WO2001059124A1

WO2001059124A1 - Microplate fluorescent screening method for detecting gene anomaly allowing convenient and less expensive treatment of specimens on mass scale

Info

Publication number: WO2001059124A1
Application number: PCT/JP2000/000693
Authority: WO
Inventors: Akihiro Yamaguchi; Kokichi Kikuchi; Kenji Nakamura
Original assignee: Sapporo Immuno Diagnostic Laboratory
Priority date: 2000-02-09
Filing date: 2000-02-09
Publication date: 2001-08-16

Abstract

A DNA extraction method whereby a number of specimens of biological samples can be conveniently and economically treated, thereby enabling introduction of gene diagnosis into a community screening test program; and a highly reliable DNA test method which comprises a specific and highly sensitive method for detecting a target gene. More particularly speaking, (a) a convenient and economical method for extracting DNAs from various scraped mucosal cells and tissue slide pieces fixed with formalin and embedded in paraffin; (b) a method comprising amplifying target and control DNAs by the specific nucleic acid amplification method, adding a double-stranded DNA intercalater and then measuring the fluorescence intensities of a number of specimens; and (c) a widely usable screening method which is applicable to genopathies in general, characterized in that the relative quantitative ratio of a target DNA on the total DNAs can be evaluated by calculating the fluorescent intensity ratio thereof.

Description

Specification

FIELD OF THE INVENTION Field of the Invention Microplate fluorescence screening method for gene abnormalities that can easily and inexpensively process large amounts of samples

The present invention is intended for screening tests for genetic diseases such as infectious diseases, congenital hereditary diseases, and cancers in a wide population, and is effective for efficient and economical processing of large amounts of DNA from DNA extraction. And a series of systematic DNA testing methods that lead to detection and identification. Examples of the above diseases are as follows:

Infectious diseases: human papillomavirus, chlamydia, etc.

Congenital hereditary diseases: cystic fibrosis, mitochondrial encephalomyopathy, etc.

Cancer: cervical cancer, colorectal cancer, etc.

Background of the Invention

Genetic detection technology has already occupied an important position in genetic diseases caused by the invasion of foreign genes or abnormalities in self genes. Above all, extremely large numbers of various biological sample DNA extraction kits, mainly for infectious diseases and cancers遺伝子 Genetic testing kits based on nucleic acid amplification methods such as PCR have been developed. However, all of these are mainly intended to ascertain the etiology and pathology of individual patients or diseases, and involve complicated steps of separation and analysis by electrophoresis, or are expensive. Because of the need for specially labeled primers and expensive dedicated measuring equipment, it is difficult to use it for screening tests in general populations that require large-volume sample processing due to technical or cost constraints. is there.

Conventional technology

Classically, DNA extraction from biological samples required extremely complicated procedures including phenol / chloroform extraction and purification by alcohol precipitation after proteolytic enzyme treatment. At present, as a simple DNA extraction method from various biological samples, a spin column-chromatographic separation method using a special support having DNA binding ability is widely used.

(Merkelbach et al., Am. J. Pathol. 150 (1997), 1537-46; QIAGEN QIAampKit series However, the cost per sample is high, and it is virtually impossible to process many samples continuously in daily tests.

On the other hand, it is clear that PCR is the most suitable method for amplifying a small amount of DNA obtained from a small amount of a biological sample. However, the detection method for PCR products is usually performed after separation analysis such as agarose electrophoresis. It is common to add a single-stranded DNA intercalator and detect it as a fluorescent substance (Molecular Cloning vol. L, chap. 6). However, although separation analysis by electrophoresis allows accurate detection of PCR products, it is not a method originally intended for processing a large number of analytes, so the processing capacity is naturally limited. When emphasis is placed on the processing of a large number of samples, dot blot hybridization using immobilized PCR products on a membrane and labeled probes (Evans et al., J. Virol.

Methods 73 (1998), 41-52 .; Sugita et al., Oncol. Rep. 6 (1999), 145-7) and enzyme immunoassays using probes immobilized on micropre-towels (Sabine et al., Diagn. Mol. Pathol. 8 (1999), 32-8; Sander et al., J. Clin. Microbiol. 37 (1999), 3097-101) are often used, but probe preparation, B / F separation and washing procedures, The detection system itself, such as the enzymatic reaction step, becomes very complicated, and lacks the simplicity and stability that are important factors in screening tests.

Recently, a multichannel capillary electrophoresis apparatus has also been developed for the purpose of DNA sequencing of a large number of samples (Zhang et al., Anal. Chem. 71 (1999), 5018-25; PE Biosystems ABI Prism 3700; Hitachi). CS-1000, etc.) and monitoring the PCR reaction using a specially labeled primer and a laser excitation detector (Glaab et al., Mutat. Res.

430 (1999), 1-12; Ryncarz et al., J. Clin. Microbiol. 37 (1999), 1941-7 .; PE Biosystems TaqMan system, etc.) Measurement method (Rautenberg et al., J. Clin. Virol. 13 (1999), 81-94 .; Chen et al., Appl. Environ. Microbiol. 64 (1998), 4210-6 .; Biotronics Technologies AmpliSensor system, etc.) New detection methods other than separation analysis have also appeared, and it is possible to physically process large amounts of samples. However, in reality, we considered the investment in equipment for expensive equipment and the running cost of expensive reagents. At times, the application of these systems will be very limited. The major difference between the detection method of the present invention and these recent methods is that PCR amplification is used. The simplicity of adding a double-stranded DNA to the broadened double-stranded DNA and measuring it with a general-purpose fluorescent plate reader requires optimization of the labeled primers and additional reactions for subsequent detection. The point is that this method is superior in operability, cost, and versatility as a screening method for genetic abnormalities for the purpose of processing large amounts of samples. -Summary of the invention

The present invention provides a simple and economical DNA extraction method for a large number of biological samples, and a special and highly sensitive target gene identification method, in order to enable the introduction of a genetic test into the screening test of the general population described above. It provides a highly reliable DNA testing method consisting of detection methods.

Description of the invention

As a simple and economical method for extracting mucosal scraped cell DNA, hemoglobin that interferes with PCR, a widely used nucleic acid amplification method, is washed and removed with erythrocyte lysed blood (RCLB). A method was developed in which a TE (10 mM Tris-1 mM EDTA, pH 8.0) solution of Proteinase K was added and incubated, and the boiled centrifuged supernatant was used directly as a template for PCR. This method is a highly versatile method that can be applied to other cells obtained by abrasion or resection, including the oral mucosa and uterine mucosa.

Focusing on fecal samples containing intestinal mucosal cells as a DNA test material for colorectal cancer, developed a simple and economical DNA extraction method using a dry filter paper stool collection kit for the purpose of colorectal cancer screening, and separately filed a patent application. did.

Formalin-fixed, paraffin-embedded tissue slides of a very small amount of biopsy material using an endoscope, such as when a follow-up examination is performed in a screening test, can be used for genetic diagnosis as well as other general surgical materials with a huge accumulation. It is very useful as a sample for the test. Conventional DNA extraction from formalin-fixed paraffin-embedded tissue slides requires deparaffinization with an organic solvent such as xylene, and is not suitable for processing large numbers of samples, including health problems. . In the DNA extraction method according to the present invention, a tissue slide section is scraped off with a microspatula in a paraffin-embedded state, and is treated with a proteinase K-pol. Of high quality and quantity of DNA. This is based on the fact that paraffin can be easily separated from DNA extract as thin slices by cooling after boiling.

We have developed a specific and highly sensitive target gene detection method that can process large amounts of various types of easily extracted DNA samples obtained as described above. The target and the appropriate control DNA are each amplified by nucleic acid amplification methods such as hot-start PCR that suppresses non-specific reactions such as TaqStart antibody (Glontech), followed by addition of double-stranded DNA intercalation and fluorescence. By measuring the intensities and calculating the ratio of the two intensities (target / control), the relative amount ratio of target DNA to the total amount of DNA can be evaluated. All operations can be performed on a microplate scale with 96 or 384 wells. High-specificity nucleic acid amplification products obtained by hot-start PCR, etc., and direct double-stranded DNA intercalation are added to detect fluorescence by fluorescence microplate reader, capillary electrophoresis, or high-performance liquid chromatography. Measure with the device. This method does not require special reagents and can be widely applied to general genetic disease screening, which enables simple and economical processing of large amounts of samples, and is highly versatile. Genetic diagnosis of infectious diseases, congenital hereditary diseases, and genetic diseases such as cancer in general healthy persons has been difficult to apply to disease screening due to technical or cost constraints. A series of practical genetic testing systems from DNA extraction to amplification, detection, and identification according to the present invention enable efficient and economical processing of large amounts of samples, and are extremely useful for disease screening. It can be expected to contribute to preventive medicine, which has become increasingly important from above and economically.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the results of detection of human papillomavirus (HPV) in uterine mucosal cells by PCR / microplate fluorescence using common sequence primers for cervical cancer screening;

Figure 2 shows the results of typing using agarose gel electrophoresis of HPV-DNA amplified by PCR using HPV type-specific primers;

Figure 3 shows a formalin-fixed paraffin wrapper for application to colorectal cancer screening. Detection of common mutations at codons 12 and 13 of oncogene K-ras using embedded tissue slide sections (primary screening);

Figure 4 shows the detection results (identification of mutations) of codon 12 and 13 common mutations of the oncogene K-ras using formalin-fixed paraffin-embedded tissue slide sections for application to colorectal cancer screening.

Description of preferred embodiments

Details of the simple DNA extraction method from mucosal scraped cells will be described in detail. The mucosal cells obtained by scraping the mucosal part with a spatula are washed away by portex mixing into 10 mL of PBS solution in a 50 mL centrifuge tube. Under this condition, DNA is stable for at least one month when stored at 4 ° C. Next, after centrifugation at 3,000 rpm for 5 minutes, discard the supernatant, add 500 L of PBS solution to the pellet, vortex, and transfer the entire amount of the mucosal cell suspension to a 1.5 mL centrifuge tube. If long-term storage is necessary, store at -20 in this state. For DNA extraction, transfer 100 / iL of this to another 0.5 mL tube, centrifuge at 10,000 rpm for 5 minutes, discard the supernatant, and add erythrocyte lysed blood (RCLB: 10 mM Tris-5 mM MgCI2- Add 400 L of 10 mM NaCI, pH 7.6) and perform portex mixing. If hemoglobin is present in the cell pellet in a single washing of a sample with a large amount of blood, repeat the washing operation with RCLB again. To the cell pellet obtained in this manner, 100_iL of 100 zg / mL Proteinase K-TE solution is added, and the cells are dispersed by vortex mixing. Incubate for 15 minutes at ° C. Next, centrifuge at 15,000 rpm for 5 minutes, and transfer the supernatant to a new 0.5 mL tube to obtain the final DNA solution.

The details of the simple DNA extraction method from formalin-fixed paraffin-embedded tissue slide sections are described in detail. The sampled tissue site (usually about 5 mm square with a thickness of 3 to 5 m of tissue pieces) is scraped together with the paraffin using a microspatula, and the thin piece on the spatula is attached to the bottom of a 0.5 mL tube using a pointed tip such as a toothpick. Move. Add 60/100 / g / mL Proteinase K-TE solution same as the one used for the above mucosal scraping cells, centrifuge for 1 min at 10,000φΐη, then incubate at 55 ° C for 30 minutes, then 95 ° C Boil for -15 minutes and finally cool to 4 ° C. Next, the supernatant after centrifugation at 15,000 rpm for 5 minutes is renewed. Dispense into a 0.5 mL tube to obtain the final DNA solution.

As an application example of a specific and highly sensitive target gene detection method capable of processing a large amount of samples, a method for detecting human papillomavirus (HPV) in uterine mucosal cells and screening for colon cancer for screening of uterine cancers. The method for detecting common mutations in the oncogene K-s using dried filter paper stool and formalin-fixed paraffin-embedded tissue slides is described below.

There have been many reports on consensus sequence primers for PCR detection of human papillomavirus (HPV) DNA associated with cervical cancer. In general, the main purpose is to classify HPVs, which are currently known as close to 100 types, so a wide range of detections is prioritized. A method combining oligohybridization with sacrificing PCR specificity Although HPV is often used, it is not suitable for screening because the HPV type, which is not closely related to canceration, is detected and the procedure is complicated. On the other hand, a method using a common sequence primer in the L1 region targeting about 10 high-risk groups and intermediate-risk groups HPV closely related to canceration iYoshikawa et al., Jpn J Cancer Res 82 (1991), 524-31} A method using common sequence primers in the E6 and 7 regions {Fujinaga et al., J General Virol 72 (1991), 1039-44} has also been reported. However, in the design of common sequence primers, base sequence-specific PCR was used. Since the strictness of the .3 'end has not been sufficiently considered, the use of low-concentration and incompletely purified DNA samples for screening purposes, at least in the former, high-risk group HPV16, In the latter case, sufficient sensitivity was not obtained for the high-risk group HPV18, which proved to be fatal for application to screening. For this reason, it is necessary to design a new common sequence primer. As a result of various studies, the forward primer shown as SEQ ID NO: 1 in the sequence listing below; E6CF4 (from the common forward primer of Fujinaga et al.) By using E6 region (80 bp upstream) and the reverse primer shown as SEQ ID NO: 2; E7CR3 (extending 1 bp at the same position as the common Reverse primer of Fujinga et al.), At least high-risk groups HPV-16 and 18 were obtained. HPV-31, 33, 52 and 58 in the middle-risk group could all be detected with high sensitivity by a single PCR.

On the other hand, with regard to HPV type identification, PGR products using common sequence primers were restricted. The method of cutting by enzyme and performing typing (RFLP) is generally used, but more complicated operations are required after PCR using expensive restriction enzymes. Therefore, it is possible to identify the main HPV only by PCR and the fluorescence measurement according to the present invention; HPV-16 and 18 of high-risk group and HPV-31, 52 and 58 of high frequency in the middle-risk group, respectively. Five types of forward primers (16SF1, 18SF2, 31SF1, 52SF1 and 58SF1) shown in the sequence listing as SEQ ID NOs: 3, 4, 5, 6 and 7 were designed, and a method was developed to use them together with a reverse primer; E7CR3. Furthermore, since these primers are designed so that each HPV type gives a different amplification band length, if agarose electrophoresis is used after PCR of a single tube containing all primers, no restriction enzyme treatment is required. Another advantage is that the HPV type can be identified.

The amount of DNA in each sample in the HPV-DNA test was estimated using the primer-pair-pair (Glob-F, Glob-R) of [Villa et al., Gastroenterology 110 (1996), 1346-53]. Amplification of the globin gene was detected by the method according to the present invention and used as a DNA control. Common mutation three oncogene K-ras using a dry filter paper flights and formalin-fixed paraffin-embedded tissue slides colon cancer screening aimed; codon _{12 g g → ga1: (12A} ), 12ggt → gt (12T) and This section describes how to detect codon 13ggc → gac (13A) simultaneously or individually. This method detects K-ras gene mutations contained in DNA in colorectal cancer cells by the arrell-specific nested PCR method. The following three points are used to obtain highly sensitive, specific, and reliable results. Something has been devised.

—The first is to amplify both normal and abnormal alleles across the codons 12 and 13 of the K-ras gene by IstPCR, and to raise the amount of the trace amount of mutated DNA quantitatively. The point is that the amount of sample DNA can be estimated by measuring the stPCR product. Reverse primer for K-ras I stPCR (Kras-R) is as described in the previous report.For non-exon forward primer, amplification band was not obtained in some samples which may be caused by polymorphism. Therefore, a new primer (KrasF-1st shown as SEQ ID NO: 8 in the sequence listing below) was designed in the transcription initiation boundary region containing exon 1.

Second, by introducing appropriate mismatches into allele-specific primers in 2nd PCR, it is possible to improve specificity while maintaining sensitivity. That is the point. K-ras mutation; For 12A and 12T, a C mismatch at 3 bp upstream from the 3 'end of the primer of Takeda et al., Hum Mutaion 2 (1993), 1 12-7} For the Kras12A-3g and Kras12T-3g shown as SEQ ID NOs: 9 and 10 in the sequence listing, respectively, and for 13A, newly designed primers were used, and G at 3 bp upstream from the 3 'end was regarded as an A mismatch. (Kras13A-1a shown as SEQ ID NO: 11 in the sequence listing below), when used alone or simultaneously, reverse primer; Kras-R {Takeda et al., Hum Mutaion 2 (1993), 112 -7}, good amplification results were obtained.

The third point, which is particularly important to ensure specificity, is that the number of allele-specific PCR cycles in the 2nd PCR is deliberately set to a low value of 15 cycles, and the PCR reaction reaches a plateau. This is an important condition for specifically detecting mutations in the previous state. These conditions are based on various balances, and are closely related to the number of cycles of IstPCR (35 cycles) and the dilution ratio of the IstPCR product used as a template for 2ndPCR (10-fold dilution).

Example

Detection of human papillomavirus (HPV) in uterine mucosal cells for cervical cancer screening based on the above methodology (Examples 1 and 2) and dried filter paper and formalin fixation for colorectal cancer screening A more specific application example of the method for detecting a common mutation of the oncogene K-ras using paraffin-embedded tissue slides (Example 3) will be described below, but the present invention is not limited thereto.

Example 1 Detection of human papillomavirus (HPV) in uterine mucosal cells using common sequence primers for cervical cancer screening:

Transfer the uterine mucosal scraping cell DNA solution 2i obtained by the simple DNA extraction method for mucosal scraping cells described above to the wall of a 0.2 mL PCR tube in a 96-well microplate array. Next, purified water: 10x PCR Buffer: 2mM dNTPmix: 50βM HPV Common Rooster Primer mix (100M each)

E6CF4 + E7CR3 2 1 + 1 mix): 2.5u / L Taq mix (5u / ji L Taq Polymerase (Pharmacia) +7 μ, M

Add 8 L of PCR reaction mixture containing Taq Start Ab (Clontech) = 1 +1 mix} at a ratio of 5.7: 1.0: 1.0: 0.2: 0.1 to each well. After capping and mixing by inversion, spin down at 1,500 rpm and PCR. To start. At this time, in order to enhance the specificity of the PCR reaction, set the aluminum block of the PCR device to 94 ° C in advance and set it in the PCR device. The PCR conditions were: preheating at 94 ° C for 5 minutes, followed by 35 temperature cycles of 94 ° C for 30 seconds / 48 ° C for 30 seconds / 72 ° C for 30 seconds, and post-heating at 72 ° C for 7 minutes. Perform the extension.

After spinning down at 1,500 µm, add 100 L of Intercalate overnight solution (here, 5,000-fold dilution of SYBR Green I (Molecular Probe) stock solution with TAE solution) to each well using a multi-channel micropip. After addition, mix by pouring and transfer 100 XL into a microplate for fluorescence measurement. At this time, if the pumping cleaning is performed between the samples using an intercalating solution, the carryover is negligible, so that chip replacement is not necessary. After standing at room temperature for 5 minutes, measure the fluorescence intensity at an excitation / emission wavelength of 480/520 nm using a fluorescence microplate reader {here, a combination of Fluorolight 1000 (Dynatech) and a 96-well black plate}. For DNA control, prepare a separate reaction mixture in which only the 12.5 M primer mix (Glob-F + Glob-R) for amplifying the 3-globin gene was replaced with the HPV common sequence primer for DNA control. Then, perform the same procedure from PCR to fluorescence measurement under the same conditions. Detection was performed when the fluorescence intensities of HPV and) 3-globin were above a certain value, respectively. When / 3-globin was below the detection limit, determination was impossible due to insufficient DNA in the sample. The value of the net obtained by subtracting the fluorescence intensity of the reagent blank from the fluorescence intensity of HPV obtained in this way and the fluorescence intensity of) 3-globin was calculated, and the ratio (HPV / i3-globin) was calculated. It was possible to estimate the relative amount of HPV in the medium (Figure 1).

Example 2 A method for typing HPV in uterine mucosal cells using a type-specific primer for HPV-positive samples

Transfer 2/1 L of the DNA solution of uterine mucosal scraping cells, which were confirmed to be HPV positive in Example 1, to the wall of a 0.2 ml_ PCR tube in a 96-well microphone plate plate array. Next, purified water: 10x PCR Buffer: 2mM dNTPmix: HPV type-specific Primer mix (each 100 z M 16SF1 + 18SF2 + 3 ^! I SF1 + 52SF1 + 58SF1 + E7CR3 + TE = 10 + 10 + 15 + 1 + 3 + 10 + 1 mix): 2.5u / L Taq mix {5u / ji L Taq Polymerase (Pharmacia) +7 M Taq Start Ab (Clontech) = 1 +1 mix} with 12,8: 2.0: 2.0: 1.0: Add 0.2 parts of the PGR reaction mixture to each well. The following is an example of the first embodiment. Change the ring temperature from 48 ° C to 55 ° C and perform PCR under the same other conditions.

^ 0 L · is separated and detected by agarose electrophoresis according to a conventional method. HPV typing was possible easily from the obtained amplification band length (Fig. 2). In addition, if each type-specific forward primer is used individually in combination with E7CR3, it is possible to determine the type by fluorescence measurement alone without the need for electrophoresis.

Example 3 Oncogene K-ras common mutations using dry filter paper stool and formalin-fixed paraffin-embedded tissue slide sections for colorectal cancer screening 3 types; codon 1 2ggt → gat (1 2A), 1 2ggt → gtt ( 1 2T) and codon 13 3ggc → gac (13 A)

A DNA sample developed by the applicant of the present invention and obtained separately by a dry filter paper collection kit and a simple DNA extraction method, or a DNA sample obtained by the simple DNA extraction method of a formalin-fixed, baraffin-embedded tissue slide section of the present invention. An example in which colorectal cancer K-ras gene mutation was detected using the obtained DNA sample will be described. Transfer the above-mentioned simple extracted DNA sample solution 2 to the wall of a 0.2 mL PCR tube with a 96-well microplate array. Next, purified water: 10x PCR BufFer: 2mM dNTPmix: 50 jl M Kras 1st PCR primer mix (100M KrasF-1 st + Kras-R = 1 +1 mix), 2.5u / L Taq mix (5u / PCR reaction mixture containing ji L Taq Polymerase (Pharmacia) +7 M Taq Start Ab (Clontech) = l + 1 mix} in the ratio of 13.6: 20: 2.0: 0.2: 0 Add x L to each well. After capping and mixing by inversion, spin down at 1,500ΦΓΠ to start PCR. The conditions of 1st PCR are the same as in Example 1 except that the annealing temperature is 60 ° C, the number of cycles is 38 cycles. A 10 iL portion of this PCR product was aliquoted into each well of a microplate for fluorescence measurement using a multichannel micropip, and the plastic seal was performed together with the subsequent 2nd PCR product until fluorescence quantification was performed. And store at room temperature. Add 90 L of TE solution to the remaining 10 parts of the Kras-1st PCR product, cover with a new cap, mix by inversion, and spin down. Transfer 2 fl L of the 10-fold diluted solution of this 1st PCR product to the wall of a new 0.2 mL PCR tube. Purified water: 10x PCR Buffer: 2 mM dNTPmix: 25 β M Kras 2nd PCR primer mix (100 ml each) / i M

Krasl 2A-3g + Kras1 2T-3g + Kras13A-1 a + Kras-R = 1 +1 +1 +1 mix): 2.5u / L Taq mix (5u / ji L

Taq Polymerase (Pharmacia) +7 [1 M Taq Start Ab (Clontech) = 1 +1 mix}

5.8: 1.1.0: 1.0: 0.1: 0.1 harmful ij mix P〇R reaction mixture 8 L per well, same as 1st PCR Start 2nd PCR. The conditions for 2nd PCR are the same as for IstPCR, except that the number of cycles is reduced to 15. When identifying three types of K-ras mutation individually, prepare 2nd PCR primer mix and perform 2nd PCR separately. Fluorescence measurements were in accordance Kras 1st and 2nd protocol of PCR products 10 L Example 1 obtained as described above, the fluorescence intensity ratio obtained _{_{(K raS 2nd / K raS "}} l S t) from the Kras gene mutation Determine the presence or absence of the mutation or identify the type of mutation (Figures 3 and 4).

Claims

The scope of the claims

1. Using DNA obtained by simple extraction from biological samples such as mucosal scraping cells and formalin-fixed paraffin-embedded tissue slide sections, the target region is specifically amplified by nucleic acid amplification methods such as PCR. A method for screening for genetic diseases, characterized in that direct fluorescence measurement is performed by adding a double-stranded DNA intercalator.

2. The method according to claim 1, wherein the method is characterized by a quantitative discriminating method using a ratio of a fluorescence intensity of a nucleic acid amplification product of each of a target and a control DNA as an index.

3. The method according to claim 1 or 2, characterized by an easy DNA extraction method comprising a step of removing hemoglobin from rubbed mucosa cells by erythrocyte hemolysis.

4. The method according to claim 1 or 2, characterized by a simple DNA extraction method that does not require deparaffinization treatment from formalin-fixed paraffin-embedded tissue slide sections.

5. In the detection of human papillomavirus (HPV) DNA associated with uterine facial cancer, nucleotides having the HPV common nucleotide sequence shown in SEQ ID NOs: 1 and 2 are used as a pair of primers for PCR amplification. The method according to any one of Items 1 to 4.

6. In the detection of human papillomavirus (HPV) DNA associated with cervical cancer, type-specific amplification products with different lengths for HPV-16, 18, 18, 31, 52 and 58 shown in SEQ ID NOs: 3 to 7, respectively. The method according to any one of claims 1 to 4, wherein each nucleotide having a nucleotide sequence that gives a primer set is used as a primer set for PCR amplification.

7. common mutation three oncogenes K-ras; codon _{12 g gt → gat (12A)} , in the detection of 12ggt → gtt (12T) Contact and codon 13ggc → _g ac (13A), SEQ ID NO: 8 to show the 11 The method according to any one of claims 1 to 4, wherein each nucleotide having the nucleotide sequence is used as a primer set for PCR amplification.

8. Fluorescent substance obtained by specifically amplifying only the target nucleic acid and adding double-stranded DNA intercalation is converted to a fluorescent microplate reader, fluorescence detection capillary electrophoresis, or fluorescence detection high-performance liquid chromatography. The method according to any one of claims 1 to 7, wherein the measurement is performed with a fluorescence measuring device such as one, and a large amount of samples can be processed easily and economically. Or the method of paragraph 1.