WO2021092795A1 - Nucleic acid detection method and device - Google Patents

Abstract

A platform technology for nucleic acid detection and quantitative analysis that uses DNA intercalating dyes (DIDs) to directly achieve visual colorimetry. On the basis of the discovery of photochemical and photocatalytic properties of DIDs, a fast light-activated substrate color development (FLASH) technology is proposed, thereby enabling nucleic acids to be quantified according to color development results. The FLASH technology may be combined with PCR, LAMP and other nucleic acid detection means to perform quantitative analysis of nucleic acids. In order to perform high-throughput FLASH detection, an array-type FLASH array device dedicated for nucleic acid detection is designed and proposed, and said device uses a Flash reader for detection. On the basis of a FLASH system, a paper-based FLASH tape is further designed and proposed for portability, and only needs to be irradiated with a light beam for a few seconds to perform quantitative nucleic acid detection by means of colorimetric readings.

Description

Nucleic acid detection method and device Technical field

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.

Background technique

Nucleic acid testing (NATs) 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), requires a large number of samples and expensive detection equipment. Therefore, nucleic acid detection is mainly carried out in clinical laboratories. Converting standard nucleic acid detection into real-time detection will help doctors directly perform rapid diagnosis in the ward or consulting room. In low- and middle-income countries, 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. According to estimates by the World Health Organization (WHO), more than half of the deaths in low-income countries in 2016 were caused by lower respiratory tract infections, human immunodeficiency virus (HIV) and malaria and other infectious diseases. At present, more than 1.5 billion people are often ignored Affected by tropical diseases, such as soil-borne nematode infection (STH). Although researchers in recent years have tried to combine standardized nucleic acid detection methods with advanced methods such as microfluidics, nanotechnology, and synthetic biology, how to quickly and widely utilize these technological innovations is still challenging. Standardized nucleic acid detection methods may increase the upfront cost for professional knowledge, infrastructure, reagents, and complex operating steps. At the same time, a lot of work needs to be done to ensure that standardized methods can be continuously and stably implemented across different laboratories and platforms. Many detection platforms only perform qualitative detection and have low sensitivity, such as lateral flow equipment. The detection methods limit their accuracy and quantitative analysis capabilities. Therefore, there is still an urgent need to develop a nucleic acid detection method that can be carried out in real time on the spot. This method not only requires fast detection speed, high detection sensitivity, and quantitative measurement, but also considers how to reduce the technical obstacles encountered in rapid application.

The new demand for implementation of standard nucleic acid testing in a laboratory with concentrated equipment to an environment with limited resources has promoted many technological innovations in portable device manufacturing, microfluidics, nanotechnology, and synthetic biology. Commercial, portable, smart phone-controllable thermal cyclers can effectively replace large-scale laboratory-grade nucleic acid amplification equipment. Recently, TINY (TINY Isothermal Nucleic Acid Quantitative System) technology was introduced to perform LAMP detection through multiple energy sources without the need for electricity. Based on the breakthrough of paper-based microfluidic technology, there are already fully integrated devices that can simplify nucleic acid extraction, amplification, and detection in a page of paper "machine" or origami. Although progress has been made in equipment and equipment manufacturing, there is still an urgent need for a visual and sensitive quantitative sensing detection system suitable for on-site nucleic acid detection. The qualitative analysis of plasma nanoparticles and lateral flow bands enhances the performance of advanced quantitative nucleic acid analysis and detection methods, such as the nucleic acid detection platform SHERLOCK (specific high sensitivity enzymatic diagnosis tool) based on CRISPR cas. Recently, paper-based synthetic gene networks can achieve visualization, quantification and sequence-specific detection of viral nucleic acids. However, the implementation of this strategy is challenged by expertise and lengthy programs that require the design, screening, and freeze-drying of toehold switch sensors and synthetic gene networks on paper.

Summary of the invention

In order to overcome the obstacles in current technological innovation and application, the present invention reinvents a standard nucleic acid detection technology, which focuses on a widely accepted and applicable detection probe-a dye probe capable of inserting DNA that has not yet been studied. (DIDs). 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. In this article, 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. Kind of relaxation pathway. Because this relaxation process produces singlet oxygen, 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 . Based on the above principles, 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. In order to supplement the adaptability of laboratory-level and field applications, 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.

In one aspect, 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.

Furthermore, in the application of the present invention, 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.

Further, in the application of the present invention, the nucleic acid intercalating dyes (DIDs) include SYBR Green 1 (SG-I), PicoGreen (PG), thioflavonoid T (ThT), thiazole orange (TO), and the like.

Furthermore, in the application of the present invention, the chromogenic substrates include 3,3,5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD) and the like.

In the second aspect, the present invention provides the application of nucleic acid intercalating dyes (DIDs) as chromogens or photosensitizers. 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.

Further, in the application of the present invention, the nucleic acid intercalating dyes (DIDs) include but are not limited to SYBR Green 1 (SG-I), PicoGreen (PG), thioflavonoid T (ThT), thiazole orange (TO), etc. .

Furthermore, in the application of the present invention, the chromogenic substrates include 3,3,5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD) and the like.

In the third aspect, the present invention provides a nucleic acid detection method, including the following steps:

(1) Provide double-stranded nucleic acid;

(2) Add nucleic acid intercalating dyes (DIDs) and chromogenic substrates to double-stranded nucleic acids;

(3) The color of the substrate is developed by irradiation with excitation light.

Furthermore, 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.

Furthermore, 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.

Further, the method of the present invention, wherein the step (1) obtains double-stranded nucleic acid by PCR, LAMP or RPA (recombinase polymerase amplification).

Further, in the method of the present invention, in the step (3), the excitation light with a wavelength of 480-495 nm is irradiated for more than 5 seconds.

Further, in the method of the present invention, when step (2) uses TMB as a color developing substrate, in step (4), a wavelength of 650 nm is selected for spectrophotometric detection.

In a fourth aspect, 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.

Further, in the FLASH reader of the present invention, 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. Corresponding to the holes on the porous microporous plate, it is preferable that the light emitting diode generates high-intensity blue light.

Further, in the FLASH reader of the present invention, 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. Preferably, the transmitted light source is white light.

In a fifth aspect, 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. Layer or deposition layer, the paper layer is cellulose paper.

Further, 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.

In a sixth aspect, the present invention provides a method for detecting nucleic acid using a paper-based FLASH detection tape, the method comprising:

(1) Obtain double-stranded nucleic acid;

(2) Mix the double-stranded nucleic acid with the nucleic acid insertion dye;

(3) Loading the mixed product of step (2) to the sample loading area of the paper-based FLASH detection tape of claim 15 or 16;

(4) Light-driven color rendering;

(5) Read the test result.

Further, in the method for detecting nucleic acid using a paper-based FLASH detection band according to the present invention, the double-stranded nucleic acid is a PCR product or a LAMP product.

Further, 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.

Further, 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.

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.

First of all, 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. 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.

Secondly, compared to other detection technology developers, such as plasma nanoparticles and lateral flow strip technology, quantitative, high sensitivity and stability are the biggest features of FLASH. Through comparison with quantitative dsDNA fluorescence analysis methods and direct comparison with commercial qPCR reagents and equipment used in different samples, the quantitative accuracy and stability of FLASH are verified. More importantly, unlike signal readout using additional enzyme amplification and/or DNA hybridization, the high sensitivity of FLASH is given by the chemical properties of nucleic acid dyes, so it does not affect the simplicity and speed of detection , To complete the test in a mixed-reading manner.

Third, FLASH technology also has high chemical diversity. The present invention found that the photocatalytic performance is not limited to SG-I, and other intercalating dyes such as PicoGreen (PG) can also produce colorimetric readings on dsDNA. Dyes that can make tertiary DNA structures emit light, such as G-quadruplex and i-motif, can also mediate FLASH reactions. 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.

Although 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. To achieve this goal, 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. Finally, through the detection performance highlighted in this article, 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.

Description of the drawings

Figure 1 Photophysical characteristics of SG-I and DNA insertion

a. Schematic diagram of the interaction between SG-I and dsDNA. b. Fluorescence (bottom) and phosphorescence (top) enhance SG-I (1μM) (λex=480nm) in 5nM dsDNA. c. Phosphorescence decay (τ = 4.97 ms) of SG-I (1 μM) when dsDNA (5 nM) is present. 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 θ. e. The potential energy function of the dihedral angle θ 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.

Figure 2 Theoretical calculation and photochemical characterization of SYBR Green I

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. [SG-I]=4μM in 1×PBS buffer (pH=7.4). Consistent with the previous report, 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).

Figure 3 Phosphorescence enhancement is also quantitatively dependent on dsDNA concentration

(a) The 2D evolution over time emission spectrum of a mixture containing 4 μM SG-I and 1 μM herring sperm dsDNA (hsDNA). The excitation wavelength is fixed at 480nm. (b) The relationship between SG-I phosphorescence and dsDNA concentration at room temperature. The results show that the insertion of dsDNA in an oxygen-free environment can also enhance the phosphorescence of SG-I. Similar to fluorescence enhancement, phosphorescence enhancement is also quantitatively dependent on dsDNA concentration.

Figure 4 Restricting the rotation of the bond can enhance fluorescence and phosphorescence

(a) SG-I HOMO (highest occupied molecular orbital)/LUMO (lowest unoccupied molecular orbital) distribution diagram (calculated at the B3LYP/6-31G(d) level). HOMO and LUMO are mainly distributed on 1,4-dihydroquinoline and benzothiazolyl, respectively. (b,c) When the temperature drops from 298k to 77K, the fluorescence spectrum (b) and luminescence duration (c) of free SG-I. The detection system is SG-I (4μM) dissolved in 2-MeTHF (melting point: 136k). (d,e) Phosphorescence spectra (d) and luminescence duration (e) at different temperatures. (f,g) Fluorescence spectrum (f) and luminescence duration (e) of free SG-I in 100% glycerol (red) and 0% glycerol (blue) solutions. The present invention observes that by freezing or increasing the viscosity of the medium to restrict the rotation of the bond, the fluorescence and phosphorescence can be enhanced, which may be due to the distortion of the intramolecular charge transfer (TICT) inhibiting the fluorescence of SG-I and the intersystem crossing (ISC) .

Figure 5 Theoretical estimation of the energy barrier between the singlet state S1 and the triplet excited state T1 with a double-sided angle θ of 0°

In theory, ΔEST is estimated to be 0.411 eV, which is very consistent with experimental observations, where ΔEST is estimated to be 0.378 eV.

Figure 6 DNA-specific light-driven color development mediated SG-I

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 4μM SG-I, 200 mg/ml TMB, 100 nM dsDNA (ssDNA) solution system. d. The λDNA standard concentration of the standard curve ranges from 1pg/μL to 500ng/μL, which are FLASH (blue), QuantiFluor dsDNA quantitative kit (red), and A260 (green). In order to facilitate quantitative comparison, 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.

Figure 7 Photochemical characteristics of SG-I

The photooxidation of TMB using the SG-I-dsDNA complex is mediated _{by the generation of singlet oxygen 1O 2.} Therefore, SG-I-dsDNA complex is a type II photosensitizer. (a) Photooxidation is only observed when oxygen is dissolved in the solution. (b) The effect of different scavengers on TMB photooxidation. Only the 1O2 scavenger tryptophan was found to inhibit the reaction, indicating that 1O2 plays a key role in SG-I-mediated photooxidation. [dsDNA]=100nM, [SG-I]=4μM; [TMB]=200mg/L, [mannitol]=2.5M; [SOD]=200U/mL, [catalase]=200U/mL,[ Tryptophan]=12M, the irradiation time is fixed at 1min.

Figure 8 Photochemical and photocatalytic performance of PicoGreen (PG)

(a) Another widely used dsDNA intercalating dye PicoGreen (PG). (b) Fluorescence spectra of 3μM PG with 5nM dsDNA and no dsDNA. (c) The phosphorescence of PG is enhanced after dsDNA insertion. (d) PG-dsDNA complex can also mediate the photosensitive oxidation of TMB.

Figure 9 Photochemical and photocatalytic characterization of thioflavonoid T (ThT)

(a) ThT is a highly efficient sensor that has been discovered recently. (b) ThT can selectively bind to DNA g-tetramer (g-4) and enhance fluorescence. (c) (Concentration) ThT binds to G-4 and the fluorescence enhancement of G-4 in the presence of (concentration) K+. (d) ThT fluorescence enhancement in the presence of G-4 and G-4/K+. (e) Through the colorless to blue transition, it is proved that the ThT/G4 binding complex can also mediate the photosensitive oxidation of TMB.

Figure 10 Photochemical and photocatalytic characterization of Thiazole Orange (TO) Thioflavone T (ThT)

a) The chemical structure of TO. (b) The specific binding of TO and i-motif is known to enhance fluorescence. (c) Under acidic (pH=4.5) and neutral (pH=7.5) conditions, the fluorescence of (concentration) TO and (concentration) DNA i-motif is enhanced. A higher fluorescence enhancement is observed under acidic conditions, which is due to the favorable i-motif formation under acidic conditions. (d) At pH values of 4.5 and 7.5, the presence of i-motif enhances the phosphorescence of TO. (e) The combination between TO and i-motif also mediates the photocatalytic reaction, resulting in the transition from colorless to blue.

Figure 11 FLASH PCR

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. ***P<0.05, P<0.01, ***P<0.001, ****P<0.0001 two-tailed t test. Each error bar represents one standard deviation obtained from three parallel experiments. d. FLASH PCR is used for bacterial culture detection. Five colonies were isolated from Escherichia coli transformed with HBV 1.3-mer WT replicon plasmid. Three colonies were selected from the E. coli culture without transformation as a negative control. Perform FASH PCR directly on each colony without any purification steps. ****P<0.0001 adopts two-tailed t test. e. Directly use FLASH PCR method to detect HBV genomic DNA in undiluted human serum samples. ****P<0.0001 adopts two-tailed t test. Each error bar represents one standard deviation obtained from three parallel experiments.

Figure 12 Design and characterization of 96-channel FLASH array

Schematic (a) and photo (b) of the 96-way array. (c) Circuit design. (d) Emission spectra and SG-I absorbance spectra of different LEDs. (e) FLASH response mediated by different LED irradiation time of 1 min. [dsDNA]=[ssDNA]=100nM; [SG-I]=4.0μM; [TMB]=200mg/L; pH 4.5 citrate buffer. Each error bar represents a standard deviation obtained from three sets of parallel experiments.

Figure 13 The influence of SG-I concentration on FLASH PCR analysis performance

[HBV-S]=1pM, [primer]=125nM, PCR cycles=35, [TMB]=200mg/L. Each error bar represents a standard deviation obtained from three sets of parallel experiments.

Figure 14 The influence of TMB concentration on FLASH PCR analysis performance

[HBV-S]=1pM, [primer]=125nM, PCR cycles=35, [SG-I]=2μM. Each error bar represents a standard deviation obtained from three sets of parallel experiments.

Figure 15 The impact of the pre-concentration step on FLASH PCR

[HBV-S]=1pM, [primer]=125nM, PCR cycles=35, TMB=300mg/L, [SG-I]=2μM. Each error bar represents a standard deviation obtained from three sets of parallel experiments.

Figure 16 The influence of primer concentration on FLASH PCR

[HBV-S]=1pM, PCR cycles=35, TMB=300mg/L, [SG-I]=2μM. Each error bar represents a standard deviation obtained from three sets of parallel experiments.

Figure 17 The impact of the number of PCR cycles on FLASH PCR. [HBV-S]=1pM, [primer]=125nM, TMB=300mg/L, [SG-I]=2μM. Each error bar represents a standard deviation obtained from three sets of parallel experiments.

Figure 18 Using qPCR (a) and electrophoresis (b) to detect and analyze IL-6 gene expression

Figure 19 FLASH PCR is used for clone screening

Using FLASH method for colony PCR screening, and visual screening (a) and traditional electrophoresis detection (b). 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 DH5α.

Figure 20 FLASH PCR used for serum HBV detection

Use conventional protocol (protocol-1) to analyze HBV genomic DNA in human serum samples, including total DNA extraction, and then perform PCR (b) and direct PCR protocol (protocol-2), in which genomic HBV DNA is amplified by direct PCR kit It is then purified by PCR purification step (c).

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 22 FLASH reader

Schematic diagram of the design of the housing (a) and circuit (b).

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]=2μM, [TMB]=300mg/L.

Figure 24 Application of PCR and PAGE to analyze clinical parasite samples

Figure 25 FLASH LAMP

a. Schematic diagram of LAMP principle b. 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.

Figure 26 Application of FLASH LAMP to detect HBV

Visual detection of HBV genomic DNA in human serum with FLASH LAMP (a), spectral analysis (b) and PAGE analysis (c) comparison. 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.

Figure 27 Paper-based FLASH tape

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. As a result, 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. Once eluted to the test area coated with TMB, a FLASH reaction will occur, and blue stripes will be formed under light irradiation.

Figure 28 Use of paper-based FLASH tape for visual and quantitative detection

(a) Schematic diagram of the distance-based HBV detection workflow in undiluted human serum samples using direct PCR and FLASH strips. (b) Distance development and observation under ultraviolet light. (c) Use FLASH reaction to develop color. The 1fM HBV vector was added to the serum of healthy blood donors to prepare HBV+ human serum samples. Each sample was subjected to 35 PCR cycles, and then purified with a fast PCR cleanup column. Load the sample on the FLASH belt for distance development (10 minutes) and color development (1 minute).

Detailed ways

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. In this article, 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. The present invention found that after the dye is inserted into the DNA, the fluorescence coloring effect is not only enhanced, but also the crossover between systems is promoted, which is a relaxation pathway for generating singlet oxygen and subsequent photosensitive oxidation. Based on this photocatalytic property, 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. By integrating two new components, a portable electronic FLASH reader and a paper-based FLASH strip, the present invention further confirms the adaptability of FLASH field detection.

The technical solution of the present invention will be described in further detail below through the embodiments. The present invention is not limited to the specific methods and solutions described herein, as they can be changed. In addition, the terms used herein are only used for the purpose of describing specific embodiments, not for limiting the scope of the present invention.

Unless there are different definitions, all technical and scientific terms and any abbreviations herein have the same meanings as commonly understood by those of ordinary skill in the art of the present invention. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, illustrative methods, devices, and materials are described herein.

Unless otherwise specified, the terms of the present invention have the meanings commonly used in the art.

Reagents and materials

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). 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×) and 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 (>18.0MΩ), 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.

Example 1 Photophysical properties of nucleic acid dyes

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), exhibit strong fluorescence enhancement after binding to adjacent double-stranded DNA (dsDNA) base pairs (Figure 1a). For example, when SG-I (4μM) is mixed with different concentrations of dsDNA, the present invention observes significant fluorescence enhancement and prolonged fluorescence emission time (Figure 1b and Figure 2). In addition to fluorescence, 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 (4μM) with or without hsDNA (1μM) was incubated for 10 minutes. The measured absorbance of hsDNA at 260nm is the molar absorbance coefficient ε260=6600Lmol-1cm-1. The absorbance spectrum was measured with a UV-1750 spectrophotometer (Shimadzu, Japan). Fluorolog-3 fluorescence spectrometer (Horiba Jobin Yvon) was used to measure the fluorescence (FL)/phosphorescence (Phos) spectrum and luminescence duration. The excitation wavelength is 485nm. The phosphorescence of the solution is measured in an anaerobic test tube. 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).

Using density functional theory (DFT) to perform geometric optimization calculations on SYBR Green I (SG-I), and establish its electronic ground state model. Establish the initial structure model of SG-I in the graphical perspective (Gaussian, Inc.). Based on the supercomputing service of Suzhou High-end Materials High Performance Computing Co., Ltd., DFT geometry optimization was carried out in the Gaussi-09 software package. All DFT calculations use B3LYP density functional functions, 6-31G basis functions and strict SCF convergence criteria. The calculation of vibration frequency shows that after SG-I optimization, the potential energy surface reaches a local minimum. These calculations provide a starting point for the study of the SG-I ground state electronic structure as a function of rotation angle. Using the same density functional function and basis function mentioned above, through a series of geometric optimizations, a DFT model with SG-I as the turning angle function is established. For each flexible potential energy scan, the dihedral angle of the molecular rotor remains unchanged, while other bond lengths and bond angles have sufficient room for change. In SG-I, 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. For the TDDFT calculation of SG-I as the rotor angle function, the same density functional function and basis function as above are used. In addition, 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.

Density functional theory (DFT) and time-dependent DFT calculation methods further support the above experimental results. From the structural point of view, 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, θ (Figure 1d), is the lowest energy configuration of the ground state and excited state of SG-I at 0° and 90°, respectively (Figure 1e) . When SG-I is in the most stable ground state (S0, θ=0°), it is activated by S1 light irradiation. During the release process, a free bond in SG-I is rotated to θ=90°(S'1, the most stable Excited state), intramolecular torsional charge transfer (TICT) will occur. When the energy gap between S’1 and S’0 is the smallest (Figure 1e), θ=90° (S’1→S’0), the relaxation process is the fastest. Therefore, the excited state duration of SG-I is very short. By studying the fluorescence and phosphorescence characteristics of SG-I at ultra-low temperature and high viscosity, the key role of bond rotation was verified (Figure 4). The observations of the present invention indicate that phosphorescence is static when SG-I is free in solution. However, in the presence of dsDNA, the bond rotation of the excited state is hindered by the insert, resulting in the extension of the duration of the excited state S1 (Figure 1f). Therefore, ISC (S1→T1) becomes a feasible way of relaxation (Figure 5).

Example 2 Utilizing the optical properties of nucleic acid dyes for nucleic acid detection

Although phosphorescence detection is not an ideal indicator in nucleic acid analysis, 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). When a chromogenic substrate is used, a visual colorimetric reading can be generated. In order to verify this hypothesis, 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 (4μM) as the photosensitizer, TMB (200mg/L) as the substrate, and dsDNA as the target (100nM). Irradiated under blue light for 30 seconds (3W LED, λ=490nm), a strong colorless to blue transition process observed in the present invention indicates that photosensitive oxidation produces an oxidized TMB product (oxTMB) (Figure 6c). More importantly, this light-driven color reaction has a high degree of dsDNA specificity and sensitivity. By plotting the absorbance function of the dsDNA concentration at 650nm and comparing it with the commercial fluorescent dye dsDNA quantification kit (QuantiFluor), the present invention finds that the colorimetric measurement and commercial kit detection method are compared with the method of detecting dsDNA absorbance at 260nm, and its detection limit (LOD) is 1000 times lower (Figure 6d). 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). In a word, 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.

In a classic FLASH analysis experiment, the reaction mixture (100μL) contains 20μl synthetic standard DNA, PCR or LAMP amplicons, 2μM SG-I and 300μg/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. When using a 96LED array for analysis, use a multi-mode microplate reader (SpectraMax i3, Molecular Devices) to measure the absorbance at 650 nm after irradiation every 5 seconds. Analyze the sample with a FLASH reader, and use the RGB color sensor to measure the colorimetric signal at the red channel in situ after each 5s of irradiation. The design and production of 96-column FLASH arrays, FLASH readers and FLASH strips are introduced in the supplementary materials.

Example 3 Establishment of FLASH PCR method

In order to use FLASH as a simple visual detection method for nucleic acid detection and quantification, it is important to evaluate its wide adaptability to standard nucleic acid amplification techniques (such as PCR). Therefore, the next step of the present invention is to integrate FLASH as an "additional component" step of PCR (Figure 11a). In order to facilitate rapid, high-throughput characterization of FLASH PCR, 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. In order to achieve flexible control of the array, 96 LED lights are grouped and controlled by 6 independent switches (Figure 12c). Use 96 light-emitting diodes to weld the circuit board to make a 96FLASH array (Figure 12). Choose a blue LED (3V) with a maximum wavelength of 495nm to maximize the photocatalytic activity of SG-I (Figure S4). Then 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. Then use double-sided silicone heat transfer tape to stack the LED board on the aluminum cooling block (heat sink).

In order to reflect the advantages of FLASH PCR, 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. 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). As expected, 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. But unlike qPCR, which requires expensive instruments to measure fluorescence, FLASH PCR is a colorimetric method, and the instrument requirements and costs are much lower.

Real-time quantitative PCR. Real-time qPCR 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 ΔCt method was used for qPCR data analysis.

Example 4 FLASH PCR is used to detect cytokine cDNA

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.

Cultivation and activation of mast cells

Isolate bone marrow from tibia and femur of wild-type C57BL/6 mice (Charles River), 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). For IgE-mediated activation, 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. Then the cells were separated at different time points (0, 15, 30, 60, 120 and 300 minutes), and 100ng/ml TNP-BSA (Biosearch Technologies, Novato, CA) was used in 100ng/ml SCF (Peprotech, Dollard des Ormeaux, QC) Stimulate the cells under enhancement.

RNA isolation and cDNA preparation

2-3×10 ⁶ BMMCs were activated, flushed, and stimulated according to the above methods; RNA was isolated according to the manufacturer’s recommended manual using the RNeasy Plus kit (Qiagen, 74136). At each time point, the cells were centrifuged at 1500 rpm and 4°C for 5 minutes, after which the cells were lysed in 350 μL RLT Plus lysis buffer with 10 μL/mL 2-mercaptoethanol; RNA was eluted in RNase-free water and stored in- 80°C. Then EcoDry RNA to cDNA double-primed Reverse Transcriptase (Clontech) was used to prepare cDNA.

In summary, 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 wide application of FLASH PCR in the diagnosis of infectious diseases and the rapid screening of successful constructs after cloning (such as colony PCR) make the present invention face challenges in bacterial culture detection. In particular, 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. After culture, the colony containing the HBV vector was subjected to colony PCR, followed by FLASH detection, without any purification steps (Figure 11d). E. coli DH5α was used as a negative control (empty vector group). All HBV-positive colonies showed visible detection signals under 5s flash illumination, and HBV-negative colonies had the smallest signal, which was close to the negative control (Figure 11d & 19a). The present invention further verified this result using a standard colony PCR method, in which polyacrylamide gel electrophoresis (PAGE) was used for the final reading (Figure 18b). Both methods showed consistent results, but the FLASH test method reads simpler and faster.

Polyacrylamide gel electrophoresis. PCR or LAMP products were electrophoresed in 6% PAGE at 110v voltage. The gel was stained with ethidium bromide (0.5 μg/ml) and ^{detected with Gel Doc™} XR+Gel Documentation System (Biorad, Mississauga, ON, Canada).

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. Prepare HBV-containing human serum samples to simulate real clinical serum samples. Different concentrations of HBV vectors or synthetic genome fragments were added to human serum samples obtained from healthy donors. In a typical standard PCR test, according to the operation manual, the QIAamp DNA Blood Mini kit is used to extract total DNA from human serum samples containing HBV. Briefly, mix 200 μL of serum sample with 20 μL of protease and 200 μL of lysis buffer, and incubate at 56°C for 10 minutes. Then 200 μL 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 (6μL-50μL).

Standard PCR. Each 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. However, 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 2μL 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.

It can be determined that when FLASH is combined with direct PCR, the present invention found a higher HBV vector detection signal (1fM) in undiluted human serum samples (Figure 11e). The present invention also observes that the residual part of the serum sample after direct PCR inhibits the reading of FLASH, and adding a simple PCR cleaning step before the reading of FLASH can successfully solve this problem. It is worth noting that due to the increase in amplicons after direct PCR amplification, this new method has a higher recovery rate (over 80%, Figure 20c) and has a much smaller impact on detection sensitivity.

Example 7 FLASH PCR is used to detect parasitic infections in samples

In order to achieve the purpose of carrying out nucleic acid detection on site, the present invention transfers experiments to environmental conditions with limited resources. For example, 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. However, 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.

Parasite samples

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. The National Autonomous University of Honduras and the University of Brooke received ethical approval. 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.

All parasite samples, including Trichuirs trichiura (TT) and Ascaris lumbricoides (AL), were collected on-site in the rural La Hicaca area of Honduras, where the prevalence of STH in children is estimated to exceed 50%. In order to facilitate on-site detection, both PCR amplification and FLASH detection should be carried out on a portable platform. Since the controllable portable thermal cycle smart phone has been commercialized and popularized, the present invention develops a portable electronic reader (FLASH reader), which is an accessible and inexpensive supporting technology that provides a stable and quantitative measurement path.

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 Arduino 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 Arduino 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 Arduino 5V pin (purple wire), and the input circuit of the relay is connected to the Arduino ground (GND) pin (yellow wire). Circuit 2 uses a red LED as an indicator to connect to the Arduino ground pin (black wire) and 5v Arduino digital pin 4 (blue wire) through a resistor (165Ω) 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 Arduino digital pin 8 (red wire) through another resistor (165Ω). Before pressing the tact switch, 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. Once the tact switch is pressed, the loop through Arduino digital pin 8 to GND is set to open, and the voltage of digital pin 8 will drop from 5V to close to 0V. The Arduino board is pre-programmed, once the voltage drop of digital pin 8 is detected, the power of digital pin 4 will be turned off for 5 seconds. Since there is no power supply for digital pin 4, the GND of the relay drops to 0V, VCC remains at 5V, the red LED will be turned off, and the power relay will be turned on. Except for the electronics and optical system, all other components, including sample holders, LED brackets, mechanical components and housings, are made by 3D printing. It has been verified that the performance of the FLASH reader is equivalent to that of a commercial qPCR instrument and two commercial kits (SYBR Green Master Mix Bio-Rad and Luna Universal qPCR Master Mix, NEB).

The analysis function of the reader uses 164bp synthetic standard DNA as the evaluation standard, corresponding analysis of the β-tubulin gene fragment containing 200 codons, identification of effective genetic markers of TT and detection of drug resistance. A portable thermal cycler (mini PCR ^™ 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. Commercial qPCR systems are comparable. In order to finally verify the system of the present invention, 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 β-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.

Example 8 FLASH LAMP

Due to the low instrument requirements, various forms of isothermal nucleic acid amplification technology are ideal alternatives to PCR for nucleic acid detection. The integration of FLASH with this amplification system may result in a simpler and cheaper nucleic acid detection platform. Therefore, the present invention finally proves the adaptability of FLASH to loop-mediated isothermal amplification (LAMP), which is one of the most widely used isothermal nucleic acid amplification techniques. As a proof of principle, a set of 6 primers was designed to selectively amplify the 192bp HBV-s gene fragment at positions 1081-1272 on the HBV vector (Figure 25a).

LAMP. LAMP reaction system (25μL) including 1x

LAMP Kit, 1.6μM 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 μM LF (SEQ ID NO.18: 5'-GGTAGTTCCC CCTAGAAA ATTGAG-3') and LB (SEQ ID NO.19: 5'-AATTTGTCC TGGTTAT CGCTGG-3') primers, 0.2 μM 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 incubated at 65°C for 30 min.

Table 2. LAMP amplification target sequence

Since each LAMP reaction produces a large number of dsDNA amplicons in size and concentration, a strong color change was immediately observed after light-driven color development using the FLASH array for 5 seconds (Figure 26). In addition to high sensitivity, LAMP is also considered a stable amplification system, which is far less sensitive to interference than PCR. In fact, the present invention found that FLASH LAMP can be directly performed in undiluted human serum samples without any extraction or purification steps. More interestingly, FLASH LAMP induced a large amount of precipitation in HBV-positive serum samples, which significantly increased the colorimetric reading; while HBV-negative serum was still clear (Figure 26).

In order to facilitate on-site detection, the present invention then implemented FLASH LAMP on two portable detection platforms, including a FLASH reader and a paper-based FLASH tape. In order to prove the quantitative effect of the FLASH reader on LAMP amplification, 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).

Example application 9 FLASH-based microfluidic nucleic acid test strips

Furthermore, 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. Then, it was heated on a hot plate at 110°C for 30 seconds to bond the sandwich device. Finally, the 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). In the previous research of the present invention, the reading of this distance-based DNA test strip was fluorescent, so a UV lamp was needed to facilitate visual inspection 29 (Figure 28). Using the TMB-coated FLASH tape, 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). After 1 minute of light-driven color development, after 10 minutes of migration changes, the blue transfer to the test area can be observed, and a quantitative reading is provided to clearly distinguish the hepatitis B virus DNA (100aM, 100aM, 100aM) from the blank area (dM=12 mm). dM=29mm) without any additional reader (Figure 25d).

Those skilled in the art should understand that although the specific embodiments of the present invention are described herein for the purpose of illustration, various modifications can be made to them without departing from the spirit and scope of the present invention. Therefore, the specific embodiments and examples of the present invention should not be regarded as limiting the scope of the present invention. The present invention is only limited by the appended claims.

references

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Claims (20)

1. The application of nucleic acid intercalating dyes (DIDs) in chemiluminescence is characterized in that the chemiluminescence is the phosphorescence of nucleic acid intercalating dyes under excitation light.
2. The application according to claim 1, characterized in that the phosphorescence generated when nucleic acid intercalating dyes (DIDs) are inserted into nucleic acid is enhanced, and the phosphorescence enhancement depends quantitatively on the concentration of nucleic acid. Preferably, the nucleic acid is single-stranded, double-stranded, Or high-level structures formed by nucleic acids.
3. Nucleic acid intercalating dyes (DIDs) are used as chromogens or photosensitizers. The nucleic acid intercalating dyes photo-driven catalyzes the photosensitive oxidation of a chromogenic substrate in the presence of nucleic acid, and then produces a color change. Preferably, the nucleic acid It is single-stranded, double-stranded, or high-level structure formed by nucleic acid.
4. The application according to any one of claims 1-3, characterized in that the nucleic acid intercalating dyes (DIDs) include SYBR Green 1 (SG-I), PicoGreen (PG), thioflavonoid T (ThT), thiazole orange ( TO) etc.
5. The application according to claim 4, characterized in that the chromogenic substrate includes 3,3,5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD) and the like.
6. A nucleic acid detection method includes the following steps:

   (1) Provide nucleic acid, which is single-stranded, double-stranded, or high-level structure formed by nucleic acid;

   (2) Add nucleic acid intercalating dyes (DIDs) and chromogenic substrates to nucleic acids;

   (3) The color of the substrate is developed by irradiation with excitation light.
7. 8. The method according to claim 6, characterized in that it further comprises the optional step (4), selecting an appropriate wavelength for spectrophotometric detection according to the type of chromogenic substrate.
8. 8. The method according to claim 7, characterized in that it further comprises an optional step (5) of quantitatively analyzing the content or concentration of the nucleic acid based on the result of the color development of the substrate.
9. The method according to claims 6-8, characterized in that in step (1), the nucleic acid is obtained by PCR, LAMP or RPA.
10. The application according to claims 6-8, characterized in that in step (3), the excitation light is irradiated for more than 5s.
11. The application according to claim 6, characterized in that when TMB is used as the color substrate in step (2), the wavelength of 650 nm is selected for spectrophotometric detection in step (4).
12. A FLASH reader 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 switch between the irradiation module and the color sensor module.
13. The FLASH reader of claim 12, wherein the light irradiation module comprises 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 Corresponding to the holes on the porous microporous plate, it is preferable that the light-emitting diodes produce high-intensity blue light.
14. The FLASH reader according to claim 12, wherein the color sensor module comprises an RGB color sensor for detecting the absorbance value of the transmitted light source after passing through the test sample, preferably the transmitted light source is white light.
15. A paper-based FLASH detection belt 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 or a deposition layer. The paper layer is cellulose paper.
16. The paper-based FLASH detection tape according to claim 15, characterized in that it 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 device for measuring the migration distance Scale mark.
17. A method for detecting nucleic acid using a paper-based FLASH detection tape, the method comprising:

    (1) Obtain nucleic acid;

    (2) Mix the nucleic acid with the nucleic acid insertion dye;

    (3) Loading the mixed product of step (2) to the sample loading area of the paper-based FLASH detection tape of claim 15 or 16;

    (4) Light-driven color rendering;

    (5) Read the test result.
18. The method for detecting nucleic acid using a paper-based FLASH detection tape according to claim 17, wherein the nucleic acid is a PCR product, a LAMP product or an RPA product.
19. The method for detecting nucleic acid using a paper-based FLASH detection tape according to claim 17 or 18, characterized in that the step (5) quantitatively detects nucleic acid by visualizing the distance of color migration.
20. The method of claims 17-19, wherein the nucleic acid intercalating dyes (DIDs) include SYBR Green 1 (SG-I), PicoGreen (PG), thioflavonoid T (ThT), and thiazole orange (TO) Wait.

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