WO2017179085A1 - Procédé de détermination de l'emplacement d'une sonde dans un microréseau et kit pour réaction de marquage - Google Patents

Procédé de détermination de l'emplacement d'une sonde dans un microréseau et kit pour réaction de marquage Download PDF

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Publication number
WO2017179085A1
WO2017179085A1 PCT/JP2016/002005 JP2016002005W WO2017179085A1 WO 2017179085 A1 WO2017179085 A1 WO 2017179085A1 JP 2016002005 W JP2016002005 W JP 2016002005W WO 2017179085 A1 WO2017179085 A1 WO 2017179085A1
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WIPO (PCT)
Prior art keywords
probe
microarray
polynucleotide
marker polynucleotide
reaction
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PCT/JP2016/002005
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English (en)
Japanese (ja)
Inventor
聡史 古川
和輝 中島
淳憲 一色
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東洋製罐グループホールディングス株式会社
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Priority to PCT/JP2016/002005 priority Critical patent/WO2017179085A1/fr
Priority to JP2018511544A priority patent/JPWO2017179085A1/ja
Publication of WO2017179085A1 publication Critical patent/WO2017179085A1/fr

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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the labeling reaction kit of the present invention is a labeling reaction kit used for a labeling reaction in the probe position determination method in the microarray, and includes the marker polynucleotide.
  • a nucleic acid amplification method can be suitably used.
  • the nucleic acid amplification method include a PCR method, a LAMP method, an SDA method, and an LCA method.
  • the “labeling reaction” includes a reaction that does not involve nucleic acid amplification, and includes, for example, a reaction in which labeled RNA is synthesized by a reverse transcriptase reaction using a sample RNA and a fluorescent dye.
  • the PCR method is mainly used as the “labeling reaction” will be described as an example.
  • the microorganisms are sampled from food and the environment, and a plurality of microorganisms are enriched and cultured at the same time. It is preferable to extract DNA from the same.
  • the present invention attempts to eliminate the occurrence of non-specific amplification by making a special contrivance not to cause non-specific amplification, as will be described later, while trying this. It is possible.
  • the marker polynucleotide is chemically modified so that it does not serve as a PCR synthesis starting point so that it does not function as a primer even when the marker polynucleotide is hybridized to the target polynucleotide. That is, when the marker polynucleotide becomes the starting point of PCR synthesis instead of the primer, a non-specific amplification product is generated. Therefore, it is preferable to process the marker polynucleotide so that it does not function as a primer.
  • a PCR reaction solution was prepared with the following composition.
  • Primer and marker polynucleotides were outsourced to Sigma Aldrich Japan GK, and other reagents were manufactured by Takara Bio Inc.
  • ⁇ Buffer (10 ⁇ Ex Taq buffer) 2.0 ⁇ l
  • Nucleic acid synthesis substrate dNTP Mixture, 2.5mM each
  • Template 1.0 ⁇ l
  • Primer mix (8 types of primer pairs in Fig. 1)
  • 1.0 ⁇ l Nucleic acid synthase (TaKaRa Ex Taq Hot Start Version)
  • Marker polynucleotide (3 'end unmodified in Fig. 3) (160nM final conc.)
  • Sterile water (remaining amount) Total 20 ⁇ l)
  • a PCR reaction solution was prepared with the following composition.
  • Primer and marker polynucleotides were outsourced to Sigma Aldrich Japan GK, and other reagents were manufactured by Takara Bio Inc.
  • the marker polynucleotide is Cy5 modified at the 5 ′ end and phosphorylated at the 3 ′ end.
  • ⁇ Buffer (10 ⁇ Ex Taq buffer) 2.0 ⁇ l
  • Nucleic acid synthesis substrate dNTP Mixture, 2.5mM each
  • Template 1.0 ⁇ l
  • Primer mix 8 types of primer pairs in Fig.
  • a primer and a marker polynucleotide for detecting S. aureus were added to the PCR reaction solution, amplification products by S. aureus were detected, and non-specific amplification products were also detected.
  • Marker polynucleotides typically do not anneal to the target polynucleotide, but in rare cases this may cause the marker polynucleotide to anneal to the target polynucleotide, resulting in a non-specific amplification product.
  • a stock solution (100 ⁇ M) of a marker polynucleotide (3 ′ end unmodified in FIG. 3) was diluted 1000-fold by adding it to 999 ⁇ L of sterile water to prepare a 100 nM solution.
  • 4 ⁇ L of a 100 nM solution of this marker polynucleotide was mixed with 96 ⁇ L of a buffer solution (3 ⁇ SSC / 0.3% SDS citrate-saline-sodium dodecyl sulfate) used for hybridization to prepare a 100 ⁇ L mixture.
  • Example 8 Marker polynucleotide (3 ′ terminal unmodified) is added to the PCR reaction solution.
  • a culture solution obtained by adding a medium for enrichment to a commercial scallion in which Staphylococcus aureus was detected and culturing overnight at 37 ° C. was used as a specimen.
  • 1 mL of the culture solution was collected, and a DNA extract was obtained in the same manner as in Experiment 1. This DNA extract was used as a template for PCR.
  • the present invention is not limited to the above embodiments and examples, and various modifications can be made within the scope of the present invention.
  • the base sequences of the target polynucleotide, probe, marker polynucleotide, reference position probe, and primer are not essential and arbitrary. Can be made.
  • experiments are performed on microorganisms, but the present invention can be applied without limiting the types of target organisms.
  • the present invention can be suitably used for detecting various targets using a microarray in food inspection, epidemiological environmental inspection, environmental inspection, clinical test, livestock hygiene, and the like.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention permet de réduire le nombre d'opérations ainsi que les pertes de réactif lors de la détermination de l'emplacement d'une sonde dans un microréseau. Plus spécifiquement, l'invention concerne un procédé de détermination de l'emplacement d'une sonde dans un microréseau, lequel procédé comprend une étape, après réaction de marquage d'un polynucléotide cible, consistant à détecter l'hybridation par le microréseau d'une sonde et du polynucléotide cible. Ce procédé comporte une étape dans laquelle, dans un microréseau qui possède à la fois plusieurs points pour la détection sur lesquels des sondes sont fixées, et un point d'emplacement de référence sur lequel une sonde d'emplacement de référence est fixée, un polynucléotide cible marqué et un polynucléotide marqueur qui s'hybride avec la sonde d'emplacement de référence sont mis en contact. Avant le début de la réaction de marquage, un polynucléotide de marquage est ajouté au liquide de réaction qui sera utilisé dans la réaction.
PCT/JP2016/002005 2016-04-13 2016-04-13 Procédé de détermination de l'emplacement d'une sonde dans un microréseau et kit pour réaction de marquage WO2017179085A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/JP2016/002005 WO2017179085A1 (fr) 2016-04-13 2016-04-13 Procédé de détermination de l'emplacement d'une sonde dans un microréseau et kit pour réaction de marquage
JP2018511544A JPWO2017179085A1 (ja) 2016-04-13 2016-04-13 マイクロアレイにおけるプローブの位置決定方法、及び標識反応用キット

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2016/002005 WO2017179085A1 (fr) 2016-04-13 2016-04-13 Procédé de détermination de l'emplacement d'une sonde dans un microréseau et kit pour réaction de marquage

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WO2017179085A1 true WO2017179085A1 (fr) 2017-10-19

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WO (1) WO2017179085A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021184708A (ja) * 2019-12-31 2021-12-09 ゼノヘリックス カンパニー リミテッド 低分子ｒｎａを検出する方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003307518A (ja) * 2002-02-14 2003-10-31 Ngk Insulators Ltd プローブ反応性チップ、試料解析装置および試料解析方法
WO2010147167A1 (fr) * 2009-06-16 2010-12-23 東洋鋼鈑株式会社 Procédé de positionnement d'une sonde dans un microréseau
JP2016101093A (ja) * 2014-11-27 2016-06-02 東洋製罐グループホールディングス株式会社 マイクロアレイにおけるプローブの位置決定方法、及び標識反応用キット

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003307518A (ja) * 2002-02-14 2003-10-31 Ngk Insulators Ltd プローブ反応性チップ、試料解析装置および試料解析方法
WO2010147167A1 (fr) * 2009-06-16 2010-12-23 東洋鋼鈑株式会社 Procédé de positionnement d'une sonde dans un microréseau
JP2016101093A (ja) * 2014-11-27 2016-06-02 東洋製罐グループホールディングス株式会社 マイクロアレイにおけるプローブの位置決定方法、及び標識反応用キット

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HWANG, B. H. ET AL.: "Quantitative oligonucleotide microarray data analysis with an artificial standard probe strategy", BIOSENSORS AND BIOELECTRONICS, vol. 23, no. 11, 2008, pages 1738 - 1744, XP022625065 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021184708A (ja) * 2019-12-31 2021-12-09 ゼノヘリックス カンパニー リミテッド 低分子ｒｎａを検出する方法
JP7242081B2 (ja) 2019-12-31 2023-03-20 ゼノヘリックス カンパニー リミテッド 低分子ｒｎａを検出する方法

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