KR20200029958A - Screening and qualitative methods of HPV types and type 16 related to oral cencer using Real-time PCR and Melting Curve Analysis - Google Patents

Abstract

The present invention relates to a composition for detecting an oral cancer-related human papillomavirus (HPV) and an HPV detection method using the same and, more specifically, to one or more oral cancer-related HPV detection compositions selected from a group consisting of HPV types 6, 31, 33, 35, 52, and 58 closely related to occurrence of an oral cancer, and an HPV detection method using the same. According to the present invention, when the composition or method of the present invention is used, qualification analysis for one or more kinds selected from a group consisting of HPV types 6, 31, 33, 35, 52, and 58 closely related to occurrence of an oral cancer, and a qualification and quantification analysis for the most important HPV type 16 related to the oral cancer can be simultaneously performed, thereby quickly and easily performing clinical diagnosis of the oral cancer with high reliability.

Description

Screening and qualitative methods of HPV types and type 16 related to oral cencer using Real-time PCR and Melting Curve Analysis}

The present invention relates to a composition for detecting oral papillomavirus (HPV) related to oral cancer and a method for detecting human papillomavirus using the same, and more specifically, to HPV types 6, 31, 33, 35, 52 and 58 closely related to the development of oral cancer It relates to a composition for detecting one or more HPV selected from the group consisting of and HPV detection method using the same.

Oral cancer is a malignant tumor that occurs in the mouth, and includes squamous cell carcinoma, bovine salivary gland carcinoma, sarcoma, lymphoma, melanoma, etc. Among them, squamous cell carcinoma in the oral cavity is the highest with about 90%. According to the World Health Organization, more than 600,000 oral cancer patients occur worldwide each year, and 300,000 deaths occur. Oral cancer is a inflammatory disease that causes 40% of deaths within 5 years of onset, and early diagnosis is difficult because early symptoms are not clear, and treatment rates of 80% or more can be expected if detected early.

Until 20 years ago, the biggest cause of oral cancer was smoking and drinking, but the number of patients with oral cancer, which is presumed to be caused by oral sex, has increased rapidly in the past 10 years, and the cause is human papillomavirus (HPV), which has been identified as the cause of cervical cancer. Was confirmed. Human papillomavirus (HPV) is known to be an important cause of cervical cancer, and among the 100 known virus types, 16 and 18 are found in more than 70% of cervical cancers worldwide. In addition, carcinogenic human papillomavirus types include 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 69, 73, etc. It is reported that carcinogenic human papillomaviruses including 16 and 18 are found in more than 99.7% of cervical cancer patients.

 As a result of the meta-analysis of the International Cancer Research Organization (IARC) conducted in 2005, 35.6% of oral cancers were identified with human papillomavirus (HPV), of which 87% were identified as type 16. Studies show that HPV is found in 65-70% of oral cancer patients found in the United States, and it is expected that oral cancer will outperform cervical cancer by 2020, the most common cancer associated with HPV. According to the presentation of the 28th International HPV Society, a study has been reported that oral cancer and oropharyngeal cancer are closely related to the alpha genus 9 genotype (types 16, 31, 33, 35, 52, 58) belonging to HPV type 16. And documents that report a deep connection between HPV and type 16 have been searched (Oral Oncology 69 (2017) 56–61).

Human papillomavirus (HPV) is recognized as a new causative factor for oral cancer because it has good chemotherapy and radiation therapy, low metastasis and recurrence of cancer, and a different clinical picture from virus-negative cancer patients. The importance of diagnosis for (HPV) is emphasized.

The present invention has been devised in the above-mentioned background, and the present inventors simultaneously screened for human papillomavirus (HPV) types 16, 31, 33, 35, 52, and 58 associated with the development of oral cancer and type 16 To qualitatively and quantitatively, specific primer sets and fluorescent probes were designed based on the E6E7 gene, which is closely related to cancer induction, and real-time polymerase chain reactions were performed using the primers, probes, and compositions or kits containing the same. By performing the melting curve analysis method, it was confirmed that the human papillomavirus (HPV) related to oral cancer can be screened with high specificity and sensitivity, and that the present invention was completed by confirming that HPV type 16 single qualitative analysis can be simultaneously performed.

Accordingly, an object of the present invention is to provide a composition for detecting human papillomavirus (HPV) comprising at least one primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a primer of SEQ ID NO: 4.

Another object of the present invention is to provide a composition for diagnosing oral cancer comprising the composition for detecting human papillomavirus.

Another object of the present invention is to provide a kit for detecting human papillomavirus (HPV) comprising at least one primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a primer of SEQ ID NO: 4.

Another object of the present invention (a) extracting the DNA from the clinical sample; (b) amplifying the gene with the obtained DNA using a primer set comprising at least one primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a primer of SEQ ID NO: 4; And (c) analyzing the amplification product to provide a method for detecting human papillomavirus (HPV).

To achieve the above object, the present invention provides a composition for detecting human papillomavirus (HPV) comprising at least one primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a primer of SEQ ID NO: 4.

In order to achieve another object of the present invention, the present invention provides a composition for diagnosing oral cancer comprising the composition for detecting human papillomavirus.

In order to achieve another object of the present invention, the present invention provides a kit for detecting human papillomavirus (HPV) comprising at least one primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a primer of SEQ ID NO: 4.

In order to achieve another object of the present invention, the present invention comprises the steps of: (a) extracting DNA from a clinical sample; (b) amplifying the gene with the obtained DNA using a primer set comprising at least one primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a primer of SEQ ID NO: 4; And (c) analyzing the amplification product to detect human papillomavirus (HPV).

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for detecting human papillomavirus (HPV) comprising at least one primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a primer of SEQ ID NO: 4.

The term “primer” used in the present invention is a nucleic acid sequence having a short free 3 'hydroxyl group, which can form a complementary template and a base pair, and for copying the template strand. A short nucleic acid sequence that functions as a starting point. Primers can initiate DNA synthesis in the presence of reagents for polymerization (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphospates (dNTPs) at appropriate buffers and temperatures.

Primers can incorporate additional features that do not change the basic properties of the primer, which serves as the starting point for DNA synthesis. In the present invention, each of the primers represented by SEQ ID NOs: 1 to 4 is a concept including a base sequence having 95% or more sequence homology. For example, when the primer is 20 bp, if two or more of the 20 bp are wrong, the melting temperature decreases by 5 degrees or more, and the result is not good. However, if only one is wrong, the annealing temperature is increased in the PCR process. This is because if you experiment a little down, you can get the same result.

The term "human papillomavirus (HPV)" used in the present invention is a circular double helix virus composed of 8 kb, and is largely divided into an early gene and a late gene according to the expression time of the gene. The functions are known for E1, E2, E3, E4, E5, E6, and E7, which are the initial genes of about 4.5 kb, respectively, and L1, L2, respectively, for the 2.5 kb late genes encode the main and protein envelopes, respectively. It is known as a gene. Recently, as molecular biological techniques have been developed, the genome sequence of HPV has been revealed, and HPV is classified into several types according to differences in ORF (Open Reading Frame) sequences of E6, E7, and L1 genes. That is, when the nucleotide sequence of the ORF is 10% different, it is defined as a new type, and when it is more than 2% and less than 10%, it is defined as a subtype, and when it shows a difference of less than 2%, it is a variant. Is defined as According to this classification, more than 120 types of HPV are known to date. Preferably, the composition for detecting HPV of the present invention can detect HPV by detecting the E6 and E7 genes.

According to an embodiment of the present invention, the primers represented by SEQ ID NO: 1 can specifically amplify the E6E7 gene regions of HPV types 16 and 35, and the primers represented by SEQ ID NO: 2 are HPV types 31, 33 And the E6E7 gene region of No. 58 can be specifically amplified, and the primer represented by SEQ ID NO: 3 can specifically amplify the E6E7 gene region of HPV type 52. The primers represented by SEQ ID NO: 4 may act as reverse primers in common with each of the forward primers represented by SEQ ID NOS: 1 to 3.

According to another embodiment of the present invention, a composition comprising one forward primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a reverse primer of SEQ ID NO: 4 is specific only for oral cancer-related HPV types, each of which can be detected. It was confirmed that the specificity was very excellent by confirming that the gene of HPV of various types not related to oral cancer and various oral flora were not amplified.

Further, according to another embodiment of the present invention, the composition comprising all of the primers of SEQ ID NOs: 1 to 4 does not cause interference with each other and is associated with oral cancer HPV types 16, 31, 33, It was confirmed that only 35, 52 and 58 can be detected with very high specificity.

Therefore, the composition for detecting HPV of the present invention can be utilized by combining at least one forward primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a reverse primer of SEQ ID NO: 4 according to the type of HPV to be detected. That is, SEQ ID NOs: 1 and 4; SEQ ID NOs: 2 and 4; SEQ ID NOs: 3 and 4; SEQ ID NOs: 1, 2 and 4; SEQ ID NOs: 1, 3 and 4; SEQ ID NOs: 2, 3 and 4; Alternatively, HPV can be detected by combining with a composition comprising SEQ ID NOs: 1, 2, 3 and 4.

Most preferably, the composition for detecting HPV of the present invention is SEQ ID NO: 1 to simultaneously detect HPV types 16, 31, 33, 35, 52, and 58 having a very high relationship with the development of oral cancer It may include all of 4 to.

Since the composition of the present invention is characterized in that it specifically detects one or more selected from the group consisting of HPV types 16, 31, 33, 35, 52 and 58, which have a very close relationship with the invention of oral cancer. It can have the effect of accurately distinguishing individuals who have had or are likely to develop oral cancer.

The composition of the present invention may further include a probe of SEQ ID NO: 5.

The term "probe" of the present invention means a nucleic acid fragment, such as RNA or DNA, which corresponds to a few bases to hundreds of bases in short, capable of specific binding with a gene or mRNA, an oligonucleotide probe, It can be manufactured in the form of a single stranded DNA probe, a double stranded DNA probe, an RNA probe, and the like. The probe may include a label used for detection at the 5 'and 3' ends of the corresponding base sequence, respectively, and such a fluorophore or quencher may be labeled at the 5 'and 3' ends, respectively. Accordingly, when the fluorophore and the quencher are combined with each other in the probe, light emission is suppressed due to mutual interference, and when the probe is decomposed through the polymerase chain reaction, the interference between the fluorophore labeled on the probe and the quencher disappears, thereby emitting light. Is done. It is possible to determine the presence or absence of HPV from the sample by such a reaction. As for the fluorophore capable of being labeled at the 5 'end, a fluorescent substance commonly used in the art can be used without limitation, for example, fluorescein, 6-carboxyfluorescein, nuclear sacro-6-carboxyfluoro Lecein, tetrachloro-6-carboxyfluorescein, VIC, JOE, 5- (2'-aminoethyl) amino naphthalene-1-sulfonic acid, coumarin and derivatives thereof, cyanine-5, lucifer yellow, texas red, tetra Methylodamine, Yakima Yellow, Carl Fluored 610 and the like can be used. The quencher that can be labeled at the 3 'end can also be used without limitation, a quencher commonly used in the art, for example, tetramethylrodamine, 6-carboxytetramethyl-rhodamine, BHQ-1, 2, 3, 4'-benzoic acid, 4-dimethylaminophenyl azophenyl-4'-maleimide and the like can be used. As such, a fluorescent polymer-labeled probe can be used to perform real-time polymerase chain reaction and monitor the amplified product in real time, enabling accurate quantification of DNA and RNA, and also fast, simple, and accurate diagnosis without the need for electrophoresis. It has the advantage that it is possible.

In the present invention, the probe of SEQ ID NO: 5 may specifically bind to HPV type 16, including a sequence complementary to HPV type 16. Therefore, when the probe of SEQ ID NO: 5 is added to the composition for detecting HPV according to the present invention, additional qualitative and quantitative determination for HPV type 16 may be possible.

Preferably, the probe of SEQ ID NO: 5 is in the composition comprising the primer of SEQ ID NO: 1 that can amplify HPV type 16 because the probe of SEQ ID NO: 5 contains a specific sequence for HPV type 16 Can be added. That is, the primers of SEQ ID NO: 1 and 4; Primers of SEQ ID NOs: 1, 2 and 4; Primers of SEQ ID NOs: 1, 3 and 4; Or it is preferred that the probe of SEQ ID NO: 5 is added to the composition comprising the primers of SEQ ID NO: 1, 2, 3 and 4.

Primers of SEQ ID NOs: 1 and 4; Primers of SEQ ID NOs: 1, 2 and 4; Primers of SEQ ID NOs: 1, 3 and 4; Alternatively, the composition in which the probe of SEQ ID NO: 5 is added to the composition comprising the primers of SEQ ID NO: 1, 2, 3 and 4 is at least one selected from the group consisting of HPV types 16, 31, 33, 35, 52 and 58 In addition to being able to detect, it is possible to simultaneously detect the presence or absence and the amount of type 16, which is most closely related to the development of oral cancer.

The primer or probe of the present invention can be chemically synthesized using a phosphoramidite solid support method, or other well-known method. In addition, such nucleic acid sequences can be modified within a range that does not change the basic properties of the primer or probe and also using a number of means known in the art. Non-limiting examples of such modifications include methylation, “capsulation”, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages (eg methyl phosphonate, phosphotriester, phos. Poroamidate, carbamate, etc.) or a modified linkage (eg, phosphorothioate, phosphorodithioate, etc.). Nucleic acids are one or more additional covalently attached residues, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (e.g., acridine, psoralen, etc.) ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents.

Further, the primer nucleic acid sequence of the present invention may include a label that can be directly or indirectly detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means, if necessary. Examples of labels include enzymes (e.g. horseradish peroxidase, alkaline phosphatase), radioactive isotopes (e.g. 32P), fluorescent molecules, chemical groups (e.g. biotin), etc. have.

The composition of the present invention may further include an additional primer sequence or probe sequence as an intrinsic standard to minimize false positives and false negatives. When using the composition for detecting HPV of the present invention, it is possible to detect in real time whether the target gene and the intrinsic standard are amplified in a single reaction vessel (tube).

In addition, the composition for detecting HPV of the present invention may additionally include components necessary for an amplification reaction. The components that can be included can be used without limitation, as long as they are materials commonly used in the art in the amplification reaction, for example, buffer solutions, DNA polymerase, dNTP and sterile distilled water, etc., but can be added by the above examples. The possible components are not limited.

In the present invention, the detection may be by melting curve analysis. The term "melting" refers to dissociating double-stranded DNA into single-stranded DNA. Fusion can be performed by heating a sample containing double-stranded DNA. The melting may be performed, for example, at 50 ° C to 95 ° C, but is not limited thereto.

According to one embodiment of the present invention, the composition comprising the primers of SEQ ID NOs: 1 to 4 and the primers of SEQ ID NO: 5 is utilized in the melting curve analysis method to be composed of HPV types 16, 31, 33, 35, 52 and 58 It was confirmed that at least one qualitative detection selected from the group was possible.

On the other hand, according to one embodiment of the present invention, the primer and probe set of the present invention does not induce oral cancer, but does not bind to HPV type 18, etc., which are known to be closely related to the development of cervical cancer, and solely to cause oral cancer Since the related types 16, 31, 33, 35, 52, and 58 human papillomaviruses can be detected with very high specificity and sensitivity, they can be very useful in the field of diagnosis of oral cancer and the possibility of developing oral cancer.

Accordingly, the present invention provides a composition for diagnosing oral cancer comprising the composition.

The present invention also provides a kit for detecting human papillomavirus (HPV) comprising at least one primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a primer of SEQ ID NO: 4.

The kit of the present invention may use one or more primers selected from the group consisting of SEQ ID NOs: 1 to 3 and components used in PCR compositions in addition to the primers of SEQ ID NO: 4, for non-limiting examples of components of these compositions Buffer solution, dNTP, Mg2 + ion, DNA polymerase may be included.

As the buffer solution for the reaction, 1 to 10 mM TrisHCl, 10 to 40 mM KCl (pH 9.0) may be used, and the dNTP may use any one or more selected from dATP, dTTP, dGTP, and dCTP.

The kit may include stabilizers and / or non-reactive dyes to improve experimental convenience, stabilization and reactivity.

The non-reactive dye material should be selected from materials that do not affect the polymerase chain reaction, and is intended to be used for analysis or identification using polymerase chain reaction products. Materials satisfying these conditions may be used as water-soluble dyes such as rhodamine, tamra, lacs, bromophenol blue, xylene cyanol, bromocresol red, cresol red. The non-reactive dye material may be included in an amount of 0.0001 to 0.01% by weight based on the total weight of the composition, and is preferably included in an amount of 0.001 to 0.005% by weight. When added in an amount of more than 0.01% by weight relative to the total weight of the composition, there is a problem that a high concentration of water-soluble dye can act as a reaction inhibitor during the polymerase chain reaction.

In addition, the polyalcohols may be used as a stabilizing material for further stabilizing the kit components of the present invention, and it is preferable to use at least one of glucose, glycerol, mannitol, galactitol, glucitol, and sorbitol. . The polyalcohols may be included in a content of 10 to 500 mM, and preferably 50 to 300 mM. If the polyhydric alcohol exceeds 500mM, it is difficult to dissolve in an aqueous solution due to the solubility of the water-soluble polymer itself, it is difficult to achieve sufficient mixing due to the high viscosity, the volume becomes larger than necessary, and sterile distilled water is used for the polymerase chain reaction. When dissolving with furnace, dissolution is not easy. In addition, there is a problem that a high concentration of water-soluble polymer may act as a reaction inhibitor during the polymerase chain reaction. And, if it is less than 10mM, it is difficult to expect an effective enzyme stabilization effect because the target enzyme and the surface water molecule cannot be sufficiently coated and protected. In addition, the viscosity of the solution is too low to spread widely over the entire bottom surface of the tube and not dry in an ideal form. There are problems that can not be protected and enzymes are not sufficiently protected. In the present invention, it is possible to use gelatin, small blue albumin, Thesit, and PEG-8000 as stabilizing substances in addition to polyhydric alcohols.

The kit components may be provided in a liquid form, and desirably dried to increase stability, ease of storage, and long-term storage. The drying may be performed by a known drying method such as general room temperature drying, warming drying, freeze drying, and vacuum drying, and any drying method may be used as long as the components of the composition are not lost.

In the present invention, various DNA polymerases can also be used in the amplification step of the present invention, and include “Klenow” fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, which includes Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Includes. Most of the polymerases can be isolated from the bacteria themselves or commercially available. In addition, the polymerase used in the present invention can be obtained from cells expressing high levels of the cloning gene encoding the polymerase.

The kit of the present invention may further include a probe of SEQ ID NO: 5 as described above.

The present invention also (a) extracting the DNA from the clinical sample; (b) amplifying the gene with the obtained DNA using a primer set comprising at least one primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a primer of SEQ ID NO: 4; And (c) analyzing the amplification product to detect human papillomavirus (HPV).

In the step (a), the clinical sample may be used without limitation as long as it is a sample predicted to contain the sequence of HPV, preferably a saliva obtained from an individual or a sample taken in the oral cavity, and extract the DNA Methods can be carried out according to conventional methods known in the art (see Sambrook, J. etal., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001); Tesniere, C. etal ., Plant Mol. Biol. Rep., 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al., Anal. Biochem 162: 156 (1987)).

In the present invention, the object is a concept including all of primates including humans, horses, sheep, pigs, goats, dog cats, rodents, and various kinds of birds, and may be preferably humans.

In step (b), amplification is well known in the art, and a target nucleic acid is increased by repeating the process of denaturation, annealing, and elongation reaction using a target nucleic acid as a template and a primer specific to the target nucleic acid. It means the process. In the present invention, the amplification may be a method capable of amplifying a nucleic acid sequence that can be conventionally used in the art, but may preferably be a PCR reaction, and more preferably, real time polymerase chain reaction (real time PCR). Can be

This real-time polymerase chain reaction analysis can be performed using a commercially available real-time polymerase chain reaction device, and the real-time polymerase chain reaction device is, for example, SLAN real time PCR detection system (LG Life Science, Korea), LightCycler 96/480 (Roche, Germany), ABI 7500 fast / 7500 / PRISMTM 7000/7700 (Applied Biosystems, USA), iCyclerTM / CFX96TM (Bio-Rad, USA), Rotor-GeneTM (Corbett, Australia), OpticonTM (PharmaTech , USA), but is not limited thereto.

In the present invention, when the amplification is a real-time polymerase chain reaction (real time PCR), the step of (b) may further include a step of obtaining a melting curve by melting the amplification product after melting. have.

In the present invention, the primer set of step (b) is as described above, and can be used to successfully amplify the HPV gene by real-time polymerase chain reaction without interference between each primer constituting it.

Therefore, when performing a real-time polymerase chain reaction using the primer set, the group consisting of HPV types 16, 31, 33, 35, 52, and 58 that are very reliable and rapidly related to oral cancer Qualitative detection of one or more selected from is possible.

The method for analyzing the amplification product in step (c) of the present invention can use methods known in the art without limitation. For example, in the case where the amplification is a general polymerase chain reaction, the presence or absence of amplification products on the agarose gel can be confirmed through electrophoresis. When the amplification is a real-time polymerase chain reaction, the melting curve is analyzed. The presence or absence of amplification products can be confirmed by analyzing the melting peak.

On the other hand, the method for detecting the HPV of the present invention further amplifies the gene by further including the probe of SEQ ID NO: 5 in step (b), and further qualitative or quantitative analysis of HPV type 16 in step (c) It may be a method characterized by performing.

In this case, in the clinical target, HPV type 16, 31, 33, 35, 52, and 58, which are deeply related to oral cancer, detected at least one qualitative group and at the same time, only for type 16 Qualitative and quantitative detection may be possible. This screening qualitative analysis and type 16 qualitative or quantitative analysis can be confirmed at the same time by using the melting curve analysis method, which is very useful in that it is possible to more accurately check the clinical diagnosis results for HPV related to oral cancer.

As described above, while specifically detecting only human papillomavirus (HPV) types associated with oral cancer, compositions capable of detecting each microorganism with excellent sensitivity by a real-time polymerase chain reaction method and a melting curve analysis method have been reported to date. There is no bar.

Qualitative analysis of one or more selected from the group consisting of HPV types 16, 31, 33, 35, 52 and 58, which are highly related to oral cancer when using the composition or method according to the present invention; And since it is possible to perform qualitative and quantitative analysis on the most important HPV type 16 related to oral cancer at the same time, it is possible to conduct oral cancer clinical diagnosis quickly and easily with very high reliability.

Figure 1 shows the results of real-time polymerase chain reaction by concentration for human papillomavirus type 16 (HPV type 16) E6E7 gene clone to confirm the detection sensitivity of the composition of the present invention (A), the results confirming the linearity of the results ( B) is.
2 is a real-time polymerase chain reaction for each type of oral cancer-associated human papillomavirus (HPV) using the composition of the present invention, and then analyzing the melting curve using the EvaGreen fluorescent material (A), and thus the melt peak. It is the result (B).
3 to 6 are results obtained by proceeding a real-time polymerase chain reaction using the composition of the present invention for DNA obtained in clinical samples (A), and deriving a melting curve using EvaGreen fluorescent material (B).

Hereinafter, the present invention will be described in detail.

However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

<Preparation for Experiment>

1. Preparation of primers and probes

The primers and probes used in the present invention are analyzed by using the DNAsis program of Hitachi Software, Genebank sequences registered in the gene bank (www.ncbi.nlm.nih.gov) operated by NCBI under the National Institutes of Health. The base sequence was determined and analyzed again with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Oral cancer-related human papillomavirus (HPV) type (types 16, 31, 33, 35, 52) And 58).

Human papillomavirus (HPV) type As a result of analysis using 30 E6E7 base sequences using MultAlin (http://multalin.toulouse.inra.fr/multalin/multalin.html), a multi-base sequence sequencing program, oral cancer-related human papillomavirus type It was confirmed that other types are not complementary to the base sequences of the designed primers and probes.

The sequences of the primers and probes designed in the present invention are shown in Tables 1 and 2 below.

In addition, the primers and probes used in the present invention confirm the E6E7 gene sequence related to cancer induction of human papillomavirus (HPV) in Genebank's entrez, and then analyze the conserved site using the multinucleate sequence analysis program clustal W2. The base sequence was determined and analyzed again with BLAST (www.ncbi.nlm.nih.gov/BLAST/), and only oral cancer-related human papillomavirus (HPV) type (types 16, 31, 33, 35, 52 and 58) It was confirmed that it is a primer base sequence that can be amplified.

2. Synthesis of primers

The primers analyzed in “Preparation for Experiment 1” were prepared using the method of synthesizing oligonucleotides described in 10.42 of molecular cloning 3rd edition (sambrook and rusell, cold spring harborlaboratory press, New York, USA, 2001). Korea).

3. Extraction of DNA from microorganisms

Oral microorganisms were obtained commercially, and genomic DNA was extracted using ExgeneTM Cell SV DNA extraction kit (Jinol Biotechnology, Korea) according to the manufacturer's instructions. The extracted genomic DNA was measured using a NanoDrop 2000 (Thermo Scientific co., USA) spectrophotometer.

4. Oral cancer related human papillomavirus (HPV) gene cloning and extraction of plasmid DNA

Genomic DNA was extracted from the oral clinical sample using the Exgene ^TM Clinic SV DNA extraction kit (Jinol Biotechnology, Korea) according to the manufacturer's instructions. The extracted DNA was amplified with E6E7 sites of human papillomavirus types 16, 18, 31, 33, 35, 45, 52, and 58 with amplifiable primer sets, followed by sequencing using amplification products. Analysis, cloning service and plasmid DNA extraction service (Cosmogenetech, Korea) were commissioned. Each type of identified plasmid DNA was measured using a NanoDrop 2000 (Thermo Scientific co., USA) spectrophotometer.

<Example 1>

Check the specificity of the primer

Human papillomavirus (HPV) of the primers represented by SEQ ID NOs: 1 to 4 prepared in the experimental preparation step type, No. 16, 31, 33, 35, 52 and 58 to confirm the specificity as follows The experiment was conducted.

PCR reaction solutions used to verify the primer specificity of human papillomavirus (HPV) were 2ul (1ng / ul) of template DNA, 0.5ul (10 pmol/ul) of primers of SEQ ID NOs: 1 to 4, 0.4ul (1 unit) ) SolgTM h-Taq DNA Polymerase (Solgent co., Korea), 2ul (10X concentration with 15 mM MgCl2 solution) h-Taq reaction buffer (Solgent co., Korea), 0.1ul (0.1 to prevent contamination unit) SolGent ^TM Uracil-DNA Glycosylase (Solgent co., Korea), 1ul (dATP, dCTP, dGTP 2mM and dUTP 6mM) dNTP (Solgent co., Korea) mixed solution of DNase, RNase free water (Sigma-Aldrich) co., USA) so that the total reaction solution was 20 ul. PCR reaction was performed by reacting Uracil-DNA Glycosylase at 50 ° C for 5 minutes at 50 ° C using a Peltier thermal cycler (Model PTC-100 DNA EngineTM, MJ Research Inc., USA). -The process of denaturation and hot start Taq was performed, the process of denaturation at 94 ° C for 20 seconds and annealing and extension for 40 seconds at 72 ° C were repeated 40 times, and finally stretched at 72 ° C for 5 minutes (post -extension). As for the amplified PCR reaction product, the presence or absence of the amplification reaction and the size of the amplified DNA were confirmed using a Mupid-2plus electrophoresis device (Mupid, Japan).

The results are shown in Table 3 below.

As shown in Table 3 below, the primer set of SEQ ID NOs: 1 to 4 that can specifically detect the human papillomavirus (HPV) type (types 16, 31, 33, 35, 52 and 58) associated with oral cancer is human papilloma Viruses (HPV) 16, 31, 33, 35, 52 and 58 were specifically detected for microorganisms, and other human papillomavirus (HPV) types (HPV types 18 and 45) and oral microorganisms It did not show any reaction, and it was confirmed that the specificity was very excellent.

In particular, as can be seen in Table 3, in the case of a composition comprising a primer set of SEQ ID NO: 1 to 4, any one of the forward primer selected from the group consisting of SEQ ID NO: 1 to 3 and the reverse primer of SEQ ID NO: 4 It was confirmed that each of the primer pairs including the HPV types that can be detected can be detected.

That is, even if the primers of SEQ ID NOs: 1 to 4 were mixed and applied to the amplification reaction, it was found that the HPV type that each primer can detect can be detected with very good specificity without interfering with each other.

<Example 2>

Detection sensitivity evaluation

In order to confirm the detection sensitivity of the real-time polymerase chain reaction, the “experiment preparation. Fluorescence amplification curves were confirmed for samples serially diluted 10 times starting with 1 ng / ul concentration of plasmid DNA for each type of human papillomavirus (HPV) produced in 4 ”.

The reaction solution of the real-time polymerase chain reaction is screened for human papillomavirus (HPV) and 2ul (1 ng / ul) template DNA, 0.5 ul (10 pmol / ul) of SEQ ID NO: 1 to confirm the qualitative reaction of type 16 Primer of 4 and probe of SEQ ID NO: 5 of 0.2 ul (10 pmol / ul), 0.2 ul of 20X EvaGreen (Biofact, Korea), 0.4 ul (1 unit) of Solg ^TM h-Taq DNA Polymerase (Solgent co., Korea) ), 2ul (10X concentration with 15 mM MgCl ₂ solution) h-Taq reaction buffer (Solgent co., Korea), 0.1ul (0.1 unit) SolGent ^TM Uracil-DNA Glycosylase (Solgent co. , Korea), 1ul (dATP, dCTP, dGTP, respectively, 2mM and dUTP 6mM) dNTP (Solgent co., Korea) mixed solution with DNase, RNase free water (Sigma-Aldrich co., USA), total reaction solution is 20ul It was made possible. PCR reaction was carried out by reacting Uracil-DNA Glycosylase at 50 ℃ for 5 minutes at 50 ℃ using a Real-time PCR Thermal Cycler (Model CFX96 Real-time PCR Detection system, Bio-Rad Inc., USA) at 94 ℃ After 10 minutes of initial denaturation (pre-denature) and hot start Taq, the process was performed for 40 seconds of denaturation at 94 ° C for 20 seconds and annealing and extension for 40 seconds at 72 ° C. Thereafter, for the melting curve analysis method, a Melting curve analysis step was performed in which the fluorescence value was measured while increasing by 0.2 degrees from 65 ° C to 95 ° C. The fluorescence value was measured at 70 ° C., and Bio-Rad CFX Manager 2.1 was used for data analysis. The cycle threshold (Ct) started with a logarithmic amplifier with a known number of DNA concentrations, and qualitative analysis was performed according to the presence or absence of a reaction.

The results are shown in Figure 1 and Table 4.

As shown in Figure 1 and Table 4, the composition according to the invention was able to detect HPV associated with oral cancer up to 31.3 copy, it was confirmed that the sensitivity is very excellent.

On the other hand, the gene amplification reaction was performed in the same manner as the above experimental method, and the detection sensitivity for HPV types 31, 33, 35, 52 or 58 was evaluated by performing a melting curve analysis method using EvaGreen. As shown, it was confirmed that the sensitivity of the composition of the present invention to the HPV types is very excellent.

<Example 3>

Qualitative evaluation of the melting curve analysis method

When the composition of the present invention in which the primer set of SEQ ID NOs: 1 to 4 and the probe of SEQ ID NO: 5 are mixed is subjected to a real-time polymerase chain reaction and a melting curve analysis method using EvaGreen, for oral cancer-related human papillomavirus (HPV) types To confirm whether the screening qualitative reaction is possible, a detection experiment was performed for each HPV type gene clone related to oral cancer with the same composition as the real-time polymerase chain reaction solution of Example 2 above.

In this experiment, the melt peak result of the melting curve analysis method for HPV type related to oral cancer is 82 ℃, and the response by non-specific reaction or dimer has a different melt peak value or low fluorescence intensity. Therefore, in the analysis result, when the melt peak value was 82 ° C, the screening was positive, and when the result of the melt peak or None at a different temperature was shown, the screening was negative.

The results are shown in Figure 2 and Table 6.

As can be seen in Figure 2 and Table 6, the results of the melting curve analysis of the real-time polymerase chain reaction performed using the composition of the present invention in which the primer set of SEQ ID NO: 1 to 4 and the probe of SEQ ID NO: 5 are mixed, melt It was confirmed that HPV types 16, 31, 33, 35, 52 and 58 related to oral cancer were detected at a peak of 82 ° C.

Meanwhile, as a result of performing a real-time polymerase chain reaction for the HPV type 18 gene clone using the composition of the present invention, the melt peak was confirmed to be 80 ° C, and for the HPV type 45, the melt peak did not appear, so the composition of the present invention It was confirmed that the non-specific amplification reaction did not occur for HPV type not related to oral cancer.

<Example 4>

Evaluation of clinical sample application of real-time polymerase chain reaction and melting curve analysis method

When the composition of the present invention in which the primer set of SEQ ID NOs: 1 to 4 and the probe of SEQ ID NO: 5 were mixed was subjected to a real-time polymerase chain reaction and a melting curve analysis method using EvaGreen fluorescent material, HPV type related to oral cancer was also found in clinical samples. In order to confirm whether it can be detected, the experiment was conducted with the same composition as the real-time polymerase chain reaction solution of Example 2 above, but the DNA obtained from the clinical sample was subjected to amplification.

The clinical sample was a sample of saliva through oral gargling for patients with high concentrations of microbes related to oral diseases such as dental caries or periodontal disease among patients visiting the dentist, and used the stored clinical sample.

The results are shown in FIGS. 3 to 6 and Table 7 below.

Referring to Table 5 and FIG. 3, in clinical sample # 1, HPV type 16 was qualitatively confirmed by the probe represented by SEQ ID NO: 5, and the melt peak was formed at 82 ° C even in the melting curve. It was confirmed that HPV type 16, which is most closely related to the development of oral cancer, was included in clinical sample # 1. Likewise, referring to Table 5 and FIG. 4, the same result as in clinical sample # 1 was observed in clinical sample # 2.

On the other hand, referring to Table 5 and FIG. 5, in the clinical sample # 3, the Cy5 type and the CT value by the probe represented by SEQ ID NO: 5 are not detected, so HPV type 16 is not included in the sample, but the melting curve From the fact that the melt peak is formed at 82 ° C, it can be seen that at least one HPV type is included in clinical sample # 3 in the group consisting of HPV types 31, 33, 35, 52 and 58, excluding HPV type 16. there was.

On the other hand, referring to Tables 5 and 6 above, in clinical sample # 4, CyV type and CT value by the probe represented by SEQ ID NO: 5 are not detected, and thus HPV type 16 is not included in the sample. However, it was found that the melting peak did not form in the melting curve, and therefore clinical trial # 4 could be judged not to contain HPV type related to oral cancer.

As described above, the composition of the present invention comprising a primer represented by SEQ ID NOs: 1 to 4 and a probe represented by SEQ ID NO: 5 not only enables qualitative examination of HPV types related to induction of oral cancer, but also causes the most powerful oral cancer It was confirmed that qualitative and quantitative tests for the factor HPV type 16 were possible.

In particular, the composition of the present invention can be used very effectively in the field of oral cancer diagnosis that requires fast and accurate results because each primer can detect oral cancer-related HPV types with very high sensitivity and specificity without causing interference reactions with each other. It can be judged that this can be.

Qualitative analysis of one or more selected from the group consisting of HPV types 16, 31, 33, 35, 52 and 58, which are highly related to oral cancer when using the composition or method according to the present invention; And since it is possible to perform qualitative and quantitative analysis on the most important HPV type 16 related to oral cancer at the same time, it is possible to conduct oral cancer clinical diagnosis quickly and easily with very high reliability, and thus has excellent industrial applicability.

<110> HEO, DAE WOOK          HEO, JUNG UK          KIM, SUNG HYUN <120> Screening and qualitative methods of HPV types and type 16          related to oral cencer using Real-time PCR and Melting Curve          Analysis <130> NP18-0034 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for HPV type 16 and 35 <400> 1 tgtcaaaagc cgctgtgtcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for HPV type 31, 33 and 58 <400> 2 tgtcaaagac cgttgtgtcc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for HPV type 52 <400> 3 tgtcaaacgc cattatgtcc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for HPV <400> 4 tcmtcctcmt ctgagctgtc 20 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Probe for HPV type 16 <400> 5 atcatcaaga acacgtagag aaacccagct 30

Claims (12)

Human papillomavirus (HPV) detection composition comprising at least one primer selected from the group consisting of SEQ ID NO: 1 to 3 and the primer of SEQ ID NO: 4.
The composition of claim 1, wherein the composition comprises primers of SEQ ID NOs: 1-4.
The composition of claim 1, wherein the human papillomavirus is at least one selected from the group consisting of types 16, 31, 33, 35, 52 and 58.
The composition of claim 1, further comprising a probe of SEQ ID NO: 5.
The method according to claim 4, wherein the composition comprises at least one qualitative detection selected from the group consisting of human papillomavirus types 16, 31, 33, 35, 52 and 58; And type 16 qualitative and quantitative detection is possible at the same time.
The composition according to any one of claims 1 to 5, wherein the detection is by melting curve analysis.
A composition for diagnosing oral cancer, comprising the composition of any one of claims 1 to 5.
Kit for detecting human papillomavirus (HPV) comprising at least one primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a primer of SEQ ID NO: 4.
(a) extracting DNA from the clinical sample;
(b) amplifying the gene with the obtained DNA using a primer set comprising at least one primer selected from the group consisting of SEQ ID NOs: 1 to 3 and a primer of SEQ ID NO: 4; And
(c) A method for detecting human papillomavirus (HPV) by analyzing the amplification product.
10. The method of claim 9, The amplification is a real-time polymerase chain reaction (real time PCR) and further comprising the step of obtaining a melting curve by melting the amplification product after step (b). Method characterized in that.
10. The method of claim 9, wherein the step (b) further comprises a probe of SEQ ID NO: 5 to amplify the gene, characterized in that further performing qualitative or quantitative analysis of human papillomavirus type 16 in step (c) How to.
The method of claim 9, wherein the clinical sample is a sample obtained from the oral cavity.

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