CN112195280B - Probe for detecting human papilloma virus HPV39 and kit thereof - Google Patents

Abstract

The invention discloses a probe for detecting human papillomavirus HPV39 and a kit thereof, wherein the probe group comprises probes capable of specifically capturing HPV39 genome; the invention has comprehensive detection range, greatly improved detection efficiency and accurate result.

Description

Probe for detecting human papilloma virus HPV39 and kit thereof

Technical Field：

The invention relates to a probe and a kit thereof, in particular to a probe and a kit for capturing and sequencing human papillomavirus HPV39 genes.

Background：

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

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

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

Disclosure of Invention：

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

The invention adopts the following technical scheme:

the invention provides a probe for detecting HPV39 typing and integration of human papillomavirus, the probe information is shown in Table 1

The invention also provides a kit for detecting human papillomavirus HPV39 typing and integration. The kit comprises the probe for detecting human papillomavirus HPV39 typing and integration; preferably, the kit further comprises a library buffer 1, a library buffer 2, an enzyme 1, an enzyme 2, a linker, a PCR amplification primer 1, a PCR amplification primer 2, a PCR reaction mixture, a blocking sequence 1, a blocking sequence 2, a blocking reagent, a hybridization buffer, a PCR amplification primer 3, a PCR amplification reaction solution, a positive control, a negative control, a capture buffer, a cleaning buffer 1, a cleaning buffer 2 and a capture magnetic bead; preferably, the blocking sequence 1 is Cot-1 DNA and the blocking sequence 2 is a double-stranded oligonucleotide.

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

The invention also discloses a method for using the probe for detecting human papillomavirus HPV39 typing and integration and a kit thereof, which mainly comprises the following steps:

(1) Design of Capture probes for HPV39

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

(3) Library target region targeted capture and quantification

(4) High throughput sequencing

(5) Bioinformatic analysis of gene sequences

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

The invention has the following beneficial effects:

(1) The invention designs the probe capable of rapidly and accurately capturing the complete sequence of HPV39 genome, and can effectively improve the detection specificity. The traditional methods such as fluorescent quantitative PCR only aim at a certain section of the HPV sequence, and the detection range of the probe can cover the whole length of the HPV genome.

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

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

Drawings：

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

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

The specific embodiment is as follows:

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

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

According to the base complementary pairing principle, oligonucleotide probes complementary to the DNA sequence of HPV39 are designed. Because of the complex structure of the genome sequence, irregular regions such as high GC, high AT regions and the like exist, and meanwhile, because of the limitation of the second generation sequencing read length, the design of the probe becomes particularly critical, and a plurality of factors such as coverage rate, uniformity, capture efficiency and the like need to be comprehensively balanced. The specific principle of probe design is as follows:

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

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

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

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

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

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

The kit reagent for HPV detection described in the example.

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

the using method of the kit comprises the following steps:

1. sample library preparation

(1) Fragmenting: 1000 ng of DNA was removed and thawed, shaken and mixed well, added with water to 100. Mu.L, and broken by a Biorupter Pico ultrasonic breaker. The specific parameters are set as follows: setting parameters of ON 30 seconds and OFF 30 seconds as 1 cycle after the temperature of the cold circulation instrument is reduced to 4 ℃, wherein every 10 cycles are one round, and carrying out 3 rounds in total; 1. Mu.L of the sample was subjected to fragment detection using Qsep1, and the main peak of the sample detection after normal disruption was about 150bp-200 bp.

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

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

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

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

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

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

2. Sample liquid phase hybridization

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

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

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

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

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

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

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

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

3. Capturing

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

4. Capture library quality control

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

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

5. Library qPCR quantification

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

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

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

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

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

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

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

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

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

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

6. Sequencing on machine

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

(1) Reagent Cartridge, HT1 and FlowCell are taken out in advance, the FlowCell is placed in a room temperature balance 30min,Reagent Cartridge and placed in a room temperature water bath for 1h, and the HT1 is placed at room temperature for thawing and then is mixed by shaking.

(2) Mixing all sample libraries to be sequenced according to the requirement of sequencing data, 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 centrifuge tube with equal volume, taking each library for mixing, and keeping the total volume after mixing at least 5 mu L.

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

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

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

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

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

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

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

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

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

(2) And (3) reading record information of an InterOp catalog in the sequencing file by using the iltiming v0.6 software, and carrying out quality analysis and evaluation on the sequencing. Fastpvc 0.20.0 for quality control requires Q30 mass values greater than 80%.

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

(4) Sequence alignment: the base sequences in fastq files were aligned to human reference genome hg38 (GRCh 38) and HPV genomes to generate bam files (based on BWA v0.7.17, samroolsv 1.9 and picard v2.20.6 software: alignment).

(5) HPV type panel analysis: the numbers and proportions of reads for each HPV in the samples were analyzed by sequence alignment files using a typing detection module (based on hpvhhpvkittyper v1.0 software).

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

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

Example 3: use effect verification of the kit

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

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

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

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SEQUENCE LISTING

<110> Kadetweisi biotechnology Co., ltd

<120> a probe for detecting human papillomavirus HPV39 and kit therefor

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<160> 14

<170> PatentIn version 3.5

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<211> 28053

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe type HPV39

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ggacagtgtc gacggtgctg gaccacaaaa cgggaggacc gcagactaac agcaggaagc 2100

tatacaggac agtgtcgacg gtgctggacc acaaaacggg aggaccgcag actaacacga 2160

agagaaaccc aagtataaca tcagatcgta gctggaccac aaaacgggag gaccgcagac 2220

taacacgaag agaaacccaa gtataacatc agatcgtagt ggaccaaagc ccaccttgca 2280

ggaaattgta acgaagagaa acccaagtat aacatcagat cgtagtggac caaagcccac 2340

cttgcaggaa attgtattag atttatgtcc ttacaatgaa atacagccgg gcgtggacca 2400

aagcccacct tgcaggaaat tgtattagat ttatgtcctt acaatgaaat acagccggtt 2460

gaccttgtat gtcacgagca attaggagag ggaccaaagc ccaccttgca ggaaattgta 2520

ttagatttat gtccttacaa tgaaatacag ccggttgacc ttgtatgtca cgagcaatta 2580

ggagagtcag tatgtcctta caatgaaata cagccggttg accttgtatg tcacgagcaa 2640

ttaggagagt cagaggatga aatagatgaa cccgacccgt aagttagttg accttgtatg 2700

tcacgagcaa ttaggagagt cagaggatga aatagatgaa cccgacccgt aagttaatca 2760

ccaacatcaa ctactagcca gacggggacc ttgtatgtca cgagcaatta ggagagtcag 2820

aggatgaaat agatgaaccc gacccgtaag ttaatcacca acatcaacta ctagccagac 2880

gggcagagga tgaaatagat gaacccgacc cgtaagttaa tcaccaacat caactactag 2940

ccagacggga tgaaccacag cgtcacacaa tacagtgttc gtggttaatc accaacatca 3000

actactagcc agacgggatg aaccacagcg tcacacaata cagtgttcgt gttgtaagtg 3060

taacaacaca ctgcagctgg tagcaccaac atcaactact agccagacgg gatgaaccac 3120

agcgtcacac aatacagtgt tcgtgttgta agtgtaacaa cacactgcag ctggtagtag 3180

gatgaaccac agcgtcacac aatacagtgt tcgtgttgta agtgtaacaa cacactgcag 3240

ctggtagtag aagcctcacg ggatactctg cgacaactac gtgttgtaag tgtaacaaca 3300

cactgcagct ggtagtagaa gcctcacggg atactctgcg acaactacag cagctgttta 3360

tggactcact aggatttgtg gtagaagcct cacgggatac tctgcgacaa ctacagcagc 3420

tgtttatgga ctcactagga tttgtgtgtc cgtggtgtgc aactgcaaac cagtaacctg 3480

acagcagctg tttatggact cactaggatt tgtgtgtccg tggtgtgcaa ctgcaaacca 3540

gtaacctgct atggccaatc gtgaaggtac agacggggat tgtccgtggt gtgcaactgc 3600

aaaccagtaa cctgctatgg ccaatcgtga aggtacagac ggggatgggt cgggatgtaa 3660

cggatggttt ctagtacagg accagtaacc tgctatggcc aatcgtgaag gtacagacgg 3720

ggatgggtcg ggatgtaacg gatggtttct agtacaggca atagtagata aacaaacagg 3780

gctatggcca atcgtgaagg tacagacggg gatgggtcgg gatgtaacgg atggtttcta 3840

gtacaggcaa tagtagataa acaaacaggc gacacagtgt gggtcgggat gtaacggatg 3900

gtttctagta caggcaatag tagataaaca aacaggcgac acagtgtcgg aggatgagga 3960

tgaaacgtaa acagatacag ggcaatagta gataaacaaa caggcgacac agtgtcggag 4020

gatgaggatg aaacgtaaac agatacaggt tcagacctgg cagactttat tgatgattcc 4080

caatagtaga taaacaaaca ggcgacacag tgtcggagga tgaggatgaa acgtaaacag 4140

atacaggttc agacctggca gactttattg atgattccac cggaggatga ggatgaaacg 4200

taaacagata caggttcaga cctggcagac tttattgatg attccacaga tatttgtgta 4260

caggcagagc gtgagacagc cggaggatga ggatgaaacg taaacagata caggttcaga 4320

cctggcagac tttattgatg attccacaga tatttgtgta caggcagagc gtgagacagc 4380

ggttcagacc tggcagactt tattgatgat tccacagata tttgtgtaca ggcagagcgt 4440

gagacagcac aggtactttt acatcgtaaa gaggcccaaa gttcagacct ggcagacttt 4500

attgatgatt ccacagatat ttgtgtacag gcagagcgtg agacagcaca ggtactttta 4560

catcgtaaag aggcccaaag ccacagatat ttgtgtacag gcagagcgtg agacagcaca 4620

ggtactttta catcgtaaag aggcccaaag ggcgtaacaa gcagtgcgtg ccttaaaacg 4680

cagatatttg tgtacaggca gagcgtgaga cagcacaggt acttttacat cgtaaagagg 4740

cccaaagggc gtaacaagca gtgcgtgcct taaaacgaaa gcacaggtac ttttacatcg 4800

taaagaggcc caaagggcgt aacaagcagt gcgtgcctta aaacgaaagt atacagacag 4860

cagtggcgac actagaccgt gggcgtaaca agcagtgcgt gccttaaaac gaaagtatac 4920

agacagcagt ggcgacacta gaccgtatgg aaaaaaagta ggcaggaata ccaggggaac 4980

gtatacagac agcagtggcg acactagacc gtatggaaaa aaagtaggca ggaataccag 5040

gggaacacta caggaaattt cattaaatgt aagcagtacg tatggaaaaa aagtaggcag 5100

gaataccagg ggaacactac aggaaatttc attaaatgta agcagtacgc aggcaacaca 5160

aacggtgtat tccgtgccag cactacagga aatttcatta aatgtaagca gtacgcaggc 5220

aacacaaacg gtgtattccg tgccagacag cggatatggc aatatggaag tggaaacagc 5280

cgcaggcaac acaaacggtg tattccgtgc cagacagcgg atatggcaat atggaagtgg 5340

aaacagctga agtggaggag gtaactgtag caactaatac gacagcggat atggcaatat 5400

ggaagtggaa acagctgaag tggaggaggt aactgtagca actaatacaa atggggcgta 5460

tgaaggggaa catggcggca agctgaagtg gaggaggtaa ctgtagcaac taatacaaat 5520

ggggcgtatg aaggggaaca tggcggcagt gtacgggagg agtgcagtag tgtggatagt 5580

acaaatgggg cgtatgaagg ggaacatggc ggcagtgtac gggaggagtg cagtagtgtg 5640

gatagtgcta tagatagtga aaaccaggat cccaaatctc cagtgtacgg gaggagtgca 5700

gtagtgtgga tagtgctata gatagtgaaa accaggatcc caaatctcca actgcacaaa 5760

ttaaattatt gttacaatcc gtgtggatag tgctatagat agtgaaaacc aggatcccaa 5820

atctccaact gcacaaatta aattattgtt acaatccaat aacaaaaagg ctgcacgtat 5880

gctatagata gtgaaaacca ggatcccaaa tctccaactg cacaaattaa attattgtta 5940

caatccaata acaaaaaggc tgcacgtata acacaattta ccaactgcac aaattaaatt 6000

attgttacaa tccaataaca aaaaggctgc acgtataaca caatttaaag aaacatatgg 6060

actatccttt actgacctgg ccaataacaa aaaggctgca cgtataacac aatttaaaga 6120

aacatatgga ctatccttta ctgacctggt acgtacgttt aaaagtgata aaacaacatg 6180

taacacaatt taaagaaaca tatggactat cctttactga cctggtacgt acgtttaaaa 6240

gtgataaaac aacatgtaca gactgggtgg cagccatatt aagaaacata tggactatcc 6300

tttactgacc tggtacgtac gtttaaaagt gataaaacaa catgtacaga ctgggtggca 6360

gccatatttg gagtacatcc ttactgacct ggtacgtacg tttaaaagtg ataaaacaac 6420

atgtacagac tgggtggcag ccatatttgg agtacatcca actattgcag aaggatttaa 6480

ggtacgtacg tttaaaagtg ataaaacaac atgtacagac tgggtggcag ccatatttgg 6540

agtacatcca actattgcag aaggatttaa aacattaatc acagactggg tggcagccat 6600

atttggagta catccaacta ttgcagaagg atttaaaaca ttaatcaaca aatcgtactt 6660

atatacacat atacaaagct ccaactattg cagaaggatt taaaacatta atcaacaaat 6720

cgtacttata tacacatata caaagcttag acacaaaaca aggagtacta attttacgta 6780

acaaatcgta cttatataca catatacaaa gcttagacac aaaacaagga gtactaattt 6840

tacgtataat aagatataca tgtggaaaaa atagggttac ttagacacaa aacaaggagt 6900

actaatttta cgtataataa gatatacatg tggaaaaaat agggttactg taggaaaggg 6960

attaagtaca ttgttacatg gctaataaga tatacatgtg gaaaaaatag ggttactgta 7020

ggaaagggat taagtacatt gttacatgtt ccagaaagtt gtcgtattct ggagcctcct 7080

actgtaggaa agggattaag tacattgtta catgttccag aaagttgtcg tattctggag 7140

cctcctaaac tgcgcagccc tgtagcagca ctatattggt gttccagaaa gttgtcgtat 7200

tctggagcct cctaaactgc gcagccctgt agcagcacta tattggtatc gcacaggtat 7260

atccaatatt agtgtggtaa aactgcgcag ccctgtagca gcactatatt ggtatcgcac 7320

aggtatatcc aatattagtg tggtaacagg ggatacgcca gaatggatac aacgattaac 7380

acaggggata cgccagaatg gatacaacga ttaactgtta tacaacatgg aatagatgat 7440

agtgtatttg acctatcgga catggtacaa tgggcatttg actgttatac aacatggaat 7500

agatgatagt gtatttgacc tatcggacat ggtacaatgg gcatttgaca atgaatatac 7560

tgatgaaagt gacatagcat tttgacctat cggacatggt acaatgggca tttgacaatg 7620

aatatactga tgaaagtgac atagcattta attcgtaaat gttagcagat tgtaacagta 7680

atgggcattt gacaatgaat atactgatga aagtgacata gcatttaatt cgtaaatgtt 7740

agcagattgt aacagtacgt atgcagcctt tttaaaaagg acaatgaata tactgatgaa 7800

agtgacatag catttaattc gtaaatgtta gcagattgta acagtacgta tgcagccttt 7860

ttaaaaagta actgccaggg tgacatagca tttaattcgt aaatgttagc agattgtaac 7920

agtacgtatg cagccttttt aaaaagtaac tgccaggcaa aatatgtaaa agattgtgct 7980

ttaattcgta aatgttagca gattgtaaca gtacgtatgc agccttttta aaaagtaact 8040

gccaggcaaa atatgtaaaa gattgtgcaa caatgtgtaa acagtacgta tgcagccttt 8100

ttaaaaagta actgccaggc aaaatatgta aaagattgtg caacaatgtg taaacattac 8160

aagcgagcac aaatgctgca gcctttttaa aaagtaactg ccaggcaaaa tatgtaaaag 8220

attgtgcaac aatgtgtaaa cattacaagc gagcacaaaa aaggcaaatg tccaaacatt 8280

acaagcgagc acaaaaaagg caaatgtcca tgtctcaatg gataaaattt aggtgtagta 8340

aatgtgatga aggcggggac tggagaccca taggtccatg tctcaatgga taaaatttag 8400

gtgtagtaaa tgtgatgaag gcggggactg gagacccata gtacaattct taagatatca 8460

aggaatagaa tttaaatgtg atgaaggcgg ggactggaga cccatagtac aattcttaag 8520

atatcaagga atagaattta tatccttttt atgtgcatta aaggaatttt taagtacaat 8580

tcttaagata tcaaggaata gaatttatat cctttttatg tgcattaaag gaatttttaa 8640

agggtactcc caaaaaaaac tgtatagtta tattatcctt tttatgtgca ttaaaggaat 8700

ttttaaaggg tactcccaaa aaaaactgta tagttatata tggacctgcg aatacaggaa 8760

agtcacattt ttgaagggta ctcccaaaaa aaactgtata gttatatatg gacctgcgaa 8820

tacaggaaag tcacattttt gtatgagcct tcgtaatttt ttacagggca cagggacctg 8880

cgaatacagg aaagtcacat ttttgtatga gccttcgtaa ttttttacag ggcacagtta 8940

tttcatatgt aaactccacc agccactttt ggcgttattt catatgtaaa ctccaccagc 9000

cacttttggc tagaaccact tgcagcgtaa aaactagcaa tgttagatgc gtaaaccggt 9060

acctgctggt catggctaga accacttgca gcgtaaaaac tagcaatgtt agatgcgtaa 9120

accggtacct gctggtcata tttcgataat tatatgagaa cgtaattaga tggtgttaga 9180

tgcgtaaacc ggtacctgct ggtcatattt cgataattat atgagaacgt aattagatgg 9240

gtcgtaaata agtttagata ggaaatataa aagtatttcg ataattatat gagaacgtaa 9300

ttagatgggt cgtaaataag tttagatagg aaatataaaa gtttactaca aatgaaatgt 9360

ccaccattat taacgtaatt agatgggtcg taaataagtt tagataggaa atataaaagt 9420

ttactacaaa tgaaatgtcc accattatta ataacctcca atacgggtcg taaataagtt 9480

tagataggaa atataaaagt ttactacaaa tgaaatgtcc accattatta ataacctcca 9540

ataccaatcc tgtggaagac gataagttta ctacaaatga aatgtccacc attattaata 9600

acctccaata ccaatcctgt ggaagacgat aggtggccat atttacgtag taggctaaca 9660

gtgttaataa cctccaatac caatcctgtg gaagacgata ggtggccata tttacgtagt 9720

aggctaacag tgtttaaatt tcctacgtaa tttccataac ctccaatacc aatcctgtgg 9780

aagacgatag gtggccatat ttacgtagta ggctaacagt gtttaaattt cctacgtaat 9840

ttccatttga ccaaagaaga cgataggtgg ccatatttac gtagtaggct aacagtgttt 9900

aaatttccta cgtaatttcc atttgaccaa aacaggaatc cagtgtacac aatcaggtgg 9960

ccatatttac gtagtaggct aacagtgttt aaatttccta cgtaatttcc atttgaccaa 10020

aacaggaatc cagtgtacac aatcaatgat aaaaggctaa cagtgtttaa atttcctacg 10080

taatttccat ttgaccaaaa caggaatcca gtgtacacaa tcaatgataa aaactggaaa 10140

tgtttttttg aaaaccattt gaccaaaaca ggaatccagt gtacacaatc aatgataaaa 10200

actggaaatg tttttttgaa aagacttggt gcagattaga cttgcagcag gacgtcaatg 10260

ataaaaactg gaaatgtttt tttgaaaaga cttggtgcag attagacttg cagcaggacg 10320

aggatgaagg agacaatgat gaaaacactt tcacagactt ggtgcagatt agacttgcag 10380

caggacgagg atgaaggaga caatgatgaa aacactttca caacgtttaa atgtgttaca 10440

ggacaaaata ctaggcagat tagacttgca gcaggacgag gatgaaggag acaatgatga 10500

aaacactttc acaacgttta aatgtgttac aggacaaaat actagaatac tatgaggatg 10560

aaggagacaa tgatgaaaac actttcacaa cgtttaaatg tgttacagga caaaatacta 10620

gaatactatg aacaagacag taaatgacaa tgatgaaaac actttcacaa cgtttaaatg 10680

tgttacagga caaaatacta gaatactatg aacaagacag taaatcaata tatgatcaaa 10740

ttaattgtgt tacaggacaa aatactagaa tactatgaac aagacagtaa atcaatatat 10800

gatcaaatta attattggaa atgtgtgcga atggaaacgt aaatatatga acaagacagt 10860

aaatcaatat atgatcaaat taattattgg aaatgtgtgc gaatggaaac gtaaatattt 10920

tcgtaagcac gagaacgtga caagacagta aatcaatata tgatcaaatt aattattgga 10980

aatgtgtgcg aatggaaacg taaatatttt cgtaagcacg agaacgtggc cgtaatacta 11040

ttaattattg gaaatgtgtg cgaatggaaa cgtaaatatt ttcgtaagca cgagaacgtg 11100

gccgtaatac tattgaccac caggtggtgc cttattggaa atgtgtgcga atggaaacgt 11160

aaatattttc gtaagcacga gaacgtggcc gtaatactat tgaccaccag gtggtgccaa 11220

ccataaacat tgaaacgtaa atattttcgt aagcacgaga acgtggccgt aatactattg 11280

accaccaggt ggtgccaacc ataaacattt caaaatgtaa agcatatcaa gttttcgtaa 11340

gcacgagaac gtggccgtaa tactattgac caccaggtgg tgccaaccat aaacatttca 11400

aaatgtaaag catatcaagc tattgaactg cggccgtaat actattgacc accaggtggt 11460

gccaaccata aacatttcaa aatgtaaagc atatcaagct attgaactgc agatggcact 11520

agactattga ccaccaggtg gtgccaacca taaacatttc aaaatgtaaa gcatatcaag 11580

ctattgaact gcagatggca ctagaaagtg ttgcacaaac tgacaggtgg tgccaaccat 11640

aaacatttca aaatgtaaag catatcaagc tattgaactg cagatggcac tagaaagtgt 11700

tgcacaaact gaatacaata aacatttcaa aatgtaaagc atatcaagct attgaactgc 11760

agatggcact agaaagtgtt gcacaaactg aatacaatac agaggagtgg acttcaaaat 11820

gtaaagcata tcaagctatt gaactgcaga tggcactaga aagtgttgca caaactgaat 11880

acaatacaga ggagtggaca ttaaaagaca ctgctattga actgcagatg gcactagaaa 11940

gtgttgcaca aactgaatac aatacagagg agtggacatt aaaagacact agtaatgaac 12000

tgtggcatac agcagatggc actagaaagt gttgcacaaa ctgaatacaa tacagaggag 12060

tggacattaa aagacactag taatgaactg tggcatacac agccaaaaca aatacaatac 12120

agaggagtgg acattaaaag acactagtaa tgaactgtgg catacacagc caaaacaatg 12180

ttttaaaaaa caaggaacta cagtggaggt gtagtaatga actgtggcat acacagccaa 12240

aacaatgttt taaaaaacaa ggaactacag tggaggtgtg gtatgatggg gacaaatgta 12300

cgtatatgaa catgttttaa aaaacaagga actacagtgg aggtgtggta tgatggggac 12360

aaatgtacgt atatgaacta tgtattatgg ggtgctatat attataaaaa tgtggtatga 12420

tggggacaaa tgtacgtata tgaactatgt attatggggt gctatatatt ataaaaataa 12480

tatagacata tggtgtaaaa cagaagggtg tctatgtatt atggggtgct atatattata 12540

aaaataatat agacatatgg tgtaaaacag aagggtgtgt ggactattgg ggtatatatt 12600

atatgaacga gaatatagac atatggtgta aaacagaagg gtgtgtggac tattggggta 12660

tatattatat gaacgagcac ctaaaagtat actatgaagt gtttattcaa ggtggactat 12720

tggggtatat attatatgaa cgagcaccta aaagtatact atgaagtgtt tattcaagcg 12780

taggaaaggt atgggactag tggcaaatgg ggcacctaaa agtatactat gaagtgttta 12840

ttcaagcgta ggaaaggtat gggactagtg gcaaatggga agtgcattat aatggcaaca 12900

taattcattg tgcgtaggaa aggtatggga ctagtggcaa atgggaagtg cattataatg 12960

gcaacataat tcattgtcct gactctatgt gcagtaccag tgacggatcg ggggaagtgc 13020

attataatgg caacataatt cattgtcctg actctatgtg cagtaccagt gacggatcgg 13080

tacccactac tgaacttact accgaattat caattcattg tcctgactct atgtgcagta 13140

ccagtgacgg atcggtaccc actactgaac ttactaccga attatcaaac accaccgcga 13200

cccattccac ctcctgactc tatgtgcagt accagtgacg gatcggtacc cactactgaa 13260

cttactaccg aattatcaaa caccaccgcg acccattcca ccgcaacaac cggtacccac 13320

tactgaactt actaccgaat tatcaaacac caccgcgacc cattccaccg caacaacccc 13380

cgtaacccaa aaaacaatcc cgccgccgtc taaacaccac cgcgacccat tccaccgcaa 13440

caacccccgt aacccaaaaa acaatcccgc cgccgtctcg aaagcgacct cgacagtgtg 13500

cagtcacaga gccccgtaac ccaaaaaaca atcccgccgc cgtctcgaaa gcgacctcga 13560

cagtgtgcag tcacagagcc cactgagccc gacggagtgt ccctggacca ttcgaaagcg 13620

acctcgacag tgtgcagtca cagagcccac tgagcccgac ggagtgtccc tggaccatct 13680

taacaaccca ctccacagta acagtacagg cagcccactg agcccgacgg agtgtccctg 13740

gaccatctta acaacccact ccacagtaac agtacaggcc acaacacaag acggtacctc 13800

agttgtggta atcttaacaa cccactccac agtaacagta caggccacaa cacaagacgg 13860

tacctcagtt gtggtaacac tacgcctata atacatttaa aaggtgacaa aggccacaac 13920

acaagacggt acctcagttg tggtaacact acgcctataa tacatttaaa aggtgacaaa 13980

aatggtttaa aatgtttaag atatagacta ccactacgcc tataatacat ttaaaaggtg 14040

acaaaaatgg tttaaaatgt ttaagatata gactacaaaa atatgacaca ttgtttgaaa 14100

atatttcatg taaatggttt aaaatgttta agatatagac tacaaaaata tgacacattg 14160

tttgaaaata tttcatgtac ctggcattgg atacggggta agggaaccaa aaaatatgac 14220

acattgtttg aaaatatttc atgtacctgg cattggatac ggggtaaggg aaccaaaaac 14280

gctggcatat taactgttac atcgtacaca ggtacctggc attggatacg gggtaaggga 14340

accaaaaacg ctggcatatt aactgttaca tcgtacacag agtcacaacg ccaaaaattt 14400

ttggacactg taaacgctgg catattaact gttacatcgt acacagagtc acaacgccaa 14460

aaatttttgg acactgttaa aataccttct agtgtacatg tttcattggg tagagtcaca 14520

acgccaaaaa tttttggaca ctgttaaaat accttctagt gtacatgttt cattgggtta 14580

catgacattg taaagtatac tatggatatt gtaaaatacc ttctagtgta catgtttcat 14640

tgggttacat gacattgtaa agtatactat ggatattgtg tatgtatatt gtatacatac 14700

tacatagatg agttacatga cattgtaaag tatactatgg atattgtgta tgtatattgt 14760

atacatacta catagatgat attattggta tttttggtgt ggtttggtgt ggtgtatgta 14820

tattgtatac atactacata gatgatatta ttggtatttt tggtgtggtt tggtgtgtgt 14880

atatatatat gttgcaatgt cccgcttttg cgatattatt ggtatttttg gtgtggtttg 14940

gtgtgtgtat atatatatgt tgcaatgtcc cgcttttgcc gtctgtgcat gtgtgtgcgt 15000

atgtgtggat agtatatata tatgttgcaa tgtcccgctt ttgccgtctg tgcatgtgtg 15060

tgcgtatgtg tggataattg tgtttgtgtt tattcttata cgtaccacac cgccgtctgt 15120

gcatgtgtgt gcgtatgtgt ggataattgt gtttgtgttt attcttatac gtaccacacc 15180

attggaggtg ttttttgtat atttactatt tttgtgtttg tgtttattct tatacgtacc 15240

acaccattgg aggtgttttt tgtatattta ctattttttg tattgcccat gtggttgttg 15300

catagactgg cccattggag gtgttttttg tatatttact attttttgta ttgcccatgt 15360

ggttgttgca tagactggca atggatatga tatagtactg tatatgtatg ttttgtattg 15420

cccatgtggt tgttgcatag actggcaatg gatatgatat agtactgtat atgtatgtgc 15480

attgtgcata actactgtac atagcttttt agcaatggat atgatatagt actgtatatg 15540

tatgtgcatt gtgcataact actgtacata gctttttata tttttttttg ttactaataa 15600

acatggtttc cgtgcattgt gcataactac tgtacatagc tttttatatt tttttttgtt 15660

actaataaac atggtttccc accgtgctgc caggcgtaag cgtgcatctg catatttttt 15720

tttgttacta ataaacatgg tttcccaccg tgctgccagg cgtaagcgtg catctgcaac 15780

tgacctatat agaacctgta aacaatcggg tccaccgtgc tgccaggcgt aagcgtgcat 15840

ctgcaactga cctatataga acctgtaaac aatcgggtac ctgtccacca gacgttgttg 15900

ataaagttga gggcgtaagc gtgcatctgc aactgaccta tatagaacct gtaaacaatc 15960

gggtacctgt ccaccagacg ttgttgataa agttgagggt actacacttg caactgacct 16020

atatagaacc tgtaaacaat cgggtacctg tccaccagac gttgttgata aagttgaggg 16080

tactacactt gctgacaaaa ttttacagtg gtacctgtcc accagacgtt gttgataaag 16140

ttgagggtac tacacttgct gacaaaattt tacagtggac tagtttaggt atatttttgg 16200

gtgggttagg cgggtactac acttgctgac aaaattttac agtggactag tttaggtata 16260

tttttgggtg ggttaggcat aggcacaggt actggtactg ggggacgcac agactagttt 16320

aggtatattt ttgggtgggt taggcatagg cacaggtact ggtactgggg gacgcacagg 16380

atatataccc ctggggggta ggcctaatac tcataggcac aggtactggt actgggggac 16440

gcacaggata tatacccctg gggggtaggc ctaatactgt tgtagatgtg tctcctgcac 16500

gtccacctgt acaggatata tacccctggg gggtaggcct aatactgttg tagatgtgtc 16560

tcctgcacgt ccacctgtag ttattgaacc tgttggtcct tctgagccat cctgttgtag 16620

atgtgtctcc tgcacgtcca cctgtagtta ttgaacctgt tggtccttct gagccatcta 16680

ttgtgcaatt ggtggaggac tcaagtgtta tagttattga acctgttggt ccttctgagc 16740

catctattgt gcaattggtg gaggactcaa gtgttataac ctctggaaca ccagtaccaa 16800

catttacagg cctattgtgc aattggtgga ggactcaagt gttataacct ctggaacacc 16860

agtaccaaca tttacaggca cctctggatt tgaaattact tcttcttcta caacctctgg 16920

aacaccagta ccaacattta caggcacctc tggatttgaa attacttctt cttctactac 16980

tacgcctgcg gtattggata ttacaccctc cggcacctct ggatttgaaa ttacttcttc 17040

ttctactact acgcctgcgg tattggatat tacaccctcc tctgggtctg tacaaataac 17100

ctctactagt tactacgcct gcggtattgg atattacacc ctcctctggg tctgtacaaa 17160

taacctctac tagttatact aaccctgcct ttacggatcc ttccttaatt gcctctgggt 17220

ctgtacaaat aacctctact agttatacta accctgcctt tacggatcct tccttaattg 17280

aggttcccca aacaggtgaa acctcgggta actaaccctg cctttacgga tccttcctta 17340

attgaggttc cccaaacagg tgaaacctcg ggtaatatat ttgtcagtac ccctacatca 17400

ggtacacatg gtgaggttcc ccaaacaggt gaaacctcgg gtaatatatt tgtcagtacc 17460

cctacatcag gtacacatgg ctatgaggaa atacctatgg aagtgtttgc ctatatttgt 17520

cagtacccct acatcaggta cacatggcta tgaggaaata cctatggaag tgtttgccac 17580

acatggcaca ggtaccgaac ctattagcag ctggctatga ggaaatacct atggaagtgt 17640

ttgccacaca tggcacaggt accgaaccta ttagcagcac acctacacct ggaatcagtc 17700

gtgtggcagg acacacatgg cacaggtacc gaacctatta gcagcacacc tacacctgga 17760

atcagtcgtg tggcaggacc acgtttatat agtagagcac atcagcaggt tggtaccgaa 17820

cctattagca gcacacctac acctggaatc agtcgtgtgg caggaccacg tttatatagt 17880

agagcacatc agcaggttcg tgttagtaat tgcacaccta cacctggaat cagtcgtgtg 17940

gcaggaccac gtttatatag tagagcacat cagcaggttc gtgttagtaa ttttgatttt 18000

gtaactcacc cagtcgtgtg gcaggaccac gtttatatag tagagcacat cagcaggttc 18060

gtgttagtaa ttttgatttt gtaactcacc cttcatcatt tgtaacggac cacgtttata 18120

tagtagagca catcagcagg ttcgtgttag taattttgat tttgtaactc acccttcatc 18180

atttgtaaca tttgataatc ctgcttgtag agcacatcag caggttcgtg ttagtaattt 18240

tgattttgta actcaccctt catcatttgt aacatttgat aatcctgctt ttgagcctgt 18300

tgatacgatt ttgtaactca cccttcatca tttgtaacat ttgataatcc tgcttttgag 18360

cctgttgata ctacattaac atatgaagct gctgacatag ctccagttca tcatttgtaa 18420

catttgataa tcctgctttt gagcctgttg atactacatt aacatatgaa gctgctgaca 18480

tagctccaga tccggatttt ctggaccatt tgataatcct gcttttgagc ctgttgatac 18540

tacattaaca tatgaagctg ctgacatagc tccagatccg gattttctgg acattgttcg 18600

tttacagagc ctgttgatac tacattaaca tatgaagctg ctgacatagc tccagatccg 18660

gattttctgg acattgttcg tttacatagg cctgccttaa cctcgcacat atgaagctgc 18720

tgacatagct ccagatccgg attttctgga cattgttcgt ttacataggc ctgccttaac 18780

ctcgcgtaaa ggaacagtaa gtgctgacat agctccagat ccggattttc tggacattgt 18840

tcgtttacat aggcctgcct taacctcgcg taaaggaaca gtaaggttta gtaggcttgg 18900

cgatccggat tttctggaca ttgttcgttt acataggcct gccttaacct cgcgtaaagg 18960

aacagtaagg tttagtaggc ttggcaaaaa ggctaccatg cattgttcgt ttacataggc 19020

ctgccttaac ctcgcgtaaa ggaacagtaa ggtttagtag gcttggcaaa aaggctacca 19080

tggttacccg gcgtggcaca gcgtaaagga acagtaaggt ttagtaggct tggcaaaaag 19140

gctaccatgg ttacccggcg tggcacacaa attggagcgc aagtacatta ttaccatgac 19200

ggcaaaaagg ctaccatggt tacccggcgt ggcacacaaa ttggagcgca agtacattat 19260

taccatgaca ttagtagtat tgctcctgct gaaagcattg caaattggag cgcaagtaca 19320

ttattaccat gacattagta gtattgctcc tgctgaaagc attgaattac agcccctagt 19380

tcacgctgag ccctctgatg cattagtagt attgctcctg ctgaaagcat tgaattacag 19440

cccctagttc acgctgagcc ctctgcgtat tcagcgtaat tatttgatat atcgtatgat 19500

attacagccc ctagttcacg ctgagccctc tgcgtattca gcgtaattat ttgatatatc 19560

gtatgatgtg gacaataaca catatttaga tactgcattt tgcttcagcg taattatttg 19620

atatatcgta tgatgtggac aataacacat atttagatac tgcatttaat aatacaaggg 19680

attcgggcac tacatataac tgtggacaat aacacatatt tagatactgc atttaataat 19740

acaagggatt cgggcactac atataacaca ggctcactac cttctgtggc ttcttcagca 19800

taataataca agggattcgg gcactacata taacacaggc tcactacctt ctgtggcttc 19860

ttcagcatct actaaatcgt acaatacaac tattcctttt cacaggctca ctaccttctg 19920

tggcttcttc agcatctact aaatcgtaca atacaactat tccttttagt acctcatgga 19980

atcgtactgt aaatactggt atctactaaa tcgtacaata caactattcc ttttagtacc 20040

tcatggaatc gtactgtaaa tactggtcct gatattgctt taccaagtac tactccacag 20100

tagtacctca tggaatcgta ctgtaaatac tggtcctgat attgctttac caagtactac 20160

tccacagttg ccattggtgc cttctggacc aatagacaca ggtcctgata ttgctttacc 20220

aagtactact ccacagttgc cattggtgcc ttctggacca atagacacaa catcgtaaat 20280

aaccattcag ggttccaatt gttgccattg gtgccttctg gaccaataga cacaacatcg 20340

taaataacca ttcagggttc caattattat ttgttgccat tattgtattt tttcctaaaa 20400

catcgtaaat aaccattcag ggttccaatt attatttgtt gccattattg tattttttcc 20460

taaaaaaacg taaacgtatt ccctattttt tttcagatgg ttatttgttg ccattattgt 20520

attttttcct aaaaaaacgt aaacgtattc cctatttttt ttcagatggc tatgtggcgg 20580

tctagtgaca gcatggtgta aaaaaacgta aacgtattcc ctattttttt tcagatggct 20640

atgtggcggt ctagtgacag catggtgtat ttgcctccac cttctgtggc aaaacgtaaa 20700

cgtattccct attttttttc agatggctat gtggcggtct agtgacagca tggtgtattt 20760

gcctccacct tctgtggcga aggttgtcaa tggctatgtg gcggtctagt gacagcatgg 20820

tgtatttgcc tccaccttct gtggcgaagg ttgtcaatac tgatgattat gttacacgca 20880

caggcatata gtgacagcat ggtgtatttg cctccacctt ctgtggcgaa ggttgtcaat 20940

actgatgatt atgttacacg cacaggcata tattattcgt atggatttgc ctccaccttc 21000

tgtggcgaag gttgtcaata ctgatgatta tgttacacgc acaggcatat attattcgta 21060

tggcagctct agattattaa cagtgtggcg aaggttgtca atactgatga ttatgttaca 21120

cgcacaggca tatattattc gtatggcagc tctagattat taacagtagg acatccatat 21180

tttacgaagg ttgtcaatac tgatgattat gttacacgca caggcatata ttattcgtat 21240

ggcagctcta gattattaac agtaggacat ccatatttta tactgatgat tatgttacac 21300

gcacaggcat atattattcg tatggcagct ctagattatt aacagtagga catccatatt 21360

ttaaagtggg tatgaatgga ttattcgtat ggcagctcta gattattaac agtaggacat 21420

ccatatttta aagtgggtat gaatggtggt cgcaagcagg acattccaaa ggtgtctgcc 21480

tagattatta acagtaggac atccatattt taaagtgggt atgaatggtg gtcgcaagca 21540

ggacattcca aaggtgtctg catatcaata ggacatccat attttaaagt gggtatgaat 21600

ggtggtcgca agcaggacat tccaaaggtg tctgcatatc aatatagggt atttcgcgtg 21660

acattgcccg gtggtcgcaa gcaggacatt ccaaaggtgt ctgcatatca atatagggta 21720

tttcgcgtga cattgcccga tcctaataaa ttcagtattc cagcgtaatc catatcaata 21780

tagggtattt cgcgtgacat tgcccgatcc taataaattc agtattccag cgtaatcctt 21840

atataatcca gaaacacaac gtttagtatg cccgatccta ataaattcag tattccagcg 21900

taatccttat ataatccaga aacacaacgt ttagtatggg cttgtgtagg ggtggaggtg 21960

ggcaggggcc ccttatataa tccagaaaca caacgtttag tatgggcttg tgtaggggtg 22020

gaggtgggca ggggccagcc attgggtgtt ggtattagtg gacacccatt ggcttgtgta 22080

ggggtggagg tgggcagggg ccagccattg ggtgttggta ttagtggaca cccattatat 22140

aatagacagg atgatactga aaactcacca cagccattgg gtgttggtat tagtggacac 22200

ccattatata atagacagga tgatactgaa aactcaccat tttcatcaac caccaataag 22260

gacagtaggg atataataga caggatgata ctgaaaactc accattttca tcaaccacca 22320

ataaggacag tagggataat gtgtctgtgg attataaaca gacacagttg ccattttcat 22380

caaccaccaa taaggacagt agggataatg tgtctgtgga ttataaacag acacagttgt 22440

gcattatagg ctgtgttccc gccattgggg ggataatgtg tctgtggatt ataaacagac 22500

acagttgtgc attataggct gtgttcccgc cattggggag cactggggta agggaaaggc 22560

cgtaaagccc tgtgcattat aggctgtgtt cccgccattg gggagcactg gggtaaggga 22620

aaggccgtaa agcccaataa tgtatctacg ggggactgtc ctcctttgga ggagcactgg 22680

ggtaagggaa aggccgtaaa gcccaataat gtatctacgg gggactgtcc tcctttggaa 22740

ctagtaaaca cccctattga ggatggtgat ataatgtatc tacgggggac tgtcctcctt 22800

tggaactagt aaacacccct attgaggatg gtgatatgat tgatactggc tatggagcta 22860

tggactttgg ggaactagta aacaccccta ttgaggatgg tgatatgatt gatactggct 22920

atggagctat ggactttggt gcattgcagg aaaccaaaag tgaggtgcct tgattgatac 22980

tggctatgga gctatggact ttggtgcatt gcaggaaacc aaaagtgagg tgcctttaga 23040

tatttgtcaa tccatttgta aatatcctga ggtgcattgc aggaaaccaa aagtgaggtg 23100

cctttagata tttgtcaatc catttgtaaa tatcctgatt atttgcaaat gtctgcagat 23160

gtgtatgggg agatatttgt caatccattt gtaaatatcc tgattatttg caaatgtctg 23220

cagatgtgta tggggacagt atgttcttct gtttacgtag ggaacaactg atcctgatta 23280

tttgcaaatg tctgcagatg tgtatgggga cagtatgttc ttctgtttac gtagggaaca 23340

actgtttgca agacattttt ggaattattt gcaaatgtct gcagatgtgt atggggacag 23400

tatgttcttc tgtttacgta gggaacaact gtttgcaaga catttttgga atcgtggtgg 23460

tatggtgttt gcaagacatt tttggaatcg tggtggtatg gtgggtgacg ccattcctgc 23520

ccaattgtat attaagggca cagatatacg tgcaaacccc ggtagtggtg ggtgacgcca 23580

ttcctgccca attgtatatt aagggcacag atatacgtgc aaaccccggt agttctgtat 23640

actgcccctc tcccagcggt tccatttaag ggcacagata tacgtgcaaa ccccggtagt 23700

tctgtatact gcccctctcc cagcggttcc atggtaacct ctgattccca gttatttaat 23760

aagccgttct gtatactgcc cctctcccag cggttccatg gtaacctctg attcccagtt 23820

atttaataag ccttattggc tacataaggc ccagggccac aacaatggta acctctgatt 23880

cccagttatt taataagcct tattggctac ataaggccca gggccacaac aatggtatat 23940

gttggcataa tcaattattt cttaccccag ttatttaata agccttattg gctacataag 24000

gcccagggcc acaacaatgg tatatgttgg cataatcaat tatttcttac tgttgtggat 24060

tatttaataa gccttattgg ctacataagg cccagggcca caacaatggt atatgttggc 24120

ataatcaatt atttcttact gttgtggaca ctacccgctt attggctaca taaggcccag 24180

ggccacaaca atggtatatg ttggcataat caattatttc ttactgttgt ggacactacc 24240

cgtagtacca actttacgcc cagggccaca acaatggtat atgttggcat aatcaattat 24300

ttcttactgt tgtggacact acccgtagta ccaactttac attatctacc tctaatggta 24360

tatgttggca taatcaatta tttcttactg ttgtggacac tacccgtagt accaacttta 24420

cattatctac ctctatagag tcttccatac cttcctgttg tggacactac ccgtagtacc 24480

aactttacat tatctacctc tatagagtct tccatacctt ctacatatga tccttctaag 24540

tttaaggaat ataccattat ctacctctat agagtcttcc ataccttcta catatgatcc 24600

ttctaagttt aaggaatata ccaggcacgt ggaggagtat gatttacaat ttatctacat 24660

atgatccttc taagtttaag gaatatacca ggcacgtgga ggagtatgat ttacaattta 24720

tatttcaact gtgtactgtc acattaacaa ctgaccaggc acgtggagga gtatgattta 24780

caatttatat ttcaactgtg tactgtcaca ttaacaactg atgttatgtc ttatattcac 24840

actatgaatt cctctatttc aactgtgtac tgtcacatta acaactgatg ttatgtctta 24900

tattcacact atgaattcct ctatattgga caattggaat tttgctgtag ctccatgtta 24960

tgtcttatat tcacactatg aattcctcta tattggacaa ttggaatttt gctgtagctc 25020

ctccaccatc tgccagtttg gtagacactt acagctatat tggacaattg gaattttgct 25080

gtagctcctc caccatctgc cagtttggta gacacttaca gatacctaca gtctgcagcc 25140

attacatgtc aaaacctcca ccatctgcca gtttggtaga cacttacaga tacctacagt 25200

ctgcagccat tacatgtcaa aaggcgtatc cagcacctga aaagaaagat ccatgatacc 25260

tacagtctgc agccattaca tgtcaaaagg cgtatccagc acctgaaaag aaagatccat 25320

atgacggtct aaagttttgg aatgttgact taagaggcgt atccagcacc tgaaaagaaa 25380

gatccatatg acggtctaaa gttttggaat gttgacttaa gggaaaagtt tagtttggaa 25440

cttgatcaat tcccgacggt ctaaagtttt ggaatgttga cttaagggaa aagtttagtt 25500

tggaacttga tcaattccct ttgggacgta aatttttgtt gcaggccagg gtccgggaaa 25560

agtttagttt ggaacttgat caattccctt tgggacgtaa atttttgttg caggccaggg 25620

tccgcaggcg ccctactata ggtccccgaa agcgtttggg acgtaaattt ttgttgcagg 25680

ccagggtccg caggcgccct actataggtc cccgaaagcg gcctgctgca tccacttcct 25740

cgtcctcagc tacttccgca ggcgccctac tataggtccc cgaaagcggc ctgctgcatc 25800

cacttcctcg tcctcagcta ctaaacacaa acgtaaacgt gtgtctaaat aatgggcctg 25860

ctgcatccac ttcctcgtcc tcagctacta aacacaaacg taaacgtgtg tctaaatacg 25920

taatgtgtcg tacttgttat gtgtgtgtat gttgctaaac acaaacgtaa acgtgtgtct 25980

aaatacgtaa tgtgtcgtac ttgttatgtg tgtgtatgtt gtttgtttcc ttatgtgttg 26040

agtgtatatg tgtagcatgt gtcgtacttg ttatgtgtgt gtatgttgtt tgtttcctta 26100

tgtgttgagt gtatatgtgt atgtttgtag gtatgtgtgt atatgttttt gttagtttgt 26160

ttccttatgt gttgagtgta tatgtgtatg tttgtaggta tgtgtgtata tgtttttgtt 26220

aataaagtat gtatgacagt ttcatgtgtg attgatgttt gtaggtatgt gtgtatatgt 26280

ttttgttaat aaagtatgta tgacagtttc atgtgtgatt gcacaccctg tgactaacag 26340

tgtatttgtt ttacaataaa gtatgtatga cagtttcatg tgtgattgca caccctgtga 26400

ctaacagtgt atttgtttta catataatag gtctgcaaca tttcatacat aatcgcacac 26460

cctgtgacta acagtgtatt tgttttacat ataataggtc tgcaacattt catacataat 26520

ctatcgtacc taccctaagg tgtgtttact acctcatata ataggtctgc aacatttcat 26580

acataatcta tcgtacctac cctaaggtgt gtttactacc taatatgtaa tttttacatt 26640

gttgtcgtag tttcctatcg tacctaccct aaggtgtgtt tactacctaa tatgtaattt 26700

ttacattgtt gtcgtagttt ctacatttta tacttcgcca ttttgtggcg accgtaatat 26760

gtaattttta cattgttgtc gtagtttcta cattttatac ttcgccattt tgtggcgacc 26820

gaagtcggtc gtgggttgag catttttttt aaacctacat tttatacttc gccattttgt 26880

ggcgaccgaa gtcggtcgtg ggttgagcat tttttttaaa ctagtggaaa ccacctttct 26940

cagcaaaaac atgtgaagtc ggtcgtgggt tgagcatttt ttttaaacta gtggaaacca 27000

cctttctcag caaaaacatg tctttacctt aggttcaccc tgcatagttg gcacctagtg 27060

gaaaccacct ttctcagcaa aaacatgtct ttaccttagg ttcaccctgc atagttggca 27120

ctggtaacag ttttactggc gcgccttatt actcgtcttt accttaggtt caccctgcat 27180

agttggcact ggtaacagtt ttactggcgc gccttattac tcatcatcct gtccaggtgc 27240

actgcaacaa tactctggta acagttttac tggcgcgcct tattactcat catcctgtcc 27300

aggtgcactg caacaatact ttggcaacat ccatatctcc accctatgta ataatactgg 27360

cgcgccttat tactcatcat cctgtccagg tgcactgcaa caatactttg gcaacatcca 27420

tatctccacc ctatgtaata aaactgcttt tagcatcatc ctgtccaggt gcactgcaac 27480

aatactttgg caacatccat atctccaccc tatgtaataa aactgctttt aggcatatat 27540

tttagctgtt tttccaggtg cactgcaaca atactttggc aacatccata tctccaccct 27600

atgtaataaa actgctttta ggcatatatt ttagctgttt ttacttgctt aatttggcaa 27660

catccatatc tccaccctat gtaataaaac tgcttttagg catatatttt agctgttttt 27720

acttgcttaa ttaaatagtt ggcctgtata actaaactgc ttttaggcat atattttagc 27780

tgtttttact tgcttaatta aatagttggc ctgtataact actttttgat tcaggaatgt 27840

gtcttacagt atatacttgc ttaattaaat agttggcctg tataactact ttttgattca 27900

ggaatgtgtc ttacagtata agttatacaa gtgactaatg tagcacacaa tagacttttt 27960

gattcaggaa tgtgtcttac agtataagtt atacaagtga ctaatgtagc acacaatagt 28020

ttcgtaaacc gaaataggtt gggcatacat acc 28053

<210> 2

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 2

caagcagaag acggcatacg agattgcatg acgtgactgg agttcagacg tgt 53

<210> 3

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 3

caagcagaag acggcatacg agattgctat cggtgactgg agttcagacg tgt 53

<210> 4

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 4

caagcagaag acggcatacg agatcacaag cagtgactgg agttcagacg tgt 53

<210> 5

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 5

caagcagaag acggcatacg agattcgctg atgtgactgg agttcagacg tgt 53

<210> 6

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 6

caagcagaag acggcatacg agatcgcttt gtgtgactgg agttcagacg tgt 53

<210> 7

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 7

caagcagaag acggcatacg agatccgatg aagtgactgg agttcagacg tgt 53

<210> 8

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 8

caagcagaag acggcatacg agattgacca cagtgactgg agttcagacg tgt 53

<210> 9

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 9

caagcagaag acggcatacg agatgaagcc atgtgactgg agttcagacg tgt 53

<210> 10

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 10

caagcagaag acggcatacg agatttctgg tggtgactgg agttcagacg tgt 53

<210> 11

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 11

caagcagaag acggcatacg agatccgaaa acgtgactgg agttcagacg tgt 53

<210> 12

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 12

caagcagaag acggcatacg agatcgaaaa gggtgactgg agttcagacg tgt 53

<210> 13

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 13

caagcagaag acggcatacg agataaccag ctgtgactgg agttcagacg tgt 53

<210> 14

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> joint

<400> 14

ctacaataat tcatgtataa aactaagggc tagtataaaa gcagacattt tcgtaaccaa 60

Claims (3)

1. A probe for detecting human papillomavirus HPV39, which is characterized in that the nucleotide sequence of the probe is shown as SEQ ID NO. 1; the probe is of a shingled design and consists of a plurality of probes connected end to end.

2. A kit for detecting human papillomavirus HPV39, comprising the probe of claim 1.

3. The probe of claim 1 for use in non-diagnostic non-therapeutic human papillomavirus HPV detection.