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WO2024222958A1 - Puce d'amplification rapide pour acide nucléique - Google Patents

Puce d'amplification rapide pour acide nucléique Download PDF

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Publication number
WO2024222958A1
WO2024222958A1 PCT/CN2024/090409 CN2024090409W WO2024222958A1 WO 2024222958 A1 WO2024222958 A1 WO 2024222958A1 CN 2024090409 W CN2024090409 W CN 2024090409W WO 2024222958 A1 WO2024222958 A1 WO 2024222958A1
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WO
WIPO (PCT)
Prior art keywords
amplification
nucleic acid
chamber
amplification chip
acid amplification
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/090409
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English (en)
Chinese (zh)
Inventor
糜军
张洁莹
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Individual
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Individual
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Publication of WO2024222958A1 publication Critical patent/WO2024222958A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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 present application relates to the field of nucleic acid amplification, and in particular to a rapid nucleic acid amplification chip.
  • the detection methods for pathogenic microorganisms mainly include microscopy, culture, immunological testing, and nucleic acid amplification testing.
  • the microscopy method is low-cost, simple, and convenient, but the positive rate is low and it is easy to miss detection; the results of the culture method are highly reliable, but the cycle is long and the cost is high; the operation of immunological testing is relatively simple, but there is a window period, so it is easy to miss detection.
  • nucleic acid testing are equivalent to those of the culture method, but there are also the following shortcomings: 1 Sample preparation (nucleic acid extraction) and nucleic acid amplification/detection are carried out separately, the operation steps are cumbersome, and professional personnel are required to perform, which increases the operation error; 2 Open nucleic acid extraction is also prone to cross-contamination of samples, resulting in false positive test results; 3 Although the operation cycle of nucleic acid testing is shorter than that of pathogenic microorganism culture, it is also difficult to meet the requirements of bedside testing (POCT). How to quickly amplify nucleic acids, shorten detection time, and reduce patient waiting time has become a top priority.
  • POCT bedside testing
  • the purpose of the present application is to provide a rapid nucleic acid amplification chip, which enables 40 cycles of nucleic acid amplification reactions to be completed within 10 minutes.
  • the present application discloses a rapid nucleic acid amplification chip, which comprises: an amplification chamber, a sample loading hole, a liquid storage chamber, a heating module and a precision pump; wherein, the number of the amplification chambers is not less than two, and the plurality of amplification chambers are arranged in parallel and spaced apart, and the interior of the amplification chambers is filled with a second medium, which is incompatible with reaction droplets, and the liquid storage chamber is pre-filled with a nucleic acid amplification reaction solution, and the liquid storage chamber is wide at the top and narrow at the bottom; the plurality of heating modules are not less than two, and are spaced apart and arranged below the amplification chamber and perpendicular to the amplification chamber, and the temperatures of the plurality of heating modules are different and can be configured according to requirements; the sample loading hole is arranged at the upper part of the amplification chamber and connected to the amplification chamber, and the precision pump is connected to both sides of the amplification chamber through a precision
  • the amplification chip further comprises a light source, wherein the light source is arranged on the Below the reaction chamber.
  • the amplification chip further includes an optical probe, which is arranged below the reaction chamber and is used to collect the fluorescence signal excited by the sample in the amplification chip.
  • the heating module is a semiconductor heating sheet
  • the amplification chip is in close contact with the semiconductor heating sheet to achieve temperature control.
  • the number of fluorescent signals inside the reaction droplet is 2-8.
  • the amplification chamber is made of plastic and has a flat rectangular shape.
  • the height of the rectangle is 0.2-0.8 mm.
  • a heat conducting plate is provided between the amplification chamber and the heating module, and the heat conducting plate is selected from a material with high thermal conductivity.
  • both ends of the amplification chamber are connected to precision pneumatic or hydraulic pumps, and the precision pumps drive the reaction medium to move back and forth.
  • an incompletely closed cover plate is provided above the liquid storage chamber.
  • the second medium is oil.
  • the instrument itself has a simple structure and relatively low cost
  • the operation time is short, and 40 PCR cycles take no more than 10 minutes.
  • This instrument can be powered by batteries to achieve portable nucleic acid detection function
  • feature A+B+C is disclosed
  • feature A+B+D+E is disclosed
  • features C and D are equivalent technical means that play the same role.
  • Feature E can be combined with feature C technically. Then, the solution of A+B+C+D should not be deemed to have been recorded because it is technically infeasible, and the solution of A+B+C+E should be deemed to have been recorded.
  • FIG1 is a perspective view of a nucleic acid amplification chip according to an embodiment of the present invention.
  • FIG2 is a side cross-sectional view of a nucleic acid amplification chip according to an embodiment of the present invention.
  • FIG. 3 is a schematic plan view of a nucleic acid amplification chip according to an embodiment of the present invention.
  • Figure numerals 1-precision pump connector; 2-amplification chamber, 3-sample loading hole, 4-liquid storage chamber, 5-piston, 6-heating module, 7-amplification reaction droplet, 8-oil.
  • the device itself has a simple structure and relatively low cost; it is simple to operate, has good repeatability, and has high accuracy of results; it has a short operation time, and 40 PCR cycles take no more than 10 minutes; and it has extremely low requirements for operators and supporting hardware facilities, and has good applicability, which can simultaneously meet the sample testing needs of centralized central laboratories and primary medical institutions.
  • the instrument can be powered by batteries to achieve portable nucleic acid detection functions.
  • a nucleic acid amplification chip of the present embodiment is shown in Figures 1 to 3, and the nucleic acid amplification chip includes: an amplification chamber 2, a sample loading hole 3, a heating module and a precision pump and a precision pump connector 1 for connecting the precision pump, wherein the number of the amplification chambers 2 is 2-10, and the interior thereof is filled with a second medium, and the second medium is incompatible with the reaction droplets.
  • the second medium is oil 8.
  • the material of the amplification chamber 2 is plastic, and the shape is a flat rectangle, and the height of the rectangle is 0.2-0.8 mm.
  • the amplification chamber 2 is connected to the sample loading hole 3, and the nucleic acid amplification reaction solution is pre-filled in the liquid storage chamber 4 below the sample loading hole 3, and a piston 5 is provided at the lower part of the liquid storage chamber 4, and the piston 5 is used to separate the liquid storage chamber 4 from the amplification chamber 2 and achieve a unidirectional effect.
  • the heating module 6 is a semiconductor heating sheet, and the amplification chip is in close contact with the semiconductor heating sheet to achieve temperature control.
  • the sample loading hole 3 is arranged on the upper part of the amplification chamber 2 and connected to the amplification chamber 2.
  • the precision pump is connected to both sides of the amplification chamber 2 through the precision pump connector 1 to push the sample loading hole 3.
  • the reaction droplets in the amplification chamber 2 reciprocate.
  • the precision pump is a pneumatic pump.
  • the precision pump is a hydraulic pump.
  • the amplification chip further comprises a light source, which is arranged below the reaction chamber.
  • the amplification chip further comprises an optical probe, which is arranged below the reaction chamber to collect the fluorescence signal excited by the sample in the amplification chip.
  • the number of the fluorescence signals inside the reaction droplet is 2-8.
  • the amplification chamber is made of plastic and has a flat rectangular shape.
  • the height of the rectangle is 0.2-0.8 mm.
  • a heat conducting plate is provided between the amplification chamber 2 and the heating module 6.
  • the heat conducting plate is made of glass.
  • an incompletely closed cover plate is provided above the liquid storage chamber 4 .
  • a reaction droplet is added through the sample addition hole 3, and the reaction droplet is pre-treated. After the reaction droplet enters the liquid storage chamber 4, it reacts with the pre-stored liquid inside the liquid storage chamber 4 and then enters the amplification chamber through the piston 5 with an inclined surface. Then, the precision pump pushes the reaction droplet to move back and forth between the multiple heating modules 6 with different temperatures to realize the amplification process. Then, a light source is used to generate incident light acting on the bottom of the test tube, and the optical probe is used to collect the light signal fed back from the amplification chip sample.
  • the nucleic acid amplification chip of the present invention is as shown in FIG. 3 , which has no liquid storage chamber 4 and piston 5 , and the reaction droplets after completing the amplification reaction externally are directly added to the amplification chamber 2 through the sample addition hole 3 to achieve amplification.
  • an action is performed according to an element, it means that the action is performed at least according to the element, which includes two situations: performing the action only according to the element, and performing the action according to the element and other elements.
  • Expressions such as multiple, multiple, and multiple include 2, 2 times, 2 kinds, and more than 2, more than 2 times, and more than 2 kinds.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Sustainable Development (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Puce d'amplification rapide pour acide nucléique. La puce d'amplification comprend : des cavités d'amplification, des orifices pour l'ajout d'échantillons, une cavité de stockage de liquide, des modules de chauffage et une pompe de précision. Le nombre de cavités d'amplification n'est pas inférieur à deux, les multiples cavités d'amplification sont agencées en parallèle à intervalles et sont remplies d'un deuxième milieu, le deuxième milieu est incompatible avec les gouttes de réaction, et les modules de chauffage sont agencés sous les cavités d'amplification à intervalles. La pompe de précision est reliée à deux côtés des cavités d'amplification au moyen de connecteurs de pompe de précision et utilisée pour pousser les gouttes de réaction dans les cavités d'amplification à effectuer un mouvement de va-et-vient; et un piston à biseau est agencé sous la cavité de stockage de liquide. Par comparaison avec les techniques existantes, l'instrument présente une structure simplifiée et un coût relativement faible; la sensibilité est élevée et atteint la sensibilité du test PCR quantitatif par fluorescence classique, ce qui permet d'obtenir des résultats précis; l'instrument est facile à utiliser, présente des exigences extrêmement faibles en matière d'opérateurs et d'installations matérielles d'appui, présente une bonne applicabilité et peut répondre aux exigences en matière de tests d'échantillons des laboratoires centraux et des institutions médicales de premier recours.
PCT/CN2024/090409 2023-04-28 2024-04-28 Puce d'amplification rapide pour acide nucléique Pending WO2024222958A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202310481279.9 2023-04-28
CN202310481279 2023-04-28

Publications (1)

Publication Number Publication Date
WO2024222958A1 true WO2024222958A1 (fr) 2024-10-31

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Family Applications (1)

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WO (1) WO2024222958A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130260447A1 (en) * 2006-05-11 2013-10-03 Darren R. Link Systems and methods for handling microfluidic droplets
CN108485909A (zh) * 2018-03-21 2018-09-04 苏州锐讯生物科技有限公司 微流控芯片及其应用
US20200023360A1 (en) * 2017-03-22 2020-01-23 The Board Trustees Of The University Of Illinois System for Rapid, Portable, and Multiplexed Detection and Identification of Pathogen Specific Nucleic Acid Sequences
CN115703986A (zh) * 2021-08-06 2023-02-17 北京航空航天大学 一种用于核酸扩增检测的微流控芯片、制备方法及用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130260447A1 (en) * 2006-05-11 2013-10-03 Darren R. Link Systems and methods for handling microfluidic droplets
US20200023360A1 (en) * 2017-03-22 2020-01-23 The Board Trustees Of The University Of Illinois System for Rapid, Portable, and Multiplexed Detection and Identification of Pathogen Specific Nucleic Acid Sequences
CN108485909A (zh) * 2018-03-21 2018-09-04 苏州锐讯生物科技有限公司 微流控芯片及其应用
CN115703986A (zh) * 2021-08-06 2023-02-17 北京航空航天大学 一种用于核酸扩增检测的微流控芯片、制备方法及用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI HUANAN, ZHANG HAOQING, XU YING, TURECKOVA ALZBETA, ZAHRADNÍK PAVEL, CHANG HONGLONG, NEUZIL PAVEL: "Versatile digital polymerase chain reaction chip design, fabrication, and image processing", SENSORS AND ACTUATORS B: CHEMICAL, ELSEVIER BV, NL, vol. 283, 1 March 2019 (2019-03-01), NL , pages 677 - 684, XP093227269, ISSN: 0925-4005, DOI: 10.1016/j.snb.2018.12.072 *
S. MOHR ; Y.-H. ZHANG ; A. MACASKILL ; P. J. R. DAY ; R. W. BARBER ; N. J. GODDARD ; D. R. EMERSON ; P. R. FIELDEN: "Numerical and experimental study of a droplet-based PCR chip", MICROFLUIDICS AND NANOFLUIDICS, SPRINGER, BERLIN, DE, vol. 3, no. 5, 10 February 2007 (2007-02-10), Berlin, DE , pages 611 - 621, XP019522732, ISSN: 1613-4990, DOI: 10.1007/s10404-007-0153-8 *

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