WO2013168832A1 - Kit d'analyse des gènes du papillomavirus humain à haut risque, et procédé d'analyse associé - Google Patents

Kit d'analyse des gènes du papillomavirus humain à haut risque, et procédé d'analyse associé Download PDF

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WO2013168832A1
WO2013168832A1 PCT/KR2012/003588 KR2012003588W WO2013168832A1 WO 2013168832 A1 WO2013168832 A1 WO 2013168832A1 KR 2012003588 W KR2012003588 W KR 2012003588W WO 2013168832 A1 WO2013168832 A1 WO 2013168832A1
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seq
nos
hpv
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detecting
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PCT/KR2012/003588
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Korean (ko)
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신지영
신수경
민경태
조우재
유왕돈
김수옥
홍선표
김석준
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(주)진매트릭스
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57411—Specifically defined cancers of cervix
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

Definitions

  • the present invention relates to a kit for genetic analysis of high risk human papillomavirus and a method of analyzing the same, and more particularly, to a method for simple and effective analysis of high risk human papillomavirus, and is particularly classified as a high risk group because it is associated with cervical cancer among various genotypes of HPV.
  • the present invention relates to a kit and a method for detecting only a specific genotype.
  • Cervical cancer is the second most common female cancer after breast cancer worldwide. Epidemiologic investigations have shown that cervical cancer is caused by human papillomavirus (HPV) infection, and HPV DNA has been detected in 99.7% of patients with cervical dysplasia and cervical cancer.
  • HPV human papillomavirus
  • the HPV genome is about 8 kb of double-stranded circular DNA, the open reading frame (ORF) that only differentiates with the E1, E2, E4, E5, E6, and E7 genes that act on its DNA replication and cell transformation. It consists of the L1 and L2 genes that only express epithelial cells to form capsid proteins.
  • ORF open reading frame
  • the classification of HPV genotypes is based on the similarity of DNA sequences, and if the sequences of E6, E7, L1 ORFs differ by more than 10% from the existing sequences, they are defined as new genotypes and show 90-98% similarity. Subtype, defined as a variant at or near 98%.
  • HPV subtypes are known, of which about 40 subtypes are known to be associated with cervical cancer.
  • HPV genotypes are classified into high risk group, probable high risk group and low risk group on the basis of patient / control comparative epidemiological studies.
  • Genotypes belonging to the high-risk group with high incidence of invasive cervical cancer include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 and are established epidemiologically.
  • Genotypes of 26, 53, and 66 genotypes which have not been reported, but have been reported to be associated with the development of cervical cancer, are 6, 11, 40, 42, 43, 44, 54 for low-risk groups that are not associated with carcinogenesis. , 61, 70, 72, 81, and the like.
  • HPV-positive cases were distributed in the order of 16, 52, 53, 62, 58, 81, 56, 66, 18, 44, and invasive squamous cervical cancer (SCC) patients.
  • SCC invasive squamous cervical cancer
  • high-risk HPV were detected in 96.2% of patients and 86% of patients with high grade squamous intraepithelial lesions (HSIL).
  • High-risk HPV infection occurs in the 10s and 20s with the onset of sexual life, and epithelial dysplasia occurs 10 years later and cervical cancer occurs in the 40s and 50s.
  • the risk of developing cervical cancer in women with persistent high-risk HPV infections was 12-350 times higher, with the presence of specific high-risk HPVs detected continuously every six months.
  • the odds ratio of the high-risk HPV group and cervical cancer was 260.63
  • the likelihood ratio of the high-risk and latent high-risk HPV group was 464.1, which supports why the high-risk HPV group should be included in the screening of cervical cancer.
  • current commercial high risk screening products do not include potential risk groups.
  • screening tests for cervical cancer are considered to be very important. Screening tests can be divided into cytology and high-risk HPV DNA tests.
  • Liquid-based Pap smears can be used to observe the morphological changes of cells with the naked eye, thereby reconstructing the cytopathological stages of atypical squamous cells of undetermined significance (ASSCUS) and low grade squamous intraepithelial dysplasia.
  • lesions LSIL
  • high grade squamous intraepithelial lesions HSIL HSIL
  • invasive squamous cervical cancer SCC
  • HPV DNA screening method a hybrid capture method (Qiagen) method and a PCR method are known.
  • Hybrid capture (Qiagen) method is a method of detecting HPV using the hybridization reaction of the probe, which is convenient because there is no separate DNA extraction process, but it requires a large amount of DNA and shows a detection limit when the sample is small Because of the use of RNA probes (probe), the stability is low, considering the screening test equipment is expensive. In addition, false positives due to cross activity, which has been reported continuously since the release, has been pointed out as a big problem, and a probe detecting a high risk group may show false positives in response to HPV DNA rather than a high risk group.
  • the PCR detection method is a method for amplifying a small amount of DNA by producing oligomers that specifically react to HPV nucleic acid, which enables rapid, simple and large-scale testing, and has higher specificity than the cytogenetic method or hybrid capturing method.
  • PCR detection methods have been reported to show differences in analytical sensitivity according to genotypes and reaction compositions and conditions, and amplification products can be obtained using commonly used PGMY11 / 09 and GP5 + / 6 + primer sets. If there is no separate genotyping process, it is not possible to determine whether HPV is a high risk group associated with cervical cancer.
  • secondary PCR (nested PCR method) is used to improve the sensitivity at present, but since the two PCR processes require more time than the first PCR, the false positive rate may increase.
  • the present invention has been made in view of the above-mentioned problems and the necessity of the above, and an object of the present invention is to identify high-risk HPV infected or potentially infected subjects with high sensitivity and specificity, which can be quickly and easily analyzed. It is to provide a method for detecting HPV high risk group.
  • Another object of the present invention is to provide a primer that specifically detects only genotypes of 18 high-risk human papillomaviruses highly associated with cervical cancer, and a kit comprising the same.
  • the present invention provides a primer pair for detecting HPV type 16 of SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, or SEQ ID NO: 7 and 8; SEQ ID NO: 9 and 10, sequence Primer pairs for detecting HPV type 18 of SEQ ID NOs: 11 and 12, or SEQ ID NOs: 13 and 14; pairs of primers for detecting HPV type 31 of SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, or SEQ ID NOs: 19 and 20; And primer pairs for detecting HPV type 33 of SEQ ID NOs: 23 and 24, or SEQ ID NOs: 25 and 26; primer pairs for detecting HPV type 35 of SEQ ID NOs: 27 and 28, SEQ ID NOs: 29 and 30, or SEQ ID NOs: 31 and 32; Primer pairs for detecting HPV type 39 of SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, or SEQ ID NOs: 37 and 38; HPV 45 type
  • the present invention provides a composition for detecting a high risk group HPV virus gene comprising the primer pair of the present invention.
  • the composition is HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, one selected for each HPV subtype of the primer pair of the present invention And combinations of 18 primer pairs for types 68, 73, 82, 26, 53, and 66, but are not limited thereto.
  • the concentration of the HPV 16, 51 and 26 type primer is HPV 18, 33, 35, 39, 45, 52, 56, 58, 68 type , Twice the concentration of primers of type 73, 82, 53, and 66
  • the concentration of the HPV 59 and 31 primer is the HPV type 18, 33, 35, 39, 45, 52, 52 It is preferable that the concentration of the primers of type 56, 58, 68, 73, 82, 53, and 66 is three times the concentration, but is not limited thereto.
  • the present invention provides a kit for detecting a high risk group HPV virus gene comprising the primer pair of the present invention.
  • the kit preferably further comprises a buffer, DNA polymerase, and dNTP, but is not limited thereto.
  • the present invention comprises the steps of obtaining a sample from the subject; And it provides a method for detecting a high risk group HPV gene comprising the step of detecting the polymerase chain reaction and high risk group HPV virus gene using the primer pair according to the present invention.
  • the annealing temperature in the polymerase chain reaction is preferably carried out at 65 to 70 °C but is not limited thereto.
  • the present invention is a genotype 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, genotype of HPV classified as high risk group, low risk group, high risk group associated with high incidence of invasive cervical cancer
  • the high risk groups used in the present invention are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26, 53, including genotypes of HPV high risk group and latent high risk group. And 66.
  • High risk primer (HRP) used in the present invention refers to primers that specifically amplify the HPV high risk group.
  • HPV high risk groups 16 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26, 53, and 66 Genotypes can be analyzed quickly, simply, sensitively, accurately, and efficiently, with no detection of other genotypes, other viruses or other microorganisms, except HPV high risk.
  • HRP Mix a mixture of reaction at a high temperature of 65 °C or more.
  • the present invention to achieve the object described in the problem to be solved, a) amplifying a specific HPV nucleic acid using a primer that can select each genotype from the primers represented by SEQ ID NO: 1 ⁇ 108; b) providing a step of detecting the high-risk HPV DNA from the amplified reaction (PCR product).
  • the HRP used in the present invention has a nucleotide sequence that specifically amplifies only the HPV high risk group gene, and is characterized by a primer formulation that specifically hybridizes under stringent hybridization conditions.
  • the HRP is not present in DNA viruses or microorganisms of humans or other species, and is characterized by having a sequence that exists only in 18 high-risk groups of HPV.
  • HRP is composed of 108 forward or reverse nucleotides, characterized in that the specific reaction at 65 ⁇ 70 °C sequencing, RFMP method, real-time polymerase chain reaction (Real- time PCR), HPV DNA chip can also be used or applied.
  • primers used are designed to operate at 65 ° C. or lower, but the HRP used in the present invention is characterized by the arrangement of guanine and cytosine in the primer sequence and temperature of Tm (Melting Temperature). It is characterized by a special design above °C.
  • the HPV high risk group detection kit of the present invention selects one primer set for each genotype among the primers of SEQ ID NOs: 1 to 108 and includes HRP Mix and DNA polymerase, dNTPs, and buffer containing 18 sets of primers in total. It features.
  • the positive diagnosis rate is more effective than the conventional HPV high risk group detection method. Furthermore, given all of the HPV diagnoses detected in infected patients, it is a robust detection method with more sensitive, specific, and high throughput processing than conventional HPV high risk group assays.
  • FIG. 1 shows annealing temperature by diluting 18 HPV plasmid DNA with TE (Tris-EDTA) buffer (including 1 ng / ml Human Genomic DNA) to find the optimum annealing temperature in HRP.
  • TE Tris-EDTA
  • the amplified product was verified using an automatic electrophoresis equipment.
  • Lane 1 is 50 ° C
  • lane 2 is 55 ° C
  • lane 3 is 60 ° C
  • lane 4 is 65 ° C
  • lane 5 is 70 ° C
  • lane 6 is a negative control and L represents the size marker.
  • 1-1 is 16, 1-2 is 18, 1-3 is 31, 1-4 is 33, 1-5 is 35, 1-6 is 39, 1-7 is 45 1-8 is 51, 1-9 is 52, 1-10 is 56, 1-11 is 58, 1-12 is 59, 1-14 is 73 and 1-14 is 73 , 1-15 is 82, 1-16 is 26, 1-17 is 53, and 1-18 is 66.
  • FIG. 2a of FIG. 2 is a photograph of verifying an amplification product using an automated electrophoresis apparatus after producing a HRP mix at 0.05 ⁇ M, which is less than 0.2 ⁇ M to 0.5 ⁇ M of primer concentration used in a conventional polymerase reaction. .
  • L is the size marker
  • lane 1 is 16, lane 2 is 18, lane 3 is 31, lane 4 is 33, lane 5 is 35, lane 6 is 39, lane 7 is 45, lane 8 51 type, lane 9 is 52 type, lane 10 is 56 type, lane 11 is 58 type, lane 12 is 59 type, lane 13 is 68 type, lane 14 is 73 type, lane 15 is 82 type, lane 16 is 26 type Type, lane 17 is type 53, lane 18 is type 66, Figure 2b is a HRP of 0.025 ⁇ M at the same concentration of the sample diluted with 18 HPV plasmid DNA in PBS buffer (including 1ng / ml Human Genomic DNA) The results of the polymerase reaction using the Mix using the automatic electrophoresis equipment were verified photos.
  • L is the size marker
  • lane 1 is 16, lane 2 is 18, lane 3 is 31, lane 4 is 33, lane 5 is 35, lane 6 is 39, lane 7 is 45, lane 8 51 type, lane 9 is 52 type, lane 10 is 56 type, lane 11 is 58 type, lane 12 is 59 type, lane 13 is 68 type, lane 14 is 73 type, lane 15 is 82 type, lane 16 is 26 type Type, lane 17 is type 53, lane 18 is type 66, and FIGS. 2C to 2G are samples prepared by diluting 18 HPV plasmid DNAs with PBS buffer (containing 1 ng / ml of human genomic DNA) in 10 steps.
  • PBS buffer containing 1 ng / ml of human genomic DNA
  • the amplification product was verified by the automated electrophoresis equipment by performing the polymerase reaction with the same concentration of 0.025 ⁇ M HRP Mix.
  • 5 kinds of sensitivity were significantly lower.
  • 2c represents 16 type
  • 2d represents 31 type
  • 2e represents 51 type
  • 2f represents 59 type
  • 2g 26 type.
  • Lane 1 is also 5x10 3 copies / PCR
  • lane 2 is 2.5x10 3 copies / PCR
  • lane 3 is 1x10 3 copies / PCR
  • lane 4 is 5x10 2 copies / PCR
  • lane 5 is 2.5x10 2 copies / PCR
  • lane 6 is 1x10 2 copies / PCR
  • lane 7 is 5x10 One copies / PCR
  • lane 8 is 2.5x10 One copies / PCR
  • lane 9 is 1x10 One copies / PCR
  • lane 10 is 1 Represents copies / PCR.
  • 3a to 3e of the three HPV genotypes of significantly lower sensitivity among the 18 kinds of HPV genotype HRP Mix was prepared by varying the magnification according to the genotype, 18 kinds of HPV plasmid DNA PBS buffer (1ng / ml of After the polymerase reaction was carried out using a sample diluted 10 consecutively with human genomic DNA), the amplification product was verified using automatic electrophoresis equipment.
  • 16 and 31 types were significantly lower in sensitivity. , 51 type, 59 type, 26 type sensitivity is increased.
  • 3a represents 16 type
  • 3b represents 31 type
  • 3c represents 51 type
  • 3d represents 59 type
  • 3e represents 26 type.
  • Lane 1 is also 5x10 3 copies / PCR
  • lane 2 is 2.5x10 3 copies / PCR
  • lane 3 is 1x10 3 copies / PCR
  • lane 4 is 5x10 2 copies / PCR
  • lane 5 is 2.5x10 2 copies / PCR
  • lane 6 is 1x10 2 copies / PCR
  • lane 7 is 5x10 One copies / PCR
  • lane 8 is 2.5x10 One copies / PCR
  • lane 9 is 1x10 One copies / PCR
  • lane 10 is 1 Represents copies / PCR.
  • FIG. 4A of FIG. 4 is a method for determining HPV high risk group results.
  • L is the size marker, lane 1 positive for HPV high risk group, lane 2 negative for HPV high risk group, and lane 3 did not show an internal control band, so DNA extraction failed or samples were insufficient.
  • FIG. 4B is a spectrum of lane 1 shown in FIG. 4A
  • FIG. 4C is a spectrum of lane 2 shown in FIG. 4A to determine positive or negative HPV high risk groups, respectively.
  • FIG. 4D is a spectrum of lane 3 shown in FIG. 4A, and DNA sampling has failed or the sample is insufficient. Therefore, sampling should be repeated.
  • FIG. 5 shows HPV 6, 11, 40, 42, 44, 54, 55, 61, 62, 67, 68, 70, 71, which were identified as low-risk HPV groups associated with the occurrence of carcinogenesis in the sequencing method.
  • Clinical samples 72, 74, 81, 83, 84, 86, 87, 89, and 102 were subjected to polymerase reaction using HRP Mix, and then the amplification products were verified using automatic electrophoresis equipment. Is a size marker and each lane represents genotypes identified as HPV low risk groups in the sequencing method, PC represents positive control, and NC represents negative control.
  • Figure 6 is a photograph of the amplification product verification using automatic electrophoresis equipment after the examination of 564 women who visited the obstetrics and gynecology department of Konkuk University Hospital. Only 21 of the 564 patients were shown, and all 21 patients showed the corresponding band at 520 bp, an amplification product of human-derived ⁇ -golbin, an internal control. The band appeared around 300bp, a high-risk amplification product.
  • L is the size marker, lanes 1, 2, 3, 5, 6, 9, 10, 11, 15, 16, 17, 20, 21 are considered HPV high risk groups, lanes 4, 7, 8, 12, 13, 14 , 18 and 19 were negative for HPV high risk group.
  • HPV high risk groups known to be closely associated with causing human cervical cancer ie 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26
  • HPV genome sequences were obtained by NCBI GeneBank (Aceession No.
  • the sequence of the primer (HRP) for amplifying the high risk group HPV gene to be analyzed is as follows.
  • Table 1 is a table showing primer sequences and designs.
  • the primers of Table 1 are primers capable of selectively amplifying type-specific HPV DNA by complementarily binding to specific types of HPV DNA through computer simulations, and thus all of the corresponding primers of Table 1 generate specific reactions. The same results are obtained using any of the primers for HPV genotypes indicated in.
  • HRP Mix used in the present invention is HPV 16 type SEQ ID NO: 3 and 4, HPV 18 type 13, 14, HPV 31 type 17, 18, HPV 33 type 25, 26, 29 and 30 for HPV 35, 33 and 34 for HPV 39, 41 and 42 for HPV 45, 47 and 48 for HPV 51, 53 and 54 for HPV 52, HPV 56 Types 57, 58, HPV 58, 67, 68, HPV 59, 69, 70, HPV 68, 77, 78, HPV 73, 83, 84, HPV 82, 89, 90 No. 93, 94 for HPV 26, 99, 100 for HPV 53, and 107, 108 for HPV 66 can complete the present invention.
  • the final reaction solution was a multi-nano (MCE-202, Shimadzu) equipment and automated electrophoresis equipment and DNA-1000Kit capable of analyzing the size of 100bp-1,000bp.
  • Prepare the separation buffer which is a component of the kit, by mixing 62 ⁇ l for each sample, 0.63 ⁇ L of the fluorescent dye solution, 6.2 ⁇ L of the marker solution, and 5 ⁇ L of the PCR product, and confirm the PCR product according to the MultiNA user protocol. As shown in Figs. 1-1 to 1-18, the validity of the bands appearing in each lane of the photograph was determined to match the expected size of the amplification products of Table 1 above.
  • Amplification products were formed in all lanes except negative control of all 18 HPV genotypes, and the size of the amplification products was the same as the expected size of Table 1, as shown in Table 2, 50 °C, 55 depending on the genotype Nonspecific bands were observed at °C and 60 ° C, and no nonspecific bands were found in all genotypes at 65 ° C and 70 ° C.
  • Table 2 is a table showing the presence or absence of non-specific band generation with temperature.
  • HRP Mix was prepared with each primer as 0.05 ⁇ M lower than 0.2 ⁇ M to 0.5 ⁇ M, the primer concentration used in the existing polymerase reaction.
  • the template was diluted with 18 HPV plasmid DNA in PBS buffer (including 1 ng / ml Human Genomic DNA).
  • the final reaction solution was identified by PCR using MultiNA (MCE-202, Shimadzu) (Fig. 2a). As shown in Figure 2a, amplification products were formed in all lanes of the 18 HPV genotypes, and the size of the amplification products was approximately 300 bp, which is consistent with the expected size of the amplification products of Table 1 above, but with non-specific products. It confirmed that it appeared.
  • MultiNA MultiNA
  • the HRP Mix was reduced from 0.05 ⁇ M to 1/2, 1/4, 1/5, 1/10, and the concentration of HRP Mix was measured as described above. It was confirmed that the non-specific product disappeared at 0.025 ⁇ M without decreasing the DNA amplification amount of 18 HPV high risk groups.
  • Fig. 2b In the HRP Mix at a concentration lower than 0.025 ⁇ M, although the same concentration of HPV DNA was used, it was confirmed that the DNA amplification amount of the 18 high HPV risk groups was reduced, so it is not preferable to use the HRP Mix lower than 0.025 ⁇ M. It was judged not.
  • an experimental control group internal control capable of confirming that the DNA extraction failed or the amount of the sample was insufficient was amplified by adding to the HRP Mix.
  • the internal control primer consists of a Forward 5'-AGGGAGGGCAGGAGCCAGGG-3 '(SEQ ID NO: 109) nucleotide and a reverse 5'-CGTGCAGCTTGTCACAGTGCAGC -3' (SEQ ID NO: 110) nucleotide.
  • the experimental control is a primer that can detect human-derived ⁇ -golbin, the amplification product is 520bp, and if the band does not appear, it means that DNA extraction failed or the sample was insufficient.
  • amplification products were formed in all lanes of 18 HPV genotypes, and the size of the amplification products was approximately 300 bp, which is consistent with the prediction result of Table 1 above. In particular, in each lane of every picture, no band was found that was difficult to distinguish from the effective band.
  • the primer concentrations of 16, 31, 51, 59, and 26 in the HRP Mix were increased by 2, 3, and 4 times, respectively.
  • primer sets 2, 24 and 47 were doubled (0.05 ⁇ M) and primer sets 9 and 35 were tripled (0.075 ⁇ M).
  • the HRP Mix was prepared, it was confirmed that all 18 HPV genotypes showed a sensitivity of less than 300copies / PCR.
  • the result of the method of the present invention was determined using the multi-nano (MCE-202, Shimadzu) equipment of the automated electrophoresis equipment and DNA-1000Kit capable of analyzing the size of 100bp-1,000bp.
  • Prepare the separation buffer which is a component of the kit, by mixing 62 ⁇ l for each sample, 0.63 ⁇ L of the fluorescent dye solution, 6.2 ⁇ L of the marker solution, and 5 ⁇ L of the PCR product, and confirm the PCR product according to the MultiNA user protocol. do.
  • the validity of the band or spectrum appearing in each lane of the photograph is determined as the HPV high risk group when the corresponding band or spectrum of about 300 bp, which is the expected size of the PCR product of Table 1, is determined (lane 1 of FIG.
  • HPV plasmid DNAs were serially diluted in PBS buffer (containing 1 ng / ml of human genomic DNA) for 10 steps and then repeated 20 times.
  • Table 3 shows the minimum detection limit of 16 HPV genotypes with repeated 16 test results.
  • Table 3 shows the HPV Type 16 minimum detection limit.
  • Table 4 HPV genotype Minimum detection limit (copy number / PCR) according to the method of the present invention 16 26.91 (95% CI: 14.93-70.33) 18 78.48 (95% CI: 46.23-183.02) 31 42.78 (95% CI: 28.78-93.90) 33 49.63 (95% CI: 27.87-129.93) 35 58.75 (95% CI: 43.10-104.86) 39 57.35 (95% CI: 40.43-113.18) 45 72.84 (95% CI: 48.74-157.48) 51 91.46 (95% CI: 56.81-207.41) 52 34.24 (95% CI: 18.58-92.57) 56 134.10 (95% CI: 91.38-265.27) 58 50.97 (95% CI: 28.96-125.43) 59 129.74 (95% CI: 93.70-238.61) 68 40.20 (95% CI: 22.51-101.79) 73 57.48 (95% CI: 33
  • Table 4 shows the minimum detection limit according to the HPV genotype.
  • the detection method of HPV high risk group according to the present invention is 10 times more sensitive than the conventional method of Hybrid Capture 2 in analyzing HPV high risk group (Lorincz, AT Hybrid Capture TM method for detection of human papillomavirus DNA). in clinical specimens: a tool for clinical management of equivocal Pap smears and for population screening.J. Obstet.Gynaecol.Res. 1996a; 22 (6): 629-636.)
  • HPV 6, 11, 40, 42, 44, 54, 55, 61, 62, 67, 69, 70, 71, 72, 74 identified as low-risk HPV groups with less carcinogenesis in the sequencing method , 81, 83, 84, 86, 87, 89, 102 clinical polymerase reaction was carried out by the method established in Examples 1 and 2.
  • HPV high-risk genotype was not amplified in all 22 genotypes identified as low-risk HPV by sequencing (FIG. 5).
  • Table 5 is a table verifying the specificity of the method of the present invention.
  • a probe detecting a high risk group may show false positives in response to HPV DNA rather than a high risk group in the hybrid capture 2, whereas the detection method according to the present invention detects only a high risk group of HPV. It was confirmed that the false positives due to the crossover error did not appear.
  • hybrid capture 2 a conventional method, has a sensitivity of 81.3%, a specificity of 73.5%, a positive predictive value of 15.6%, and a negative predictive value of 98.5%, and the sequencing method has a sensitivity of 93.8%, a specificity of 73.7%, and a positive predictive value of 17.7%. And 99.5% of the voice predictions were confirmed.
  • the method of the present invention confirmed sensitivity 93.8%, specificity 73.3%, positive predictive value 17.4%, and negative predictive value 99.5%.
  • Table 6 is a table comparing the clinical effectiveness of the conventional method and the method according to the present invention.
  • Table 7 is a table comparing the experiments between the method of the present invention and the conventional method.
  • This experimental example is a genotype of human papillomavirus in order to identify the cause of the inconsistency between the hybrid capture 2 and the sequencing method and the method of the present invention and the method and the sequencing method, which showed 2.1% inconsistency in the experimental example 4 and 2.1% inconsistency. The analysis was performed.
  • hybridcaptures2 positive And sequence negative samples 40 hybridCapture2 negatives And human papillomavirus genotypes of sequencing positive samples.
  • hybrid capture 2 positive Eleven low-risk and unknown risk groups and four high-risk groups of the sequencing negative samples were identified, and the remaining 21 did not identify human papillomaviruses.
  • 39 of 40 hybridcapture2 negative and sequencing positive samples were identified as high risk and one low risk, and one did not identify human papillomavirus.
  • Table 8 is a table confirming the inconsistency between the method of the present invention and the conventional method.

Abstract

Cette invention concerne un kit d'analyse des gènes du papillomavirus humain à haut risque, et un procédé d'analyse associé. Plus spécifiquement, cette invention concerne un procédé d'analyse simple et efficace des gènes du papillomavirus humain à haut risque, et en particulier un kit et un procédé permettant de détecter spécifiquement, parmi les divers types de gènes du papillomavirus, uniquement les types de gènes qui sont étroitement associés au cancer du col de l'utérus et par conséquent classés dans un groupe à haut risque.
PCT/KR2012/003588 2012-05-08 2012-05-08 Kit d'analyse des gènes du papillomavirus humain à haut risque, et procédé d'analyse associé WO2013168832A1 (fr)

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CN108929869A (zh) * 2018-08-09 2018-12-04 亚能生物技术（深圳）有限公司 Hpv全长基因组质控品的制备方法、扩增引物及检测试剂
CN112575123A (zh) * 2021-01-05 2021-03-30 郑州安图生物工程股份有限公司 引物组合、探针组合以及人乳头瘤病毒核酸检测试剂盒

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CN108929869B (zh) * 2018-08-09 2022-03-08 亚能生物技术（深圳）有限公司 Hpv全长基因组质控品的制备方法、扩增引物及检测试剂
CN112575123A (zh) * 2021-01-05 2021-03-30 郑州安图生物工程股份有限公司 引物组合、探针组合以及人乳头瘤病毒核酸检测试剂盒
CN112575123B (zh) * 2021-01-05 2024-02-20 郑州安图生物工程股份有限公司 引物组合、探针组合以及人乳头瘤病毒核酸检测试剂盒

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