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WO2023109032A1 - Système de détection d'acides nucléiques multiples, son procédé de préparation et son utilisation - Google Patents

Système de détection d'acides nucléiques multiples, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2023109032A1
WO2023109032A1 PCT/CN2022/097203 CN2022097203W WO2023109032A1 WO 2023109032 A1 WO2023109032 A1 WO 2023109032A1 CN 2022097203 W CN2022097203 W CN 2022097203W WO 2023109032 A1 WO2023109032 A1 WO 2023109032A1
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WIPO (PCT)
Prior art keywords
nucleic acid
probe
target
sequence
primer
Prior art date
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Ceased
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PCT/CN2022/097203
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English (en)
Chinese (zh)
Inventor
陈嘉昌
李楚明
刘向东
唐海辉
王维世
王辉芳
张源明
张乾毅
胡朝晖
柳俊
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Guangzhou Jinqirui Biotechnology Co Ltd
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Guangzhou Jinqirui Biotechnology Co Ltd
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Application filed by Guangzhou Jinqirui Biotechnology Co Ltd filed Critical Guangzhou Jinqirui Biotechnology Co Ltd
Publication of WO2023109032A1 publication Critical patent/WO2023109032A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the sequence of the 5' end region of the Target probe is reversely complementary to the loop region of the Beacon probe, and the Beacon probe is naturally coiled in a free state, because the reporter group (R) and the 5-terminal multiple (1-8 1)
  • the distance between the G bases is short and there is no fluorescence (self-quenching molecular beacon probe).
  • the end region is cut off and is in a free state.
  • the temperature is lower than the Tm value, it can hybridize with the loop region of the Beacon probe, and under the action of the 5' ⁇ 3' polymerase activity of DNA polymerase, it can be extended and amplified.
  • the inventor combined the design of the PCR program, through the design of the PCR program, combined with the use of the landing PCR program, and also proposed a multiple nucleic acid detection method, which can further reduce the number of non-specific nucleic acids in the PCR reaction. Heterosexual amplification improves detection sensitivity.
  • a multiple nucleic acid detection method which can further reduce the number of non-specific nucleic acids in the PCR reaction. Heterosexual amplification improves detection sensitivity.
  • the real-time fluorescent quantitative PCR instrument can be used for testing in a real-time fluorescent quantitative PCR instrument with a simpler reaction system and lower detection cost. Accurate and qualitative detection can be achieved for each target gene in at most 20 kinds of target nucleic acid sequences to be tested in the sample, and the specificity of detection can be guaranteed by interpreting the specific melting peak in the melting curve.
  • Fig. 3 is the agarose gel electrophoresis figure of the influenza A virus PCR product that adopts different PCR programs to amplify in embodiment 2.
  • the number n of continuous guanines at the 5' end of the 5' end region sequence of the above-mentioned Beacon probe is an integer of 3-5. It is further preferred that n is 4, so as to effectively provide the fluorescence quenching function.
  • the above-mentioned diseases are infectious diseases caused by cross-infection caused by various pathogens.
  • the aforementioned infectious diseases are respiratory tract infection, digestive tract infection, blood infection and/or urinary tract infection and the like. More preferably, it is used for the detection and determination of pathogens in respiratory tract infections with similar symptoms such as cough, which can be influenza viruses such as influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus , Mycoplasma pneumoniae, parainfluenza virus and other infections.
  • influenza viruses such as influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus , Mycoplasma pneumoniae, parainfluenza virus and other infections.
  • Primer preparation dissolved in TE, the concentration of each universal primer is the same, the concentration of each CLO primer is also the same, the final concentration of each universal primer is 10 times the final concentration of a single CLO primer, for example: the final concentration of a single CLO primer 1.6pmol/ ⁇ L, the final concentration of a single universal primer is 16pmol/ ⁇ L, and the mixture formed is labeled as HXD-T.
  • Combination 1 and combination 2 were used to detect influenza A virus quality control products, and the amplification curve and melting curve of the detection results are shown in Figure 2 below.
  • the kit contains a negative quality control and a positive quality control, and the negative and positive quality controls and the samples to be tested need to be processed synchronously.
  • Positive quality control products consist of pseudoviruses containing influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae, parainfluenza virus and internal reference gene fragments; negative quality control
  • the product consists of a pseudovirus containing internal reference gene fragments. Pseudoviruses were purchased from Fubaiao (Suzhou) Biotechnology Co., Ltd. and prepared.

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

Abstract

La présente invention concerne un système de détection d'acides nucléiques multiples, comprenant un ensemble d'amorces d'amplification et un ensemble de sondes de détection pour une séquence d'acide nucléique cible. Le système de détection comprend une sonde cible modifiée par LNA et une sonde Beacon permettant d'obtenir un effet d'extinction de la fluorescence au moyen de 1 à 8 bases G consécutives. La présente invention concerne en outre un procédé de détection d'acides nucléiques multiples combiné au système de détection d'acides nucléiques multiples susmentionné et à un programme de PCR par essais. Le procédé permet en outre de réduire l'amplification non spécifique dans une réaction par PCR et d'améliorer la sensibilité de la détection. La présente invention permet de surmonter les limites de la technique traditionnelle de typage par PCR quantitative fluorescente en temps réel, de réaliser un typage multiple sur un seul tube au moyen de signaux spéciaux et d'une analyse de la courbe de fusion, et de détecter des acides nucléiques cibles dans un échantillon avec un système de réaction plus simple et des coûts de détection plus faibles.
PCT/CN2022/097203 2021-12-14 2022-06-06 Système de détection d'acides nucléiques multiples, son procédé de préparation et son utilisation Ceased WO2023109032A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111537083.4A CN114134219A (zh) 2021-12-14 2021-12-14 一种多重核酸检测系统及其制备方法与应用
CN202111537083.4 2021-12-14

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CN (1) CN114134219A (fr)
WO (1) WO2023109032A1 (fr)

Cited By (1)

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CN116694743A (zh) * 2023-06-29 2023-09-05 山东迪曼生物科技有限公司 一种利用荧光探针检测多靶标基因序列的方法

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CN114134219A (zh) * 2021-12-14 2022-03-04 广州市金圻睿生物科技有限责任公司 一种多重核酸检测系统及其制备方法与应用
WO2023236037A1 (fr) * 2022-06-07 2023-12-14 广州市金圻睿生物科技有限责任公司 Kit de détection d'acide nucléique du hpv, son procédé de préparation et son utilisation
CN117230250B (zh) * 2022-06-07 2025-03-14 广州市金圻睿生物科技有限责任公司 一种hpv核酸检测试剂盒及其制备方法与应用
CN117802237B (zh) * 2023-10-20 2025-03-21 艾普拜生物科技(苏州)有限公司 信标探针及其组合、用于检测flt3-itd基因突变的pcr扩增试剂及试剂盒
CN117363767B (zh) * 2023-12-07 2024-04-05 上海美吉生物医药科技有限公司 一种用于靶基因实时荧光pcr检测的探针组合、引物组、试剂盒及其应用
CN118547113B (zh) * 2024-05-20 2025-03-28 广州市金圻睿生物科技有限责任公司 多重核酸的检测系统及其应用

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CN102959092A (zh) * 2011-01-11 2013-03-06 Seegene株式会社 借助于探测和标记的寡核苷酸切割以及延长试验的靶核酸序列检测
CN104145029A (zh) * 2012-02-02 2014-11-12 Seegene株式会社 利用探测和标记寡核苷酸切割及延伸-依赖性信号传导寡核苷酸杂交的靶核酸序列的检测
CN105483285A (zh) * 2015-12-10 2016-04-13 湖北民族学院 一种基于鸟嘌呤的超猝灭分子信标的构建及应用
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CN114134219A (zh) * 2021-12-14 2022-03-04 广州市金圻睿生物科技有限责任公司 一种多重核酸检测系统及其制备方法与应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116694743A (zh) * 2023-06-29 2023-09-05 山东迪曼生物科技有限公司 一种利用荧光探针检测多靶标基因序列的方法
CN116694743B (zh) * 2023-06-29 2024-02-02 果然基因科技(山东)股份有限公司 一种利用荧光探针检测多靶标基因序列的方法

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