KR20000055412A - Development of HPV quantification and Typing method using Polymerase Chain Reaction and Hybridization - Google Patents

Development of HPV quantification and Typing method using Polymerase Chain Reaction and Hybridization Download PDF

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KR20000055412A
KR20000055412A KR1019990004009A KR19990004009A KR20000055412A KR 20000055412 A KR20000055412 A KR 20000055412A KR 1019990004009 A KR1019990004009 A KR 1019990004009A KR 19990004009 A KR19990004009 A KR 19990004009A KR 20000055412 A KR20000055412 A KR 20000055412A
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hpv
dna
reaction
pcr
patient
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김희태
김병성
안웅식
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김희태
김병성
안웅식
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    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • 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

Abstract

PURPOSE: A fixed quantity and a type of HPV are provided to improve the limitation, a defect and inefficiency of general detecting methods. CONSTITUTION: A fixed quantity and a type of HPV(human papilloma virus) are confirmed by the following sequences. A standard DNA competitively reacting to HPV Genome in a sample of a patient in the reaction of HPV PCR(polymerase chain-reaction) while not interfering to HPV typing is developed. The amount of HPV Genome in the DNA of the patient is confirmed after performing PCR reaction by mixing the DNA extracted from the sample with the HPV standard DNA previously fixed in the quantity. Herein, a biotin is attached on a primer for HPV typing. DNA probes available for unusual reaction with respective HPV types are developed. The respective DNA probes are fixed on the surface of a membrane. The PCR result is spread on the membrane, hybridized and cleansed. An enzyme reagent attachable by discriminating a reaction biotin is spread on the cleansed membrane, attached thereon and cleansed. A coloring reaction is performed by spreading a coloring reactive base material of the enzyme reagent on the cleansed membrane for the discrimination.

Description

중합효소 연쇄반응와 디엔에이/디엔에이 서열 특이결합을 이용한 에이치브이의 정량 및 타이핑{Development of HPV quantification and Typing method using Polymerase Chain Reaction and Hybridization}Development of HPV quantification and Typing method using Polymerase Chain Reaction and Hybridization

HPV( Human Papilloma Virus )에 의한 감염은 여성들의 자궁경부암을 유발하는 주요한 인자로 밝혀졌으며 이에 따라 다양한 검출기법이 개발 활용되어지고있다.Infection with HPV (Human Papilloma Virus) has been found to be a major cause of cervical cancer in women, and various detection methods have been developed and utilized.

현재 HPV의 DNA ( 또는 Genome )을 확인하는 기법은 두가지 방법이 주로 사용되고 있으며 각 기법의 특징과 단점은 아래와 같다.Currently, two methods are used to identify DNA (or genome) of HPV. The characteristics and disadvantages of each method are as follows.

첫 번째 HPV의 DNA와 HPV에 특이적으로 결합 할 수 있는 RNA간의 결합으로 HPV의 감염여부 확인하는 기법.( HybridCapture, Digene, USA )A technique to confirm the infection of HPV by binding between the DNA of the first HPV and RNA that can specifically bind to HPV. (HybridCapture, Digene, USA)

이 방법은 자궁 경부의 세포로부터 추출된 DNA를 NaOH용액으로 변성 시켜준후 여기에 HPV에 대하여 특이적으로 결합반응( Hybridization )할 수 있는 RNA 탐침자( 이하 Probe )를 함께 섞어주어 결합시켜준 뒤 여기에 DNA/RNA의 결합체에 특이적으로 반응하는 항체를 결합시켜주어 이를 효소 반응으로 확인하는 기법이다.In this method, DNA extracted from cervical cells is denatured with NaOH solution, and then mixed with an RNA probe (hereinafter referred to as probe) that can specifically hybridize to HPV. This is a technique that binds an antibody that specifically reacts to a DNA / RNA conjugate and binds it to an enzyme reaction.

본 기법의 경우 기본적으로 많은 양의 HPV Genome을 필요로 하며 이러한 이유로 검체내에 HPV의 Genome이 미량으로 존재하는 경우에는 이를 확인하는 것이 불가능하다.This technique basically requires a large amount of HPV genome. For this reason, it is impossible to check if there is a small amount of HPV genome in the sample.

또한 본 기법은 HPV Type을 고위험군과 저위험군으로만 구분 검출하기에 구체적인 Type의 확인은 불가능하다.In addition, this technique can distinguish HPV type into high risk group and low risk group, so it is impossible to identify specific type.

두 번 째 방법은 HPV에 대하여 특이적인 PCR을 수행하여 HPV에 의한 감염여부를 확인하고 이후 2차 PCR을 추가 수행하여 고위험군 Type인 HPV 16과 HPV18를 구분, 확인하는 기법.The second method is to identify HPV 16 and HPV 18 by performing a specific PCR for HPV and then checking whether it is infected by HPV.

본 기법은( 이하 Co-Nested PCR ) 시료로부터 추출된 DNA를 많은 Type HPV에 대하여 반응할수 있도록 고안된 Primer로 PCR을 수행하여 HPV에 의한 감염 여부를 확인한 이후 PCR결과물 일부를 PCR 시료로 하여 2차 PCR을 추가적으로 수행하여HPV 16과 HPV 18을 검출, 확인하는 기법이다. 본 기법의 경우 PCR기법의 최대 단점인 PCR 결과물에 의한 오염과 이에 의한 위양섬 결과의 산출로부터 벗어날 수 없으며 이로 인하여 높은 위양성율 나타내는 것으로 확인되어지고 있다.This technique (hereinafter referred to as Co-Nested PCR) performs PCR with a primer designed to react DNA extracted from a sample against many types of HPV, and then confirms the infection by HPV. This is an additional technique to detect and identify HPV 16 and HPV 18. In case of this technique, it is confirmed that it can not escape from the contamination of PCR result, which is the biggest disadvantage of PCR technique, and the calculation of false positive result.

위와 같은 HPV 검출법이 현재 시행되고 있으나 각 기법들의 검출 감도와 소요시간 및 비용상의 비효율성과 같은 한계성으로 인하여 새롭고 충실한 검출법의 개발이 요구되어 왔다. 따라서 본 발명은 기존의 각검출법들이 가지고 있던 한계성과 단점, 그리고 비효율성을 개선하기 위하여 개발되었다.Although the above-mentioned HPV detection methods are currently being implemented, new and faithful detection methods have been required due to limitations such as detection sensitivity, time required, and cost inefficiency of each technique. Therefore, the present invention was developed to improve the limitations, disadvantages, and inefficiencies of the conventional angle detection methods.

본발명에 의해 개발된 기법은 기존에 활용되어오던 분자생물학적 기초기술인 PCR기법과 HPV 정량용 표준 Genome 그리고 각각의 HPV Type을 구분하여 특이적으로 결합반응 할 수 있는 Probe를 활용하여 기존 검사법에 비하여 높은 감도로 HPV의 환부내의 HPV 정량과 HPV Type의 동시 확인을 가능케 하였다.The technique developed by the present invention is higher than the conventional test method by using the PCR technique, which is the basic molecular biology technology, the standard genome for quantifying HPV, and the probe that can specifically bind and distinguish each HPV type. Sensitivity allowed simultaneous determination of HPV and HPV type in the affected area of HPV.

본 발명을 살펴보면 다음과 같다.Looking at the present invention is as follows.

1) HPV PCR 반응 시 환자의 시료내의 HPV Genome과 경쟁반응할 수 있으면서 HPV Typing에 간섭하지 않는 표준DNA의 개발.1) Development of a standard DNA that can compete with HPV Genome in a patient's sample during the HPV PCR reaction and does not interfere with HPV Typing.

2) 환자의 시료로부터 추출된 DNA와 이미 정량된 HPV 표준 DNA를 함께 섞어준 뒤 PCR 반응을 수행한 뒤 환자의 DNA내의 HPV Genome의 량을 확인한다.(그림1.)이때 사용된 Primer에는 HPV Typing을 위하여 표지자( Biotin )를 부착하여 사용한다.2) After mixing the DNA extracted from the patient's sample with the already quantified HPV standard DNA, perform a PCR reaction and check the amount of HPV genome in the patient's DNA (Fig. 1). Attach a marker (Biotin) for typing.

3) 각각의 HPV Type에 특이적으로 반응할수 있는 DNA 탐침자의 개발.3) Development of DNA probes that can specifically react to each HPV type.

4) 3)에서 개발된 각 Probe DNA를 Membrane 표면에 각각 고정하여 준다.(그림 2.)4) Fix each Probe DNA developed in 3) to the surface of Membrane (Figure 2.)

5) 2)에서 수행된 PCR 결과물을 Membrane에 도말하여 준 뒤 Hybridization 시켜주고 세척한다.5) Apply the PCR result in 2) to Membrane, hybridize and wash.

6) 세척된 Membrane에 반응 표지자를 인식하여 부착될 수 있는 효소시약을 도말하여 부착시켜주고 세척한다.6) Plate the enzyme reagent that can be attached to the washed marker by attaching it to the washed membrane.

7) 세척된 Membrane에 효소시약의 발색반응기질을 도말하여 발색반응을 시켜주어 확인한다.7) Make a color reaction by smearing the color reaction substrate of enzyme reagent on the washed membrane.

본 PCR에 사용된 PCR 시발체 ( 이하 Primer )는 환자 DNA와 표준 DNA에 함께작용할수 있도록 고안되었다. Lane 2에서 Lane 6에는 각각 환자의 DNA와 표준 DNA를 함께 넣어 반응 시켜준 것으로 반응시 두 DNA는 HPV Primer에 경쟁적으로 반응하며 이의 산물을 위와 같이 전기영동으로 분석할 수 있다.The PCR primers (hereinafter Primer) used in this PCR were designed to work together with patient DNA and standard DNA. In Lane 2 and Lane 6, the DNA and standard DNA of each patient were put together and reacted. In response, the two DNAs reacted competitively with HPV Primer, and their products can be analyzed by electrophoresis as above.

위의 전기영동 결과를 보면 Lane 2의 경우 환자의 DNA 내에 HPV Genome이 너무 적어 PCR 반응에 참여하지 못하여 표준 DNA의 PCR 산물만이 발생하였다. Lane 3의 경우 환자의 DNA내의 HPV Genome이 PCR 반응에 참여하여 PCR 산물을 발생시켯으나 표준 DNA에 비하여 미약하다. Lane 4의 경우 표준 DNA와 환자 DNA가 동등하게 PCR에 참여하여 유사한 정도로 PCR 산물을 생성시켰다. Lane 5와 Lane 6의 경우 환자의 HPV DNA내의 Genome이 표준 DNA에 비하여 상대적으로 많은 반응을 이루었다.이러한 결과를 분석하여 볼시 환자의 DNA내의 HPV Genome은 약 10 fmol이 존재하고 있음을 유추할 수 있다.The electrophoresis results showed that Lane 2 had only a small amount of HPV genome in the DNA of the patient. In Lane 3, the HPV genome in the patient's DNA participated in the PCR reaction to generate a PCR product, but it was weaker than the standard DNA. In lane 4, the standard DNA and the patient DNA participated in the PCR equally to generate a PCR product to a similar degree. In Lane 5 and Lane 6, the genomes in the patient's HPV DNA responded relatively more than the standard DNA. .

본 발명은 2단계의 HPV 확인으로 구성되는데 1차 확인은 표준 HPV DNA와의 경쟁 PCR에 의한 HPV 정량, 그리고 2차의 확인은 이미 수행된 PCR 결과물을 위의(그림 2.)Membrane을 사용하여 환자가 보균중인 HPV의 Type을 확인하는 것으로 구성되어있다.The present invention consists of two stages of HPV confirmation, wherein the primary confirmation is HPV quantification by competitive PCR with standard HPV DNA, and the secondary confirmation is performed on the patient using the above-described PCR result (Figure 2). Consists of identifying the type of HPV that is present.

이하, 본 발명을 실시 예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail by examples.

[실시예 1]Example 1

자궁경부암이 의심되거나 HPV에의 감염이 우려되는 환자의 환부세포로부터 DNA를 추출하여 본 발명에 의해 준비된 경쟁 PCR을 수행하여 HPV Genome의 양을 확인한다.DNA is extracted from affected cells of patients suspected of cervical cancer or infected with HPV, and competition PCR prepared by the present invention is performed to confirm the amount of HPV genome.

위의 결과를 분석하여 볼 경우 환자의 DNA에는 100 fmol이상의 HPV Genome이 존재하고 있는 것으로 유추되어 관리와 치료가 요망된다.The analysis of the above results suggests that HPV genomes of 100 fmol or more are present in the DNA of the patient.

본 환자는 High risk group에 속하는 HPV 16과 Low risk group에 속하는 HPV 11에 동시 감염되어 있어 자궁경부암의 발병이 우려됨으로 관리와 치료가 요망된다.The patient is concurrently infected with HPV 16 in the high risk group and HPV 11 in the low risk group.

[실시예 2]Example 2

자궁경부암이 의심되거나 HPV에의 감염이 우려되는 환자의 환부세포로부터DNA를 추출하여 본 발명에 의해 준비된 경쟁 PCR을 수행하여 HPV Genome의 양을 확인한다.DNA is extracted from affected cells of patients suspected of cervical cancer or infected with HPV, and competition PCR prepared by the present invention is performed to confirm the amount of HPV genome.

위의 결과를 분석하여 볼 경우 환자의 DNA에는 1 nmol이상의 HW Genome이 존재하고 있는 것으로 유추되어 관리와 치료가 요망된다.The analysis of the above results suggests that more than 1 nmol of HW genome is present in the patient's DNA.

본 환자는 High risk group에 속하는 HPV 18, Intermediate risk group에 속하는 HPV 31 그리고 Low risK group에 속하는 HPV 11에 동시 감염되어 있어 자궁경부암의 발병이 우려됨으로 관리와 치료가 요망된다.The patient is concurrently infected with HPV 18 in the high risk group, HPV 31 in the Intermediate risk group and HPV 11 in the Low risK group.

[실시예 3]Example 3

자궁경부암이 의심되거나 HPV에의 감염이 우려되는 환자의 환부세포로부터 DNA를 추출하여 본 발명에 의해 준비된 경쟁 PCR을 수행하여 HPV Genome의 양을 확인한다.DNA is extracted from affected cells of patients suspected of cervical cancer or infected with HPV, and competition PCR prepared by the present invention is performed to confirm the amount of HPV genome.

위의 결과를 분석하여 볼 경우 환자의 DNA에는 1 fmol이상의 HPV Genome이 존재하고 있는 것으로 유추되어 관리와 치료가 요망된다.Based on the above results, it is inferred that more than 1 fmol HPV genome is present in the patient's DNA.

본 환자는 낮은 농도이지만 High risk group에 속하는 HPV 16과 18 그리고 Low risk group에 속하는 HPV 11이 동시 감염되어 있어 자궁경부암의 발병이 우려됨으로 관리와 치료가 요망된다.This patient is low in concentration but is infected with HPV 16 and 18 in the high risk group and HPV 11 in the low risk group.

[실시예 4]Example 4

자궁경부암이 의심되거나 HPV에의 감염이 우려되는 환자의 환부세포로부터 DNA를 추출하여 본 발명에 의해 준비된 경쟁 PCR을 수행하여 HPV Genome의 양을 확인한다.DNA is extracted from affected cells of patients suspected of cervical cancer or infected with HPV, and competition PCR prepared by the present invention is performed to confirm the amount of HPV genome.

위의 결과를 분석하여 볼 경우 환자의 DNA에는 100 fmol이상의 HPV Genome이 존재하고 있는 것으로 유추되어 관리와 치료가 요망된다.The analysis of the above results suggests that HPV genomes of 100 fmol or more are present in the DNA of the patient.

본 환자는 Intermediate risk group에 속하는 HPV 31과 35 그리고 Low risk group에 속하는 HPV 11에 높은 농도로 동시 감염되어 있어 자궁경부암의 발병이 우려됨으로 관리와 치료가 요망된다.The patient is infected with high concentrations of HPV 31 and 35 in the Intermediate risk group and HPV 11 in the Low risk group.

[비교예 1]Comparative Example 1

자궁경부암이나 HPV 감염이 의심되는 환자의 환부로부터 DNA를 추출하여 기존의 이중 PCR 기법( Co-Nested PCR )과 본 발명기법으로 함께 검사하였다.DNA was extracted from the lesions of patients suspected of cervical cancer or HPV infection and tested together with the existing double PCR technique (Co-Nested PCR) and the present invention.

이중 PCR 기법 결과 : 비교대상 환자는 HPV 감염되어있는 것이 확인되었으나 HPV16 또는 18에는 감염되지 않은 것으로 확인되었다.Results of double PCR technique: Patients to be compared were confirmed to be infected with HPV but not HPV16 or 18.

발명기법 결과 : 비교대상 환자는 고농도의 HPV 45와 31에 감염되어 있는 것으로 확인되어 각별한 관리와 치료가 요망되었다.Results of Invention Technique: The patients to be compared were found to be infected with high concentrations of HPV 45 and 31. Therefore, special care and treatment were required.

위의 결과를 분석하여 볼 경우 환자의 DNA에는 1 nmol이상의 HPV Genome이 존재하고 있는 것으로 유추되어 관리와 치료가 요망된다.Based on the above results, it is inferred that more than 1 nmol of HPV genome is present in the patient's DNA.

검출결과 분석 : 본 환자는 낮은 감염율을 나타내고 있는 high risk group HPV 45와 Intermediate risk group HPV 31에 높은 농도로 감염되어 있는 것이 확인되었는데 이는 기존의 이중 PCR 검사기법으로는 HPV의 감염 농도 확인과 HPV Type 16과 18이외에는 확인이 불가능하였기에 단순 감염자로 처리 될 수밖에 없었으나 본 발명기법에 의한 검사 결과로 고위험군 HPV Type인 45 그리고 중위 위험군 31에 감염된 것을 확인할 수 있었다.Analysis of the results: The patient was infected with high concentrations in the high risk group HPV 45 and the intermediate risk group HPV 31, which showed low infection rates. The existing double PCR method confirmed the HPV infection level and HPV type. Since it was impossible to confirm other than 16 and 18, it was inevitable to be treated as a simple infection, but the test results of the present invention showed that the high-risk HPV type 45 and the median risk group 31 were infected.

[비교예 2]Comparative Example 2

자궁경부암이나 HPV 감염이 의심되는 환자의 환부로부터 DNA를 추출하여 기존의 RNA Probe 특이결합 검출법( HybridCapture )과 본 발명기법으로 함께 검사하였다.DNA was extracted from the lesions of patients suspected of cervical cancer or HPV infection and tested together with the existing RNA probe specific binding detection method (HybridCapture).

HybridCapture : 비교대상 환자는 HPV에 감염되지 않은 것으로 확인되었다.HybridCapture: Patients to be compared were confirmed not to be infected with HPV.

발명기법 결과 : 비교대상 환자는 매우 낮은 농도로 HPV 16와 11에 감염되어 있는 것으로 확인되어 각별한 관리와 치료가 요망되었다.Results of Invention Technique: The patients to be compared were found to be infected with HPV 16 and 11 at very low concentrations.

위의 결과를 분석하여 볼 경우 환자의 DNA에는 1 fmol이하의 HPV Genome이 존재 하고 있는 것으로 유추되어 관리와 치료가 요망된다.The analysis of the above results suggests that less than 1 fmol of HPV genomes are present in the patient's DNA.

검출결과 분석 : 본 환자는 낮은 농도이지만 High risk group에 속하는 HPV 16 그리고 Low risk group에 속하는 HPV 11이 동시 감염되어 있는 것이 본 발명 기법으로 확인되어 지궁경부암의 발병이 우려됨으로 관리와 치료가 요망된다.Analysis of detection results: The patient is at low concentration but HPV 16 belonging to high risk group and HPV 11 belonging to low risk group are confirmed by the present technique. .

그러나 기존의 HybridCapture 검사결과는 감염 음성으로 처리되었는데 이는 본 기법의 검출 감도가 매우 낮은데 기인하는 것으로 보인다.However, the existing HybridCapture test result was treated as negative of infection, which may be due to the very low detection sensitivity.

Claims (1)

본 발명은 경쟁 PCR과 특이 probe를 사용한 Hybridization을 함께 수행하여 HPV의 정량과 Typing 결과를 함께 확인할 수 있는 기법이다.The present invention is a technique that can confirm the quantification and Typing results of HPV by performing a hybridization using a competitive PCR and a specific probe together.
KR1019990004009A 1999-02-05 1999-02-05 Development of HPV quantification and Typing method using Polymerase Chain Reaction and Hybridization KR20000055412A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100382703B1 (en) * 2000-03-15 2003-05-09 주식회사 바이오메드랩 diagnosis kit for genotyping of Human Papillomavirus and manufacturing method for thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100382703B1 (en) * 2000-03-15 2003-05-09 주식회사 바이오메드랩 diagnosis kit for genotyping of Human Papillomavirus and manufacturing method for thereof

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