WO2021092795A1 - Procédé et dispositif de détection d'acide nucléique - Google Patents

Procédé et dispositif de détection d'acide nucléique Download PDF

Info

Publication number
WO2021092795A1
WO2021092795A1 PCT/CN2019/118033 CN2019118033W WO2021092795A1 WO 2021092795 A1 WO2021092795 A1 WO 2021092795A1 CN 2019118033 W CN2019118033 W CN 2019118033W WO 2021092795 A1 WO2021092795 A1 WO 2021092795A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
flash
detection
pcr
paper
Prior art date
Application number
PCT/CN2019/118033
Other languages
English (en)
Chinese (zh)
Inventor
李峰
董天彧
王冠
Original Assignee
李峰
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 李峰 filed Critical 李峰
Priority to CN201980100017.5A priority Critical patent/CN114341627A/zh
Priority to PCT/CN2019/118033 priority patent/WO2021092795A1/fr
Publication of WO2021092795A1 publication Critical patent/WO2021092795A1/fr

Links

Images

Classifications

    • 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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence

Definitions

  • the invention relates to the field of biological detection, in particular to a rapid light-activated substrate color development (FLASH) detection platform for on-site instant nucleic acid detection.
  • FLASH rapid light-activated substrate color development
  • Nucleic acid testing play an indispensable role in disease diagnosis and testing, and various molecular biological information can be obtained from trace genetic material.
  • Nucleic acid detection such as polymerase chain reaction (PCR)
  • PCR polymerase chain reaction
  • Converting standard nucleic acid detection into real-time detection will help doctors directly perform rapid diagnosis in the ward or consulting room.
  • simplified nucleic acid testing methods are also essential for on-site diagnosis, monitoring and control of viral, bacterial, and parasitic infections. In these countries, large-scale infections often lead to serious morbidity and even death.
  • TINY TINY Isothermal Nucleic Acid Quantitative System
  • DIDs are universal probes for nucleic acid staining used in standard nucleic acid detection such as quantitative PCR (qPCR); however, existing tests only use the fluorescence enhancement properties of these dyes.
  • qPCR quantitative PCR
  • the present invention systematically studied the photochemical properties of DIDs, and found that the insertion of dye-DNA also enhanced the process of inter-spin forbidden system crossover (ISC), which is a kind of singlet state from excited state to triplet state.
  • ISC inter-spin forbidden system crossover
  • the present invention tries to transform traditional fluorinated DIDs into high-efficiency photosensitizers that can trigger color reaction, which can be used for nucleic acid detection and quantitative analysis to directly obtain visual colorimetric readings .
  • the present invention further introduces the fast-activated substrate color development (FLASH) technology, which uses a universal light-excited pigment developer, which can be applied to all fluorescent dye nucleic acid detection methods (such as qPCR) and convert it into Portable detection, you can obtain visual colorimetric readings by simply irradiating a light source for a few seconds.
  • FLASH fast-activated substrate color development
  • the present invention is also equipped with three detection platforms for FLASH detection, including 96-channel FLASH arrays for high-throughput analysis, portable electronic FLASH readers and field-based applications Paper FLASH strips.
  • the present invention provides the application of nucleic acid intercalating dyes (DIDs) in chemiluminescence, which is characterized in that the chemiluminescence is phosphorescence at a wavelength of 625 nm.
  • DIDs nucleic acid intercalating dyes
  • the phosphorescence generated when nucleic acid intercalating dyes (DIDs) are inserted into double-stranded nucleic acid is enhanced, and the phosphorescence enhancement depends quantitatively on the concentration of the double-stranded nucleic acid.
  • the nucleic acid intercalating dyes include SYBR Green 1 (SG-I), PicoGreen (PG), thioflavonoid T (ThT), thiazole orange (TO), and the like.
  • the chromogenic substrates include 3,3,5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD) and the like.
  • the present invention provides the application of nucleic acid intercalating dyes (DIDs) as chromogens or photosensitizers.
  • DIDs nucleic acid intercalating dyes
  • the nucleic acid intercalating dyes photo-drive the chromogenic substrate to undergo photosensitive oxidation in the presence of double-stranded nucleic acid, and then produce The color changes.
  • the nucleic acid intercalating dyes include but are not limited to SYBR Green 1 (SG-I), PicoGreen (PG), thioflavonoid T (ThT), thiazole orange (TO), etc. .
  • the chromogenic substrates include 3,3,5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD) and the like.
  • the present invention provides a nucleic acid detection method, including the following steps:
  • DIDs nucleic acid intercalating dyes
  • the color of the substrate is developed by irradiation with excitation light.
  • the method of the present invention further includes the optional step (4), selecting an appropriate wavelength according to the type of the color-developing substrate for spectrophotometric detection.
  • the method of the present invention further includes an optional step (5), which is to quantitatively analyze the content or concentration of the double-stranded nucleic acid according to the color development result of the substrate.
  • step (1) obtains double-stranded nucleic acid by PCR, LAMP or RPA (recombinase polymerase amplification).
  • the excitation light with a wavelength of 480-495 nm is irradiated for more than 5 seconds.
  • step (2) uses TMB as a color developing substrate
  • step (4) a wavelength of 650 nm is selected for spectrophotometric detection.
  • the present invention provides a FLASH reader, which includes a housing, a light irradiation module, a transmission light source, a color sensor module, a controller, and a circuit.
  • the reader can be programmed to realize the irradiation module and the color sensor module Switch between.
  • the light irradiation module includes a circuit board welded with a plurality of light-emitting diodes, and the circuit board is matched with the porous plate, so that the light-emitting diodes on the circuit board are parallel to each other.
  • the light emitting diode generates high-intensity blue light.
  • the color sensing module includes an RGB color sensor for detecting the absorbance value of the transmitted light source after passing through the test sample.
  • the transmitted light source is white light.
  • the present invention provides a paper-based FLASH detection tape, which includes a paper layer provided with a pattern, the paper layer includes a sample loading area and a test area, and is characterized in that the test area of the paper layer has a TMB coating.
  • the paper layer is cellulose paper.
  • the paper-based FLASH detection tape of the present invention is made by wax printing process, and the hydrophobic wax barrier forms the sample loading area and the test area; preferably, one side of the test area also includes a scale for measuring the migration distance logo.
  • the present invention provides a method for detecting nucleic acid using a paper-based FLASH detection tape, the method comprising:
  • the double-stranded nucleic acid is a PCR product or a LAMP product.
  • the method for detecting nucleic acid using a paper-based FLASH detection tape of the present invention wherein said step (5) quantitatively detects nucleic acid by visualizing the distance of color migration.
  • the method for detecting nucleic acid using a paper-based FLASH detection band of the present invention wherein the nucleic acid intercalating dyes (DIDs) include SYBR Green 1 (SG-I), PicoGreen (PG), and thioflavonoid T (ThT) , Thiazole Orange (TO) and so on.
  • DIDs nucleic acid intercalating dyes
  • SG-I SYBR Green 1
  • PG PicoGreen
  • ThT thioflavonoid T
  • TO Thiazole Orange
  • FLASH technology proposes an innovative "chemical” method to solve the current on-site nucleic acid detection needs.
  • the present invention does not create a new nano or biosensing platform, but focuses on the research of widely used nucleic acid detection chemical probes. "Unexplored” feature, so the method of the present invention has several advantages.
  • FLASH is a simple plug-and-play detection system that can easily realize on-site nucleic acid detection.
  • the color reaction only needs to directly mix the nucleic acid amplicon with two chemical substances, SG-I and TMB, and then irradiate it with light. Since SG-I and TMB are one of the most commonly used reagents in clinical and biological laboratories, there is basically no or almost no intellectual and technical obstacles to using these two chemicals and FLASH.
  • RT-PCR, colony PCR, direct PCR Using different PCR (RT-PCR, colony PCR, direct PCR) and LAMP methods, the simplicity and wide applicability of FLASH have been successfully verified.
  • the compatibility of FLASH with various engineering platforms such as portable devices and paper-based microfluidics has also been proven through the development and adoption of FLASH readers and strips for step-by-step nucleic acid detection.
  • FLASH technology also has high chemical diversity.
  • the photocatalytic performance is not limited to SG-I, and other intercalating dyes such as PicoGreen (PG) can also produce colorimetric readings on dsDNA.
  • This chemical diversity may further expand the flexibility of FLASH, enabling it to design new detection methods in strategies that rely on nucleic acid dyes other than SG-I and/or utilize more complex DNA structures other than dsDNA.
  • the present invention only uses FLASH as the end reader of PCR and LAMP in this work, it is also possible to realize a real-time FLASH PCR or LAMP system in the future.
  • the current ongoing work is to carry out research and development at the molecular and equipment level, including screening FLASH reactants and fully complying with the conditions of real-time PCR or LAMP reactions, and equipping FLASH readers with heating modules.
  • the present invention expects that FLASH technology will be easily used in fast, stable and sensitive step-by-step nucleic acid detection equipment, and open up new ways for POC diagnosis and instant detection applications.
  • a Schematic diagram of the interaction between SG-I and dsDNA.
  • d The rotation of the axial bond between the quinoline (electron donor) and benzothiazole (electron acceptor) groups in SG-I and the definition of the dihedral angle ⁇ .
  • e The potential energy function of the dihedral angle ⁇ in the ground state (S0) and the excited state (S1).
  • f The Jablonski diagram shows the energy level and the electronic transition of SG-I photochemistry.
  • the absorption spectrum (a), fluorescence spectrum (b) and time-resolved fluorescence spectrum (c) were used to characterize the optical properties of SG-I and SG-I inserted into dsDNA.
  • the fluorescence enhancement of SG-I after inserting dsDNA is related to the concentration.
  • the fluorescence emission time is also related to the dsDNA concentration (c).
  • ⁇ EST is estimated to be 0.411 eV, which is very consistent with experimental observations, where ⁇ EST is estimated to be 0.378 eV.
  • a Schematic diagram of the light-driven color development (FLASH) reaction mediated by SG-I and DNA insertion.
  • b. Jablons Keto shows the possible mechanism of FLASH reaction.
  • c. Image and absorbance spectrum of 4 ⁇ M SG-I, 200 mg/ml TMB, 100 nM dsDNA (ssDNA) solution system.
  • d. The ⁇ DNA standard concentration of the standard curve ranges from 1pg/ ⁇ L to 500ng/ ⁇ L, which are FLASH (blue), QuantiFluor dsDNA quantitative kit (red), and A260 (green).
  • the signal readings of each method are standardized for the positive control that produces the maximum signal and the negative control that is the blank control.
  • Each error bar represents a standard deviation calculated from three parallel experiments.
  • SG-I-dsDNA complex is a type II photosensitizer.
  • Photooxidation is only observed when oxygen is dissolved in the solution.
  • ThT is a highly efficient sensor that has been discovered recently.
  • ThT can selectively bind to DNA g-tetramer (g-4) and enhance fluorescence.
  • g-4 DNA g-tetramer
  • g-4 Enhancement
  • ThT binds to G-4 and the fluorescence enhancement of G-4 in the presence of (concentration) K+.
  • ThT fluorescence enhancement in the presence of G-4 and G-4/K+.
  • the ThT/G4 binding complex can also mediate the photosensitive oxidation of TMB.
  • a Schematic diagram of a typical workflow for analyzing clinical or biological samples using FLASH PCR.
  • b Synthetic DNA standard amplification PCR reaction, the concentration ranges from 1aM to 1pM. The absorbance parameters measured at 650nm are constructed according to the irradiation time (above, 35 cycles) or the PCR cycle (bottom, 5 seconds of irradiation) The function. Each error bar represents one standard deviation obtained from three parallel experiments.
  • c Use FLASH PCR (blue) and qPCR (green) to quantitatively analyze IL-6 gene expression kinetics after 100ng/mL synthetic allergen TNP-BSA stimulates IgE-sensitized BMMCs.
  • C1 to C5 were selected from Escherichia coli transformed into HBV 1.3-mer WT replicon.
  • C6 to C8 are clones selected from untransfected E. coli DH5 ⁇ .
  • Figure 21 Portable FLASH reader for detecting STH infection
  • a The picture shows the fully assembled FLASH reader (left) and the key unit (right).
  • b Schematic diagram of the working principle of the FLASH reader.
  • c Use DNA standards with initial concentrations ranging from 10aM to 100fM for PCR amplification and evaluate the FLASH reader. Compare the quantitative capability of the FLASH reader with a commercial qPCR instrument (below). Each error bar is a standard deviation derived from three parallel experiments.
  • d By analyzing samples of parasites excreted from school-age children in rural Honduras after treatment, including Trichuirs trichiura (TT) and Ascaris lumbricoides (AL), to evaluate the value of FLASH readers for on-site diagnosis. ****P ⁇ 0.0001 adopts two-tailed t test. NS, not significant (P ⁇ 0.05 is the significant level).
  • Each error bar is a standard deviation derived from three parallel experiments.
  • Figure 23 The characteristics of the FLASH reader for quantitative detection of PCR amplicons
  • R, G, and B respectively represent the signals of the RGB color sensor at the red channel, the green channel, and the blue channel.
  • [Primer] 125nM
  • [SG-I] 2 ⁇ M
  • [TMB] 300mg/L.
  • a Schematic diagram of LAMP principle
  • FLASH LAMP is used to directly analyze HBV genomic DNA in undiluted human serum samples without any sample preparation steps. HBV positive serum samples can be quantitatively detected visually (upper image) or using a FLASH reader (bottom).
  • c Schematic diagram of the production process of paper FLASH tape.
  • d Detection of HBV genomic DNA based on LAMP and FLASH bands with varying distances.
  • Lane 1 is a 20bp DNA ladder
  • lanes 2 and 3 are the 1fM HBV vector bands amplified by LAMP in human serum samples
  • lanes 4 and 5 are the LAMP amplified serum sample mixture as a blank control, and there is no band.
  • the production legend (a) and principle diagram (b) of FLASH tape based on paper The design concept of the FLASH band is based on the results of the previous research of the present invention.
  • SG-I interacts with cellulose paper, and the binding strength is stronger than SG-I-ssDNA, but weaker than SG-I-dsDNA8.
  • SG-I will be captured in the sample loading area of the strip, and only dsDNA can precipitate SG-I to the test area for distance visualization.
  • TMB Once eluted to the test area coated with TMB, a FLASH reaction will occur, and blue stripes will be formed under light irradiation.
  • the miniaturization of nucleic acid testing makes it a portable and inexpensive detection platform, which is helpful for on-site diagnosis of diseases and expansion of applicable scenarios.
  • the present invention reports a nucleic acid detection and quantitative analysis technology that uses DNA intercalating dyes (DIDs) to directly achieve visual colorimetry, that is, fast light-activated substrate color development (FLASH) technology.
  • DIDs DNA intercalating dyes
  • FLASH fast light-activated substrate color development
  • the FLASH system of the present invention can be inserted into almost any fluorescent nucleic acid detection and converted into a portable detection, and the colorimetric reading can be seen only by irradiating with a light beam for a few seconds.
  • the present invention successfully proves that the FLASH technology is widely applicable to the detection of a variety of biological and clinical samples by combining different nucleic acid amplification technologies.
  • the FLASH method is highly sensitive and can perform quantitative detection. Its detection performance is basically the same as that of commercial quantitative PCR reactions, but its equipment requirements are much lower.
  • the present invention further confirms the adaptability of FLASH field detection.
  • SYBR Green I (SG-I) dye 3,3' 5,5'-tetramethylbenzidine (TMB), Thioflavin T (ThT), fluorescent dye Thiazole Orange (TO), 10 ⁇ phosphate buffer ( 10 ⁇ PBS), TWEEN 20, polyethylene glycol (PEG) 100,000, sodium citrate (Na3C6H5O7), citric acid solution (H3C6H5O7), hydrochloric acid (HCl), herring sperm DNA (hsDNA), Whitman filter paper (Grade 1 ), microscope slide, Sigma paraffin film (Oakville, ON, Canada).
  • Taq 2 ⁇ PCR Master Mix iTaqTM Universal SYBR Green Supermix, N, N, N′, N′-methylethylenediamine (TEMED) , Ammonium persulfate (APS), 40% acrylamide/bisacrylamide solution, DNA loading buffer, and 20bp DNA ladder purchased from Bio-Rad Laboratories, Inc. (Mississauga, ON, Canada).
  • TEMED N, N, N′, N′-methylethylenediamine
  • APS Ammonium persulfate
  • 40% acrylamide/bisacrylamide solution DNA loading buffer
  • 20bp DNA ladder purchased from Bio-Rad Laboratories, Inc. (Mississauga, ON, Canada).
  • Luna Universal qPCR Master Mix and Monarch PCR&DNA Cleanup Kit were purchased from New England BioLabs (Whitby, ON, Canada).
  • Pico Green (PG) dye 10,000 ⁇
  • Phusion Blood Direct PCR kit were purchased from Thermo Fisher Scientific (Whitby, ON, Canada).
  • QIAamp Circulating Nucleic Acid Kit and QIAprep Spin Miniprep Kit were purchased from Qiagen Inc. (Toronto, ON, Canada). QuntiFluor dsDNA quantification kit was purchased from Promega (Madison, WI). NANO pure water H2O (>18.0M ⁇ ), purified by Ultrapure Milli-Q water system and used in the overall experiment. All synthetic DNA templates and primers (Table 1 and S2) were purchased from Integrated DNA Technologies (Coralville, IA) and purified by standard desalting.
  • Fluorescent compounds can achieve the labeling effect by inserting DNA into light, which has been widely used in the detection and visualization of nucleic acids in vivo and in vitro.
  • Many DNA insertion dyes such as SYBR Green I (SG-I)
  • SG-I SYBR Green I
  • dsDNA adjacent double-stranded DNA
  • Figure 1a For example, when SG-I (4 ⁇ M) is mixed with different concentrations of dsDNA, the present invention observes significant fluorescence enhancement and prolonged fluorescence emission time ( Figure 1b and Figure 2).
  • the present invention speculates that the excited state electrons in the DNA inserted into the dye may also be released through another ISC pathway, which can be detected by phosphorescence.
  • the solution containing SG-I (4 ⁇ M) with or without hsDNA (1 ⁇ M) was incubated for 10 minutes.
  • the absorbance spectrum was measured with a UV-1750 spectrophotometer (Shimadzu, Japan).
  • Fluorolog-3 fluorescence spectrometer Horiba Jobin Yvon
  • the excitation wavelength is 485nm.
  • the phosphorescence of the solution is measured in an anaerobic test tube.
  • the present invention By analyzing SG-I in an oxygen-free environment, the present invention observes unique phosphorescence at the maximum wavelength of 625 nm (Fig. 1b and Fig. 3), and the phosphorescence lifetime is about 4.97 ms (Fig. 1c and Fig. 3). Like fluorescence, dsDNA quantitatively enhanced the phosphorescence of SG-I ( Figure 1b and Figure 3b).
  • DFT density functional theory
  • a DFT model with SG-I as the turning angle function is established.
  • the dihedral angle of the molecular rotor remains unchanged, while other bond lengths and bond angles have sufficient room for change.
  • the rotor dihedral angle changes in 10° increments, generating 36 different geometric forms.
  • the total energy of SCF optimized by each geometry is used to evaluate the variation of SG-I ground state electron energy with rotor angle.
  • the same density functional function and basis function as above are used.
  • TDDFT was used to scan the S1 excited state flexible potential energy of SG-I, and the scan started from the minimum value of the rotor potential energy surface.
  • SG-I Density functional theory (DFT) and time-dependent DFT calculation methods further support the above experimental results.
  • SG-I is a typical molecular motor, which contains an electron donor (quinoline) and an electron acceptor (benzothiazole) (Figure 1d).
  • the potential energy scan shows that the dihedral angle of the quinoline and benzothiazole groups, ⁇ ( Figure 1d), is the lowest energy configuration of the ground state and excited state of SG-I at 0° and 90°, respectively ( Figure 1e) .
  • Example 2 Utilizing the optical properties of nucleic acid dyes for nucleic acid detection
  • the excited triplet state may also generate singlet oxygen (1O2) through energy transfer and cause a photosensitive oxidation reaction, which only occurs when SG-I is inserted into dsDNA ( Figure 6 and Figure 7).
  • a visual colorimetric reading can be generated.
  • the present invention chose 3,3,5,5'-tetramethylbenzidine (TMB), a reagent widely used in immunoassays, as the substrate for photosensitive oxidation. After that, the present invention selects SG-I (4 ⁇ M) as the photosensitizer, TMB (200mg/L) as the substrate, and dsDNA as the target (100nM).
  • TMB 3,3,5,5'-tetramethylbenzidine
  • the present invention also found that phosphorescence enhancement and photosensitive oxidation activity are not limited to SG-I, other dsDNA fluorescent dyes are also applicable, such as PicoGreen ( Figure 8), and organic dyes specifically designed for tertiary DNA structure, g-fourplex ( Figure 9) And i-motif ( Figure 10).
  • the research of the present invention on the photochemical properties of fluorescent dyes establishes a solid theoretical framework for FLASH detection.
  • the goal of the present invention is to realize a nucleic acid detection and quantification method of a chromogen with high sensitivity and high efficiency.
  • the reaction mixture (100 ⁇ L) contains 20 ⁇ l synthetic standard DNA, PCR or LAMP amplicons, 2 ⁇ M SG-I and 300 ⁇ g/ml TMB dissolved in pH 4 citric acid buffer (33mM citric acid , 17mM sodium citrate millimeter, 10mM MgCl2, 0.1% Tween 20), the system uses 96FLASH array irradiation or portable FLASH reader.
  • pH 4 citric acid buffer 33mM citric acid , 17mM sodium citrate millimeter, 10mM MgCl2, 0.1% Tween 20
  • the system uses 96FLASH array irradiation or portable FLASH reader.
  • a multi-mode microplate reader (SpectraMax i3, Molecular Devices) to measure the absorbance at 650 nm after irradiation every 5 seconds.
  • the next step of the present invention is to integrate FLASH as an "additional component" step of PCR ( Figure 11a).
  • the present invention also fabricated a 96-plex LED array (FLASH array) on a standard 96-well plate to drive parallel photosensitive oxidation reactions ( Figure 12).
  • the FLASH array realizes rapid photosensitive oxidation and rapid heat dissipation by welding 96 high-intensity LEDs (3W) to an aluminum cooling block.
  • 96 LED lights are grouped and controlled by 6 independent switches (Figure 12c).
  • the LEDs are uniformly welded on the plate, 8 rows ⁇ 12 rows, with an interval of 9.0mm between each row, which matches the 96-well microplate ( Figure S5). Every two columns of LEDs are connected and powered by an LED driver (42-68VDC, 600mA output). All LED drivers are controlled by a main switch connected to 110V AC.
  • the present invention first selects a 184bp synthetic DNA template as a model target. This sequence is consistent with the hepatitis B virus surface protein gene (HBV-S) sequence. After that, the present invention systematically studied and compared different FLASH PCR operating conditions, including primer concentration ( Figure 13), SG-I ( Figure 14), TMB ( Figure 15) and the pre-incubation time of PCR amplicons and FLASH reagents ( Figure 16). The present invention finds that FLASH PCR has strong adaptability to different detection conditions, and color can be developed without pre-incubation. In addition, the light-driven color rendering efficiency is high and easy to control.
  • HBV-S hepatitis B virus surface protein gene
  • the present invention finds that 5s irradiation is sufficient to produce colorimetric readings for nucleic acid quantification, and the color development can be started or stopped by turning on or off the irradiation LED (the upper part of Fig. 11b).
  • the fluctuation range of FLASH PCR depends on the number of cycles of PCR (bottom of Figure 11b and Figure 17).
  • the detection limit of 1aM HBV-S template (approximately 10 copies per reaction) can be detected after 35 cycles, which is comparable to the results of qPCR.
  • FLASH PCR is a colorimetric method, and the instrument requirements and costs are much lower.
  • Real-time quantitative PCR uses BioRad CFX96 TM IVD Real-time PCR detection system and BioRad iTaq TM Universal Green Supermix and Luna Universal qPCR Master Mix were tested. The cycle threshold (Ct) was recorded and the ⁇ Ct method was used for qPCR data analysis.
  • Example 4 FLASH PCR is used to detect cytokine cDNA
  • the present invention After verifying FLASH PCR using the synthetic DNA target, the present invention then uses different PCR reagents and protocols to test its applicability in nucleic acid analysis in various biological and clinical samples. Gene expression analysis is one of the most common uses of qPCR.
  • the present invention first uses FLASH PCR to analyze the changes of interleukin 6 (IL-6) during the allergen-mediated activation of mast cells. Rapid quantitative level of expression of IL-6 or other inflammatory factors in understanding and diagnosis of allergic reactions mastocytosis, allergic reactions and asthma and other diseases has great potential 23.
  • IL-6 interleukin 6
  • Isolate bone marrow from tibia and femur of wild-type C57BL/6 mice induce differentiation with IL-3 (Wehi-3b cells, American Type Culture Collection-ATCC) and PGE2 (Sigma), and culture to establish primary hypertrophy Cell line.
  • Bone marrow-derived mast cells BMMCs were collected every two weeks of culture, centrifuged (Avanti J-15R, Beckman-Coulter), and resuspended in fresh medium. NucBlue staining (Life Technologies, R37605) was used to determine the cell survival rate.
  • Countess II FL (Life Technologies Inc., AMQAF1000) measured cell viability.
  • the cell density was maintained at 0.5 ⁇ 10 6 cells/ml, 37°C, and incubated with 5% carbon dioxide (CO2).
  • CO2 carbon dioxide
  • TNP-specific IgE from TIB-141 cells ATCC was used to stimulate and activate BMMCs overnight. The next day, the unbound IgE was washed with RPMI 1640 (Gibco) and added to RPMI 1640. Resuspend cells in 10% FBS (Sigma) and 1% Pen/Strep.
  • TNP-BSA Biosearch Technologies, Novato, CA
  • SCF Seprotech, Dollard des Ormeaux, QC
  • TNP-specific IgE was first used to activate wild-type C57BL/6 mouse bone marrow-derived mast cells (BMMCs), and then TNP-BSA (100ng/mL) was used to activate BMMCs.
  • Total RNA was isolated at different time points (0, 15, 30, 60, 120, and 300 minutes) and converted into cDNA using reverse transcription. Then, use FLASH PCR and commercial qPCR kits to amplify and quantify the cDNA samples. In both detection methods, IL-6 expression levels increased after activation of mast cells, reaching a peak in 60-120 minutes, which is consistent with previous studies. More importantly, almost the same kinetic curve was obtained using these two techniques ( Figure 11c and Figure 18), confirming that the colorimetric reading of FLASH PCR can be quantified and can be equivalent to a fluorescence-based measurement.
  • Example 5 FLASH PCR is used for colony PCR detection
  • the present invention is selected HBV 1.3-mer WT replicon, an E. coli 1.3 HBV genome coding units as experimental test platform 26 of the present invention.
  • Two sets of primers were designed to specifically amplify the HBV-S gene at positions 1221-1444 and 1345-1564, respectively.
  • the colony containing the HBV vector was subjected to colony PCR, followed by FLASH detection, without any purification steps (Figure 11d).
  • E. coli DH5 ⁇ was used as a negative control (empty vector group).
  • Example 6 FLASH PCR is used to detect the viral load of clinical samples
  • the present invention uses FLASH PCR to detect the HBV virus load.
  • the detection sample is a preclinical sample prepared by mixing the HBV vector with human serum obtained from a healthy donor.
  • the use of pre-clinical samples for validation is essential to verify whether FLASH PCR is applicable to patient samples in a clinical environment.
  • Preparation of human serum samples containing hepatitis B virus and isolation of DNA Preparation of human serum samples containing hepatitis B virus and isolation of DNA.
  • the QIAamp DNA Blood Mini kit is used to extract total DNA from human serum samples containing HBV. Briefly, mix 200 ⁇ L of serum sample with 20 ⁇ L of protease and 200 ⁇ L of lysis buffer, and incubate at 56°C for 10 minutes. Then 200 ⁇ L of ethanol was added to the mixture and applied to the column. After washing the column, the total serum DNA is dissolved in the desired volume of TE buffer (6 ⁇ L-50 ⁇ L).
  • PCR reaction mixture 25mL contains 1 ⁇ Taq Master Mix, the final concentration of primers is 125nM, and the target nucleic acid template. These reactions were all carried out using BioRad T100 TM thermal cycler.
  • a typical PCR reaction includes an initial incubation at 94°C for 3 minutes, followed by 35 PCR cycles (denaturation at 94°C for 3 minutes, annealing for 15 seconds, and extension at 72°C for 15 seconds), followed by an extension at 72°C for 5 minutes.
  • the present invention first uses a commercial kit (QIAamp Cycle Nucleic Acid Kit, Qiagen) to extract total serum DNA, and then uses FLASH PCR to amplify and detect the HBV-S gene at positions 1221-1444 and 1345-1564 ( Figure 20).
  • the present invention finds that FLASH PCR is completely suitable for serum DNA extraction scheme and related reagents.
  • the peak of the detected signal of 1fM HBV vector in undiluted human serum is only about 30% of the detected signal of the positive control (1fM HBV vector in buffer), indicating that significant sample loss occurred during the DNA extraction process (Figure 20b) .
  • a feasible solution to this is to use a direct PCR program (Phusion Blood Direct PCR Test Kit, Fisher Scientific), in which PCR can be performed directly from unpurified whole blood samples 27 .
  • Direct PCR to detect human serum HBV. Serum samples can be directly analyzed using direct PCR methods, without the need to extract DNA.
  • a typical reaction mixture contains 1 ⁇ Phusion blood direct PCR reaction mixture, the final concentration of primers is 125nM per tube, and 2 ⁇ L of human serum sample containing hepatitis B virus.
  • a typical direct PCR includes a cell lysis step at 98°C for 5 minutes, followed by 35 PCR cycles (denaturation at 98°C for 15 seconds, annealing for 5 seconds, extension at 72°C for 15 seconds), and finally extension at 72°C for 1 minute.
  • Use Monarch PCR&DNA Cleanup Kit to purify PCR products to remove interference factors in subsequent analysis.
  • Example 7 FLASH PCR is used to detect parasitic infections in samples
  • the present invention transfers experiments to environmental conditions with limited resources.
  • STH infection is a global health problem that affects more than 1.5 billion people. It is one of the most important causes of malnutrition and cognitive impairment in children.
  • classic microscopy methods do not have clinical sensitivity and specificity to accurately diagnose STH, and nucleic acid detection based on qPCR is expensive and cannot be implemented in most impoverished and resource-limited infectious areas. Therefore, the objective of the present invention is to use FLASH PCR as a low-cost qPCR method for on-site diagnosis and control of STH infection.
  • Soil-borne parasites (STH) samples were recovered from 8 school-age children infected with Trichuris trichiura in the rural La Hicaca area in northwestern Honduras.
  • Eight participants received a treatment regimen based on pyrantyl-pamoate and ocantyl-pamoate (Commet) for the first 3 days and albendazole treatment regimen on the fourth day.
  • the adult worms excreted in the feces were washed with salt water and stored in 70% ethanol. After the sample is recovered, use the Automate Express DNA Extraction System (Thermo Fisher Scientific Inc.) and the commercial kit PrepFiler Express BTA to extract DNA according to the operation manual.
  • the FLASH reader integrates the light irradiation module (high-intensity blue LED) and the color sensor module (Figure 21a and Figure 21b) to measure the color reaction under light drive in real time ( Figure 21a and Figure 21b). Both modules are encapsulated in a 3D printed shell and operated using an electrician controller and open source code ( Figure 22). The reader can switch between the two modules of programmable irradiation and sensing interval, so as to monitor the color reaction in real time ( Figure 21b). Among all the RGB color channels, the red (R) channel is the most sensitive to the transition from colorless to blue of TMB ( Figure 23a).
  • the FLASH reader uses a high-power blue LED (495nm) as the FLASH illumination device, and an in-situ color detection system modified by an IO Rodeo colorimeter.
  • the housing of the reader uses 3D MAX design, and the 3D printing uses Stratasys Object30Pro printer and UV curing acrylic material.
  • the blue LED is temporarily programmable using the PC control board to achieve precise activation and termination of the FLASH response.
  • the control system consists of two related loops.
  • Circuit 1 includes an LED driver (output: 4V-5V, 600mA, direct current) connected to 110V alternating current, a 495nM wavelength LED connected to a relay (model: SRD-05VDC-SL-C) as a switch for irradiating the LED.
  • the voltage common collector (VCC) of the relay is connected to the chicken 5V pin (purple wire), and the input circuit of the relay is connected to the chicken ground (GND) pin (yellow wire).
  • Circuit 2 uses a red LED as an indicator to connect to the chicken ground pin (black wire) and 5v chicken digital pin 4 (blue wire) through a resistor (165 ⁇ ) and connect to the relay ground pin (green wire).
  • a tactile switch is also connected to the red LED light through the black wire, and connected to the 5v chicken digital pin 8 (red wire) through another resistor (165 ⁇ ).
  • the red LED loop is set to open. Since this loop is also connected to the relay GND, the relay in circuit 1 is closed, so both VCC and GND have a 5V power supply.
  • the analysis function of the reader uses 164bp synthetic standard DNA as the evaluation standard, corresponding analysis of the ⁇ -tubulin gene fragment containing 200 codons, identification of effective genetic markers of TT and detection of drug resistance.
  • a portable thermal cycler mini PCR TM mini8 was used to successfully amplify synthetic DNA standards of different concentrations from 10aM to 100fM, and a FLASH reader was used for detection ( Figure 21c, top).
  • the standard curve established by the FLASH reader (1min irradiation) is very similar to the standard curve obtained by the standard qPCR method ( Figure 21c, bottom figure and Figure 23c), which confirms that the reader of the present invention can perform quantitative nucleic acid detection, and the results are similar to those obtained by the standard qPCR method.
  • the present invention collected and tested clinical STH worm samples excreted from school-age children who had received deworming treatment (Figure 21d). It was confirmed by microscope that 5 trichocarpus and 2 roundworms were isolated from the fecal specimen. Genomic DNA was extracted on site in Honduras, using a standard magnetic separation protocol, and then transferred to Canada for analysis. A pair of Trichuris trichocarpa-specific primers were used to amplify the 164bp ⁇ -tubulin gene fragment, and the FLASH reader and electrophoresis were used for analysis. The test results using the FLASH reader are identical to those confirmed under the microscope and electrophoresis analysis ( Figures 21d and 24), showing the potential of FLASH technology in field disease diagnosis and monitoring.
  • LAMP LAMP reaction system (25 ⁇ L) including 1x LAMP Kit, 1.6 ⁇ M FIP (SEQ ID NO.16: 5'-GTTGGGGACT GCGAATTTTG GCTTTTTAGA CTCGTGGTGG ACTTCT-3') and BIP (SEQ ID NO.17: 5'-TCACTCACCAA CCTCTTGTCC TTTTTAAAAC GCCGCAGACA CAT-3') primers, 0.4 ⁇ M LF (SEQ ID NO.18: 5'-GGTAGTTCCC CCTAGAAA ATTGAG-3') and LB (SEQ ID NO.19: 5'-AATTTGTCC TGGTTAT CGCTGG-3') primers, 0.2 ⁇ M of F3 (SEQ ID NO.20 : 5'-TCCTCACAATA CCGCAGAGT-3') and B3 (SEQ ID NO.21: 5'-GCAGCAGGATG AAGAGGAAT-3') primers, and different concentrations of HBV vector or human serum specimens were
  • the present invention implemented FLASH LAMP on two portable detection platforms, including a FLASH reader and a paper-based FLASH tape.
  • the present invention performs 30-minute LAMP amplification detection on HBV amplicons in undiluted human serum samples. After 5 minutes of color development in the reader, the concentration of HBV positive serum samples ranged from 10aM to 100fM, which can be visually identified from the negative serum control ( Figure 25b). The real-time monitoring of the color rendering can be quantified from 10aM to 1fM ( Figure 25b).
  • a paper-based FLASH tape was introduced to further eliminate the need for instruments using FLASH technology (Figure 25c and Figure 25d).
  • the paper tape is made by wax printing process, and the hydrophobic wax barrier forms a clear circular sample loading area and linear test area (Figure 27a). Then deposit a thin layer of TMB evenly on the color test area ( Figure 5c).
  • the FLASH tape is designed to include a circular sample loading area (6mm inner diameter) and a linear test area (2.0mm width and 36mm length).
  • the production method is: first print the designed pattern on the XEROX ColorQube 8580 solid ink printer. On cellulose chromatograph paper, then heat at 150°C for 40s. Then superimpose patterned paper and a layer of paraffin film on the microscope slide to make a strip.
  • TMB is coated on the test area of the strip. Before coating, TMB is first dissolved in acetonitrile, and then deposited on the cellulose paper by fast solvent evaporation.
  • the working principle of the FLASH strip is that in the previous research of the present invention, it was found that SG-I will be tightly retained in the sample loading area, and can only be eluted to the test area in the presence of dsDNA. Therefore, a quantitative relationship can be established between the migration distance of SG-I and the concentration of dsDNA ( Figure 27b).
  • the reading of this distance-based DNA test strip was fluorescent, so a UV lamp was needed to facilitate visual inspection 29 ( Figure 28).
  • the present invention can convert the inconvenient fluorescence reading system into a permanent blue color. Amplicons generated by PCR or LAMP can be visually and quantitatively detected by measuring the migration distance (dM) ( Figure 28).

Landscapes

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

Abstract

L'invention concerne une plate-forme technologique pour la détection et l'analyse quantitative d'acides nucléiques qui utilise des colorants intercalant l'ADN (DID) pour directement accomplir une colorimétrie visuelle. Sur la base de la découverte de propriétés photochimiques et photocatalytiques des DID, une technologie de développement rapide de couleur de substrat activé par la lumière (FLASH) est proposée, ce qui permet à des acides nucléiques d'être quantifiés selon des résultats de développement de couleur. La technologie FLASH peut être combinée avec une amplification en chaîne par polymérase (PCR), une amplification isotherme médiée par boucle (LAMP) et d'autres moyens de détection d'acides nucléiques pour réaliser une analyse quantitative d'acides nucléiques. Afin de réaliser une détection FLASH à haut débit, un dispositif de réseau FLASH de type réseau dédié à la détection d'acides nucléiques est conçu et proposé, et ledit dispositif utilise un lecteur Flash pour une détection. Sur la base d'un système FLASH, une bande FLASH à base de papier est en outre conçue et proposée pour une portabilité, et doit seulement être irradiée avec un faisceau de lumière pendant quelques secondes pour réaliser une détection quantitative d'acides nucléiques au moyen de lectures colorimétriques.
PCT/CN2019/118033 2019-11-13 2019-11-13 Procédé et dispositif de détection d'acide nucléique WO2021092795A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201980100017.5A CN114341627A (zh) 2019-11-13 2019-11-13 核酸检测方法及装置
PCT/CN2019/118033 WO2021092795A1 (fr) 2019-11-13 2019-11-13 Procédé et dispositif de détection d'acide nucléique

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/118033 WO2021092795A1 (fr) 2019-11-13 2019-11-13 Procédé et dispositif de détection d'acide nucléique

Publications (1)

Publication Number Publication Date
WO2021092795A1 true WO2021092795A1 (fr) 2021-05-20

Family

ID=75911577

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/118033 WO2021092795A1 (fr) 2019-11-13 2019-11-13 Procédé et dispositif de détection d'acide nucléique

Country Status (2)

Country Link
CN (1) CN114341627A (fr)
WO (1) WO2021092795A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113583467A (zh) * 2021-08-20 2021-11-02 四川大学 一种核酸荧光染料及其制备和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009111072A1 (fr) * 2008-03-05 2009-09-11 Clean Earth Technologies, Llc Dosage par cytométrie en flux avec des billes à marquage pna et avec couplage pcr multiplexé pour la détection simultanée de plusieurs agents biologiques
US20100221703A1 (en) * 2005-06-03 2010-09-02 Blind Pig Proteomics, Inc. Chemiluminescence proximity nucleic acid assay
WO2011026182A1 (fr) * 2009-09-02 2011-03-10 Accugen Pty Ltd Procédé perfectionné de quantification d'acides nucléiques

Family Cites Families (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3189000B2 (ja) * 1994-12-01 2001-07-16 東ソー株式会社 特定核酸配列の検出方法
KR100538669B1 (ko) * 1996-01-23 2005-12-26 퀴아진 지노믹스, 인코포레이티드 리간드 쌍의 결합 검출에 사용되는 화합물, 이를 포함하는조성물 및 키트
CN1464071A (zh) * 2002-06-27 2003-12-31 赵翀 镶嵌式高通量基因芯片检测技术及试剂盒
DE10316159A1 (de) * 2003-04-09 2004-10-28 Klapproth, Holger, Dr. 5`-3`-Exonuklease Assay zur Bestimmung der Konzentration von Nukleinsäuren
CN100400667C (zh) * 2006-06-27 2008-07-09 浙江大学 脂质体包裹弓形虫pGRA4真核表达质粒的方法及用途
CN101631796B (zh) * 2007-03-09 2013-08-07 独立行政法人理化学研究所 具有由单核苷或单核苷酸衍生的结构的化合物、核酸、标记物以及核酸检测方法和试剂盒
ES2399318T3 (es) * 2008-10-29 2013-03-27 Check-Points Holding B.V. Método para la detección e identificación rápidas de microorganismos
EP2566979B1 (fr) * 2010-05-05 2015-02-25 Check-Points Holding B.V. Dosages, compositions et méthodes de détection de micro-organismes pharmacorésistants
WO2011138402A1 (fr) * 2010-05-05 2011-11-10 Check-Points Holding B.V. Dosages, compositions et méthodes de détection de micro-organismes pharmacorésistants
US20120064528A1 (en) * 2010-06-01 2012-03-15 The Macfarlane Burnet Institute For Medical Research And Public Health Ltd Methods and reagents for quantifying nucleic acid fragmentation and apoptosis (qlm-pcr, cell number qpcr and apoqpcr)
WO2012014778A1 (fr) * 2010-07-26 2012-02-02 オリンパス株式会社 Procédé de détection de particules diluées dans une solution en utilisant une sonde luminescente
KR101572682B1 (ko) * 2013-05-30 2015-11-30 케이맥(주) 바이오 물질의 실시간 정량 및 정성 분석 방법
CN104278079A (zh) * 2013-07-08 2015-01-14 嘉兴朝云帆生物科技有限公司 用核酸层析技术检测核酸的试纸条和方法
EP3224383B1 (fr) * 2014-11-25 2020-02-12 Quest Diagnostics Investments Incorporated Amplification et détection d'acides nucléiques dans un échantillon biologique
JP6766804B2 (ja) * 2015-04-07 2020-10-14 コニカミノルタ株式会社 核酸プローブ
CN105136786A (zh) * 2015-08-04 2015-12-09 南京邮电大学 一种利用金纳米颗粒和硫黄素t检测富g核酸序列的方法
CN107024583B (zh) * 2016-01-29 2019-03-08 中国科学院苏州纳米技术与纳米仿生研究所 硫黄素t适配体、其筛选方法及应用
CN106119343B (zh) * 2016-06-20 2019-05-24 南昌大学 一种基于Hg2+辅助及双聚合酶等温核酸扩增技术的microRNA检测方法
CN107523611A (zh) * 2016-06-22 2017-12-29 南京大学 一种非扩增型核酸杂交捕获体系及其在核酸检测中的应用
CN106755593B (zh) * 2017-02-21 2020-08-14 北京易活生物科技有限公司 一种hpv分型检测的核酸组合及其应用和试剂盒
CN106399536B (zh) * 2016-10-24 2019-01-29 江苏科德生物医药科技有限公司 体液循环dna定量检测方法及试剂盒
CN107384769B (zh) * 2017-08-24 2021-04-09 李峰 基于纸的核酸检测装置及其应用
CN109628557A (zh) * 2019-01-07 2019-04-16 济南大学 双重信号增强纸基生物传感器在miRNA检测中的应用
CN110241180A (zh) * 2019-06-05 2019-09-17 成都理工大学 一种基于dsDNA-SYBR Green I光敏催化的化学发光传感方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100221703A1 (en) * 2005-06-03 2010-09-02 Blind Pig Proteomics, Inc. Chemiluminescence proximity nucleic acid assay
WO2009111072A1 (fr) * 2008-03-05 2009-09-11 Clean Earth Technologies, Llc Dosage par cytométrie en flux avec des billes à marquage pna et avec couplage pcr multiplexé pour la détection simultanée de plusieurs agents biologiques
WO2011026182A1 (fr) * 2009-09-02 2011-03-10 Accugen Pty Ltd Procédé perfectionné de quantification d'acides nucléiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DONG TIANYU: "Development of Field-deployable Nucleic Acid Testing Platforms", MASTER'S THESIS, FACULTY OF MATHEMATICS AND SCIENCE, BROCK UNIVERSITY, 1 January 2019 (2019-01-01), XP055812207 *
DONG TIANYU; WANG GUAN A.; LI FENG: "Shaping up field-deployable nucleic acid testing using microfluidic paper-based analytical devices", ANALYTICAL AND BIOANALYTICAL CHEMISTRY, SPRINGER BERLIN HEIDELBERG, DE, vol. 411, no. 19, 1 February 2019 (2019-02-01), DE, pages 4401 - 4414, XP036827227, ISSN: 1618-2642, DOI: 10.1007/s00216-019-01595-7 *
LIANG CHANGNENG, ZHANG JIE, XU WEN, DING LAN, QIAN WEI, LI XUAN: "Photoluminescence Properties of Single-Stranded Nucleic Acids with Different Base Sequences", LASER & OPTOELECTRONICS PROGRESS, no. 3, 1 January 2018 (2018-01-01), pages 454 - 459, XP055812212, DOI: 10.3788/LOP55.033004 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113583467A (zh) * 2021-08-20 2021-11-02 四川大学 一种核酸荧光染料及其制备和应用

Also Published As

Publication number Publication date
CN114341627A (zh) 2022-04-12

Similar Documents

Publication Publication Date Title
Li et al. A comprehensive summary of a decade development of the recombinase polymerase amplification
ES2639938T3 (es) Métodos y composiciones para la secuenciación de ácidos nucleicos
ES2689676T3 (es) Procedimiento para la detección de modificaciones de ADN por manipulación de una sola molécula
JP2021503969A (ja) 出現（ｅｍｅｒｇｅｎｃｅ）による核酸のシーケンシング
ATE450623T1 (de) Modifikatoren der lichtemission und ihre verwendungen zur detektion, amplifikation und analyse von nukleinsäuren
HRP20100414T1 (hr) Postupak detekcije mutacija egfr u uzorcima krvi
US20200123594A1 (en) Methods and devices for sequencing
TWI622580B (zh) 多次甲基化合物及其作爲螢光標記之用途
US20230159986A1 (en) Methods for detecting and sequencing a target nucleic acid
WO2021092795A1 (fr) Procédé et dispositif de détection d'acide nucléique
CN114929891A (zh) 样品制备系统和方法
CN114929889A (zh) 使用盒进行测序的方法和装置
US20200362397A1 (en) Oligonucleotide probe array with electronic detection system
Zhang et al. A rapid label-and enzyme-free G-quadruplex-based fluorescence strategy for highly-sensitive detection of HIV DNA
CA2811294C (fr) Stabilisation de colorants fluorescents labiles a l'ozone par une thiouree
ES2371664T3 (es) Ensayo de ácidos nucleicos de proximidad de quimioluminiscencia.
ES2338662T3 (es) Pcr en tiempo real con adicion de pirofosfatasa.
KR20190011706A (ko) 핵산 복합체 페어, 핵산 복합체 페어를 포함하는 pcr용 키트, 및 핵산 복합체 페어를 이용한 타겟 검출 방법
JP5206047B2 (ja) 核酸解析方法
Chen et al. DNA sequencing by denaturation: experimental proof of concept with an integrated fluidic device
Weng et al. Development of Detection Equipment for a Polymerase Chain Reaction with a Loop‐Mediated Isothermal Amplification Reaction
JP4320188B2 (ja) 一塩基置換検出方法及び一塩基置換検出用キット
AU2015224467B2 (en) Stabilization of ozone-labile fluorescent dyes by thiourea
JPWO2017158840A1 (ja) 試料中の複数種類の短鎖核酸の検出方法、組み合わせ分析キットおよび分析キット供給管理方法
Chen Nucleic Acid-Based Detection of Small Molecules and Biomolecules Using Time-Resolved Luminescent Spectroscopy With Iridium (III) Metal Complexes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19952372

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19952372

Country of ref document: EP

Kind code of ref document: A1