KR100576610B1 - Primers and Method for Detecting High Risk Human Papillomavirus by PCR - Google Patents

Abstract

The present invention uses a primer capable of selectively amplifying high-risk HPV type DNA, which is found to have a high incidence of cervical cancer by polymerase chain reaction, and selectively amplifies a high-risk HPV DNA present in a sample using the primer. The present invention relates to a method for analyzing the presence of HPV and effectively detecting the high risk HPV.

HPV PCR kit (kit) containing the primer according to the present invention is economical, accurate and efficient compared to the conventional method, it is possible to detect high risk group HPV DNA simply and quickly.

Human Papillomavirus (HPV), Uterine Cervical Cancer, Primer,

Description

Primers and Methods for Detecting High Risk Human Papillomavirus by PCR for Detection of High Risk Human Papilloma Virus by Polymerase Chain Reaction             

1A to 1D show three sets of primers synthesized in the present invention using CaSki, an HPV-16 infected cell line, HeLa, an HPV-18 infected cell line, and K562, an HPV uninfected cell line, and a combination of two of these primers. The electrophoresis photographs after PCR using the set.

2a to 2d are HPV plasmids of HPV-16, HPV-18, HPV-31, HPV-43, HPV-2a, HPV-6b, HPV-11 and HPV-1a types as target DNA, respectively. Electrophoresis is performed after PCR using three sets of primers synthesized and two sets of primers used in combination.

Figures 3a to 3y using three sets of primers synthesized in the present invention and the combined primer set, respectively, cervical abnormalities [(microscopic invasive cancer (SCC), unidentified atypical cells (ASCUS), high grade lesions (HSIL) )] Is a picture of electrophoresis after PCR on DNA obtained from the human body.

Figures 4a to 4o after the PCR of the DNA obtained from a low-grade lesion (LSIL) patients and normal humans in cervical abnormalities using the three sets of primers and the combination primer set synthesized in the present invention, respectively It is an electrophoretic picture.

The present invention relates to a primer that can be used for a polymerase chain reaction (hereinafter, referred to as "PCR") and an amplification method using the primer, and more particularly to a high risk human papillomavirus. The present invention relates to a nucleotide capable of complementarily binding to and amplifying a nucleic acid and a method for detecting the presence of a high risk human papillomavirus using the nucleotide as a primer.

Human Papillomavirus (HPV) is an 8kb circular double-helix virus. All types of HPV viruses have open reading frames (ORFs), which are DNA fragments that synthesize proteins of similar nature, and are largely early genes. It is divided into (early gene) and late gene (late gene). The initial gene, about 4.5 kb, is responsible for viral DNA replication (E1), inducing or inhibiting the action of DNA to form proteins that cause malignant transformation of host cells (E2), and for proteins involved in host cell and viral growth. Synthesis (E4), effector activation of epidermal growth factor (EGF) and colony stimulator factor (CSF) receptors (E5), permanent survival of cells and malignant transformation (E7) due to the mechanism of cancer gene activity and inactivation of cancer suppressor Can be divided into 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.

On the other hand, the 2.5 kb late gene is 1 kb in size, which is the DNA responsible for the synthesis of the viral envelope protein (L1) and the envelope protein (L2), and a non-coding region that controls their transcription and translation functions. It consists of LCR (long control region).

Recently, with the rapid development of molecular biological techniques, the genetic structure of HPV has been revealed, and many types of (genotype) HPV genome sequences have been revealed. HPV is determined by the difference in the ORF sequence of the E6, E7, and L1 genes.If the nucleotide sequence of the ORF differs by more than 10%, it is defined as a new type. subtype), where less than 2% is defined as a variant. According to this classification, more than 120 types of HPV are known so far.

HPV has not only been associated with genital cancer, laryngeal cancer and tongue cancer, but has also been identified as an essential maintenance factor for onset of cancer and cancer, particularly in 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.

Among them, HPV-16, -18, -26, -30, -31, -33, -34, -35, -39, -45, -51, -52, -53, -56, -58, -59 HPV types such as -61, -66, -67, -68, -69, -70, and -73 have been shown to have a relatively high rate of progression to cervical cancer, leading to a 'high-risk' HPV. Are classified, such as HPV -2, -3, -6, -7, -10, -11, -13, -32, -40, -42, -43, -44, -55, -54 and -57 Types of HPV are classified as 'low-risk' HPV due to their low progression to 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. And molecular biological methods.

The cytological method of observing HPV-infected cells for morphological changes has been a standard test for HPV infection since the 1940's and has greatly reduced mortality from cervical cancer. Cytological methods of diagnosing cervical cancer include cytopathology (Papanicolaou [Pap] Smear) system, CIN (cervical intraepithelial neoplasia) system, World Health Organization World health organization (WHO) and TBS (The bethesda system).

Although cytological methods have advantages in economics, ease of operation, and safety, they are not diagnosed by the shape of the cells, which results in low objectivity of diagnosis and inconsistent accuracy due to high false negative rate (15-45%). It is pointed out.

On the other hand, the molecular biological methods can be divided into the method of directly identifying the HPV DNA and the PCR method by amplification. The molecular biological method has the advantage that it is possible to objectively detect the viral DNA causing cervical cancer and to make the diagnosis accurate. Therefore, in cases where the diagnosis is unclear in cytology, histology, etc., cervical cancer can be diagnosed early before the pathological diagnosis of cervical cancer. .

For direct identification of HPV DNA, methods such as Southern blotting, dot blotting and Filter in situ hybridization (FISH) using conventional HPV type-specific probes have been used. System (Hybrid Capture System, Digene) has been developed and widely used. Hybrid capture has the advantage of high accuracy and high risk / low risk compared to the existing HPV DNA diagnosis method.However, since it requires a large amount of HPV DNA, the initial capture or insufficient sampling of HPV is insufficient. The detection limits and the use of RNA probes not only result in low safety, but also result in relatively expensive and insensitive sensitivity of the equipment.

The PCR method is a technology capable of amplifying a trace amount of a pathogen nucleic acid by using a complementary nucleic acid (oligomeric primer) that specifically binds to a nucleic acid sequence of a target pathogen and detecting the presence thereof (US Pat. Nos. 4683195 and 4683202). ), Compared with the above-described cytological method and direct HPV DNA test, the relative superiority in terms of accuracy, ease and cost is evaluated.

To date, primers used in PCR-based HPV detection methods are either general primers capable of amplifying the DNA regardless of the HPV type or type-specific primers capable of selectively amplifying only the type-specific HPV DNA. primers have been developed.

However, even if amplification products are obtained by PCR reaction using a common primer, it is difficult to confirm the exact progression of cervical cancer because it is not possible to determine whether it is a high-risk HPV type closely related to cervical cancer. In other words, using a common primer that can be complementarily bound to all types results in amplification consistently regardless of HPV type, making it difficult to accurately predict the progression of HPV infection or the spread of cervical cancer. In order to solve the problem, a method such as the hybrid capture described above must be used.

On the other hand, when using the type specific primers that can specifically bind to each type, there is a need to use different primers to confirm the presence of HPV in the sample. In other words, even if a patient is infected with HPV and is infected with a type of HPV that cannot bind complementarily with a type-specific primer, the amplification product is not obtained by the PCR reaction. It can be diagnosed as if it is a normal patient, and in the end, various primers have to be used at the same time, and thus, there is a limit to wide use clinically.

In conclusion, in order to increase the practical efficiency of the diagnosis of cervical cancer according to the amplification of HPV DNA using PCR, the high-risk HPV type, which is found to have a high progression to cervical cancer, is selectively included and amplified. There is an urgent need for the development of primers that can detect early detection and simple diagnosis of cervical cancer caused by high-risk HPV.

The present invention has been proposed to solve the above problems, and an object of the present invention is to perform a single PCR reaction on a high-risk HPV type DNA that is closely related to the progression of cervical cancer among more than 100 different types of HPV. It is to provide a primer that can be detected by the amplification according.

Another object of the present invention is to amplify a target DNA using the primer and to provide a method for analyzing the presence or absence of high-risk HPV type according to the amplification.

Another object of the present invention is to provide a method for identifying the presence of a high risk group HPV type in a sample quickly, accurately, efficiently and economically.

In order to achieve the above object, the present invention provides a primer composed of oligonucleotides capable of amplifying a high-risk human Papillomavirus (HPV).

The oligonucleotide is a nucleotide consisting of SEQ ID NO: 1 and SEQ ID NO: 2; Nucleotides consisting of SEQ ID NO: 3 and SEQ ID NO: 4; And at least one trillion or more high-risk HPV DNA selected from the group consisting of nucleotides consisting of SEQ ID NO: 5 and SEQ ID NO: 6.

In another aspect, the present invention provides a method for amplifying a target DNA according to a PCR reaction using the above-described primer and detect the presence of a high risk group HPV type according to the amplification.

The present invention also provides a kit for PCR capable of amplifying a specific type of HPV DNA, including the primers, dNTP, heat resistant polymerase and PCR buffer.

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

Detection of high-risk HPV by conventional hybrid capturing has a lower number of detectable types and a lower sensitivity depending on HPV types compared to the number of 100 or more HPV types detected to date. However, in the present invention, many high-risk HPV types could be detected by synthesizing primers capable of quickly and accurately detecting high-risk HPV types and performing one-time PCR amplification by the primers.

As described above, more than 120 types of HPV have been identified to date, and are classified into high-risk HPV type and low-risk HPV type according to the relevance of cervical cancer. High-risk HPV types include HPV -16, -18, -26, -30, -31, -33, -34, -35, -39, -45, -51, -52, -53, -56, and -58 , -59, -61, -66, -67, -68, -69, -70, and -73. Low-risk HPV types include HPV -2, -3, -6, -7, -10 , -11, -13, -32, -40, -42, -43, -44, -55, -54 and -57.

Therefore, in the present invention, a primer capable of detecting a high risk group HPV type with high progression to cervical cancer was designed, and a single PCR was performed by the primer to detect more high risk group HPV types. To this end, in the present invention, PCR primers specific to the high risk group were designed using a computer program based on the high risk group HPV DNA sequence having high progression to cervical cancer among all 72 HPV types. The designed primers were used to confirm the degree of amplification for various HPV types using different computer programs, and finally, three 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 identified by HPS-type specific amplification by PCR using the representative HPV infected cell lines CaSki (HPV-16) and HeLa (HPV-18) DNA as target DNA.

Subsequently, PCR amplification was performed by targeting the various types of HPV plasmid DNA using the three sets of primers and one set of combination primers. As a result, all of the corresponding specific types of HPV DNA were selectively amplified. It was confirmed.

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. That is, in the present invention, cervical cancer-related abnormal patients [(microinvasive cancer (CFC)) through professional examinations such as cervicography, biopsy, and pap smear under the responsibility of the clinician. SCC), unidentified atypical cells (ASCUS), low grade lesions (LSIL), high grade lesions (HSIL)] and the hybrid capture II method (hybrid capture II, Digene) in eight healthy individuals who were found to be unrelated to cervical cancer. As a result, PCR experiments were performed using the primers identified in the present invention to detect more types of HPV than the hybrid capture II method, thereby providing accurate HPV infection and early diagnosis. It was confirmed that possible.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1

In this embodiment, three sets of primers identified through computer simulations were synthesized, and CaSki, an HPV-16 infected cell line, HeLa, an HPV-18 infected cell line, and K562, an uninfected HPV line, were used, respectively, using the primers and a set of combination primers. PCR was performed.

(1) Design and Screening of PCR Primers

In this example, nucleotides as PCR primers capable of comprehensively amplifying the DNA by complementarily binding to high-risk HPV DNA as a major inducer of cervical cancer were designed. First, HPV-1a, -2a, -3, -4, -5, -6b, -7, -8, -9, -10 from the National Center for Biotechnology Information (NCBI) and the US National 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 A total of 72 HPV types, including -63, -65, -66, -67, -68, -70, -72, -73, -75, -76, -77, and -80, were obtained. . The obtained DNA sequence is subjected to pairwise alignment and multiple sequence alignment using the ClustalW method using the computer program 'DNA STAR (MegAlign ^TM 5, DNASTAR Inc.)' and then the Phylogenetic tree ), Each of the groups selected sequences representing the high-risk HPV type, and then designed a PCR primer that amplifies only the high-risk HPV type by using the computer program primer premier 5 (PreMIER Biosoft International Co.). . At this time, the primer length was set to oligonucleotides of 27 ± 3 and 31 ± 2, and the PCR product was designed by limiting the size of the PCR product to approximately 300bp.

Three sets of primers designed in this way were selected and named as AlbioHRP1, AlbioHRP2 and AlbioHRP3, respectively.

(2) Virtual Amplification of Selected PCR Primers

Amplification degree was confirmed using a different computer program (Amplify 1.2, University of Wisconsin) for a total of three sets of primers selected in the above process. To this end, virtual amplification experiments were performed on a total of 72 different HPV types already acquired in the design process.

In this example, HPV-2a, -3, -6b, -7, -10, -11, -13, -16, -18, -26, -30, -31, -32, -33 associated with cervical cancer , -34, -35, -39, -40, -42, -44, -45, -51, -52, -53, -54, -55, -56, -57, -58, -59,- Focusing on the amplification of the high-risk HPV type out of 36 of 61, -66, -67, -68, -70, and -73, the sequence number, amplification type, and size of the amplification products according to the selected primers , Predicted amplification position, the results are shown in Table 1 below.

Table 1. Primers selected for specific amplification of high risk HPV DNA

primer SEQ ID NO: Amplified HPV Type Amplification Product Size (bp) AlbioHRP1 SEQ ID NO: 1 SEQ ID NO: 2 16, 31, 33, 34, 35, 35H, 52, 53, 58, 59, 66, 67, 73 300-314 AlbioHRP2 SEQ ID NO: 3 SEQ ID NO: 4 26, 30, 33, 35, 35H, 51, 52, 53, 56, 58, 66 297-312 AlbioHRP3 SEQ ID NO: 5 SEQ ID NO: 6 18, 39, 45, 59, 68, 70 277-286

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

(3) Synthesis of Selected PCR Primers

Three primers of AlbioHRP1, AlbioHRP2, and AlbioHRP3 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 having nucleosides immobilized at the 3 'end of the oligonucleotide, basically detritylation, coupling, capping, and oxidation reactions. The oligonucleotide polymerization reaction of the selected PCR 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

Using three sets of primers synthesized in the synthesis process and two sets of primers (AlbioHRP1 and AlbioHRP3), Caski (ATCC CRL-1550), an HPV-16 infected cell line, and HeLa (ATCC), an HPV-18 infected cell line PCR amplification test was performed on CCL-2) and non-infected cell line K562 (KCLB-10243, Korean Cell Line Bank).

First, CaSki and HeLa were incubated according to the manufacturer's instructions, and then removed from the culture flask by adding 0.25% trypsin solution and centrifuged. The cells were washed once with Dulbecco's phosphate-buffered saline (Gibco), and the number of cells was measured under a microscope. Then, 2 × 10 ⁴ cells were suspended in 150 μl of distilled water and heated at 100 ° C. for 15 minutes to extract cellular DNA. Finally, they were completely cooled at room temperature, centrifuged at 12000 rpm for 5 minutes, and the supernatant was taken as a template DNA solution.

PCR experiments were performed by using the following procedure: 2.5 μl of 10 × buffer, 3.75 μl of 10 mM MgCl ₂ , 0.5 μl of 10 mM dNTP, 0.5 μl of Taq polymerase (5 unit), 1 μl of primer (50 pmoles), and distilled water from SuperBio. The reaction solution consisting of 11.5 μl of 5.2 μl template DNA was sufficiently denatured by reaction at 94 ° C. for 5 minutes, and then 94 ° C. 1 minute-58 ° C. 1 minute-72 ° C. 1 minute was repeated 30 times and finally extended at 72 ° C. for 5 minutes. . 5 μl of the final reaction solution was subjected to electrophoresis by imposing on a 2% agarose gel along with a DNA size standard marker. At this time, the electrophoresis gel was stained with 0.00005% ethidium bromide solution, and the validity of the band appeared in each path of the gel was determined by comparing with the expected size of the PCR product obtained in the virtual amplification experiment of Table 1. It was.

1A to 1D show HPV-16 infected cell line CaSki, HPV-18 infected cell line HeLa, and uninfected using three sets of primers synthesized in the present invention and two sets of them (AlbioHRP1 and AlbioHRP3). Electrophoresis after PCR using cell line K562 as a target DNA.

In the figure, M is a standard marker, lane 1 is CaSki, lane 2 is HeLa, lane 3 is K562 as the target DNA, and the number on the side indicates the size of the marker. As can be seen in the figure, amplification of AlbioHRP1 primers was performed on CaSki, an HPV-16 infected cell line (FIG. 1A), and amplification of AlbioHRP2 primers did not occur on both infected and non-infected cell lines (FIG. 1B), and AlbioHRP 3 Primers were amplified against HeLa, an HPV-18 infected cell line (FIG. 1C). In addition, it was confirmed that the amplification occurred only for CaSki and HeLa as a result of using a primer set using two sets of them (AlbioHRP1, AlbioHRP3) (FIG. 1D). It can be seen that this result is consistent with the virtual amplification result of Table 1.

Example 2

In this example, PCR was performed on E. coli strains containing various types of HPV plasmid DNA using three sets of primers identified in Example 1 and two of them in combination.

The strain used in this example is as follows.

HPV-1a (ATCC 45021);

HPV-2a (ATCC 45022);

HPV-6b (ATCC 45150);

HPV-11 (ATCC 45151);

HPV-16 (ATCC 45113);

HPV-18 (ATCC 45152);

HPV-31 (ATCC 65446);

HPV-43 (ATCC 40338);

In other words, in the present embodiment, HPV-16, HPV-18, and HPV- confirmed to be amplified by the primers synthesized in the present invention through the virtual amplification of Example 1 among the high risk group HPV types closely related to the progression of cervical cancer. PCR amplification was performed on strains containing 31 DNA and strains containing HPV-43, HPV-2a, HPV-6b, HPV-11 and HPV-1a DNA among low risk group HPV types.

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 μl, and these were also used as template DNA solutions. PCR amplification was performed independently for each of the constituent primers for each template DNA according to the same reactions and procedures as in Example 1. The validity of the bands that appeared on each pathway of the gel was determined by computer hypothesis of each primer for HPV-16, HPV-18 HPV-31, HPV-43, HPV-2a, HPV-6b, HPV-11, HPV-1a DNA. The amplification experiment was compared with the expected size of the PCR product obtained in Example 1 (see Table 2 below).

2a to 2d are three sets of primers synthesized in the present invention for HPV-16, HPV-18 HPV-31, HPV-43, HPV-2a, HPV-6b, HPV-11, HPV-1a, respectively; Electrophoresis after PCR using a combination primer set (AlbioHRP1, AlbioHRP3). In the figure, M at the bottom of the figure is a standard marker, and other numbers indicate a target HPV type used in this embodiment. The number on the side indicates the size of the marker.

As can be seen in the figure AlbioHRP1 of the present invention was amplified for HPV-16 and HPV-18 DNA (Fig. 2a), AlbioHRP2 was not amplified anywhere in the target HPV DNA used in this example (FIG. 2B), AlbioHRP3 was amplified against HPV-18 DNA (FIG. 2C). Also, when two groups (AlbioHRP1 and AlbioHRP3) of the three sets of primers synthesized in the present invention were used in combination, HPV-16, HPV It can be seen that amplification occurred for −18 and HPV-31 (FIG. 2D). These results were consistent with the prediction results of Table 2 below.

Table 2. Expected Product Size of HPV PCR Amplification by Albio HRP

Amplification type Size of amplification products of Albio HRP primers (bp) Albio HRP1 Albio HRP2 Albio HRP3 HPV-16 314 -* - HPV-18 - - 277 HPV-26 - 308 - HPV-30 - 313 - HPV-31 314 - - HPV-33 314 298 - HPV-35 314 297 - HPV-39 - - 286 HPV-45 - - 280 HPV-51 - 300 - HPV-52 314 303 - HPV-53 313 310 - HPV-56 - 312 - HPV-58 314 301 - HPV-59 314 - 285 HPV-66 313 312 - HPV-67 314 - - HPV-68 - - 288 HPV-70 - - 286 HPV-73 314 - -

^* Non-amplified

These results indicate that the AlbioHRP1, AlbioHRP2, and AlbioHRP3 primers are capable of PCR amplification for high-risk HPV DNA, respectively, and do not show non-selective amplification. Therefore, this result is consistent with the selective specific amplification of AlbioHRP1, AlbioHRP2, and AlbioHRP3 primers predicted in computer virtual amplification experiments. The virtual amplification results are expected to be of sufficient significance in the 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.

First of all, 40 patients were randomly selected from the outpatient clinic of the women's cancer center of the Pochon CHA University College of Gynecology. The cervicography, biopsy and Pap-smear Specialized examinations such as inspections were conducted. The results of examination through a cell test of a total of 30 patients are shown in Table 3 below.

As shown in Table 3 below, the cervical cancer-related abnormal patients were subjects with low grade squamous intraepithelial lesions (LSIL) in cytology, and subjects with unidentified atypical cells (ASCUS). , 9, 11, 12, 19, 20 and 23, subjects with high grade squamous epithelial lesions (HSIL) 1, 2, 5, 7, 8, 13, 14, 16, 17, 18, 24 and 25 and microinfiltration Cancer (SCC) patients 3, 4, 10, 15, 21, and 22 were included, and the remaining subjects 26, 27, 28, 30, 32, 33, 36, 38, 39, and 40 were normal individuals unrelated to cervical cancer. Separated.

Table 3. Clinical Trial Results by Cell Screening

Subject No. Cell test Subject No. Cell test Subject No. Cell test Subject No. Cell test One HSIL ^* 11 ASCUS 21 SCC 31 LSIL 2 HSIL 12 ASCUS 22 SCC 32 normal 3 SCC ^** 13 HSIL 23 ASCUS 33 normal 4 SCC 14 HSIL 24 HSIL 34 LSIL 5 HSIL 15 SCC 25 HSIL 35 LSIL 6 ASCUS ^*** 16 HSIL 26 normal 36 normal 7 HSIL 17 HSIL 27 normal 37 LSIL 8 HSIL 18 HSIL 28 normal 38 normal 9 ASCUS 19 ASCUS 29 LSIL ^**** 30 normal 10 SCC 20 ASCUS 30 normal 40 normal

^* High-grade squamous intraepithelial lesions (High Squamous Intraepithelial lesion)

^** Squamous Cell Carcinoma

^*** Atypical Squamous Cell Undetermined significance

^**** Low Squamous Intraepithelial Lesions

Comparative Example 2

In this comparative example, the hybrid capture II method (Hybrid Capture II, Digene Co., Ltd.) was performed on 30 cervical cancer-related abnormal patients and 10 normal cervical cancer-related patients. The examination results according to the hybrid capture II method is briefly shown in Table 4 below.

As shown in Table 5, among the total 25 patients screened as abnormal in Comparative Example 1, the subjects 1-11, 13-18, 21-25 was found to be a high-risk HPV infection, subjects 29, 31, 34, 35 and 37 were found to be low risk HPV infections. In addition, the test subjects 26, 27, 28, 30, 32, 33, 36, 38, 39, and 40, which were determined to be normal in Comparative Example 1, were also found to be HPV uninfected. However, Subject 1, Subject 19, and Subject 20 determined to have unidentified atypical cells (ASCUS) in Comparative Example 1 were found to be normal in this Comparative Example.

Table 4. Clinical Trial Results for HPV Infection Using Hybrid Capture II

Subject No. Examination Results (High / Low) Subject No. Examination Results (High / Low) Subject No. Examination Results (High / Low) Subject No. Examination Results (High / Low) One +/- 11 +/- 21 +/- 31 -/ + 2 +/- 12 -/- 22 +/- 32 -/- 3 +/- 13 +/- 23 +/- 33 -/- 4 +/- 14 +/- 24 +/- 34 -/ + 5 +/- 15 +/- 25 +/- 35 -/ + 6 +/- 16 +/- 26 -/- 36 -/- 7 +/- 17 +/- 27 -/- 37 -/ + 8 +/- 18 +/- 28 -/- 38 -/- 9 + / + 19 -/- 29 -/ + 39 -/- 10 +/- 20 -/- 30 -/- 40 -/-

Example 3 PCR Amplification Test of Selected HR Primers for Clinical Samples

In the present embodiment, cervical cancer-related abnormal patients through Comparative Example 1 using the set of three primers (AlbioHRP1, AlbioHRP2, AlbioHRP3) and the set of combination primers (AlbioHRP1 + AlbioHRP3) identified in Example 1 and Example 2 above We also examined the amplification of DNA from normal subjects not associated with cervical cancer and whether they were infected with high-risk HPV and whether early causes of cervical cancer could be identified.

To this end, electrophoresis was performed after PCR was performed on DNA samples obtained from subjects using the three sets of primers identified in Example 1 and the combination primer set. DNA from the subjects was obtained according to the specific method or the same method as in Example, PCR reaction conditions were carried out in the same procedure as in Example 1.

The primer of the present invention is clear for a patient sample diagnosed with high-risk HPV infection in a hybrid capture test among DNA samples obtained from abnormal cervical cancer-related patients (subjects 1 to 25 and 29, 31, 34, 35, 37, 38 and 40). It was confirmed that amplification occurred in the DNA samples of patients (test subjects 12, 19 to 20) diagnosed as normal in some hybrid capture tests.

On the other hand, patients who were diagnosed with low-risk HPV infection in hybrid capture (29, 31, 34, 35, 37, 38 to 40) and those who were not associated with cervical cancer (26, 27, 28, 30, 32, 33, 36) It was confirmed that the amplification did not occur for the DNA sample diagnosed with 39). This is consistent with the etiological reasoning of 30 patients with cervical cancer and 10 patients with cervical cancer.

3A to 3Y are infected with high risk group HPV such as high risk group lesion (HSIL), microinvasive cancer (SCC) or unidentified atypical cells (ASCUS) in Comparative Example 1 using the 3 sets of primers according to the present invention and the combination primer set. DNA samples obtained from the determined patients were subjected to PCR and electrophoresed. FIG. 3A shows a subject 1, FIG. 3B shows a subject 2, FIG. 3C shows a subject 3, FIG. 3D shows a subject 4, FIG. 3E shows a subject 5, FIG. 3F shows a subject 6, FIG. 3G shows a subject 7, FIG. 3H shows a subject 8, and FIG. 3I. FIG. 3J is a subject 10, FIG. 3K is a subject 11, FIG. 3L is a subject 12, FIG. 3M is a subject 13, FIG. 3N is a subject 14, FIG. 3O is a subject 15, FIG. 3P is a subject 16, and FIG. 3Q is a subject. 17, FIG. 3R shows the subject 18, FIG. 3S shows the subject 19, FIG. 3T shows the subject 20, FIG. 3U shows the subject 21, FIG. 3V shows the subject 22, FIG. 3W shows the subject 23, FIG. 3X shows the subject 24, and FIG. 3Y shows the subject 25. DNA obtained from the patient was targeted. Where M is a standard marker, lane 1 represents an AlbioHRP 1 primer, lane 2 represents an Albio HRP 2 primer, lane 3 represents an Albio HRP 3 primer, and lane 4 represents a combination of Albio HRP 1 and Albio HRP 3. The number on the left side shows the size of the DNA marker.

As shown, amplification occurred in at least one set of primers synthesized in the present invention on DNA obtained from all the subjects (Subject 1 to Subject 24) determined to be infected with the high risk group HPV through Comparative Example 1. It was confirmed. That is, subject 1 is Albio HRP 1 primer, Albio HRP 2 primer and combination primer (Fig. 3a), subject 2 is Albio HRP 2 primer (Fig. 3b), subject 3 is Albio HRP 1 primer and combination primer (Fig. 3c), subject 4 is Albio HRP 3 primer and combination primer (FIG. 3d), subject 5 is Albio HRP 2 primer (FIG. 3e), subject 6 is Albio HRP 1 primer and combination primer (FIG. 3f), and subject 7 is Albio HRP 1 primer and combination Primer (FIG. 3G), subject 8 is Albio HRP 1 primer, Albio HRP 2 primer and combination primer (FIG. 3H), subject 9 is Albio HRP 2 primer (FIG. 3I), and subject 10 is Albio HRP 1 primer and combination primer (FIG. 3j), subject 11 is Albio HRP 2 primer (FIG. 3K), subject 12 is Albio HRP 1 primer, Albio HRP 2 primer and combination primer (FIG. 3L), subject 13 is Albio HRP 2 primer (FIG. 3M), and subject 14 is Albio HRP 1 primer and combination primer (FIG. 3N), subject 15 is Albio HRP3 primer and combination primer (subject 3o), subject 16 is Albio HRP 1 primer and combination primer (Fig. 3p), subject 17 is Albio HRP 1 primer, Albio HRP 2 primer and combination primer (Fig. 3q), subject 18 Silver Albio HRP 2 primer (FIG. 3R), subject 19 is Albio HRP 2 primer (FIG. 3S), subject 20 is Albio HRP 1 primer and combination primer (FIG. 3T), and subject 21 is Albio HRP 3 primer and combination primer (FIG. 3U ), Subject 22 is Albio HRP 1 primer and combination primer (FIG. 3v), subject 23 is Albio HRP 1 primer and combination primer (FIG. 3w), subject 24 is Albio HRP 2 primer (FIG. 3x), and subject 25 is Albio HRP 1) In the case of using the primer, Albio HRP 2 primer and a combination primer (Fig. 3y) it can be seen that the amplification product of the size predicted in the virtual amplification is obtained.

There are a variety of high-risk HPV types that are closely related to the progression of cervical cancer, and samples from patients determined to be infected with high-risk HPV in Comparative Example 1 may be infected by different high-risk HPVs, Depending on the DNA samples obtained, the product amplified by the primers used in this example may vary, and it can be seen that amplification has occurred for only some of the primers used in this example.

In particular, in some of the subjects diagnosed as normal in the hybrid capture system (test subjects 12, 19 and 20), it was confirmed that amplification occurred when PCR was performed using the primers according to the present invention. This means that the PCR amplification method using the primer of the present invention is somewhat higher in sensitivity or specificity than the hybrid capture system.

4a to 4o are Comparative Example 1 using a set of three primers (Albio HRP 1 primer, Albio HRP 2 primer, Albio HRP 3 primer) and a combination primer (Albio HRP 1 primer + Albio HRP 3 primer) set according to the present invention And the result of the examination of Comparative Example 2 PCR and electrophoresis was performed on DNA samples obtained from patients with a low risk group HPV infection (LSIL) and normal people in the hybrid capture test among cervical cancer-related abnormal patients.

4A is a subject 26, FIG. 4B is a subject 27, FIG. 4C is a subject 28, FIG. 4D is a subject 29, FIG. 4E is a subject 30, FIG. 4F is a subject 31, FIG. 4G is a subject 32, FIG. 4H is a subject 33, FIG. 4I. FIG. 4 shows subject 34, FIG. 4J shows subject 35, FIG. 4K shows subject 36, FIG. 4L shows subject 37, FIG. 4M shows subject 38, FIG. 4N shows subject 39 and FIG. .

Where M is a standard marker, lane 1 represents Albio HRP 1 primer, lane 2 represents Albio HRP 2 primer, lane 3 represents Albio HRP 3 primer and lane 4 represents Albio HRP 1 + Albio HRP 3 combination primer. The number on the left side shows the size of the DNA marker. As can be seen in the drawings, PCR amplification was carried out using primers synthesized in this example on DNA obtained from a subject determined as normal or low risk lesions (LSIL) in Comparative Examples 1 and 2 above. As a result, it was confirmed that the amplification product was not produced.

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.

Since the oligonucleotide synthesized in the present invention can specifically amplify the high-risk HPV type by complementarily binding to the high-risk HPV type DNA, it is possible to detect whether the high-risk HPV type is closely related to the progression of cervical cancer. Diagnosis can be made.

In other words, when PCR is performed on the primers synthesized in the present invention and samples obtained from humans, cervical cancer can be diagnosed quickly and economically and accurately because amplification occurs only for samples infected with high-risk HPV.

In particular, by using a primer synthesized in the present invention and a known common primer (general Primer) it can be confirmed whether more efficient HPV infection.

In other words, by performing PCR using a common primer first to determine whether HPV infection according to amplification, and by performing PCR using the primers synthesized in the present invention only for a sample obtained amplification products, more accurate HPV Early diagnosis can be made of infection and cervical cancer progression.

In addition, since the primer of the present invention can be complementarily bound to the genome of high-risk HPV, it can be utilized as a probe that can directly detect high-risk HPV DNA.

<110> ALBIOMED Co., LTD <120> Primers and Method for Detecting High Risk Human Papillomavirus          by Multiplex PCR <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 1 gagtacctaa gacatgggga ggaatatga 29 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 2 cgacctaaag gaaactgatc taaatctgc 29 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 3 caaacagaaa ttgacctaca gtgctatgag caa 33 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 4 accccttccc ctcctcatct gtaccttca 29 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 5 ataacatagc tggaaactat acaggacag 29 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 6 acgctgtggt tcggctcgtc tggctagta 29

Claims (12)

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; or A synthesized primer for amplifying a high risk group HPV DNA selected from the group consisting of nucleotides (third primers) consisting of SEQ ID NO: 5 and SEQ ID NO: 6. The method of claim 1, The first primer is HPV-16, HPV-31, HPV-33, HPV-34, HPV-35, HPV-35H, HPV-52, HPV-53, HPV-58, HPV-59, HPV-66, HPV A primer for amplifying DNA of HPV type selected from -67 or HPV-73, The second primer is selected from HPV-26, HPV-30, HPV-33, HPV-35, HPV-35H, HPV-51, HPV-52, HPV-53, HPV-56, HPV-58 or HPV-66 Is a primer for amplifying DNA of HPV type, The third primer was synthesized to amplify DNA of high risk group HPV type, which is a primer for amplifying DNA of HPV type selected from HPV-18, HPV-39, HPV-45, HPV-59, HPV-68 or HPV-70. primer. The method according to claim 1 or 2, Wherein said first primer is a primer for amplifying at least one type of HPV DNA from HPV-16 or HPV-31 DNA. The method according to claim 1 or 2, Wherein said third primer is a primer that amplifies HPV-18 DNA. 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, or Nucleotide (third primer) consisting of SEQ ID NO: 5 and SEQ ID NO: 6 Using as a primer at least one trillion nucleotides selected from the group consisting of A method of amplifying a high risk HPV type DNA 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 primer is HPV-16, HPV-31, HPV-33, HPV-34, HPV-35, HPV-35H, HPV-52, HPV-53, HPV-58, HPV-59, HPV-66, HPV A primer for amplifying DNA of HPV type selected from -67 or HPV-73, The second primer is selected from HPV-26, HPV-30, HPV-33, HPV-35, HPV-35H, HPV-51, HPV-52, HPV-53, HPV-56, HPV-58 or HPV-66 Is a primer for amplifying DNA of HPV type, Wherein said third primer is a primer for amplifying a DNA of HPV type selected from HPV-18, HPV-39, HPV-45, HPV-59, HPV-68, or HPV-70. The method according to any one of claims 5 to 6, Wherein said first primer is a primer for amplifying at least one type of HPV DNA from HPV-16 or HPV-31 DNA. The method according to any one of claims 5 to 6, Wherein said third primer is a primer for amplifying HPV-18 DNA. 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, or Nucleotide (third primer) consisting of SEQ ID NO: 5 and SEQ ID NO: 6 Nucleotides selected from the group consisting of at least one trillion; dNTP mixtures (dATP, dCTP, dGTP, dTTP); Heat resistant polymerase; And Kit for DNA detection of high risk group HPV type comprising PCR buffer solution. The method of claim 9, The first primer is HPV-16, HPV-31, HPV-33, HPV-34, HPV-35, HPV-35H, HPV-52, HPV-53, HPV-58, HPV-59, HPV-66, HPV A primer for amplifying DNA of HPV type selected from -67 or HPV-73, The second primer is selected from HPV-26, HPV-30, HPV-33, HPV-35, HPV-35H, HPV-51, HPV-52, HPV-53, HPV-56, HPV-58 or HPV-66 Is a primer for amplifying DNA of HPV type, Wherein said third primer is a primer for amplifying DNA of HPV type selected from HPV-18, HPV-39, HPV-45, HPV-59, HPV-68 or HPV-70.  The method according to any one of claims 9 to 10, The first primer is a high-risk HPV type DNA detection kit is a primer for amplifying at least one type of HPV DNA from HPV-16 or HPV-31 DNA. The method according to any one of claims 9 to 10, Wherein the third primer is a primer for amplifying HPV-18 DNA high-risk HPV type DNA detection kit.

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