KR100616548B1 - Novel PCR General Primers and Method for Detecting Diverse Types of Human Papillomavirus by PCR - Google Patents

Abstract

The present invention is a novel method for detecting DNA fragments of specific sequences present in various human papillomavirus (HPV) genotypes that cause cervical cancer through polymerase chain reaction (PCR). The present invention relates to a PCR common primer and a method for detecting the presence or absence of various HPV types using the same. More specifically, the oligonucleotide sequence capable of amplifying various types of HPV nucleic acid and a single oligonucleotide using the oligonucleotide as a primer A method for detecting the presence of various HPV types through PCR analysis is proposed.

Human Papillomavirus (HPV), PCR General Primer, Cervical Cancer, Polymerase Chain Reaction (PCR)

Description

Novel PCR General Primers and Method for Detecting Diverse Types of Human Papillomavirus by PCR for Detection of Various Types of Human Papilloma Virus by Polymerase Chain Reaction             

1A to 1H show CaSki (ATCC CRL-1550), an HPV-16 infected cell line, HeLa (ATCC CCL-2), an HPV-18 infected cell line, and K562 (KCLB-10243, Korean Cell Line Bank), an HPV-18 infected cell line, respectively. After electrophoresis after PCR using the 8 sets of primers synthesized in the present invention as a target DNA.

Figures 2a to 2g is a picture of the electrophoresis after PCR for a variety of HPV Plasmid DNA using a set of seven primers consisting of SEQ ID NO: 1 to SEQ ID NO: 14 synthesized in the present invention.

Figure 3 is a photograph of the electrophoresis after PCR for a variety of HPV Plasmid DNA using AlbioGP primer consisting of SEQ ID NO: 15 and SEQ ID NO: 16 synthesized in the present invention.

4a to 4t are electrophoresed images of a DNA obtained from a human body diagnosed as being related to cervical cancer using a set of 7 primers consisting of SEQ ID NOs: 1 to 14 synthesized in the present invention. to be.

5a to 5j are electrophoresed after PCR on DNA obtained from a human body diagnosed as not related to cervical cancer using 7 sets of primers consisting of SEQ ID NOs: 1 to 14 synthesized in the present invention. It is a photograph.

The present invention relates to a primer capable of amplifying a human papilloma virus (hereinafter referred to as "HPV") through a polymerase chain reaction ("PCR") and a method for detecting HPV using the same. More specifically, by combining complementary with HPV DNA divided into various genotypes (genotype), amplification of various types of HPV DNA by using HPV common primers and amplification of HPV DNA through a single PCR process The present invention relates to the presence of HPV in a sample and to a method of diagnosing cervical cancer.

Cervical cancer is a malignant tumor occurring in the cervix, accounting for more than 95% of all uterine cancer incidence, and the highest incidence of cancer among women in the world after breast cancer, about 440,000 cases are reported each year. In Korea, the Ministry of Health and Welfare's Cancer Registration Survey in 2000 reports about 5,000 new patients per year. The number of cervical cancers (10.6%) among women was 3803, the third most common among cancers among stomach cancers (15.8%) and breast cancers (15.1%). In particular, the infection rate of young women in their 20s and 30s has increased significantly, accounting for 32% of all patients, and has become a serious health problem.

Cervical cancer usually develops as a final invasive cancer after HPV infects cervical epithelial basal cells through long-term progenitor stages such as low epithelial dysplasia, advanced epithelial dysplasia, and intraepithelial cancer. Since stage lesions are present, early treatment of these lesions will enable the prevention of cervical cancer. Indeed, in Israel, where early screening programs for cervical cancer and precancerous lesions are established, the incidence of cervical cancer is about 3.8 out of 100,000 people, compared to 48.2 in 100,000 people in developing countries such as Colombia.

To date, studies of cervical cancer have shown that HPV, which is infected by sexual contact, is the main factor. HPV is a circular double-stranded DNA virus of about 8 kb in length. The HPV genome contains the E1 to E7 genes involved in the initial process and the L1 and L2 genes involved in the late process after infecting the host cell. In particular, oncogenic proteins E6 and E7, which are expressed after HPV infects host epithelial cells, bind to host cell tumor suppressor proteins p53 and pRB to inhibit the function of these tumor suppressor proteins. It has been found to develop into tumorigenesis following transformation of infected cells.

To date, more than 120 different types of HPV are known depending on the differences in open reading frame (ORF) sequences of the E6, E7, and L1 genes. If the nucleotide sequence of the ORF is more than 10% different, it is defined as a new type, if it is more than 2% and less than 10%, it is defined as a subtype, and less than 2% is defined as a variant. Of these, about 37 HPV types (HPV-2, -3, -6, -7, -10, -11, -13, -16, -18, -26, -30, -31, -32,- 33, -35, -39, -40, -42, -43, -44, -45, -51, -52, -53, -54, -55, -56, -57, -58, -59, -61, -66, -67, -68, -69, -70, -73) have been shown to cause cervical cancer. Due to the close relationship between HPV infection and cervical cancer and high mortality due to cervical cancer, a method for early diagnosis of cervical cancer has been sought.

Currently, Pappanicolaou (Pap) smear based on the cytological morphology of cervical papules for the primary screening of cervical cancer and progenitor lesions has been performed as a standard test since the 1940s, greatly reducing mortality from cervical cancer. It has been reported that it still has a high false negative rate of 30-40%.

On the other hand, HPV hybrid capture Ⅱ detects the liquid complementary binding between HPV DNA and RNA probe of cervical cytology by antibody enzyme development, and has been used as a method of assisted screening or follow-up of cervical cancer and progenitor lesions since its development in 1994. It has recently been approved by the US FDA for use in primary screening. However, despite these analytical methods, there is still a high need for the development of a universal method for comprehensive early detection of cervical cancer and progenitor lesions, as evidenced by the number of deaths in the world of 5 to 500,000 deaths from cervical cancer.

In recent years, in order to detect a large number of HPV type infections, PCR techniques that can quickly and simply amplify DNA fragments having a specific sequence have become popular, and efforts have been actively made to comprehensively detect all HPVs that have been identified. .

The PCR technique adds a complementary nucleic acid (oligomeric primer) that specifically binds to the nucleic acid sequence of the target pathogen, and repeats the process of denaturation, annealing and polymerization depending on the temperature to remove the trace pathogen nucleic acid. As a technique capable of amplifying and detecting the presence (US Pat. Nos. 4683195 and 4683202), it is one of the representative techniques used to diagnose HPV infection.

Unlike the Pap smear cell test, the PCR technique can be objectively large-scaled in terms of the practicality of clinical diagnosis, and it has a comparative advantage in terms of test cost, experimental procedure, detection sensitivity, and specificity compared to cell test or liquid hybrid capture. It is pointed out to have. In light of these facts, HPV PCR screening is expected to be an attractive method for population exploration programs for comprehensive primary screening of cervical cancer and progenitor lesions. However, due to the lack of a general primer (General Primer) to search for a wide range of primary and primary HPV infection, there are many difficulties in its active clinical application.

Representative common primers that are widely used in clinical practice all over the world are the MY09 / MY11 primer set mainly used in North America, South America and Asia, GP5 ⁺ / GP6 ⁺ primer set and SPF1 / SPF2 primer set mainly used in Europe. Can be distinguished. In addition, primer sets such as FAP59 / FAP64 and PGMYO9 / PGMY11, which are newly developed and used recently, are now widely used.

 However, as a method for the primary screening of cervical cancer, the existing PCR primer sets are limited to the detection of a limited number of genotypes compared to the number of HPV types. In spite of the value and necessity that it possesses, the reality is that it is very limited in practical use.

Therefore, there is a growing need to develop HPV common primers that can be detected by simultaneously amplifying dozens of types of oncogenic HPV DNA as described above, and capable of early screening for cervical cancer by improving sensitivity according to HPV types.

The present invention has been proposed to solve the above problems, and an object of the present invention is to bind nucleotides capable of comprehensively amplifying many types of HPV through a single PCR process by complementarily binding to various types of HPV DNA. It is to provide.

Another object of the present invention is to amplify HPV DNA by using the nucleotides as a primer, thereby analyzing the presence of HPV, and to provide a method for early diagnosis of cervical cancer infection.

On the other hand, another object of the present invention to provide a kit for PCR that can amplify a variety of HPV type DNA by including the primer.

In order to achieve the above object, according to an aspect of the present invention provides a nucleotide (primer) capable of comprehensively amplifying HPV DNA which is divided into various types at the same time.

The primer is a nucleotide consisting of SEQ ID NO: 1 and SEQ ID NO: 2; Nucleotides consisting of SEQ ID NO: 2 and SEQ ID NO: 4; Nucleotides consisting of SEQ ID NO: 5 and SEQ ID NO: 6; Nucleotides consisting of SEQ ID NO: 7 and SEQ ID NO: 8; Nucleotides consisting of SEQ ID NO: 9 and SEQ ID NO: 10; Nucleotides consisting of SEQ ID NO: 11 and SEQ ID NO: 12; Nucleotides consisting of SEQ ID NO: 13 and SEQ ID NO: 14; And it is characterized in that the primer selected from nucleotides consisting of SEQ ID NO: 15 and SEQ ID NO: 16.

The primers according to the invention are HPV type 10, 11, 13, 16, 18, 30, 31, 32, 33, 35, 39, 40, 43, 44, 45, 52, 54, 55, 56, 58, 59, 61, 66, 68, 70 can be amplified, and other types of HPV DNA can be further amplified.

In another aspect of the present invention can perform amplification of various types of HPV DNA by performing PCR using the nucleotides selected above as a primer, and provides a method for detecting the presence of HPV DNA in the sample depending on whether or not amplification.

Hereinafter, the present invention will be described in more detail.

Common oligonucleotide primers that can hybridize with various types of HPV by conventional PCR methods have fewer detectable types and are less sensitive according to HPV types compared to the existing number of HPV types. However, in the present invention, a primer capable of hybridizing with more HPV types than conventional PCR common primers was designed, and one-time PCR was performed by the primers to detect various HPV types.

To this end, in the present invention, a representative PCR common primer was designed using a computer program based on a total of 72 HPV types. The designed primers were used to confirm the degree of amplification for various HPV types using different computer programs, and finally, 8 sets of primers were selected.

The primers selected above were synthesized using a nucleic acid autosynthesizer (Expedite ^™ 8909 synthesizer, ABI). The primers thus synthesized were first confirmed to amplify HPV by PCR using DNA of representative HPV infected cell lines as target DNA.

In addition, PCR primer amplification of various types of HPV plasmid DNA using the primers confirmed that all of the corresponding DNA amplification.

Furthermore, PCR amplification can be performed on samples obtained from the human body using the primers identified in the present invention, thereby prematurely diagnosing the infection of HPV depending on the amplification. In the present invention, 20 patients who were diagnosed as abnormal through clinical pathology examination such as colposcopy and 10 patients who were determined to be normal were selected as hybrid capture II (digene). DNA chip test (biomedlab) was performed. Finally, PCR test was performed using 7 sets of primers out of a total of 8 sets of common primers identified in the present invention. By performing the amplification by the common primer identified in the present invention, the same results as the above-described cytology, hybrid capture II, and DNA chip diagnosis were obtained, and it was confirmed that accurate HPV DNA infection and early diagnosis were possible. .

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only for describing the present invention in detail, and the present invention is not limited to the following examples.

Example 1

In this example, a total of eight sets of primers identified through computer simulations were synthesized, and Caski (ATCC CRL-1550), an HPV-16 infected cell line, HeLa (ATCC CCL-2), an HPV-18 infected cell line, and K562 (non HPV infected cell line). PCR amplification test was performed on KCLB-10243, Korean Cell LIne Bank).

(1) Design and Screening of PCR Primers

Nucleotide as a PCR common primer capable of comprehensively amplifying the HPV DNA was designed by binding complementarily with various HPV DNAs for the purpose of the present invention.

First, HPV-1a, -2a, -3, -4, -5, -6b, -7, -8, -9, -10, from the National Center for Biotechnology Information (NCBI) and the American Los Alamos HPV database. -11, -12, -13, -15, -16, -16r, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27 -28, -29, -30, -31, -32, -33, -34, -35, -35h, -36, -37, -38, -39, -40, -41, -42,- 44, -45, -47, -48, -49, -50, -51, -52, -53, -54, -55, -56, -57, -58, -59, -60, -61, Total DNA sequences of 72 HPV types were obtained: -63, -65, -66, -67, -68, -70, -72, -73, -75, -76, -77, and -80. The obtained DNA sequence is performed using the computer program 'DNASTAR (MegAlign ^TM 5, DNASTAR Inc.)' to perform pairwise alignment and multiple sequence alignment using the ClustalW method, followed by a Phylogenetic tree. Then, after sequencing the base sequences of each group, the PCR common primers were designed using the computer program primer premier 5 (PreMIER Biosoft International Co.). At this time, the primer length was set to oligonucleotides of 24 ± 3 and 33 ± 2, and PCR common primers were selected by limiting the size of the PCR product to 200bp to 550bp, and finally, eight sets of primers were selected.

(2) Virtual Amplification of Selected PCR Primers

Using a computer program (Amplify 1.2, University of Wisconsin) on a total of eight sets of primers designed in the above process, a virtual amplification experiment was performed on a total of 72 different HPV types already acquired in the design process. In particular, in this embodiment, HPV-2a, -3, -6b, -7, -10, -11, -13, -16, -18, -26, -30, -31, which have been found to be closely related to cervical cancer -32, -33, -34, -35, -39, -40, -42, -44, -45, -51, -52, -53, -54, -55, -56, -57, -58 8 sets of common primers that prioritize 36 HPV types, -59, -61, -66, -67, -68, -70, and -73 and amplify many other HPV types. The final screening was named AlbioGP1, AlbioGP2, AlbioGP3, AlbioGP4, AlbioGP5, AlbioGP6 and AlbioGP7, respectively. Marked on.

Table 1. Common primers selected for comprehensive amplification of various types of HPV DNA

primer SEQ ID NO: Amplified HPV Type Amplification Product Size (bp) Amplification Position (HPV 16) AlbioGP1 SEQ ID NO: 1 SEQ ID NO: 2 3, 6b, 7, 10, 11, 13, 16, 18, 26, 29, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 66, 68, 70, 73, 74, 75, 76, 77 388-406 6379 ~ 6773 AlbioGP2 SEQ ID NO: 3 SEQ ID NO: 4 2a, 3, 6b, 7, 10, 11, 13, 16, 18, 26, 27, 28, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 65, 66, 67, 68, 70, 72, 73, 74, 77 322-334 6226 ~ 6548 AlbioGP3 SEQ ID NO: 5 SEQ ID NO: 6 2a, 3, 6b, 10, 11, 13, 16, 18, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 39, 40, 43, 44, 45, 48, 49, 50, 51, 52, 54, 55, 56, 57, 58, 59, 61, 62, 64, 66, 67, 68, 70, 72, 73, 75, 76, 77 533-542 6230-6762 AlbioGP4 SEQ ID NO: 7 SEQ ID NO: 8 2a, 3, 6b, 7, 10, 11, 13, 16, 18, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 39, 40, 43, 44, 45, 51, 52, 54, 55, 56, 57, 58, 59, 61, 62, 64, 66, 68, 70, 73, 74, 77 210-242 6547-6762 AlbioGP5 SEQ ID NO: 9 SEQ ID NO: 10 2a, 7, 10, 11, 13, 16, 18, 28, 29, 30, 31, 32, 33, 35, 39, 40, 42, 43, 44, 45, 52, 53, 54, 55, 56, 58, 59, 61, 63, 66, 67, 68, 70, 72, 75, 76, 77 432-438 6119-6555 AlbioGP6 SEQ ID NO: 11 SEQ ID NO: 12 2a, 3, 6b, 7, 10, 11, 13, 16, 18, 26, 29, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 65, 66, 67, 68, 70, 73, 74, 77 298-305 5947-6618 AlbioGP7 SEQ ID NO: 13 SEQ ID NO: 14 2a, 3, 6b, 7, 10, 11, 13, 16, 18, 26, 29, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 65, 66, 67, 68, 70, 73, 74, 77 301-312 5947 ~ 6254 AlbioGP8 SEQ ID NO: 15 SEQ ID NO: 16 3, 6b, 7, 10, 11, 13, 16, 18, 26, 29, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 65, 66, 67, 68, 70, 73, 74 171-172 6379 ~ 6548

Odd sequence numbers of the sequence numbers in Table 1 are used as forward primers and even sequence numbers are used as reverse primers.

(3) Synthesis of Selected PCR Common Primers

Eight sets of primers including AlbioGP1, AlbioGP2, AlbioGP3, AlbioGP4, AlbioGP5, AlbioGP6, AlbioGP7 and AlbioGP8 selected in the above procedure were synthesized. The primer was synthesized using an Expedite ^™ 8909 nucleic acid synthesizer (ABI) equipped with a solid phase synthesis technique based on oligonucleotide phosphoramidite synthesis chemistry.

First, the synthesis reaction was carried out on a CPG column immobilized with nucleosides located at the 3 'end of the oligonucleotide, and basically detritylation, coupling, capping and oxidation reactions. The oligonucleotide polymerization reaction of the selected primers was carried out in a repeating cycle. After completion of the synthesis, 30% ammonia water was added to the CPG column to isolate the oligomer, and then deprotected at 55 ° C. for at least 12 hours, concentrated to dryness by Speed Vac., And then reversed phase liquid chromatography and anion exchange chromatography. Pure water purification was carried out. The final purified oligomer was quantified by measuring absorbance at 260 nm.

(4) PCR amplification test by synthesized PCR common primer

A total of eight sets of primers synthesized in the synthesis process were used to confirm amplification of HPV DNA.

In the present embodiment, Caski (ATCC CRL-1550), an HPV-16 infected cell line, HeLa (ATCC CCL-2), an HPV-18 infected cell line, and K562 (KCLB-10243, Korean Cell Line Bank), a non-HPV infected cell line, were used. PCR amplification tests were performed.

First, Caski and HeLa were incubated according to the manufacturer's instructions and then removed from the culture flask by 0.25% trypsin solution and centrifuged. K562 was also incubated according to the manufacturer's instructions and washed with Dulbecco's phosphate-buffered saline (Gibco). After measuring the number of cells under a microscope, 2 × 10 ⁶ cells were isolated and purified using Genomic DNA isolation Kit (Cat. No. K-3032, Bioneer) and finally dissolved in 200ul DW to use as template DNA solution. . In addition, each E. coli strain containing plasmid DNA of each HPV type was shaken for 16 hours in a 37 ° C. LB liquid medium and separated and purified by 'Qiafilter Plasmid Maxi Kit (Cat. No. 12263, QIAGEN)'. The concentration was adjusted to 100ng / μl and these were also used as template DNA solution.

The reaction composition for PCR experiments was obtained by using a 10 × buffer 2.5 μl, 10 mM MgCl ₂ 3.75 μl, 10 μm dNTP 0.5 μl, Taq polymerase 0.5 μl (1 unit) and 1 μl each of the primers purchased from Super Bio. 20 pmoles), 5.2 µl of distilled water and 8.0 µl of template DNA were adjusted to 25 µl in total.

The PCR cycle was pre-heated for 5 minutes at 94 ° C, followed by 50 cycles of 1 minute at 94 ° C, 1 minute at 50 ° C, and 1 minute at 72 ° C. 5 μl of the final reaction solution was added to a 2% agarose gel along with a DNA size standard marker for electrophoresis. At this time, the electrophoresis gel was stained with 0.00005% ethidium bromide solution, and the validity of the band appearing on each path of the gel was compared with the expected size of the PCR product obtained in the virtual amplification experiment of Table 1 above. Determined.

1A to 1H are electrophoresed images after PCR using Caski, HeLa, and K562 as targets, respectively, using eight sets of primers independently synthesized in the present invention. In the figure, M is a standard marker, and GP1 to GP8 at the top of the figure each represent AlbioGP1 primer to AlbioGP8 primer used in this example. Lane 1 is for Caski, lane 2 is for HeLa, lane 3 is for K562, and the number on the side indicates the size of the amplification product.

As can be seen in the figure, a total of eight sets of primers synthesized in the present Example was obtained for amplification products of the same size as predicted in Table 1 for Caski and HeLa, HPV infected cell lines, but for K562, a non-infected cell line It can be seen that amplification did not occur.

Example 2

In this example, seven sets of primers, such as AlbioGP 1 primer, AlbioGP 2 primer, AlbioGP 3 primer, AlbioGP 4 primer, AlbioGP 5 primer, AlbioGP 6 primer, and AlbioGP 7 primer, were used. PCR experiments were performed on E. coli strains containing various types of HPV plasmid DNA.

The strain used in this example is as follows.

HPV-2a (ATCC 45202);

HPV-6b (ATCC 45150);

HPV-11 (ATCC 45151);

HPV-16 (ATCC 45113);

HPV-18 (ATCC 45152);

HPV-31 (ATCC 65446);

HPV-43 (ATCC 40338);

Each E. coli strain containing HPV DNA of this type was incubated for 16 hours in a 37 ° C. LB liquid medium and then separated and purified by 'Qiafilter Plasmid Maxi Kit (Cat. No. 12263, QIAGEN)'. The concentration was adjusted to / μl, they were also used as template DNA solution.

PCR amplification was performed independently for each constituent primer for each template DNA according to the same reaction and procedure as in Example 1. The validity of the band appearing on each pathway of the gel was synthesized in Example 1 on the DNA of HPV-16, HPV-18, HPV-31, HPV-43, HPV-2a, HPV-6b, HPV-11. It was determined by comparing the size of the PCR product obtained in the computer virtual amplification experiment using the 7 sets of primers (see Table 2 below).

Figures 2a to 2g is a picture of the electrophoresis after performing a PCR on a variety of HPV strains using the 7 sets of primers synthesized in this example, respectively, Figure 2a is a ATCC 45113 strain, HPV-16 DNA is inserted, Figure 2b shows the ATCC 45142 strain inserted with HPV-18 DNA, FIG. 2c shows the ATCC 65446 strain inserted with the HPV-31 DNA, FIG. 2d shows the ATCC 40338 strain inserted with the HPV-43 DNA, and FIG. 2e shows the HPV-2a DNA inserted. The ATCC 45202 strain, FIG. 2F shows the ATCC 45140 strain inserted with the HPV-6b DNA, and FIG. 2G shows the ATCC 45151 strain inserted with the HPV-11 DNA as the target DNA. At the bottom of the figure, M is the standard marker, lane 1 is AlbioGP1, lane 2 is AlbioGP2, lane 3 is AlbioGP3, lane 4 is AlbioGP4, lane 5 is AlbioGP5, lane 6 is AlbioGP6, lane 7 is AlbioGP7 as a primer. It is. In addition, the number of a side shows the magnitude | size of the marker used in this Example.

As shown in the figure, the seven sets of primers (AlbioGP1 primer to AlbioGP7 primer) used in this example are the same size of amplification products as predicted in Table 2 in all lanes of all types of HPV used in this example. It can be seen that no band appears to be difficult to distinguish from the effective band in each path of all electrophoretic photographs.

 Table 2. Estimated Sizes of Selected Primer Amplification Products

primer Expected size of the amplification product (bp) HPV-16 HPV-18 HPV-31 HPV-43 HPV-2a HPV-6b HPV-11 AlbioGP1 397 400 397 391 395 397 392 AlbioGP2 325 325 325 325 322 323 325 AlbioGP3 534 537 534 537 539 531 531 AlbioGP4 215 215 213 214 214 215 215 AlbioGP5 438 438 438 438 432 438 438 AlbioGP6 298 298 298 298 297 293 297 AlbioGP7 301 301 301 301 302 301 301

Therefore, the seven sets of primers used in this example are capable of PCR amplification for HPV-16, HPV-18, HPV-31, HPV-43, HPV-2a, HPV-6b and HPV-11 DNA, respectively. It can be confirmed that no selective amplification is shown. This result is consistent with the selective specific amplification of 7 sets of primers predicted in the computer virtual amplification experiment.

Based on this, the results of computer virtual amplification (Table 2) for many HPV types not covered in this example were expected to be sufficient in the actual PCR amplification experiment.

Example 3

In this example, PCR experiments were performed on E. coli strains containing plasmids of various HPV types using AlbioGP 8 primers selected and synthesized in Example 1.

The strain used in this example is the same as the strain used in Example 2, and the isolation and PCR reaction of the strain DNA was carried out under the same conditions as in Examples 1 and 2.

However, in the present Example, unlike Example 2, only one PCR primer (AlgioGP 8 primer) was used, and electrophoresis was performed on different template plasmid DNA according to each path of the gel.

The validity of the bands that appeared on each pathway of the gel was simulated using AlbioGP 8 primers on the DNA of HPV-16, HPV-18, HPV-31, HPV-43, HPV-2a, HPV-6b and HPV-11. The amplification test was performed by comparing the size of the obtained PCR product (see Table 3 below).

Figure 3 is a photoelectrophoresis after the PCR for a variety of HPV strains using the AlbioGP8 primer. M is a standard marker and the number on the side indicates the size of the marker. Lane 1 shows ATCC 45113 strain with HPV-16 DNA inserted, lane 2 shows ATCC 45142 strain with HPV-18 DNA inserted, lane 3 shows ATCC 65446 with HPV-31NA inserted and lane 4 inserts HPV-43 DNA ATCC 40338 strain, lane 5 is ATCC 45202 strain inserted with HPV-2a DNA, lane 6 is ATCC 45140 strain inserted with HPV-6b DNA, lane 7 is ATCC 45151 strain inserted HPV-11 DNA as target DNA It is.

As shown in the figure, it can be seen that the AlbioGP8 primer used in this example had the same size amplification products as those predicted in Table 3 below for all HPV types of DNA selected as target DNA in this example. In each path of all electrophoretic photographs no bands were found which were difficult to distinguish from the effective bands.

Table 3. Estimated Sizes of AlbioGP8 Primer Amplification Products

primer Expected size of the amplification product (bp) HPV-16 HPV-18 HPV-31 HPV-43 HPV-2a HPV-6b HPV-11 AlbioGP8 171 172 172 171 171 172 172

Therefore, AlbioGP8 primers through the present embodiment are recognized for PCR amplification ability against HPV-16, HPV-18, HPV-31, HPV-43, HPV-2a, HPV-6b and HPV-11 DNA, respectively, non-selective amplification It was confirmed that not shown. The results were consistent with the selective specific amplifications predicted in the computer virtual amplification experiment.

Based on this, computer virtual amplification results (Table 3) for many HPV types not covered in this example are expected to have sufficient significance in actual PCR amplification experiments.

Comparative Example 1

In this comparative example, HPV infection was confirmed according to a conventional cell test method for clinical samples.

For women who visited the outpatient clinic of the Women's Cancer Center, Pochon College of Medicine, Korea, under the responsibility of a clinician, colposcopy, cervicography, biopsy, HPV DNA testing, and smear smear Specialized examinations such as pap-smear were conducted. The results of the examination through a cell test of a total of 30 patients are shown in Table 4 below.

As shown in Table 4 below, abnormal cervical cancer-related abnormal patients were 6, 9, 11, 12, 19, and 20 patients with unidentified atypical cells (ASCUS) in cytology. 1, 2, 5, 7, 8, 13, 14, 16, 17, and 18 and 3, 4, 10, and 15 patients with microinvasive cancer (SCC), and the remaining 21–30 patients were not associated with cervical cancer. Classified as normal.

Table 4. Clinical Trial Results by Cytometry

Subject No. Cytology Subject No. Cytology Subject No. Cytology One HSIL ^* 11 ASCUS 21 normal 2 HSIL ^** 12 ASCUS 22 normal 3 SCC ^*** 13 HSIL 23 normal 4 SCC 14 HSIL 24 normal 5 HSIL 15 SCC 25 normal 6 ASCUS 16 HSIL 26 normal 7 HSIL 17 HSIL 27 normal 8 HSIL 18 HSIL 28 normal 9 ASCUS 19 ASCUS 29 normal 10 SCC 20 ASCUS 30 normal

HSIL ^* : High Squamous Intraepithelial Lesions

SCC ^** : Squamous Cell Carcinoma

ASCUS ^*** : Atypical Squamous Cell Undetermined significance

Comparative Example 2

In this comparative example, the hybrid capture II method (Hybrid Capture II, Digene Co., Ltd.) was performed on 20 cervical cancer-related abnormal patients and normal persons not related to cervical cancer. The examination results according to the hybrid capture II method are briefly shown in Table 5 below.

As shown in Table 5, out of a total of 20 patients diagnosed as abnormal in Comparative Example 1, the subjects 1 to 11 and 13 to 19 were found to be high-risk HPV infection, and the subjects determined to be normal in Comparative Example 1 21-30 turned out to be non-HPV infection also in this comparative example. However, Subject 12 and Subject 20 determined to have unidentified atypical cells (ASCUS) in Comparative Example 1 were found to be normal in this Comparative Example.

Table 5. Clinical Trial Results for HPV Infection Using Hybrid Capture II

Subject No. Examination Results (High / Low) Subject No. Examination Results (High / Low) One +/- 16 +/- 2 +/- 17 +/- 3 +/- 18 +/- 4 +/- 19 +/- 5 +/- 20 +/- 6 +/- 21 -/- 7 +/- 22 -/- 8 +/- 23 -/- 9 +/- 24 -/- 10 +/- 25 -/- 11 +/- 26 -/- 12 -/- 27 -/- 13 +/- 28 -/- 14 +/- 29 -/- 15 +/- 30 -/-

Example 4

In this example, the clinical test was performed on the patient DNA obtained through the comparative example using 7 sets of primers (AlbioGP 1 primer to AlbioGP 7 primer) whose amplification ability was confirmed in Examples 1 and 2. In addition, HPV infection, as well as cervical cancer, could be the first screening test depending on DNA amplification.

First, DNA samples obtained from the subjects obtained for the clinical trials of Comparative Examples 1 and 2 were subjected to PCR and electrophoresis using a total of 7 sets of primer sets identified in Examples 1 and 2 above. DNAs of the subjects were obtained according to a conventional method, and PCR reactions were carried out under the same conditions and procedures as in Example 1 and Example 2 above.

As a result of the PCR reaction, a total of 7 sets of primer sets synthesized in Example 1 were amplified products were obtained only for DNA samples obtained from patients (Subjects 1 to 20) found to be abnormal in Comparative Example 1, It was confirmed that amplification did not occur for DNA samples obtained from patients found to be normal in (21 to 30 subjects). This was consistent with the etiological reasoning of 20 patients with cervical cancer and 10 patients with unrelated cervical cancer.

4A to 4T are photographs obtained by conducting PCR and electrophoresis on DNA samples obtained from abnormal patients determined to be related to cervical cancer as a result of the screening of Comparative Example 1 using the 7 sets of primer sets according to the present invention. . 4A is a subject 1, FIG. 4B is a subject 2, FIG. 4C is a subject 3, FIG. 4D is a subject 4, FIG. 4E is a subject 5, FIG. 4F is a subject 6, FIG. 4G is a subject 7, FIG. 4H is a subject 8, FIG. 4I FIG. 9 is a subject 9, FIG. 4J is a subject 10, FIG. 4K is a subject 11, FIG. 4L is a subject 12, FIG. 4M is a subject 14, FIG. 4N is a subject 14, FIG. 4O is a subject 15, FIG. 4P is a subject 16, and FIG. 4Q is a subject. 17, FIG. 4R shows subject 18, FIG. 4S shows subject 19 and FIG. 4T shows DNA obtained from a patient of subject 20. FIG. Where M is the standard marker, lane 1 is AlbioGP1 primer, lane 2 is AlbioGP2 primer, lane 3 is AlbioGP3 primer, lane 4 is AlbioGP4 primer, lane 5 is AlbioGP5 primer, lane 6 is AlbioGP6 primer and lane 7 is AlbioGP7 primer The number on the side is the size of the marker.

As shown, it can be seen that the amplification occurred for all the subjects identified as being related to cervical cancer by cytology, the size was found to be consistent with the virtual amplification result of Table 1 above.

5A to 5J are photographs obtained by conducting PCR and electrophoresis on DNA samples obtained from normal patients determined to be unrelated to cervical cancer as a result of the screening of Comparative Example 1 using the 7 sets of primer sets according to the present invention. . 5A is a subject 21, FIG. 5B is a subject 22, FIG. 5C is a subject 23, FIG. 5D is a subject 24, FIG. 5E is a subject 25, FIG. 5F is a subject 26, FIG. 5G is a subject 27, FIG. 5H is a subject 28, FIG. 5I. Figure 2J, Figure 5J is for the DNA obtained from the subject of the subject 30. Where M is the standard marker, lane 1 is AlbioGP1 primer, lane 2 is AlbioGP2 primer, lane 3 is AlbioGP3 primer, lane 4 is AlbioGP4 primer, lane 5 is AlbioGP5 primer, lane 6 is AlbioGP6 primer and lane 7 is AlbioGP7 primer The number on the side is the size of the marker.

As shown, it can be seen that no amplification products were obtained for all the subjects identified by cytology not related to cervical cancer.

While the embodiments of the present invention have been described above, these are only examples, and it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit of the present invention, but all of them belong to the scope of the present invention. The claims will be clarified.

As described above, the oligonucleotides synthesized in the present invention can be used as novel PCR consensus primers that can complementarily bind a variety of HPV types to comprehensively amplify the HPV types.

In addition, the primer can be used as a probe that can directly detect a variety of HPV, and can be used to detect the presence of HPV type by performing PCR and amplification of the primer and the target HPV to HPV It can be used for the early examination of cervical cancer caused by.

       Genetic approach using this genome can diagnose the infection early, so that it is possible to carry out continuous and long-term examinations later, to detect it early, and to prevent the occurrence of cervical cancer by blocking the occurrence factors through prior education. You can expect the effect.

       In particular, by conducting HPV diagnosis on a low budget, it is possible to maximize the efficiency of the project and build satisfaction and reliability that the examinee can receive advanced results ahead of others.                     

       Moreover, it can play a part in promoting the health of the whole nation at the national level, and is expected to be a new opportunity for the public relations effect and the change of public awareness.

<110> ALBIOMED Co., LTD <120> Novel PCR General Primers and Method for Detecting Diverse Types          of Human Papillomavirus by PCR <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 1 ttcttcttac gaagggaaca actgtttgtt agaca 35 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 2 cataattgaa acataaactg taaatcatat tcctc 35 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 3 gatggtgata tggtagatac aggatttgg 29 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 4 gcgtcagagg ttaccataga gccactagg 29 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 5 tgatatggtt catacaggat ttgg 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 6 aataaactgt aaatcatatt cctc 24 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 7 gcacaactat ttaataagcc atattgg 27 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 8 aataaactgt aaatcatatt cctc 24 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 9 tgtacctgct attggggaac actgggctaa ggg 33 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 10 taattgggaa tcagaagtaa ccatagagcc act 33 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 11 gaggtgggcc ggggncarcc nyt 23 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 12 aagccggtgt cgaccatrtc nccrtcyt 28 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 13 gaggtgggcc ggggncarcc nyt 23 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 14 ccgaagccgg tgtcgaycat rtcnccrt 28 <210> 15 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 15 ttcttcttac gaagggaaca actgtttgtt agaca 35 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amplifying HPV <400> 16 gcgtcagagg ttaccataga gccactagg 29

Claims (13)

Nucleotides (first primers) consisting of SEQ ID NO: 1 and SEQ ID NO: 2; Nucleotides (second primer) consisting of SEQ ID NO: 3 and SEQ ID NO: 4; Nucleotides consisting of SEQ ID NO: 5 and SEQ ID NO: 6 (third primer); Nucleotides (fourth primer) consisting of SEQ ID NO: 7 and SEQ ID NO: 8; Nucleotides (fifth primer) consisting of SEQ ID NO: 9 and SEQ ID NO: 10; Nucleotide (sixth primer) consisting of SEQ ID NO: 11 and SEQ ID NO: 12; Nucleotide (seventh primer) consisting of SEQ ID NO: 13 and SEQ ID NO: 14; or A synthesized primer for amplifying a variety of HPV DNA selected from the group consisting of nucleotides (eighth primer) consisting of SEQ ID NO: 15 and SEQ ID NO: 16, at least one trillion or more. The method of claim 1, The first to eighth primers are HPV type 10, 11, 13, 16, 18, 30, 31, 32, 33, 35, 39, 40, 43, 44, 45, 52, 54, 55, 56 As a primer for amplifying HPV DNA selected from 58, 59, 61, 66, 68 and 70, The first primer can further amplify HPV DNA selected from HPV types 3, 6b, 7, 29, 34, 42, 51, 53, 60, 62, 64, 73, 74, 75, 76 and 77, The second primer will further amplify HPV DNA selected from HPV types 2a, 3, 6b, 7, 26, 27, 28, 34, 35, 51, 53, 57, 60, 67, 72, 73, 74 and 77. Can, The third primer is selected from HPV type 2a, 3, 6b, 26, 27, 28, 29, 34, 48, 49, 50, 51, 57, 62, 64, 67, 72, 73, 75, 76 and 77 Can further amplify the HPV DNA, The fourth primer further comprises HPV DNA selected from HPV types 2a, 3, 6b, 7, 26, 27, 28, 29, 34, 43, 44, 45, 51, 57, 62, 64, 73, 74 and 77. Can be amplified, The fifth primer can further amplify HPV DNA selected from HPV types 2a, 7, 28, 29, 42, 53, 63, 66, 67, 68, 72, 75, 76 and 77, The sixth primer can further amplify HPV DNA selected from HPV types 2a, 3, 6b, 7, 26, 29, 34, 42, 51, 53, 60, 65, 67, 73, 74 and 77, The seventh primer can further amplify DNA selected from HPV types 2a, 3, 6b, 26, 29, 34, 51, 53, 60, 65, 67, 73, 74 and 77, The eighth primer can further amplify HPV DNA selected from HPV types 2a, 3, 6b, 7, 26, 29, 34, 42, 51, 53, 60, 65, 67, 70, 73 and 74. Synthesized primers to amplify various HPV DNA. The method of claim 1, The first primer can amplify at least one HPV DNA selected from HPV type 6b, 11, 16, 18, 31, 43, The second to fourth primers, sixth primers, and seventh primers may amplify at least one HPV DNA selected from HPV types 2a, 6b, 11, 16, 18, 31, and 43, respectively, Wherein said fifth primer is capable of amplifying at least one HPV DNA selected from HPV type 2a, 11, 16, 18, 31, 43. The method of claim 1, Wherein said first to eighth primers amplify various HPV DNAs capable of amplifying at least one DNA of HPV-16 or HPV-18 DNA, respectively. Nucleotide (first primer) consisting of SEQ ID NO: 1 and SEQ ID NO: 2, Nucleotide (second primer) consisting of SEQ ID NO: 3 and SEQ ID NO: 4, Nucleotide (third primer) consisting of SEQ ID NO: 5 and SEQ ID NO: 6, Nucleotide (fourth primer) consisting of SEQ ID NO: 7 and SEQ ID NO: 8, Nucleotide (fifth primer) consisting of SEQ ID NO: 9 and SEQ ID NO: 10, Nucleotide (sixth primer) consisting of SEQ ID NO: 11 and SEQ ID NO: 12, A nucleotide (seventh primer) consisting of SEQ ID NO: 13 and SEQ ID NO: 14 or Using as a primer at least one nucleotide selected from the group consisting of a nucleotide (eighth primer) consisting of SEQ ID NO: 15 and SEQ ID NO: 16, A method of amplifying DNA of various HPV by performing a polymerase chain reaction (PCR) using a nucleic acid obtained from a biological sample as a target DNA. The method of claim 5, The first to eighth primers are HPV type 10, 11, 13, 16, 18, 30, 31, 32, 33, 35, 39, 40, 43, 44, 45, 52, 54, 55, 56 As a primer for amplifying HPV DNA selected from 58, 59, 61, 66, 68 and 70, The first primer can further amplify HPV DNA selected from HPV types 3, 6b, 7, 29, 34, 42, 51, 53, 60, 62, 64, 73, 74, 75, 76 and 77, The second primer will further amplify HPV DNA selected from HPV types 2a, 3, 6b, 7, 26, 27, 28, 34, 35, 51, 53, 57, 60, 67, 72, 73, 74 and 77. Can, The third primer is selected from HPV type 2a, 3, 6b, 26, 27, 28, 29, 34, 48, 49, 50, 51, 57, 62, 64, 67, 72, 73, 75, 76 and 77 Can further amplify the HPV DNA, The fourth primer further comprises HPV DNA selected from HPV types 2a, 3, 6b, 7, 26, 27, 28, 29, 34, 43, 44, 45, 51, 57, 62, 64, 73, 74 and 77. Can be amplified, The fifth primer can further amplify HPV DNA selected from HPV types 2a, 7, 28, 29, 42, 53, 63, 66, 67, 68, 72, 75, 76 and 77, The sixth primer can further amplify HPV DNA selected from HPV types 2a, 3, 6b, 7, 26, 29, 34, 42, 51, 53, 60, 65, 67, 73, 74 and 77, The seventh primer can further amplify DNA selected from HPV types 2a, 3, 6b, 26, 29, 34, 51, 53, 60, 65, 67, 73, 74 and 77, The eighth primer is a primer capable of further amplifying HPV DNA selected from HPV types 2a, 3, 6b, 7, 26, 29, 34, 42, 51, 53, 60, 65, 67, 70, 73, and 74. How to amplify various HPV DNA. The method of claim 5, The first primer can amplify at least one HPV DNA selected from HPV type 6b, 11, 16, 18, 31, 43, The second to fourth primers, sixth primers, and seventh primers may amplify at least one HPV DNA selected from HPV types 2a, 6b, 11, 16, 18, 31, and 43, respectively, The fifth primer is capable of amplifying at least one HPV DNA selected from HPV type 2a, 11, 16, 18, 31, 43. The method of claim 5, Wherein the first to eighth primers amplify a variety of HPV DNA that can amplify at least one DNA of HPV-16 or HPV-18 DNA, respectively. The method of claim 5, The target DNA is a method for amplifying a variety of HPV DNA, characterized in that obtained from human. Nucleotide (first primer) consisting of SEQ ID NO: 1 and SEQ ID NO: 2, Nucleotide (second primer) consisting of SEQ ID NO: 3 and SEQ ID NO: 4, Nucleotide (third primer) consisting of SEQ ID NO: 5 and SEQ ID NO: 6, Nucleotide (fourth primer) consisting of SEQ ID NO: 7 and SEQ ID NO: 8, Nucleotide (fifth primer) consisting of SEQ ID NO: 9 and SEQ ID NO: 10, Nucleotide (sixth primer) consisting of SEQ ID NO: 11 and SEQ ID NO: 12, Nucleotide (seventh primer) consisting of SEQ ID NO: 13 and SEQ ID NO: 14, or At least one group selected from the group consisting of a nucleotide (eighth primer) consisting of SEQ ID NO: 15 and SEQ ID NO: 16; dNTP mixtures (dATP, dCTP, dGTP, dTTP); Heat resistant polymerase; And Kits for detecting a variety of HPV types, including PCR buffers. The method of claim 10, The first to eighth primers are HPV type 10, 11, 13, 16, 18, 30, 31, 32, 33, 35, 39, 40, 43, 44, 45, 52, 54, 55, 56 As a primer for amplifying at least one HPV DNA selected from 58, 59, 61, 66, 68 and 70, The first primer can further amplify HPV types 3, 6b, 7, 29, 34, 42, 51, 53, 60, 62, 64, 73, 74, 75, 76 and 77 DNA, The second primer can further amplify HPV types 2a, 3, 6b, 7, 26, 27, 28, 34, 35, 51, 53, 57, 60, 67, 72, 73, 74 and 77 DNA, The third primer contains HPV types 2a, 3, 6b, 26, 27, 28, 29, 34, 48, 49, 50, 51, 57, 62, 64, 67, 72, 73, 75, 76 and 77 DNA. You can amplify it further, The fourth primer can further amplify HPV types 2a, 3, 6b, 7, 26, 27, 28, 29, 34, 43, 44, 45, 51, 57, 62, 64, 73, 74 and 77 DNA. There is, The fifth primer can further amplify HPV types 2a, 7, 28, 29, 42, 53, 63, 66, 67, 68, 72, 75, 76 and 77 DNA, The sixth primer can further amplify HPV types 2a, 3, 6b, 7, 26, 29, 34, 42, 51, 53, 60, 65, 67, 73, 74 and 77 DNA, The seventh primer can further amplify HPV types 2a, 3, 6b, 26, 29, 34, 51, 53, 60, 65, 67, 73, 74 and 77 DNA, The eighth primer is a variety of HPV types that are primers that can further amplify HPV types 2a, 3, 6b, 7, 26, 29, 34, 42, 51, 53, 60, 65, 67, 70, 73 and 74 DNA. Kit to detect. The method of claim 10, The first primer can amplify at least one HPV DNA selected from HPV type 6b, 11, 16, 18, 31, 43, The second to fourth primers, sixth primers, and seventh primers may amplify at least one HPV DNA selected from HPV types 2a, 6b, 11, 16, 18, 31, and 43, respectively, The fifth primer is a kit for detecting a variety of HPV types, characterized in that the amplification of at least one HPV DNA selected from HPV type 2a, 11, 16, 18, 31, 43. The method of claim 10, Wherein the first to eighth primers detect various HPV types capable of amplifying at least one DNA of HPV-16 or HPV-18 DNA, respectively.

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