WO2001059124A1 - Procede de criblage par fluorescence de microplaques, aux fins de detection d'anomalies genetiques, permettant un traitement pratique et moins cher, a grande echelle, de specimens - Google Patents

Procede de criblage par fluorescence de microplaques, aux fins de detection d'anomalies genetiques, permettant un traitement pratique et moins cher, a grande echelle, de specimens Download PDF

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Publication number
WO2001059124A1
WO2001059124A1 PCT/JP2000/000693 JP0000693W WO0159124A1 WO 2001059124 A1 WO2001059124 A1 WO 2001059124A1 JP 0000693 W JP0000693 W JP 0000693W WO 0159124 A1 WO0159124 A1 WO 0159124A1
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dna
pcr
hpv
detection
fluorescence
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PCT/JP2000/000693
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English (en)
Japanese (ja)
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Akihiro Yamaguchi
Kokichi Kikuchi
Kenji Nakamura
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Sapporo Immuno Diagnostic Laboratory
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Priority to PCT/JP2000/000693 priority Critical patent/WO2001059124A1/fr
Publication of WO2001059124A1 publication Critical patent/WO2001059124A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • 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.
  • cystic fibrosis cystic fibrosis
  • mitochondrial encephalomyopathy etc.
  • Cancer cervical cancer, colorectal cancer, etc.
  • Genetic detection technology has already occupied an important position in genetic diseases caused by the invasion of foreign genes or abnormalities in self genes.
  • extremely large numbers of various biological sample DNA extraction kits mainly for infectious diseases and cancers ⁇ ⁇ ⁇
  • Genetic testing kits based on nucleic acid amplification methods such as PCR have been developed.
  • 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.
  • DNA extraction from biological samples required extremely complicated procedures including phenol / chloroform extraction and purification by alcohol precipitation after proteolytic enzyme treatment.
  • 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.
  • PCR is the most suitable method for amplifying a small amount of DNA obtained from a small amount of a biological sample.
  • 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).
  • 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.
  • dot blot hybridization using immobilized PCR products on a membrane and labeled probes (Evans et al., J. Virol.
  • 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.
  • 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. .
  • 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.
  • 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.
  • nucleic acid amplification methods such as hot-start PCR that suppresses non-specific reactions such as TaqStart antibody (Glontech)
  • TaqStart antibody GaqStart antibody
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 10,000 ⁇ , 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.
  • a method for detecting human papillomavirus (HPV) in uterine mucosal cells and screening for colon cancer for screening of uterine cancers 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.
  • HPV human papillomavirus
  • HPV human papillomavirus
  • E6 region 80 bp upstream
  • E7CR3 extending 1 bp at the same position as the common Reverse primer of Fujinga et al.
  • 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.
  • 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.
  • 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.
  • 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 1 Detection of human papillomavirus (HPV) in uterine mucosal cells using common sequence primers for cervical cancer screening:
  • Example 2 A method for typing HPV in uterine mucosal cells using a type-specific primer for HPV-positive samples
  • ⁇ 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.
  • An example in which colorectal cancer K-ras gene mutation was detected using the obtained DNA sample will be described.
  • 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.

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Abstract

L'invention concerne un procédé d'extraction d'ADN dans lequel on peut traiter de manière pratique et économique un certain nombre de spécimens d'échantillons biologiques, de façon à permettre l'introduction d'un diagnostic génétique dans le programme collectif d'essai de criblage. L'invention concerne également un procédé d'essai d'ADN hautement fiable, consistant en une technique de détection, spécifique et très sensible, d'un gène cible. L'invention concerne notamment (a) un procédé économique et pratique d'extraction d'ADN à partir de diverses cellules de muqueuses obtenues par raclage et de morceaux tissulaires fixés à l'aide de formaline et enfermés dans de la paraffine, (b) un procédé consistant à amplifier les ADN cibles et de régulation, au moyen d'une technique d'amplification spécifique d'acides nucléiques, à ajouter un agent d'intercalation d'ADN double brin et à mesurer ensuite les intensités de fluorescence d'un certain nombre de spécimens, et (c) un procédé de criblage pouvant être largement utilisé, s'appliquant en général à des génopathies, et caractérisé en ce que le rapport quantitatif relatif d'un ADN cible et des ADN totaux peut être évalué par calcul du rapport d'intensité fluorescente de ceux-ci.
PCT/JP2000/000693 2000-02-09 2000-02-09 Procede de criblage par fluorescence de microplaques, aux fins de detection d'anomalies genetiques, permettant un traitement pratique et moins cher, a grande echelle, de specimens WO2001059124A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103966314A (zh) * 2014-03-19 2014-08-06 新乡学院 猪外周血单核淋巴细胞pd-1重组质粒的构建、基因丰度实时检测方法及其应用

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JPH0279980A (ja) * 1988-09-17 1990-03-20 Tonen Corp ヒトパピローマウイルス５２ｂ遺伝子
JPH02145198A (ja) * 1988-11-17 1990-06-04 Univ New York State ヒト膵臓癌の特異的検出法
EP0373352B1 (fr) * 1988-11-11 1996-01-31 BEHRINGWERKE Aktiengesellschaft Méthode pour détecter de l'ARNm épissée
US5679509A (en) * 1993-09-28 1997-10-21 University Of New Mexico Methods and a diagnostic aid for distinguishing a subset of HPV that is associated with an increased risk of developing cervical dysplasia and cervical cancer
US5702886A (en) * 1994-10-05 1997-12-30 The Johns Hopkins University Chromosome I8Q loss and prognosis in colorectal cancer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0279980A (ja) * 1988-09-17 1990-03-20 Tonen Corp ヒトパピローマウイルス５２ｂ遺伝子
EP0373352B1 (fr) * 1988-11-11 1996-01-31 BEHRINGWERKE Aktiengesellschaft Méthode pour détecter de l'ARNm épissée
JPH02145198A (ja) * 1988-11-17 1990-06-04 Univ New York State ヒト膵臓癌の特異的検出法
US5679509A (en) * 1993-09-28 1997-10-21 University Of New Mexico Methods and a diagnostic aid for distinguishing a subset of HPV that is associated with an increased risk of developing cervical dysplasia and cervical cancer
US5702886A (en) * 1994-10-05 1997-12-30 The Johns Hopkins University Chromosome I8Q loss and prognosis in colorectal cancer

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Title
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GOLDSBOROUGH M. D. ET. AL.: "Nucleotide sequence of human papillomavirus type 31: A cervival neoplasia-associated virus", VIROLOGY, vol. 171, no. 1, 1989, pages 306 - 311, XP002928265 *
J. J. O. LEARLY ET. AL.: "The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization", HISTOCHEMICAL JOURNAL, vol. 26, no. 4, 1994, pages 337 - 346, XP002928263 *
K. YASUYUKI KIRII ET. AL.: "Human papillomavirus type 58 DNA sequence", VIROLOGY, vol. 185, no. 1, 1991, pages 424 - 427, XP002928266 *
P. SPEISER ET. AL.: "Microdissection as a means to verify allelic imbalance in tumour Biology samples", ANTICANCER RESEARCH, vol. 16, no. 1, 1996, pages 461 - 464, XP002928267 *
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Y. FUJINAGA ET. AL.: "Simultaneous detection and typing genital human paplillomavirus DNA using the polymerase chain reaction", JOURNAL OF GENERAL VIROLOGY, vol. 72, no. 5, 1991, pages 1039 - 1044, XP002928264 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103966314A (zh) * 2014-03-19 2014-08-06 新乡学院 猪外周血单核淋巴细胞pd-1重组质粒的构建、基因丰度实时检测方法及其应用
CN103966314B (zh) * 2014-03-19 2016-04-20 新乡学院 猪外周血单核淋巴细胞pd-1重组质粒的构建、基因丰度实时检测方法及其应用

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