KR20100120760A - Primer and probe for detection of human papilloma virus and method for detecting human papilloma virus using the same - Google Patents

Abstract

PURPOSE: A primer and primer for detecting human papilloma virus(HPV) and a method for detecting HPV are provided to quickly and accurately detect the presence and type of HPV-1, 2, 3, 4, 27 and 57. CONSTITUTION: A primer or probe for detecting human papilloma virus(HPV) comprises a primer pair containing sequence numbers 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10 or 11 and 12. A microarray for detecting HPV comprises primer or probe fixed on a substrate. A kit for detecting HPV contains the priemr or probe. The HPV is detected by PCR using the primer. The HPV is HPV-1, 2, 3, 4, 27 or 57.

Description

Primer and probe for detection of human papilloma virus and method for detecting human papilloma virus using the same}

The present invention relates to a primer for detecting human papillomavirus, a probe and a method for detecting human papillomavirus using the same.

Human Papilloma virus (HPV) is a cyclic double helix DNA virus belonging to the Papovaviridae family that infects not only humans but also mammalian right-sided epithelial cells and causes various malignant tumors such as warts. It is closely related and is known to be an important cause of cervical cancer.

About 40 of the 100 known HPVs to date have been found in the reproductive organs, of which the high-risk groups HPV-16 and -18 have been shown to progress about 40% faster than other HPV types. It is important, accounting for about 70% of the HPV found in. HPV-26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 61, 66, 67, 68, 69, 70, 73, etc. Are relatively high and classified as "high risk groups" and are classified as HPV-1, 2, 3, 6, 7, 10, 11, 13, 32, 34, 40, 42, 43, 44, 54, 55, 57, 72 and 81 are classified as "low risk groups".

On the other hand, the most common cause of infection in total mucosal HPV infection is low-risk HPVs (HPV-1, 2, 3, 4), rather than high-risk HPVs associated with cervical cancer, and these low-risk HPVs generally have wart lesions on mucous membranes and skin. Cause

Warts are a very common viral infection that occurs when HPV is infected with the skin or mucous membranes. Excessive proliferation of the epidermis results in bumpy surface papules. It can occur on the skin of any part, but occurs mainly on the exposed hands, feet, legs, face, etc., and can also occur on the genitals through sexual contact.

In general, warts are classified into common warts, flat warts, plantar warts, and genital warts according to clinical types. Among these, genital warts are related to cervical cancer, and research on these has been relatively active. Recently, a vaccine to prevent this has been commercialized. However, common skin diseases, such as warts, have not been associated with direct death, so they have been alienated by doctors and scientists, and there is little basic research on this.

The diagnosis of HPV infection is largely divided into cytological and molecular biological methods. The cytological method for determining the morphological changes of HPV infected cells is the Pap smear (Papanicolaou (Pap) Smear), but the problem of not having consistent accuracy due to low objectivity of diagnosis and high false negative rate (15-45%). There is this.

On the other hand, the molecular biological method is to examine the presence and genotype of HPV using the DNA of the virus, it is largely divided into a method of directly identifying the HPV DNA and amplification by PCR. This method has the advantage of being able to objectively detect the HPV DNA and to make the diagnosis accurate, so that the disease can be diagnosed early if the diagnosis is unclear by the cytological method.

For direct identification of HPV DNA, conventional methods such as southern blotting, dot blotting and filter in situ hybridization (FISH) using HPV type-specific probes have been used. Hybrid capture II method is widely used, but because it requires a large amount of HPV DNA, it shows a limit of detection when HPV early infection or sampling is insufficient. The equipment is relatively expensive and has a low sensitivity.

On the other hand, PCR is a technique that can detect the presence of a variety of types of HPV infection by amplifying a trace amount of the pathogen nucleic acid using a complementary oligonucleotide (primer) that specifically binds to the nucleic acid sequence of the target pathogen. It is one of the representative techniques used to diagnose the disease. These PCR techniques are capable of objective large-scale testing, and are evaluated to have a relative advantage in accuracy, ease, and cost compared to the above-described cytological methods and direct HPV DNA tests.

To date, primers used in HPV detection by PCR have been developed by common primers that amplify the DNA regardless of the HPV type, or by type-specific primers that bind specifically to each type to selectively amplify only the type-specific HPV DNA. It became.

However, these prior arts can only diagnose a very limited type of HPV compared to the various types of HPV that have been discovered so far, and have a problem that the sensitivity of detection according to the HPV type is also greatly reduced. There is a restriction. In particular, even if amplification products are obtained by PCR reaction using a common primer, it is difficult to accurately determine whether a newly identified and classified HPV type is a high risk group or a low risk group.In the case of using a type specific primer, to confirm the presence of HPV in a sample The use of different primers at the same time has the limitation of using a wide range of clinical applications.

Therefore, in order to increase the practical efficiency of disease diagnosis according to whether HPV DNA amplification by PCR, HPV type can be selectively comprehensively amplified, so that HPV can be diagnosed early and the disease caused by HPV can be diagnosed at once. Development of a primer that can be urgently urgent.

Therefore, the present inventors have found a novel PCR primer pair for specifically detecting six subtypes of human papillomavirus, which is the cause of mucosal skin warts, and can detect human papillomavirus by type in a specific and rapid manner. The present invention has been completed by developing a method.

Accordingly, an object of the present invention is to provide a primer and probe for detecting human papillomavirus, which is designed to effectively detect human papillomavirus.

Another object of the present invention is to provide a microarray for detecting human papillomavirus, wherein the probe is integrated on a substrate.

Another object of the present invention is to provide a method for detecting human papillomavirus using the primer or probe.

Furthermore, another object of the present invention is to provide a kit for detecting human papillomavirus comprising the primer or probe.

In order to achieve the object of the present invention as described above, the present invention comprises a first primer pair consisting of the nucleotide sequence of SEQ ID NO: 1 and 2; A second primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4; A third primer pair consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6; A fourth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8; A fifth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 9 and 10; And a sixth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 11 and 12; It provides a primer for detecting human papillomavirus comprising at least one primer pair selected from the group consisting of.

In addition, the present invention comprises a first primer pair consisting of the nucleotide sequence of SEQ ID NO: 1 and 2; A second primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4; A third primer pair consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6; A fourth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8; A fifth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 9 and 10; And a sixth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 11 and 12; It provides a probe for detecting human papillomavirus comprising at least one primer pair selected from the group consisting of.

In one embodiment of the present invention, the first primer pair is a primer for amplifying DNA of HPV-1, the second primer pair is a primer for amplifying DNA of HPV-2, and the third primer pair is HPV A primer for amplifying DNA of -3, the fourth primer pair is a primer for amplifying DNA of HPV-4, the fifth primer pair is a primer for amplifying DNA of HPV-27, and the sixth primer pair is The primer for amplifying the DNA of HPV-57 may be a primer for detecting human papillomavirus.

The present invention also provides a microarray for detecting human papillomavirus, wherein the primer or probe is immobilized on a substrate.

In addition, the present invention provides a human papillomavirus detection kit comprising the primer or probe.

The present invention also provides a method for detecting human papillomavirus comprising the step of detecting human papillomavirus by polymerase chain reaction using the primer for detecting human papillomavirus.

In one embodiment of the invention, the human papillomavirus may be selected from the crowd consisting of HPV-1, 2, 3, 4, 27 and 57.

Human papillomavirus detection primer or probe according to the present invention and human papillomavirus detection method using the same is the human papilloma of six subtypes (HPV-1, 2, 3, 4, 27, 57) that causes skin mucosal warts It is effective to quickly and accurately determine the presence and type of the virus, and can effectively diagnose by reducing the time and cost of detecting human papilloma virus infected with warts.

The inventors of the present invention devised a primer and probe that can specifically detect human papillomavirus that causes skin mucosal warts, and a method for detecting human papillomavirus using the primer or probe and human papilloma including the primer or probe. Virus detection kits and microarrays were developed.

Therefore, the present invention is characterized in that it provides a primer for detecting human papilloma virus of six subtypes of human papillomavirus, specifically, the present invention comprises a first primer pair consisting of the nucleotide sequence of SEQ ID NO: 1 and 2; A second primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4; A third primer pair consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6; A fourth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8; A fifth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 9 and 10; And a sixth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 11 and 12; It provides a primer for detecting human papillomavirus comprising at least one primer pair selected from the group consisting of.

Human papillomavirus in the present invention is a causative factor for developing skin mucosal warts, preferably human papillomavirus of subtype HPV-1, 2, 3, 4, 27, 57.

The primer for detecting human papillomavirus according to the present invention refers to a primer that can hybridize complementarily to a gene of human papillomavirus, and preferably a primer, primer pair or primer that can amplify a specific gene of the human papillomavirus. It can be a set.

As used herein, the term “primer” refers to a nucleic acid sequence having a short free 3 ′ ydroxyl group, which forms a complementary template and base pair and serves as a starting point for template strand copying. Refers to a nucleic acid sequence. That is, the primers can initiate template-directed DNA synthesis under appropriate conditions (eg, four different nucleoside triphosphates and polymerizers such as DNA, RNA polymerase or reverse transcriptase) in appropriate buffers and at appropriate temperatures. Refers to a single stranded oligonucleotide.

In addition, the primers of the present invention may be chemically synthesized using phosphoramidite solid support methods, or other well known methods. In addition, such primers may be modified (e.g., added, deleted, substituted) using a number of means known in the art to the extent that they do not affect the specific detection of human papillomavirus, and are completely compatible with the template. It does not have to be complementary, but it must be sufficiently complementary to hybridize with the template. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, phosphoroami Date, carbamate, etc.) or charged linkages such as phosphorothioate, phosphorodithioate and the like. Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, proralene, etc.). ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents. Nucleic acid sequences of the invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.

In the present invention, primers capable of detecting human papillomavirus may be primers that specifically amplify specific regions of HPV-1, HPV-2, HPV-3, HPV-4, HPV-27 and HPV-57. have. In addition, the primers of the present invention may be a pair of forward and reverse primers having 10 to 40, preferably 20 to 30 nucleotide sequences complementary to the gene.

As the primer of the present invention, more preferably, the first primer pair consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2, the second primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4, the nucleotide sequences of SEQ ID NOs: 5 and 6 A third primer pair consisting of a fourth primer pair consisting of a nucleotide sequence of SEQ ID NOs: 7 and 8, a fifth primer pair consisting of a nucleotide sequence of SEQ ID NOs: 9 and 10, and a sixth primer consisting of a nucleotide sequence of SEQ ID NOs: 11 and 12 It may be one or more primer pairs selected from the group consisting of pairs.

Specifically, HPV-1 is a primer pair having a nucleotide sequence of SEQ ID NO: 1 and 2, HPV-2 is a primer pair having a nucleotide sequence of SEQ ID NO: 3 and 4, HPV-3 is a base sequence of SEQ ID NO: 5 and 6 Primer pair having, and HPV-4 is a primer pair having the nucleotide sequence of SEQ ID NO: 7 and 8, HPV-27 is a primer pair having the nucleotide sequence of SEQ ID NO: 9 and 10, HPV-57 is the base of SEQ ID NO: 11 and 12 Specific regions of each gene can be specifically amplified by primer pairs having sequences.

The present invention provides primer pairs for amplification of specific genes such as HPV-1, 2, 3, 4, 27, and 57 for detecting subtype specific DNA sequences, and in particular, human papilloma virus of six subtypes. Provide primer sets with all primer pairs mixed. Six PCR primer pairs can be designed and synthesized to simultaneously detect the specific DNA base sequences of the six subtypes of human papillomaviruses of different sizes.

According to an embodiment of the present invention, in the human papillomavirus detection primer pair, the first primer pair T1-5 / T1-3 consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2 described below is specific for HPV-1. The gene was designed to be detected and amplified to about 575 bp.

SEQ ID NO: 1 (T1-5): 5'-GTGAACCATCATTTACAATAGTGA-3 '

SEQ ID NO: 2 (T1-3): 5'-ACGACCAGATAAGTTTGGCAGCA-3 '

In addition, the second primer pair T2-5 / T2-3 consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4 described below was designed to specifically detect HPV-2, and amplified the specific gene to about 222 bp.

SEQ ID NO: 3 (T2-5): 5'-ATACATTTCAAGGCCTGCCTCCGCA-3 '

SEQ ID NO: 4 (T2-3): 5'-ACGTATGGCGAAATAATAACGACTA-3 '

In addition, the third primer pair T3-5 / T3-3 consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6 described below was designed to specifically detect HPV-3, and amplified the specific gene to about 348 bp.

SEQ ID NO: 5 (T3-5): 5'-TACTATTTGTGCAGGCTTTCTCTGT-3 '

SEQ ID NO: 6 (T3-3): 5'-ACAGGTGACCTGCAACTACAACCT-3 '

In addition, the fourth primer pair T4-5 / T4-3 consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8 described below was designed to specifically detect HPV-4, and the specific gene was amplified to about 437 bp.

SEQ ID NO: 7 (T4-5): 5'-TCTGGAATGTTTTATTCTGCCAGGA-3 '

SEQ ID NO: 8 (T4-3): 5'-ATTTTCCACCACTCCCGGTGCAAA-3 '

In addition, the fifth primer pair T27-5 / T27-3 consisting of the nucleotide sequences of SEQ ID NOs: 9 and 10 described below was designed to specifically detect HPV-27, and amplified the specific gene to about 174 bp.

SEQ ID NO: 9 (T27-5): 5'-TAATTGTGACATATCGCCACCAT-3 '

SEQ ID NO: 10 (T27-3): 5'-TAAGCCAATGAGGGTGAGAA-3 '

In addition, the sixth primer pair T57-5 / T57-3 consisting of the nucleotide sequences of SEQ ID NOs: 11 and 12 described below was designed to specifically detect HPV-57, and amplified the specific gene to about 511 bp.

SEQ ID NO: 11 (T57-5): 5'-GYAACATTTCACAGCCTGTATCAT-3 '

SEQ ID NO: 12 (T57-3): 5'-GTTTATTGACACGYAGCAATACA-3 '

Therefore, the primer pair for detecting human papillomavirus of the present invention not only has specificity for each of six types of human papillomaviruses, ie, HPV-1, 2, 3, 4, 27, 57, but also amplifies specific genes at different sizes. Since it is possible to detect each of the six types of human papillomavirus, it is possible to detect the six types of human papillomavirus at the same time. Furthermore, the primer of the present invention can be effectively used for HPV infection, identification of infected HPV genotype, HPV epidemiological investigation, efficacy and risk investigation of HPV vaccine.

The present invention also provides a probe capable of detecting human papillomavirus.

The probe capable of detecting human papillomavirus according to the present invention preferably comprises a first primer pair consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2, a second primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3, 4, SEQ ID NO: 5 And a third primer pair consisting of a nucleotide sequence of 6, a fourth primer pair consisting of a nucleotide sequence of SEQ ID NOs: 7 and 8, a fifth primer pair consisting of a nucleotide sequence of SEQ ID NOs: 9 and 10, and a nucleotide sequence of SEQ ID NOs: 11 and 12 It may be composed of one or more primer pairs selected from the group consisting of a sixth primer pair consisting of.

In addition, the first primer pair is a primer for amplifying DNA of HPV-1, the second primer pair is a primer for amplifying DNA of HPV-2, and the third primer pair is for amplifying DNA of HPV-3 Primer, the fourth primer pair is a primer to amplify DNA of HPV-4, the fifth primer pair is a primer to amplify DNA of HPV-27, and the sixth primer pair to amplify DNA of HPV-57 It is preferable that it is a primer.

In the present invention, the "probe" refers to a nucleic acid fragment corresponding to several to several hundred bases that can specifically bind to DNA or RNA, and the probe is labeled so that the specific DNA or RNA may be You can check the presence. Probes may be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, RNA probes, and the like, and may be labeled with biotin, FITC, rhodamine and DIG, or labeled with radioisotopes.

The present invention also provides a microarray for detecting human papillomavirus, wherein the probe or primer of the present invention is immobilized on a substrate.

The microarray may include DNA or RNA polynucleotides. The microarray consists of conventional microarrays except that the polynucleotide of the invention is included in the polynucleotide of a probe or primer.

Methods of preparing microarrays by immobilizing probe polynucleotides on substrates are well known in the art. In the present invention, the "probe polynucleotide" refers to a polynucleotide capable of hybridization, and refers to an oligonucleotide capable of sequence-specific binding to the complementary strand of a nucleic acid. Such probes may include peptide nucleic acids described in Nielsen et al., Science 254, 1497-1500 (1991).

The process of immobilizing a probe polynucleotide associated with a human papillomavirus according to the present invention on a substrate can also be easily prepared using the prior art. In addition, the hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection may, for example, label a nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent substance such as Cy3 and Cy5, and then hybridize onto a microarray and from the labeling substance. By detecting the generated signal, the hybridization result can be detected.

The present invention also provides a method for detecting human papillomavirus using a primer or probe for detecting human papillomavirus according to the present invention.

As a method for detecting the human papilloma virus of the present invention, but not limited to, multiplex polymerase chain reaction, polymerase chain reaction, reverse transcriptase polymerase chain reaction transcriptase PCR (DNA), DNA sequencing, hybridization by microarray, restriction fragment length polymerphism (PCR-RFLP), randomly amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF), and AP-PCR (arbitrarily) primed PCR, sequence tagged site (STS), expressed sequence tag (EST), sequence characterized amplified regions (SCAR), inter-simple sequence repeat amplication (ISSR), amplified fragment length polymorphism (AFLP), cleaved amplified polymorphic sequence ), Single-strand conformation polymorphism (PCR-SSCP), Northern blot, and Southern blot. The.

The term “polymerase chain reaction (PCR)” in the present invention is one of the representative nucleic acid amplification techniques (NATs), in which a specific DNA region is amplified using a enzyme in vitro. Developed by Mullis et al. In 1985, any part of a DNA molecule can be amplified using this method only by knowing its bounding sequence. PCR basically consists of three stages: denaturation, binding, and extension. DNA is amplified as this process is repeated. The first step of PCR, the denaturation step, denatures the template DNA into a single chain, and the second step, the binding step, binds two types of single-stranded DNA with the desired region in between. In the third step, the synthesis step synthesizes DNA by DNA polymerase resistant to high heat from the primer, and repeats the above cycle 25 to 30 times.

Primers or probes that can be used in the human papillomavirus detection method of the present invention, the first primer pair for detecting HPV-1 consisting of the nucleotide sequence of SEQ ID NO: 1 and 2; A second primer pair for detecting HPV-2 consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4; A third primer pair for detecting HPV-3 consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6; A fourth primer pair for detecting HPV-4 consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8; A fifth primer pair for detecting HPV-27 consisting of the nucleotide sequences of SEQ ID NOs: 9 and 10; And at least one primer pair or probe selected from the group consisting of a sixth primer pair for detecting HPV-57 consisting of the nucleotide sequences of SEQ ID NOs: 11 and 12.

Specifically, in one embodiment of the present invention, a method of specifically detecting only six subtypes of human papillomaviruses by the method of multiple polymerase chain reaction (M-PCR) using a primer according to the present invention is illustrated. That is, six subtypes of human papillomaviruses can be detected using primer pairs capable of amplifying six subtype specific DNA sequences of HPV-1, 2, 3, 4, 27, and 57.

In another embodiment of the present invention, a DNA polymerized by amplifying one or more types of HPV-1, 2, 3, 4, 27, 57 by multiple polymerase chain reaction using a primer set including all six pairs of primer pairs. It can be detected by dividing by size.

According to one embodiment of the present invention, first, the wart skin tissue is treated with proteinase K to extract DNA from the tissue, and then, as a template DNA, a polymerase chain reaction using a PCR primer mixture is performed. To perform. After the completion of the multipolymerase chain reaction, the amplified DNA was electrophoresed on a 1.5% agarose gel to confirm and determine the subtype according to the specific DNA sequence amplification yu and its size (see FIGS. 1 to 3).

As described above, as a result of performing a polymerase chain reaction using a primer set capable of amplifying DNA specific for HPV-1, 2, 3, 4, 27 and 57, primer pairs specific for each subtype are different. It was confirmed that it does not amplify the type of human papillomavirus DNA.

Therefore, the detection method of the present invention can not only specifically detect each human papillomavirus subtype using one type of primer pair selected from the first to sixth primer pairs, but also all six types of primer pairs. 6 types (HPV-1, 2, 3, 4, 27, 59) of human papillomaviruses can be detected at once by carrying out the multiple polymerase chain reaction using the primer set containing .

In addition, the present invention can more easily and quickly identify subtypes of various warts occurring on the skin or mucous membranes, and can increase diagnostic value based on the types of warts that are most commonly encountered in practice, rather than high-risk warts, which have a very low prevalence. There is a characteristic.

Furthermore, the present invention provides a human papillomavirus detection kit comprising a primer or probe for detecting human papillomavirus according to the present invention.

The detection kit may comprise a primer or probe for detecting human papillomavirus according to the present invention, the kit may include a conventional component of the virus detection kit, the conventional components include reaction buffer, DNA polymerase, dNTP, and the like.

Human papillomavirus detection kit according to an embodiment of the present invention is six pairs of primer pairs having the nucleotide sequence of SEQ ID NO: 1-12 according to the present invention (T1-5 / T1-3; T2-5 / T2-3 T3-5 / T3-3, T4-5 / T4-3, T27-5 / T27-3, T57-5 / T57-3), and buffer buffers and Taq DNA polymerase.

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Example  1>

Six subtypes DNA  For sequence amplification Of primer  Design and synthesis

In order to detect HPV-1, 2, 3, 4, 27, 57, which cause skin mucosal warts without cross-reaction with each other, the present inventors have found an intergenic sequence between HPV genes as shown in Table 1 below. ) Was designed and synthesized as a PCR primer for detection as a base sequence specific to each type.

SEQ ID NO: primer Detection type Sequence Sequence position
(Nucleotide number) SEQ ID NO: 1 T1-5 HPV 1 5'-GTGAACCATCATTTACAATAGTGA-3 ' Between L1-3 'ends (7101) SEQ ID NO: 2 T1-3 HPV 1 5'-ACGACCAGATAAGTTTGGCAGCA-3 ' Between L1-3 'ends (7676) SEQ ID NO: 3 T2-5 HPV 2 5'-ATACATTTCAAGGCCTGCCTCCGCA-3 ' Between E2-L2 (3801) SEQ ID NO: 4 T2-3 HPV 2 5'-ACGTATGGCGAAATAATAACGACTA-3 ' Between E2-L2 (4113) SEQ ID NO: 5 T3-5 HPV 3 5'-TACTATTTGTGCAGGCTTTCTCTGT-3 ' Between E2-L2 (3907) SEQ ID NO: 6 T3-3 HPV 3 5'-ACAGGTGACCTGCAACTACAACCT-3 ' Between E2-L2 (4255) SEQ ID NO: 7 T4-5 HPV 4 5'-TCTGGAATGTTTTATTCTGCCAGGA-3 ' Between L1-3 'ends (7346) SEQ ID NO: 8 T4-3 HPV 4 5'-ATTTTCCACCACTCCCGGTGCAAA-3 ' Between L1-3 'ends (6909) SEQ ID NO: 9 T27-5 HPV 27 5'-TAATTGTGACATATCGCCACCAT-3 ' Between E2-L2 (4091) SEQ ID NO: 10 T27-3 HPV 27 5'-TAAGCCAATGAGGGTGAGAA-3 ' Between E2-L2 (4264) SEQ ID NO: 11 T57-5 HPV 57 5'-GYAACATTTCACAGCCTGTATCAT-3 ' Between E2-L2 (3843) SEQ ID NO: 12 T57-3 HPV 57 5'-GTTTATTGACACGYAGCAATACA-3 ' Between E2-L2 (4354)

< Example  2>

Human Papilloma Virus  For detection Kit  Produce

The present inventors prepared a kit for detection by making a multipolymerase chain reaction mixture containing the six pairs of PCR primers (SEQ ID NOs (1-12) synthesized in Example 1. Here, the detection kit is a 10X reaction buffer, It consists of a multiplex primer mixture (SEQ ID NOs: 1-12) and Taq DNA polymerase (5U / μl).

< Example  3>

Each of the six subtypes primer  Paired Multiple Polymerase Chain Reaction  Perform

The present inventors carried out the multiple polymerase chain reaction with six subtypes of standard HPV DNA and primer pairs for amplification of each subtype.

First, the wart skin tissue was collected, treated with proteinase K, and DNA was extracted from the tissue. Then, the polymerase chain reaction was performed using the PCR primer mixture as the template DNA. After the completion of the multiple polymerase chain reaction, the amplified DNA was electrophoresed on 1.5% agarose gel to confirm and determine the subtype according to the specific DNA sequence amplification and its size, and the results are shown in FIG. 1.

As a result, the first primer pair consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2 amplifies the DNA of HPV-1, the second primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4 amplifies the DNA of HPV-2 , The third primer pair consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6 amplifies the DNA of HPV-3, the fourth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8 amplifies the DNA of HPV-4, and The fifth primer pair consisting of the nucleotide sequences of Nos. 9 and 10 amplified the DNA of HPV-27, and the sixth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 11 and 12 amplified the DNA of HPV-57. (See Figure 1).

< Example  4>

Of the present invention primer  Set By multiple polymerase chain reaction Human papilloma bar Detection of viruses

The present inventors confirmed that each primer pair designed in the present invention can specifically amplify each type of human papillomavirus through Example 3, and further, by using each primer pair as one primer set, We investigated whether six subtypes of human papillomaviruses can be detected at one time. To this end, a standard HPV DNA mixture was used as a template, and a multipolymerase chain reaction was performed using a primer set in which 6 pairs of primers were mixed. Experimental method was carried out in the same manner as in Example 3, the experimental results are shown in FIG.

As a result, when using the primer set of the present invention including primer pairs specific for each subtype of HPV-1, 2, 3, 4, 27, 57, each primer pair amplified DNA of a different type of human papillomavirus. Instead, the primer pairs specifically amplified human papillomavirus, which can be amplified. Therefore, the above results show that when the inventors use the primer set of the present invention including six pairs of primer pairs, It was found that the papillomavirus can be easily and accurately amplified.

< Example  5>

Of the present invention primer  Set By multiple polymerase chain reaction Human papilloma bar Detection of viruses

The present inventors performed a multiplex polymerase chain reaction with a primer set containing 6 pairs of primers, using the standard human papillomavirus subtype DNA of each of the six subtypes as a single template. The experimental method was performed in the same manner as in Example 3, and the results are shown in FIG.

As a result, it was confirmed that the primer pair of the present invention specific for each subtype of HPV-1, 2, 3, 4, 27, 57 did not amplify other types of human papillomavirus DNA.

< Example  6>

Multiple polymerase chain reaction  From common wart tissue Human papilloma virus Detection

Using DNA extracted from the wart tissue as a template, the polymerase chain reaction was performed with the multipolymerase chain reaction mixture prepared in Example 2 to amplify specific DNA sequences of subtypes of human papillomavirus. After amplified DNA was electrophoresed on agarose gel, the presence and subtype of human papillomaviruses were determined according to the presence or absence of specific DNA amplification and the size of the amplified DNA.

<6-1> from wart tissue DNA  detach

Six tissues determined as different warts were collected and placed in a 1.5 ml centrifuge tube, and 400 µl of cytolysis buffer solution was added thereto, followed by 16-18 hours at 37 ° C. After confirming that all the tissues are destroyed, and heated for 10 minutes at 95 ℃ to eliminate the activity of proteinase K (proteinase K) and to the same volume of phenol was added and centrifuged. The supernatant was transferred to another centrifuge tube, to which twice ethanol was added and stored at -20 ° C for 2 hours. This was centrifuged for 15 minutes, the supernatant was discarded, 50 μl of distilled water was added to the DNA precipitate, and the DNA was dissolved by separating DNA.

<6-2> PCR  Preparation of Reaction Liquid

2 μl of each wart tissue DNA, 5 μl of 10X reaction buffer, 4 μl of 2.5 mM dNTP, multiplex primer mixture, that is, 12 μl of primer mixture containing 6 pairs of primers of the present invention, and Taq DNA polymerase (5 U / μl). ) After adding 0.5 μl, the total reaction solution volume was 50 μl with sterile distilled water to prepare a PCR reaction solution.

<6-3> Of multiple polymerase chain reaction  Perform

Multiple polymerase chain reaction was performed with the PCR reaction solution prepared in <6-2>. The conditions of the polymerase chain reaction were performed after initial denaturation at 94 ° C. for 1 minute and 30 seconds, denaturation at 94 ° C. for 30 seconds, and annealing at 55 ° C. for 30 seconds, followed by 72 A total of 30 repetitions were performed at 30 ° C. for 30 seconds.

<6-4> Human Papilloma Virus  Presence and Subtype Determination

The reaction product of PCR completed in <6-3> was subjected to electrophoresis on 1.8% agarose gel to confirm the presence and subtype of HPV by DNA size, and the results are shown in FIG. 4.

As a result, it was confirmed that the DNA products amplified by multiple polymerase chain reaction using primer pairs according to the present invention for each wart tissue sample were 437 bp, 575 bp, 222 bp, 174 bp, 511 bp and 222 bp, respectively. Therefore, the size of the amplified product was found to be 437bp HPV-4, 575bp HPV-1, 222bp HPV-2, 174bp HPV-27, 511bp HPV-57 and 222bp HPV-27.

Therefore, through the above results, it can be seen that the primers of the present invention described in Table 1 are primers capable of specifically amplifying HPV-1, 2, 3, 4, 27, 57, and using the primers of the present invention. Genetic amplification methods such as PCR were able to quickly and accurately determine the presence and type of major human papillomaviruses that cause skin mucosal warts.

In addition, the present invention can more easily determine the subtypes of skin mucosa warts, whether or not self-inoculation of various warts, identifying infection paths, identifying recurrences, confirming reinfection, investigating the prevalence according to subtypes, and various wart epidemiological studies. Can be. In addition, through the present invention, as well as the research base composition for the alienated study, it is possible to more easily and easily diagnose precisely the differences in the clinical features and the mechanism of occurrence according to the subtypes of the warts, the resistance of treatment, and the study of various prognostic factors. have.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Figure 1 is a photograph showing the results of performing a multiplex polymerase chain reaction with a primer pair for amplification of each subtype with standard HPV DNA of six subtypes according to an embodiment of the present invention. Here, lane M represents a size marker, lane 1 is HPV-1, lane 2 is HPV-2, lane 3 is HPV-3, lane 4 is HPV-4, lane 5 is HPV-27, lane 6 represents HPV-57.

Figure 2 is a photograph showing the results of performing a multiplex polymerase chain reaction with a primer set of six primers mixed with the standard HPV DNA of six subtypes according to another embodiment of the present invention as a template. Here, lane M represents a size marker, and lane 1 represents HPV-1, 2, 3, 4, 27, 57 mixed solution.

Figure 3 is a photograph showing the results of performing a multiplex polymerase chain reaction with each standard HPV subtype DNA as a single template in a primer set mixed with six primers according to another embodiment of the present invention. Here, lane M represents a size marker, lane 1 is HPV-1, lane 2 is HPV-2, lane 3 is HPV-3, lane 4 is HPV-4, lane 5 is HPV-27, lane 6 represents HPV-57.

Figure 4 is a photograph showing the result of confirming the presence and subtype of the HPV by DNA size by electrophoresis of the reaction product PCR completed according to another embodiment of the present invention on 1.8% agarose gel. Here, lane M represents a size marker of a 100 bp ladder.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Primer and probe for detection of human papilloma virus and          method for detection human papilloma virus using <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> T1-5 <400> 1 gtgaaccatc atttacaata gtga 24 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> T1-3 <400> 2 acgaccagat aagtttggca gca 23 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> T2-5 <400> 3 atacatttca aggcctgcct ccgca 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> T2-3 <400> 4 acgtatggcg aaataataac gacta 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> T3-5 <400> 5 tactatttgt gcaggctttc tctgt 25 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> T3-3 <400> 6 acaggtgacc tgcaactaca acct 24 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> T4-5 <400> 7 tctggaatgt tttattctgc cagga 25 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> T4-3 <400> 8 attttccacc actcccggtg caaa 24 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> T27-5 <400> 9 taattgtgac atatcgccac cat 23 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T27-3 <400> 10 taagccaatg agggtgagaa 20 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> T57-5 <400> 11 gyaacatttc acagcctgta tcat 24 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> T57-3 <400> 12 gtttattgac acgyagcaat aca 23  

Claims (6)

A first primer pair consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2; A second primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4; A third primer pair consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6; A fourth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8; A fifth primer pair consisting of the nucleotide sequences of SEQ ID NOs: 9 and 10; And Primer or probe for detecting human papillomavirus comprising at least one primer pair selected from the group consisting of a sixth primer pair consisting of the nucleotide sequences of SEQ ID NO: 11 and 12. The method of claim 1, The first primer pair is a primer for amplifying the DNA of HPV-1, The second primer pair is a primer for amplifying the DNA of HPV-2, The third primer pair is a primer for amplifying the DNA of HPV-3, The fourth primer pair is a primer for amplifying the DNA of HPV-4, The fifth primer pair is a primer for amplifying the DNA of HPV-27, The sixth primer pair is a primer or probe for detecting human papillomavirus, characterized in that the primer for amplifying the DNA of HPV-57. A microarray for detecting human papillomavirus, wherein the primer or probe according to claim 1 or 2 is immobilized on a substrate. Human papillomavirus detection kit comprising a primer or probe according to claim 1. A method for detecting human papillomavirus comprising the step of detecting a human papillomavirus by a polymerase chain reaction (PCR) using the primer for detecting a human papillomavirus according to claim 1. The method of claim 5, wherein the human papillomavirus is selected from the group consisting of HPV-1, 2, 3, 4, 27, and 57. 7.

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