KR101331170B1 - A probe for identifying hpv genotype and a method for identifying hpv genotype - Google Patents

Abstract

The present invention relates to a HPV genotype diagnostic probe and an analysis method thereof.

Description

HPP genotyping probe and analysis method {A PROBE FOR IDENTIFYING HPV GENOTYPE AND A METHOD FOR IDENTIFYING HPV GENOTYPE}

The present invention relates to a HPV genotype diagnostic probe and an analysis method thereof.

High risk (HPV) -HPV DNA is present in 99.7% of cervical cancer patients worldwide, and the persistence of such HR-HPV infection is known to cause metastasis to invasive cervical cancer and cervical precancerous cancer. Cervical cancer causes 500,000 cases worldwide and 6,000 new cases in Korea each year, with 50% fatal outcomes. Human Papilloma Virus (HPV) is known to be an important cause of the progression of cervical tumors, and HPV DNA is used for early diagnosis of cervical cancer (Bernard, HU, IE Calleja-Macias, and ST Dunn. 2006. Int J Cancer 118: 1071-6). HPV is a double-stranded DNA virus of about 7.9 kb, belonging to the papovavirus, which contains more than 100 subtypes, of which 40 are known to infect the genitals. The HPV virus also contains 10 viral proteins, two late gene products, L1 and L2, which are involved in the synthesis of structural proteins that make up the capsid of the virus, and the replication of the virus, which determines the activity and latency of the virus's infection. It expresses eight early gene products, E1, E2, E3, E4, E5, E6, E7, and E8, which regulate protein synthesis. In particular, E6 and E7 encode precancerous proteins and are known to be associated with cancer (Sedman, SA, et al. 1991. J Virol 65: 4860-6). And is closely associated with various malignancies (Godfroid, E., et al. 1998. J Virol Methods 75: 69-81).

Cervical cancer in women is the second highest incidence in the world after breast cancer, with more than 1,000 deaths annually in Korea, making efforts for early diagnosis of cervical cancer very important (Wui, JH, etc.). 2006. Journal of Gynecologic oncology and Coloscopy 17: 39-4711.

There are currently over 100 known genotypes of HPV (Likes, WM, and J. Itano. 2003. Clin J Oncol Nurs 7: 271-6), of which about 30 are known to cause disease in humans. . The genotypes were high risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82) and low risk (6, 11, 40, 42, 43, 44). , 54, 61, 70, 72, 81), and potential risk groups (34, 57, 83) (Munoz, N., et al. 2003. N Engl J Med 348: 518-27). HPV was found to be genotype-specific depending on the location of the lesion and the progression of the lesion, thereby recognizing the biological diversity of HPV infection.

The most commonly used method for diagnosing HPV infection is Papnicolaou Smear (Pap) method. Currently, histological diagnosis of cervical cancer is diagnosed by this test. It has a lot of dependence and has the disadvantage of poor accuracy (Kurman, RJ, DE Henson, AL Herbst, KL Noller, and MH Schiffman. 1994. JAMA 271: 1866-9). In addition, the vaginal magnification test is more accurate than the Pap smear, but requires a skilled technician and expensive equipment, and the HPV genotype cannot be distinguished. The disadvantage is that the risk of malignancy cannot be classified between different HPV genotypes.

In order to make up for the above drawbacks, studies of methods for diagnosing cervical cancer precancerous stage and confirming genotype through HPV-specific DNA amplification using PCR, HC, DNA chip, etc. are being conducted.

In related prior patents, Korean Patent Publication No. 10-2006-0015669 relates to a human papillomavirus type diagnostic kit, and includes a probe for detecting a specific HPV type and an oligo DNA chip for identifying HPV type using the same and the DNA chip. A kit for diagnosing HPV type is described.

In another related prior art, Korean Patent Publication No. 10-2003-0027178 relates to a genotyping kit for diagnosing infection of a human papilloma virus, comprising a probe that binds to DNA or RNA of a human papilloma virus, and a DNA chip comprising the same. A genotyping kit of human papillomavirus that diagnoses infection and genotype of human papillomavirus, and a method of diagnosing human papillomavirus infection using an electrical analysis kit are described.

The present invention solves the above problems, and the object of the present invention is to provide a probe having a specificity and sensitivity for HPV better than the conventional method.

Another object of the present invention to provide a method for analyzing HPV genotype using the probe.

In order to achieve the above object, the present invention provides an HPV diagnostic oligonucleotide probe composition consisting of the nucleotide sequences of SEQ ID NOs: 8, 22 to 24, 27 to 28, and 32.

In one embodiment of the present invention, the composition preferably further comprises a probe consisting of the nucleotide sequence of SEQ ID NO: 5 to 7, 9 to 21, 25 to 26, 29 to 31, 33 to 37, but is not limited thereto.

Technical details, including probe sequences additionally used in the present invention, are well described in Korean Patent Publication No. 10-2009-0129639.

In another aspect, the present invention provides a kit for diagnosing HPV comprising the probe composition and primer of the present invention.

In one embodiment of the present invention, the primer is preferably a primer described in SEQ ID NOs: 1 to 4, but is not limited thereto.

In another embodiment of the present invention, the kit preferably further includes a labeling means for detecting a PCR product, but is not limited thereto.

In another aspect, the present invention is to attach the probe of the present invention to a solid support;

b) hybridizing the amplified HPV PCR product to the probe; And

c) providing a method for identifying HPV genotype comprising detecting the hybridized product.

In the present invention, the solid support is preferably a membrane, a slide, or a well plate, but is not limited thereto.

In one embodiment of the present invention, the detecting step is preferably detected using a luminescence or color reaction.

In the present invention, nucleic acid was extracted from a sample and PCR amplification was performed. Methods of extracting nucleic acids from cells and PCR methods were performed through methods well known in the art, such as those described in Molecular cloning (1989) by Sambrook et al.

The nucleic acid amplification method of the present invention used a primer. Generally, the primer is preferably about 10 to 25 bp in length oligonucleotide, but may be longer. The primer is preferably single stranded but may be present in double or single stranded form. It described in the Example.

The primers of the present invention were prepared by conventional oligonucleotide synthesis methods, and such synthesis methods are disclosed in US Pat.

Sequences such as primers of the invention can be used to form a double helix optionally with complementary sequences, such as DNA derived from a sample. One skilled in the art can alter the hybridization conditions to change the selectivity of the primer for the target sequence.

In some embodiments, nucleic acids of defined sequences of the invention may be employed in combination with appropriate means such as labels to determine hybridization. Suitable labeling means are various labeling means including fluorescence, radioisotope enzymes or other ligands.

Various template dependent processes are possible to amplify specific gene products present in a given cell sample. One of the most known amplification methods is polymerase chain reaction (PCR), which is described in US Pat. Nos. 4,683,202 and 4,800,159 and the like.

It is generally desirable to separate the amplification product from the template or excess primer or other amplification product in one or another step. For example, the amplification products can be separated using agarose, agarose-acrylamide or PAGE using the standard method described in Molecular cloning (1989) by Sambrook et al.

Chromatography, such as adsorption, partitioning and ion exchange, can also be employed as an effective separation method. These separations can be adapted to function in clinical settings to process large volumes of samples, and new methods for separating or detecting thousands of samples at once, FMAT (fluorometric microvolume assay technique), chemiluminescence, and sequence detection systems (Applied) Biosystems) and mass spectroscopy.

Nucleic acids according to the invention will be detected and quantified. In one embodiment the detection can be performed by visual means. Typical visual means method is to dye the gel with ethidium bromide and visualize the bands under UV light. If the amplification product is a nucleotide that is labeled with radiation or fluorescence, the isolated amplification product performs radiosynthesis or fluorescence detection of the inserted radioisotope label.

The present invention includes a kit for carrying out the method. In a non-limiting example, the kit of the present invention may include primers, enzymes for amplification and additional reagents. Thus, the kits will include these one or more reagents in suitable container means. The kits may also include reagents for isolating nucleic acids and purifying amplification products. The components of the kits may be packaged in an aqueous medium or lyophilized form, and suitable container means of the kit include at least one vial, test tube, flask, bottle, etc. into which the components may be placed. If one or more components are present in the kit, they may include other additional containers, such as second, third, etc., where the additional components may be located separately. However, a combination of components may be included in one container.

The nested PCR method used in the present invention generally means a two-tube nested PCR. 'Two-tube nested PCR' usually has a very limited amount of DNA in a sample, so the second PCR cannot be detected by a single PCR. In order to perform the amplification using the inner primer, four primers should be attached in order to amplify the desired part well.The first polymerase chain reaction using the outer primers is performed and the second amplification is performed with the product. In this case, the reaction can occur smoothly because new reagents are added again, and a pair of primers have a relatively small number of reactions, which has an advantage of increasing sensitivity and specificity compared to conventional PCR. The problem of contamination often occurs during the amplification process. To solve this problem, two pairs of primers are used in one tube. The attempt to put a one-tube nested PCR, could simultaneously reduce the problem of pollution in the sensitive area is better than the existing Two-tube PCR check it appears (see Example 8 and Figure 8 and 9).

In the HPV genotyping method of the present invention, PCR amplification of HPV L1 gene, a probe capable of specifically binding to each HPV type in its region, and reverse hybridization hybridization using ECL-Southern hybridization of amplified DNA on a membrane attached thereto As a method using torch, the end purpose is to develop a probe with better specificity and sensitivity than a conventional DNA chip and to develop a more economic method.

In particular, the present invention is to provide a new method for detecting HPV by observing the detection rate of HPV using one-tube nested PCR amplification by putting two pairs of primers in the same tube and comparing the results of PCR with other diagnostic methods.

As can be seen from the configuration and drawings of the present invention, HPV genotyping method of the present invention is superior in specificity and sensitivity compared to the existing method, and expensive equipment (scanner, MALDI-) compared to the existing DNA chip or other methods TOF, etc.) is not required, and convenience can be provided through linkage with automation equipment (REBA processor, REBA scanner), and there is a limit in detecting direct sequencing of two or more HPV genotypes, but two REBA products Multiple infection samples can be detected, and one tube nested PCR type can minimize contamination between samples compared to the existing nested PCR type, and β-globin probe, which is an internal control, is fixed to the REBA strip together to improve PCR and DNA extraction process. Errors can be identified, and hybridization positive control for detecting errors in hybridization is also fixed to the REBA strip. There is no effect on the subject's view of the actual experimenter because no equipment is needed.

1 is an explanatory diagram for the basic principle of Reverse Blot Hybridization mentioned in the present invention. That is, ① the amine-labeled probe is bound to the membrane. ② the target gene is amplified with a biotin-labeled primer and then reacted with the probe bound to the membrane. ③ Reacts with Streptavidin-conjugated alkaline phosphatase. ④ Detects defects of probe and amplified target gene by treating substrate of Alkaline phosphatase.
Figure 2 shows the Membrane by PCR amplifying the HPV L1 gene from HPV type 26, 69 belonging to the high-risk group and HPV type 73, 34 belonging to the low-risk group HPV type 73, 34 clones of ATCC and KFDA This is a picture showing detection by color by reacting reverse blot hybridization with a probe bound to
Figure 3 is a photograph showing the REBA results of the sample subjected to Capture2 High-Risk HPV DNA Test ™ (HC2) Test. In other words, 100 cases of Digene (HC2) were developed, and 7 samples were negatively confirmed as a result of 32 detection kits that were added to the existing 25 types of HPV kits. , 43, single, 16, 18, 26, 35, 39, 45, 51, 53, 56, 58, 66, 69, 70 and 6 other genotypes infected, 22 samples of two or more genotypes 78 REBA (Reverse Blot Hybridization Coloring Experiment) is a photograph showing the results.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are set forth to illustrate the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1: Obtaining HPV Positive Clones

Plasma sets corresponding to all seven HPV genotypes were obtained from KFDA and secured, and sequencing data for each plasmid was also confirmed. All these plasmids were transformed into E. coli for expansion and stored in a large amount of plasmids and live cell storage, so plasmid DNA was extracted after cultivation as needed (GeneAll, Seoul, Korea) to polymerase chain amplification ( Polymerase Chain Reaction, PCR). In particular the double HPV-26 plasmid was used as a positive control for the entire experiment.

Example 2: Obtaining HPV Clinical Specimens

Clinical samples used in the present invention were collected from 78 patients who had already been tested with Hybrid Capture 2 High-Risk HPV DNA Test ^TM (HC2, Digene, Gaithersburg, USA) at Wonju Christian Hospital for 6 months from November 2007. In order to verify the reliability of this study, HPV type high risk group 26 low risk group 54 potential risk group 73, 34 which were genotyping by sequencing among clinical specimens isolated from patients at Wonju Christian Hospital, Korea were secured. Medium HPV type 69 and low risk HPV type 32 and 40 were obtained from Gangneung Asan Hospital and Wonju German Hospital.

Example  3: from clinical specimen DNA  extraction

(1) Place 1 ml ~ 1.5 ml clinical test LBC sample into 1.5ml tube.

(2) Centrifuge at 8,000 rpm for 5 minutes at room temperature.

(3) Carefully remove the supernatant.

(4) Add 1 ml PBS (pH 7.2) and vortex for 10 seconds.

(5) Centrifuge at 8,000 rpm for 5 minutes at room temperature.

(6) Repeat steps 4 and 5.

(7) Add 300 ~ 500 μl of sterile distilled water and vortex for 10 seconds.

(8) Centrifuge at 13,000 rpm for 10 minutes at room temperature.

(9) Carefully remove the supernatant.

(10) Add 50 µl of the extraction solution, vortex for 1 minute, and heat for 10 minutes.

(11) Centrifuge at 13,000 rpm for 3 minutes.

(12) Transfer the supernatant to the tube carefully.

(13) Use 3-5 ul as a template for PCR reaction.

Example  4: PCR

The PCR reaction was performed after beta-5PCR was performed to confirm whether there was an inhibitor in the DNA product. The reaction conditions were preliminarily modified for 5 minutes at a denaturation temperature of 94 ° C, followed by 30 seconds of denaturation at 94 ° C, 30 seconds of annealing at 55 ° C, and 30 seconds of extension at 72 ° C. The final elongation reaction was repeated for 7 minutes at 72 ° C. After the reaction, one-tube nested PCR was performed using DNA of amplified product (positive sample). 20 mM Tris-HCl (pH8.0), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween-20, 0.5% Nonidet P-40 and 10 mM dNTP, 25 mM Outer primer of MgCl ₂ , 2 pmole concentration, inner primer of 5 pmole concentration, and 1 unit of Taq polymerase were used. Total reaction amount was 20 μl.

The reaction conditions were premodified for 5 minutes at a denaturation temperature of 94 ° C., followed by 30 cycles of degeneration at 94 ° C., and 15 cycles of reaction at 55 ° C. for 30 seconds, followed by 30 seconds of denaturation at 94 ° C. again. , 45 cycle was reacted by annealing at 52 ° C. for 30 seconds, and finally, a final elongation reaction was performed at 72 ° C. for 10 minutes. In this case, the primer used is MY11 / 09-GP5-1 / 6 +, and thus amplification products of 150bp can be obtained. The base sequence of each primer is as follows. Biotin is labeled at the 5 'end of reverse primer (My09, GP6) for binding to Streptavidin in the subsequent REBA process.

Name order density MY11 (A) GCM CAG GGW CTA TAA YAA TGG (SEQ ID NO: 1) 2 pmole/ul MY09 (B *) CGT CCM ARR GGA WAC TGA TC (SEQ ID NO: 2) 2 pmole/ul GP5-1 (E) TTTGTTACWGTKGTRGATAC (SEQ ID NO: 3) 5 pmole/ul GP6 (D *) GAA AAA TAA ACT GTA AAT CAT ATT C (SEQ ID NO: 4) 5 pmole/ul

Table 1 shows the HPV primer sequences and concentrations. In Table 1 the MY11, MY09, GP5-1 and GP6 + sequences are set forth in SEQ ID NOs: 1-4, respectively.

Table 2 shows the PCR conditions.

Example  5: Probe Available as 자이클립 Wow probe Of Membrane with Oxide

(1) The membrane was cut into 14X 12 cm, and then reacted with 1.6g / 10ml EDAC for 10 minutes and washed with 100ml sterile distilled water for 1 minute.

(2) Place the activated membrane on Miniblotter-MN45 (Immunetics, Cambridge, Mass.) And completely remove water from the slot using an aspirator.

(3) The oligonucleotide to be used as a probe was commissioned to Bioneer (Korea) to add an amine group at the 5 'end to have a positive potential to bind a carboxyl group with a negative potential of the membrane, and to attach a 100 pmole / ul prepared probe to the membrane. To this end, dilute 0.5M NaHCO3 solution to the probe concentration shown in the table below.

(4) After loading the probe into each slot, it was reacted at room temperature for 1 hour.

(5) and then reacted with 100 mM NaOH for 9 minutes.

(6) Incubate at 60 ° C for 5 minutes with HS solution (2X SSPE, 0.1% SDS) warmed to 60 ° C.

(7) Incubate for 5 minutes at 60 ° C with 1% SDS solution heated to 60 ° C.

(8) Add 20 mM EDTA solution and wash at room temperature for 15 minutes.

Probe Name Operating concentration (pmoles / ul) Autoclaved D.W. 100 μM probe Probe Volume (μl) 0.5 M NaHCO ₃ (μl) hyb pc 10 900 100 10 140 Universal 20 800 200 10 140 Type 16 20 800 200 10 140 Type 18 5 950 50 10 140 Type  26 25 975 25 5 145 Type 31 10 900 100 10 140 Type 33 25 750 250 10 140 Type 35 25 750 250 10 140 Type 39 15 850 150 10 140 Type 45 5 950 50 10 140 Type 51 20 800 200 10 140 Type 52 5 950 50 10 140 Type 53 20 800 200 10 140 Type 56 10 900 100 10 140 Type 58 15 850 150 10 140 Type 66 10 900 100 10 140 Type 59 20 800 200 10 130 Type 68 10 900 100 10 Type  69 2.5 975 25 5 145 Type  73 2.5 975 25 5 Type  34 2.5 975 25 5 Type 6 25 750 250 10 140 Type 11 15 850 150 10 140 Type  32 2.5 975 25 5 Type  40 2.5 975 25 5 Type 42 25 750 250 10 140 Type 43 10 900 100 10 140 Type 44 10 900 100 10 140 Type  54 2.5 975 5 Type 70 15 850 150 10 140 Type 72 15 850 150 10 140 Type 84 15 850 150 10 140 Type 81 20 800 200 10 130 Type 87 15 850 150 10

Table 3 is a table showing the probe concentration. The boldfaces are the probe concentrations for the seven additional species.

Example 6 : Reverse Blot Hybridization  ( REBA )

(1) Mix 15 µl and 15 µl Denaturation solution (DS) (1: 1 ratio) to denature PCR products.

(2) After tapping or weakly vortex the mixture, quick-spin and leave at 25 ℃ for 5 minutes.

(3) During the reaction, place a specific oligo-probe-attached Membrane strip for REBA on the tray well and dispense 500 µl of hybridization solution (HS) into each well.

(4) Pipetting DNA denaturate with 470 μl of Hybridization Solution (HS), mix well, and sprinkle on membrane.

(5) Hybridize for 30 minutes at 60 ~ 80 rpm in 50 ℃ constant temperature water bath.

(6) Preheat the Washing Solution (WS) to 50 ° C.

(7) Completely remove the hybridization mixture from the well with an aspirator, dispense 1 ml of pre-warmed Washing Solution (WS), and repeat twice for 10 min at 60 ~ 80 rpm in a 50 ℃ constant temperature water bath.

(8) 1-2 minutes before the second wash is completed, dilute the AP conjugate with Conjugate Dilution Solution (CDS) by 1: 2500 (0.4 μl of AP / 1 ml of CDS).

(9) Completely remove the Wash solution, dispense 1 ㎖ of AP conjugate into each well, react for 30 minutes at 60 ~ 80 rpm at 25 ℃, and completely remove the reaction solution.

(10) Dispense 1 ml of CDS into each well and repeat twice for 1 minute at 25 ° C with Dancer (Shaker).

For the color reaction, the membranes with AP conjugate reaction were washed twice with TBS (50 mM Tis-HCl, 150 mM NaCl, pH 7.5) for 1 min and NBT / BCIP (Nitro Blue Tertrazolium Chloride and 5-Bromo- 4-Chloro-3-indolyl Phosphate, Toluidine salt in 67% DMSO (v / v), Roche Applied Science, Germany) was added to TBS (100 mM Tis-HCl, 100 mM NaCl, 50 mM MgCl ₂ , pH 9.5). After diluting to 1:50 (v / v) and reacting for 5 minutes at room temperature, the desired color development was completed and the color development solution was completely removed and tertiary distilled water was added to stop the reaction.

name Sequence (amine label at 5 ') Universal 5'GNCATGNNGARGAATATGA-3 '(SEQ ID NO: 5) Type 16 5'CAT TAT GTG CTG CCA TAT C-3 '(SEQ ID NO: 6) Type 18 5'TGC TTC TAC ACA GTC TCC T-3 (SEQ ID NO: 7) Type  26 5'TCTGCAGCATCTGCATCC-3 (SEQ ID NO: 8) Type 31 5 'GCA ATT GCA AAC AGT GAT AC-3' (SEQ ID NO: 9) Type 33 5'TGCACACAAGTAACTAGTGA-3 (SEQ ID NO: 10) Type 35 5'CTGCTGTGTCTTCTAGTGA-3 '(SEQ ID NO: 11) Type 39 5'ATAGAGTCTTCCATACCTTC-3 '(SEQ ID NO: 12) Type 45 5'TAATTTAACATTATGTGCCTC-3 '(SEQ ID NO: 13) Type 51 5'TGCTGCGGTTTCCCCAA-3 '(SEQ ID NO: 14) Type 52 5'GAATACCTTCGTCATGGC-3 '(SEQ ID NO: 15) Type 53 5'AACCACACAGTCTATGTCTACA -3 (SEQ ID NO: 16) Type 56 5'CAGAACAGTTAAGTAAATATG-3 '(SEQ ID NO: 17) Type 58 5'TATGCACTGAAGTAACTAAG-3 (SEQ ID NO: 18) Type 66 5'AGCTAAAAGCACATTAACTAA-3 (SEQ ID NO: 19) Type 59 5'TCTACTACTTCTTCTATTCC-3 (SEQ ID NO: 20) Type 68 5'CAGACTCTACTGTACCAGC-3 '(SEQ ID NO: 21) Type  69 5'ACAATCTGCATCTGCCACT-3 (SEQ ID NO: 22) Type  73 5'TAGGTACACAGGCTAGTAGCTCTA-3 (SEQ ID NO: 23) Type  34 5'CCACAAGTACAACTGCACCA-3 (SEQ ID NO: 24) Type 6 5'TCCGTAACTACATCTTCCA-3 '(SEQ ID NO: 25) Type 11 5'TCTGTGTCTAAATCTGCTAC-3 '(SEQ ID NO: 26) Type  32 5'GCTACTGTAACAACTGAAGACACA-3 (SEQ ID NO: 27) Type  40 5'CTTATGTGCTGCCACACAGT-3 (SEQ ID NO: 28) Type 42 5'TGGTGATACATATACAGCTG-3 '(SEQ ID NO: 29) Type 43 5'TCTACTGACCCTACTGTG-3 '(SEQ ID NO: 30) Type 44 5'TACTAGTGAACAATATAAGCA-3 '(SEQ ID NO: 31) Type  54 5'ACAGCATCCACGCAGGA-3 (SEQ ID NO: 32) Type 70 5'GAAACGGCCATACCTG-3 '(SEQ ID NO: 33) Type 72 5'GCGTCCTCTGTATCAGA-3 (SEQ ID NO: 34) Type 84-1 5'AACACCGAATCAGAATATAAACCTACC-3 (SEQ ID NO: 35) Type 81 5'CATCTGCTGCTGCAGA-3 (SEQ ID NO: 36) Type 87-2 5'AACCACTGAATATGACCCCACA-3 (SEQ ID NO: 37)

Table 4 is a table showing the probe sequence. Probe sequences for seven species added in bold.

Infection Reverse Blot hybridization  result Relative frequency (%) HPV type n Negative - 7 8.97 Total 7 8.97 Single 16 14 17.95 18 3 3.85 26 6 7.69 69 One 1.28 53 3 3.85 45 One 1.28 56 2 2.56 39 One 1.28 70 2 2.56 66 One 1.28 35 3 3.85 58 4 5.13 51 One 1.28 33 One 1.28 Total 43 55.13 Double
(19 cases) 58, 26 3 3.85 26, 84 One 1.28 43, 84 One 1.28 16, 66 One 1.28 58, 54 One 1.28 53, 70 One 1.28 53, 58 One 1.28 26, 69 One 1.28 58, 84 One 1.28 16, 26 2 2.56 26, 33 One 1.28 16, 53 One 1.28 18, 35 One 1.28 35, 38 One 1.28 59, 68 One 1.28 35, 58 One 1.28 Total 19 24.36 Multi 16, 18, 26, 45, 52,66, 59/68, 6 One 1.28 18, 26, 52, 59/68 2 2.56 Total 3 3.85 Universal Unknown HPV type 6 7.69 Total 6 7.69 Total 100

Table 5 is a table showing the probability of occurrence according to genotype of the clinical sample using the seven additional HPV kit.

As a result of quantifying the results of REBA on 78 clinical specimens received from Gangneung Asan Hospital, HPV type 16 was the highest with 17.95%, and the probability of occurrence for other genotypes is shown in Table 5.

Attach an electronic file to a sequence list

Claims (9)

HPV diagnostic oligonucleotide probe composition consisting of the nucleotide sequence of SEQ ID NO: 5 to 37. delete HPV diagnostic kit comprising the probe composition of claim 1 and a primer. The kit for diagnosing HPV according to claim 3, wherein the primers are primers set forth in SEQ ID NOs: 1-4. The kit for diagnosing HPV according to claim 3, wherein the kit further comprises labeling means for detecting a PCR product. a) attaching the probe composition of claim 1 to a solid support;
b) hybridizing the amplified HPV PCR product to the probe; And
c) HPV genotyping method comprising the step of detecting the hybridized product. 7. The method of claim 6, wherein said solid support is a membrane, slide, or well plate. 7. The HPV genotyping method according to claim 6, wherein the PCR product is amplified through the primers set forth in SEQ ID NOs: 1-4. 7. The HPV genotyping method according to claim 6, wherein the detecting step is performed using a luminescence or color reaction.

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