960

tagttagatg gcaatccaat aagtattgat agaaagcaca aaccattaat acaactaaaa 10020

tgtcctccaa tactactaac cacaaatatg tattgataga aagcacaaac cattaataca 10080

actaaaatgt cctccaatac tactaaccac aaatatacat ccagcaaagg ataatagatg 10140

gccatatttc taaaatgtcc tccaatacta ctaaccacaa atatacatcc agcaaaggat 10200

aatagatggc catatttaga aagtagaata acagtatttg aatttccaat acatccagca 10260

aaggataata gatggccata tttagaaagt agaataacag tatttgaatt tccaacgtaa 10320

tttccatttg ataaaaatgg caatccagtg caaaggataa tagatggcca tatttagaaa 10380

gtagaataac agtatttgaa tttccaacgt aatttccatt tgataaaaat ggcaatccag 10440

tatatgaaat agaaagtaga ataacagtat ttgaatttcc aacgtaattt ccatttgata 10500

aaaatggcaa tccagtatat gaaataaatg acaaaaattg gaaatgtttg tagaataaca 10560

gtatttgaat ttccaacgta atttccattt gataaaaatg gcaatccagt atatgaaata 10620

aatgacaaaa attggaaatg tatttgaatt tccaacgtaa tttccatttg ataaaaatgg 10680

caatccagta tatgaaataa atgacaaaaa ttggaaatgt ttttttgaaa gccatttgat 10740

aaaaatggca atccagtata tgaaataaat gacaaaaatt ggaaatgttt ttttgaaagg 10800

acatggtcca gattagattt gcacgaggaa gaatccagta tatgaaataa atgacaaaaa 10860

ttggaaatgt ttttttgaaa ggacatggtc cagattagat ttgcacgagg aagaggaagc 10920

gtataaatga caaaaattgg aaatgttttt ttgaaaggac atggtccaga ttagatttgc 10980

acgaggaaga ggaagcgtaa gacaccgaag gaaacccttt cggggacatg gtccagatta 11040

gatttgcacg aggaagagga agcgtaagac accgaaggaa accctttcgg aacgtttaag 11100

tgcgttgcag gacaaaatga agcgtaagac accgaaggaa accctttcgg aacgtttaag 11160

tgcgttgcag gacaaaatca tagaccacta tgaaaatgac agtaaagaca tagacagcac 11220

cctttcggaa cgtttaagtg cgttgcagga caaaatcata gaccactatg aaaatgacag 11280

taaagacata gacagccaaa tacagtattg gcaactaaga acgtttaagt gcgttgcagg 11340

acaaaatcat agaccactat gaaaatgaca gtaaagacat agacagccaa atacagtatt 11400

ggcaactaat acgttgggat catagaccac tatgaaaatg acagtaaaga catagacagc 11460

caaatacagt attggcaact aatacgttgg gaaacgtaaa tattctttgc agcaagggga 11520

ccactatgaa aatgacagta aagacataga cagccaaata cagtattggc aactaatacg 11580

ttgggaaacg taaatattct ttgcagcaag ggaacatggc caaatacagt attggcaact 11640

aatacgttgg gaaacgtaaa tattctttgc agcaagggaa catggcatac agacattaaa 11700

ccaccaggtg gtgcctacgt tgggaaacgt aaatattctt tgcagcaagg gaacatggca 11760

tacagacatt aaaccaccag gtggtgccag cctataacat ttcaaaaagt aaagcaggga 11820

acatggcata cagacattaa accaccaggt ggtgccagcc tataacattt caaaaagtaa 11880

agcacataaa gctattgaac tgcaaatggc cctacaccac caggtggtgc cagcctataa 11940

catttcaaaa agtaaagcac ataaagctat tgaactgcaa atggccctac aaggccttgc 12000

acaaagtggt gccagcctat aacatttcaa aaagtaaagc acataaagct attgaactgc 12060

aaatggccct acaaggcctt gcacaaagtg catacaaaac cgagggcaca taaagctatt 12120

gaactgcaaa tggccctaca aggccttgca caaagtgcat acaaaaccga ggattggaca 12180

ctgcaagaca ccgtagagga actatgctat tgaactgcaa atggccctac aaggccttgc 12240

acaaagtgca tacaaaaccg aggattggac actgcaagac accgtagagg aactatggaa 12300

tacagcaagg ccttgcacaa agtgcataca aaaccgagga ttggacactg caagacaccg 12360

tagaggaact atggaataca gaacctactc actgctttaa aaaagggatt ggacactgca 12420

agacaccgta gaggaactat ggaatacaga acctactcac tgctttaaaa aaggtggcca 12480

aacagtacaa gtatattttg atggcctgca agacaccgta gaggaactat ggaatacaga 12540

acctactcac tgctttaaaa aaggtggcca aacagtacaa gtatattttg atggcaacaa 12600

agacatggaa tacagaacct actcactgct ttaaaaaagg tggccaaaca gtacaagtat 12660

attttgatgg caacaaagac aattgtatga cctatgtagc atgggggtgg ccaaacagta 12720

caagtatatt ttgatggcaa caaagacaat tgtatgacct atgtagcatg ggacagtgtg 12780

tattatatga ctgcgtaagg aacatacaaa gacaattgta tgacctatgt agcatgggac 12840

agtgtgtatt atatgactgc gtaaggaaca tgggacaaaa cggctacctg tgtaagtcac 12900

aggggggaca gtgtgtatta tatgactgcg taaggaacat gggacaaaac ggctacctgt 12960

gtaagtcaca ggggattgta ttatgtaaag gaagggtaca acacgtggga caaaacggct 13020

acctgtgtaa gtcacagggg attgtattat gtaaaggaag ggtacaacac gttttatata 13080

gaatttaaaa gtgaatgtga aaaatggatt gtattatgta aaggaagggt acaacacgtt 13140

ttatatagaa tttaaaagtg aatgtgaaaa atatgggaac acaggtacgt gggaagtaca 13200

ttttgttata tagaatttaa aagtgaatgt gaaaaatatg ggaacacagg tacgtgggaa 13260

gtacattttg ggaataatgt aattgattgt aatgactcta tgtgcaattt aaaagtgaat 13320

gtgaaaaata tgggaacaca ggtacgtggg aagtacattt tgggaataat gtaattgatt 13380

gtaatgactc tatgtgcagt accatatggg aacacaggta cgtgggaagt acattttggg 13440

aataatgtaa ttgattgtaa tgactctatg tgcagtacca gtgacgacac ggtatccgct 13500

actctacgtg ggaagtacat tttgggaata atgtaattga ttgtaatgac tctatgtgca 13560

gtaccagtga cgacacggta tccgctactc agcttgttag ggaataatgt aattgattgt 13620

aatgactcta tgtgcagtac cagtgacgac acggtatccg ctactcagct tgttaaacag 13680

ctacagcaca ccccctcact gcagtaccag tgacgacacg gtatccgcta ctcagcttgt 13740

taaacagcta cagcacaccc cctcaccgta ttccagcacc gtgtccgtgg gcaccgcaat 13800

cagcttgtta aacagctaca gcacaccccc tcaccgtatt ccagcaccgt gtccgtgggc 13860

accgcaaaga cctacggcca gacgtcggct gctacacgac cgtattccag caccgtgtcc 13920

gtgggcaccg caaagaccta cggccagacg tcggctgcta cacgacctgg acactgtgga 13980

ctcgcggaga agcagcatta agacctacgg ccagacgtcg gctgctacac gacctggaca 14040

ctgtggactc gcggagaagc agcattgtgg acctgtcaac ccacttctcg gtgcagctac 14100

ctggacactg tggactcgcg gagaagcagc attgtggacc tgtcaaccca cttctcggtg 14160

cagctacacc tacaggcaac aacaaaagac ggaaactctt gtggacctgt caacccactt 14220

ctcggtgcag ctacacctac aggcaacaac aaaagacgga aactctgtag tggtaacact 14280

acgcctataa tacatttaaa cacctacagg caacaacaaa agacggaaac tctgtagtgg 14340

taacactacg cctataatac atttaaaagg tgacagaaac agtttaaaat gtttacggtt 14400

gtagtggtaa cactacgcct ataatacatt taaaaggtga cagaaacagt ttaaaatgtt 14460

tacggtacag attgcgaaaa catagcgacc actatagaga ggtgacagaa acagtttaaa 14520

atgtttacgg tacagattgc gaaaacatag cgaccactat agagatatat catccacctg 14580

gcattggaca ggtgcaggcg atatatcatc cacctggcat tggacaggtg caggcaatga 14640

aaaaacagga atactgactg taacatacca tagtgaaaca caaagaacaa aatttttaag 14700

gcaatgaaaa aacaggaata ctgactgtaa cataccatag tgaaacacaa agaacaaaat 14760

ttttaaatac tgttgcaatt ccagatagtg tacaaatata ccatagtgaa acacaaagaa 14820

caaaattttt aaatactgtt gcaattccag atagtgtaca aatattggtg ggatacatga 14880

caatgtaata catcgtatga atactgttgc aattccagat agtgtacaaa tattggtggg 14940

atacatgaca atgtaataca tcgtatgtag taccaatatg ttatcactta tttttttatt 15000

tggtgggata catgacaatg taatacatcg tatgtagtac caatatgtta tcacttattt 15060

ttttattttg cttttgtgtc gtaatgtatg tgtgctgccg tagtaccaat atgttatcac 15120

ttattttttt attttgcttt tgtgtcgtaa tgtatgtgtg ctgccatgtc ccgcttttgc 15180

catctgtctg tatgtgtgct tttgcttttg tgtcgtaatg tatgtgtgct gccatgtccc 15240

gcttttgcca tctgtctgta tgtgtgcgtc gtaatgggta ttggtatttg tgtatattgc 15300

atgtcccgct tttgccatct gtctgtatgt gtgcgtcgta atgggtattg gtatttgtgt 15360

atattgtggt aataacgtcc cctgccacag cattcacagg cgtcgtaatg ggtattggta 15420

tttgtgtata ttgtggtaat aacgtcccct gccacagcat tcacagtata tgtattttgt 15480

tttttattgc ccatgttacg tggtaataac gtcccctgcc acagcattca cagtatatgt 15540

attttgtttt ttattgccca tgttactatt gcatataccg tatatattgt ctttacagtg 15600

tatatgtatt ttgtttttta ttgcccatgt tactattgca tataccgtat atattgtctt 15660

tacagtaatt gtataggttg ttttatacag tgtattgtac tattgcatat accgtatata 15720

ttgtctttac agtaattgta taggttgttt tatacagtgt attgtacatt gtatattttg 15780

ttttatacct tttcgtattt tgtataggtt gttttataca gtgtattgta cattgtatat 15840

tttgttttat accttttcgt atttttgtat ttttgtaata aaagtatggt atcccaccgt 15900

tttgtatttt tgtaataaaa gtatggtatc ccaccgtgcc gcacgacgca aacgggcttc 15960

ggtaactgac ttatataaaa catgtaaaca atctggtacc cgtgccgcac gacgcaaacg 16020

ggcttcggta actgacttat ataaaacatg taaacaatct ggtacatgtc cacctgatgt 16080

tgttcctaag gtggagggcg ccgcacgacg caaacgggct tcggtaactg acttatataa 16140

aacatgtaaa caatctggta catgtccacc tgatgttgtt cctaaggtgg agctgactta 16200

tataaaacat gtaaacaatc tggtacatgt ccacctgatg ttgttcctaa ggtggagggc 16260

accacgttag cagataaaat attgcaatgg tccatgtcca cctgatgttg ttcctaaggt 16320

ggagggcacc acgttagcag ataaaatatt gcaatggtca agccttggta tatttttggg 16380

tggacttggc atgcaccacg ttagcagata aaatattgca atggtcaagc cttggtatat 16440

ttttgggtgg acttggcata ggtactggca gtggtacagg gggtcgtaca ggcaagcctt 16500

ggtatatttt tgggtggact tggcataggt actggcagtg gtacaggggg tcgtacaggg 16560

tacattccat tgggtgggcg ttccaataca gttaggtact ggcagtggta cagggggtcg 16620

tacagggtac attccattgg gtgggcgttc caatacagtg gtggatgttg gtcctacacg 16680

tcccccagtg gtgtacattc cattgggtgg gcgttccaat acagtggtgg atgttggtcc 16740

tacacgtccc ccagtggtta ttgaacctgt gggccccaca gacccatcta tttggtggat 16800

gttggtccta cacgtccccc agtggttatt gaacctgtgg gccccacaga cccatctatt 16860

gttacattaa tagaggactc cagtgtggtt actattgaac ctgtgggccc cacagaccca 16920

tctattgtta cattaataga ggactccagt gtggttacat caggtgcacc taggcctacg 16980

tttactggca cgttgttaca ttaatagagg actccagtgt ggttacatca ggtgcaccta 17040

ggcctacgtt tactggcacg tctgggtttg atataacatc tgcgggtaca accatcaggt 17100

gcacctaggc ctacgtttac tggcacgtct gggtttgata taacatctgc gggtacaact 17160

acacctgcgg ttttggatat cacaccttcg tccgtctggg tttgatataa catctgcggg 17220

tacaactaca cctgcggttt tggatatcac accttcgtct acctctgtgt ctatttccac 17280

aaccaatttt acctacacct gcggttttgg atatcacacc ttcgtctacc tctgtgtcta 17340

tttccacaac caattttacc aatcctgcat tttctgatcc gtccattatt gactacctct 17400

gtgtctattt ccacaaccaa ttttaccaat cctgcatttt ctgatccgtc cattattgaa 17460

gttccacaaa ctggggaggt ggcaggtaat gtccaatcct gcattttctg atccgtccat 17520

tattgaagtt ccacaaactg gggaggtggc aggtaatgta tttgttggta cccctacatc 17580

tggaacacat ggaagttcca caaactgggg aggtggcagg taatgtattt gttggtaccc 17640

ctacatctgg aacacatggg tatgaggaaa tacctttaca aacatttgct tcccacaaac 17700

tggggaggtg gcaggtaatg tatttgttgg tacccctaca tctggaacac atgggtatga 17760

ggaaatacct ttacaaacat ttgctcaaac tggggaggtg gcaggtaatg tatttgttgg 17820

tacccctaca tctggaacac atgggtatga ggaaatacct ttacaaacat ttgcttcttc 17880

tggttgttgg tacccctaca tctggaacac atgggtatga ggaaatacct ttacaaacat 17940

ttgcttcttc tggtacgggg gaggaaccca ttagtagtac cccaaatacc tttacaaaca 18000

tttgcttctt ctggtacggg ggaggaaccc attagtagta ccccattgcc tactgtgcgg 18060

cgtgtagcag gtccccgcct ttattctggt acgggggagg aacccattag tagtacccca 18120

ttgcctactg tgcggcgtgt agcaggtccc cgcctttaca gtagggccta ccaacaagtg 18180

tcaaccccat tgcctactgt gcggcgtgta gcaggtcccc gcctttacag tagggcctac 18240

caacaagtgt cagtggctaa ccctgagttt cttacacgtc catccccgcc tttacagtag 18300

ggcctaccaa caagtgtcag tggctaaccc tgagtttctt acacgtccat cctctttaat 18360

tacatatgac aacccggcct ttgcagtggc taaccctgag tttcttacac gtccatcctc 18420

tttaattaca tatgacaacc cggcctttga gcctgtggac actacattaa catttgatcc 18480

tcgcctcttt aattacatat gacaacccgg cctttgagcc tgtggacact acattaacat 18540

ttgatcctcg tagtgatgtt cctgattcag attttatgga tatgcctgtg gacactacat 18600

taacatttga tcctcgtagt gatgttcctg attcagattt tatggatatt atccgtctac 18660

ataggcctgc tttaacatcc aggcgtagtg atgttcctga ttcagatttt atggatatta 18720

tccgtctaca taggcctgct ttaacatcca ggcgtgggac tgttcgcttt agtagattag 18780

gtcttatccg tctacatagg cctgctttaa catccaggcg tgggactgtt cgctttagta 18840

gattaggtca acgggcaact atgtttaccc gcagcggtac acaggcgtgg gactgttcgc 18900

tttagtagat taggtcaacg ggcaactatg tttacccgca gcggtacaca aataggtgct 18960

agggttcact tttatcatga tatcaacggg caactatgtt tacccgcagc ggtacacaaa 19020

taggtgctag ggttcacttt tatcatgata taagtcctat tgcaccttcc ccagaatata 19080

ttgcaaatag gtgctagggt tcacttttat catgatataa gtcctattgc accttcccca 19140

gaatatattg aactgcagcc tttagtatct gccacggagg acagtcctat tgcaccttcc 19200

ccagaatata ttgaactgca gcctttagta tctgccacgg aggacaatga cttgtttgat 19260

atatcgtaag atgacatgga cccactgcag cctttagtat ctgccacgga ggacaatgac 19320

ttgtttgata tatcgtaaga tgacatggac cctgcagtgc ctgtaccatc gcgttctact 19380

accatgactt gtttgatata tcgtaagatg acatggaccc tgcagtgcct gtaccatcgc 19440

gttctactac ctcctttgca ttttttaaat attcgcccac tatgaccctg cagtgcctgt 19500

accatcgcgt tctactacct cctttgcatt ttttaaatat tcgcccacta tatcttctgc 19560

ctcttcctat agtaatgtaa cggcctcctt tgcatttttt aaatattcgc ccactatatc 19620

ttctgcctct tcctatagta atgtaacggt ccctttaacc tcctcttggg atgtgcctgt 19680

ataatcttct gcctcttcct atagtaatgt aacggtccct ttaacctcct cttgggatgt 19740

gcctgtatac acgggtcctg atattacatt accatctact acctcccttt aacctcctct 19800

tgggatgtgc ctgtatacac gggtcctgat attacattac catctactac ctctgtatgg 19860

cccattgtat cacccacggc cccacacggg tcctgatatt acattaccat ctactacctc 19920

tgtatggccc attgtatcac ccacggcccc tgcctctaca cagtatattg gtatacatgg 19980

taccctctgt atggcccatt gtatcaccca cggcccctgc ctctacacag tatattggta 20040

tacatggtac acattattat ttgtggccat tatattattt tatcacatta ttatttgtgg 20100

ccattatatt attttattcc taagaaacgt aaacgtgttc cctatttttt tgcagatggc 20160

tttgtggcgg cctagtgaca atattcctaa gaaacgtaaa cgtgttccct atttttttgc 20220

agatggcttt gtggcggcct agtgacaata ccgtatatct tccacctcct tctgtggcaa 20280

gagaagaaac gtaaacgtgt tccctatttt tttgcagatg gctttgtggc ggcctagtga 20340

caataccgta tatcttccac ctccttctgt ggcaagagtt gtgcagatgg ctttgtggcg 20400

gcctagtgac aataccgtat atcttccacc tccttctgtg gcaagagttg taaataccga 20460

tgattatgtg actcgcacaa gcgatggctt tgtggcggcc tagtgacaat accgtatatc 20520

ttccacctcc ttctgtggca agagttgtaa ataccgatga ttatgtgact cgcacaagca 20580

taaccgtata tcttccacct ccttctgtgg caagagttgt aaataccgat gattatgtga 20640

ctcgcacaag catattttat ccgtatggca gctctagatt attatcttcc acctccttct 20700

gtggcaagag ttgtaaatac cgatgattat gtgactcgca caagcatatt ttatccgtat 20760

ggcagctcta gattattaac tggttgtaaa taccgatgat tatgtgactc gcacaagcat 20820

attttatccg tatggcagct ctagattatt aactgttggt aatccatatt ttagttgtaa 20880

ataccgatga ttatgtgact cgcacaagca tattttatcc gtatggcagc tctagattat 20940

taactgttgg taatccatat tttagggttc ctgagcatat tttatccgta tggcagctct 21000

agattattaa ctgttggtaa tccatatttt agggttcctg caggtggtgg caataagcag 21060

gatattccta aggttaactg ttggtaatcc atattttagg gttcctgcag gtggtggcaa 21120

taagcaggat attcctaagg tttctgcata ccaatataga gtatttaggg tgcgcaggtg 21180

gtggcaataa gcaggatatt cctaaggttt ctgcatacca atatagagta tttagggtgc 21240

agttacctga cccaaataaa tttggtttac ctgggtttct gcataccaat atagagtatt 21300

tagggtgcag ttacctgacc caaataaatt tggtttacct gatactagta tttataatcc 21360

tgaaacacaa cgtcagttac ctgacccaaa taaatttggt ttacctgata ctagtattta 21420

taatcctgaa acacaacgtt tagtgtgggc ctgtgctgga gtggaaattg gccgatacta 21480

gtatttataa tcctgaaaca caacgtttag tgtgggcctg tgctggagtg gaaattggcc 21540

gtggtcagcc tttaggtgtt ggccttagtg ggctagtgtg ggcctgtgct ggagtggaaa 21600

ttggccgtgg tcagccttta ggtgttggcc ttagtgggca tccattttat aataaattag 21660

atgacactga aagcgtggtc agcctttagg tgttggcctt agtgggcatc cattttataa 21720

taaattagat gacactgaaa gttccccgta cgccacgtct aatgtttctg aggcatccat 21780

tttataataa attagatgac actgaaagtt ccccgtacgc cacgtctaat gtttctgagg 21840

acgttaggga caatgtgtct gtagattata agcgttcccc gtacgccacg tctaatgttt 21900

ctgaggacgt tagggacaat gtgtctgtag attataagca gacacagtta tgtattttgg 21960

gctgtgcccc tgcggacgtt agggacaatg tgtctgtaga ttataagcag acacagttat 22020

gtattttggg ctgtgcccct gctattgggg aacactgggc taaaggcact gctcagacac 22080

agttatgtat tttgggctgt gcccctgcta ttggggaaca ctgggctaaa ggcactgctt 22140

gtaaatcgcg tcctttatca cagggcgatt gccgctattg gggaacactg ggctaaaggc 22200

actgcttgta aatcgcgtcc tttatcacag ggcgattgcc cccctttaga acttaaaaac 22260

acagttttgg aagtgtaaat cgcgtccttt atcacagggc gattgccccc ctttagaact 22320

taaaaacaca gttttggaag atggtgatat ggtagatact ggatatggtg ccaccccctt 22380

tagaacttaa aaacacagtt ttggaagatg gtgatatggt agatactgga tatggtgcca 22440

tggactttag tacattgcaa gatactaaat gtggatggtg atatggtaga tactggatat 22500

ggtgccatgg actttagtac attgcaagat actaaatgtg aggtaccatt ggatatttgt 22560

cagtctattt gtatggactt tagtacattg caagatacta aatgtgaggt accattggat 22620

atttgtcagt ctatttgtaa atatcctgat tatttacaaa tgtctgcaga tccgaggtac 22680

cattggatat ttgtcagtct atttgtaaat atcctgatta tttacaaatg tctgcagatc 22740

cttatgggga ttccatgttt ttttgcttac ggccagtcta tttgtaaata tcctgattat 22800

ttacaaatgt ctgcagatcc ttatggggat tccatgtttt tttgcttacg gcgtgagcag 22860

ctttttgcta ggcaatatcc tgattattta caaatgtctg cagatcctta tggggattcc 22920

atgttttttt gcttacggcg tgagcagctt tttgctaggc atttttggaa tagccttatg 22980

gggattccat gtttttttgc ttacggcgtg agcagctttt tgctaggcat ttttggaata 23040

gagcaggtac tatgggtgac actgtgcctc aatggcgtga gcagcttttt gctaggcatt 23100

tttggaatag agcaggtact atgggtgaca ctgtgcctca atccttatat attaaaggca 23160

caggtcgtag tgcgagcagg tactatgggt gacactgtgc ctcaatcctt atatattaaa 23220

ggcacaggtc gtagtgcttc acctggcagc tgtgtgtatt ctccctctcc aagtccttat 23280

atattaaagg cacaggtcgt agtgcttcac ctggcagctg tgtgtattct ccctctccaa 23340

gtggctctat tgttacctct gactcccagt tgtcacctgg cagctgtgtg tattctccct 23400

ctccaagtgg ctctattgtt acctctgact cccagttgtt taataaacca tattggttac 23460

ataaggcaca gggagtggct ctattgttac ctctgactcc cagttgttta ataaaccata 23520

ttggttacat aaggcacagg gtcataacaa tggtgtttgc tggcataatc aatgtttaat 23580

aaaccatatt ggttacataa ggcacagggt cataacaatg gtgtttgctg gcataatcaa 23640

ttatttgtta ctgtggtaga tactaataaa ccatattggt tacataaggc acagggtcat 23700

aacaatggtg tttgctggca taatcaatta tttgttactg tggtagatac cactcgcagt 23760

accggtcata acaatggtgt ttgctggcat aatcaattat ttgttactgt ggtagatacc 23820

actcgcagta ccaatttaac aatatgtgct tctacacagt ctcttatttg ttactgtggt 23880

agataccact cgcagtacca atttaacaat atgtgcttct acacagtctc ctgtacctgg 23940

gcaatatgcg tataccaaat ttaaccaatt taacaatatg tgcttctaca cagtctcctg 24000

tacctgggca atatgcgtat accaaattta agcagtatag cagacatgtt gaggaatatg 24060

attcctgtac ctgggcaata tgcgtatacc aaatttaagc agtatagcag acatgttgag 24120

gaatatgatt tgcagtttat ttttcagttg tgtactatta ctttttaagc agtatagcag 24180

acatgttgag gaatatgatt tgcagtttat ttttcagttg tgtactatta ctttaactgc 24240

agatgttatg tcctatattc ataagcagta tagcagacat gttgaggaat atgatttgca 24300

gtttattttt cagttgtgta ctattacttt aactgcagat gttatgtcct atattcatag 24360

tatgatttgc agtttatttt tcagttgtgt actattactt taactgcaga tgttatgtcc 24420

tatattcata gtatgaatag cagtatttta gaggattgga atgcagttta tttttcagtt 24480

gtgtactatt actttaactg cagatgttat gtcctatatt catagtatga atagcagtat 24540

tttagaggat tggaactttg gttaactgca gatgttatgt cctatattca tagtatgaat 24600

agcagtattt tagaggattg gaactttggt gttccccccc cgccaactac tagtttggtg 24660

gagtatgaat agcagtattt tagaggattg gaactttggt gttccccccc cgccaactac 24720

tagtttggtg gatacatatc gttttgtaca atctgttgct gaatagcagt attttagagg 24780

attggaactt tggtgttccc cccccgccaa ctactagttt ggtggataca tatcgttttg 24840

tacaatctgt tgctattacc ggtgttcccc ccccgccaac tactagtttg gtggatacat 24900

atcgttttgt acaatctgtt gctattacct gtcaaaaggc gtatgcaccg gctgaaaata 24960

gatacatatc gttttgtaca atctgttgct attacctgtc aaaaggcgta tgcaccggct 25020

gaaaataagg atccctatga taagttaaag ttttggaatg acctgtcaaa aggcgtatgc 25080

accggctgaa aataaggatc cctatgataa gttaaagttt tggaatgtgg atttaaagga 25140

aaagttttct ttagacttag aaggatccct atgataagtt aaagttttgg aatgtggatt 25200

taaaggaaaa gttttcttta gacttagatc aatatcccct tggacgtaaa tttttggttc 25260

gtggatttaa aggaaaagtt ttctttagac ttagatcaat atccccttgg acgtaaattt 25320

ttggttcagg ctggattgcg tcgcaagccc accataggcc gatcaatatc cccttggacg 25380

taaatttttg gttcaggctg gattgcgtcg caagcccacc ataggccctc gcaaacgttc 25440

tgctccatct gccactacgt tcaggctgga ttgcgtcgca agcccaccat aggccctcgc 25500

aaacgttctg ctccatctgc cactacgtct tctaaacctg ccaagcgtgt gcgtgtacgt 25560

ggccctcgca aacgttctgc tccatctgcc actacgtctt ctaaacctgc caagcgtgtg 25620

cgtgtacgtg ccaggaagta atatgtgtgt gtgtatatat cgtcttctaa acctgccaag 25680

cgtgtgcgtg tacgtgccag gaagtaatat gtgtgtgtgt atatatatat acatctattg 25740

ttgtgtttgt atgtcctgtg gccaggaagt aatatgtgtg tgtgtatata tatatacatc 25800

tattgttgtg tttgtatgtc ctgtgtttgt gtttgttgta tgattgcatt gtatggtatg 25860

atacatctat tgttgtgttt gtatgtcctg tgtttgtgtt tgttgtatga ttgcattgta 25920

tggtatgtat ggttgttgtt gtatgttgta tgttactata ttgtgtttgt tgtatgattg 25980

cattgtatgg tatgtatggt tgttgttgta tgttgtatgt tactatattt gttggtatgt 26040

ggcattaaat aaaatatgtt gtatggttgt tgttgtatgt tgtatgttac tatatttgtt 26100

ggtatgtggc attaaataaa atatgttttg tggttctgtg tgttatgtgg ttgcgcccta 26160

ttgttggtat gtggcattaa ataaaatatg ttttgtggtt ctgtgtgtta tgtggttgcg 26220

ccctagtgag taacaactgt atttgtgttt gtggtatggg tttgtggttc tgtgtgttat 26280

gtggttgcgc cctagtgagt aacaactgta tttgtgtttg tggtatgggt gttgcttgtt 26340

gggctatata ttgtcctgta gtgagtaaca actgtatttg tgtttgtggt atgggtgttg 26400

cttgttgggc tatatattgt cctgtatttc aagttataaa actgcacacc ttacagcatc 26460

gggtgttgct tgttgggcta tatattgtcc tgtatttcaa gttataaaac tgcacacctt 26520

acagcatcca ttttatccta caatcctcca ttttgctgtg atttcaagtt ataaaactgc 26580

acaccttaca gcatccattt tatcctacaa tcctccattt tgctgtgcaa ccgatttcgg 26640

ttgcctttgg cttatgtctg tccattttat cctacaatcc tccattttgc tgtgcaaccg 26700

atttcggttg cctttggctt atgtctgtgg ttttctgcac aatacagtac gctggcacta 26760

gcaaccgatt tcggttgcct ttggcttatg tctgtggttt tctgcacaat acagtacgct 26820

ggcactattg caaactttaa tcttttgggc actgctccta gtggttttct gcacaataca 26880

gtacgctggc actattgcaa actttaatct tttgggcact gctcctacat attttgaaca 26940

attggcgcgc ctctttggcg attgcaaact ttaatctttt gggcactgct cctacatatt 27000

ttgaacaatt ggcgcgcctc tttggcgcat ataaggcgca cctggtatta gtcattttcc 27060

catattttga acaattggcg cgcctctttg gcgcatataa ggcgcacctg gtattagtca 27120

ttttcctgtc caggtgcgct acaacaattg cttgcataac cacctggtat tagtcatttt 27180

cctgtccagg tgcgctacaa caattgcttg cataactata tccactccct aagtaataaa 27240

actgctttta gctgtccagg tgcgctacaa caattgcttg cataactata tccactccct 27300

aagtaataaa actgctttta ggcacatatt ttagtttgtt tttacttaag cactatatcc 27360

actccctaag taataaaact gcttttaggc acatatttta gtttgttttt acttaagcta 27420

attgcatact tggcttgtac aactactttc attaggcaca tattttagtt tgtttttact 27480

taagctaatt gcatacttgg cttgtacaac tactttcatg tccaacattc tgtctaccct 27540

taacatgaac tgctaattgc atacttggct tgtacaacta ctttcatgtc caacattctg 27600

tctaccctta acatgaacta taatatgact aagctgtgca tacatagttt acatgtccaa 27660

cattctgtct acccttaaca tgaactataa tatgactaag ctgtgcatac atagtttcgt 27720

aaaccgaaat aggttgggca gcacatacta t 27751

<210> 3

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 3

caagcagaag acggcatacg agattgcatg acgtgactgg agttcagacg tgt 53

<210> 4

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 4

caagcagaag acggcatacg agattgctat cggtgactgg agttcagacg tgt 53

<210> 5

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 5

caagcagaag acggcatacg agatcacaag cagtgactgg agttcagacg tgt 53

<210> 6

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 6

caagcagaag acggcatacg agattcgctg atgtgactgg agttcagacg tgt 53

<210> 7

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 7

caagcagaag acggcatacg agatcgcttt gtgtgactgg agttcagacg tgt 53

<210> 8

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 8

caagcagaag acggcatacg agatccgatg aagtgactgg agttcagacg tgt 53

<210> 9

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 9

caagcagaag acggcatacg agattgacca cagtgactgg agttcagacg tgt 53

<210> 10

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 10

caagcagaag acggcatacg agatgaagcc atgtgactgg agttcagacg tgt 53

<210> 11

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 11

caagcagaag acggcatacg agatttctgg tggtgactgg agttcagacg tgt 53

<210> 12

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 12

caagcagaag acggcatacg agatccgaaa acgtgactgg agttcagacg tgt 53

<210> 13

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 13

caagcagaag acggcatacg agatcgaaaa gggtgactgg agttcagacg tgt 53

<210> 14

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 14

caagcagaag acggcatacg agataaccag ctgtgactgg agttcagacg tgt 53

<210> 15

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> joint

<400> 15

ctacaataat tcatgtataa aactaagggc tagtataaaa gcagacattt tcgtaaccaa 60

Claims (7)

1. A probe set for detecting human papillomaviruses HPV16 and HPV18, characterized by comprising the following probes: SEQ ID NO.1 and/or SEQ ID NO. 2.

2. A kit for detecting human papillomaviruses HPV16 and HPV18, characterized by comprising a set of probes according to 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 the probe set of human papillomavirus HPV16 and HPV18 according to claim 1, characterized in that it comprises the following steps:

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

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

(3) and a high-throughput sequencing method,

(4) and bioinformatic analysis of gene sequences.

7. The panel of claim 1 for use in human papillomavirus HPV typing and integration detection of non-diagnostic non-therapeutic interest.

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