WO2015008885A1 - Method for diagnosing cervical cancer and diagnostic kit therefor - Google Patents

Method for diagnosing cervical cancer and diagnostic kit therefor Download PDF

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WO2015008885A1
WO2015008885A1 PCT/KR2013/006512 KR2013006512W WO2015008885A1 WO 2015008885 A1 WO2015008885 A1 WO 2015008885A1 KR 2013006512 W KR2013006512 W KR 2013006512W WO 2015008885 A1 WO2015008885 A1 WO 2015008885A1
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seq
undetermined
hpv
set forth
amplifying
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French (fr)
Korean (ko)
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이혜영
박광화
이동섭
왕혜영
김성현
김연
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엠앤디(주)
연세대학교 원주산학협력단
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Priority to PCT/KR2013/006512 priority Critical patent/WO2015008885A1/en
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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a novel method for diagnosing cervical cancer and a kit for diagnosis thereof.
  • Cervical cancer is the second most common cancer among women worldwide (Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM.Int J Cancer. 2010. 127: 2893-2917. ),
  • the 5-year survival rate of cervical cancer was about 80% or more according to the National Cancer Registration Project. Therefore, the earlier the early detection, the higher the survival rate.
  • the gene of human papillomavirus (HPV) was found in 99.7% of patients with cervical cancer, and the persistence of this HPV infection is known to cause metastasis to invasive cervical cancer and cervical precancerous cancer.
  • genotypes of HPV There are over 100 known genotypes of HPV, and 30 of them are known to cause disease in humans.
  • the genotypes are high risk groups (16, 18, 31, 33, 35, 39, 45, 51, 52). , 56, 58, 59, 68, 73, 82) and low risk groups (6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81) and potential risk groups (34, 57, 83).
  • the genotype-specific HPVs were identified according to the location of the lesion and the progression of the lesion, thereby recognizing the biological diversity of HPV infection.
  • Pap Pap smear (Papanicolaou smear), which performs cytological examination using shedding cells obtained from the cervix.
  • Pap Pap smear Papanicolaou smear
  • it has a disadvantage of having a very high false negative rate with sensitivity of about 20-50% (Hwang TS, Jeong JK, Park M, Han HS, Choi HK, Park TS Gynecol Oncol. 2003. 90: 51-56).
  • HSIL high-grade squamous intraepithelial lesions
  • vaginal magnification test method is more accurate than uterine Pap smear, but requires a skilled technician and expensive equipment, there is a disadvantage that can not distinguish between HPV infection.
  • HPV HPV DNA
  • Pap test a cytological test that has been used for cervical cancer.
  • HPV genotyping is based on the detection of HPV DNA from the cervix and the L1 gene encoding HPV capsid proteins.
  • Many HPV DNA assays have been developed and commercialized, including the HPV genotyping assay.
  • these molecular diagnostic methods are used together as an adjunct to Pap smears (Thomas I, Liesje G, Ryan S. J Clin Microbiol. 1999. 37: 2508-2517), and its use is also increasing domestically (Cho EJ, Do JH, Kim YS, Bae S, Ahn WS. J Med Microbiol. 2011. 60: 162-171).
  • the HPV DNA test is typically a method of detecting the presence of HPV DNA using a polymerase chain reaction (PCR) or a method of testing the genotype of HPV using a DNA chip or reverse blot hybridization assay (REBA). This is known.
  • PCR polymerase chain reaction
  • REBA reverse blot hybridization assay
  • HPV DNA and HPV Genotyping tests have the advantage of being highly sensitive and revealing the genotypes of infected HPV, but their analytical sensitivity is so high that they are not only found in high-grade lesions SCC and HSIL, but also in low-grade lesions and normal findings. There is a limit of detection at a high rate of about 40 to 50%, and the presence of HPV DNA alone can detect infection of HPV, but has the disadvantage of not being able to diagnose cervical cancer immediately.
  • the region coding for the largest l1 capsid protein of HPV nucleic acid has been developed, but rather, it is E6 / E7, a protein expressed when inducing cancer. Detecting the degree of expression of the gene by targeting the mRNA coding for the cervical cancer may be more useful in determining the prognosis of cervical cancer and cervical cancer.
  • HPV has been developed to target mRNAs encoding E6 and E7 genes, which are HPV high-risk carcinogens, and HPV, which is already known to be the most common in cervical cancers worldwide.
  • real-time NASBA assays that detect 16, 18, 31, 33, and 45 types of E6 and E7 mRNAs are commercially available, these test kits are quite expensive (price: 30,000 won / test), In order to do this, a dedicated analysis machine is needed, but since the current large and medium-sized hospitals do not have such analytical machines, it is difficult to apply them to clinical tests.
  • the commercialized E6 and E7 mRNA assays are available worldwide.
  • HPV isolates from domestic cervical cancer because they contain five genotypes of HPV (HPV 16, 18, 31, 33, 45) Because the type and indicate the differences there are limitations to apply to domestic patients.
  • the present invention has been made in view of the above problems and the need to provide a method for diagnosing cervical cancer quickly and accurately.
  • Another object of the present invention is to provide a rapid and accurate kit for diagnosing cervical cancer.
  • the present invention to achieve the above object is a) separating the full-length RNA from the cells obtained from the blood of the patient; b) synthesizing cDNA from the isolated full-length RNA; c) a primer pair and a primer pair capable of amplifying HPV types 16, 35, 31, 58, 33, and 52, and a primer pair capable of amplifying HPV types 18, 45, 39, 68, and 59 And performing real-time PCR using one or more primer pairs and probes selected from the group consisting of probes and primer pairs and probes capable of amplifying HPV types 53, 56, 66, 51, 69, 26, and 30 ; And d) comparing the expressed amount with the amount expressed for a normal person. It provides an information providing method for diagnosing cervical cancer.
  • Primers of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, “capsulation”, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosph Modifications to poroamidates, carbamates, etc.) or charged linkers (eg, phosphorothioates, phosphorodithioates, etc.).
  • Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, psoralene, etc.). ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. Nucleic acid sequences of the invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.
  • the amplified target sequence may be labeled with a detectable labeling substance.
  • the labeling material may be a fluorescent, phosphorescent, chemiluminescent or radioactive material, but is not limited thereto.
  • the labeling substance may be fluorescein, phycoerythrin, rhodamine, lissamine Cy-5 or Cy-3.
  • real-time RT-PCR may be performed by labeling Cy-5 or Cy-3 at the 5'-end and / or 3 'end of the primer to label the target sequence with a detectable fluorescent label. .
  • the label using radioactive material is added to the PCR reaction solution by adding radioactive isotopes such as 32 P or 35 S to the PCR reaction solution during real-time RT-PCR, and the amplification product is radioactively incorporated into the amplification product.
  • radioactive isotopes such as 32 P or 35 S
  • the amplification product is radioactively incorporated into the amplification product.
  • One or more sets of oligonucleotide primers used to amplify a target sequence can be used.
  • Labeling is carried out in a variety of ways conventionally practiced in the art, such as nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, "Amersham” (1989)) and chination methods (Maxam & Gilbert, Methods). in Enzymology, 65: 499 (1986)). Labels provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetric, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, nanocrystals.
  • the present invention is to measure the expression level at the mRNA level via RT-PCR.
  • a novel primer pair and a fluorescently labeled probe that specifically bind to the HPV gene are required.
  • the primers and probes specified by specific nucleotide sequences may be used, but are not limited thereto. It can be used without limitation as long as it can perform real-time RT-PCR by specifically binding to provide a detectable signal.
  • FAM and Quen (Quencher) means a fluorescent dye.
  • Real-time RT-PCR method applied to the present invention can be carried out through known procedures commonly used in the art.
  • the step of measuring the mRNA expression level can be used without limitation as long as it is a method capable of measuring the normal mRNA expression level, depending on the type of probe label used can be performed by radiometric measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. It doesn't work.
  • the fluorescence measurement method uses Cy-5 or Cy-3 at the 5'-end of a primer to perform real-time RT-PCR to label a target sequence with a detectable fluorescent label.
  • the labeled fluorescence may be measured using a fluorimeter.
  • the radioactivity measuring method is to add a radioactive isotope such as 32 P or 35 S to the PCR reaction solution when real-time RT-PCR is performed to label the amplification product, and then radioactive measuring apparatus, for example, Geiger counter (Geiger Radioactivity can be measured using a counter or a liquid scintillation counter.
  • a radioactive isotope such as 32 P or 35 S
  • radioactive measuring apparatus for example, Geiger counter (Geiger Radioactivity can be measured using a counter or a liquid scintillation counter.
  • a fluorescent-labeled probe is attached to the PCR product amplified by the realtime RT-PCR to emit fluorescence of a specific wavelength, and at the same time, the fluorescence measuring device of the realtime PCR device The mRNA expression level of the genes are measured in real time, and the measured values are calculated and visualized through a PC so that the examiner can easily check the expression level.
  • the step of comparing the amplified amount with the amplified amount for a normal person is preferably performed by a cutoff value, it is marked as positive for a sample whose Ct value is detected below 35 as the cutoff. More preferably, but not limited to.
  • primer pairs capable of amplifying the HPV types 16, 35, 31, 58, 33, and 52 are set forth in SEQ ID NOs: 1 and 2, and the probes are set forth in SEQ ID NO: 3 It is preferable to have
  • the primer pairs capable of amplifying the HPV types 18, 45, 39, 68, and 59 are set forth in SEQ ID NOs: 4 and 5, and the probe has a nucleotide sequence set forth in SEQ ID NO: 6 Is preferred,
  • the primer pairs capable of amplifying the HPV types 53, 56, 66, 51, 69, 26, and 30 are described in SEQ ID NOs: 7 and 8, or 10 and 11, probes Preferably has a nucleotide sequence set forth in SEQ ID NO: 9 or 12, but is not limited thereto.
  • the present invention is a probe having a primer pair shown in SEQ ID NO: 1 and 2 and a nucleotide sequence shown in SEQ ID NO: 3 capable of amplifying HPV type 16, 35, 31, 58, 33, and 52; A primer pair as set forth in SEQ ID NOs: 4 and 5 and a probe as set forth in SEQ ID NO: 6 capable of amplifying HPV types 18, 45, 39, 68, and 59; And a primer pair as set forth in SEQ ID NOs: 7 and 8 or 10 and 11 capable of amplifying HPV types 53, 56, 66, 51, 69, 26, and 30 and a probe as set forth in SEQ ID NOs: 9 or 12
  • a primer pair and a probe for the diagnosis of cervical cancer comprising the above primer pair and probe.
  • the present invention provides a composition for diagnosing cervical cancer comprising the primer pair and probe of the present invention.
  • the present invention provides a kit for diagnosing cervical cancer comprising the composition of the present invention.
  • the diagnostic kit may be a kit for diagnosing cancer, which includes an essential element necessary for performing reverse transcriptase.
  • the reverse transcription polymerase kit may comprise each primer pair specific for the gene of the present invention.
  • the primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and may be about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length, and more preferably, SEQ ID NO: It may include a novel primer pair and a fluorescently labeled probe represented by.
  • reverse transcriptase kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), enzymes such as deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC - May include DEPC-water, sterile water, and the like.
  • reaction buffers pH and magnesium concentrations vary
  • enzymes such as deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase
  • DNAse DNAse
  • RNAse inhibitor DEPC - May include DEPC-water, sterile water, and the like.
  • the term "information providing method for diagnosing cancer” in the present invention is to provide objective basic information necessary for diagnosing cancer as a preliminary step for diagnosis and excludes the clinical judgment or findings of the doctor.
  • primer refers to a short nucleic acid sequence that is capable of forming base pairs with complementary templates with nucleic acid sequences having short free 3-terminal hydroxyl groups and that serves as a starting point for template strand copying.
  • Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.
  • Primers of the invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis.
  • probe It is a single chain nucleic acid molecule and comprises a sequence that is complementary to a target nucleic acid sequence.
  • realtime RT-PCR refers to a target primer and label using cDNA produced after reverse transcription of RNA into complementary DNA (cDNA) using reverse transcriptase. It is a molecular biological polymerization method that amplifies a target using a target probe and simultaneously detects a signal generated from a label of a target probe on the amplified target.
  • the technical aspects of the diagnostic methods to be developed are expected to overcome the limitations of the existing HPV DNA test by providing a faster and more accurate population of patients who need clinical and preventive treatment, and it is possible to automate the extraction of RNA from fallen cells.
  • result analysis is made possible by software, which will provide more objective and accurate results more quickly.
  • Figure 1 shows the results of clinical sample GAPDH mRNA real-time RT-PCR
  • the collection and acquisition of clinical specimens of the present invention is performed in patients who underwent cervical fluid cytology (Thin Prep PAP TEST, PreservCyt Solution, Hologic Inc. Marlborough, MA, USA) Dropped cells in the cervix were collected and stored in 100% anhydrous ethanol for RNA extraction.
  • cervical fluid cytology Thin Prep PAP TEST, PreservCyt Solution, Hologic Inc. Marlborough, MA, USA
  • Table 1 shows the concentration and purity of total RNA extracted from clinical samples.
  • Tris-HCl pH 8.3 50 mM
  • KCl 75 mM MgCl 2 3 mM
  • DTT 8 mM and MMLV Add 200 units of reverse transcriptase (Invitrogen), add DEPC treated DW to the final volume of 20 ul, mix well, and mix the reaction solution at 25 °C for 10 minutes at thermocycler (ABI), 50 minutes at 37 °C, 70 °C The reaction was carried out for 15 minutes to synthesize cDNA.
  • ABSI thermocycler
  • cDNA was found to be suitable for use in the present invention (FIG. 1).
  • Table 2 shows the Primer and Probe sequences used in the present invention.
  • the composition of the real-time PCR was 25 mM TAPS (pH 9.3 at 25 ° C), 50 mM KCl, 2 mM MgCl 2 , 1 mM 2-mercaptoethanol, 200 ⁇ M each dNTP, 1 unit Taq polymerase (TAKARA), Forward primer and Reverse 10pmole was added to each primer, and 10pmole was added to the probe, and 2ul of the synthesized cDNA was added to a final volume of 20ul.
  • the PCR reaction was performed once using CFX 96 (BioRad-USA) at a denaturation temperature of 94 ° C. for 5 minutes, followed by 40 cycles of 30 seconds at denaturation temperature of 95 ° C. and 20 seconds at annealing temperature of 55 ° C. In addition, the fluorescence was measured after each annealing process, and the fluorescence value increased with each cycle was measured.
  • Set II detected HPV types 18, 39, 45, 59 and 68 using Cy5 dye.
  • HPV types 53, 56, 66, 51, and 69 were detected using HEX dye (Table 3).
  • Table 3 shows the detection of multiplex real-time PCR according to HPV type.
  • Real-time NASBA can detect E6 / E7 mRNA for each target genotype in specimens positive for internal control (IC).
  • IC internal control
  • 38 samples showed IC-positive positive for real-time NASBA and detected E6 / E7 mRNA for each target genotype, showing 28/38 (73.7%) positive rate.
  • 5/38 (13.2%) of the genotypes could not be detected in real-time NASBA, and 5/38 (13.2%) were detected in real-time NASBA but not detected.
  • Table 4 compares the detection of Real-time NASBA and Multiplex Real-time PCR using HPV mRNA.
  • Cytopathological examination showed high grade lesions and multiplex real-time PCR was performed using DNA samples of 75 HPV-infected patients on DNA genotyping (DNA chip). The positive rate was 98.7% and the negative rate was 1.3%. In addition, multiplex real-time PCR was performed using 110 samples uninfected with HPV in DNA pathotyping and showed normality in cytopathologic tests. The positive rate was 0% and the negative rate was 100%. As a result, the specificity of the test was 100% and the sensitivity was 98.7%, which was very high compared to the conventional test method (Table 5).
  • Table 5 shows the results of multiplex real-time PCR using HPV mRNA isolated from clinical specimens.
  • HPV DNA positive rate in the cervix of normal people is very high. Therefore, to confirm the usefulness of the mRNA-targeted test, 66 samples infected with HPV high-risk group were detected by DNA pathology. Multiplex real-time PCR was performed using the result, and as a result, 6.3% (6/95) was detected and it was confirmed that it could reduce the false positive problem for DNA chip (Table 6).
  • Table 6 compares DNA chip results with DNA and multiplex real-time PCR using mRNA.

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Abstract

The present invention relates to a novel method for diagnosing cervical cancer and a diagnostic kit therefor.

Description

자궁경부암 진단 방법 및 그 진단용 키트Cervical cancer diagnostic method and kit for diagnosis thereof
본 발명은 신규한 자궁경부암 진단 방법 및 그 진단용 키트에 관한 것이다.The present invention relates to a novel method for diagnosing cervical cancer and a kit for diagnosis thereof.
자궁경부암은 세계적으로 여성에게 발생하는 암 중 두 번째로 많이 발생하는 암이며(Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM.Int J Cancer. 2010. 127: 2893-2917.), 국가암등록사업에 의해 자궁경부암의 5년 생존율이 약 80%이상으로 나타났고 이에 따라 조기에 발견 할수록 생존율이 높아짐을 알 수 있다. 자궁경부암 환자의 99.7%에서 인유두종바이러스(Human Papillomavirus, HPV)의 유전자가 존재하는 것이 밝혀졌으며, 이러한 HPV 감염의 존속이 침윤성 자궁경부암, 자궁경부 전암으로의 전이를 일으키는 것으로 알려져 있다. Cervical cancer is the second most common cancer among women worldwide (Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM.Int J Cancer. 2010. 127: 2893-2917. ), The 5-year survival rate of cervical cancer was about 80% or more according to the National Cancer Registration Project. Therefore, the earlier the early detection, the higher the survival rate. The gene of human papillomavirus (HPV) was found in 99.7% of patients with cervical cancer, and the persistence of this HPV infection is known to cause metastasis to invasive cervical cancer and cervical precancerous cancer.
현재 HPV의 유전자형은 100가지 이상이 알려져 있고 이 중에서 사람에게 질병을 일으킬 수 있는 유전자형은 30가지 정도로 알려선져 있으며 그 유전자형은 고위험군 (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82) 과 저위험군 (6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81), 잠재위험군(34, 57, 83)으로 분류하고 있으며, 각각의 유전자형을 지닌 HPV가 병소의 위치와 병변의 진행정도에 따라 유전자형 특이적으로 발견됨으로써 HPV 감염의 생물학적 다양성을 인지하게 되었다.There are over 100 known genotypes of HPV, and 30 of them are known to cause disease in humans. The genotypes are high risk groups (16, 18, 31, 33, 35, 39, 45, 51, 52). , 56, 58, 59, 68, 73, 82) and low risk groups (6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81) and potential risk groups (34, 57, 83). The genotype-specific HPVs were identified according to the location of the lesion and the progression of the lesion, thereby recognizing the biological diversity of HPV infection.
HPV의 감염여부를 진단하기 위하여 가장 많이 쓰이는 방법은 자궁경부에서부터 얻은 탈락세포를 이용해 세포진단학적 검사를 수행하는 자궁경부 세포진검사 (Papanicolaou smear, 이하 Pap 도말검사) 가 있으며, 이는 검사방법이 간단하고 비용이 적게 드는 경제적인 이유로 자궁경부암 선별 검사로서 유용성이 높다고 알려져 있지만, 민감도가 약 20~50% 로 위음성률이 매우 높다는 단점이 있다 (Hwang TS, Jeong JK, Park M, Han HS, Choi HK, Park TS. Gynecol Oncol.2003. 90: 51-56). 특히 High-grade squamous intraepithelial lesion (이하 HSIL)에 대한 낮은 민감도와 예측도 (predictive value)가 보고되고 있고, 편평세포암에 비해 선병변 (grandular lesion)이나 선암 (adenocarcinoma)에 대한 낮은 선별력으로 인해 자궁경부암의 조기 진단이 어렵다는 등 자궁경부암 진단에 있어 Pap 도말검사의 여러 가지 제한점이 나타나고 있다 (Kim YS, Lee HJ, Lee GG. Korean J Clin Pathol. 2001. 21: 210-214;Kwon HS, Kim YT, Kim JW, Kim SH. Korean J Gynecol Oncol Colposc. 2002. 13: 327-335.).The most commonly used method for diagnosing HPV infection is Pap Pap smear (Papanicolaou smear), which performs cytological examination using shedding cells obtained from the cervix. Although it is known to be useful as a screening test for cervical cancer because of its low cost, it has a disadvantage of having a very high false negative rate with sensitivity of about 20-50% (Hwang TS, Jeong JK, Park M, Han HS, Choi HK, Park TS Gynecol Oncol. 2003. 90: 51-56). In particular, low sensitivity and predictive value for high-grade squamous intraepithelial lesions (HSIL) have been reported, and due to the low screening capacity for grandular lesions or adenocarcinoma compared to squamous cell carcinoma, Several limitations of Pap smear have been shown in the diagnosis of cervical cancer such as early diagnosis of cervical cancer (Kim YS, Lee HJ, Lee GG.Korean J Clin Pathol. 2001. 21: 210-214; Kwon HS, Kim YT , Kim JW, Kim SH.Korean J Gynecol Oncol Colposc. 2002. 13: 327-335.).
또한 질확대경을 이용한 검사 방법은 자궁세포진 검사보다 정확한 결과를 얻을 수는 있지만, 숙련된 기술자와 고가의 장비가 필요하며, HPV의 감염유무를 구별할 수 없다는 단점이 있다.In addition, vaginal magnification test method is more accurate than uterine Pap smear, but requires a skilled technician and expensive equipment, there is a disadvantage that can not distinguish between HPV infection.
자궁경부암으로 진행하는 과정에 HPV가 중요한 원인으로 알려지면서 기존에 자궁경부암검사에 사용되던 세포학적 검사법인 Pap test와 더불어 자궁암의 조기 진단에 HPV DNA의 존재 유무를 밝히는 것이 중요하게 여겨지고 있다.As HPV is known to be an important cause of cervical cancer, it is important to identify the presence of HPV DNA in the early diagnosis of cervical cancer along with Pap test, a cytological test that has been used for cervical cancer.
자궁경부암으로 발전함에 있어서 인유두종바이러스 (human papillomavirus; 이하 HPV)의 감염이 연관이 있다는 점이 알려지면서, 자궁경부에서부터 HPV DNA를 검출하는 방법부터 HPV의 캡시드 단백질을 암호화하는 L1 유전자를 기초로 HPV 유전형을 검사하는 HPV 유전형 검사법 등 HPV DNA 검사법들이 많이 개발되어 상용화되었고, 현재 이러한 분자진단학적 방법들은 Pap 도말검사의 보조적인 검사로 함께 사용되고 있으며 (Thomas I, Liesje G, Ryan S. J Clin Microbiol. 1999. 37: 2508-2517), 그 이용이 국내에서 또한 증가하고 있다 (Cho EJ, Do JH, Kim YS, Bae S, Ahn WS. J Med Microbiol.2011. 60: 162-171).Knowing that infection of human papillomavirus (HPV) is involved in the development of cervical cancer, HPV genotyping is based on the detection of HPV DNA from the cervix and the L1 gene encoding HPV capsid proteins. Many HPV DNA assays have been developed and commercialized, including the HPV genotyping assay. Currently, these molecular diagnostic methods are used together as an adjunct to Pap smears (Thomas I, Liesje G, Ryan S. J Clin Microbiol. 1999. 37: 2508-2517), and its use is also increasing domestically (Cho EJ, Do JH, Kim YS, Bae S, Ahn WS. J Med Microbiol. 2011. 60: 162-171).
HPV DNA 검사법으로는 대표적으로 중합효소연쇄반응(Polymerase chain reaction, PCR)을 이용해 HPV의 DNA 존재 유무를 알아볼 수 있는 방법 또는 DNA chip나 reverse blot hybridization assay(REBA)를 이용해 HPV의 유전자형을 검사하는 방법이 알려져 있다.The HPV DNA test is typically a method of detecting the presence of HPV DNA using a polymerase chain reaction (PCR) or a method of testing the genotype of HPV using a DNA chip or reverse blot hybridization assay (REBA). This is known.
HPV DNA 검사와 HPV Genotyping 검사는 민감도가 매우 높고, 감염되어 있는 HPV의 유전형을 알 수 있다는 장점이 있지만, 분석적 민감도가 너무 높아 고등급 병변인 SCC와 HSIL에서 뿐만 아니라 저등급 병변과 정상 소견에서도 약 40~50% 가량 높은 비율로 검출이 된다는 제한점이 있으며 HPV DNA의 존재만으로는 HPV의 감염을 검출할 수는 있지만, 자궁겸부암을 바로 진단할 수 없다는 단점이 있다.HPV DNA and HPV Genotyping tests have the advantage of being highly sensitive and revealing the genotypes of infected HPV, but their analytical sensitivity is so high that they are not only found in high-grade lesions SCC and HSIL, but also in low-grade lesions and normal findings. There is a limit of detection at a high rate of about 40 to 50%, and the presence of HPV DNA alone can detect infection of HPV, but has the disadvantage of not being able to diagnose cervical cancer immediately.
자궁경부가 HPV에 감염되면, HPV의 발암유전자인 E6/E7가 과발현되어 p53과 pRB와 같은 암 억제 단백질의 기능을 억제해 암을 유도하게 된다. When the cervix is infected with HPV, E6 / E7, the oncogene of HPV, is overexpressed, leading to cancer by inhibiting the function of cancer suppressor proteins such as p53 and pRB.
하지만, 최근 몇 년간의 연구결과에 따르면, 자궁경부암환자 뿐만 아니라 암으로 발생하기 이전인 염증단계 또는 정상인의 자궁경부에서도 HPV DNA 양성률이 매우 높게 나오고 있기 때문에, HPV DNA가 검출되더라도 임상적인 자궁경부암의 치료 및 예방을 하기에 매우 어려운 실정에 있다.However, recent years have shown that HPV DNA positive rates are high not only in cervical cancer patients but also in inflammatory stages or normal cervical cervix before cancer. The situation is very difficult to treat and prevent.
그동안 HPV DNA 검출 및 유전자형 검출을 위해서 HPV의 핵산 중 가장 큰 크기의 L1(late gene 1) capsid 단백질을 암호화하는 부위를 표적으로 개발되어 왔지만, 이 보다는 암을 유도할 때 발현되는 단백질인 E6/E7을 암호화하는 mRNA를 표적으로 그 유전자 발현양의 정도를 검출하는 것이 자궁경부암 및 자궁경부암의 예후를 판정하는데 보다 유용성이 있다고 본다.For the detection of HPV DNA and genotype detection, the region coding for the largest l1 capsid protein of HPV nucleic acid has been developed, but rather, it is E6 / E7, a protein expressed when inducing cancer. Detecting the degree of expression of the gene by targeting the mRNA coding for the cervical cancer may be more useful in determining the prognosis of cervical cancer and cervical cancer.
현재까지 알려진 바로는 국내에서는 HPV 발암유전자 E6, E7 mRNA를 표적으로 한 HPV mRNA검사법의 개발이 미미한 상황이며 국내 중대형병원에서의 HPV DNA 유전형검사의 수행은 증가하고 있는 추세에 있지만, HPV DNA 양성율이 자궁경부암뿐만 아니라 정상에서의 양성율이 모두 높게 나타났기 때문에 직접적인 임상적 치료나 예방치료에 바로 적용하기는 힘든 상황이다.As far as is known, the development of HPV mRNA test targeting HPV oncogenes E6 and E7 mRNA is insignificant in Korea and the performance of HPV DNA genotyping in medium and large hospitals in Korea is increasing. As well as cervical cancer as well as the positive rate in both normal, it is difficult to apply directly to direct clinical treatment or preventive treatment.
최근에는 HPV의 DNA를 이용한 검사가 아닌 HPV 고위험군의 발암유전자인 E6와 E7유전자를 암호화 하고 있는 mRNA를 표적으로 한 검사법이 개발되고 있는 추세이며, 이미 전 세계적으로 자궁경부암에서 가장 많이 존재한다고 알려진 HPV 16, 18, 31, 33, 45형의 E6, E7 mRNA를 검출할 수 있는 real-time NASBA 검사법이 상용화되었지만 이러한 검사키트들은 상당히 고가(단가:3만원/검사)이며, real-time NASBA를 하기위해서는 전용분석기계가 필요하지만, 현재 중대형 병원에서는 이러한 전용분석기계를 보유하고 있지 않기 때문에, 실질적으로 임상검사에 적용하기는 힘든 상황이며, 또한, 현재 상용화되어있는 E6, E7 mRNA 검사법의 경우 전 세계적으로 유행하는 HPV의 5개 유전형(HPV 16, 18, 31, 33, 45)을 포함하고 있기 때문에 국내에서 발생하는 자궁경부암으로부터 분리되는 HPV의 유전형과는 차이를 나타내기 때문에 국내의 환자들에게 적용하기에는 제한점이 있다.In recent years, HPV has been developed to target mRNAs encoding E6 and E7 genes, which are HPV high-risk carcinogens, and HPV, which is already known to be the most common in cervical cancers worldwide. Although real-time NASBA assays that detect 16, 18, 31, 33, and 45 types of E6 and E7 mRNAs are commercially available, these test kits are quite expensive (price: 30,000 won / test), In order to do this, a dedicated analysis machine is needed, but since the current large and medium-sized hospitals do not have such analytical machines, it is difficult to apply them to clinical tests. In addition, the commercialized E6 and E7 mRNA assays are available worldwide. HPV isolates from domestic cervical cancer because they contain five genotypes of HPV ( HPV 16, 18, 31, 33, 45) Because the type and indicate the differences there are limitations to apply to domestic patients.
[선행 특허문헌][Previous Patent Document]
대한민국특허공개번호 제1020040078506호 Korean Patent Publication No. 1020040078506
본 발명은 상기의 문제점을 해결하고 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 신속하고 정확한 자궁경부암 진단 방법을 제공하는 것이다.SUMMARY OF THE INVENTION The present invention has been made in view of the above problems and the need to provide a method for diagnosing cervical cancer quickly and accurately.
본 발명의 다른 목적은 신속하고 정확한 자궁경부암 진단용 키트를 제공하는 것이다.Another object of the present invention is to provide a rapid and accurate kit for diagnosing cervical cancer.
상기의 목적을 달성하기 위하여 본 발명은 a) 환자의 혈액에서 얻은 세포로부터 전장 RNA를 분리하는 단계;b) 상기 분리된 전장 RNA로부터 cDNA를 합성하는 단계; c) 상기 합성된 cDNA를 HPV 타입 16, 35, 31, 58, 33, 및 52를 증폭할 수 있는 프라이머쌍 및 프로브, HPV 타입 18, 45, 39, 68, 및 59를 증폭할 수 있는 프라이머쌍 및 프로브, 및 HPV 타입 53, 56, 66, 51, 69, 26, 및 30을 증폭할 수 있는 프라이머 쌍 및 프로브로 구성된 군으로부터 선택된 하나 이상의 프라이머쌍 및 프로브를 이용하여 실시간-PCR을 수행하는 단계; 및 d) 상기 발현된 양을 정상인에 대해 발현된 양과 비교하는 단계;를 포함하는 자궁경부암의 진단을 위한 정보제공방법을 제공한다.The present invention to achieve the above object is a) separating the full-length RNA from the cells obtained from the blood of the patient; b) synthesizing cDNA from the isolated full-length RNA; c) a primer pair and a primer pair capable of amplifying HPV types 16, 35, 31, 58, 33, and 52, and a primer pair capable of amplifying HPV types 18, 45, 39, 68, and 59 And performing real-time PCR using one or more primer pairs and probes selected from the group consisting of probes and primer pairs and probes capable of amplifying HPV types 53, 56, 66, 51, 69, 26, and 30 ; And d) comparing the expressed amount with the amount expressed for a normal person. It provides an information providing method for diagnosing cervical cancer.
일반적으로 사용되는 전장 RNA(Total RNA)를 분리하는 방법 및 이로부터 cDNA를 합성하는 방법은 공지된 방법을 통해 수행될 수 있으며, 이 과정에 대한 자세한 설명은 Joseph Sambrook 등, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); 및 Noonan, K. F. 등에 개시되어 있어 본 발명의 참조로서 삽입될 수 있다.A method for separating totally used full RNA and synthesizing cDNA therefrom can be carried out through a known method. For a detailed description of this process, see Joseph Sambrook et al., Molecular Cloning, A Laboratory Manual. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And Noonan, K. F. et al., Which may be incorporated by reference of the present invention.
본 발명의 프라이머는 포스포르아미다이트 고체 지지체 방법, 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비제한적인 예로는 메틸화, "캡화", 천연 뉴클레오타이드 하나 이상의 동족체로의 치환, 및 뉴클레오타이드 간의 변형, 예를 들면, 하전되지 않은 연결체(예: 메틸 포스포네이트, 포스포트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다. 핵산은 하나 이상의 부가적인 공유 결합된 잔기, 예를 들면, 단백질(예: 뉴클레아제, 독소, 항체, 시그날 펩타이드, 폴리-L-리신 등), 삽입제(예: 아크리딘, 프소랄렌 등), 킬레이트화제(예: 금속, 방사성 금속,철, 산화성 금속 등), 및 알킬화제를 함유할 수 있다. 본 발명의 핵산 서열은 또한 검출 가능한 시그널을 직접 또는 간접적으로 제공할 수 있는 표지를 이용하여 변형시킬 수 있다. 표지의 예로는 방사성 동위원소, 형광성 분자, 바이오틴 등이 있다.Primers of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, “capsulation”, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosph Modifications to poroamidates, carbamates, etc.) or charged linkers (eg, phosphorothioates, phosphorodithioates, etc.). Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, psoralene, etc.). ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. Nucleic acid sequences of the invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.
본 발명의 방법에 있어서, 상기 증폭된 표적 서열은 검출가능한 표지 물질로 표지될 수 있다. 일 구현예에서, 상기 표지 물질은 형광, 인광, 화학발광단 또는 방사성을 발하는 물질일 수 있으나, 이에 제한되지 않는다. 바람직하게는, 상기 표지 물질은 루오리신(fluorescein), 피코에리트린 (phycoerythrin), 로다민, 리사민 (lissamine) Cy-5 또는 Cy-3일 수 있다. 표적 서열의 증폭시 프라이머의 5'-말단 및/또는 3' 말단에 Cy-5 또는 Cy-3를 표지하여 real-time RT-PCR을 수행하면 표적 서열이 검출가능한 형광 표지 물질로 표지될 수 있다. In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be a fluorescent, phosphorescent, chemiluminescent or radioactive material, but is not limited thereto. Preferably, the labeling substance may be fluorescein, phycoerythrin, rhodamine, lissamine Cy-5 or Cy-3. When amplifying the target sequence, real-time RT-PCR may be performed by labeling Cy-5 or Cy-3 at the 5'-end and / or 3 'end of the primer to label the target sequence with a detectable fluorescent label. .
또한, 방사성 물질을 이용한 표지는 real-time RT-PCR 수행시 32P 또는 35S 등과 같은 방사성 동위원소를 PCR 반응액에 첨가하면 증폭 산물이 합성되면서 방사성이 증폭 산물에 혼입되어 증폭 산물이 방사성으로 표지될 수 있다. 표적 서열을 증폭하기 위해 이용된 하나 이상의 올리고뉴클레오티드 프라이머 세트를 이용할 수 있다.In addition, the label using radioactive material is added to the PCR reaction solution by adding radioactive isotopes such as 32 P or 35 S to the PCR reaction solution during real-time RT-PCR, and the amplification product is radioactively incorporated into the amplification product. Can be labeled. One or more sets of oligonucleotide primers used to amplify a target sequence can be used.
표지는 당업계에서 통상적으로 실시되는 다양한 방법, 예컨대, 닉 트랜스레이션 (nick translation) 방법, 무작위 프라이밍 방법 (Multiprime DNA labelling systems booklet, "Amersham"(1989)) 및 카이네이션 방법 (Maxam & Gilbert, Methods in Enzymology, 65:499(1986))을 통해 실시될 수 있다. 표지는 형광, 방사능, 발색 측정, 중량 측정, X-선 회절 또는 흡수, 자기, 효소적 활성, 매스 분석, 결합 친화도, 혼성화 고주파, 나노크리스탈에 의하여 검출할 수 있는 시그널을 제공한다.Labeling is carried out in a variety of ways conventionally practiced in the art, such as nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, "Amersham" (1989)) and chination methods (Maxam & Gilbert, Methods). in Enzymology, 65: 499 (1986)). Labels provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetric, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, nanocrystals.
본 발명의 한 측면에 따르면, 본 발명에서는 RT-PCR을 통해 mRNA 수준에서 발현수준을 측정하게 된다. 이를 위하여 상기 HPV 유전자에 특이적으로 결합하는 신규한 프라이머 쌍과 형광이 표지된 프로브가 요구되며, 본 발명에서 특정한 염기서열로 특정된 해당 프라이머 및 프로브를 사용할 수 있으나 이에 제한되는 것은 아니며, 이들 유전자에 특이적으로 결합하여 검출가능한 시그널을 제공하여 real-time RT-PCR을 수행할 수 있는 것이면, 제한 없이 사용될 수 있다. 상기에서 FAM과 Quen(Quencher)는 형광염료를 의미한다.According to one aspect of the invention, the present invention is to measure the expression level at the mRNA level via RT-PCR. To this end, a novel primer pair and a fluorescently labeled probe that specifically bind to the HPV gene are required. In the present invention, the primers and probes specified by specific nucleotide sequences may be used, but are not limited thereto. It can be used without limitation as long as it can perform real-time RT-PCR by specifically binding to provide a detectable signal. In the above FAM and Quen (Quencher) means a fluorescent dye.
본 발명에 적용되는 real-time RT-PCR 방법은 당업계에서 통상적으로 사용되는 공지의 과정을 통해 수행될 수 있다.Real-time RT-PCR method applied to the present invention can be carried out through known procedures commonly used in the art.
mRNA 발현수준을 측정하는 단계는 통상의 mRNA 발현수준을 측정할 수 있는 방법이면 제한 없이 사용될 수 있으며, 사용한 프로브 표지의 종류에 따라 방사성 측정, 형광 측정 또는 인광 측정을 통해 수행될 수 있으나, 이에 제한되지 않는다. 증폭 산물을 검출하는 방법 중의 하나로서, 형광 측정 방법은 프라이머의 5'-말단에 Cy-5 또는 Cy-3를 표지하여 real-time RT-PCR을 수행하면 표적 서열이 검출가능한 형광 표지 물질로 표지되며, 이렇게 표지된 형광은 형광 측정기를 이용하여 측정할 수 있다. 또한, 방사성 측정 방법은 real-time RT-PCR 수행시 32P 또는 35S 등과 같은 방사성 동위원소를 PCR 반응액에 첨가하여 증폭 산물을 표지한 후, 방사성 측정기구, 예를 들면, 가이거 계수기(Geiger counter) 또는 액체섬광계수기(liquid scintillation counter)를 이용하여 방사성을 측정할 수 있다.The step of measuring the mRNA expression level can be used without limitation as long as it is a method capable of measuring the normal mRNA expression level, depending on the type of probe label used can be performed by radiometric measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. It doesn't work. As one of the methods for detecting amplification products, the fluorescence measurement method uses Cy-5 or Cy-3 at the 5'-end of a primer to perform real-time RT-PCR to label a target sequence with a detectable fluorescent label. The labeled fluorescence may be measured using a fluorimeter. In addition, the radioactivity measuring method is to add a radioactive isotope such as 32 P or 35 S to the PCR reaction solution when real-time RT-PCR is performed to label the amplification product, and then radioactive measuring apparatus, for example, Geiger counter (Geiger Radioactivity can be measured using a counter or a liquid scintillation counter.
본 발명의 바람직한 일구현예에 따르면, 상기 realtime RT-PCR을 통해 증폭된 PCR 산물에 형광이 표지된 프로브가 붙어 특정 파장의 형광을 내게 되고, 증폭과 동시에 realtime PCR 장치의 형광 측정기에서 본 발명의 유전자들의 mRNA 발현수준을 실시간으로 측정하고, 측정된 값이 계산되어 PC를 통해 시각화 되게 되어 검사자는 쉽게 그 발현정도를 확인할 수 있다.According to a preferred embodiment of the present invention, a fluorescent-labeled probe is attached to the PCR product amplified by the realtime RT-PCR to emit fluorescence of a specific wavelength, and at the same time, the fluorescence measuring device of the realtime PCR device The mRNA expression level of the genes are measured in real time, and the measured values are calculated and visualized through a PC so that the examiner can easily check the expression level.
본 발명의 일 구현예에 있어서, 상기 증폭된 양을 정상인에 대해 증폭된 양과 비교하는 단계는 컷오프 값에 의하여 수행되는 것이 바람직하며, 컷오프로 Ct값이 35이하로 검출되는 검체에 대해서는 양성으로 표기하는 것이 더욱 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the step of comparing the amplified amount with the amplified amount for a normal person is preferably performed by a cutoff value, it is marked as positive for a sample whose Ct value is detected below 35 as the cutoff. More preferably, but not limited to.
본 발명의 일 구현예에 있어서, 상기 HPV 타입 16, 35, 31, 58, 33, 및 52를 증폭할 수 있는 프라이머쌍은 서열번호 1 및 2에 기재되고, 프로브는 서열번호 3에 기재된 염기서열을 가지는 것이 바람직하고,In one embodiment of the present invention, primer pairs capable of amplifying the HPV types 16, 35, 31, 58, 33, and 52 are set forth in SEQ ID NOs: 1 and 2, and the probes are set forth in SEQ ID NO: 3 It is preferable to have
본 발명의 다른 구현예에 있어서, 상기 HPV 타입 18, 45, 39, 68, 및 59를 증폭할 수 있는 프라이머쌍은 서열번호 4 및 5에 기재되고, 프로브는 서열번호 6에 기재된 염기서열을 가지는 것이 바람직하며,In another embodiment of the present invention, the primer pairs capable of amplifying the HPV types 18, 45, 39, 68, and 59 are set forth in SEQ ID NOs: 4 and 5, and the probe has a nucleotide sequence set forth in SEQ ID NO: 6 Is preferred,
본 발명의 또 다른 구현예에 있어서, 상기 HPV 타입 53, 56, 66, 51, 69, 26, 및 30을 증폭할 수 있는 프라이머쌍은 서열번호 7 및 8, 또는 10 및 11에 기재되고, 프로브는 서열번호 9 또는 12에 기재된 염기서열을 가지는 것이 바람직하나 이에 한정되지 아니한다.In another embodiment of the invention, the primer pairs capable of amplifying the HPV types 53, 56, 66, 51, 69, 26, and 30 are described in SEQ ID NOs: 7 and 8, or 10 and 11, probes Preferably has a nucleotide sequence set forth in SEQ ID NO: 9 or 12, but is not limited thereto.
또한 본 발명은 HPV 타입 16, 35, 31, 58, 33, 및 52를 증폭할 수 있는 서열번호 1 및 2에 기재된 프라이머쌍과 서열번호 3에 기재된 염기서열을 가지는 프로브; HPV 타입 18, 45, 39, 68, 및 59를 증폭할 수 있는 서열번호 4 및 5에 기재된 프라이머쌍과 서열번호 6에 기재된 프로브; 및 HPV 타입 53, 56, 66, 51, 69, 26, 및 30을 증폭할 수 있는 서열번호 7 및 8 또는 10 및 11에 기재된 프라이머쌍과 서열번호 9 또는 12에 기재된 프로브로 구성된 군으로부터 선택된 하나 이상의 프라이머쌍 및 프로브를 포함하는 자궁경부암의 진단을 위한 프라이머쌍 및 프로브를 제공한다.In another aspect, the present invention is a probe having a primer pair shown in SEQ ID NO: 1 and 2 and a nucleotide sequence shown in SEQ ID NO: 3 capable of amplifying HPV type 16, 35, 31, 58, 33, and 52; A primer pair as set forth in SEQ ID NOs: 4 and 5 and a probe as set forth in SEQ ID NO: 6 capable of amplifying HPV types 18, 45, 39, 68, and 59; And a primer pair as set forth in SEQ ID NOs: 7 and 8 or 10 and 11 capable of amplifying HPV types 53, 56, 66, 51, 69, 26, and 30 and a probe as set forth in SEQ ID NOs: 9 or 12 Provided are a primer pair and a probe for the diagnosis of cervical cancer comprising the above primer pair and probe.
또 본 발명은 상기 본 발명의 프라이머 쌍 및 프로브를 포함하는 자궁경부암 진단용 조성물을 제공한다.In another aspect, the present invention provides a composition for diagnosing cervical cancer comprising the primer pair and probe of the present invention.
또한 본 발명은 상기 본 발명의 조성물을 포함하는 자궁경부암 진단용 키트를 제공한다.In another aspect, the present invention provides a kit for diagnosing cervical cancer comprising the composition of the present invention.
본 발명의 다른 측면에 따르면 상기 진단 키트는 역전사 중합효소반응을 수행하기 위해 필요한 필수 요소를 포함하는 것을 특징으로 하는 암 진단용 키트일 수 있다. 역전사 중합효소반응 키트는 상기 본 발명의 유전자에 대한 특이적인 각각의 프라이머 쌍을 포함할 수 있다. 프라이머는 각 마커 유전자의 핵산서열에 특이적인 서열을 가지는 뉴클레오타이드로서, 약 7 bp 내지 50 bp의 길이, 보다 바람직하게는 약 10 bp 내지 30 bp 의 길이일 수 있으며 보다 바람직하게는 본 발명의 서열번호로 표시되는 신규한 프라이머 쌍 및 형광표지된 프로브를 포함할 수 있다.According to another aspect of the present invention, the diagnostic kit may be a kit for diagnosing cancer, which includes an essential element necessary for performing reverse transcriptase. The reverse transcription polymerase kit may comprise each primer pair specific for the gene of the present invention. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and may be about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length, and more preferably, SEQ ID NO: It may include a novel primer pair and a fluorescently labeled probe represented by.
그 외 역전사 중합효소반응 키트는 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드(dNTPs), Taq-폴리머라아제 및 역전사효소와 같은 효소, DNAse, RNAse 억제제 DEPC-수(DEPC-water), 멸균수 등을 포함할 수 있다.Other reverse transcriptase kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), enzymes such as deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -May include DEPC-water, sterile water, and the like.
본 발명에서 용어 "암 진단을 위한 정보제공방법"은 진단을 위한 예비적 단계로서 암의 진단을 위하여 필요한 객관적인 기초정보를 제공하는 것이며 의사의 임상학적 판단 또는 소견은 제외된다.The term "information providing method for diagnosing cancer" in the present invention is to provide objective basic information necessary for diagnosing cancer as a preliminary step for diagnosis and excludes the clinical judgment or findings of the doctor.
용어 "프라이머"는 짧은 자유 3말단 수산화기를 가지는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응(즉, DNA 중합효소 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성이 개시할 수 있다. 본 발명의 프라이머는, 각 마커 유전자 특이적인 프라이머로 7개 내지 50개의 뉴클레오타이드 서열을 가진 센스 및 안티센스 핵산이다. 프라이머는 DNA 합성의 개시점으로 작용하는 프라이머의 기본 성질을 변화시키지 않는 추가의 특징을 혼입할 수 있다.The term "primer" refers to a short nucleic acid sequence that is capable of forming base pairs with complementary templates with nucleic acid sequences having short free 3-terminal hydroxyl groups and that serves as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. Primers of the invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis.
용어 "프로브"는 단일 연쇄 핵산 분자이며, 타깃 핵산 서열에 상보적인 서열을 포함한다.The term "probe"  It is a single chain nucleic acid molecule and comprises a sequence that is complementary to a target nucleic acid sequence.
용어 "실시간 역전사 중합효소 반응(realtime RT-PCR)"이라 함은 역전사효소를 이용하여 RNA를 상보적인 DNA(cDNA)로 역전사 시킨 후에 만들어진 cDNA를 주형(template) 으로하여 타겟 프라이머와 표지를 포함하는 타겟 프로브를 이용해 타겟을 증폭함과 동시에 증폭된 타겟에 타겟 프로프의 표지에서 발생하는 신호를 정량적으로 검출해 내는 분자생물학적 중합방법이다.The term "realtime RT-PCR" refers to a target primer and label using cDNA produced after reverse transcription of RNA into complementary DNA (cDNA) using reverse transcriptase. It is a molecular biological polymerization method that amplifies a target using a target probe and simultaneously detects a signal generated from a label of a target probe on the amplified target.
이하 본 발명을 설명한다.Hereinafter, the present invention will be described.
본 발명에서는 세계적으로 자궁경부암과 연관이 있다고 알려진 HPV 16, 18, 31, 33, 45 유전형과 더불어 국내 및 주변국들에서 자궁경부암과 연관이 있다고 알려진 HPV 35, 52, 56, 58, 39, 51, 59, 68, 53, 66, 69 유전형을 표적으로 E6, E7 발암유전자의 mRNA 발현양상을 정확히 측정할 수 있는 국내실정에 맞는 TaqMan 프로브를 이용한 Multiplex real-time RT-PCR검사법을 개발하였다.In the present invention, HPV 16, 18, 31, 33, 45 genotypes known to be associated with cervical cancer worldwide HPV 35, 52, 56, 58, 39, 51, We have developed a multiplex real-time RT-PCR assay using TaqMan probes that can accurately measure mRNA expression of E6 and E7 oncogenes targeting 59, 68, 53, 66 and 69 genotypes.
개발하고자 하는 진단법의 기술적 측면으로 임상적 치료 및 예방치료가 필요한 환자군을 보다 신속하고 정확하게 제공하여 기존 HPV DNA검사법의 제한점을 극복할 수 있을 것으로 예상되며, 탈락세포로부터의 RNA 추출의 자동화가 가능하고 real-time RT-PCR을 사용함으로써, 결과분석이 소프트웨어에 의해 가능하기 때문에 보다 객관적이고 정확한 결과를 보다 신속하게 제공할 수 있을 것이다.The technical aspects of the diagnostic methods to be developed are expected to overcome the limitations of the existing HPV DNA test by providing a faster and more accurate population of patients who need clinical and preventive treatment, and it is possible to automate the extraction of RNA from fallen cells. By using real-time RT-PCR, result analysis is made possible by software, which will provide more objective and accurate results more quickly.
도 1은 임상검체 GAPDH mRNA real-time RT-PCR 결과Figure 1 shows the results of clinical sample GAPDH mRNA real-time RT-PCR
도 2 내지 3은 주요 고위험군 16종 HPV의 E6/E7 표준염기서열 분석 및 프라이머와 TaqMan 프로브 위치2-3 show E6 / E7 standard sequencing and primers and TaqMan probe positions of 16 major high risk groups HPV
도 4 내지 7은 HPV mRNA를 이용한 E6/7에서의 민감도4 to 7 shows sensitivity at E6 / 7 using HPV mRNA
이하, 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 의도로 기재된 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are intended to illustrate the invention and the scope of the present invention is not to be construed as limited by the following examples.
본 발명의 임상검체의 수집 및 확보는 연세대학교 원주기독병원 병리과로부터 자궁경부 액상세포검사 (Thin Prep PAP TEST, PreservCyt Solution, Hologic Inc.Marlborough, MA, USA)를 시행한 환자를 대상으로 남아있는 자궁경부의 탈락세포를 가지고 RNA추출을 위해 100% 무수에탄올에 보관하여 수집하여 확보하였다.The collection and acquisition of clinical specimens of the present invention is performed in patients who underwent cervical fluid cytology (Thin Prep PAP TEST, PreservCyt Solution, Hologic Inc. Marlborough, MA, USA) Dropped cells in the cervix were collected and stored in 100% anhydrous ethanol for RNA extraction.
실시예 1:임상검체(자궁경부 탈락세포)로부터 total RNA추출Example 1 Total RNA Extraction from Clinical Specimens (Cervix Fallen Cells)
임상검체로부터 total RNA를 분리하기위해 자동핵산추출장비인 MagNA Pure LC 2.0(Roche)와 MagNA Pure LC RNA Isolation Kit High Performance (Roche)를 사용하였다.In order to separate total RNA from clinical specimens, we used MagNA Pure LC 2.0 (Roche) and MagNA Pure LC RNA Isolation Kit High Performance (Roche).
추출한 total RNA의 상태를 평가하기 위해 1.0% TBE Agarose gel에 전기영동하여 확인한 결과 18S, 28S rRNA의 밴드가 뚜렷이 나타나는 것을 확인하여 임상검체로부터 자동추출장비를 이용한 total RNA의 추출이 본 과제진행을 위해 적합한 것을 확인하였다(표 1).In order to evaluate the state of extracted total RNA, electrophoresis on 1.0% TBE Agarose gel confirmed that the bands of 18S and 28S rRNAs were clearly observed. It was found to be suitable (Table 1).
표 1
순서 샘플번호 RNA농도(ng/ul) 260/280 Ratio
1 11-1203 not extracted 　
2 11-1234 192.72 2.03
3 11-1254 185.28 1.97
4 11-1259 201.28 2.01
5 11-1260 514.88 1.98
6 11-1261 195.28 2.06
7 11-1274 172.56 2.01
8 11-1283 244.96 1.99
9 11-1364 157.52 2.03
10 11-1372 205.52 2.07
11 11-1373 176.16 2.14
12 11-1374 669.6 1.96
13 11-1483 206.88 2
14 11-1321 325.6 2.04
15 11-1312 461.84 2.04
16 11-1296 331.76 2.1
17 11-1285 159.52 2.06
18 11-1273 708.88 1.98
19 11-1226 243.28 2.08
20 11-1225 248.88 1.99
Table 1
order Sample number RNA concentration (ng / ul) 260/280 Ratio
One 11-1203 not extracted
2 11-1234 192.72 2.03
3 11-1254 185.28 1.97
4 11-1259 201.28 2.01
5 11-1260 514.88 1.98
6 11-1261 195.28 2.06
7 11-1274 172.56 2.01
8 11-1283 244.96 1.99
9 11-1364 157.52 2.03
10 11-1372 205.52 2.07
11 11-1373 176.16 2.14
12 11-1374 669.6 1.96
13 11-1483 206.88 2
14 11-1321 325.6 2.04
15 11-1312 461.84 2.04
16 11-1296 331.76 2.1
17 11-1285 159.52 2.06
18 11-1273 708.88 1.98
19 11-1226 243.28 2.08
20 11-1225 248.88 1.99
표 1은 임상검체로부터 추출한 total RNA의 농도 및 순도Table 1 shows the concentration and purity of total RNA extracted from clinical samples.
실시예 2:추출된 total RNA로부터 cDNA의 합성 Example 2 Synthesis of cDNA from Extracted Total RNA
임상검체로부터 분리한 total RNA 2 ug, random primer (Invitrogen) 0.2 5 ug, dNTP(Cosmo gene tech) 250 uM, Tris-HCl(pH 8.3) 50 mM, KCl 75 mM, MgCl2 3mM,DTT 8mM와 MMLV 역전사 중합효소 200 units (Invitrogen)을 첨가하고 최종부피를 20 ul가 되도록 DEPC treated DW를 넣고 잘 섞은 후 합성 반응액을 thermocycler (ABI)에서 25℃에서 10 분, 37℃에서 50 분, 70℃에서 15 분간 반응시켜 cDNA를 합성하였다. 합성된 cDNA가 real-time RT-PCR을 수행하기에 적절한지를 확인하기위해 human gyceraldehyde 3-phosphate dehydrogenase(GAPDH)를 endogenous control로 하여 real-time RT-PCR을 수행한 결과 임상검체로부터 추출하여 합성된 cDNA가 본 발명에 사용하기에 적합한 것으로 확인하였다 (도 1). Total RNA 2 ug, random primer (Invitrogen) 0.2 5 ug, dNTP (Cosmo gene tech) 250 uM, Tris-HCl (pH 8.3) 50 mM, KCl 75 mM, MgCl 2 3 mM, DTT 8 mM and MMLV Add 200 units of reverse transcriptase (Invitrogen), add DEPC treated DW to the final volume of 20 ul, mix well, and mix the reaction solution at 25 ℃ for 10 minutes at thermocycler (ABI), 50 minutes at 37 ℃, 70 ℃ The reaction was carried out for 15 minutes to synthesize cDNA. In order to confirm that the synthesized cDNA is suitable for real-time RT-PCR, real-time RT-PCR was performed using human gyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. cDNA was found to be suitable for use in the present invention (FIG. 1).
실시예 3:Multiplex RT-qPCR을 위한 고위험군 HPV 주요 16종 유전형의 E6/E7 mRNA 염기서열 분석 및 프라이머 TaqMan 프로브 디자인 Example 3 E6 / E7 mRNA Sequencing and Primer TaqMan Probe Design of High Risk Group HPV Major 16 Genotypes for Multiplex RT-qPCR
자궁경부암에서 가장 많이 발견되는 고위험군 HPV 주요 16종의 E6/E7 mRNA의 NCBI 표준 염기서열을 기초로 확보하였고, 이러한 염기서열들을 multi-align system을 통해 적절한 프라이머와 TaqMan 프로브 위치를 선정하고 이를 디자인하였다(도 2). Based on the NCBI standard sequences of the 16 high-risk HPV major E6 / E7 mRNAs most commonly found in cervical cancer, these sequences were selected and designed using the multi-align system to select appropriate primers and TaqMan probes. (FIG. 2).
표 2
Set I 검출 type 16, 35, 31, 58, 33, 52 　 　
F1 TTAGATTTRBADCCHGARVCAACTGAYCT(서열번호 1) 10p 　
R1 CYGGTTBTGCTTGTCCAKCTGG(서열번호 2) 10p 　
P1-2 CTGYTATGAGCAATTRNVYGRCAGCTCAGA(서열번호 3) 10p FAM-BHQ1
Set II 검출 type 18, 45, 39, 68, 59 　 　
F2-4 TGCARGAMATTGTDTTRSATTTRKRDCC(서열번호 4) 10p
R2-1 TGTGACGYTGTKGTTCRKCYCGTCKRGCT(서열번호 5) 10p 　
P2-2 TTGACYTKBWRTGYYACGAGCAATT(서열번호 6) 10p Cy5-BHQ2
Set III 검출 type 53, 56, 66, 51, 69, 26, 30 　 　
F3-1 TRTWTTAGAACTDRYACCDCAAAC(서열번호 7) 10p 　
R3-2 GTCTAYTTCATCCTCATCCTCYTCCTCTG(서열번호 8) 10p 　
P3-3 TTGACCTRCADTGCHATGAGCAATTGRAC(서열번호 9) 10p HEX-BHQ1　
51-69F GATGTWRTATTRSATTTRRYRCC(서열번호 10) 10p 　
26-51-69R ACGCAYATTATCTRYTTCATCCTCMTC(서열번호 11) 10p 　
51-69-P TTGACYTRCAVTGYTACGARCAATTKGAC(서열번호 12) 10p HEX-BHQ1
TABLE 2
Set I detection type 16, 35, 31, 58, 33, 52
F1 TTAGATTTRBADCCHGARVCAACTGAYCT (SEQ ID NO: 1) 10p
R1 CYGGTTBTGCTTGTCCAKCTGG (SEQ ID NO: 2) 10p
P1-2 CTGYTATGAGCAATTRNVYGRCAGCTCAGA (SEQ ID NO: 3) 10p FAM-BHQ1
Set II detection type 18, 45, 39, 68, 59
F2-4 TGCARGAMATTGTDTTRSATTTRKRDCC (SEQ ID NO: 4) 10p
R2-1 TGTGACGYTGTKGTTCRKCYCGTCKRGCT (SEQ ID NO: 5) 10p
P2-2 TTGACYTKBWRTGYYACGAGCAATT (SEQ ID NO: 6) 10p Cy5-BHQ2
Set III detection type 53, 56, 66, 51, 69, 26, 30
F3-1 TRTWTTAGAACTDRYACCDCAAAC (SEQ ID NO: 7) 10p
R3-2 GTCTAYTTCATCCTCATCCTCYTCCTCTG (SEQ ID NO: 8) 10p
P3-3 TTGACCTRCADTGCHATGAGCAATTGRAC (SEQ ID NO: 9) 10p HEX-BHQ1
51-69F GATGTWRTATTRSATTTRRYRCC (SEQ ID NO: 10) 10p
26-51-69R ACGCAYATTATCTRYTTCATCCTCMTC (SEQ ID NO: 11) 10p
51-69-P TTGACYTRCAVTGYTACGARCAATTKGAC (SEQ ID NO: 12) 10p HEX-BHQ1
표 2는 본 발명에 사용된 Primer와 Probe sequenceTable 2 shows the Primer and Probe sequences used in the present invention.
실시예 4: Real-time PCR 수행Example 4: Real-time PCR
Real-time PCR의 반응물의 조성은 25 mM TAPS (pH 9.3 at 25°C), 50 mM KCl, 2 mM MgCl2,1mM 2-mercaptoethanol,200μM 각 dNTP,1unit Taq polymerase(TAKARA)와 Forward primer와 Reverse primer를 각각 10pmole을 넣어주고, probe 또한 10pmole을 넣어 주고, 합성된 cDNA를 2ul를 넣고 최종 부피를 20ul가 되게 수행하였다. PCR 반응은 CFX 96(BioRad-USA)를 이용하여 변성 온도 94℃에서 5분 동안 1회 수행하고, 변성 온도 95℃에서 30초, 어닐링 온도 55℃에서 20초인 사이클을 40회 반복하여 수행하였다. 또한 각각의 어닐링 과정 후 형광을 측정하는 과정을 추가하여, 각 사이클 별로 증가되는 형광 값을 측정하였다. The composition of the real-time PCR was 25 mM TAPS (pH 9.3 at 25 ° C), 50 mM KCl, 2 mM MgCl 2 , 1 mM 2-mercaptoethanol, 200 μM each dNTP, 1 unit Taq polymerase (TAKARA), Forward primer and Reverse 10pmole was added to each primer, and 10pmole was added to the probe, and 2ul of the synthesized cDNA was added to a final volume of 20ul. The PCR reaction was performed once using CFX 96 (BioRad-USA) at a denaturation temperature of 94 ° C. for 5 minutes, followed by 40 cycles of 30 seconds at denaturation temperature of 95 ° C. and 20 seconds at annealing temperature of 55 ° C. In addition, the fluorescence was measured after each annealing process, and the fluorescence value increased with each cycle was measured.
상기 실시예의 결과는 다음과 같다.The result of the above example is as follows.
1. 각 타입별 민감도1. Sensitivity for each type
각 타입별 민감도를 확인해 본 결과 100~10fg에서 검출됨을 확인할 수 있었다(도 3).As a result of checking the sensitivity for each type, it could be confirmed that the detection was performed at 100 to 10 fg (FIG. 3).
2. Multiplex real-time PCR을 이용한 16종의 HPV검출2. Detection of 16 HPVs using Multiplex real-time PCR
Set I은 FAM dye를 이용하여 HPV type 16, 31, 33, 35, 52, 58이 검출 확인하였으며Set I was detected by HPV type 16, 31, 33, 35, 52, 58 using FAM dye.
Set II은 Cy5 dye를 이용하여 HPV type 18, 39, 45, 59, 68을 검출하였으며Set II detected HPV types 18, 39, 45, 59 and 68 using Cy5 dye.
Set III는 HEX dye를 이용하여 HPV type 53, 56, 66, 51, 69이 검출 확인됨을 알 수 있었다(표 3).In Set III, HPV types 53, 56, 66, 51, and 69 were detected using HEX dye (Table 3).
표 3
　 　 　F2-2(3)mix/R2-1　 F2-4/R2 F2-4/R2-1
　 　 set 1 set 2 set 3 set 1 set 2 set 3 set 1 set 2 set 3
1 HPV-16 19.35 N/A N/A 20.45 24.89 N/A 20.73 N/A N/A
2 HPV-18 N/A 25.29 N/A 22.46 22.57 N/A N/A 22.07 N/A
3 HPV-31 19.13 13.84 N/A 18.4 N/A N/A 20.43 N/A N/A
4 HPV-33 19.98 N/A N/A 19.85 N/A N/A 19.82 N/A N/A
5 HPV-35 19.62 N/A N/A 19.36 N/A N/A 21.48 N/A N/A
6 HPV-39 N/A 28.98 N/A N/A 24.72 N/A N/A 26.06 N/A
7 HPV-45 N/A 26.28 N/A N/A 23.46 N/A N/A 24.36 N/A
8 HPV-51 N/A N/A 28.82 N/A N/A 34.89 N/A N/A 29.56
9 HPV-52 17.57 N/A N/A 18.97 N/A 1.84 16.81 N/A 1.93
10 HPV-53 N/A N/A 23.72 N/A N/A 22.71 N/A N/A 22.8
11 HPV-54 N/A N/A N/A N/A N/A N/A N/A N/A N/A
12 HPV-56 N/A N/A 22.78 N/A N/A 23.21 N/A N/A 22.77
13 HPV-58 19.77 N/A N/A 19.39 N/A N/A 20.74 N/A N/A
14 HPV-59 N/A 30.26 N/A N/A 25.22 N/A N/A 26.48 N/A
15 HPV-66 N/A N/A 22.27 N/A N/A 19.49 N/A N/A 20.2
16 HPV-68 N/A 26.5 N/A N/A 25.86 N/A N/A 26.58 N/A
17 HPV-69 12.55 N/A 25.4 N/A N/A 26.24 N/A N/A 26.56
18 HPV-06 N/A N/A N/A N/A N/A N/A N/A N/A N/A
19 HPV-11 N/A N/A N/A N/A N/A N/A N/A N/A N/A
20 HPV-84 N/A N/A N/A N/A N/A N/A N/A N/A N/A
21 HPV-87 N/A N/A N/A N/A N/A N/A N/A N/A N/A
TABLE 3
F2-2 (3) mix / R2-1 F2-4 / R2 F2-4 / R2-1
set 1 set 2 set 3 set 1 set 2 set 3 set 1 set 2 set 3
One HPV-16 19.35 N / A N / A 20.45 24.89 N / A 20.73 N / A N / A
2 HPV-18 N / A 25.29 N / A 22.46 22.57 N / A N / A 22.07 N / A
3 HPV-31 19.13 13.84 N / A 18.4 N / A N / A 20.43 N / A N / A
4 HPV-33 19.98 N / A N / A 19.85 N / A N / A 19.82 N / A N / A
5 HPV-35 19.62 N / A N / A 19.36 N / A N / A 21.48 N / A N / A
6 HPV-39 N / A 28.98 N / A N / A 24.72 N / A N / A 26.06 N / A
7 HPV-45 N / A 26.28 N / A N / A 23.46 N / A N / A 24.36 N / A
8 HPV-51 N / A N / A 28.82 N / A N / A 34.89 N / A N / A 29.56
9 HPV-52 17.57 N / A N / A 18.97 N / A 1.84 16.81 N / A 1.93
10 HPV-53 N / A N / A 23.72 N / A N / A 22.71 N / A N / A 22.8
11 HPV-54 N / A N / A N / A N / A N / A N / A N / A N / A N / A
12 HPV-56 N / A N / A 22.78 N / A N / A 23.21 N / A N / A 22.77
13 HPV-58 19.77 N / A N / A 19.39 N / A N / A 20.74 N / A N / A
14 HPV-59 N / A 30.26 N / A N / A 25.22 N / A N / A 26.48 N / A
15 HPV-66 N / A N / A 22.27 N / A N / A 19.49 N / A N / A 20.2
16 HPV-68 N / A 26.5 N / A N / A 25.86 N / A N / A 26.58 N / A
17 HPV-69 12.55 N / A 25.4 N / A N / A 26.24 N / A N / A 26.56
18 HPV-06 N / A N / A N / A N / A N / A N / A N / A N / A N / A
19 HPV-11 N / A N / A N / A N / A N / A N / A N / A N / A N / A
20 HPV-84 N / A N / A N / A N / A N / A N / A N / A N / A N / A
21 HPV-87 N / A N / A N / A N / A N / A N / A N / A N / A N / A
표 3은 HPV 타입에 따른 Multiplex real-time PCR의 검출Table 3 shows the detection of multiplex real-time PCR according to HPV type.
3. 임상에서 기존방법(Real-time NASBA)과의 검출 비교 민감도 확인3. Check the sensitivity of detection in clinical trials with real-time NASBA
기존 DNA chip의 결과가 있는 임상검체 68검체와 특이도 검사를 위한 정상인 검체 49의 총 117검체를 대상으로 기존에 나와 있는 Real-time NASBA와의 비교테스트를 진행하였다.A total of 117 samples from 68 clinical specimens with existing DNA chip results and 49 normal specimens for specificity testing were compared with the existing Real-time NASBA.
그 결과 정상인 검체에서는 모두 나오지 않음을 확인할 수 있었고, 세포병리학적으로 고등급 병변을 나타내는 총 68개의 임상검체에서 Real-time NASBA(Biometriux, HPV 16, 18, 31, 33, 45의 5개 타입만 검출이 가능)를 수행하였다. Real-time NASBA는 Internal control (IC)에서 양성이 나온 검체에서 각 타겟 유전형별 E6/E7 mRNA를 검출할 수 있다. 68개의 임상검체 중 38검체에서 real-time NASBA의 IC 양성이 나와 각 타겟 유전형별 E6/E7 mRNA를 검출 해 본 결과 28/38 (73.7%)의 양성률을 보였으며, 음성이 나온 10개의 검체 중 Real-time NASBA에서 검출할 수 없는 유전형에 대한 검체는 5/38 (13.2%)였으며, Real-time NASBA에서 검출 할 수 있으나 검출되지 않은 경우는 5/38(13.2%)로 나타났다. 반면에 Multiplex Real-time PCR의 경우에서는 real-time NASBA에서 양성이 나온 38개의 검체에서 38/38 (100%)의 양성률을 확인 하였다. 또한, Real-time NASBA에서 IC가 음성이 나와 HPV E6/E7 mRNA를 검출하지 하지 못했던 30검체에 대해서도 동일하게 Real-time PCR을 수행한 결과 24/30 (80%)에서 양성이 나옴을 확인할 수 있었다. 이와 같이 Multiplex real-time PCR (62/68, 91.2%)을 수행할 경우 Real-time NASBA (28/68, 41.2%)를 수행하였을 때 보다 50% (34/68) 의 민감도가 높아짐을 확인 할 수 있었다(표 4).As a result, it was confirmed that none of the normal samples appeared, and only five types of Real-time NASBA (Biometriux, HPV 16, 18, 31, 33, 45) were found in 68 clinical specimens showing cytopathologically high grade lesions. Detection is possible). Real-time NASBA can detect E6 / E7 mRNA for each target genotype in specimens positive for internal control (IC). Of the 68 clinical samples, 38 samples showed IC-positive positive for real-time NASBA and detected E6 / E7 mRNA for each target genotype, showing 28/38 (73.7%) positive rate. 5/38 (13.2%) of the genotypes could not be detected in real-time NASBA, and 5/38 (13.2%) were detected in real-time NASBA but not detected. On the other hand, in the case of multiplex real-time PCR, 38/38 (100%) positive rates were confirmed in 38 samples that were positive in real-time NASBA. In addition, the same real-time PCR was performed on 30 samples in which IC was negative in real-time NASBA and failed to detect HPV E6 / E7 mRNA. The result was positive in 24/30 (80%). there was. When the multiplex real-time PCR (62/68, 91.2%) is performed, the sensitivity of 50% (34/68) is higher than that of the real-time NASBA (28/68, 41.2%). (Table 4).
표 4
Cytology results DNA chip (Goodgen) Sample No. Real-time NASBA (biomeriux)U1A, 16, 18, 31, 33, 45 type Real time E6-7 PCRPositive rate (%)
Positive rate (%) Negative rate (%)
SCC &ADC High risk 15 11 (73.3%) 4 (26.7%) 15 (100%)
HSIL High risk 23 17 (73.9%) 6 (26.1%) 23 (100%)
SCC* High risk 9 9 (100%) 9 (100%)
HSIL* High risk 21 21 (100%) 15 (71.4%)
Normal Normal 60 0 (0.0%) 60 (100.0%)　 0 (0%)
Table 4
Cytology results DNA chip (Goodgen) Sample No. Real-time NASBA (biomeriux) U1A, 16, 18, 31, 33, 45 type Real time E6-7 PCRPositive rate (%)
Positive rate (%) Negative rate (%)
SCC & ADC High risk 15 11 (73.3%) 4 (26.7%) 15 (100%)
HSIL High risk 23 17 (73.9%) 6 (26.1%) 23 (100%)
SCC * High risk 9 9 (100%) 9 (100%)
HSIL * High risk 21 21 (100%) 15 (71.4%)
Normal Normal 60 0 (0.0%) 60 (100.0%) 0 (0%)
표 4는 HPV mRNA를 이용한 Real-time NASBA와 Multiplex Real-time PCR의 검출비교Table 4 compares the detection of Real-time NASBA and Multiplex Real-time PCR using HPV mRNA.
* Real-time NASBA Internal control 음성인 검체* Real-time NASBA Internal control negative sample
4. 임상검체를 이용한 Multiplex Real-time PCR의 양성율과 음성율4. Positive Rate and Negative Rate of Multiplex Real-time PCR Using Clinical Specimens
세포병리학적 검사에서 고등급 병변을 나타내면서 DNA 유전형 검사(DNA chip)에서 HPV 고위험군이 감염된 75개의 검체를 이용해 Multiplex Real-time PCR를 수행한 결과 양성율이 98.7%, 음성율이 1.3% 였다. 또한 세포병리학적 검사에서 정상을 나타내면서 DNA 유전형 검사(DNA chip)에서 HPV에 감염되지 않은 110개의 검체를 이용해 Multiplex Real-time PCR를 수행한 결과 양성율이 0%, 음성율이 100% 였다. 이 결과 검사의 특이도는 100%, 민감도는 98.7%로 기존의 검사법에 비해 매우 높음을 확인할 수 있었다(표 5). Cytopathological examination showed high grade lesions and multiplex real-time PCR was performed using DNA samples of 75 HPV-infected patients on DNA genotyping (DNA chip). The positive rate was 98.7% and the negative rate was 1.3%. In addition, multiplex real-time PCR was performed using 110 samples uninfected with HPV in DNA pathotyping and showed normality in cytopathologic tests. The positive rate was 0% and the negative rate was 100%. As a result, the specificity of the test was 100% and the sensitivity was 98.7%, which was very high compared to the conventional test method (Table 5).
표 5
HPV mRNA Multiplex Real-time PCR
Clinical Specimens Positive Negative Total
Cytologic Test HPV DNA Chip Test
SCC &HSIL HPV High-risk Positive 74 (98.7 %) 1 (1.3%) 75 (100%)
Normal HPV Negative 0 (0%) 110 (100%) 110 (100%)
Table 5
HPV mRNA Multiplex Real-time PCR
Clinical Specimens Positive Negative Total
Cytologic Test HPV DNA Chip Test
SCC & HSIL HPV High-risk Positive 74 (98.7%) 1 (1.3%) 75 (100%)
Normal HPV Negative 0 (0%) 110 (100%) 110 (100%)
표 5는 임상검체로부터 분리한 HPV mRNA를 이용한 Multiplex Real-time PCR의 결과Table 5 shows the results of multiplex real-time PCR using HPV mRNA isolated from clinical specimens.
5. DNA 유전형 검사 (DNA chip)과 Multiplex Real-time PCR의 특이도 비교5. Specificity comparison between DNA genotyping (DNA chip) and Multiplex Real-time PCR
정상인의 자궁경부에서도 HPV DNA 양성률이 매우 높게 나오고 있기 때문에 mRNA를 표적으로 한 검사의 유용성을 확인 하고자 세포병리학적 검사에서 정상으로 나왔으나 DNA 유전형 검사(DNA chip)에서 HPV 고위험군으로 감염된 66개의 검체를 이용해 Multiplex Real-time PCR를 수행하였으며 그 결과 6.3%(6/95)가 검출되어 DNA chip에 대한 위양성 문제를 줄일수 있음을 확인하였다 (표 6)The HPV DNA positive rate in the cervix of normal people is very high. Therefore, to confirm the usefulness of the mRNA-targeted test, 66 samples infected with HPV high-risk group were detected by DNA pathology. Multiplex real-time PCR was performed using the result, and as a result, 6.3% (6/95) was detected and it was confirmed that it could reduce the false positive problem for DNA chip (Table 6).
표 6
No. Molecular No. Cytology Result (Goodgen kit) Multiplex
1 M-11-1392 - 16 28.95
2 M-11-150 - 52 30.14
3 M-11-917 - 31 32.88
4 M-11-551 - 58 undetermined
5 M-11-672 - 16, 33 undetermined
6 M-10-1708 - 16 undetermined
7 M-11-152 - 16 undetermined
8 M-11-106 - 52 undetermined
9 M-10-1578 - 16 undetermined
10 M-11-149 - 16 undetermined
11 M-10-1299 - 58 undetermined
12 M-11-119 - 35, 66 32.54
13 M-10-993 - 33 undetermined
14 M-10-1170 - 16 undetermined
15 M-10-1517 - 58 undetermined
16 M-11-52 - 16 undetermined
17 M-11-94 - 31 undetermined
18 M-11-104 - 33, 58 undetermined
19 M-11-253 - 16 undetermined
20 M-11-286 - 16 undetermined
21 M-11-287 - 16 undetermined
22 M-11-327 - 16 undetermined
23 M-11-389 - 33, 58 undetermined
24 M-11-466 - 16, 18 undetermined
25 M-11-467 - 16, 18, 39, 58, 68 undetermined
26 M-11-486 - 16 undetermined
27 M-11-605 - 18, 58 undetermined
28 M-11-617 - 16, 18 undetermined
29 M-11-623 - 33 undetermined
30 M-11-633 - 16, 18 undetermined
31 M-11-696 - 33 undetermined
32 M-11-742 - 18, 35 undetermined
33 M-11-925 - 16 undetermined
34 M-11-858 - 58, 68 undetermined
35 M-11-948 - 16 undetermined
36 M-11-964 - 16 undetermined
37 M-11-986 - 58 undetermined
38 M-11-993 - 16, 58 undetermined
39 M-11-1027 - 16 undetermined
40 M-11-1038 - 58 undetermined
41 M-11-1042 - 16 undetermined
42 M-11-1043 - 16 undetermined
43 M-11-1057 - 16, 68 undetermined
44 M-11-1059 - 58 undetermined
45 M-11-1083 - 16, 18, 56 undetermined
46 M-11-1084 - 16 undetermined
47 M-11-1087 - 16, 18 undetermined
48 M-11-1088 - 16 undetermined
49 M-11-1416 - 16, 58 undetermined
50 M-11-1454 - 16, 18 undetermined
51 M-11-1497 - 52 undetermined
52 M-11-1536 - 16, 18, 33 undetermined
53 M-11-1543 - 16, 39, 66 undetermined
54 M-11-1562 - 16 undetermined
55 M-11-1563 - 33 undetermined
56 M-11-1637 - 16 undetermined
57 M-11-1658 - 16, 18, 39 undetermined
58 M-11-1663 - 52 undetermined
59 M-11-1669 - 16 undetermined
60 M-11-1747 - 18, 58 26.52
61 M-11-1748 - 16 undetermined
62 M-11-1798 - 16 undetermined
63 M-11-1799 - 33 undetermined
64 M-11-1812 - 16 undetermined
65 M-11-1897 - 18, 58 undetermined
66 M-11-1932 - 16 undetermined
67 M-11-47 - 18 undetermined
68 M-10-1056 - 66 33.3
69 M-10-1542 - 56 undetermined
70 M-10-1651 - 45 undetermined
71 M-11-69 - 45 undetermined
72 M-11-73 - 56 undetermined
73 M-11-239 - 59 undetermined
74 M-11-241 - 18 undetermined
75 M-11-328 - 18 undetermined
76 M-11-460 - 68 undetermined
77 M-11-472 - 18, 56 undetermined
78 M-11-523 - 18 undetermined
79 M-11-729 - 18 undetermined
80 M-11-738 - 18 undetermined
81 M-11-846 - 18 undetermined
82 M-11-919 - 18 undetermined
83 M-11-1089 - 39 undetermined
84 M-11-1154 - 18 undetermined
85 M-11-1535 - 45 undetermined
86 M-11-1654 - 69 undetermined
87 M-11-1661 - 18 undetermined
88 M-11-1718 - 66 undetermined
89 M-11-1728 - 18 undetermined
90 M-11-1793 - 66 undetermined
91 M-11-1808 - 68 undetermined
92 M-11-1809 - 56, 69 undetermined
93 M-11-1894 - 18 undetermined
94 M-11-320 - 66 undetermined
95 M-11-1880 - 56 undetermined
Table 6
No. Molecular No. Cytology Result (Goodgen kit) Multiplex
One M-11-1392 - 16 28.95
2 M-11-150 - 52 30.14
3 M-11-917 - 31 32.88
4 M-11-551 - 58 undetermined
5 M-11-672 - 16, 33 undetermined
6 M-10-1708 - 16 undetermined
7 M-11-152 - 16 undetermined
8 M-11-106 - 52 undetermined
9 M-10-1578 - 16 undetermined
10 M-11-149 - 16 undetermined
11 M-10-1299 - 58 undetermined
12 M-11-119 - 35, 66 32.54
13 M-10-993 - 33 undetermined
14 M-10-1170 - 16 undetermined
15 M-10-1517 - 58 undetermined
16 M-11-52 - 16 undetermined
17 M-11-94 - 31 undetermined
18 M-11-104 - 33, 58 undetermined
19 M-11-253 - 16 undetermined
20 M-11-286 - 16 undetermined
21 M-11-287 - 16 undetermined
22 M-11-327 - 16 undetermined
23 M-11-389 - 33, 58 undetermined
24 M-11-466 - 16, 18 undetermined
25 M-11-467 - 16, 18, 39, 58, 68 undetermined
26 M-11-486 - 16 undetermined
27 M-11-605 - 18, 58 undetermined
28 M-11-617 - 16, 18 undetermined
29 M-11-623 - 33 undetermined
30 M-11-633 - 16, 18 undetermined
31 M-11-696 - 33 undetermined
32 M-11-742 - 18, 35 undetermined
33 M-11-925 - 16 undetermined
34 M-11-858 - 58, 68 undetermined
35 M-11-948 - 16 undetermined
36 M-11-964 - 16 undetermined
37 M-11-986 - 58 undetermined
38 M-11-993 - 16, 58 undetermined
39 M-11-1027 - 16 undetermined
40 M-11-1038 - 58 undetermined
41 M-11-1042 - 16 undetermined
42 M-11-1043 - 16 undetermined
43 M-11-1057 - 16, 68 undetermined
44 M-11-1059 - 58 undetermined
45 M-11-1083 - 16, 18, 56 undetermined
46 M-11-1084 - 16 undetermined
47 M-11-1087 - 16, 18 undetermined
48 M-11-1088 - 16 undetermined
49 M-11-1416 - 16, 58 undetermined
50 M-11-1454 - 16, 18 undetermined
51 M-11-1497 - 52 undetermined
52 M-11-1536 - 16, 18, 33 undetermined
53 M-11-1543 - 16, 39, 66 undetermined
54 M-11-1562 - 16 undetermined
55 M-11-1563 - 33 undetermined
56 M-11-1637 - 16 undetermined
57 M-11-1658 - 16, 18, 39 undetermined
58 M-11-1663 - 52 undetermined
59 M-11-1669 - 16 undetermined
60 M-11-1747 - 18, 58 26.52
61 M-11-1748 - 16 undetermined
62 M-11-1798 - 16 undetermined
63 M-11-1799 - 33 undetermined
64 M-11-1812 - 16 undetermined
65 M-11-1897 - 18, 58 undetermined
66 M-11-1932 - 16 undetermined
67 M-11-47 - 18 undetermined
68 M-10-1056 - 66 33.3
69 M-10-1542 - 56 undetermined
70 M-10-1651 - 45 undetermined
71 M-11-69 - 45 undetermined
72 M-11-73 - 56 undetermined
73 M-11-239 - 59 undetermined
74 M-11-241 - 18 undetermined
75 M-11-328 - 18 undetermined
76 M-11-460 - 68 undetermined
77 M-11-472 - 18, 56 undetermined
78 M-11-523 - 18 undetermined
79 M-11-729 - 18 undetermined
80 M-11-738 - 18 undetermined
81 M-11-846 - 18 undetermined
82 M-11-919 - 18 undetermined
83 M-11-1089 - 39 undetermined
84 M-11-1154 - 18 undetermined
85 M-11-1535 - 45 undetermined
86 M-11-1654 - 69 undetermined
87 M-11-1661 - 18 undetermined
88 M-11-1718 - 66 undetermined
89 M-11-1728 - 18 undetermined
90 M-11-1793 - 66 undetermined
91 M-11-1808 - 68 undetermined
92 M-11-1809 - 56, 69 undetermined
93 M-11-1894 - 18 undetermined
94 M-11-320 - 66 undetermined
95 M-11-1880 - 56 undetermined
표 6은 DNA를 이용한 DNA chip 결과와 mRNA를 이용한 Multiplex real-time PCR의 결과비교Table 6 compares DNA chip results with DNA and multiplex real-time PCR using mRNA.

Claims (7)

  1. a) 환자의 혈액에서 얻은 세포로부터 전장 RNA를 분리하는 단계;a) separating full length RNA from cells obtained from the blood of the patient;
    b) 상기 분리된 전장 RNA로부터 cDNA를 합성하는 단계;b) synthesizing cDNA from the isolated full-length RNA;
    c) 상기 합성된 cDNA를 HPV 타입 16, 35, 31, 58, 33, 및 52를 증폭할 수 있는 프라이머쌍 및 프로브, HPV 타입 18, 45, 39, 68, 및 59를 증폭할 수 있는 프라이머쌍 및 프로브, 및 HPV 타입 53, 56, 66, 51, 69, 26, 및 30을 증폭할 수 있는 프라이머 쌍 및 프로브로 구성된 군으로부터 선택된 하나 이상의 프라이머쌍 및 프로브를 이용하여 실시간-PCR을 수행하는 단계; 및c) a primer pair and a primer pair capable of amplifying HPV types 16, 35, 31, 58, 33, and 52, and a primer pair capable of amplifying HPV types 18, 45, 39, 68, and 59 And performing real-time PCR using one or more primer pairs and probes selected from the group consisting of probes and primer pairs and probes capable of amplifying HPV types 53, 56, 66, 51, 69, 26, and 30 ; And
    d) 상기 발현된 양을 정상인에 대해 발현된 양과 비교하는 단계;를 포함하는 자궁경부암의 진단을 위한 정보제공방법.and d) comparing the expressed amount with the amount expressed for a normal person.
  2. 제 1항에 있어서, 상기 HPV 타입 16, 35, 31, 58, 33, 및 52를 증폭할 수 있는 프라이머쌍은 서열번호 1 및 2에 기재되고, 프로브는 서열번호 3에 기재된 염기서열을 가지는 것을 특징으로 하는 정보제공방법.The method of claim 1, wherein the primer pairs capable of amplifying the HPV types 16, 35, 31, 58, 33, and 52 are set forth in SEQ ID NOs: 1 and 2, and the probe has a nucleotide sequence set forth in SEQ ID NO: 3 Information providing method characterized by.
  3. 제 1항에 있어서, 상기 HPV 타입 18, 45, 39, 68, 및 59를 증폭할 수 있는 프라이머쌍은 서열번호 4 및 5에 기재되고, 프로브는 서열번호 6에 기재된 염기서열을 가지는 것을 특징으로 하는 정보제공방법.The method of claim 1, wherein the primer pairs capable of amplifying the HPV types 18, 45, 39, 68, and 59 are set forth in SEQ ID NOs: 4 and 5, and the probe has a nucleotide sequence set forth in SEQ ID NO: 6 How to provide information.
  4. 제 1항에 있어서, 상기 HPV 타입 53, 56, 66, 51, 69, 26, 및 30을 증폭할 수 있는 프라이머쌍은 서열번호 7 및 8, 또는 10 및 11에 기재되고, 프로브는 서열번호 9 또는 12에 기재된 염기서열을 가지는 것을 특징으로 하는 암의 진단을 위한 정보제공방법.The method of claim 1, wherein the primer pairs capable of amplifying the HPV types 53, 56, 66, 51, 69, 26, and 30 are set forth in SEQ ID NOs: 7 and 8, or 10 and 11, and the probe is SEQ ID NO: 9 Or a nucleotide sequence as set forth in claim 12.
  5. HPV 타입 16, 35, 31, 58, 33, 및 52를 증폭할 수 있는 서열번호 1 및 2에 기재된 프라이머쌍과 서열번호 3에 기재된 염기서열을 가지는 프로브;Probes having the primer pairs set forth in SEQ ID NOs: 1 and 2 and the base sequences set forth in SEQ ID NO: 3 capable of amplifying HPV types 16, 35, 31, 58, 33, and 52;
    HPV 타입 18, 45, 39, 68, 및 59를 증폭할 수 있는 서열번호 4 및 5에 기재된 프라이머쌍과 서열번호 6에 기재된 프로브; 및 A primer pair as set forth in SEQ ID NOs: 4 and 5 and a probe as set forth in SEQ ID NO: 6 capable of amplifying HPV types 18, 45, 39, 68, and 59; And
    HPV 타입 53, 56, 66, 51, 69, 26, 및 30을 증폭할 수 있는 서열번호 7 및 8 또는 10 및 11에 기재된 프라이머쌍과 서열번호 9 또는 12에 기재된 프로브로 구성된 군으로부터 선택된 하나 이상의 프라이머쌍 및 프로브를 포함하는 자궁경부암의 진단을 위한 프라이머쌍 및 프로브. At least one selected from the group consisting of the primer pairs set forth in SEQ ID NOs: 7 and 8 or 10 and 11 and the probes set forth in SEQ ID NOs: 9 or 12 capable of amplifying HPV types 53, 56, 66, 51, 69, 26, and 30 Primer pairs and probes for the diagnosis of cervical cancer, including primer pairs and probes.
  6. 제 5항의 프라이머 쌍 및 프로브를 포함하는 자궁경부암 진단용 조성물.A composition for diagnosing cervical cancer comprising the primer pair of claim 5 and a probe.
  7. 제6항의 조성물을 포함하는 자궁경부암 진단용 키트.Cervical cancer diagnostic kit comprising the composition of claim 6.
PCT/KR2013/006512 2013-07-19 2013-07-19 Method for diagnosing cervical cancer and diagnostic kit therefor WO2015008885A1 (en)

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