WO2009091148A2 - Amorce, sonde, puce à adn contenant ces dernières, méthode de détection du papillomavirus humain, et trousse de détection correspondante - Google Patents

Amorce, sonde, puce à adn contenant ces dernières, méthode de détection du papillomavirus humain, et trousse de détection correspondante Download PDF

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WO2009091148A2
WO2009091148A2 PCT/KR2008/007836 KR2008007836W WO2009091148A2 WO 2009091148 A2 WO2009091148 A2 WO 2009091148A2 KR 2008007836 W KR2008007836 W KR 2008007836W WO 2009091148 A2 WO2009091148 A2 WO 2009091148A2
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hpv
seq
primer
dna
testing
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WO2009091148A3 (fr
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Woon-Won Jung
Hyun-Sook Kim
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Priority claimed from KR1020080137163A external-priority patent/KR20090073987A/ko
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Priority to US12/811,280 priority Critical patent/US20100285485A1/en
Publication of WO2009091148A2 publication Critical patent/WO2009091148A2/fr
Publication of WO2009091148A3 publication Critical patent/WO2009091148A3/fr
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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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma

Definitions

  • the present invention relates to a primer, a probe and DNA chip containing them for genotyping of Human Papillomavirus (HPV). Specifically, the present invention relates to a PCR primer for amplification of HPV genes, a probe which specifically binds with the amplified genes, and a testing kit and method using them for diagnosis and/or test of HPV genes.
  • HPV Human Papillomavirus
  • HPV has been found in papilloma of cottontail rabbit, and known as one of oncoviruses, a DNA virus on a double-helix with a size of about 45-55nm and 8kb.
  • HPV belongs to Papovavirus family, and it is generally found in various kinds of animals other than rabbit, causing infections in the form of warts or condyloma in human.
  • HPV has been known to cause cervical cancer in women, and be closely related to other malignant tumors such as skin cancer, laryngeal cancer or the like. HPV infection isvery commonly occurred in women, approximately 75% of women get infected as they become sexually active. Cervical cancer can be developed 10-20 years after a certain HPV infection. 20-25% of infections may progress to a precancerous stage.
  • HPV-16 types prevalently infecting the genital area are following 45 types: HFV-16, -31, -33, -35, -52, -58, -67, -40, -43, -7, -32, -42, -6, -11, -74, -44, -55, -13, -61, -72, -62, -2, -27, -57, -3, -28, -29, -10, -54, -18, -39, -45, -59, -68, -70, -26, -69, -51, -30, -53, -56, -66, -34, -64 and -73.
  • HPV types being closely associated with cervical cancer are'- as a high-risk group of 22 types, HPV-16, -18, -26, -30, -31, -33, -35, -45, -51, -52, -53, -56, -57, -58, -59, -61, -67, -68, -70, -73 and -74 and as a low-risk group, HPV ⁇ 2a, -3, -6, -10, -11, -32, -34, -40, -43, -44, -54, -55, -66 and -69.
  • 90% HPV types belonging to the high risk group are detected in epithelial tissues of cervical tumors.
  • a DNA chip method which has been recently developed, is a method combined with DNA amplification, being capable of testing the HPV genotypes from a large amount of samples in convenient way, without problems of production economy or process time. Such advantages make this method very useful in early diagnosis, prevention and prognosis of cervical cancer.
  • US FDA has recommended both PAP smear testing and HPV infection test at the same time for testing cervix cancer.
  • vaccines for HPV-16 and HPV-18 have been developed and now available.
  • a device (kit) or a method for diagnosing HPV genotype is needed.
  • a HPV diagnosis device (kit) and a test method for determining HPV genotypes are further required, at present.
  • the present invention is to provide a primer used in PCR for HPV amplification. ⁇ 13> Further, the present invention is to provide a probe for testing HPV genotypes with enhanced specificity. ⁇ 14> Still further, the present invention is to provide a DNA chip and kit for testing several tens of HPV genotypes in rapid way. ⁇ i5> Further, the present invention is to provide a method for testing several tens of HPV genotypes at once through a single test.
  • the present invention it is possible to identify 36 HPV types present in cervical cells with enhanced specificity and further to detect multiple infections by different HPV types.
  • the present invention can test HPV genotypes in a reduced time, taking about 6 hours or less from DNA extractionfrom cervical cell specimen to identification of the HPV genotype by using a DNA chip.
  • Fig. l is a view showing a DNA chip for a HPV genotype test according to one embodiment of the present invention.
  • FIG. 2 is a view showing the gene amplification result by using a primer according to the present invention, JKl set.
  • HPV genotypes are as follows-' from left to right, 6,11,16,18,31,32,33,34,35,39,40,42,43,44,45,51,52,53,54,56,58,59,61,62,66,67, 68,69,70,71,72,81,82,83,84,90, positive control, and negative control.
  • Fig. 3 is a view showing the gene amplification result by using a primer according to the present invention, JK2 set.HPV genotypes are as follows: from left to right, 6,11,16,18,31,32,33,34,35,39,40,42,43,44,45,51,52,53,54,56,58,59,61,62,66,67, 68,69,70,71,72,81,82,83,84,90, positive control, and negative control.
  • Fig. 4 is a view showing the gene amplification result by using a control primer set.HPV genotypes are as follows: from left to right,6,11, 16, 18,31,32,33,34,35,39,40,42,43,44,45,51,52,53,54,56,58,59,61,62, 66,67,68,69,70,71,72,81,82,83,84,90, positive control, and negative control.
  • Figs. 5 and 6 are the results of HPV genotype tests with a DNA chip according to the present invention, appeared by a chip scanner.
  • Fig. 7 is a DNA extraction reagent kit according to one embodiment of the present invention.
  • Fig. 8 is a reagent kit containing a PCR primer according to one embodiment of the present invention.
  • Fig. 9 is a DNA chip kit for testing HPV genotype according to one embodiment of the present invention.
  • Figs. 10 to 22 are views showing the base sequencing results of PCR products according to each HPV genotype, obtained from Example 1-1.
  • the present invention provides a primer for HPV (Human Papilloma Virus) amplification, which contains at least one base sequence selected from the group consisting of SEQ. ID.NO:1 to SEQ.ID.N0:6.
  • HPV Human Papilloma Virus
  • the base sequence may have a fluorescent dye attached to 5' end thereof.
  • the fluorescent dye can be selected from the group consisting of Cy3, Cy5, a biotin binding material, Alexa 647, Alexa 555, 5-(2- aminoethyl)amino-l-naphthalene sulfonic acid (EDANS), tetramethyl rhodamine (TMR), tetramethyl rhodamine isocyanate (TMRITC), x-rhodamine, fluorescein isocyanate(FITC)and Texas red.
  • the present invention provides aprobe which forms complementary binding with HPV, containing at least one oligonucleotide selected from the group consisting of oligonucleotide having base sequences represented by SEQ.ID.N0:9 to SEQ.ID.N0:44, and oligonucleotidehaving base sequences complementary to said sequences.
  • the oligonucleotide may correspond to the following HPV types: HPV- 6,11,16,18,31,32,33,34,35,39,40,42,43,44,45,51,52,53,54,56,58,59,61,62,66,67, 68,69,70,71,72,81,82,83,84 and 90.
  • the present invention provides a DNA chip for testing HPV genotypes, containing the above-described probe.
  • the present invention provides a kit for detecting or testing HPV genotype, containing:
  • ⁇ 36> a reagent for DNA extraction
  • the primer for PCR can contain at least one base sequence selected from the group consisting of SEQ. ID.NO: 1 to SEQ.ID.N0:6.
  • the primer for PCR can be a set selected from: a set consisting of
  • SEQ.ID.NO:l and SEQ.ID.N0:2 (referred to 'JKl set'); a set consisting of
  • SEQ.ID.N0:3 and SEQ.ID.N0:4 (referred to 'JK2 set); and a set consisting of
  • the present invention provides a method for testing HPV genotype, comprising the steps of•' ⁇ 42> extracting DNA from cells taken from the cervix; ⁇ 43> amplifying the DNA by PCR involving a primer for HPV amplification; ⁇ 44> hybridization of the amplified DNA to a DNA chip containing a HPV probe which containsat least one oligonucleotide selected from the group consisting of oligonucleotide having base sequences represented bySEQ.ID.N0:9 to
  • the method can further include, in the PCR amplification, a beta-globin primer set represented by SEQ.ID.N0:7 and SEQ.ID.N0:8, as a control.
  • an amine group can be attached to the 5' end of the base sequences represented in SEQ.ID.N0:9 to
  • the HPV genotype can be selected from the group consisting of HPV-
  • the present invention is comprised of the following steps: amplification of DNA by PCR involving a primer for HPV amplification; hybridization of the amplified DNA to a DNA chip containing a HPV probe; and determining HPV genotype, by assaying the product specifically hybridized with the probe.
  • the present invention provides a novel primer, a probe, a DNA chip and a test kit.
  • the present invention is further illustrated in detail.
  • the primer contains at least one base sequenceselected from the group consisting of SEQ. ID.N(Kl to SEQ.ID.N0:6.
  • the primer is 5' primer or 3' primer specific to HPV genes, as represented in Table 1 below.
  • the base sequence can be easily detected, owing to a fluorescent dye being attached to 5' end thereof.
  • the fluorescent dye may be selected from the group consisting of Cy3, Cy5, a biotin binding material, Alexa 647, Alexa 555, 5-(2-aminoethyl)amino-l-naphthalene sulfonic acid (EDANS), tetramethyl rhodamine (TMR), tetramethyl rhodamine isocyanate (TMRITC), ⁇ -rhodamine, fluorescein isocyanate(FITC) and Texas red, without being limited to these.
  • the primer can be one or moreselected from the group consisting of SEQ. ID.NO: 1 to SEQ.ID.N0:6.
  • the primer is used as a primer set selected from: JKl set consisting of SEQ. ID.NO:1 and SEQ.ID.N0:2; JK2 set consistingof SEQ.ID.N0:3 and SEQ.ID.N0:4; and JK3 set consisting of SEQ.ID.N0:5 and SEQ.ID.NO'- ⁇ .
  • the present invention may further include a beta-globin primer set represented by SEQ.ID.N0:7 and SEQ.ID.N0:8, as a control gene.
  • a fluorescent dye can be attached to the end of the 5' primer of beta-globin.
  • the probe of the present invention contains at least one oligonucleotide selected from the group consisting of oligonucleotide having base sequences represented as SEQ.ID.N0:9 to SEQ. ID.N0:44, and oligonucleotide having base sequences complementary to said sequences, as shown in Table 2. As seen from the following Table 2, the probes of the present invention can specifically bind with 36 types of HPV genotypes in total.
  • an amine group can be attached so as to be usedin DNA chip fabrication.
  • Such technique is well known in this field of art to which the present invention pertains, thereby eliminating detaileddescription thereof in this specification.
  • the primer as described in the above, amplifies HPV gene (DNA), and then thus amplified gene isspecifically hybridized with the probe, resulting a product by which HPV genotype can be determined.
  • a DNA chip for a genotype test may be fabricated by using said probe (Fig. 1). It will be further illustrated through the following examples.
  • the present invention provides a kit for detecting or testing HPV genotype, containing: a reagent for DNA extraction; a PCR primer for amplification of the extracted DNA; and a DNA chip for testing HPV genotype, containing the above-described probe.
  • the presentinvention provides a method for testing HPV genotype, including the steps of: extracting DNA from cells taken from the cervix; amplifying the DNA by PCR involving a primer for HPV amplification; hybridization of the amplified DNA toa DNA chip containing the above- described HPV probe.' and determining the HPV genotype through the product specifically hybridized with the probe.
  • a method for detecting HPV genes and/or testing HPV genotypes may include the following procedure, however it is not just limited to this example.
  • cervix cells taken from a vaginal swab to collect cells and lysing cells with a solutionfor cell lysis so as to obtain DNA
  • ⁇ 76> 2 amplifying a portion of the resulted DNA by using the 5' primer and 3' primer of the present invention, which are the primers specific to HPV genes, for HPV gene amplification, and
  • a method for testing HPV genotypes by using a DNA chip according to one embodiment of the present invention may include the following procedure, however it is not limited to this example.
  • cervix cells taken from a vaginal swab to collect cells and lysing cells with a solution for cell lysis so as to obtain DNA
  • ⁇ 8i> 2 amplifying a portion of the resulted DNA by using the 5' primer and 3' primer of the present invention, which are primers specific to HPV genes, for HPV gene amplification, ⁇ 82> 3) fabricating a DNA chip for testing genotypes of the amplified HPV genes, by synthesizing oligonucleotides of 36 types of HPV probes which specifically bind to the 36 types of HPV genes, to the size as much as about
  • kits for testing HPV genotypes may include a reagent, a device and equipment as follows, however it is not just limited to this example.
  • washing solution I (2X SSC, 0.2% SDS)
  • washing solution II (0.1X SSC)
  • a DNA chip for a HPV genotype test By fabricating a DNA chip for a HPV genotype test from the above- described reagents, devices and equipment, it is possible to determine HPV genotype of samples taken from the cervix. Further, based on such DNA chip, a kit for HPV gene detection or a HPV genotype test can be formed by the following construction. However, the following description is only one embodimentof the present invention, and the present invention is by no means limited by such examples.
  • ⁇ ii5> (2) a 30ml volume tube containing a 1OX washing solution (20OmM Tris- HCl, 5OmM KCl, 5OmM NaH 2 PO 4 , 5OmM Na 2 HPO 4 , pH 7.4)
  • kits 1) and 2) may be used, and for a HPV genotype test, kits 1), 2) and 3) may be used. According to the present invention, it takes about 6 hours from extraction of DNA from the cervix cells and HPV gene amplification by PCR to HPV genotype assay by using the HPV DNA chip. With one HPV DNA chip, 8 samples can be tested simultaneously.
  • ⁇ i30> A cytobrush sample preserved in 5 ill of a washing solution (PBS) was severely vortexed to separate the cervix cells therefrom. The cells were centrifugedat 1,30Og for 10 minutes, and the supernatant was removed therefrom. The cells were re-suspended in a washing buffer (20OmM NaCl, 5OmM KCl, 5OmM NaH2P04, 5OmM Na2HP04, pH 7.4), transferred to a 1.5m£-volume microtube, and centrifuged at 1,30Og for 5 minutes.
  • a washing buffer (20OmM NaCl, 5OmM KCl, 5OmM NaH2P04, 5OmM Na2HP04, pH 7.4
  • a chelex buffer (7% chelex, 2OmM Tris HCl, 100 /zg/ml proteinase K, 1OmM CaCb, pH 8.0)were added thereto for re-suspension.
  • the suspension was heated in a water bath at 55°C for 3 hours, and then further heated at 95 ° C for 20 minutes so as to eliminate the activity of proteinase K, finally obtaining DNA from the cervix cell.
  • ⁇ i33> For amplifyingthe DNA obtained from the above 1-1, JKl and JK2 primer sets as shown in Table 1 were used independently. As a control, a ⁇ -globin primer represented in SEQ. ID.NO: 7 and SEQ. ID.NO: 8 was used, wherein Cy5 fluorescent dye was attached to the 5' end of the 3' primer (5'-Cy5 ⁇ CAA GAC AGG TTT AAG GAG ACC A-375'- GGT TGG CCA ATC TAC TCC CAG G-3 1 ).
  • PCR thereof was conducted at 95°C for 60 seconds, 53"C for 60 seconds, and 72°C for 60 seconds, and this cycle was repeated 40 times. Finally, it was treated at 72°C for 30 minutes (9700, Applied Biosystems).
  • ⁇ 135> The amplification of ⁇ -globin genes that was used as a control, was conducted under the conditions as same as above, except the different primer. After completing the PCR amplification, S.O ⁇ Jt of the amplified gene products was applied to a 2.5% agarose gel for electrophoresis, and the image analysis was carried out (imageanalyzer, alphascan). The time elapsed was one and half hour or so.
  • ⁇ i4i> The glass slide attached with the probes was kept in a dark place for 2 days; then baked at 80°C for 4 hours or more; and subjected to shaking incubation in a blocking buffer (5XSSC, 1%BSA, 0.1%SDS) at 42 0 C for 45 minutes. Isopropanol was added thereto and the mixture was subjected to shaking incubation for 1 minute. After removing the supernatant (reacted solution), shaking incubation in distilled water was carried out for 1 minute and it was repeated 5 times. The resulted slide was dried and kept in a closed container before use.
  • a blocking buffer 5XSSC, 1%BSA, 0.1%SDS
  • PCR product 8fd and 2X hybridization buffer 32 ⁇ Jt were added into a 1.5ml tube, wherein the PCR product was denatured at 95°C for 5 minutes, and the hybridization buffer 32/ ⁇ S was allowed for reaction at 48°C for 10 minutes just right before use.
  • the hybridization buffer and the PCR product were well mixed together, and carefully added to each opening of a well on the DNA chip, (1 sample per well). The opening of the well was covered with Cellotape for preventing the hybridizationmixture from being dried out, allowing hybridization to occur at 48°C for 1 hour.
  • a kit for a HPV genotype test according to the present was composed of 3 parts: a first part including a reagent for DNA extraction from cells taken from the cervix (Fig. 7) a second part including a reagent containing a primer for PCR of HPV genes (Fig. 8); and a third part including a DNA chip for a HPV genotypetest which could determine genotype of the PCR products of HPV genes.
  • kits were formed, wherein the kit was comprised of ⁇ a 30ml volume tube of 1OX washing solution, which contained 20ml PBS solution, and (2) a 30ml volume tube containing a proteinase K (Sigma, US) at the concentration of 100 ⁇ g of the DNA extraction solution/ml, 7% chelex, both of them being kept at room temperature.
  • the kit can process 80 cervical cell samples, and the method for operation and use the same was as described in the examples 1 and 2.
  • reagents for PCR of HPV genes were provided as a kit .
  • the kit was comprised of: (D 20pmol// ⁇ of primer selected from SEQ. ID.NO: 1 to SEQ.ID.N0:6, these are PCR primers for HPV genes, ⁇ a 1OX PCR buffer solution, (3) a mixed solution of 2.5mM dNTP (dATP, dGTP, dCTP, dUTP), ⁇ an 1.5ml volume tube containing lOOul of a beta-globin primer at the concentration of 3pmol/ ⁇ t, ( B a premix of primers for a HPV test, wherein the premix contained uracil-N-glycosylase (IU/ ⁇ l) 20ul and template DNA 5ul , and as a control, ⁇ a 1.5ml tube containing Taq.
  • DNA polymerase 250 units Solgent, South Korea
  • a 1.5ml tube containing a 2X hybridization solution (8) a 1.5ml tube containing distilled water ImI.
  • the kit containing all the above-described reagents was kept in a freezer at -20°C, and the method of use was the same as described in example 2.
  • kits with a DNA chip of HPV genes ⁇ 165> With one HPV DNA chip fabricated as in example 2, it was possible to test 8 samples.
  • a kit was composed of 10 pieces of such DNA chips.
  • a hybridization chamber was attached to the kit, for ready use, and the method of use was the same as described in example 2.
  • the DNA chip kit for a HPV genotype test was composed of 10 DNA chips, being capable of processing 80 cervical cell samples (Fig. 9).
  • each HPV genotype was determined.
  • a base sequence analysis was carried out. To 5 ⁇ £ of the PCR product of each sample corresponding to the HPV genotype of the HPV DNA chip (PCR products of Example 1-1), 8 ⁇ i of a reagent (Bigdye), I ⁇ i of 5 primer of each genes and the like were added, making a mixture to have the total volume of 2Q ⁇ i. The mixture was denatured at 95°C for 2 minutes.

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Abstract

La présente invention concerne une amorce, une sonde, une puce à ADN et une trousse de test pour diagnostiquer et génotyper un virus du papillome humain (HPV) ainsi qu'une méthode de test du génotype HPV. Selon la présente invention, il est possible de cribler les génotypes HPV avec une sensibilité et une spécificité élevées et de diagnostiquer l'infection par plusieurs types HPV, ce qui n'était pas possible dans les autres méthodes de test HPV classiques.
PCT/KR2008/007836 2007-12-31 2008-12-31 Amorce, sonde, puce à adn contenant ces dernières, méthode de détection du papillomavirus humain, et trousse de détection correspondante Ceased WO2009091148A2 (fr)

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Application Number Priority Date Filing Date Title
US12/811,280 US20100285485A1 (en) 2007-12-31 2008-12-31 Primer, probe, dna chip containing the same and method for detecting human papillomavirus and detection kit thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2007-0141257 2007-12-31
KR20070141257 2007-12-31
KR10-2008-0137163 2008-12-30
KR1020080137163A KR20090073987A (ko) 2007-12-31 2008-12-30 인간유두종바이러스 유전자형 검사를 위한 프라이머, 프로브 및 이를 포함하는 ｄｎａ칩, 이의 검사 방법 및 검사 키트

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011080068A1 (fr) * 2009-12-21 2011-07-07 Pea Genetics Ab Procédé d'hybridation de polynucléotides
WO2011088573A1 (fr) 2010-01-19 2011-07-28 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Jeu de sondes pour la détection et le typage de 46 papillomavirus humains de types muqueux
CN102367487A (zh) * 2011-09-09 2012-03-07 中国医学科学院病原生物学研究所 一种高精确度检测人类乳头状瘤病毒基因型的方法
CN103276107A (zh) * 2013-05-13 2013-09-04 中国医学科学院病原生物学研究所 一种高灵敏度检测和鉴定人多瘤病毒的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7670774B2 (en) * 2004-10-04 2010-03-02 Goodgene Inc. Probe of human papillomavirus and DNA chip comprising the same

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011080068A1 (fr) * 2009-12-21 2011-07-07 Pea Genetics Ab Procédé d'hybridation de polynucléotides
WO2011088573A1 (fr) 2010-01-19 2011-07-28 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Jeu de sondes pour la détection et le typage de 46 papillomavirus humains de types muqueux
EP2526221A4 (fr) * 2010-01-19 2013-11-06 Ca Minister Health & Welfare Jeu de sondes pour la détection et le typage de 46 papillomavirus humains de types muqueux
CN102367487A (zh) * 2011-09-09 2012-03-07 中国医学科学院病原生物学研究所 一种高精确度检测人类乳头状瘤病毒基因型的方法
CN103276107A (zh) * 2013-05-13 2013-09-04 中国医学科学院病原生物学研究所 一种高灵敏度检测和鉴定人多瘤病毒的方法
CN103276107B (zh) * 2013-05-13 2014-11-19 中国医学科学院病原生物学研究所 一种高灵敏度检测和鉴定人多瘤病毒的方法

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