KR101335483B1

KR101335483B1 - Method of Detecting Human Papilloma Virus and Genotyping Thereof

Info

Publication number: KR101335483B1
Application number: KR1020110045957A
Authority: KR
Inventors: 안웅식; 배수미; 김윤선
Original assignee: 주식회사 진진바이오
Priority date: 2010-05-14
Filing date: 2011-05-16
Publication date: 2013-12-05
Also published as: KR20110126076A; WO2011142646A3; WO2011142646A2; WO2011142646A9

Abstract

The present invention amplifies a single strand of L1 gene of human papillomavirus without a separate labeling PCR process, and hybridizes with the amplified L1 gene single strand and human papillomavirus detection probe to detect and identify genotype of human papillomavirus with high detection sensitivity. It is about a method. According to the present invention, it is possible to detect the human papillomavirus present in trace amounts before the propagation of the human papillomavirus, with high accuracy, reliability and simple high sensitivity.

Description

Method of Detecting Human Papilloma Virus and Genotyping Thereof}

The present invention amplifies a single strand of L1 gene of human papillomavirus without a separate labeling PCR process, and hybridizes a probe specific to each genotype capable of distinguishing the genotype of the L1 gene from the amplified L1 gene single strand, thereby detecting sensitivity. Relates to high human papillomavirus detection and genotyping methods. The present invention also relates to a method for providing information for diagnosing HPV infection and infecting genotypes using the above method, and a screening method for treating cervical cancer.

Every two minutes, one woman dies of cervical cancer worldwide. Cervical cancer, which occurs worldwide annually, is the second most common cancer among women worldwide, affecting about half a million people each year, and among women, the third leading cause of cancer deaths after breast and lung cancer. Human papilloma virus infections are found in more than 99% of cervical cancers, including more than 100 of these, 30-40 of which cause mucosal tissue infections, most of which are papilloma types. Viral infections usually heal within six months to two years, but persistent infection with carcinogenic HPV can lead to cervical cancer. Cervical cancer is closely related to sexual contact, and HPV infection is known to be related to cervical tumor development. To date, about 100 human papillomavirus genotypes have been reported. Among them, about 30 human papillomaviruses can cause disease in humans, which are largely high risk (16, 18, 31, 33, 35, etc.) and low risk ( 6, 11, 42, 43, 44, etc.).

Currently, the most commonly used screening method is a Pap smear (PAP smear), which is disadvantageous in that the accuracy of the test depends on the skill of the examiner. Although colposcopy can diagnose up to 70% of human papillomavirus infections, it requires a trained specialist and expensive equipment and has the disadvantage of not being able to classify genotypes of human papillomaviruses. PCR-RFLP using a restriction enzyme after amplifying the L1 region of the human papillomavirus gene by PCR has a simple and easy result, but has a disadvantage in that the restriction enzyme cannot be analyzed if the restriction enzyme is not recognized. In addition, the efficiency of the PCR amplification may vary depending on the HPV genotype, which may be a problem in the accuracy of the test. Commercially available hybrid capture kits (Digene, USA) can be identified without PCR amplification, but they can be classified into high-risk and low-risk groups and cannot distinguish between high-risk types 16 and 18 and other high-risk groups. . The recently developed human papillomavirus genotyping kit (Micromedlab, Korea) using the microchip technology is a two-dimensional method that reacts on a slide, and it is cumbersome to undergo three steps after the hybridization reaction. In addition, although a human papillomavirus detection kit of a method using a suspension array has been developed in a similar manner, the kit has a limitation in that the signal value required for detection is very low so that it can be detected in reality. Also, if more than one virus is infected, a low signal may not recognize a low concentration of the virus. In addition, the inventors of the present invention (application number: 10-2006-0020684) has a disadvantage in that it takes too long time to go through a separate PCR labeling process.

Thus, the inventors have used L1 using a primer labeled with a first marker, which is common to two or more genotypes of the L1 gene of a sample, and a second primer labeled with a second marker recognized by an exonuclease. PCR amplification of the gene and digestion of only the B-fluorescent-labeled strands with enzymes that recognize the B-fluorophore, yielding highly-labeled strands with high sensitivity quickly with only one PCR without a separate labeling PCR process It was confirmed that it can be done. In addition, by using a probe specific to each genotype capable of distinguishing two or more genotypes of the L1 gene, the present invention is confirmed by not only detection of human papillomavirus, but also the identification of various genotypes of human papillomavirus with high sensitivity. Completed.

An object of the present invention is to provide a human papillomavirus detection and genotyping method contained in the sample to be analyzed.

Another object of the present invention is to provide an information providing method for diagnosing human papillomavirus infection and infecting genotype.

It is another object of the present invention to provide a method for screening a cervical cancer therapeutic agent.

Another object of the present invention is to provide a method for amplifying a single strand of L1 gene of human papillomavirus.

In order to achieve the object of the present invention described above, in one aspect, the present invention provides a method for labeling the first marker, which is commonly specific to two or more genotypes of human papillomavirus (HPV) L1 gene, from a sample to be analyzed. PCR amplifying the L1 gene using a first primer and a second primer labeled with a second marker recognized by an exonuclease; 2) by reacting the double-stranded L1 gene product amplified in the first step by adding an exonuclease that recognizes the second marker, and decomposing the single-stranded L1 gene product labeled with the second marker. Obtaining a labeled single stranded L1 gene product; And 3) using a single strand of the L1 gene product labeled with the first marker, obtained in the second step, using at least two probes specific to each genotype capable of distinguishing two or more genotypes of the L1 gene. Provided is a human papillomavirus detection and genotyping method, characterized in that it comprises a third step of identifying the genotype.

The present invention can quickly determine the presence or genotype of the L1 gene of human papillomavirus in a sample to be analyzed quickly through a single PCR without a labeling PCR process. In addition, through the process of steps 1) and 2), by obtaining only a single strand of the L1 gene labeled with the first marker to increase the signal value required for detection, the accuracy and sensitivity of genotyping of human papillomaviruses having various genotypes Can increase.

Step 1) is amplification of the L1 gene of human papillomavirus by PCR using a set of forward and reverse primers specific for the L1 gene from the sample DNA, in which the L1 gene is amplified exponentially. In this case, the primer set is labeled with a first marker and a second marker, respectively.

In the present invention, the term "first marker" is intended to label a gene with a first marker by PCR using a first primer labeled with a first marker as the first primer marker. The first marker is not limited as long as it can be used for gene labeling without interfering with the hybridization of the probe with the gene product, and may use various materials known in the art. In a specific embodiment of the present invention, biotin is used, but is not limited thereto. Primers labeled with the first marker in the forward or reverse direction are used for amplification into strands labeled with the first marker. When the hybridizing probe is an antisense strand, the first primer is labeled on the forward primer to amplify the sense strand. When the hybridizing probe is the sense strand, the first marker is labeled on the reverse primer to amplify the antisense strand. Let's do it.

In the present invention, the term "second marker" is intended to label a gene with a second marker by PCR using a second primer labeled with a second marker as a second primer marker. The second marker may label genes other than the first marker, but may be various materials known in the art without being limited as long as they can be recognized by exonuclease. In a specific embodiment of the present invention used phosphate, but is not limited thereto. Primers labeled with a second marker in the forward or reverse direction are used for amplification with the strand labeled with the second marker. When the hybridizing probe is an antisense strand, the antisense strand is amplified by labeling the second marker on the reverse primer, and when the hybridizing probe is the sense strand, the antisense strand is labeled by the first primer on the forward primer. Let's do it.

As used herein, the term “primer” refers to a nucleic acid sequence having a short free 3 ′ hydroxyl group, which may form complementary templates and base pairs and is a starting point for copying a template. By short nucleic acid sequence is meant to function. Primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In the present invention, PCR amplification is performed using the sense and antisense primers of the marker polynucleotide of the present invention to diagnose the exposure of electromagnetic waves through the generation of a desired product. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art. The first primer provided by the present invention is a primer that is common to two or more genotypes of the HPV L1 gene. The second primer is a primer labeled with a second marker recognized by an exonuclease, and may be common to two or more genotypes of the L1 gene, but is not limited thereto. In a specific embodiment of the present invention, a first primer of SEQ ID NO: 1 labeled with biotin and a second primer of SEQ ID NO: 2 labeled with phosphate were used.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosphoro Amidate, carbamate, etc.) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).

In addition, in the PCR of step 1), denaturation, binding, synthesis reaction time and temperature, the number of cycles and the like can be adjusted. In order to increase the sensitivity of the detection of the human papillomavirus L1 gene, an optimal PCR condition was established. In an embodiment of the present invention, the PCR was performed by setting the annealing temperature to 50, 53, 55, or 57 ° C., and then, the biotin-labeled single strand was obtained, reacted with the probe, and the signal value was measured. As a result, the PCR products were more specific in all types compared to the results at other temperatures when performed under 53 ℃ annealing conditions, and HPV 11, 40, 51 when the reaction temperature was raised to 57 ℃. PCR products of 58, 70 and 70 were not identified. Therefore, it was found that the appropriate PCR annealing temperature is 53 ℃ (Figs. 3 to 6).

Based on the above embodiment, in the PCR amplification of the present invention, the annealing temperature is preferably set to 52 to 55 ℃.

In addition, in one embodiment of the present invention after PCR using the primer concentration of 0.0125, 0.025, 0.05 or 0.1μM, respectively, the results were confirmed by electrophoresis. As a result, it was confirmed that the best response at 0.025μM, PCR reaction was not good at 0.1 and no band was identified at HPV 51 type at 0.0125μM.

In addition, PCR amplification of step 1) is preferably performed using dCTP labeled with a first marker.

Based on the above embodiment, the concentration of the primer used in PCR amplification of the present invention is preferably 0.02 to 0.03μM.

Step 2) is to leave only a single strand of L1 gene labeled with the first marker in the HPV amplified L1 gene in step 1), wherein the L1 gene labeled with the second marker in this step is determined by exonuclease. Decompose That is, since only the strands complementary to the probe are amplified by the template activation of the exponentially amplified gene product through the PCR reaction of step 1), the hybridization efficiency between the probe and the gene product Is increased, and there is an effect that the detection signal is increased.

The exonuclease is not limited as long as it recognizes the second marker and can decompose the gene labeled by the second marker, and various enzymes known in the art may be used. In a specific embodiment of the present invention, the second marker may be a phosphate. The exonuclease that recognizes the phosphate and degrades the phosphate-labeled gene was used lambda exonuclease.

The time and temperature at which the template activation reaction occurs in step 2) can be controlled.

According to the present invention, when the human papillomavirus gene in the sample of the present invention is processed into a single-stranded L1 gene labeled with the first marker, it is possible to save time about 4 hours than the conventional labeling PCR process separately. It was confirmed. In addition, only a single strand of the L1 gene labeled with the first marker can be obtained, increasing the signal for detection of human papillomavirus (FIG. 8). Accordingly, the present invention provides a method for detecting HPV genotypes that can detect human papillomavirus very quickly and has high accuracy and reliability and simple high sensitivity.

Step 3) is a step of performing a hybridization reaction of a single-stranded L1 gene product labeled with the first marker prepared by the method of steps 1) and 2 with a probe for detecting HPV.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which is short to several bases to hundreds of bases, which is capable of specific binding with mRNA. You can check the presence. The probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. Probes of the present invention may be prepared in consideration of each type of HPV L1 gene sequence, and hybridization reactions with samples using the probe include all probes of any sequence as long as the HPV can be specifically detected.

The probe provided in the present invention preferably uses two or more probes specific to each genotype capable of distinguishing two or more genotypes of the L1 gene, and is preferably provided in the form of a bead array bound to the beads. The kind of beads to be combined with the probe is not particularly limited and the preparation of the bead array is in accordance with general methods known in the art.

The two or more probes used in step 3) may be the same site or different sites in the L1 gene.

In step 3), a single strand of L1 gene product labeled with the first marker is hybridized with two or more L1 genotype detection probes to be distinguished by L1 genotype, and then, the L1 gene product is specific to the first marker. The human papillomavirus detection and genotyping may be performed by binding to and reacting the binder to which the fluorescent substance is attached and measuring the fluorescence value.

As a method for increasing the detection sensitivity of the HPV L1 gene in step 3), in an embodiment of the present invention, an optimal temperature for hybridizing a single-stranded L1 gene product and a probe labeled with the first marker was set. The fluorescence value (MFI, mean fluorescence intensity) was measured by setting the hybridization temperature to 30, 37 or 40 ° C for HPV 11, 16, 18, 51, 58, 70, Caski or C33A. At 30 ° C, the HPV MFI values detected at Caski were detected at HPV 18 in addition to HPV 16. At 40 ° C, the overall background signal was reduced, but GAPDH of C33A and Caski, the standards, were not detected because they did not exceed the cut-off value. 9 to 11).

Based on the embodiment, the temperature at the time of hybridization with the probe of step 3) is preferably 35 to 38 ° C.

In addition, in one embodiment of the present invention, in order to set the optimal PCR product dose for hybridization, 10, 20 or 50 μl of the PCR product for HPV 11, 16, 18, 51, 58, 70, Caski or C33A. And hybridized with the probe. As a result, the results of using 50 μl showed a tendency to lower the MFI value of HPV slightly (FIGS. 15 to 17).

Based on the above embodiment, the PCR product for hybridization in step 3) is preferably 10 to 30 μl.

In step 3), a single-stranded L1 gene product labeled with the first marker is hybridized with two or more L1 genotype detection probes to be distinguished by L1 genotype, and then specifically bound to the first marker. Fluorescence is confirmed by treating the binder to which the fluorescent substance is attached. Treatment of a specific binding agent with the first marker to which the fluorescent substance is attached binds specifically to the first marker, so that the L1 gene present in the sample can be measured by fluorescence.

The specific binding agent to the first marker to which the fluorescent substance is attached may be used in a variety of materials known in the art, and may be used without limitation as long as it has a specific binding force with the first marker and attaches the fluorescent material. . In a specific embodiment of the present invention, streptavidin was used as a specific binding agent of biotin as a first marker and a first marker to which a fluorescent substance was attached, but is not limited thereto.

Fluorescent materials may include, but are not particularly limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. In a specific embodiment of the present invention, phycoerythrin was used.

In the present invention, when the human papillomavirus gene in the sample of the present invention is processed into a single-stranded L1 gene labeled with a first marker, the signal value is about 10 times stronger than the direct fluorescent labeling of a conventional double-stranded PCR product. It was confirmed experimentally that it can be obtained. Accordingly, the present invention provides a high-accuracy, simple and highly sensitive detection method capable of early diagnosis of human papillomaviruses present in trace amounts before the propagation of human papillomaviruses.

In addition, as a result of comparing the detection sensitivity, specificity and accuracy of the human papillomavirus type disclosed in the Republic of Korea Patent (10-0702415) devised by the present inventors with the detection sensitivity, specificity and accuracy of the L1 gene, the present invention is compared with the known method. Virus detection sensitivity and accuracy was found to be significantly higher (Fig. 19).

In another embodiment, the present invention provides a method for the detection of 1) a first primer labeled by a first marker, an exonuclease, which is common to two or more genotypes of the human papillomavirus (HPV) L1 gene. The L1 gene using a second primer labeled with a second marker, a third primer labeled with a first marker specific for the GAPDH gene, and a fourth primer labeled with a second marker recognized by the exonuclease; A first step of PCR amplifying the GAPDH gene; 2) by reacting the double-stranded gene product amplified in the first step by adding an exonuclease that recognizes the second marker to decompose the single-stranded gene product labeled with the second marker to label the first marker. Obtaining a single strand of gene product; And 3) using at least two probes specific to each genotype capable of distinguishing two or more genotypes of the L1 gene from the single-stranded L1 gene product labeled with the first marker, obtained in the second step, and using the GAPDH gene. It provides a human papillomavirus detection and genotyping method, characterized in that it comprises a third step of identifying the genotype of the L1 gene using a probe specific for the.

Anything already described and repetitive matters will be omitted to avoid repetition.

GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is a gene located at human chromosome No. 12 and is an enzyme that is involved in glycolysis, an essential metabolic process in cells. Therefore, in the present invention, the GAPDH gene was used as an internal control.

GADPH primers are designed to specifically amplify the GADPH gene, which is always expressed as an endogenous gene in a sample. In a specific embodiment of the present invention, a primer set having a nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4 was used. SEQ ID NO: 3 is the reverse primer labeled with the first marker and SEQ ID NO: 4 is the forward primer labeled with the second marker.

A GAPDH gene detection probe for detecting a GADPH gene is simultaneously hybridized with the probe for detecting the L1 gene. This process can immediately determine whether the PCR reaction is successful in the beads array analyzer can significantly reduce the number of undetected cases due to PCR amplification failure.

In still another aspect, the present invention provides a method for preparing a protein comprising 1) a first primer labeled with a first marker, and an exonuclease, which are commonly specific to two or more genotypes of the human papillomavirus (HPV) L1 gene. A first step of PCR amplifying the L1 gene using a second primer labeled with a recognized second marker; 2) by reacting the double-stranded L1 gene product amplified in the first step by adding an exonuclease that recognizes the second marker, and decomposing the single-stranded L1 gene product labeled with the second marker. Obtaining a labeled single stranded L1 gene product; And 3) using a single strand of the L1 gene product labeled with the first marker, obtained in the second step, using at least two probes specific to each genotype capable of distinguishing two or more genotypes of the L1 gene. Characterized in that it comprises a third step of identifying the genotype, the present invention relates to a method for providing information for the diagnosis of human papillomavirus infection and the infected genotype.

When the HPV is diagnosed by the above method, the diagnosis time is significantly reduced because the gene is detected by one PCR process, and only the first marker can obtain only a single strand of the labeled L1 gene and hybridize with the probe. The presence of human papillomavirus infection and the infected genotype can be diagnosed.

In still another aspect, the present invention provides a method of treating cancer, comprising the steps of: 1) administering a candidate therapeutic agent to a subject infected with HPV; And 2) separating the DNA from the subject, and detecting the genotype of the human papillomavirus by the method described above.

The therapeutic agent for cervical cancer can be screened by administering a candidate therapeutic agent and confirming that human papillomavirus is detected. Since the screening method can detect human papillomavirus with high sensitivity or confirm genotype, it is possible to screen an accurate cervical cancer therapeutic agent in a short time.

Such candidate therapeutics include any molecule such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides and a wide variety of compounds. Such candidates also include both natural and synthetic materials.

The individual used in the present invention means an entire mammal including a dog, cow, horse, rabbit, mouse, rat, chicken or human, but the mammal of the present invention is not limited to the above examples.

In still another aspect, the present invention provides a method for the preparation of 1) a first marker labeled with a first marker, and an exonuclease, which are commonly specific to two or more genotypes of the human papillomavirus (HPV) L1 gene. PCR amplifying the L1 gene using a second primer labeled with a second marker recognized by the first step; And 2) reacting the double-stranded L1 gene product amplified in the first step by adding an exonuclease that recognizes the second marker to decompose the single-stranded L1 gene product labeled with the second marker. The second step of obtaining a single-stranded L1 gene product labeled; relates to a method for amplifying a single-stranded L1 gene of human papillomavirus.

In the single-strand amplification method of the gene, since a single strand of the gene labeled with the first marker can be obtained without a separate labeling PCR process, the time required for PCR is shortened.

According to the present invention, it is possible to quickly obtain a strand labeled with fluorescent material by only one PCR without going through a separate labeling PCR process, so that pathogens to be detected such as human papilloma virus can be detected quickly, and conventional double-stranded PCR A signal value 10 times stronger than a direct fluorescent label on the product can be obtained. In addition, it is possible to distinguish between 16 and 18 types that are highly correlated with cervical cancer and other high-risk groups. Accordingly, the present invention provides a high-accuracy, simple and highly sensitive detection method capable of early diagnosis of human papillomaviruses present in trace amounts before the propagation of human papillomaviruses.

Figure 1 shows a schematic diagram of the PCR process and the template activation (Template activation) process.
Figure 2 is a chart confirming the detection of HPV using the present invention CaSki and HeLa cell line known to be infected HPV negative cell line C33A and HPV 16 type and HPV 18 type.
Figure 3 is an electrophoresis picture showing the PCR product performed at 50 ℃.
Figure 4 is an electrophoresis picture showing the PCR product performed at 53 ℃.
Figure 5 is an electrophoresis picture showing the PCR product performed by setting at 55 ℃.
Figure 6 is an electrophoresis picture showing the PCR product performed at 57 ℃.
Figure 7 is an electrophoresis picture showing the PCR product performed by varying the concentration of the primer.
8 is a chart showing MFI values before and after lambda exonuclease treatment.
9 is a graph showing MFI values measured when the hybridization temperature is 30 ° C.
10 is a chart showing the MFI value measured when the hybridization temperature is 37 ℃.
11 is a chart showing MFI values measured when the hybridization temperature is 40 ° C.
12 is a chart showing MFI values measured when the hybridization time is 1 hour.
FIG. 13 is a chart showing MFI values measured when the hybridization time is 2 hours.
14 is a chart showing MFI values measured when the hybridization time is 3 hours.
FIG. 15 is a chart showing MFI values measured when 10 μl of PCR product was used. FIG.
FIG. 16 is a chart showing MFI values measured when 20 μl of PCR product was used. FIG.
17 is a chart showing MFI values measured when 50 μl of PCR product was used.
18 is a graph showing changes in relative MFI values according to fluorescent materials.
19 shows the sensitivity, specificity and accuracy of HPV detection and genotyping using the present invention. In addition, the results are compared with the conventional HPV genotype detection method is shown together.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Method of Confirming Infection and Genotype of HPV by Performing One PCR

Example 1-1: DNA Extraction from Samples

To separate the DNA of the sample, scrape the inner wall of the vagina with a brush to obtain a cell sample. To the cell sample, 400 μl of lysis solution and 10 μl of Proteinase K (20 mg / mL) were added, followed by reaction at 58 ° C. for 2 hours. , 15 minutes was heated to 95 ℃ to obtain a cell lysate. DNA contained in the cell lysate was extracted by a known method.

Example 1-2 Amplification of Human Papilloma Virus (HPV) Gene in DNA

Human papillomavirus (HPV) gene was amplified by PCR using 5 μg of DNA extracted in Example 1 as a template, using biotin-labeled dCTP, and modified HPV primer set or GAPDH primer set. The base sequence of each primer set used at this time is as follows, the forward primer (Forward primer) was labeled with phosphate at 5 'and the reverse primer was labeled with biotin at 5'.

Modified HPV Primer Set:

HPV forward: 5'phosphate-GCMCAGGGWCATAAYAATGG 3 '(SEQ ID NO: 1)

HPV reverse: 5'biotin-CGTCCMARRGGAWACTGATC 3 '(SEQ ID NO: 2)

Modified GAPDH Primer Set:

GAPDH forward: 5'phosphate-GGGCAGCCCCTTCATACCCTCA 3 '(SEQ ID NO: 3)

GAPDH reverse: 5'biotin-CCCAAGGGAGCCACACCATCCT 3 '(SEQ ID NO: 4)

In addition, PCR conditions are as follows.

PCR conditions Cycles Time Temp Denaturation / Activation One 5min 95 ℃ Denaturation
Annealing
Elongation 40 1min
1min
1min 94 ℃
55 ℃
72 ℃ Final Extension One 7min 72 ℃ Cooling One ∞ 4 ℃

Example 1-3 Screening of PCR Products

The PCR product obtained in Example 2 was treated with lambda exonuclease, reacted at 37 ° C. for 30 minutes, heated at 80 ° C. for 15 minutes, and the PCR product labeled with phosphate at 5 ′ was obtained from the PCR product. By digestion, PCR products labeled with biotin at 5 'were selected from the obtained PCR products.

Example 1-4 Reaction of Bead-bound Probe with PCR Product Labeled with Biotin

Probe or each HPV genotype (HPV 6, 11, 16, 18, 31, 33, 35, 39, 40, 42) designed to bind the GAPDH gene to the 5 'biotin-labeled PCR product selected in Example 3 , 51, 52, 53, 55, 56, 58, 59, 66, 68 and 70) was added to the probe-bound beads and Hybrisol ^® and reacted for 5 minutes at 95 ℃ and 30 minutes at 37 ℃. At this time, the bead has a unique MFI value of each bead, as a MFI value of each bead can determine which genotype HPV can be detected by the probe bound to each bead, probes designed to recognize each HPV genotype The combined beads were votexed before use, and Hybrisol ^® was used after incubation at 60 ° C for 5 minutes.

Each reaction was then placed in each well of a 96-well plate, filtered to remove liquid components, and then washed three times. Then, streptavidin labeled with phycoerythrin (PE) was added to each well and mixed at 250 rpm for 10 minutes.

Example 1-5: Signal Detection

The reaction product of Example 4 was added to Luminex 100, using two lasers, one detecting the MFI value of the beads, and one detecting the value of PE. Among the reaction products detected by the Luminex 100, a sample in which the MFI value and the PE value of the beads were detected at the same time was selected to identify which HPV genotypes the sample exhibited. As a result, it was confirmed that only GAPDH was detected in the sample without HPV infection, and that GAPDH and HPV16 were simultaneously detected or that both GAPDH and HPV18 were simultaneously detected in the HPV-infected sample (FIG. 2). Figure 2 is a chart confirming the detection of HPV using the present invention CaSki and HeLa cell line known to be infected HPV negative cell line C33A and HPV 16 type and HPV 18 type. As shown in Figure 2, using the method of the present invention, it can be seen that whether the HPV is infected by performing a single PCR, it is possible to determine the genotype of the infected HPV.

Example 2: Establish Optimal Conditions

Example 2-1: PCR Annealing Optimization of temperature

Genes of HPV 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 51, 52, 53, 55, 56, 58, 59, 66, 68 and 70 are used as templates, and the annealing temperature PCR was performed under the same conditions as in Example 1-2 except for setting 50, 53, 55, or 57 ° C., and the results were confirmed by electrophoresis (FIGS. 3 to 6). Figure 3 is an electrophoretic picture showing the PCR product performed at 50 ℃, Figure 4 is an electrophoretic picture showing the PCR product performed at 53 ℃, Figure 5 is a PCR product performed at 55 ℃ It is an electrophoretic picture showing, Figure 6 is an electrophoretic picture showing the PCR product performed by setting at 57 ℃. As shown in FIGS. 3 to 6, when PCR was performed at 53 ° C. annealing conditions, more specific PCR products were observed in all types compared to the results at other temperatures, and the reaction temperature was raised to 57 ° C. In case no PCR products of HPV 11, 40, 51, 58 and 70 were identified. Therefore, the appropriate PCR annealing temperature was found to be 53 ℃.

Example 2-2: PCR primer Optimization of concentration

Examples 1-2 except genes of HPV 11, 16, 18, 51, 58, 70, Caski and C33A were used as templates and the concentration of primers was used at 0.0125, 0.025, 0.05 or 0.1 μM, respectively PCR was performed under the same conditions as above, and the results were confirmed by electrophoresis (FIG. 7). Figure 7 is an electrophoresis picture showing the PCR product performed by varying the concentration of the primer. As shown in Figure 7, it was found that the appropriate primer concentration is 0.025μM.

Example 2-3: Exonuclease According to before and after treatment MFI Compare values

To determine the effect of the single-stranded digestion of the phosphate-labeled L1 gene on lambda exonuclease treatment on human papillomavirus detection and genotyping, the C33A and HPV positive samples were used as negative samples. The experiment was carried out using Caski as a standard. Lambda exonuclease was reacted by adding 40 µl of the PCR product, and the reaction was hybridized with a probe specific for the genotype of the human papillomavirus L1 gene to measure MFI values. When comparing before and after lambda exonuclease treatment, it was not detected in Caski when not treated, and when treated, it was confirmed that the MFI value of HPV detected in Caski was high (FIG. 8).

Example 2-4: hybridization Optimization of temperature conditions

Example 1-4 except setting the hybridization temperature to 30, 37 or 40 ° C for HPV 11, 16, 18, 51, 58, 70, Caski or C33A to set the optimum temperature for hybridization Hybridization was performed under the same conditions as, and the MFI values measured as a result were compared (FIGS. 9 to 11). FIG. 9 shows the MFI value measured when the hybridization temperature is 30 ° C, FIG. 10 shows the MFI value measured when the hybridization temperature is 37 ° C, and FIG. 11 shows the MFI value measured when the hybridization temperature is 40 ° C. . As shown in FIGS. 9 to 11, the MFI values of HPV detected in all results were similar, but at 30 ° C., the HPV MFI values detected at Caski were detected at HPV 18 as well as HPV 16, and at 40 ° C., the overall background signal was detected. Although it was reduced but was not detected because the standard C33A and Caski's GAPDH did not exceed the cut off value, 37 ° C was determined to be suitable.

Example 2-5: hybridization Optimization of time condition

Except setting the hybridization time to 1, 2 or 3 hours at 37 ° C for HPV 11, 16, 18, 51, 58, 70, Caski or C33A to set the optimal time for hybridization Hybridization was performed under the same conditions as in 1-4, and the measured MFI values were compared as a result (FIGS. 12 to 14). 12 shows the MFI value measured when the hybridization time is 1 hour, FIG. 13 shows the MFI value measured when the hybridization time is 2 hours, and FIG. 14 shows the MFI value measured when the hybridization time is 3 hours. . As shown in Figures 12 to 14, the MFI value of HPV detected in all the results was similar and determined to 2 hours.

Example 2-6: hybridization PCR Product capacity optimization

In order to set the optimal PCR product dose for hybridization, except for setting the PCR product to 10, 20 or 50 μL for HPV 11, 16, 18, 51, 58, 70, Caski or C33A Hybridization was performed under the same conditions as 1-4, and the measured MFI values were compared as a result (FIGS. 15 to 17). Figure 15 shows the MFI value measured when using the PCR product 10μl, Figure 16 shows the MFI value measured when using 20μl PCR product, Figure 17 measured MFI when using 50μl PCR product Indicates a value. As shown in Figure 15 to 17, when using the PCR product 10μl and 20μL MV value of HPV was similar, the results using 50μL showed a tendency to decrease the MFI value of HPV slightly 20μL was used It was determined that it was appropriate to perform a hybridization reaction using 20 µl of PCR product.

Example 2-7: hybridization Probe Optimization of concentration

Experiments were performed using the following hypotheses to determine the concentration of the probe that binds to the beads. Theoretically, the number of COOH (carboxyl groups) per bead is known to be 1 × 10 ⁶ , and when binding probes to beads, about 1 × 10 ⁶ beads are attached at one time. Therefore, in theory, the number of possible reactions is 1 x 10 ¹² . It is known to insert about 10 times more probe than reactor to react with all carboxyl groups at once in all beads. It is recommended to use 0.04-0.1 nmole of probe per 1 X 10 ⁶ beads. Doing. Therefore, in order to determine the degree of reaction according to the probe concentration in HPV 6, 11, 16, or 18, the probe was connected to the beads using 0.04, 0.1, and 0.2 nmole concentration probes, respectively. The values were compared (Table 2).

Changes in MFI Values with Probe Concentration nmole HPV 6 HPV 11 HPV 16 HPV 18 0.04 4030.5 4317.5 3970 5020 0.1 4355 4365 4061 5106 0.2 4345 4585 4160 5088 Buffer 32 38.5 46 41

As shown in Table 2, since there was no change in MFI value according to the probe concentration, it was predicted that a high amount of MFI value was entered even at a low concentration of 0.04 nm, and the MFI value was constant even when the probe concentration was increased. It was confirmed that excess probe does not interfere with hybridization.

Therefore, when the probe is bound to 1 X 10 ⁶ beads, 0.1 nmole of probe is used, and in order to confirm this, each probe of 0.1 nmole on 21 beads (GAPDH, HPV 6, 11, 16, 18). , 31, 33, 35, 39, 40, 42, 51, 52, 53, 55, 56, 58, 59, 66, 68 or 70) were combined to determine the MFI value.

MFI value of beads bound with 0.1 nmole of probe Bead MFI Bead MFI Bead MFI GAPDH 4145 HPV 35 4867 HPV 55 4676 HPV 6 4466 HPV 39 4602 HPV 56 5292 HPV 11 4974 HPV 40 4861 HPV 58 5301 HPV 16 5313 HPV 42 5196 HPV 59 4627 HPV 18 5323 HPV 51 4757 HPV 66 4574 HPV 31 4154 HPV 52 5117 HPV 68 4591 HPV 33 5017 HPV 53 5104 HPV 70 4397

As shown in Table 3, it was confirmed that the MFI value is more than 3,000 in all the beads, it was found that it is preferable to use a 0.1 nmole probe to bind the probe to 1 X 10 ⁶ beads.

Example 2-7: Optimization of the Fluorescent Material

To select a suitable phosphor for the Luminex Analyzer, eight different phosphors (R-PE, Alexa 532, Cy3, Cryptofluor Tangerine, Alexa 546, Bodipy-TMR-X, PBXL-1 or Cryptofluor Orange) were used. MFI values were compared (FIG. 18). 18 is a graph showing changes in relative MFI values according to fluorescent materials. As shown in Figure 18, it was confirmed that Straptoavidin PE shows the best value.

Example 3: Measurement of detection sensitivity of human papillomavirus

By the method described in Examples 1 and 2, DNA was isolated from the sample to confirm HPV detection and HPV genotype. The sensitivity, specificity and accuracy of the HPV detection method of the present invention were measured and compared with the HPV type detection method disclosed in Korean Patent Registration (10-0708415).

HPV type detection method disclosed in the Korean registered patent (1) the first PCR amplification of the L1 gene using a primer set specific to the L1 gene of human papillomavirus from the sample to be analyzed; (2) second PCR amplification of the first amplified L1 gene product into a single-stranded L1 gene labeled biotin using primers; (3) hybridization of the single-stranded L1 gene product labeled with the second amplified biotin with a human papillomavirus type detection probe. In other words, the PCR process of labeling to amplify a single strand of L1 gene was additionally involved, which took about 3 hours longer.

Clinical samples were collected with Catholic University IRB approval. Cervical decidual cells of normal and patient were collected by cervical brush and centrifuged to collect only cells. Collected cells were extracted using genomic DNA extraction kit. The extracted genomic DNA was not quantified and 5ul was used in this experiment. Using 5ul DNA, 0.025uM of 12 PGMY09Biotin primers, 0.025uM of 5 PGMY 11-Phosphate primers, 0.025uM of GAPDHBiotin reverse primers, 0.025uM of GAPDH-Phosphate forward primers 0.025uM, 0.05uM of dCTPs, Biotin-dCTP 10 uM, Taq pol. After mixing 5 U of 1.5 U, 10 X PCR buffer was adjusted to 50ul using distilled water. PCR conditions were set to 95 ℃ 5 minutes, 94 ℃ 1 minutes, 53 ℃ 1 minutes, 72 ℃ 1 minutes 40 cycles, 72 ℃ 7 minutes. After the PCR, 2ul (10 units) of lambda exonuclease, 10ul of 5X buffer, and 3ul of DW were added to 40ul of the PCR solution, and reacted at 37 ° C for 30 minutes to form a single strand. After completion of the reaction, the reaction was inactivated at 80 ° C. for 10 minutes. 20ul of Hybrizol, 20ul of single strand PCR solution, 0.5ul of bead mixture solution (2,500ea / type, total 60,000ea) were mixed and hybridization reaction was performed at 37 ° C for 2 hours. After the hybridization reaction, the washing solution was washed using 1 and 2, and 0.2 μl of SAPE solution (1 mg / ml) was added thereto, followed by reaction for 10 minutes. After the reaction, the solution was washed once with Wash 2 and analyzed using Luminex equipment.

As a result, when HPV detection and genotyping were confirmed by the method of the present invention, the sensitivity was 92.94%, the specificity was 99.65%, and the accuracy was 96.03%. On the other hand, when the HPV type was detected by a known method, the sensitivity was measured to be 82.65%, specificity 99.31%, and accuracy 90.30% (FIG. 19).

From the above results, it was found that the present invention can detect HPV and identify genotype at the same time with high sensitivity and accuracy within a short time.

Attach an electronic file to a sequence list

Claims

1) From the sample to be analyzed, a first primer labeled with a first marker, and a second marker recognized by an exonuclease, which are common to two or more genotypes of the human papillomavirus (HPV) L1 gene PCR amplifying the L1 gene using the prepared second primer;
2) by reacting the double-stranded L1 gene product amplified in the first step by adding an exonuclease that recognizes the second marker, and decomposing the single-stranded L1 gene product labeled with the second marker. Obtaining a labeled single stranded L1 gene product;
3) Genotype of the L1 gene by using two or more probes specific to each genotype capable of distinguishing two or more genotypes of the L1 gene from a single-stranded L1 gene product obtained in the second step. Characterized in that it comprises a third step to confirm,
Human Papilloma Virus Detection and Genotyping Methods.

The method of claim 1, wherein the second primer is also specific for two or more genotypes of the HPV L1 gene.

The method of claim 1, wherein the two or more probes used in the third step are the same or different regions of the L1 gene.

The method of claim 1, wherein the third step comprises hybridizing the single-stranded L1 gene product labeled with the first marker with two or more L1 genotype detection probes to distinguish the L1 genotypes.
Specifically binding to the first marker and reacting a binder having a fluorescent substance attached thereto,
Human papillomavirus detection and genotyping method characterized by measuring the fluorescence value.

The method of claim 1, wherein the first marker is biotin.

The method of claim 1, wherein the second marker is phosphate.

The method of claim 1, wherein the exonuclease that recognizes the second marker is lambda exonuclease.

The method of claim 1, wherein the first primer has a nucleotide sequence of SEQ ID NO: 1, and the second primer has a nucleotide sequence of SEQ ID NO: 2, HPV detection and genotyping method.

The method of claim 1, wherein the PCR amplification of the first step is performed using dCTP, dATP, dGTP, dTTP, or a mixture thereof labeled with the first marker.

The human papillomavirus detection and genotyping method according to claim 1, wherein the probe is in a form bound to beads.

5. The human papillomavirus detection and genotyping method according to claim 4, wherein the binding agent of the third step is streptavidin.

The method of claim 4, wherein the fluorescent material of the third step is fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde or fluorescamine. Human papillomavirus detection and genotyping method.

The method of claim 1, wherein the annealing temperature is set to 52 to 55 ° C. during the PCR amplification of the first step.

The method of claim 1, wherein the primer concentration in the first step is 0.02 to 0.03μM human papillomavirus detection and genotyping method.

5. The human papillomavirus detection and genotyping method according to claim 4, wherein the temperature when hybridizing with the probe in the third step is 35 to 38 ° C.

5. The human papillomavirus detection and genotyping method according to claim 4, wherein the L1 gene product for hybridization in the third step is 10-30 mu l.

1) From the sample to be analyzed, a first primer labeled with a first marker, a second marker recognized by an exonuclease, which is common to two or more genotypes of the human papillomavirus (HPV) L1 gene. PCR amplification of the L1 gene and the GAPDH gene using a second primer, a third primer labeled with a first marker specific for the GAPDH gene, and a fourth primer labeled with a second marker recognized by the exonuclease First step;
2) by reacting the double-stranded gene product amplified in the first step by adding an exonuclease that recognizes the second marker to decompose the single-stranded gene product labeled with the second marker to label the first marker. Obtaining a single strand of gene product;
3) Using a single-stranded L1 gene product obtained in the second step labeled with a single strand of L1 gene, at least two probes specific to each genotype capable of distinguishing two or more genotypes of the L1 gene, Characterized by the third step of identifying the genotype of the L1 gene using a specific probe,
Human Papilloma Virus Detection and Genotyping Methods.

18. The method of claim 17, wherein the first marker is biotin, the second marker is phosphate, and the exonuclease that recognizes the second marker is lambda exonuclease.

18. The method of claim 17, wherein the first primer is a nucleotide sequence of SEQ ID NO: 1, the second primer is a nucleotide sequence of SEQ ID NO: 2, the third primer is a nucleotide sequence of SEQ ID NO: 3, the fourth primer is SEQ ID NO: 4 Human papillomavirus detection and genotyping method characterized in that the base sequence.

1) From the sample to be analyzed, a first primer labeled with a first marker, and a second marker recognized by an exonuclease, which are common to two or more genotypes of the human papillomavirus (HPV) L1 gene PCR amplifying the L1 gene using the prepared second primer;
2) by reacting the double-stranded L1 gene product amplified in the first step by adding an exonuclease that recognizes the second marker, and decomposing the single-stranded L1 gene product labeled with the second marker. Obtaining a labeled single stranded L1 gene product;
3) Genotype of the L1 gene by using two or more probes specific to each genotype capable of distinguishing two or more genotypes of the L1 gene from a single-stranded L1 gene product obtained in the second step. Characterized in that it comprises a third step to confirm,
Method of providing information for diagnosis of human papilloma virus infection and infected genotype.

delete

1) From the sample to be analyzed, a first primer labeled with a first marker, and a second marker recognized by an exonuclease, which are common to two or more genotypes of the human papillomavirus (HPV) L1 gene PCR amplifying the L1 gene using the prepared second primer;
2) by reacting the double-stranded L1 gene product amplified in the first step by adding an exonuclease that recognizes the second marker, and decomposing the single-stranded L1 gene product labeled with the second marker. A second step of obtaining a labeled single strand of L1 gene product; comprising, a method for amplifying a single strand of L1 gene of human papillomavirus.