KR20060014305A - Oligonucleotide probe for analyzing human papillomavirus (hpv) genotype and detecting method thereof - Google Patents

Oligonucleotide probe for analyzing human papillomavirus (hpv) genotype and detecting method thereof Download PDF

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KR20060014305A
KR20060014305A KR1020040062936A KR20040062936A KR20060014305A KR 20060014305 A KR20060014305 A KR 20060014305A KR 1020040062936 A KR1020040062936 A KR 1020040062936A KR 20040062936 A KR20040062936 A KR 20040062936A KR 20060014305 A KR20060014305 A KR 20060014305A
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박영석
박재찬
강진석
이경률
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바이오코아 주식회사
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Abstract

본 발명은 인유두종바이러스(Human Papillomavirus, HPV) 유전자형 분석을 위한 올리고뉴크레오티드 프로브 및 그 검사 방법에 관한 것이다.The present invention relates to oligonucleotide probes for human papillomavirus (HPV) genotyping and methods of testing thereof.

인유두종바이러스, 유전자형, 올리고뉴크레오티드 프로브, 서스펜션 어레이Human Papilloma Virus, Genotype, Oligonucleotide Probe, Suspension Array

Description

인유두종바이러스 유전자형 분석을 위한 올리고뉴크레오티드 프로브 및 그 검사 방법{Oligonucleotide probe for analyzing Human Papillomavirus (HPV) genotype and detecting method thereof}Oligonucleotide probe for analyzing human papillomavirus (HPV) genotype and detecting method

도 1은 HPV 유전자의 증폭을 위한 OTN(one-tube nested) 비대칭 PCR 산물의 전기영동 사진이다. 레인 1 내지 6에서 HPV 유전자가 증폭된 양성 밴드가 나타났다.1 is an electrophoretic photograph of a one-tube nested (OTN) asymmetric PCR product for amplification of the HPV gene. Positive bands with amplified HPV genes were shown in lanes 1-6.

도 2는 532㎚의 파장에서 측정한 HPV 16 유전자형에 관한 서스펜션 어레이의 형광 반응 결과로, 64개의 프로브 중에서 프로브 10번이 고정된 마이크로스피어 비드 및 교잡반응 컨트롤이 고정된 마이크로스피어 비드(표 1 참조)에서 측정된 형광값이 높은 수치를 나타내었다.FIG. 2 shows fluorescence reaction results of a suspension array of HPV 16 genotypes measured at a wavelength of 532 nm. Among 64 probes, microsphere beads with fixed probe 10 and microsphere beads with fixed hybridization control (see Table 1). The fluorescence value measured at) shows a high value.

도 3은 532㎚의 파장에서 측정한 HPV 18 유전자형에 관한 서스펜션 어레이의 형광 반응 결과로, 64개의 프로브 중에서 프로브 12번이 고정된 마이크로스피어 비드 및 교잡반응 컨트롤이 고정된 마이크로스피어 비드(표 1 참조)에서 측정된 형광값이 높은 수치를 나타내었다.FIG. 3 shows fluorescence reaction results of a suspension array of HPV 18 genotype measured at a wavelength of 532 nm, and microsphere beads with fixed probe 12 and 64 microsphere beads with fixed hybridization control among 64 probes (see Table 1). The fluorescence value measured at) shows a high value.

도 4는 532㎚의 파장에서 측정한 HPV 58 유전자형에 관한 서스펜션 어레이의 형광 반응 결과로, 64개의 프로브 중에서 프로브 45번이 고정된 마이크로스피어 비드 및 교잡반응 컨트롤이 고정된 마이크로스피어 비드(표 1 참조)에서 측정된 형광 값이 높은 수치를 나타내었다.FIG. 4 shows the fluorescence response of the suspension array for the HPV 58 genotype measured at a wavelength of 532 nm. Among the 64 probes, microsphere beads fixed with probe 45 and microsphere beads fixed with hybridization control (see Table 1). Fluorescence values measured at) show high values.

본 발명은 인유두종바이러스(Human Papillomavirus, HPV)의 유전자형 분석을 위한 올리고뉴크레오티드 프로브, 이를 포함하는 조성물, 이 올리고뉴크레오티드 프로브가 고정된 마이크로스피어(microsphere) 비드(bead) 및 상기 마이크로스피어 비드가 포함된 분석용 키트에 관한 것이다.The present invention provides an oligonucleotide probe for genotyping of a human papillomavirus (HPV), a composition comprising the same, a microsphere bead to which the oligonucleotide probe is immobilized and the microsphere beads It relates to an analysis kit containing.

본 발명은 또한 HPV의 유전자형 분석을 위한 상기 올리고뉴크레오티드 프로브를 이용한 분석방법 및 상기 올리고뉴크레오티드 프로브가 고정된 마이크로스피어 비드를 이용한 서스펜션 어레이 분석방법에 관한 것이다.The present invention also relates to an analysis method using the oligonucleotide probe for genotyping of HPV and a suspension array analysis method using microsphere beads to which the oligonucleotide probe is fixed.

HPV는 약 8Kb의 이중나선 원형의 유전자를 가진 직경 55㎚의 DNA 바이러스이며 여성의 자궁경부암을 유발하는 여러 가지 악성종양과 밀접한 관계가 있는 것으로 알려져 있다(Godfroid et al., J. Virol. Method 75:69-81, 1998). HPV 유전자는 E1, E2, E4, E5, E6, E7과 L1, L2로 구성되어 있고, E1 단백질은 바이러스 복제에 관여하며, E2 단백질은 바이러스의 복제와 전사의 조절에 관여한다(Wison et al., Virus Genes 24:275-90, 2002). E6와 E7 유전자는 암유전자 역할을 하는 것으로 각각 암억제 단백질인 p53과 pRB에 결합하는 단백질을 암호화하고 있다(Furumoto et al., J. Med. Invest. 49:124-133, 2002).HPV is a 55 nm diameter DNA virus with a double-helix circular gene of about 8 Kb and is known to be closely associated with various malignancies that cause cervical cancer in women (Godfroid et al ., J. Virol. Method 75 : 69-81, 1998). The HPV gene consists of E1, E2, E4, E5, E6, E7 and L1, L2, the E1 protein is involved in viral replication, and the E2 protein is involved in viral replication and transcription (Wison et al . , Virus Genes 24: 275-90, 2002). E6 and E7 genes act as oncogenes and encode proteins that bind to the cancer suppressor proteins p53 and pRB, respectively (Furumoto et al ., J. Med. Invest . 49: 124-133, 2002).

자궁경부암은 성접촉과 밀접한 관계가 있으며, HPV의 감염이 자궁경부 종양 발생에 관계가 있다고 알려져 있다. 현재까지 약 100여종의 HPV 유전자형이 보고되어 있는데 이 중에서 사람에게 질병을 유발시킬 수 있는 HPV는 약 30여 종이며 이를 크게 고위험군(HPV 유전자형 16, 18, 31, 33, 35 등)과 저위험군(HPV 유전자형 6, 11, 42, 43, 44 등)으로 분류하고 있다(De Villiers, J. Virol. 63:4898-4903, 1989; Jacobs et al., J. Clin. Microbiol. 33:901-905, 1995). 병소의 위치와 병변의 진행정도에 따라 각 특이한 유전자형이 발견되어 HPV 감염의 생물학적 다양성을 인식하게 되었다(Garrenstroom et al., J. Gynecol. Cancer 4:73-78, 1994).Cervical cancer is closely related to sexual contact, and HPV infection is known to be related to the development of cervical tumors. To date, about 100 HPV genotypes have been reported, of which about 30 HPV can cause disease in humans, which are largely high-risk (HPV genotypes 16, 18, 31, 33, 35, etc.) and low-risk ( HPV genotypes 6, 11, 42, 43, 44, etc. (De Villiers, J. Virol . 63: 4898-4903, 1989; Jacobs et al ., J. Clin. Microbiol. 33: 901-905, 1995). Depending on the location of the lesion and the progression of the lesion, each unique genotype was discovered to recognize the biodiversity of HPV infection (Garrenstroom et al ., J. Gynecol. Cancer 4: 73-78, 1994).

상기 HPV 감염여부를 판별하기 위해 현재 가장 많이 시행되는 검진법은 자궁세포진 검사(PAP smear)로서 검사자의 숙련도에 의존하여 검사의 정확도가 떨어지는 단점이 있다(Menezes et al., Acta Cytol. 45:919-926, 2001). 질확대경을 시행하면 HPV의 감염을 70%까지 진단할 수 있으나, 수련된 전문가와 고가의 장비가 필요하며 인유두종바이러스의 유전자형을 분류할 수 없는 단점이 있다(Reid et al., Clin Obstet Gynecol. 32:157-179, 1989).Currently, the most widely used screening method to determine whether the HPV infection is a Pap smear (PAP smear) has a disadvantage that the accuracy of the test is inferior depending on the skill of the examiner (Menezes et al ., Acta Cytol . 45: 919 -926, 2001). Although colposcopy can diagnose up to 70% of HPV infections, it requires disadvantageous training and expensive equipment and cannot classify genotypes of HPV (Reid et al ., Clin Obstet Gynecol. 32) . : 157-179, 1989).

또 L1 부위를 PCR(polymerase chain reaction)로 증폭한 후에 제한효소를 사용하는 PCR-RFLP(Restriction Fragment Length Polymorphism)는 간단하고 쉽게 결과를 얻을 수 있는 장점이 있으나 사용하는 제한 효소가 변이부분을 인식하지 못하면 분석하지 못하는 단점이 있다(Lungu et al., JAMA 267:2493-2496, 1992). 또한 HPV 유전자형에 따라서 PCR 증폭의 효율이 달라 검사의 정확도가 문제 될 수 있다(Qu et al., J. Clin. Microbiol. 35:1304-1310, 1997; Karksen et al., J. Clin. Microbiol. 34:2095-2100, 1996; Gravitt et al., J. Clin. Microbiol. 38:357- 361).In addition, PCR-RFLP (Restriction Fragment Length Polymorphism) using restriction enzymes after amplifying the L1 region by polymerase chain reaction (PCR) has a simple and easy result, but the restriction enzyme used does not recognize the mutation region. There is a drawback that cannot be analyzed (Lungu et al ., JAMA 267: 2493-2496, 1992). In addition, the efficiency of the PCR amplification may differ depending on the HPV genotype, which may cause the accuracy of the test (Qu et al ., J. Clin. Microbiol . 35: 1304-1310, 1997; Karksen et al ., J. Clin.Microbiol. 34: 2095-2100, 1996; Gravitt et al ., J. Clin.Microbiol. 38: 357-361).

상품화되어 있는 하이브리드 캡쳐 키트(Digene, 미국)는 PCR 증폭 과정없이 확인 가능하나 고위험군과 저위험군으로 분류 가능할 뿐 자궁경부암과 상관관계가 높은 HPV 유전자형 16 및 18과 다른 유전자형을 구별할 수 없다는 단점이 있다(Clavel et al., J. Clin. Pathol. 51:737-740, 1998).Commercially available hybrid capture kits (Digene, USA) can be identified without PCR amplification, but they can be classified into high- and low-risk groups and cannot distinguish HPV genotypes 16 and 18, which are highly correlated with cervical cancer, with other genotypes. (Clavel et al. , J. Clin. Pathol. 51: 737-740, 1998).

최근에 개발된 마이크로 칩 기술을 이용한 HPV 유전자형 분석키트(바이오메드랩, 한국)는 슬라이드상에서 반응하는 2차원적인 방법으로 교잡반응 시킨 후에 3차에 걸쳐서 세척과정을 거쳐야 하는 번거로움이 있으며 이미지 작업시 기준점을 정하기 어려운 단점이 있다(대한민국특허 공개번호 제2003-0027178호 참조).Recently developed HPV genotyping kit using microchip technology (Bio Med Lab, Korea) is a two-dimensional method that reacts on a slide and has to be washed three times after hybridization. There is a disadvantage that it is difficult to determine the reference point (see Korean Patent Publication No. 2003-0027178).

본 발명은 상기 문제점을 해결하고자 안출된 것으로서, 본 발명의 목적은 HPV의 유전자형 분석을 위한 올리고뉴크레오티드 프로브, 이를 포함하는 조성물 및 이 올리고뉴크레오티드 프로브가 고정된 마이크로스피어 비드를 제공하는 것이다. 본 발명의 또 다른 목적은 상기 HPV의 유전자형 분석을 위한 상기 마이크로스피어 비드를 포함하는 유전자형 분석용 키트를 제공하는 것이다.The present invention has been made to solve the above problems, an object of the present invention is to provide an oligonucleotide probe for genotyping of HPV, a composition comprising the same and a microsphere bead fixed to the oligonucleotide probe. . Still another object of the present invention is to provide a kit for genotyping comprising the microsphere beads for genotyping of the HPV.

본 발명의 또 다른 목적은 HPV의 유전자형 분석을 위한 상기 올리고뉴크레오티드 프로브를 이용한 분석방법 및 상기 올리고뉴크레오티드가 고정된 마이크로스피어 비드를 이용한 서스펜션 어레이 분석방법을 제공하는 것이다.Still another object of the present invention is to provide an analysis method using the oligonucleotide probe for genotyping of HPV and a suspension array analysis method using the microsphere beads to which the oligonucleotide is fixed.

본 발명자들은 간단한 방법으로 HPV의 감염여부를 진단하고, 감염된 HPV의 유전자형을 정확하게 판별할 수 있는 방법을 확립하고자 연구 노력한 결과 상기 언급한 문제점을 해결할 수 있는 올리고뉴크레오티드 프로브 및 이를 포함하는 조성물을 개발하였다.The present inventors have tried to establish a method for diagnosing the infection of HPV by a simple method and to accurately determine the genotype of the infected HPV. As a result, an oligonucleotide probe and a composition comprising the same can be solved. Developed.

상기 목적을 달성하기 위하여, 본 발명은 HPV 유전자형에 상보적으로 결합할 수 있는 서열번호 1에서 64까지의 염기서열을 포함하고 있는 올리고뉴크레오티드 프로브들을 제공한다(표 1 참조). 본 발명은 또한 상기 올리고뉴크레오티드 프로브를 포함하는 조성물을 제공한다. 상기 조성물은 고위험군의 HPV 유전자형 감염여부를 판별하기 위하여 고위험군의 HPV 유전자형에 상보적으로 결합하는 복수의 올리고뉴크레오티드 프로브들을 포함하는 것이 바람직하며 또 비교를 위한 기준점을 정하기 위하여 서열번호 64의 염기서열을 포함하는 올리고뉴크레오티드 프로브를 더 포함하는 것이 더욱 바람직하다.In order to achieve the above object, the present invention provides oligonucleotide probes comprising the nucleotide sequence of SEQ ID NO: 1 to 64 capable of complementary binding to HPV genotype (see Table 1). The present invention also provides a composition comprising said oligonucleotide probe. Preferably, the composition comprises a plurality of oligonucleotide probes that complementarily bind to high-risk HPV genotypes to determine whether the high-risk HPV genotype is infected, and base sequence of SEQ ID NO: 64 to establish a reference point for comparison. More preferably further comprises an oligonucleotide probe comprising a.

또한 1회의 분석으로 모든 HPV 유전자형을 검출하기 위하여 상기 서열번호 1에서 64까지의 염기서열을 포함하는 올리고뉴크레오티드 프로브들을 모두 포함하는 조성물이 가장 바람직하다. In addition, the composition containing all the oligonucleotide probes comprising the nucleotide sequence of SEQ ID NO: 1 to 64 in order to detect all the HPV genotype in one analysis is most preferred.                     

Figure 112004035725493-PAT00001
Figure 112004035725493-PAT00001

Figure 112004035725493-PAT00002
Figure 112004035725493-PAT00002

본 발명자들은 HPV 감염여부 및 상기 HPV 유전자형을 간편하게 판별하기 위하여 서스펜션 어레이 시스템을 채택하였다. 서스펜션 어레이는 색깔을 이용하여 표지된 마이크로스피어 비드에 분석하고자 하는 각각 다른 목표 프로브들을 붙이고, 여러 반응을 이용하여 시료 샘플과 목표 프로브와의 반응 정도를 측정하는 것이다. 마이크로스피어 비드는 형광 강도 레벨이 다른 염료를 부착함으로써 각각의 비드를 구분할 수 있다. 서스펜션 어레이의 가장 큰 장점은 한 시료에서 여러 가지 물질들을 동시에 분석할 수 있어 분석이 매우 빠르며, 따라서 시료와 사용되는 시약의 양을 최소한으로 줄일 수 있다.The inventors have adopted a suspension array system to easily determine whether HPV is infected and the HPV genotype. The suspension array attaches different target probes to be analyzed to labeled microsphere beads using color, and measures the reaction degree between the sample sample and the target probe using various reactions. Microsphere beads can be distinguished from each bead by attaching dyes having different fluorescence intensity levels. The main advantage of the suspension array is that it can analyze several substances in one sample at the same time, making the analysis very fast, thus minimizing the amount of sample and reagents used.

즉 한 튜부(Tube)안에서 교잡반응이 일어나는 3차원적 방법으로 반응시킨 후 Luminex 100(Luminex사, 미국)시스템과 같은 기기에서 동시에 532㎚과 633㎚ 파장을 이용하여 측정한 형광값을 표시하게 된다. 633㎚ 파장에서 측정된 형광값은 마이크로스피어 비드를 식별하여 프로브에 부착된 물질을 판별하며, 532㎚ 파장에서 측정된 형광값은 각 프로브와 반응한 PCR 산물이 Streptavidin-Cy3 또는 Streptavidin-Phycoerythrin와 결합하여 나타난 값이다(Keji et al., Cytometry 33:318-323, 1998). 서스펜션 어레이는 프로우메트리(Fluorometry)기술을 이용한 Luminex 100(Luminex사, 미국)시스템과 같은 기기를 사용함으로써 고속측정이 가능하며, 1회 측정으로 100-300개 마이크로스피어 비드의 형광값의 최대빈도를 확인하기 때문에 정확한 분석이 가능하며, 형광 반응의 결과가 수치로 나타남으로써 정량 분석 및 자동화 시스템 적용에 용이하다(Armstrong et al., Cytometry 40:102-108, 2000).In other words, after reacting in a three-dimensional manner in which hybridization reaction occurs in a tube, the fluorescence value measured using 532 nm and 633 nm wavelengths is simultaneously displayed on a device such as Luminex 100 (Luminex, USA) system. . The fluorescence value measured at 633 nm wavelength identifies the microsphere beads to determine the substance attached to the probe, and the fluorescence value measured at the 532 nm wavelength binds Streptavidin-Cy3 or Streptavidin-Phycoerythrin with PCR products reacted with each probe. (Keji et al ., Cytometry 33: 318-323, 1998). Suspension arrays can be measured at high speed by using instruments such as the Luminex 100 (Luminex, USA) system using Fluorometry technology, and the maximum frequency of fluorescence values of 100-300 microsphere beads in one measurement. As a result, accurate analysis is possible, and the result of the fluorescence reaction is shown as a numerical value, which is easy for quantitative analysis and automation system application (Armstrong et al., Cytometry 40: 102-108, 2000).

이를 위하여 상기 서열정보 1 내지 64의 올리고뉴크레오티드 프로브를 포함하는 염기서열을 마이크로스피어 비드에 고정화하였다. 본 발명의 고정화는 프로브의 아민기와 비드의 카르복실기를 반응시켜 수행하였으나 이 반응 이외에도 여러 반응이 상기 프로브를 비드에 고정하기 위해 사용될 수 있음은 본 발명이 속한 분야에서 통상의 지식을 가진 자에게 자명한 사실이다.To this end, the nucleotide sequence including the oligonucleotide probe of SEQ ID NO: 1 to 64 was immobilized on the microsphere beads. The immobilization of the present invention was carried out by reacting the amine group of the probe with the carboxyl group of the beads, but in addition to this reaction, various reactions can be used to fix the probe to the beads, which will be apparent to those skilled in the art. It is true.

보다 구체적으로는 HPV 유전자형을 결정하기 위하여 HPV L1 부위에서 63개 유전자형에 특이적으로 반응할 수 있는 63개의 올리고뉴크레오티드 프로브와 교잡반응의 기준값을 제시하는 1개의 올리고뉴크레오티드 프로브를 제작하였으며, 이를 색깔로 표지된 각각의 마이크로스피어 비드에 고정하였다.More specifically, in order to determine the HPV genotype, 63 oligonucleotide probes that can specifically react to 63 genotypes in the HPV L1 region and one oligonucleotide probe that provides reference values for hybridization were prepared. This was fixed to each microsphere bead labeled with color.

이와 같이 기존의 슬라이드상에서 반응하는 2차원적 방법이 아닌 한 튜브안에서 교잡반응이 일어나는 3차원적 방법을 이용함으로써 HPV 유전자형 분석 효율을 극대화할 수 있었다.As such, the efficiency of HPV genotyping could be maximized by using the three-dimensional method of hybridization reaction in the tube, rather than the two-dimensional method that reacts on the conventional slide.

카르복실기를 가진 마이크로스피어 비드와 공유결합을 시키기 위하여 모든 프로브의 5' 말단에 아미노 링크를 붙이고 교잡 반응시 반응을 용이하게 하기 위하여 10개의 올리고(dT)를 부착한 다음 표 1과 같은 염기서열을 붙여 합성하였다. Amino link is attached to 5 'end of all probes to covalently bond with carboxyl group with carboxyl group, and 10 oligo (dT) is attached to facilitate the reaction during hybridization reaction, and then base sequence such as Table 1 is attached. Synthesized.                     

본 발명의 HPV 감염을 진단하기 위한 유전자형 분석용 키트는 상기 프로브가 고정된 마이크로스피어 비드 이외에 시료로부터 채취한 DNA를 PCR 방법으로 증폭시키기 위한 프라이머 등을 추가로 포함할 수도 있다.The genotyping kit for diagnosing HPV infection of the present invention may further include a primer for amplifying DNA collected from a sample in a PCR method, in addition to the microsphere beads fixed with the probe.

또 본 발명자들은 상기 올리고뉴크레오티드 프로브를 이용한 분석방법으로, 질내 분비물로부터 HPV DNA를 추출하는 단계, 추출한 DNA를 이용하여 비대칭 PCR을 실시하는 단계, 상기 비대칭 PCR 산물과 프로브를 결합시키는 단계 및 상기의 결합을 확인하는 단계를 포함하는 HPV 유전자형 분석방법을 개발하였다.In addition, the present inventors in the analysis method using the oligonucleotide probe, extracting HPV DNA from the vaginal secretion, performing asymmetric PCR using the extracted DNA, the step of combining the asymmetric PCR product and the probe and the HPV genotyping method was developed that includes the step of confirming the binding.

상기 확인단계를 위하여 PCR 방법으로 증폭시키기 위한 프라이머에 바이오틴 또는 로다민(Rhodamine)을 부착하여 사용하였다. 확인을 위하여 바이오틴이 부착된 안티센스 프라이머의 경우에는 바이오틴과 결합하는 Streptavidin-Cy3 또는 Streptavidin-Phycoerythrin을 상기 비대칭 PCR 산물과 프로브를 결합시키는 단계에 첨가하여 사용하였고, 로다민이 부착된 안티센스 프라이머의 경우 로다민의 형광을 직접 측정하여 결과를 평가하였다.Biotin or rhodamine was attached to primers for amplification by PCR. For confirmation, biotin-attached antisense primers were used by adding Streptavidin-Cy3 or Streptavidin-Phycoerythrin, which binds the asymmetric PCR product and probe, to biotin-bound antisense primers. Fluorescence was measured directly to evaluate the results.

구체적으로 HPV를 유전자형에 관계없이 효과적으로 검출하기 위해서는 본 발명은 프라이머쌍을 이용하며, 종래의 네스티드 PCR에서 요구되는 2번의 PCR을 수행하지 않고 검출이 가능하여 그 절차와 시간을 대폭 단축시킬 수 있었다.Specifically, in order to effectively detect HPV regardless of genotype, the present invention uses primer pairs, and it was possible to greatly reduce the procedure and time by detecting without performing two PCRs required in conventional nested PCR. .

결과는 서스펜션 어레이 시스템인 Luminex 100(Luminex사, 미국)을 사용하여 확인하였다. 비대칭 PCR법을 사용하여 증폭시킨 HPV PCR 산물과 마이크로스피어 비드에 고정된 올리고뉴크레오티드 프로브의 교잡반응으로 형광 반응이 나타난 프로브를 분석하여 HPV에 대한 63개의 유전자형을 구분할 수 있었다. The results were confirmed using a Luminex 100 (Luminex, USA) suspension system. The hybridization of HPV PCR products amplified using asymmetric PCR and oligonucleotide probes immobilized on microsphere beads was analyzed to identify 63 genotypes for HPV.                     

이와 같이 HPV 유전자형을 분석하는데 HPV L1 부위를 비대칭 PCR로 증폭하여 HPV 유전자형에 따라 다르게 증폭되는 단점을 최소화하였으며, 마이크로스피어 비드에 고정된 프로브와 반응시키기 위하여 변성화 과정을 거쳐야 하는 번거로움을 제거하였다. 이 L1 부위에서 HPV 유전자형을 분석하는 것은 프라이머 선택이 용이하다는 장점이 있다.As described above, HPV L1 region was amplified by asymmetric PCR to minimize the disadvantage of amplifying differently according to HPV genotypes, and to eliminate the need for denaturation process to react with probes immobilized on microsphere beads. . Analyzing the HPV genotype at this L1 site has the advantage of easy primer selection.

이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 아래에서 상술하는 실시예들에 한정되는 것으로 해석되는 것은 아니다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해서 제공되어지는 것이다.
Hereinafter, the present invention will be described with reference to Examples. However, embodiments according to the present invention can be modified in many different forms, the scope of the present invention is not to be construed as limited to the embodiments described below. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

실시예 1: HPV 프라이머의 합성 및 염기서열Example 1 Synthesis and Sequence of HPV Primer

비대칭 PCR에 사용한 HPV PCR 프라이머는 Bauer(Oxford press, Oxford, 영국, 1992)가 사용한 MY09/MY11와 Jacobs(J. Clin. Microbiol. 35:791-795, 1997)가 사용한 GP5+/GP6+ 프라이머 부위를 분석하여 사용하였으며 각 프라이머의 안티센스는 바이오틴 또는 로다민을 부착하였다.HPV PCR primers used for asymmetric PCR were analyzed for GP5 + / GP6 + primer sites used by MY09 / MY11 and Jacobs ( J. Clin. Microbiol . 35: 791-795, 1997) used by Bauer (Oxford press, Oxford, UK, 1992). The antisense of each primer was attached to biotin or rhodamine.

여기서 사용한 프라이머는 Molecular cloning 3판(Sambrook과 Rusell, Cold Spring Harbor Laboratory Press, 미국, 2001년)의 10.42에 기술된 올리고뉴크레오티드 합성과 같은 방법을 사용하였으며 독일의 MWG-biotech 사에 의뢰하여 합성하였다. The primers used here were prepared using the same method as the oligonucleotide synthesis described in 10.42 of Molecular cloning 3rd edition (Sambrook and Rusell, Cold Spring Harbor Laboratory Press, USA, 2001) and commissioned by MWG-biotech of Germany. It was.                     

실시예 2: HPV DNA의 추출 및 비대칭 PCR 반응Example 2: Extraction of HPV DNA and Asymmetric PCR Reaction

1) 1.5㎖ tube에 분주된 침전물을 넣는다. PBS(Phosphate buffer saline)로 2회 세척 후 원심분리하여 침전물을 얻었다.1) Put the precipitated precipitate in 1.5ml tube. After washing twice with PBS (Phosphate buffer saline) to obtain a precipitate by centrifugation.

2) HPV DNA 추출버퍼(10% chelex resin) 50㎕를 넣었다.2) 50 μl of HPV DNA extraction buffer (10% chelex resin) was added.

3) 100℃에서 20분간 가열하였다.3) Heated at 100 ° C. for 20 minutes.

4) 5분간 원심분리한 후 분리된 상층액 4.5㎕를 PCR에 사용하였다.4) After centrifugation for 5 minutes, 4.5 µl of the separated supernatant was used for PCR.

5) 아래의 표 2와 같은 방법으로 비대칭 PCR 반응을 GeneAmp PCR system 9600 thermal cycler(Perkin Elmer사, 미국)를 이용하여 연속적으로 수행하였다.5) The asymmetric PCR reaction was performed continuously using the GeneAmp PCR system 9600 thermal cycler (Perkin Elmer, USA) as shown in Table 2 below.

6) 비대칭 PCR이 끝난 산물 5㎕에 젤 로딩 버퍼(0.25% bromophenol blue, 0.25% xylene cyanol FF 및 15% Ficoll 400) 1㎕를 넣고 1㎍/㎖ 에티디움 브로마이드(ethidium bromide, EtBr)가 함유된 2% 아가로스 젤에서 전기영동한 후 UV 트랜스일루미네이터(transilluminator)가 부착된 이미지 분석기(Image analyzer, Vilber Lourmat사, 프랑스)에서 450bp(MY09/MY11) 또는 150bp(GP5+/GP6+)의 밴드를 확인하였다. 그 결과를 도 1에 나타내었다. 6) Add 1 µl of gel loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, and 15% Ficoll 400) to 5 µl of the asymmetric PCR finished product and contain 1 µg / ml ethidium bromide (EtBr). After electrophoresis on 2% agarose gel, bands of 450 bp (MY09 / MY11) or 150 bp (GP5 + / GP6 +) were identified on an image analyzer (UV analyzer) (Vilber Lourmat, France) with a UV transilluminator. . The results are shown in FIG.                     

Figure 112004035725493-PAT00003
Figure 112004035725493-PAT00003

실시예 3: 올리고뉴크레오티드 프로브 합성Example 3: Oligonucleotide Probe Synthesis

마이크로스피어 비드의 카르복실기와 공유결합을 시키기 위하여 모든 프로브의 5' 말단에 아미노 링크를 붙이고 교잡 반응시 반응을 용이하게 하기 위하여 10개의 올리고(dT)를 부착한 다음 표 1과 같은 염기서열을 붙여 합성하였다. 즉, "아미노 링크-올리고(dT)10-프로브 염기서열"의 순서로 하여 독일의 MWG사에 의뢰하여 합성하였다. 이때 결정한 HPV 유전자형의 염기서열은 표 3에서 나타낸 것 같이 총 63종의 HPV 유전자형을 분석하여 결정하였다. Amino link is attached to the 5 'end of all probes to covalently bond the carboxyl group of the microsphere beads, and 10 oligos (dT) are attached to facilitate the reaction during the hybridization reaction, followed by the nucleotide sequence shown in Table 1 It was. That is, it synthesize | combined by the German MWG company in order of "amino link- oligo (dT) 10-probe base sequence." The base sequence of the HPV genotype determined at this time was determined by analyzing a total of 63 HPV genotypes as shown in Table 3.                     

Figure 112004035725493-PAT00004
Figure 112004035725493-PAT00004

표 1에서 나타낸 염기서열과 같이 약 15bp에서 35bp의 크기로 하여 60℃를 전후하여 Tm값을 설정하여 합성하였다.As shown in the base sequence shown in Table 1 it was synthesized by setting the Tm value around 60 ℃ around the size of about 15bp to 35bp.

실시예 4: 프로브가 고정된 마이크로스피어 비드의 제작Example 4 Fabrication of Microsphere Beads with Probe Fixed

1) 100pmole의 아미노기가 붙어 있는 프로브를 0.1M MES(M2-(N-morpholino)ethane sulfonic acid pH 4.5)에 10μM이 되도록 혼합하였다.1) A probe having an amino group of 100 pmole was mixed in 0.1 M MES (M2- (N-morpholino) ethane sulfonic acid pH 4.5) to 10 μM.

2) 마이크로스피어 비드를 제작하기 위하여 Miraibio LabMaP bead(미국)를 구입하여 사용하였다. LabMAP bead(농도 1.2×106개/㎖)의 10㎕를 1.5㎖ tube로 분주한 후 10,000g에서 1분간 원심분리 후 상층액을 조심스럽게 제거하였다.2) Miraibio LabMaP bead (USA) was purchased and used to manufacture microsphere beads. 10 μl of LabMAP bead (concentration 1.2 × 10 6 pcs / ml) was dispensed into 1.5 ml tubes, followed by centrifugation at 10,000 g for 1 minute, and the supernatant was carefully removed.

3) 침사물에 50㎕의 0.1M MES buffer(pH 4.5)를 넣고 소니케이션 하였다.3) 50μl of 0.1M MES buffer (pH 4.5) was added to the immersion and sonicated.

4) 10μM 프로브 2.5㎕를 취하여 상기 제조된 마이크로스피어 비드에 첨가하였다.4) 2.5 μl of 10 μM probe was taken and added to the prepared microsphere beads.

5) 1% EDC(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimided hydrochloride) 용액 2.5㎕를 첨가하여 암소에서 30분간 2회 반복 반응하였다. 5) 2.5 μl of 1% EDC (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimided hydrochloride) solution was added, and the reaction was repeated twice in the dark for 30 minutes.                     

6) 0.02% 트윈20 용액 200㎕를 첨가한 후 10,000g에서 1분간 원심 분리하여 상층액을 제거하였다.6) After 200 μl of 0.02% Tween20 solution was added, the supernatant was removed by centrifugation at 10,000 g for 1 minute.

7) 01.% SDS 용액 200㎕를 첨가한 후 10,000g에서 1분간 원심 분리 후 상층액을 제거하였다.7) After 200 μl of 01.% SDS solution was added, the supernatant was removed after centrifugation at 10,000 g for 1 minute.

8) 침사물을 0.1M MES(pH4.5) 100㎕에 녹인 후 암소에서 냉장보관 하였다.8) The sediment was dissolved in 100 μl of 0.1 M MES (pH 4.5) and refrigerated in the dark.

전체적인 반응을 모식도로 나타내면 다음과 같다.The overall reaction is as follows.

Figure 112004035725493-PAT00005
Figure 112004035725493-PAT00005

R : 올리고뉴크레오티드, X : 마이크로스피어 비드R: oligonucleotide, X: microsphere bead

실시예 5: HPV PCR 산물과의 결합 반응Example 5: Binding Reaction with HPV PCR Products

1) 바이오틴이 부착된 프라이머로 증폭된 HPV PCR 산물의 경우 변성화 과정 없이 하이브리다이제이션 용액(3X SSC, 0.3% SDS)과 l:4의 비율로 혼합한 후 Streptavidin-Cy3(농도 1㎎/㎖) 또는 Streptavidin-Phycoerythrin(농도 1㎎/㎖)를 1/500로 희석하여 2ng/㎕ 농도가 되도록 첨가하였다.1) For HPV PCR products amplified with biotin-attached primers, Streptavidin-Cy3 (concentration 1 mg / ml) after mixing with hybridization solution (3X SSC, 0.3% SDS) and l: 4 ratio without denaturation. ) Or Streptavidin-Phycoerythrin (concentration 1 mg / ml) Diluted to 1/500 and added to a concentration of 2 ng / μl.

로다민 형광이 부착된 프라이머로 증폭한 경우 변성화 과정없이 하이브리다이제이션 용액(3X SSC, 0.3% SDS)과 1:4의 비율로 혼합하였다.When amplified with a rhodamine fluorescence attached primers were mixed with a hybridization solution (3X SSC, 0.3% SDS) in a ratio of 1: 4 without denaturation.

2) 60℃에서 1시간 동안 반응시켰다.2) The reaction was carried out at 60 ° C. for 1 hour.

3) 0.1X SSC 75㎕를 첨가하여 반응을 중지시켰다. 3) 75 μl of 0.1 × SSC was added to stop the reaction.                     

4) Luminex 100 LabMAP(Luminex사, 미국)를 사용하여 형광을 나타내는 HPV 유전자형을 분석하여 결과를 확인하였다.4) Luminex 100 LabMAP (Luminex, USA) was used to analyze the HPV genotype fluorescence was confirmed by the results.

그 결과를 도 2 내지 도 4에 나타내었다. 도 2 내지 도 4에서 나타나는 바와 HPV 유전자형에 따라 선택적으로 해당 유전자형에 상보적으로 결합하는 올리고뉴크레오티드 프로브가 부착된 마이크로스피어 비드에서 높은 형광값을 나타냄을 알 수 있다. 이는 본 발명의 서스펜션 어레이 분석방법이 매우 정확하며 간편하다는 것을 의미한다.The results are shown in FIGS. 2 to 4. As shown in FIGS. 2 to 4, it can be seen that oligonucleotide probes that selectively bind complementarily to the genotype according to the HPV genotype show high fluorescence values. This means that the suspension array analysis method of the present invention is very accurate and simple.

본 발명은 그 절차와 시간이 대폭 단축된 HPV 유전자형 분석을 위한 올리고뉴크레오티드 프로브, 이를 포함하는 조성물, 이 프로브가 고정된 마이크로스피어 비드 및 이 비드가 포함된 HPV 유전자형 분석용 키트를 제공한다. 본 발명의 올리고뉴크레오티드 프로브는 마이크로스피어 비드에 고정된 프로브와 반응하도록 PCR 산물을 변성화시키는 번거로움이 없다.The present invention provides oligonucleotide probes for HPV genotyping, the procedure and time of which are greatly shortened, compositions comprising the same, microsphere beads fixed with the probes, and kits for HPV genotyping containing the beads. Oligonucleotide probes of the present invention are free from the hassle of denaturing PCR products to react with probes immobilized on microsphere beads.

본 발명은 또한 상기 올리고뉴크레오티드를 이용한 분석방법을 제공한다. 본 발명은 또한 1회의 분석으로 정확한 유전자형 판별이 가능한 상기 올리고뉴크레오티드가 부착된 마이크로스피어 비드를 이용한 서스펜션 어레이 분석방법을 제공한다.The present invention also provides an analysis method using the oligonucleotide. The present invention also provides a suspension array analysis method using the oligonucleotide attached microsphere beads capable of accurate genotyping in one analysis.

<110> BIOCORE. CO., LTD. <120> Oligonucleotide probe for analyzing Human Papillomavirus(HPV) genotype and detecting method thereof <130> P04-216 <160> 64 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 01) <400> 1 accatgcaag tactacatat tccaatgct 29 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 03) <400> 2 tctactgaaa cctcggctac atatgat 27 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 04) <400> 3 tcagatggtg ctaatgacaa ttatcagtat a 31 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 06) <400> 4 atccgtaact acatcttcca catacaca 28 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 09) <400> 5 tacagaggca gctcaaacag aagaa 25 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 10) <400> 6 ggcctcccct gccac 15 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 11) <400> 7 gcatctgtgt ctaaatctgc tacatacac 29 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 13) <400> 8 agccactaca tcatctcttt cagaca 26 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 15) <400> 9 tacctctgat ggtaatgcca taaagtaat 29 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 16) <400> 10 tcattatgtg ctgccatatc tacttcaga 29 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 17) <400> 11 gtctacagaa gctggggctg tta 23 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 18) <400> 12 tgcttctaca cagtctcctg tacct 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 22) <400> 13 gacggcacca cagttaatta tgatg 25 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 23) <400> 14 ccaatgacag cagtttagaa aagtatgatg 30 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 24) <400> 15 aaaatggcaa agtaacagat attaacgagt a 31 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 26) <400> 16 agtacattat ctgcagcatc tgcatcc 27 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 27) <400> 17 tgtgcagctg aggtgtctga taata 25 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 28) <400> 18 ctactgactc ttcagctacg tacga 25 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 29) <400> 19 cgttgaccac ttatgatgct accaa 25 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 30) <400> 20 atatctgcaa ccacacaaac gttatcc 27 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 31) <400> 21 gtgctgcaat tgcaaacagt gatac 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 32) <400> 22 gactgtgtgt gctactgtaa caact 25 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 33) <400> 23 tatgcacaca agtaactagt gacagtac 28 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 34) <400> 24 tccacaagta caactgcacc atatg 25 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 35) <400> 25 tgtgttctgc tgtgtcttct agtga 25 <210> 26 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 36) <400> 26 tagtatttca atatataaca ataatggggc actaa 35 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 38) <400> 27 aaaacggggg tgctcaagaa tat 23 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 39) <400> 28 tctacctcta tagagtcttc cataccttct 30 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 40) <400> 29 cccacaccaa ccccatataa taacagt 27 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 41) <400> 30 ttcctgaggg ggatgcttct 20 <210> 31 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 42) <400> 31 ctgcaacatc tggtgataca tatacagct 29 <210> 32 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 43) <400> 32 tctactgacc ctactgtgcc cag 23 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 44) <400> 33 tacacagtcc cctccgtcta catat 25 <210> 34 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 45) <400> 34 acacaaaatc ctgtgccaag tacatat 27 <210> 35 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 47) <400> 35 caggggacat aaaggatata caggattata a 31 <210> 36 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 48) <400> 36 aactgcatta actgaaaact acatagataa tgg 33 <210> 37 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 50) <400> 37 cctttggatc ctttaaatgt agcaagtt 28 <210> 38 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 51) <400> 38 tgctgcggtt tccccaacat tta 23 <210> 39 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 52) <400> 39 tgctgaggtt aaaaaggaaa gcacata 27 <210> 40 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 53) <400> 40 tttccgcaac cacacagtct atgtcta 27 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 54) <400> 41 tacagcatcc acgcaggata gctttaa 27 <210> 42 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 55) <400> 42 tgctacaact cagtctccat ctacaac 27 <210> 43 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 56) <400> 43 gtactgctac agaacagtta agtaaatatg at 32 <210> 44 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 57) <400> 44 tctctttgtg tgccactgta accac 25 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 58) <400> 45 gcactgaagt aactaaggaa ggta 24 <210> 46 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 59) <400> 46 tctactactg cttctattcc taatgtatac ac 32 <210> 47 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 61) <400> 47 aaccatttgt actgctacat ccccc 25 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 62) <400> 48 gctgcagcag aatacacggc ta 22 <210> 49 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 63) <400> 49 aaccacgatg aatataaatg ttcttaacaa agc 33 <210> 50 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 64) <400> 50 acaatccaca agtacaaatc caccata 27 <210> 51 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 65) <400> 51 agaagcagct gatgaatcct ataaatataa agc 33 <210> 52 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 66) <400> 52 actattaatg cagctaaaag cacattaact aa 32 <210> 53 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 67) <400> 53 tctgaggaaa aatcagaggc tacatac 27 <210> 54 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 68) <400> 54 tctactacta ctgaatcagc tgtaccaaat 30 <210> 55 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 69) <400> 55 cacaatctgc atctgccact tttaaac 27 <210> 56 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 70) <400> 56 attgtctgcc tgcaccgaaa cg 22 <210> 57 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 72) <400> 57 actgccacag cgtcctctg 19 <210> 58 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 73) <400> 58 agctctacta caacgtatgc caact 25 <210> 59 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 74) <400> 59 tcctacctca caatcgcctt ctg 23 <210> 60 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 77) <400> 60 gagtctcaaa ccccatctac ctatg 25 <210> 61 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 80) <400> 61 acttctgatg gtagtactat aactgaatat aacac 35 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 82) <400> 62 tgctactcca tcagttgcac aga 23 <210> 63 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(HPV type 84) <400> 63 ctgctaccaa caccgaatca gaata 25 <210> 64 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype(hydridization control) <400> 64 gaatatgatt tacagtttat ttttc 25 <110> BIOCORE. CO., LTD. <120> Oligonucleotide probe for analyzing Human Papillomavirus (HPV)          genotype and detecting method <130> P04-216 <160> 64 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 01) <400> 1 accatgcaag tactacatat tccaatgct 29 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 03) <400> 2 tctactgaaa cctcggctac atatgat 27 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 04) <400> 3 tcagatggtg ctaatgacaa ttatcagtat a 31 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 06) <400> 4 atccgtaact acatcttcca catacaca 28 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 09) <400> 5 tacagaggca gctcaaacag aagaa 25 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 10) <400> 6 ggcctcccct gccac 15 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 11) <400> 7 gcatctgtgt ctaaatctgc tacatacac 29 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 13) <400> 8 agccactaca tcatctcttt cagaca 26 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 15) <400> 9 tacctctgat ggtaatgcca taaagtaat 29 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 16) <400> 10 tcattatgtg ctgccatatc tacttcaga 29 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 17) <400> 11 gtctacagaa gctggggctg tta 23 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 18) <400> 12 tgcttctaca cagtctcctg tacct 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 22) <400> 13 gacggcacca cagttaatta tgatg 25 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 23) <400> 14 ccaatgacag cagtttagaa aagtatgatg 30 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 24) <400> 15 aaaatggcaa agtaacagat attaacgagt a 31 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 26) <400> 16 agtacattat ctgcagcatc tgcatcc 27 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 27) <400> 17 tgtgcagctg aggtgtctga taata 25 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 28) <400> 18 ctactgactc ttcagctacg tacga 25 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 29) <400> 19 cgttgaccac ttatgatgct accaa 25 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 30) <400> 20 atatctgcaa ccacacaaac gttatcc 27 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 31) <400> 21 gtgctgcaat tgcaaacagt gatac 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 32) <400> 22 gactgtgtgt gctactgtaa caact 25 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 33) <400> 23 tatgcacaca agtaactagt gacagtac 28 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 34) <400> 24 tccacaagta caactgcacc atatg 25 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 35) <400> 25 tgtgttctgc tgtgtcttct agtga 25 <210> 26 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 36) <400> 26 tagtatttca atatataaca ataatggggc actaa 35 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 38) <400> 27 aaaacggggg tgctcaagaa tat 23 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 39) <400> 28 tctacctcta tagagtcttc cataccttct 30 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 40) <400> 29 cccacaccaa ccccatataa taacagt 27 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 41) <400> 30 ttcctgaggg ggatgcttct 20 <210> 31 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 42) <400> 31 ctgcaacatc tggtgataca tatacagct 29 <210> 32 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 43) <400> 32 tctactgacc ctactgtgcc cag 23 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 44) <400> 33 tacacagtcc cctccgtcta catat 25 <210> 34 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 45) <400> 34 acacaaaatc ctgtgccaag tacatat 27 <210> 35 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 47) <400> 35 caggggacat aaaggatata caggattata a 31 <210> 36 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 48) <400> 36 aactgcatta actgaaaact acatagataa tgg 33 <210> 37 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 50) <400> 37 cctttggatc ctttaaatgt agcaagtt 28 <210> 38 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 51) <400> 38 tgctgcggtt tccccaacat tta 23 <210> 39 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 52) <400> 39 tgctgaggtt aaaaaggaaa gcacata 27 <210> 40 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 53) <400> 40 tttccgcaac cacacagtct atgtcta 27 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 54) <400> 41 tacagcatcc acgcaggata gctttaa 27 <210> 42 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 55) <400> 42 tgctacaact cagtctccat ctacaac 27 <210> 43 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 56) <400> 43 gtactgctac agaacagtta agtaaatatg at 32 <210> 44 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 57) <400> 44 tctctttgtg tgccactgta accac 25 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 58) <400> 45 gcactgaagt aactaaggaa ggta 24 <210> 46 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 59) <400> 46 tctactactg cttctattcc taatgtatac ac 32 <210> 47 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 61) <400> 47 aaccatttgt actgctacat ccccc 25 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 62) <400> 48 gctgcagcag aatacacggc ta 22 <210> 49 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 63) <400> 49 aaccacgatg aatataaatg ttcttaacaa agc 33 <210> 50 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 64) <400> 50 acaatccaca agtacaaatc caccata 27 <210> 51 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 65) <400> 51 agaagcagct gatgaatcct ataaatataa agc 33 <210> 52 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 66) <400> 52 actattaatg cagctaaaag cacattaact aa 32 <210> 53 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 67) <400> 53 tctgaggaaa aatcagaggc tacatac 27 <210> 54 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 68) <400> 54 tctactacta ctgaatcagc tgtaccaaat 30 <210> 55 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 69) <400> 55 cacaatctgc atctgccact tttaaac 27 <210> 56 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 70) <400> 56 attgtctgcc tgcaccgaaa cg 22 <210> 57 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 72) <400> 57 actgccacag cgtcctctg 19 <210> 58 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 73) <400> 58 agctctacta caacgtatgc caact 25 <210> 59 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 74) <400> 59 tcctacctca caatcgcctt ctg 23 <210> 60 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 77) <400> 60 gagtctcaaa ccccatctac ctatg 25 <210> 61 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 80) <400> 61 acttctgatg gtagtactat aactgaatat aacac 35 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 82) <400> 62 tgctactcca tcagttgcac aga 23 <210> 63 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (HPV type 84) <400> 63 ctgctaccaa caccgaatca gaata 25 <210> 64 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for analyzing HPV genotype (hydridization control) <400> 64 gaatatgatt tacagtttat ttttc 25  

Claims (9)

서열정보 1 에서 63까지 기재된 염기서열을 포함하는 올리고뉴크레오티드들로 이루어진 군으로부터 선택된 하나 이상의 HPV 유전자형 결정을 위한 올리고뉴크레오티드 프로브.Oligonucleotide probe for determining at least one HPV genotype selected from the group consisting of oligonucleotides comprising the nucleotide sequence described in SEQ ID NO: 1 to 63. 제1항에 따른 프로브들로 이루어진 군으로부터 선택된 1종 이상의 HPV 유전자형 결정을 위한 올리고뉴크레오티드 프로브를 포함하는 프로브 조성물.A probe composition comprising an oligonucleotide probe for determining at least one HPV genotype selected from the group consisting of the probes according to claim 1. 제2항에 있어서, 상기 프로브 조성물은 서열정보 64에 기재된 염기서열을 포함하는 올리고뉴크레오티드를 컨트롤로 더 포함하는 것을 특징으로 하는 프로브 조성물.The probe composition of claim 2, wherein the probe composition further comprises an oligonucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 64 as a control. 제1항 내지 제3항 중 어느 한 항에 있어서, 상기 프로브는 마이크로스피어 비드에 고정화되어 있는 것을 특징으로 하는 프로브 조성물.The probe composition according to any one of claims 1 to 3, wherein the probe is immobilized on the microsphere beads. 제4항에 있어서, 상기 고정화는 프로브의 아민기와 마이크로스피어 비드의 카르복실기를 반응시켜 수행하는 것을 특징으로 하는 올리고뉴크레오티드 프로브 조성물.The oligonucleotide probe composition according to claim 4, wherein the immobilization is performed by reacting the amine group of the probe with the carboxyl group of the microsphere beads. 제4항의 프로브가 고정된 마이크로스피어 비드를 포함하는 HPV 분석용 키트.HPV analysis kit comprising a microsphere bead fixed to the probe of claim 4. a) 혈액, 세포 및 조직 중 어느 하나로부터 HPV DNA를 추출하는 단계;a) extracting HPV DNA from any of blood, cells and tissues; b) 추출한 DNA를 이용하여 비대칭 PCR을 실시하는 단계;b) performing asymmetric PCR using the extracted DNA; c) 상기 비대칭 PCR 산물과 제2항 또는 제3항의 프로브 조성물을 결합시키는 단계; 및c) combining the asymmetric PCR product with the probe composition of claim 2 or 3; And d) 상기의 결합을 확인하는 단계를 포함하는 HPV 유전자형 분석방법.d) HPV genotyping method comprising the step of confirming the binding. 제7항에 있어서, 상기 b)의 비대칭 PCR은 바이오틴이 부착된 프라이머로 증폭시키며, 이 바이오틴은 상기 c)의 결합시키는 단계에서 첨가한 Streptavidin-Cy3 또는 Streptavidin-Phycoerythrin과 결합하고, 이를 서스펜션 어레이를 이용하여 확인하는 것을 특징으로 하는 HPV 유전자형 분석방법.8. The method of claim 7, wherein the asymmetric PCR of b) amplifies with a primer to which biotin is attached, which binds to Streptavidin-Cy3 or Streptavidin-Phycoerythrin added in the step of c), and binds the suspension array. HPV genotyping method, characterized in that confirmed using. 제7항에 있어서, 상기 b)의 비대칭 PCR은 로다민이 부착된 프라이머로 증폭시키며, 이 로다민을 서스펜션 어레이를 이용하여 확인하는 것을 특징으로 하는 HPV 유전자형 분석방법.8. The HPV genotyping method according to claim 7, wherein the asymmetric PCR of b) is amplified with a primer attached to rhodamine, and the rhodamine is identified using a suspension array.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100787108B1 (en) * 2006-11-17 2007-12-21 (주)예비티 A kit for diagnosis of leukemia
WO2011088573A1 (en) 2010-01-19 2011-07-28 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Set of probes for the detection and typing of 46 human papillomavirus mucosal types
KR101451780B1 (en) * 2007-03-06 2014-10-16 주식회사 파나진 PNA probes, kits and methods for detecting genotypes of Human Papillomavirus

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100787108B1 (en) * 2006-11-17 2007-12-21 (주)예비티 A kit for diagnosis of leukemia
KR101451780B1 (en) * 2007-03-06 2014-10-16 주식회사 파나진 PNA probes, kits and methods for detecting genotypes of Human Papillomavirus
WO2011088573A1 (en) 2010-01-19 2011-07-28 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Set of probes for the detection and typing of 46 human papillomavirus mucosal types
EP2526221A1 (en) * 2010-01-19 2012-11-28 Her Majesty The Queen In Right of Canada as represented by The Minister of Health Set of probes for the detection and typing of 46 human papillomavirus mucosal types
EP2526221A4 (en) * 2010-01-19 2013-11-06 Ca Minister Health & Welfare Set of probes for the detection and typing of 46 human papillomavirus mucosal types

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