WO2009035177A1 - Génotypage des virus du papillome humain (vph) par amplification multiplex et différenciation selon la taille - Google Patents

Génotypage des virus du papillome humain (vph) par amplification multiplex et différenciation selon la taille Download PDF

Info

Publication number
WO2009035177A1
WO2009035177A1 PCT/KR2007/004452 KR2007004452W WO2009035177A1 WO 2009035177 A1 WO2009035177 A1 WO 2009035177A1 KR 2007004452 W KR2007004452 W KR 2007004452W WO 2009035177 A1 WO2009035177 A1 WO 2009035177A1
Authority
WO
WIPO (PCT)
Prior art keywords
hpv
nitropyrrole
priming
priming portion
types
Prior art date
Application number
PCT/KR2007/004452
Other languages
English (en)
Inventor
Jong Yoon Chun
Original Assignee
Seegene, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Seegene, Inc. filed Critical Seegene, Inc.
Priority to PCT/KR2007/004452 priority Critical patent/WO2009035177A1/fr
Publication of WO2009035177A1 publication Critical patent/WO2009035177A1/fr

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method for identifying at least two types of human papilloma viruses (HPVs) by a multiplex amplification of a nucleic acid molecule of HPVs in a biological sample and a size differentiation of amplicons and a kit for conducting such method.
  • HPVs human papilloma viruses
  • HPV Human papillomavirus
  • HPV-positive women The ability to determine the type-specific profile of HPV-positive women is essential in evaluating the efficacy of HPV vaccine implementation. Therefore, it is very important to know what type of HPV is infected in patients to prevent cancer development and transmission of the disease.
  • Pap smears require a sample of cells from the uterine cervix of each woman screened. Skilled technicians then examine the specimen for cellular changes (dysplasia) known to precede the development of cervical cancer.
  • Such screening methods can be expensive, prone to error, and logistically difficult to set up.
  • the direct detection of HPV in cervical specimens therefore has been required for an alternative or complement to population-based cytological screening.
  • Amplification techniques can be further divided into three separate categories: Firstly, target amplification, in which the assay amplifies the target nucleic acids ⁇ e.g. polymerase chain reaction; PCR); Secondly, signal amplification, in which the signal generated from each probe is increased by a compound-probe or branched-probe technology; and thirdly, probe amplification, in which the probe molecule itself is amplified ⁇ e.g. ligase chain reaction). Because of a time-consuming and labor-intensive process of non-amplification techniques, target and signal amplification techniques have been more often applied to the molecular detection of HPV than non-amplification techniques.
  • target amplification in which the assay amplifies the target nucleic acids ⁇ e.g. polymerase chain reaction
  • signal amplification in which the signal generated from each probe is increased by a compound-probe or branched-probe technology
  • probe amplification in which the probe molecule itself is amplified ⁇ e.g. ligase chain reaction
  • the present inventors have made intensive researches to develop a novel approach for genotyping HPVs in more convenient and accurate manner. As a result, we have discovered that the genotyping of HPVs could be successfully performed according to multiplex amplifications using at least two primer pairs and a size differentiation of amplicons without false results.
  • HPVs human papilloma viruses
  • HPVs human papilloma viruses
  • Figs. Ia-Ie represents electropherograms of GeneScan analysis results showing 19-plex HPV PCR amplification products for 18 different HPV types run under denaturing conditions.
  • the 19-plex HPV PCR was performed in the presence of each 18 individual HPV cloned plasmids.
  • IC indicates internal control ⁇ Arabidopsis thaliana CESA3 gene).
  • X-axis length of DNA fragments, bp (base pair).
  • Fig. If shows an electropherogram of GeneScan analysis results for combined 19 single PCR reactants.
  • Figs. 2a and 2b represent analytic sensitivity of the HPV 19-plex PCR system for detecting 18 different HPV genotypes on dilution series (10-fold serial dilution of 10 4 copies down to 10 copies) of HPV-31 and HPV-42 control plasmids.
  • Figs. 3a-3d represent representative electropherograms from the HPV 19-plex PCR on 15 positive clinical samples. 30 clinical samples were evaluated using the HPV 19-plex PCR system for identifying the genotype of HPV.
  • a method for identifying at least two types of human papilloma viruses (HPVs) by a multiplex amplification of a nucleic acid molecule of HPVs in a biological sample and a size differentiation of amplicons which comprises the steps of:
  • the present inventors have made intensive researches to develop a novel approach for genotyping HPVs in more convenient and accurate manner. As a result, we have discovered that the genotyping of HPVs could be successfully performed according to multiplex amplifications using at least two primer pairs and a size differentiation of amplicons without false results. According to the present invention, the determination of genotypes of HPVs is easily made based on sizes of finally amplified products (amplicons).
  • the present method belongs to "target amplification" method.
  • the present HPV detection/genotyping method is designed to amplify only specific targets and more importantly, can be automated in detection using a suitable analysis tool ⁇ e.g., GeneScan size analysis). Therefore, the present invention is appropriate for the high throughput analysis of HPV detection and genotyping.
  • the present method is directed to genotyping of HPVs by a multiplex amplification and a size differentiation of amplicons. To our best knowledge, the present method is the first accomplishment to genotype HPVs based on multiplex amplification and amplicon size differentiation, preferably using a genescan process.
  • amplicon refers to the product of a gene amplification reaction wherein primers are employed in the presence of a template and one or more nucleotides and a template-dependent polymerizing agent to yield a nucleic acid.
  • An amplicon product of a primer extension reaction is typically double- stranded.
  • size differentiation of amplicons is intended to mean the difference in sizes of finally amplified products by the HPV type-specific primers designed to produce defined and different amplicon sizes.
  • the size differentiation of amplicons permits to easily identify the type of HPVs, preferably, by automated size analysis tools.
  • DNA molecules are obtained from biological samples.
  • the biological samples include, but not limited to, biological fluids ⁇ e.g., blood, serum, plasma and urine) and cervix scrapings (tissue and cell samples).
  • biological fluids e.g., blood, serum, plasma and urine
  • cervix scrapings tissue and cell samples.
  • the preparation of DNA molecules from biological samples may be carried out by conventional techniques including, but not limited to, extraction with a phenol/chloroform mixture and precipitation, and isolation using spin columns (Peter B. Kaufman, et al., Handbook of Molecular and Cellular Methods in Biology and Medicine, CRC Press, Inc (1995) and Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)).
  • the preparation of DNA molecules from biological samples may be conducted in three sequential steps: (i) cells (or viruses) are lysed to release their content which includes protein, lipids, RNA and DNA by use of a detergent-containing lysis solution ⁇ e.g., containing sodium dodecyl sulfate or N-Lauroyl sarcosine) or a chaotropic-containing lysis solution; (ii) ribonucleases (RNases) are optionally added to remove RNA; and (iii) non-DNA contaminants such as protein are removed to yield pure DNA by organic solvents or column chromatography.
  • a detergent-containing lysis solution e.g., containing sodium dodecyl sulfate or N-Lauroyl sarcosine
  • RNases ribonucleases
  • non-DNA contaminants such as protein are removed to yield pure DNA by organic solvents or column chromatography.
  • the isolated DNA molecules are then amplified using at least two HPV type-specific primer pairs in which the HPV type-specific primer pairs generate amplicons with defined and different sizes from each other.
  • the HPV type-specific primer has a dual priming oligonucleotide (DPO) structure represented by the following general formula I: 5'-X p -Y q -Z r -3' (I) wherein, X p represents a 5'-first priming portion having a hybridizing nucleotide sequence substantially complementary to a target sequence in the nucleic acid molecule of HPV, Y q represents a separation portion comprising at least three universal bases, Z 1 - represents a 3'-second priming portion having a hybridizing nucleotide sequence substantially complementary to a target sequence in the nucleic acid molecule of HPV, p, q and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotides or ribonucleotides; the T m of the 5'-first priming portion is higher than that of the 3'-second priming portion and the separation portion has the lowest T m in
  • the HPV type-specific primers have a unique structure or formula called as a dual specificity oligonucleotide structure.
  • This dual specificity oligonucleotide (DSO) structure was first proposed by the present inventor (see WO 2006/095981) and then its nomenclature was changed to a dual priming oligonucleotide (DPO) structure.
  • the DPO embodies a novel concept in which its hybridization or annealing is dually determined by the 5'-high T m specif icity portion (or the 5'-first priming portion) and the 3'-low T m specificity portion (or the 3'-second priming portion) separated by the separation portion, exhibiting dramatically enhanced specificity (see WO 2006/095981).
  • the DPO has eventually two primer segments with distinct annealing properties: the 5'-first priming portion that initiates stable priming, and the 3'-second priming portion that determines target-specific extension.
  • the separation portion comprising at least three universal bases delineates the boundary between the 5'-first priming portion and the 3'-second priming portion, resulting in separation of the 5'-first priming portion from the 3'-second priming portion in view of annealing events.
  • Such separation permits the annealing specificity and priming of the primers to be determined dually by the 5'-first priming portion and the 3'-second priming portion, finally dramatically increasing the overall annealing specificity of the HPV type-specific primers.
  • the universal base in the separation portion is selected from the group consisting of deoxyinosine, inosine, 7-deaza-2'- deoxyinosine, 2-aza-2'-deoxyinosine, 2'-OMe inosine, 2'-F inosine, deoxy 3- nitropyrrole, 3-nitropyrrole, 2'-OMe 3-nitropyrrole, 2'-F 3-nitropyrrole, l-(2'-deoxy- beta-D-ribofuranosyl)-3-nitropyrrole, deoxy 5-nitroindole, 5-nitroindole, 2'-0Me 5- nitroindole, 2'-F 5-nitroindole, deoxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy 4-aminobenzimidazole, 4-aminobenzimidazole, deoxy nebularine, 2'-F nebularine, 2'-F 4-nitrobenzimidazole, PNA
  • the universal base or non-discriminatory base analog is deoxyinosine, l-(2'-deoxy-beta-D- ribofuranosyl)-3-nitropyrrole or 5-nitroindole, most preferably, deoxyinosine.
  • the separation portion comprises contiguous nucleotides having at least three, more preferably at least four, most preferably at least five universal bases, preferably, deoxyinosine.
  • the 5'-first priming portion is longer than the 3'-second priming portion.
  • the 5'-first priming portion is preferably 15-40 nucleotides, more preferably 15-25 nucleotides in length.
  • the 3'-second priming portion is 3-15 nucleotides, more preferably 6-13 nucleotides in length.
  • the separation portion is preferably 3-10 nucleotides, more preferably 4-8 nucleotides, most preferably 5-7 nucleotides in length.
  • the T m of the 5'-first priming portion ranges from 40 0 C to 80 0 C, more preferably 45°C to 65 0 C.
  • the T m of the 3'-second priming portion ranges preferably from 10 0 C to 40 0 C. It is preferable that the T m of the separation portion ranges from 3°C to 15°C.
  • the HPV-specific primers used in this invention have a sequence specific to a type of HPVs and have the structure of the dual priming oligonucleotide (DPO).
  • the specific nucleotide sequence to a type of HPVs is located in the 5'-first priming portion and/or the 3'-second priming portion.
  • the separation portion may accommodate common sequences found in various types of HPVs.
  • the HPV type-specific primers are designed to amplify nucleotide sequences of human papilloma virus (HPV) types 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59 or 68.
  • HPV human papilloma virus
  • the main reference sequences for preparing HPV type-specific primers are unique sequences selected by aligning publicly-known nucleotide sequences of various types of HPVs, Among the selected sequences, a sequence suitable to design primers or probes having the DPO structure is then determined.
  • the target nucleotide sequence annealed with the HPV type-specific primers includes a nucleotide sequence of a gene or a sequence in a HPV genomic DNA.
  • the target nucleotide sequence may be selected on the basis of publicly-known nucleotide sequences.
  • the target nucleotide sequence may be selected with referring to sequences described in the following data bases: for HPV types 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51,
  • the HPV type-specific primers used in this invention are designed to hybridize with or amplify the E5 gene for HPV type 6, the L2 gene for HPV type 11, the E2 gene for HPV type 16, the E4-E5 gene for HPV type 18, the E2 gene for HPV type 31, the E4 gene for HPV type 33, the E2-E4 gene for HPV type 39, the L2 gene for HPV type 43, the E2 gene for HPV type 44, the E4-E5 gene for HPV type 45, the E2 gene for HPV type 52, the E2-E5 gene for HPV type 58, the E2-E4 gene for HPV type 59 or the L2 gene for HPV type 68.
  • the HPV type-specific primer pair comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 2, 3 and 4, 5 and
  • the HPV type-specific primers comprises not only the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-36 but also a complementary sequence to and a substantially identical nucleotide sequence to that.
  • substantially identical nucleotide sequence refers to a nucleotide sequence having some deletions, additions and/or substitutions in the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-36. Such nucleotide changes are permissible, so long as the HPV type-specific primers can be specifically hybridized with a target sequence. It will be appreciated under the doctrine of equivalency that these substantially identical nucleotide sequences fall within the scope of claims.
  • HPV type-specific primers adopting the DPO structure completely eliminate false-positive results and backgrounds associated with conventional technologies using probes or primers for detecting HPVs.
  • the HPV type-specific primer is labeled with fluorescent molecules such as fluorescein, tetramethyl rhodamine (TAMRA), 5'- or ⁇ '-carboxyfluorescein (FAM), 6'- or S'-carboxy- ⁇ ' ⁇ ' ⁇ ' ⁇ '-hexachlorofluorescein (HEX), 5'-tetrachloro-fluorescein (TET), 6'-carboxy-4',5'-dichloro-2',7'- dimethoxyfluorescein (JOE), Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7- diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1- pyrenebuty
  • the fluorescent dye is attached to the 5'-end of the HPV type-specific primers.
  • Either forward or reverse primer of the HPV type-specific primer pair may be labeled with fluorescent dyes.
  • all primers of the HPV type- specific primer pair may be labeled with fluorescent dyes.
  • primer refers to an oligonucleotide, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand (template) is induced, i.e., in the presence of nucleotides and an agent for polymerization, such as DNA polymerase, and at a suitable temperature and pH.
  • the primer is preferably single stranded for maximum efficiency in amplification.
  • the primer is an oligodeoxyribonucleotide.
  • the primer of this invention can be comprised of naturally occurring dNMP ⁇ i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotide, or non-natural nucleotide.
  • the primer can also include ribonucleotides.
  • the primer used in this invention may include nucleotides with backbone modifications such as peptide nucleic acid (PNA) (M.
  • PNA peptide nucleic acid
  • nucleotides with sugar modifications such as 2'-O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-0-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexitol DNA, and nucleotides having base modifications such as C-5 substituted pyrimidines (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-, propy
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization.
  • the exact length of the primers will depend on many factors, including temperature, application, and source of primer.
  • annealing or “priming” as used herein refers to the apposition of an oligodeoxynucleotide or nucleic acid to a template nucleic acid, whereby the apposition enables the polymerase to polymerize nucleotides into a nucleic acid molecule which is complementary to the template nucleic acid or a portion thereof.
  • hybridizing used herein refers to the formation of a double- stranded nucleic acid from complementary single stranded nucleic acids. There is no intended distinction between the terms “annealing” and “hybridizing”, and these terms will be used interchangeably.
  • the sequences of the HPV type-specific primers may comprise some mismatches, so long as they can be hybridized with templates and serve as primers.
  • the 5'-first priming portion and the 3'-second priming portion are designed to have a nucleotide sequence substantially complementary to the template DNA molecule.
  • substantially complementary is used herein to mean that the primer is sufficiently complementary to hybridize selectively to a template nucleic acid sequence under the designated annealing conditions or stringent conditions, such that the annealed primer can be extended by a polymerase to form a complementary copy of the template.
  • the 5'-first priming portion and the 3'-second priming portion have a nucleotide sequence perfectly complementary to template DNA molecules, i.e., no mismatches.
  • Suitable annealing or hybridization conditions may be routinely determined by optimization procedures. Conditions such as temperature, concentration of components, hybridization and washing times, buffer components, and their pH and ionic strength may be varied depending on various factors, including the length and GC content of primer and target nucleotide sequence.
  • the detailed conditions for hybridization can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); and M. LM. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y.(1999).
  • the annealing is performed at temperature of 45-72°C, more preferably, 50-70 0 C, most preferably 58-65 0 C.
  • the DNA molecule as templates is typically double-stranded, it is preferred to render the two strands into a single-stranded or partially single-stranded form.
  • Methods known to separate strands includes, but not limited to, heating, alkali, formamide, urea and glycoxal treatment, enzymatic methods ⁇ e.g., helicase action), and binding proteins.
  • strand separation can be achieved by heating at temperature ranging from 8O 0 C to 105 0 C.
  • General methods for accomplishing this treatment are provided by Joseph Sambrook, et al v Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001).
  • the present methods do not require that the template nucleic acid molecules have any particular sequence or length.
  • the HPV type-specific primers used for the present invention are annealed to a site on the template such that double-stranded structure is formed. Conditions of nucleic acid annealing suitable for forming such double stranded structures are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) and Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).
  • a variety of DNA polymerases can be used in the extension step of the present methods, which includes "Klenow" fragment of E. coli DNA polymerase I, a thermostable DNA polymerase, and bacteriophage T7 DNA polymerase.
  • the polymerase is a thermostable DNA polymerase which may be obtained from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Therm us filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu).
  • Taq Thermus aquaticus
  • Tth Thermus thermophilus
  • Therm us filiformis Thermis flavus
  • Thermococcus literalis Thermococcus literalis
  • Pyrococcus furiosus Pyrococcus furiosus
  • Excess in reference to components of the extension reaction refers to an amount of each component such that the ability to achieve the desired extension is not substantially limited by the concentration of that component. It is desirable to provide to the reaction mixture an amount of required cofactors such as Mg 2+ , dATP, dCTP, dGTP, and dTTP in sufficient quantity to support the degree of the extension desired.
  • Annealing or hybridization in the present method is performed under stringent conditions that allow for specific binding between the primer and the template nucleic acid. Such stringent conditions for annealing will be sequence-dependent and varied depending on environmental parameters. In the present method, the annealing step is generally performed under high stringent conditions. In the most preferable embodiment, the amplification is performed in accordance with PCR (polymerase chain reaction) which is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159.
  • PCR polymerase chain reaction
  • the present method follows multiplexing amplifications using at least two HPV type-specific primer pairs annealed to several target sequences.
  • the amplification is performed in accordance with multiplex PCR.
  • the results obtained with multiplex PCR are frequently complicated by the artifacts of the amplification procedure. These include “false-negative” results due to reaction failure and “false-positive” results such as the amplification of spurious products, which may be caused by annealing of the primers to sequences which are related to but distinct from the true recognition sequences. Therefore, elaborate optimization steps of multiplex PCR have to be conducted to reduce such false results; however, the optimization of the reaction conditions for multiplex PCR may become labor-intensive and time-consuming and unsuccessful.
  • the present method amplifies simultaneously a variety of target sequence for HPV genotyping with no false results in a single multiplex PCR reaction to completely overcome shortcomings associated with conventional multiplex PCR.
  • the step (b) for multiplex amplification reactions is carried out using at least four HPV type-specific primer pairs for identifying at least four HPV types (preferably, HPV types 16, 18, 31 and 45 accounted for 80% of cervical cancer). More preferably, the step (b) is carried out using at least eight HPV type-specific primer pairs for identifying at least eight HPV types. Still more preferably, the step (b) is carried out using at least thirteen HPV type-specific primer pairs for identifying at least thirteen HPV types (preferably, HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, a high-risk group).
  • the step (b) is carried out using at least eighteen HPV type- specific primer pairs for identifying at least eighteen HPV types comprising HPV types 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59 and 68.
  • the present method generates amplicons specific for the types of HPV even when eighteen HPV type-specific primer pairs are used, as demonstrated in Examples.
  • the step (b) for multiplex amplification is carried out using an additional internal control for evaluating the quality of the multiplex amplification.
  • the internal control sequence should show no homology with HPV genomic DNA.
  • the internal control sequence is the Arabidopsis thaliana CESA3 gene.
  • Primers for amplifying the internal control sequence comprises preferably the nucleotide sequence of SEQ ID NOs:37 and 38.
  • the present method for genotyping HPVs exhibits dramatic accuracy and reproducibility. In addition, the present method shows much higher sensitivity for genotyping HPVs in biological samples. The present inventors appreciate that the instant method could detect and identify HPVs present even in 10 copy number- According to a preferred embodiment, the nucleic acid molecule of human papilloma virus (HPV) in the biological sample is present in at least 10 copies.
  • HPV human papilloma virus
  • the amplified DNA molecules in step (b) are then analyzed based on their sizes, allowing to identify the type of HPVs in the biological sample.
  • the analysis of amplified products may be conducted by various methods or protocols.
  • the amplified products can be analyzed by electrophoresis on a suitable gel ⁇ e.g., agarose gel).
  • the amplified products could be also detected on a denaturing polyacrylamide gel by autoradiography or non-radioactive detection methods, such as silver staining (Gottschlich et al., (1997) Res. Commun. MoI. Path. Pharm. 97, 237-240; Kociok, N., et al. (1998) MoI.
  • the HPV type-specific primers used in the present invention are designed to produce amplicons with defined and different sizes for identifying HPV types; therefore the simple analysis or observation of size difference of amplified products enables to genotype HPVs in more convenient manner.
  • the analysis of the amplified DNA molecules in step (b) is conducted by a genescan process. Where the analysis is performed by the genescan process, at least one primer of the HPV type-specific primer pair has to be labeled with fluorescent dyes in step (b).
  • the term used herein "genescan process” is intended to mean a process involving (i) denaturation of the amplified DNA molecules, (ii) fluorescent capillary electrophoresis (preferably, automated fluorescent capillary electrophoresis) by sequencers (preferably, automated sequencers) and (iii) software analysis to analyze and show DNA fragments with different sizes in different peaks.
  • the denaturation of the amplified DNA molecules may be carried out by conventional techniques. According to a preferred embodiment, the denaturation is carried out by incubating the amplified product of step (b) with NaOH.
  • the concentration of NaOH ranges from 0.1 N to 0.001 N, more preferably 0.1-0.005 N, still more preferably 0.05-0.008 N, most preferably 0.02-0.009 N.
  • the amplified product of step (b) is incubated with NaOH and formamide at 90-98 0 C in step (c).
  • a fluorescent capillary electrophoresis is then performed using sequencers ⁇ e.g., ABI PRIMTM family such as 310, 377, 3100 and 3700 automated DNA sequencers available from Applied Biosystems, USA), which are described in Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001).
  • sequencers ⁇ e.g., ABI PRIMTM family such as 310, 377, 3100 and 3700 automated DNA sequencers available from Applied Biosystems, USA
  • a small portion of the amplified and denatured DNA molecules is combined with a dye-labeled size standard and electrophoresed on an automated sequencer, where the fluorescent product is sized and quantified.
  • the software analysis is to analyze DNA fragment resolution data from fluorescent capillary electrophoresis and display DNA fragments with different sizes in different peaks. This analysis may be performed using commercially available software such as GeneScanTM software purchasable from Applied Bio
  • the peaks generated by the software represent each type of HPVs.
  • the present method identifies simultaneously at least two types of human papilloma viruses (HPVs) in a single multiplex amplification reaction in step (b) and a single detection process in step (c).
  • HPVs human papilloma viruses
  • a method for identifying at least eighteen types of human papilloma viruses (HPVs) by an 18-plex amplification of a nucleic acid molecule of HPVs in a biological sample and a size differentiation of amplicons which comprises the steps of: (a) obtaining a DNA molecule from the biological sample;
  • kits for identifying at least two types of human papilloma viruses (HPVs) by a multiplex amplification of a nucleic acid molecule of HPVs in a biological sample which comprises at least two HPV type-specific primer pairs for identifying at least two types of human papilloma viruses (HPVs); wherein the HPV type-specific primer pairs generate amplicons with defined and different sizes from each other; wherein the HPV-type specific primer has the structure represented by the following general formula I:
  • X p represents a 5'-first priming portion having a hybridizing nucleotide sequence substantially complementary to a target sequence to hybridize therewith
  • Y q represents a separation portion comprising at least three universal bases
  • Z 1 - represents a 3'-second priming portion having a hybridizing nucleotide sequence substantially complementary to a target sequence to hybridize therewith
  • p, q and r represent the number of nucleotides
  • X, Y, and Z are deoxyribonucleotides or ribonucleotides
  • the T m of the 5'-first priming portion is higher than that of the 3'-second priming portion and the separation portion has the lowest T m in the three portions
  • the separation portion separates the 5'-first priming portion from the 3'-second priming portion in terms of annealing events to the template DNA molecule, whereby the annealing specificity and priming
  • HPV type-specific primers contained in the present kit are identical to those used in the present genotyping method, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.
  • the present kit may optionally include the reagents required for performing PCR reactions such as buffers, thermostable DNA polymerase, DNA polymerase cofactors, and deoxyribonucleotide-5-triphosphates.
  • the kit may also include various polynucleotide molecules, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.
  • the kits may also include reagents necessary for performing positive and negative control reactions. Optimal amounts of reagents to be used in a given reaction can be readily determined by the skilled artisan having the benefit of the current disclosure.
  • the kits typically, are adapted to contain in separate packaging or compartments the constituents afore-described.
  • the present invention identify various types of HPVs in accordance with multiplex amplification using a multitude of HPV type-specific primer pairs and a size differentiation of amplicons;
  • the HPV type-specific primer pairs used are not limited in their number and generate distinct amplicons to permit the genotyping of HPVs by a single multiplex amplification and a single identifying process;
  • the present genotyping method is carried out in accordance with relatively simple and convenient process such as multiplex amplification and analysis of amplicon sizes, enabling the high-throughput analysis of HPV detection and genotyping;
  • DPO primers specific for 18 different types of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59 and 68
  • An artificially created plasmid DNA was used as an internal control (IC) in every PCR reaction. This IC was used to monitor any possible inhibition in the PCR reaction due to the presence of inhibitory substances in the clinical specimens.
  • Primers for IC were designed from the Arabidopsis thaliana CESA3 gene which shows no homology with human or viral genomic DNA. Consequent PCR amplicon (558 bp) was subsequently cloned into pUC19 plasmid vector.
  • This recombinant plasmid (CESA3 in pUC19) and its specific primer set, IC/Df_FAM and IC/Dr (Table 1) were added to multiplex primer mix so as to serve as an internal control all through multiplex PCR.
  • the total 38 primers were designed to produce 19 PCR amplicons with different sizes, which permit to discriminate each of 18 different HPV genotypes and internal control based on PCR product sizes.
  • the forward primer of each primer set was 5'- end labeled with the fluorescent dye 6-carboxyfluorescein (6-FAM). All primers were synthesized by Bioneer (Korea), Table 1
  • Df and Dr represent DPO forward primer and DPO reverse primer, respectively.
  • bAccesion numbers are available from the GenBank.
  • the symbol "I” denotes deoxyinosine.
  • IC indicates internal control (Arabidopsis thaliana CESA3 gene). *not identified region.
  • the total 20 ⁇ l PCR reaction contained 5-10 ng viral DNA or 10 to 10000 copies of cloned plasmids, 4 ⁇ l of 5 X primer mixture (final concentration of 3 pmole per each primer and 10 5 copies of internal control plasmid in a single reaction), 10 ⁇ l of 2X Master Mix (Seegene, Korea) containing 5% DMSO.
  • the cycling conditions were as follows: denaturation for 15 min at 94 °C; amplification for 40 cycles, with denaturation for 30 sec at 94 0 C, annealing for 1.5 min at 60 0 C and extension for 1.5 min at 72 °C; and a final extension step of 72 ° C for 10 min.
  • the sizes of each PCR amplicon are listed in Table 1.
  • the positive control plasmids which were used for 19-plex HPV PCR templates were generated from clinical samples of HPV-infected patients by mono-PCR with specific primer pair for 18 individual HPV types (6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59 or 68; indicated in Table 1).
  • Each of 18 PCR amplicons was eluted using spin columns (PCR DNA Purification Kit, GeneAII Biotechnology, Korea) and ligated into TOPO ® vector (Invitrogen) respectively.
  • the resulting ligates were used for eventual transformation into chemically competent cells ⁇ e.g. DH5 ⁇ ) and subsequent clones were confirmed by complete sequencing of PCR-generated fragments.
  • the sensitivities of the GeneScan PCR assays were assessed by testing 10-fold serial dilutions of the quantified, cloned PCR amplification products of each HPV type prepared with the TOPO TA cloning kit (Invitrogen).
  • Viral genomic DNA was isolated from 200 ⁇ l aliquots of human cervical swab samples using spin columns (Viral Gene-spinTM Viral DNA/RNA extraction kit, iNtRON Biotechnology, Inc., Korea) according to the manufacturer's recommendations.
  • Viral genomic DNA samples were obtained from 30 patients of whom results were proven to be identical by two different commercially available HPV detection and/or genotyping methods ⁇ i.e. Digene's hybrid capture 2 HPV test and Roche's Linear Array HPV genotyping test), and then 19-plex PCR amplifications were conducted using 5-10 ng of viral genomic DNA samples as described above.
  • the aim of this invention is to develop a multiplex PCR system on automated fluorescent capillary electrophoresis and GeneScan software analysis for simultaneous detection of 18 different HPV genotypes; 13 high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) and 5 low-risk types (6, 11, 42, 43, 44).
  • Our results suggest that the HPV 19-plex PCR system will achieve this goal and improve the ability of laboratories to detect HPV genotypes in a highly specific manner.
  • the HPV 19-plex PCR system shows excellent type specificity.
  • the analytical sensitivity was between 10 and 100 copies HPV per reaction mixture for each of the 18 individual HPV types (Table 3).
  • the HPV 19-plex PCR system can be used on clinical material.
  • the present invention overcomes the most difficult problems with conventional multiplex PCR; incompatible primer sets and high background amplification/detection.
  • the HPV 19-plex PCR system is easy to optimize since it is based on DPO system which has much wider "optimal" range of annealing temperature and salt concentration. Furthermore, the HPV 19-plex PCR system does not require any post-PCR step such as probe hybridization step and are read directly on genetic analyzer. These features result in an assay that requires only about 3.5 hours of hands-on time. The entire procedure is amenable to automation because only reagent addition is required.
  • the 19-plex PCR system is an open platform that could theoretically be integrated with other high-throughput platforms for amplicon detection and identification. However, quality control for multiple sets of DPO primers, especially those with FAM label should be addressed to a satisfactory level of compliance by the laboratories prior to routine settings for the high throughput analysis.
  • HPV 19-plex PCR system has been developed and offers a highly specific method for genotyping 18 different HPVs in cervical swab specimens.
  • Luminex assay to detect the presence of human papillomavirus types Cancer Sc/, 98, 549-554.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode permettant l'identification d'au moins deux types de virus du papillome humain (les VPH) par amplification multiplex d'une molécule d'acide nucléique des VPH dans un échantillon biologique et différenciation des amplicons selon la taille, et une trousse permettant la mise en œuvre de ladite méthode. La présente méthode de génotypage est conduite suivant un procédé relativement simple et commode tel que l'amplification multiplex et l'analyse des tailles des amplicons, permettant l'analyse à haut débit de la détection et du génotypage des VPH. Même si la présente invention est conduite d'une manière relativement simple, les résultats obtenus pour le génotypage des VPH sont beaucoup plus précis et reproductibles que n'importe quelle méthode classique.
PCT/KR2007/004452 2007-09-14 2007-09-14 Génotypage des virus du papillome humain (vph) par amplification multiplex et différenciation selon la taille WO2009035177A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/KR2007/004452 WO2009035177A1 (fr) 2007-09-14 2007-09-14 Génotypage des virus du papillome humain (vph) par amplification multiplex et différenciation selon la taille

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2007/004452 WO2009035177A1 (fr) 2007-09-14 2007-09-14 Génotypage des virus du papillome humain (vph) par amplification multiplex et différenciation selon la taille

Publications (1)

Publication Number Publication Date
WO2009035177A1 true WO2009035177A1 (fr) 2009-03-19

Family

ID=40452164

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2007/004452 WO2009035177A1 (fr) 2007-09-14 2007-09-14 Génotypage des virus du papillome humain (vph) par amplification multiplex et différenciation selon la taille

Country Status (1)

Country Link
WO (1) WO2009035177A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102329866A (zh) * 2011-09-19 2012-01-25 泰普生物科学（中国）有限公司 一种沙眼衣原体的pcr荧光定量快速检测试剂盒及方法
US20120258447A1 (en) * 2009-12-24 2012-10-11 Seegene, Inc Real-time multiplexing detection of target nucleic acid sequences with elimination of false signals
CN105861499A (zh) * 2016-06-13 2016-08-17 湛江出入境检验检疫局检验检疫技术中心 一种检测副溶血弧菌和霍乱弧菌的多重dpo-pcr引物组合及方法
CN106834545A (zh) * 2017-03-13 2017-06-13 苏州市立医院 高危人乳头瘤病毒试剂盒及检测方法
CN109811079A (zh) * 2019-04-02 2019-05-28 丹娜（天津）生物科技有限公司 一种曲霉菌分种检测的dpo引物对、检测方法、试剂盒及其应用
WO2023195782A1 (fr) * 2022-04-08 2023-10-12 Seegene, Inc. Procédé de détection d'acides nucléiques cibles d'au moins neuf types de vhp dans un échantillon

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0425995A2 (fr) * 1989-11-03 1991-05-08 Abbott Laboratories Utilisation des amorces oligomnucléotidiques conservées pour l'amplification des séquences d'ADN du virus papilloma humain
US5447839A (en) * 1988-09-09 1995-09-05 Hoffmann-La Roche Inc. Detection of human papillomavirus by the polymerase chain reaction
US5639871A (en) * 1988-09-09 1997-06-17 Roche Molecular Systems, Inc. Detection of human papillomavirus by the polymerase chain reaction
US20030175770A1 (en) * 1996-03-15 2003-09-18 Gocke Christopher D. Methods for detecting papillomavirus DNA in blood plasma and serum
US20060160188A1 (en) * 2005-01-14 2006-07-20 Kurnit David M Systems, methods , and compositions for detection of human papilloma virus in biological samples

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5447839A (en) * 1988-09-09 1995-09-05 Hoffmann-La Roche Inc. Detection of human papillomavirus by the polymerase chain reaction
US5639871A (en) * 1988-09-09 1997-06-17 Roche Molecular Systems, Inc. Detection of human papillomavirus by the polymerase chain reaction
EP0425995A2 (fr) * 1989-11-03 1991-05-08 Abbott Laboratories Utilisation des amorces oligomnucléotidiques conservées pour l'amplification des séquences d'ADN du virus papilloma humain
US20030175770A1 (en) * 1996-03-15 2003-09-18 Gocke Christopher D. Methods for detecting papillomavirus DNA in blood plasma and serum
US20060160188A1 (en) * 2005-01-14 2006-07-20 Kurnit David M Systems, methods , and compositions for detection of human papilloma virus in biological samples

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120258447A1 (en) * 2009-12-24 2012-10-11 Seegene, Inc Real-time multiplexing detection of target nucleic acid sequences with elimination of false signals
CN102329866A (zh) * 2011-09-19 2012-01-25 泰普生物科学（中国）有限公司 一种沙眼衣原体的pcr荧光定量快速检测试剂盒及方法
CN102329866B (zh) * 2011-09-19 2013-09-18 泰普生物科学（中国）有限公司 一种沙眼衣原体的pcr荧光定量快速检测试剂盒及方法
CN105861499A (zh) * 2016-06-13 2016-08-17 湛江出入境检验检疫局检验检疫技术中心 一种检测副溶血弧菌和霍乱弧菌的多重dpo-pcr引物组合及方法
CN106834545A (zh) * 2017-03-13 2017-06-13 苏州市立医院 高危人乳头瘤病毒试剂盒及检测方法
CN109811079A (zh) * 2019-04-02 2019-05-28 丹娜（天津）生物科技有限公司 一种曲霉菌分种检测的dpo引物对、检测方法、试剂盒及其应用
WO2023195782A1 (fr) * 2022-04-08 2023-10-12 Seegene, Inc. Procédé de détection d'acides nucléiques cibles d'au moins neuf types de vhp dans un échantillon

Similar Documents

Publication Publication Date Title
US11499199B2 (en) Bordetella detection assay
JP6177844B2 (ja) 試験サンプル中のヒトパピローマウイルスおよびヒトベータグロビン配列を検出するためのプライマーおよびプローブ
US10988816B2 (en) Compositions and methods for detecting human papillomavirus nucleic acid
EP1082466B1 (fr) Procede, reactif et kit servant a determiner le genotype de papillomavirus humains
WO2012094682A2 (fr) Matériaux et procédé pour le génotypage et la quantification d'un papillomavirus humain à haut risque
WO2009035177A1 (fr) Génotypage des virus du papillome humain (vph) par amplification multiplex et différenciation selon la taille
EP3899051A1 (fr) Dosage pour la détection du papillomavirus humain (hpv)
US20150376725A1 (en) HPV Detection in Urine
EP2576841B1 (fr) Procédé de détection spécifique du virus de la peste porcine classique
EP3126530A1 (fr) Procédé de détection spécifique du virus de la peste porcine classique
Bogovac et al. Prevalence of HPV-16 genomic variant carrying a 63-bp duplicated sequence within the E1 gene in Slovenian women
JP4388061B2 (ja) Ｅ型肝炎ウイルスを検出するためのｌａｍｐ増幅用核酸プライマーセット
AU2014237563A1 (en) Detection of neisseria gonorrhoeaes
JP2001321168A (ja) Ｈｐｖ感染型の同定方法
JP2021532787A (ja) エプスタイン−バールウイルスの核酸を検出するための組成物および方法
WO2005100610A2 (fr) Procede de detection de virus, amorces correspondantes et necessaire de criblage

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07808243

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07808243

Country of ref document: EP

Kind code of ref document: A1