CN118166074A - Homogeneous nucleic acid detection box with three-mode signal response and application thereof - Google Patents

Abstract

The invention relates to a three-mode signal response homogeneous nucleic acid detection box and application thereof, belonging to the technical field of molecular biology. The invention provides a homogeneous nucleic acid detection kit, which comprises an amplification reagent and a detection reagent, wherein the amplification reagent is used for amplifying target nucleic acid to form an amplification product, the detection reagent comprises a photoelectric dual-mode biological probe and ribonuclease, the photoelectric dual-mode biological probe can be specifically combined with the amplification product, an electrochemical marker and a fluorescent marker are connected to the photoelectric dual-mode biological probe, and the ribonuclease can hydrolyze the photoelectric dual-mode biological probe to enable the photoelectric dual-mode biological probe to break, so that an electrochemical signal and a fluorescent signal are released. The homogeneous nucleic acid detection box integrates three common biosensor signal response modes of electrochemistry, fluorescence and colorimetry into a single reaction system or even a single report probe, has high signal response speed and simple structure, and can realize simple, sensitive, rapid, accurate and low-cost detection of target nucleic acid.

Description

A homogeneous nucleic acid detection kit with three-mode signal response and its application

Technical Field

The invention relates to a homogeneous nucleic acid detection box with three-mode signal response and application thereof, belonging to the technical field of molecular biology.

Background technique

In recent years, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technology has performed well in the rapid detection of viral nucleic acids, and its excellent sensitivity and accuracy have also been recognized. However, RT-qPCR usually requires complex fluorescence detection modules and temperature control systems, which ultimately makes the supporting equipment bulky and expensive, making it difficult to popularize in remote areas. On the contrary, electrochemical equipment is more suitable for the development of POCT (point-of-care testing) and application in remote areas due to its small size, simple structure and low cost. Therefore, the development of nucleic acid detection systems based on electrochemical signals has become a research hotspot.

In published studies, most nucleic acid detection systems based on electrochemical signals are immobilized or heterogeneous. Although such nucleic acid detection systems have excellent novelty and sensitivity, they have obvious shortcomings in the process of establishment or application. For example, the fixation and modification of reporter probes on the electrode surface is time-consuming and laborious. The steric hindrance and electrode surface closure brought by the immobilized probes affect the molecular diffusion rate and electron transfer rate, which greatly limits their application in POCT. The non-immobilized homogeneous nucleic acid detection system not only does not require the reporter probe to be fixed on the electrode surface, but also does not require additional rinsing during use. Therefore, it is urgent to find a non-immobilized homogeneous nucleic acid detection system based on electrochemical signals with high sensitivity, strong specificity and good signal-to-noise ratio.

Summary of the invention

To solve the above problems, the present invention provides a homogeneous nucleic acid detection kit with a three-mode signal response, wherein the homogeneous nucleic acid detection kit includes an amplification reagent and a detection reagent; the amplification reagent is used to amplify the target nucleic acid to form an amplification product; the detection reagent includes a photoelectric dual-mode biological probe and a ribonuclease (RNase); the photoelectric dual-mode biological probe can specifically bind to the amplification product, and the photoelectric dual-mode biological probe is connected to an electrochemical marker and a fluorescent marker; the ribonuclease can hydrolyze the photoelectric dual-mode biological probe, causing the photoelectric dual-mode biological probe to break, thereby releasing an electrochemical signal and a fluorescent signal.

In one embodiment of the present invention, the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is double-stranded DNA or single-stranded DNA; when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is double-stranded DNA, the detection reagent further comprises λ exonuclease and a detection reaction buffer; when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is single-stranded DNA, the detection reagent further comprises a detection reaction buffer.

In one embodiment of the present invention, the ribonuclease refers to a type of enzyme that only hydrolyzes the RNA chain in the DNA-RNA hybrid chain, but does not hydrolyze the DNA chain and the unhybridized RNA chain.

In one embodiment of the present invention, the ribonuclease comprises RNase H.

In one embodiment of the present invention, the photoelectric dual-mode biological probe is an RNA molecule with a length of 6 to 100 bp.

In one embodiment of the present invention, the electrochemical marker includes at least one of a methylene blue compound, a ferrocene compound or a daunomycin compound; the methylene blue compound includes at least one of methylene blue, a methylene blue derivative or a methylene blue modifier; the ferrocene compound includes at least one of ferrocene, a ferrocene derivative or a ferrocene modifier; the daunomycin compound includes at least one of daunomycin, a daunomycin derivative or a daunomycin modifier.

In one embodiment of the present invention, the fluorescent marker includes at least one of a fluorescein fluorescent marker, a rhodamine fluorescent marker, a cyanine dye fluorescent marker or a coumarin fluorescent marker; the fluorescein fluorescent marker includes at least one of fluorescein isothiocyanate, tetrachlorofluorescein or hydroxyfluorescein; the rhodamine fluorescent marker includes at least one of rhodamine B, rhodamine 6G or rhodamine 101; the cyanine dye fluorescent marker includes at least one of Cy5, Cy7 or Cy7.5; the coumarin fluorescent marker includes at least one of AMCA, AFC or AMC.

In one embodiment of the present invention, one end of the photoelectric dual-mode biological probe is modified with carboxyfluorescein, and the other end is modified with methylene blue.

In one embodiment of the present invention, the 5' end of the photoelectric dual-mode biological probe is modified with carboxyfluorescein, and the 3' end is modified with methylene blue.

In one embodiment of the present invention, the components of the detection reaction buffer include Tris-HCl buffer, Glycine-KOH, bovine serum albumin (BSA), KCl, MgCl ₂ and dithiothreitol (DTT).

In one embodiment of the present invention, the components of the detection reaction buffer include 10-80 mM Tris-HCl buffer, 70-150 mM Glycine-KOH, 40-100 μg/mL BSA, 40-90 mM KCl, 2-20 mM MgCl ₂ and 2-20 mM DTT.

In one embodiment of the present invention, the components of the detection reaction buffer include 50 mM Tris-HCl buffer, 100 mM Glycine-KOH, 70 μg/mL BSA, 60 mM KCl, 10 mM MgCl ₂ and 10 mM DTT.

In one embodiment of the present invention, the detection reaction buffer consists of 50 mM Tris-HCl buffer, 100 mM Glycine-KOH, 70 μg/mL BSA, 60 mM KCl, 10 mM MgCl ₂ and 10 mM DTT.

In one embodiment of the present invention, the pH of the Tris-HCl buffer is 7.5-8.5.

In one embodiment of the present invention, the pH of the Tris-HCl buffer is 7.8.

In one embodiment of the present invention, the amplification reagent includes a recombinase polymerase amplification (RPA) reagent; the recombinase polymerase amplification reagent includes an amplification primer pair targeting a target nucleic acid.

In one embodiment of the present invention, when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is double-stranded DNA, the amplification primer pair is a symmetric amplification primer pair; when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is single-stranded DNA, the amplification primer pair is an asymmetric amplification primer pair; the symmetric amplification primer pair includes an upstream primer and a downstream primer; the asymmetric amplification primer pair includes a restrictive primer and a non-restrictive primer.

In one embodiment of the present invention, the recombinase polymerase amplification reagent further includes an enzyme mixture, an RPA reaction buffer and magnesium acetate.

In one embodiment of the present invention, the components of the enzyme mixture include UvsX recombinase, Gp32 single-stranded binding protein, UvsY recombinase and strand-displacing DNA polymerase.

In one embodiment of the present invention, the enzyme mixture comprises 80-150 ng/μL UvsX recombinase, 400-800 ng/μL Gp32 single-stranded binding protein, 20-50 ng/mL UvsY recombinase, and 10-40 ng/μL strand displacement DNA polymerase.

In one embodiment of the present invention, the enzyme mixture comprises 125 ng/μL UvsX recombinase, 600 ng/μL Gp32 single-stranded binding protein, 30 ng/mL UvsY recombinase, and 20 ng/μL strand displacement DNA polymerase.

In one embodiment of the present invention, the enzyme mixture consists of 125 ng/μL UvsX recombinase, 600 ng/μL Gp32 single-stranded binding protein, 30 ng/mL UvsY recombinase, and 20 ng/μL strand displacement DNA polymerase.

In one embodiment of the present invention, the components of the RPA reaction buffer include Tris buffer, potassium acetate, magnesium acetate, dithiothreitol (DTT), polyethylene glycol, dNTP, ATP, creatine phosphate and creatine kinase.

In one embodiment of the present invention, the components of the RPA reaction buffer include 10-100 mM Tris buffer, 50-80 mM potassium acetate, 10-40 mM magnesium acetate, 0.5-5 mM DTT, 1-10% (w/v, g/100 mL) polyethylene glycol, 50-400 μM dNTP, 1-5 mM ATP, 5-50 mM creatine phosphate and 50-300 ng/μL creatine kinase.

In one embodiment of the present invention, the components of the RPA reaction buffer include 50 mM Tris buffer, 70 mM potassium acetate, 15 mM magnesium acetate, 2 mM DTT, 5% polyethylene glycol, 200 μM dNTP, 3 mM ATP, 20 mM creatine phosphate and 100 ng/μL creatine kinase.

In one embodiment of the present invention, the RPA reaction buffer consists of 50 mM Tris buffer, 70 mM potassium acetate, 15 mM magnesium acetate, 2 mM DTT, 5% polyethylene glycol, 200 μM dNTP, 3 mM ATP, 20 mM creatine phosphate and 100 ng/μL creatine kinase.

In one embodiment of the present invention, the pH of the Tris buffer is 8.0-8.5.

In one embodiment of the present invention, the pH of the Tris buffer is 8.4.

The present invention also provides a homogeneous nucleic acid detection method, which is not intended for the diagnosis and treatment of diseases. The method comprises: using the above-mentioned homogeneous nucleic acid detection kit to detect the target nucleic acid in the sample to be tested.

In one embodiment of the present invention, the method includes: mixing an amplification reagent and a sample to be tested to obtain an amplification system; incubating the amplification system to obtain an incubation solution; mixing the incubation solution and a detection reagent to obtain a detection system; reacting the detection system to obtain a reaction solution; detecting the electrochemical signal, fluorescence signal and color signal of the reaction solution; and calculating the concentration of the target nucleic acid in the sample to be tested based on the electrochemical signal, fluorescence signal and color signal obtained by the detection.

In one embodiment of the present invention, in the detection system, the concentration of the photoelectric dual-mode biological probe is 1 to 15 μM, and the concentration of the ribonuclease is 0.2 to 7 U/30 μL;

When the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is double-stranded DNA, in the detection system, the concentration of λ exonuclease is 1-5U/30μL, and the concentration of the detection reaction buffer is 0.1-2×; when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is single-stranded DNA, in the detection system, the concentration of the detection reaction buffer is 0.1-2×.

In one embodiment of the present invention, in the detection system, the concentration of the photoelectric dual-mode biological probe is 3 to 9 μM, and the concentration of the ribonuclease is 3 to 7 U/30 μL;

When the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is double-stranded DNA, in the detection system, the concentration of λ exonuclease is 1.5 to 3.5 U/30 μL, and the concentration of the detection reaction buffer is 0.5 to 1.5×; when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is single-stranded DNA, in the detection system, the concentration of the detection reaction buffer is 0.5 to 1.5×.

In one embodiment of the present invention, the reaction time is 5 to 40 minutes and the temperature is 20 to 60°C.

In one embodiment of the present invention, the reaction time is 15 to 35 minutes and the temperature is 30 to 45°C.

In one embodiment of the present invention, when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is double-stranded DNA, the concentrations of the upstream primer and the downstream primer in the amplification system are both 300 to 600 nM; when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is single-stranded DNA, the concentration of the non-restrictive primer in the amplification system is 300 to 600 nM, and the concentration ratio of the restrictive primer to the non-restrictive primer is 1:10 to 1:100;

In the amplification system, the concentration of the enzyme mixture is 0.8-1.2×, the concentration of the RPA reaction buffer is 0.5-1.1×, and the concentration of magnesium acetate is 20-35 mM.

In one embodiment of the present invention, the incubation time is 5 to 40 minutes and the temperature is 35 to 45°C.

In one embodiment of the present invention, the incubation time is 15 to 35 minutes and the temperature is 37 to 42°C.

In one embodiment of the present invention, the volume of the sample to be tested is 1-10 μL.

The present invention also provides the use of the above homogeneous nucleic acid detection kit or the above homogeneous nucleic acid detection method in target nucleic acid detection, and the application is for non-disease diagnosis and treatment purposes.

The technical solution of the present invention has the following advantages:

The present invention provides a homogeneous nucleic acid detection kit with a three-mode signal response, the homogeneous nucleic acid detection kit includes an amplification reagent and a detection reagent; the amplification reagent is used to amplify the target nucleic acid to form an amplification product; the detection reagent includes a photoelectric dual-mode biological probe and a ribonuclease (RNase); the photoelectric dual-mode biological probe can specifically bind to the amplification product, and the photoelectric dual-mode biological probe is connected to an electrochemical marker and a fluorescent marker; the ribonuclease can hydrolyze the photoelectric dual-mode biological probe, so that the photoelectric dual-mode biological probe breaks, and then releases electrochemical signals and fluorescent signals. When the homogeneous nucleic acid detection kit of the present invention is used for nucleic acid detection, a large amount of amplification products are first generated by using an amplification reagent, and then the amplification products are added to the detection reagent, the amplification products are hybridized and combined with the photoelectric dual-mode biological probe, and then the ribonuclease hydrolyzes the RNA chain in the photoelectric dual-mode biological probe in the DNA-RNA hybrid chain. As the photoelectric dual-mode biological probe breaks, the detection system electrical signal and fluorescent signal are enhanced, and at the same time, a color change is generated to achieve target reporting. The homogeneous nucleic acid detection kit of the present invention has the following advantages:

First, compared with existing electrochemical biosensors, the homogeneous nucleic acid detection kit proposed in the present invention integrates three common biosensor signal response modes of electrochemistry, fluorescence and colorimetry into a single reaction system or even a single reporter probe. It has a fast signal response speed and a simple structure, and can achieve simple, sensitive, fast, accurate and low-cost detection of target nucleic acids. It has a wide range of applications and strong portability. It is of great significance in the field of biological detection, innovates the response mode of biosensors, and provides new ideas for the design of next-generation reporter probes.

Second, the homogeneous nucleic acid detection kit proposed in the present invention eliminates the operation steps such as electrode surface modification and probe immobilization, avoiding or eliminating the common problems of poor reproducibility and cumbersome operation of heterogeneous and immobilized biosensors, making the electrochemical method for detecting pathogens simpler and more economical.

Third, compared with the patent disclosure with publication number CN116297376A, the homogeneous nucleic acid detection kit of the present invention uses more common ribonucleases to replace Cas proteins. Under the premise of ensuring that the detection limit remains unchanged, the detection time is shortened from 45 minutes to 40 minutes, and the reagent cost is reduced by 59%, which provides greater possibilities for clinical detection. At the same time, it proves that the strategy of establishing a homogeneous electrochemical reaction system based on photoelectric dual-mode biological probes is universal and scalable, and is not limited to the specificity of the CRISPR/Cas system. The photoelectric dual-mode biological probe can integrate more technologies and principles to establish a detection system with practical value, further promoting the innovative development and practical application of electrochemical biosensors.

Fourth, the use of the homogeneous nucleic acid detection kit of the present invention for nucleic acid detection breaks the limitations of previous electrode modifications on the precision, reproducibility, and repeatability of electrochemical detection technology, and realizes real-time monitoring of the electrochemical nucleic acid detection process under homogeneous conditions and repeated detection of the electrochemical nucleic acid system by electrodes under homogeneous flow.

Furthermore, the amplification reagent includes a recombinase polymerase amplification (RPA) reagent; the recombinase polymerase amplification reagent includes an amplification primer pair targeting the target nucleic acid; when the amplification product obtained by amplifying the target nucleic acid by the amplification reagent is double-stranded DNA, the amplification primer pair is a symmetric amplification primer pair; the detection reagent also includes λ exonuclease and a detection reaction buffer; when the amplification product obtained by amplifying the target nucleic acid by the amplification reagent is single-stranded DNA, the amplification primer pair is an asymmetric amplification primer pair; the detection reagent also includes a detection reaction buffer. When the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is double-stranded DNA, a symmetric amplification primer pair is first used to amplify a large amount of dsDNA, and then the amplification product is added to the detection reagent containing λ exonuclease. The dsDNA is hydrolyzed by the λ exonuclease to produce ssDNA, and the ssDNA is hybridized with the photoelectric dual-mode biological probe, and then the ribonuclease hydrolyzes the RNA chain in the photoelectric dual-mode biological probe in the DNA-RNA hybrid chain. As the photoelectric dual-mode biological probe breaks, the electrical signal and fluorescence signal of the detection system are enhanced, the color changes, and the target is reported; when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is single-stranded DNA, an asymmetric amplification primer pair is first used to amplify a large amount of ssDNA, and then the amplification product is added to the detection reagent without λ exonuclease. The ssDNA is hybridized with the photoelectric dual-mode biological probe, and then the RNase hydrolyzes the RNA chain in the photoelectric dual-mode biological probe in the DNA-RNA hybrid chain. As the photoelectric dual-mode biological probe breaks, the electrical signal and fluorescence signal of the detection system are enhanced, the color changes, and the target is reported.

Further, when the homogeneous nucleic acid detection kit of the present invention is used for nucleic acid detection, the amplification reagent and the sample to be tested are mixed to obtain an amplification system; the amplification system is incubated to obtain an incubation solution; the incubation solution and the detection reagent are mixed to obtain a detection system; the detection system is reacted to obtain a reaction solution; the electrochemical signal, fluorescence signal and color signal of the reaction solution are detected; the concentration of the target nucleic acid in the sample to be tested is calculated based on the electrochemical signal, fluorescence signal and color signal obtained by the detection; in the detection system, the concentration of the photoelectric dual-mode biological probe FAM-RNA-MB is 7 μM. At this concentration, the reaction system has a strong positive signal and signal-to-noise ratio.

Furthermore, in the detection system, the concentration of λ exonuclease is 2.5 U/30 μL. At this concentration, the reaction system has a strong positive signal and signal-to-noise ratio.

Furthermore, in the detection system, the concentration of RNase is 4 U/30 μL. At this concentration, the reaction system has a strong positive signal and signal-to-noise ratio.

Furthermore, in the detection system, the concentration of the detection reaction buffer is 1×. At this concentration, the reaction system has a strong positive signal and signal-to-noise ratio.

Furthermore, the reaction temperature of the detection system is 37° C. Within this temperature range, the reaction system has a stronger signal-to-noise ratio and a lower background signal.

Furthermore, the reaction time of the detection system is 20 min. Within this time range, the detection limit of the reaction system is as low as 0.3 aM.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Schematic diagram of the detection process of the homogeneous nucleic acid detection kit in Example 1.

Figure 2: Electrochemical signal, fluorescence signal and color signal detection results of the homogeneous nucleic acid detection method in Example 2.

Figure 3: Schematic diagram of the detection process of the homogeneous nucleic acid detection kit in Example 3.

Figure 4: Electrochemical signal, fluorescence signal and color signal detection results of the homogeneous nucleic acid detection method in Example 4.

Figure 5: Evaluation of the detection limit of the homogeneous nucleic acid detection method in Example 2.

Figure 6: Reproducibility evaluation of the homogeneous nucleic acid detection method in Example 2.

Figure 7: Experiment on the effect of photoelectric dual-mode biological probe concentration on signal response.

Figure 8: Experiment on the effect of λ exonuclease concentration on signal response.

Figure 9: Experiment on the effect of RNase concentration on signal response.

Figure 10: Experiment to detect the effect of reaction buffer concentration on signal response.

Figure 11: Experiment on the effect of reaction temperature on signal response.

Figure 12: Experiment on the effect of reaction time on signal response.

Detailed ways

The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

Example 1: A homogeneous nucleic acid detection kit with three-mode signal response

This embodiment provides a homogeneous nucleic acid detection kit with a three-mode signal response, wherein the homogeneous nucleic acid detection kit with a three-mode signal response is composed of an amplification reagent and a detection reagent;

The amplification reagent is a recombinase polymerase amplification (RPA) reagent, which consists of a symmetric amplification primer pair targeting a target nucleic acid, an enzyme mixture, an RPA reaction buffer, and magnesium acetate; the symmetric amplification primer pair consists of an upstream primer and a downstream primer; the enzyme mixture consists of 125 ng/μL UvsX recombinase, 600 ng/μL Gp32 single-strand binding protein, 30 ng/mL UvsY recombinase, and 20 ng/μL strand displacement DNA polymerase; the RPA reaction buffer consists of 50 mM Tris buffer (pH 8.4), 70 mM potassium acetate, 15 mM magnesium acetate, 2 mM DTT, 5% (w/v, g/100 mL) polyethylene glycol, 200 μM dNTP (200 μM each of A, T, G, and C), 3 mM ATP, 20 mM creatine phosphate, and 100 ng/μL creatine kinase;

The detection reagent consists of a photoelectric dual-mode biological probe, RNase H, λ exonuclease and a detection reaction buffer; the 5' end of the photoelectric dual-mode biological probe is connected to carboxyl fluorescein through a carbon chain, and the 3' end is connected to methylene blue through a carbon chain; the detection reaction buffer consists of 50mM Tris-HCl buffer (pH7.8), 100mM Glycine-KOH, 70μg/mL BSA, 60mM KCl, 10mM _MgCl2 and 10mM DTT.

Example 2: A homogeneous nucleic acid detection method

This embodiment provides a homogeneous nucleic acid detection method (see Figure 1 for the detection process), which uses the homogeneous nucleic acid detection kit with three-mode signal response of Example 1 and includes the following steps:

Mix 47 μL of amplification reagent and 3 μL of the sample to be tested to obtain an amplification system; incubate the amplification system at 40° C. for 20 minutes to obtain an incubation solution; mix 6 μL of the incubation solution and 24 μL of the detection reagent to obtain a detection system; react the detection system at 37° C. for 30 minutes to obtain a reaction solution; detect the methylene blue electrical signal, carboxyfluorescein fluorescence signal and color signal in the reaction solution; calculate the concentration of the target nucleic acid in the sample to be tested based on the electrochemical signal, fluorescence signal and color signal obtained by the detection;

In the detection system, the concentration of the photoelectric dual-mode biological probe is 7 μM, the concentration of ribonuclease is 4 U/30 μL, the concentration of λ exonuclease is 2.5 U/30 μL, and the concentration of the detection reaction buffer is 1×;

In the amplification system, the concentrations of the upstream primer and the downstream primer were both 300 nM, the concentration of the enzyme mixture was 0.8×, the concentration of the RPA reaction buffer was 0.5×, and the concentration of magnesium acetate was 20 mM.

Example 3: A homogeneous nucleic acid detection kit with three-mode signal response

This embodiment provides a homogeneous nucleic acid detection kit with a three-mode signal response, wherein the homogeneous nucleic acid detection kit with a three-mode signal response is composed of an amplification reagent and a detection reagent;

The amplification reagent is a recombinase polymerase amplification (RPA) reagent, which is composed of an asymmetric amplification primer pair targeting a target nucleic acid, an enzyme mixture, an RPA reaction buffer, and magnesium acetate; the asymmetric amplification primer pair is composed of a limiting primer and a non-limiting primer; the enzyme mixture is composed of 125 ng/μL UvsX recombinase, 600 ng/μL Gp32 single-strand binding protein, 30 ng/mL UvsY recombinase, and 20 ng/μL strand displacement DNA polymerase; the RPA reaction buffer is composed of 50 mM Tris buffer (pH 8.4), 70 mM potassium acetate, 15 mM magnesium acetate, 2 mM DTT, 5% (w/v, g/100 mL) polyethylene glycol, 200 μM dNTP (200 μM each of A, T, G, and C), 3 mM ATP, 20 mM creatine phosphate, and 100 ng/μL creatine kinase;

The detection reagent consists of a photoelectric dual-mode biological probe, RNase H and a detection reaction buffer; the 5' end of the photoelectric dual-mode biological probe is connected to carboxyfluorescein via a carbon chain, and the 3' end is connected to methylene blue via a carbon chain; the detection reaction buffer consists of 50mM Tris-HCl buffer (pH 7.8), 100mM Glycine-KOH, 70μg/mL BSA, 60mM KCl, 10mM _MgCl2 and 10mM DTT.

Example 4: A homogeneous nucleic acid detection method

This embodiment provides a homogeneous nucleic acid detection method (see Figure 3 for the detection process), which uses the homogeneous nucleic acid detection kit with three-mode signal response of Example 3, and includes the following steps:

Mix 47 μL of amplification reagent and 3 μL of the sample to be tested to obtain an amplification system; incubate the amplification system at 40° C. for 20 minutes to obtain an incubation solution; mix 6 μL of the incubation solution and 24 μL of the detection reagent to obtain a detection system; react the detection system at 37° C. for 30 minutes to obtain a reaction solution; detect the methylene blue electrical signal, carboxyfluorescein fluorescence signal and color signal in the reaction solution; calculate the concentration of the target nucleic acid in the sample to be tested based on the electrochemical signal, fluorescence signal and color signal obtained by the detection;

In the detection system, the concentration of the photoelectric dual-mode biological probe is 7 μM, the concentration of ribonuclease is 4 U/30 μL, the concentration of λ exonuclease is 2.5 U/30 μL, and the concentration of the detection reaction buffer is 1×;

In the amplification system, 30 nM of limiting primer, 300 nM of non-limiting primer, the concentration of enzyme mixture is 0.8×, the concentration of RPA reaction buffer is 0.5×, and the concentration of magnesium acetate is 20 mM.

Experimental Example 1: Performance Verification of a Homogeneous Nucleic Acid Detection Kit with Tri-Mode Signal Response

1. Experimental methods

1.1. Detection performance verification

Experiment 1: Using dsDNA with a nucleotide sequence as shown in SEQ ID NO.1 as the target nucleic acid (concentration of 300aM), designing an upstream primer (ATTTGGATCTCCATTCCCATTGAGGGCATTT) with a nucleotide sequence as shown in SEQ ID NO.2, a downstream primer (TCTCTCTATCGTTCCATCAGGCCCCCTCAAA) with a nucleotide sequence as shown in SEQ ID NO.3, and a photoelectric dual-mode biological probe FAM-RNA-MB (FAM-UGCAGUCCUCGCUCACUGGGCACG-MB) with a nucleotide sequence as shown in SEQ ID NO.4, and referring to the homogeneous nucleic acid detection method of Example 2, the target nucleic acid was detected using the homogeneous nucleic acid detection kit of Example 1. After the detection, the gold electrode (purchased from Metrohm DropSens) was connected to the electrochemical workstation (purchased from Shanghai Chenhua) and immersed in the reaction solution, and the methylene blue electrochemical signal in the reaction solution was detected by square wave voltammetry, the carboxyfluorescein fluorescence signal in the reaction solution was detected by fluorescence spectrophotometry, and the color signal in the reaction solution was detected by colorimetry. The electrochemical signal, fluorescence signal and color signal detected are shown in Figure 2.

Experiment 2: Based on Experiment 1, with reference to the homogeneous nucleic acid detection method of Example 4, the target nucleic acid was detected using the homogeneous nucleic acid detection kit of Example 3. After the detection, the gold electrode (purchased from Metrohm DropSens) was connected to the electrochemical workstation (purchased from Shanghai Chenhua), and immersed in the reaction solution. The methylene blue electrochemical signal in the reaction solution was detected by square wave voltammetry, the carboxyfluorescein fluorescence signal in the reaction solution was detected by fluorescence spectrophotometry, and the color signal in the reaction solution was detected by colorimetry. The results of the electrochemical signal, fluorescence signal and color signal obtained by detection are shown in Figure 4.

1.2. Detection limit verification

Referring to the homogeneous nucleic acid detection method of Example 2, the homogeneous nucleic acid detection kit of Example 1 was used to detect target dsDNA of different concentrations (0aM, 0.3aM, 3aM, 30aM, 300aM, 3000aM) to complete the detection limit evaluation of the kit. The results are shown in Figure 5.

1.3 Reproducibility Verification

Referring to the homogeneous nucleic acid detection method of Example 2, the homogeneous nucleic acid detection kit of Example 1 was used to construct the same detection system through multiple batches. The same negative sample (not containing any dsDNA) or the same positive sample (target dsDNA with a concentration of 300aM) was measured using this system to complete the reproducibility evaluation of the kit. The results are shown in Figure 6.

1.4 Experiment on the effect of photoelectric dual-mode biological probe concentration on signal response

On the basis of 1.1, the concentrations of the photoelectric dual-mode biological probe FAM-RNA-MB were replaced with 1, 3, 5, 7 and 10 μM, respectively. The positive signals, background signals and signal-to-noise ratios obtained at different photoelectric dual-mode biological probe concentrations were compared to clarify the effect of the photoelectric dual-mode biological probe concentration on the signal response and determine the optimal photoelectric dual-mode biological probe concentration. The results are shown in Figure 7.

1.5 Experiment on the effect of λ exonuclease concentration on signal response

On the basis of 1.1, the concentrations of λ exonuclease were replaced with 1.5, 2, 2.5, 3 and 3.5 U/30 μL, respectively. The positive signals, background signals and signal-to-noise ratios obtained at different λ exonuclease concentrations were compared to clarify the effect of λ exonuclease concentration on signal response and determine the optimal λ exonuclease concentration. The results are shown in Figure 8.

1.6 Experiment on the effect of RNase concentration on signal response

On the basis of 1.1, the concentration of RNase H was replaced with 3, 4, 5, 6 and 7 U/30 μL respectively, and the positive signal, background signal and signal-to-noise ratio obtained under different RNase H concentrations were compared to clarify the effect of RNase H concentration on signal response and determine the optimal RNase H concentration. The results are shown in Figure 9.

1.7. Experiment on the effect of reaction buffer concentration on signal response

On the basis of 1.1, the concentration of the detection reaction buffer was replaced with 0.1, 0.5, 1.0, 1.5 and 2.0× respectively, and the positive signal, background signal and signal-to-noise ratio obtained under different detection reaction buffer concentrations were compared to clarify the effect of the detection reaction buffer concentration on the signal response and determine the optimal detection reaction buffer concentration. The results are shown in Figure 10.

1.8 Experiment on the effect of reaction temperature on signal response

Based on 1.1, the reaction temperatures were replaced with 34, 37, 42, 50, 55, 58, 62 and 65°C, respectively. The positive signals, background signals and signal-to-noise ratios obtained at different temperatures were compared to clarify the effect of reaction temperature on signal response and determine the optimal reaction temperature. The results are shown in Figure 11.

1.9 Experiment on the influence of reaction time on signal response

On the basis of 1.1, the reaction time was replaced with 15, 20, 30, 40 and 50 min respectively, and the positive signal, background signal and signal-to-noise ratio obtained under different conditions were compared to clarify the effect of reaction time on signal response and determine the optimal reaction time. The results are shown in Figure 12.

2. Experimental results

2.1. Detection performance verification

As can be seen from Figures 2 and 4, with the addition of target DNA, the photoelectric dual-mode biological probe breaks, the electrical signal and fluorescence signal of the detection system are enhanced, the color changes, and the target DNA is reported. It can be seen that the homogeneous nucleic acid detection kit with three-mode signal response of Example 1 and Example 3 can simultaneously achieve three signal responses, among which the generation of color signals may be related to the enhancement of the ICT effect caused by the coordination binding of Mg2 ⁺ and the receptor group, or the complexity of the spatial structure caused by the coordination binding of ^Mg2 + and K ⁺ with the base and phosphate groups in FAM-RNA-MB.

2.2 Detection limit verification

As shown in Figure 5, in the range of 0.3 to 300 aM, the electrochemical, fluorescent and color signals increased significantly with the increase of dsDNA concentration; in the range of 300 to 3000 aM, the electrochemical, fluorescent and color signals increased slightly with the increase of dsDNA concentration. The detection limit of electrochemical and fluorescent signals is 0.3 aM, and the detection limit of color signals is 3 aM.

2.3 Reproducibility Verification

As shown in Figure 6, the oxidation peak currents of negative samples measured using the homogeneous nucleic acid detection kit were 0.565, 0.612, 0.604 and 0.570 μA, respectively, with an RSD of 4.06%, and the oxidation peak currents of positive samples measured using the homogeneous nucleic acid detection kit were 1.283, 1.244, 1.299 and 1.355 μA, respectively, with an RSD of 3.57%. The RSDs of negative and positive samples are within an acceptable range, that is, the homogeneous nucleic acid detection kit has good reproducibility.

2.4 Experiment on the effect of photoelectric dual-mode biological probe concentration on signal response

As shown in Figure 7, both the background signal and the positive signal gradually increase with the increase of the photoelectric dual-mode biological probe concentration, while the P/N value shows a trend of first increasing and then decreasing with the increase of the photoelectric dual-mode biological probe concentration. When the FAM-RNA-MB concentration is 7μM, the reaction system has a strong electrochemical signal and the strongest P/N value.

2.5 Experiment on the effect of λ exonuclease concentration on signal response

As shown in Figure 8, the positive signal and P/N value both showed a trend of first increasing and then decreasing with the increase of λ exonuclease concentration. When the λ exonuclease concentration was 2.5U/30μL, the reaction system obtained the strongest positive signal and P/N value.

2.6 Experiment on the effect of RNase concentration on signal response

As shown in Figure 9, the positive signal and P/N value both increased first and then decreased with the increase of RNase H concentration. When the RNase H concentration was 4U/30μL, the reaction system obtained the strongest positive signal and P/N value.

2.7. Experiment on the effect of reaction buffer concentration on signal response

As shown in Figure 10, the background signal gradually increases with the increase of the detection reaction buffer concentration, and the positive signal and P/N value increase first and then decrease with the increase of the detection reaction buffer concentration. When the detection reaction buffer concentration is 1.0×, the reaction system has the strongest positive signal and P/N value.

2.8 Experiment on the effect of reaction temperature on signal response

As shown in Figure 11, the background signal gradually increases with the increase of reaction temperature, and the positive signal has two peaks with the increase of temperature. The first peak is mainly contributed by λ exonuclease, and the second peak is mainly contributed by RNase H. When the temperature of the reaction system is higher than 50℃, the photoelectric dual-mode biological probe begins to break and the background signal gradually increases. After comprehensive consideration, the reaction temperature was finally determined to be 37℃ based on the P/N value.

2.9 Experiment on the influence of reaction time on signal response

As shown in Figure 12, when the reaction time is 20 minutes, 0.3aM target dsDNA has an obvious positive signal and P/N value, and when the reaction time is 40 minutes, 0.03aM target dsDNA has a certain positive signal. Based on the comprehensive consideration of the detection limit and the acceptance of personnel, 20 minutes was finally determined as the optimal reaction time.

Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the protection scope of the invention.

Claims (10)

1. A homogeneous nucleic acid detection kit with a three-mode signal response, characterized in that the homogeneous nucleic acid detection kit includes an amplification reagent and a detection reagent; the amplification reagent is used to amplify the target nucleic acid to form an amplification product; the detection reagent includes a photoelectric dual-mode biological probe and a ribonuclease; the photoelectric dual-mode biological probe can specifically bind to the amplification product, and the photoelectric dual-mode biological probe is connected to an electrochemical marker and a fluorescent marker; the ribonuclease can hydrolyze the photoelectric dual-mode biological probe, causing the photoelectric dual-mode biological probe to break, thereby releasing an electrochemical signal and a fluorescent signal. 2. The homogeneous nucleic acid detection kit as described in claim 1 is characterized in that the amplification product obtained by the amplification reagent amplifying the target nucleic acid is double-stranded DNA or single-stranded DNA; when the amplification product obtained by the amplification reagent amplifying the target nucleic acid is double-stranded DNA, the detection reagent also contains λ exonuclease and a detection reaction buffer; when the amplification product obtained by the amplification reagent amplifying the target nucleic acid is single-stranded DNA, the detection reagent also contains a detection reaction buffer. 3. The homogeneous nucleic acid detection kit according to claim 1 or 2, characterized in that the amplification reagent comprises a recombinase polymerase amplification reagent; the recombinase polymerase amplification reagent comprises an amplification primer pair targeting the target nucleic acid. 4. The homogeneous nucleic acid detection kit as described in claim 3 is characterized in that when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is double-stranded DNA, the amplification primer pair is a symmetric amplification primer pair; when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is single-stranded DNA, the amplification primer pair is an asymmetric amplification primer pair; the symmetric amplification primer pair includes an upstream primer and a downstream primer; the asymmetric amplification primer pair includes a restrictive primer and a non-restrictive primer. 5. The homogeneous nucleic acid detection kit according to claim 3 or 4, characterized in that the recombinase polymerase amplification reagent also includes an enzyme mixture, an RPA reaction buffer and magnesium acetate. 6. A homogeneous nucleic acid detection method, the method is not for the purpose of disease diagnosis and treatment, characterized in that the method comprises: using the homogeneous nucleic acid detection kit according to any one of claims 1 to 5 to detect the target nucleic acid in the sample to be tested. 7. The homogeneous nucleic acid detection method as described in claim 6 is characterized in that the method comprises: mixing an amplification reagent and a sample to be tested to obtain an amplification system; incubating the amplification system to obtain an incubation solution; mixing the incubation solution and a detection reagent to obtain a detection system; reacting the detection system to obtain a reaction solution; detecting the electrochemical signal, fluorescence signal and color signal of the reaction solution; and calculating the concentration of the target nucleic acid in the sample to be tested based on the electrochemical signal, fluorescence signal and color signal obtained by the detection. 8. The homogeneous nucleic acid detection method according to claim 7, characterized in that in the detection system, the concentration of the photoelectric dual-mode biological probe is 1 to 15 μM, and the concentration of the ribonuclease is 0.2 to 7 U/30 μL; When the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is double-stranded DNA, in the detection system, the concentration of λ exonuclease is 1-5U/30μL, and the concentration of the detection reaction buffer is 0.1-2×; when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is single-stranded DNA, in the detection system, the concentration of the detection reaction buffer is 0.1-2×. 9. The homogeneous nucleic acid detection method according to claim 7 or 8, characterized in that when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is double-stranded DNA, the concentrations of the upstream primer and the downstream primer in the amplification system are both 300 to 600 nM; when the amplification product obtained by amplifying the target nucleic acid with the amplification reagent is single-stranded DNA, the concentration of the non-restrictive primer in the amplification system is 300 to 600 nM, and the concentration ratio of the restrictive primer to the non-restrictive primer is 1:10 to 1:100; In the amplification system, the concentration of the enzyme mixture is 0.8-1.2×, the concentration of the RPA reaction buffer is 0.5-1.1×, and the concentration of magnesium acetate is 20-35 mM. 10. Use of the homogeneous nucleic acid detection kit according to any one of claims 1 to 5 or the homogeneous nucleic acid detection method according to any one of claims 6 to 9 in target nucleic acid detection, wherein the use is for non-disease diagnosis and treatment purposes.