Isothermal enzyme-free cascade amplification system based on HCR-DNAzyme technology and method for detecting tetA by adopting system

Abstract

The invention relates to the technical field of molecular biological detection, and discloses an isothermal enzyme-free cascade amplification system based on an HCR-DNAzyme technology and a method for detecting tetA by adopting the system. The system comprises an identification module, an amplification module, a feedback loop module and a detection module. According to the system and the method provided by the invention, HCR and DNAzyme technologies are integrated, a substrate is cut by DNAzyme to regenerate an initiation chain, a self-catalytic feedback loop is formed, signal exponential amplification is realized, the design of a hairpin structure (H1-H4) is optimized through NUPACK software, non-specific signal leakage is inhibited in combination with a blocking chain (B), and an identification sequence of an auxiliary hairpin (Hh) is replaced, so that the identification sequence of the auxiliary hairpin (Hh) is identified. Different ARGs detection (such as blaTEM-1 and aac3-II) can be rapidly adapted, and the repeated development cost is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology detection, and in particular to an isothermal enzyme-free cascade amplification system based on HCR-DNAzyme technology and a method for detecting tetA method. Background Art

[0002] Antibiotic Resistance Genes (ARGs), as a new type of environmental pollutants, have been widely detected in global water systems, posing a serious threat to the ecological environment and human health.

[0003] Tetracycline resistance gene ( tetA ) As a representative member of ARGs, it has become a key target for water safety monitoring due to its high abundance and horizontal transfer ability in the environment. Traditional detection methods such as quantitative polymerase chain reaction (qPCR) and digital droplet PCR (ddPCR) have high sensitivity, but rely on complex instruments, strict temperature control, and require professional operation, which makes it difficult to meet the needs of rapid on-site detection. In addition, the trace characteristics of ARGs in the water environment (concentrations as low as pM level) and complex matrix interference further limit the application of traditional technologies.

[0004] In recent years, isothermal nucleic acid amplification (INA) technology has attracted much attention because it does not require thermal cycling equipment. Existing INA technologies are enzyme-assisted (such as rolling circle amplification, loop-mediated isothermal amplification) and enzyme-free (such as hybridization chain reaction, catalytic hairpin assembly).

[0005] Enzyme-assisted methods rely on biological enzymes such as DNA polymerase and are easily interfered by environmental factors (such as pH, temperature fluctuations, inhibitors), resulting in decreased catalytic efficiency. Enzyme-free methods can achieve signal amplification through DNA chain displacement, but there are the following bottlenecks: (1) Low signal amplification efficiency: Traditional hybridization chain reaction (HCR) relies on linear amplification and has limited signal gain; (2) High background noise: Non-specific hairpin self-opening (leakage) leads to false positive signals; (3) Lack of versatility: Complex probes need to be redesigned for different targets, and have poor adaptability. Summary of the invention

[0006] The purpose of the present invention is to solve the problems of low signal amplification efficiency, high background noise and insufficient versatility of the existing enzyme-free isothermal nucleic acid amplification technology in resistance gene detection.

[0007] In order to achieve the above-mentioned object, the first aspect of the present invention provides an isothermal enzyme-free cascade amplification system based on HCR-DNAzyme technology, the system comprising: a recognition module, an amplification module, a feedback loop module, and a detection module;

[0008] The recognition module is connected to the auxiliary hairpin structure H h Specifically recognize target genes and trigger cascade reactions;

[0009] The amplification module is composed of a hairpin structure H 1 , Hairpin structure H 2 , Hairpin structure H 3 , Hairpin structure H 4 It is composed of a substrate-blocking complex and realizes exponential signal amplification through the synergistic effect of HCR and DNAzyme;

[0010] The feedback loop module is that DNAzyme cuts the substrate-blocking complex, releasing a new initiator chain (I), further activating HCR, and forming an autocatalytic feedback loop;

[0011] The detection module is a fluorescence spectrometer, which is used to detect changes in fluorescence signals and quantify the concentration of target genes.

[0012] Preferably, in the recognition module, the auxiliary hairpin structure H h The nucleotide sequence is shown in SEQ ID NO:9.

[0013] Further preferably, in the amplification module, the hairpin structure H 1 The nucleotide sequence of the hairpin structure H is shown in SEQ ID NO: 1. 2 The nucleotide sequence of is shown in SEQ ID NO: 2, the hairpin structure H 3 The nucleotide sequence of is shown in SEQ ID NO: 3, the hairpin structure H 4 The nucleotide sequence is shown in SEQ ID NO:4.

[0014] Preferably, in the amplification module, the hairpin structure H 1 It comprises an ab domain for binding to a priming chain (I), and the nucleotide sequence of the priming chain (I) is as shown in SEQ ID NO:8.

[0015] More preferably, in the amplification module, the hairpin structure H 2 Contains a c*-b domain for binding to the hairpin structure H 1 The cb* sequence binds.

[0016] Preferably, in the amplification module, the hairpin structure H3 Contains a db domain for binding to the hairpin structure H 2 db sequence combination.

[0017] Preferably, in the amplification module, the hairpin structure H 4 Contains an e*-b domain for binding to the hairpin structure H 3 The eb* sequence is bound to the 5' end and labeled with the fluorescence donor FAM at the 5' end and the fluorescence quencher TAMRA at the 3' end.

[0018] Further preferably, in the amplification module, the substrate-blocking complex includes a substrate chain S and a blocking chain B, and the substrate chain S contains a ribonucleotide rA for cutting DNAzyme, and the blocking chain B is complementary to the substrate chain S to prevent signal leakage.

[0019] Preferably, in the substrate-blocking complex, the nucleotide sequence of the substrate strand S is as shown in SEQ ID NO:5, and the nucleotide sequence of the blocking strand B is as shown in SEQ ID NO:7.

[0020] Preferably, in the substrate-blocking complex, the molar ratio of the substrate strand S to the blocking strand B is 1:1.2.

[0021] More preferably, in the feedback loop module, the DNAzyme is composed of a hairpin structure H 1 , Hairpin structure H 2 , Hairpin structure H 3 , Hairpin structure H 4 Self-assembled and formed by Mg 2+ The catalytic action of the nucleotide sequence cleaves the ribonucleotide rA site in the substrate chain S, releasing a new initiator chain (I).

[0022] Preferably, in the detection module, the excitation wavelength of the fluorescence spectrometer is 490 nm, and the emission wavelength is 508-650 nm.

[0023] The second aspect of the present invention provides a method for detecting a tetA The method comprises the following steps: (1) Filter the environmental samples to remove particulate matter, and then take 200 mL of the environmental samples and place them in sterile containers, which are respectively recorded as sample S1 to sample S15;

[0024] Extract DNA from the above sterile containers respectively, and confirm the quality and concentration of the extracted DNA by ultraviolet spectrophotometer respectively, randomly select 10 samples for establishing a linear correlation curve, record them as spare sample DNA S1 to spare sample DNA S10, and obtain information of each sample; the remaining 5 samples are used as prediction samples to evaluate the accuracy of the linear correlation curve, record them as spare sample DNA S11 to spare sample DNA S15, and obtain information of each sample;

[0025] Then they were dissolved in 1×TAE-Mg 2+ Buffer and stored at -20°C;

[0026] (2) respectively heating the standby sample DNA S1 to the standby sample DNA S15 to boiling, treating them in a boiling state for 15 minutes, and then cooling them rapidly at 0° C. for standby use, and recording them as sample DNA 1 to sample DNA 15 respectively;

[0027] Prepare 15 portions of auxiliary hairpin structure H at a concentration of 400 nmol / L h , hairpin structure H at a concentration of 400nmol / L 1 , hairpin structure H at a concentration of 400nmol / L 2 , hairpin structure H at a concentration of 400nmol / L 3 , hairpin structure H at a concentration of 400nmol / L 4 , a substrate-blocking complex (SB) with a concentration of 200 nmol / L, and mixed with the sample DNA 1 to the sample DNA 15, respectively, and incubated at 25°C for 30 minutes, respectively, to start the HCR reaction, and measure the change of the fluorescence signal by a fluorescence spectrometer to obtain the absolute fluorescence change intensity, respectively, and monitor the fluorescence recovery during the reaction in real time;

[0028] (3) Target genes in samples S1 to S15 in step (1) tetA The concentration is quantitatively analyzed, and the accuracy of the linear correlation curve is evaluated based on the linear correlation curve of the samples S1 to S10 in step (1) and the samples S11 to S15 in step (1).

[0029] Preferably, in step (2), the absolute fluorescence change intensity is calculated using the following formula:

[0030] ΔF = F_sample- F_blank, where “F_sample” represents the absolute fluorescence intensity of the sample and “F_blank” represents the absolute fluorescence intensity of the blank control sample.

[0031] Further preferably, in step (3), the target gene in the samples S1 to S15 in step (1) is tetA The concentration is quantitatively analyzed, and the quantitative analysis includes: substituting the absolute fluorescence change intensity obtained in step (2) into the linear correlation curve described in step (1) to obtain the target gene tetA concentration.

[0032] The present invention provides an isothermal enzyme-free cascade amplification system based on HCR-DNAzyme technology and a method for detecting tetA The method also has at least the following beneficial effects: 1. Enzyme-free catalysis and cascade amplification: Integrate HCR and DNAzyme technology, use DNAzyme to cut the substrate to regenerate the initiator chain, form an autocatalytic feedback loop, and achieve exponential signal amplification; 2. High stability and low background: hairpin structure optimized by NUPACK software (H 1 -H 4 ) design, combined with a blocking chain (B) to inhibit nonspecific signal leakage; 3. Modularity and versatility: By replacing the auxiliary hairpin (H h ) recognition sequence, quickly adapt to different ARGs detection (such as blaTEM-1 , aac3-II ), avoiding duplication of development costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The fluorescence intensity of the HCR-DNAzyme system of the present invention is related to the target gene tetA Linear correlation plot of concentration;

[0034] Figure 2 It is a graph of linear fitting and prediction results of environmental samples of the present invention;

[0035] Figure 3 is the fluorescence intensity and target gene tetA Concentration relationship diagram;

[0036] Figure 4 This is a schematic diagram of the isothermal enzyme-free cascade amplification system based on HCR-DNAzyme technology, in which the fluorescent donor FAM is represented by a green sphere, the fluorescent quencher TAMRA is represented by a red sphere, and the overall process: the auxiliary hairpin structure H h Identify target genes tetA → Trigger HCR → DNAzyme cuts substrate-blocking complex (SB) → Releases new initiator strand (I) → Fluorescence signal increases;

[0037] Figure 5is a schematic diagram of the DNAzyme biocatalysis of the present invention, wherein the initiator chain (I) can trigger a continuous HCR reaction between hairpin structures to form a nanowire with a complete DNAzyme, and the DNAzyme cuts the substrate-blocking complex, resulting in the regeneration of the initiator chain (I) and reversely excites the HCR reaction, further enhancing the fluorescence signal;

[0038] Figure 6 is the fluorescence spectrum of HCR under different conditions of the present invention, where “a” is H 1 +H 2 +H 3 +H 4 +I, "b" is H 1 +H 2 +H 3 +H 4 , "c" is H 1 + H 2 + H 4 +I, "d" is H 1 +H 3 +H 4 +I, "e" is H 1 +H 3 +H 4 , "f" is H 2 +H 3 +H 4 +I, "g" is H 1 +H 2 +H 4 , "h" is H 2 +H 3 +H 4 , where “H 1 " indicates a hairpin structure H 1 , "H 2 " indicates a hairpin structure H 2 , "H 3 " indicates a hairpin structure H 3 , "H 4 " indicates a hairpin structure H 4 , “I” represents the initiation chain (I), with a concentration of 200 nmol / L;

[0039] Figure 7 is the fluorescence intensity change diagram in different HCR systems, where: different HCR systems: “a” is H 1 +H 2 +H 3 +H 4 +I, "b" is H 1 +H 2 +H 3 +H 4 , "c" is H1 + H 2 + H 4 +I, "d" is H 1 +H 3 +H 4 +I, "e" is H 1 +H 3 +H 4 , "f" is H 2 +H 3 +H 4 +I, "g" is H 1 +H 2 +H 4 , "h" is H 2 +H 3 +H 4 , where “H 1 " indicates a hairpin structure H 1 , "H 2 " indicates a hairpin structure H 2 , "H 3 " indicates a hairpin structure H 3 , "H 4 " indicates a hairpin structure H 4 , “I” represents the initiation chain (I), with a concentration of 200 nmol / L;

[0040] Figure 8 The time-dependent fluorescence curves of the IR-HCR-based amplifier circuit in the absence (a) and presence (b) of 40 nmol / L initiator chain (I), where λ = 520 nm and (a) is H 1 +H 2 +H 3 +H 4 +I, (b) is H 1 +H 2 +H 3 +H 4 , where “H 1 " indicates a hairpin structure H 1 , "H 2 " indicates a hairpin structure H 2 , "H 3 " indicates a hairpin structure H 3 , "H 4 " indicates a hairpin structure H 4 , “I” represents the initiation chain (I), with a concentration of 200 nmol / L;

[0041] Fig. 9 is the fluorescence spectrum of the IR-HCR system at 120 minutes, where the system in the reaction buffer contains 400 nmol / L H 1 , H 2 , H3 , H 4 and 200 nmol / L SB, where “H 1 " indicates a hairpin structure H 1 , "H 2 " indicates a hairpin structure H 2 , "H 3 " indicates a hairpin structure H 3 , "H 4 " indicates a hairpin structure H 4 “SB” indicates substrate-blocker complex, and the error bars are derived from n = 3 experiments;

[0042] Fig.10 It is a single-chain H based HCR amplifier circuit. 1 , H 2 , H 3 , H 4 , SB or I, and H 1 +H 2 +H 3 +H 4 +SB and H 1 +H 2 +H 3 +H 4 +S-B+I complex native polyacrylamide gel electrophoresis, where “H 1 " indicates a hairpin structure H 1 , "H 2 " indicates a hairpin structure H 2 , "H 3 " indicates a hairpin structure H 3 , "H 4 " indicates a hairpin structure H 4 , “SB” indicates substrate-blocker complex, “I” indicates initiator chain (I), with a concentration of 200 nmol / L;

[0043] Fig.11 It is a schematic diagram of the IR-HCR based amplifier circuit and the nHCR circuit;

[0044] Fig.12 It is based on the IR-HCR amplification circuit and the FF under the action of nHCR initiation chain (I) at different concentrations. 0 A curve diagram, wherein the IR-HCR-based amplification circuit is represented by blue, the nHCR is represented by red, and the different concentrations of the initiator chain (I) are 5-200 nmol / L;

[0045] Fig.13It is a linear calibration curve diagram of the IR-HCR-based amplifier circuit and nHCR for detecting the initiation chain (I), wherein the IR-HCR-based amplifier circuit is represented by blue, and nHCR is represented by red, wherein the line graph shows that the IR-HCR signal intensity is 3.09 times that of nHCR (slope difference);

[0046] Fig.14 The target gene of the present invention tetA Schematic diagram of the principle of identifying target sequences;

[0047] Fig.15 It is based on the IR-HCR amplification circuit and different concentrations of target genes tetA Fluorescence spectrum of the target gene after incubation with the analyte. tetA Different concentrations range from 0 to 200 nmol / L;

[0048] Fig.16 When λ is 520nm, based on tetA Concentration fluorescence intensity change verification diagram;

[0049] Fig.17 The fluorescence intensity of the IR-HCR amplifier circuit is tetA Linear correlation plot of concentration;

[0050] Fig.18 The target gene is activated by a, b, c, d, and e respectively. tetA Fluorescence spectrum of the IR-HCR based amplifier circuit, where "a" represents the effect of 40nmol / L blank control and "b" represents 16S-rDNA The role of interfering nucleic acid, "c" role means blaTEM-1 The effect of interfering nucleic acid, "d" effect means aac3-II The effect of interfering nucleic acid, "e" indicates the target gene tetA The role of;

[0051] Fig.19 It is an IR-HCR based amplifier circuit for detecting nucleic acid fragments (FF 0 ) values, where the error bars represent the data of three repeated experiments, (FF 0 ) values ​​represent the absolute fluorescence changes of the fluorescent donor FAM;

[0052] Fig. 20 It is the linear correlation between the environmental sample detection results and qPCR in Example 2 of the present invention. DETAILED DESCRIPTION

[0053] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0054] The nucleotide sequences involved in the present invention are detailed in Table 1 of the specification.

[0055] In the present invention, the target gene tetA Specifically binds to the auxiliary hairpin structure H in the reaction system h , and inspire HCR reaction, produce long-chain DNA nanowires and form DNA enzymes, further enhance the HCR reaction by cleaving the substrate-blocking complex in the system, increase the distance between the fluorophore and the quenching group, release the fluorescence signal, and use a fluorescence spectrometer to read the fluorescence signal.

[0056] The present invention will be described in detail below by way of examples. In the following examples, unless otherwise specified, the raw materials are all commercially available.

[0057] In the following examples, unless otherwise specified, 1×TAE-Mg 2+ Preparation method of buffer: 40mmol / L Tris, 2mmol / L EDTA, 12.5mmol / L MgAc 2 Mix and adjust pH to 7.0;

[0058] Auxiliary hairpin structure H h Preparation method: Synthesized by solid phase synthesis, purified by HPLC, dissolved in 1×TAE-Mg 2+ The concentration was 400 nmol / L in the buffer solution and the secondary structure was simulated by NUPACK software to ensure that the target gene was not bound. tetA Maintain a stable hairpin structure;

[0059] Hairpin structure H 1 , Hairpin structure H 2 , Hairpin structure H 3 , Hairpin structure H 4 Preparation method: Synthesized by solid phase synthesis, purified by HPLC, dissolved in 1×TAE-Mg 2+ The concentration was 400 nmol / L in the buffer solution and the secondary structure was simulated by NUPACK software to ensure that the target gene was not bound. tetA Maintain a stable hairpin structure;

[0060] Preparation method of substrate-blocking complex: substrate chain S and blocking chain B were mixed at a molar ratio of 1:1.2 in 1×TAE-Mg 2+ Incubate in buffer for 2 h.

[0061] In the following examples, unless otherwise specified, fluorescence detection was performed using a F-2700FL spectrophotometer in a fluorescence spectrometer, and fluorescence spectra were recorded in the range of 506 to 650 nm using a fixed excitation wavelength of 490 nm.

[0062] In the following examples, unless otherwise specified, the auxiliary hairpin structure H h The nucleotide sequence is shown in SEQ ID NO: 9; in the 16S-rDNA In the interfering nucleic acid solution, 16S-rDNA is a common bacterial gene, and its nucleotide sequence is shown in SEQ ID NO:11; blaTEM-1 In the interfering nucleic acid solution, blaTEM-1 is a β-lactamase gene, the nucleotide sequence of which is shown in SEQ ID NO: 12; aac3-II In the interfering nucleic acid solution, aac3-II is an aminoglycoside resistance gene, and its nucleotide sequence is shown in SEQ ID NO: 13;

[0063] tetA The nucleotide sequence of the single-stranded DNA is shown in SEQ ID NO: 10;

[0064] In the following examples, unless otherwise specified, the target gene in Example 1 tetA The standard curve is obtained by:

[0065] Using 1×TAE-Mg 2+ The buffer was diluted 10-fold in series to prepare target genes at 0.01nmol / L, 0.1nmol / L, 0.2nmol / L, 0.5nmol / L, 1nmol / L, 2nmol / L, 5nmol / L, 10nmol / L, 20nmol / L, 40nmol / L, 100nmol / L, and 200nmol / L, respectively. tetA Concentration; 0 " as the ordinate, with the target gene tetA The concentration is the horizontal axis, and the standard curve is drawn. The result is as follows Figure 1 shown.

[0066] In the following examples, unless otherwise specified, the DNA sequences used to construct the sensor system of the present invention are shown in Table 1.

[0067] Table 1 DNA sequence list used to construct the system

[0068] Test Example 1

[0069] This example is used to provide an isothermal enzyme-free cascade amplification system based on hybridization chain reaction (referred to as HCR reaction) and deoxyribozyme technology (referred to as DNAzyme technology) to detect tetA The method comprises the following steps: (1) Take 200 mL of laboratory samples (referred to as target genes) tetA S1~target gene tetA S12, target genes in laboratory samples tetA S1~target gene tetA The concentrations of S12 were 0.01nmol / L, 0.1nmol / L, 0.2nmol / L, 0.5nmol / L, 1nmol / L, 2nmol / L, 5nmol / L, 10nmol / L, 20nmol / L, 40nmol / L, 100nmol / L, and 200nmol / L, respectively, and placed in sterile containers, and 1×TAE-Mg 2+ buffer and stored at -20°C.

[0070] (2) Prepare 12 portions of auxiliary hairpin structure H at a concentration of 400 nmol / L h , and the target gene of the series concentration gradient obtained in step (1) tetA S1~target gene tetA S12 mixture, incubate at 25°C for 30 min (to allow the auxiliary hairpin structure H h Target gene tetA Combined and exposed the initiator chain (I), and then added the hairpin structure H at a concentration of 400nmol / L 1 , Hairpin structure H 2 , Hairpin structure H 3 , Hairpin structure H 4 , and add the substrate-blocking complex at a concentration of 200nmol / L, incubate at 25°C for 2-3h to initiate the HCR reaction (in this process, the auxiliary hairpin structure H h Target gene tetA Binds to expose the initiator strand I, initiating the hairpin structure H 1 ~Hairpin structure H 4 cascade reaction), the change of fluorescence signal was measured by fluorescence spectrometer, and the absolute fluorescence change intensity was obtained according to the following formula (as shown in Table 2), and fluorescein amide (FAM) and tetramethylrhodamine (TAMRA) were used as fluorescent markers to monitor the fluorescence recovery during the reaction in real time;

[0071] ΔF = F_sample - F_blank.

[0072] Table 2 Fluorescence intensity changes and standard deviations of different samples

[0073] (3) For the target gene described in step (1) tetA S1~The target gene tetA Target genes with known concentrations in S5 tetA Conduct quantitative analysis by tetA The recovery rate of the test was used to verify the accuracy of the detection system. The results are shown in Table 3 and Figure 1 As shown (according to the results, this detection system has tetA The detection has a high recovery rate, that is, high accuracy).

[0074] The quantitative analysis is specifically as follows: the absolute fluorescence change intensity obtained in step (3) is respectively substituted into the standard curve obtained in step (2) to obtain the target gene tetA concentration.

[0075] Depend on Figure 1 It can be seen that the linear range of the standard curve is 0.01~1nmol / L, and the linear equation is (FF 0 )=9.57+127.59x(R²=0.997), and the detection limit (LOD) is 4.6pM.

[0076] The isothermal enzyme-free cascade amplification reaction time was 180 min, and the reaction temperature was set at 25 °C.

[0077] Table 3 Laboratory samples tetA Concentration test results

[0078] Test Example 1

[0079] This example is used to target gene tetA Perform sensitivity testing.

[0080] (A1) Preparation of standards: Synthetic tetA The absorbance of single-stranded DNA at 260 nm was converted according to the following formula to obtain the initial concentration; and 1×TAE-Mg 2+The buffer was diluted 10-fold to obtain target genes at concentrations of 0.01 nmol / L, -0.1 nmol / L, -0.2 nmol / L, -0.5 nmol / L, -1 nmol / L, -2 nmol / L, -5 nmol / L, -10 nmol / L, -20 nmol / L, -40 nmol / L, -100 nmol / L, and -200 nmol / L, respectively. tetA concentration;

[0081] Initial concentration (μg / mL) = A260 × 50 × dilution factor.

[0082] (A2) Construction of reaction system: Preparation of 1×TAE-Mg 2+ The buffer was used as the reaction buffer; the auxiliary hairpin structure H was prepared at a concentration of 400 nmol / L h , the concentration of hairpin structure H was 400nmol / L 1 , Hairpin structure H 2 , Hairpin structure H 3 , Hairpin structure H 4 , a substrate-blocking complex with a concentration of 200nmol / L as a reaction component; the target genes with concentrations of 0.01nmol / L, 0.1nmol / L, 0.2nmol / L, 0.5nmol / L, 1nmol / L, 2nmol / L, 5nmol / L, 10nmol / L, 20nmol / L, 40nmol / L, 100nmol / L, and 200nmol / L in step (A1) are tetA (Used to draw the standard curve).

[0083] (A3) Detection process: The auxiliary hairpin structure H described in step (A2) h Mix, pre-incubate at 25°C for 30 min, and then add the hairpin structure H 1 , the hairpin structure H 2 , the hairpin structure H 3 , the hairpin structure H 4 The substrate-blocking complex was incubated at 25° C. in the dark for 3 h, and the fluorescence signal change was detected by a fluorescence spectrometer (using a F-2700 fluorescence spectrophotometer, excitation / emission = 490 / 520 nm, and a slit width of 5 nm).

[0084] (A4) Data analysis: Using an equal volume of 1×TAE-Mg 2+ Target gene as described in the buffer replacement step (A1) tetASingle-stranded DNA was added to the reaction components as a blank sample, and the fluorescence signal change was measured 10 times in parallel by fluorescence spectrometer as a blank control test; the absolute fluorescence change intensity was calculated according to the following formula, and the results are shown in Table 2;

[0085] Among them, the absolute fluorescence change intensity ΔF = F_sample - F_blank, where "F_blank" is the mean value of the blank control test measurement;

[0086] Then "log10(target gene tetA The standard curve was drawn with "ΔF" as the horizontal axis and the LOD value was calculated according to the following formula:

[0087] LOD = 3σ / S=(3×2.8) / 127.59=0.066 log10(nM)=4.6pM, where σ is the standard deviation of ΔF of the blank sample; S is the slope of the calibration curve.

[0088] Test Example 2

[0089] This example is used to target gene tetA Perform specificity testing.

[0090] (B1) Preparation of interference samples: Prepare 10 nmol / L 16S-rDNA Interfering nucleic acid solution, concentration of 10nmol / L blaTEM-1 Interfering nucleic acid solution, concentration of 10nmol / L aac3-II The interfering nucleic acid solution was sequentially obtained to obtain interference sample I, interference sample II, and interference sample III.

[0091] (B2) Construction of reaction system: Preparation of 1×TAE-Mg 2+ The buffer was used as the reaction buffer; the auxiliary hairpin structure H was prepared at a concentration of 400 nmol / L h , the concentration of hairpin structure H was 400nmol / L 1 , Hairpin structure H 2 , Hairpin structure H 3 , Hairpin structure H 4 , a substrate-blocking complex with a concentration of 200nmol / L was used as a reaction component; 10nmol / L / 50μL of the interference sample obtained in step (B1) was used as an interference nucleic acid / negative control.

[0092] (B3) Detection process: The interference sample I, the interference sample II, the interference sample III, and the auxiliary hairpin structure H in step (B1) are h Mix, pre-incubate at 25°C for 30 min, and then add the hairpin structure H 1 , the hairpin structure H2 , the hairpin structure H 3 , the hairpin structure H 4 The substrate-blocking complex is incubated at 25° C. in the dark for 3 h, and the change of fluorescence signal is detected by fluorescence spectrometer.

[0093] (B4) Data analysis: The absolute fluorescence change intensity (ΔF) and specificity index were calculated according to the following formula. The results are shown in Table 4.

[0094] Where, ΔF = F_sample - F_blank;

[0095] Specificity index = (ΔFinterference sample / ΔFtarget gene tetA )×100%.

[0096] Table 4 Interference samples and target genes tetA Comparison of absolute fluorescence intensity changes

[0097] Example 1

[0098] This example is used to provide an isothermal enzyme-free cascade amplification system based on hybridization chain reaction (referred to as HCR reaction) and deoxyribozyme technology (referred to as DNAzyme technology) to detect tetA The method comprises the following steps: (1) Fifteen water samples, including source water samples, treated water samples (water samples from different treatment stages of the water plant, including flocculation tanks, sand filters, activated carbon filters, etc.), and outlet pipe samples (water samples collected from the water plant outlet pipes and water supply networks) from five water plants in a city in southern China, were randomly divided into 15 parts as environmental samples. Following the standardized water sample collection operation, the environmental samples were filtered and particulate matter was removed. Then, 200 mL of environmental samples were taken and placed in sterile containers (respectively recorded as samples S1 to S15);

[0099] DNA was extracted from the above sterile containers using the DNeasy PowerWater Kit (water sample DNA extraction kit) purchased from QIAGEN, Germany, and the quality and concentration of the extracted DNA were confirmed by UV spectrophotometer. Ten samples (referred to as spare sample DNA S1 to spare sample DNA S10, and the sample information is shown in Table 5) were randomly selected to establish a linear correlation curve (see Table 5). Figure 2 As shown in Table 5, the remaining 5 samples (recorded as spare sample DNA S11 to spare sample DNA S15, sample information is shown in Table 5) were used as prediction samples to evaluate the accuracy of the linear curve;

[0100] Finally dissolved in 1×TAE-Mg2+ buffer and stored at -20°C.

[0101] Table 5

[0102] (2) The standby sample DNA S1 to the standby sample DNA S15 are heated to boiling, and treated in a boiling state for 15 minutes (to denature the double-stranded DNA into single-stranded DNA), and then quenched at 0° C. for standby use, and are respectively recorded as sample DNA 1 to sample DNA 15;

[0103] Prepare 15 portions of auxiliary hairpin structure H at a concentration of 400 nmol / L h , hairpin structure H at a concentration of 400nmol / L 1 , hairpin structure H at a concentration of 400nmol / L 2 , hairpin structure H at a concentration of 400nmol / L 3 , hairpin structure H at a concentration of 400nmol / L 4 , a substrate-blocking complex with a concentration of 200 nmol / L, and mixed with the sample DNA 1 to the sample DNA 15, respectively, and incubated at 25°C for 30 min to initiate the HCR reaction (in this process, the auxiliary hairpin structure H h Target gene tetA Binds to expose the initiator strand I, initiating the hairpin structure H 1 ~Hairpin structure H 4 Cascade reaction), the change of fluorescence signal was measured by fluorescence spectrometer, and the absolute fluorescence change intensity was obtained according to the following formula (as shown in Table 6), and the labeled fluorescence donor FAM and fluorescence acceptor TAMRA were used as fluorescence markers to monitor the fluorescence recovery in real time during the reaction;

[0104] ΔF = F_sample - F_blank.

[0105] Table 6 Environmental samples tetA Changes in concentration and fluorescence intensity

[0106] (3) Target genes in samples S1 to S15 in step (1) tetA The concentration is quantitatively analyzed, and the accuracy of the linear correlation curve is evaluated based on the linear correlation curve of the samples S1 to S10 in step (1) and the samples S11 to S15 in step (1). The results are as follows: Figure 2 shown.

[0107] The quantitative analysis is specifically as follows: the absolute fluorescence change intensity obtained in step (2) is substituted into the linear correlation curve obtained in step (1), and the target gene is obtained. tetA concentration.

[0108] Depend on Figure 2 It can be seen that at a wavelength of 520 nm, the (FF 0 ) values ​​and target genes detected by qPCR tetA The concentration showed a significant positive linear relationship; the calibration curve regression coefficient R²=0.997, the corresponding equation was y=6.74x−0.67, and the linear range was 0.85×10 -2 pM~0.69pM, and the predicted samples all fall within the 95% confidence interval.

[0109] Comparative Example 1

[0110] This example provides a traditional quantitative qPCR method, which includes the following steps:

[0111] (D1) tetA Drawing of plasmid standard curve:

[0112] Dilute the known concentration with double distilled water tetA Plasmid standards were obtained at concentrations of 10 7 , 10 6 , 10 5 , 10 4 , 10 3 Copies / μL tetA Plasmid standard solution, using forward primer tetA F and reverse primer tetA R tetA The plasmid standard solution is subjected to recombinase polymerase amplification reaction to obtain an amplified product. The amplified product binds to the SYBR Green dye to generate a fluorescent signal. The fluorescent signal is read using a qPCR instrument to obtain the concentration of each tetA Fluorescence amplification curve of plasmid standard solution.

[0113] The reaction time of the recombinase polymerase amplification reaction was 22.6 min, and the fluorescence signal was collected once every 34 s, for a total of 40 cycles, and the reaction temperature was set to 60°C.

[0114] The threshold cycle number CT of each fluorescence amplification curve is used as the ordinate. tetA lg of plasmid standard solution 10 The logarithm is used as the horizontal axis, and the fitting is tetA Plasmid standard curve.

[0115] (D2) Prepare qPCR amplification reaction system:

[0116] 10 μL 2×TB Green qPCR premix (the trade name of the kit is TB Green Premix Ex Taq II (Tli RNaseH Plus)), 0.8 μL 10 μM forward primer, 0.8 μL 10 μM reverse primer, 2 μL template DNA, 6 μL sterile ultrapure water and 0.4 μL ROX Reference Dye II dye.

[0117] (D3) The qPCR reaction procedure is:

[0118] 95℃ pre-denaturation for 30s, 95℃ denaturation for 30s, 60℃ annealing and extension for 34s, a total of 40 cycles. The fluorescence amplification curve of each clinical isolate was obtained. The cycle number (Ct value) in the fluorescence amplification curve where the fluorescence intensity △Rn was greater than the threshold value of 0.032545 was substituted into tetA The tetracycline resistance gene was calculated in the ordinate of the plasmid standard curve. tetA The copy number [lg10(copy number / μL)].

[0119] By comparing the comparative example with the embodiment of the present invention, it can be seen that the embodiment of the present invention can detect tetracycline resistance genes at a low detection limit. tetA It can also perform quantitative analysis with high accuracy. The operation steps are simpler and the target gene in the sample can be detected under isothermal and enzyme-free mild experimental conditions. tetA It can perform qualitative and quantitative analysis with good specificity.

[0120] In order to more clearly describe the method of the present invention, the present invention Figure 3 The fluorescence intensity and target gene are shown tetA The relationship between concentration, Figure 4 A schematic diagram of the isothermal enzyme-free cascade amplification system based on HCR-DNAzyme technology is shown. Figure 5 A schematic diagram of the DNAzyme biocatalysis of the present invention is shown, Figure 6 The fluorescence spectra of HCR in different conditions are shown. Figure 7 The fluorescence intensity changes in different HCR systems are shown. Figure 8 The time-dependent fluorescence curves of the IR-HCR-based amplification circuit in the absence (a) and presence of 40 nmol / L initiator chain (I) (b) are shown; Fig. 9 The fluorescence spectrum of the IR-HCR system at 120 minutes is shown; Fig.10 A single-chain HCR-based amplification circuit is shown. 1 , H 2 , H 3 , H 4, SB or I, and H 1 +H 2 +H 3 +H 4 +SB and H 1 +H 2 +H 3 +H 4 Native polyacrylamide gel electrophoresis of +S-B+I complex; Fig.11 Schematic diagrams of IR-HCR based amplification circuit and nHCR circuit are shown; Fig.12 The IR-HCR based amplification circuit and the FF under the action of different concentrations of nHCR initiator chains (I) are shown. 0 curve; Fig.13 The IR-HCR based amplification circuit and the linear calibration curve of nHCR for detecting the initiation chain (I) are shown; Fig.14 The target gene of the present invention is shown tetA Schematic diagram of the principle of identifying target sequences; Fig.15 The IR-HCR based amplification circuit and different concentrations of target genes are shown. tetA Fluorescence spectra after analyte incubation; Fig.16 It shows that when λ is 520nm, based on tetA Verification of fluorescence intensity changes of concentration; Fig.17 The fluorescence intensity of the IR-HCR based amplifier circuit is shown in Figure 2. tetA Linear dependence of concentration; Fig.18 It shows that the target gene is activated under the action of a, b, c, d, and e respectively. tetA Fluorescence spectrum of the IR-HCR based amplifier circuit; Fig.19 The IR-HCR based amplification circuit is shown for detecting nucleic acid fragments (FF 0 )value; Fig. 20 The linear correlation between the environmental sample detection results and qPCR in Example 2 of the present invention is shown.

[0121] Depend on Figure 3 It can be seen that the absolute fluorescence change intensity (FF 0 )along with tetA The concentration of tetA Feasibility of detection.

[0122] Depend on Figure 4 , Figure 5 It can be seen that the amplification module consists of a hairpin structure H 1 , Hairpin structure H 2 , Hairpin structure H 3 , Hairpin structure H 4and substrate-blocking complex, triggering the HCR reaction under the action of the initiation chain (I), causing H 4 The distance between the fluorescent group and the quenching group in the HCR reaction increases, releasing the fluorescent signal. The HCR reaction produces long-chain DNA nanowires and forms DNA enzymes, which further strengthen the HCR reaction by regenerating the initiator chain (I) through the substrate-blocking complex (SB) in the cleavage system, thereby amplifying the fluorescent signal.

[0123] Depend on Figure 6~Figure 10 It can be seen that when the system is subjected to fluorescence spectroscopy detection and natural polyacrylamide gel electrophoresis analysis under different system compositions, the target fluorescence spectrum and natural polyacrylamide gel electrophoresis results can be obtained only when all hairpin structures and initiator chains (I) are present, which verifies the necessity of each DNA in the system.

[0124] Depend on Figure 11~Figure 13 It can be seen that due to the synergistic feedback effect of the regeneration ability of the initiator chain (I) and the biocatalytic effect of DNA hydrolase, the IR-HCR-based amplification system has a stronger amplification ability than the traditional nHCR system.

[0125] Depend on Figure 14~Figure 19 It can be seen that the system can detect different concentrations of tetA , the fluorescence signal intensity and tetA The system has a significant correlation with the concentration change, and there is a linear correlation in the low concentration range. At the same time, the system can effectively resist the influence of representative interfering ssDNA sequences, indicating that the system is tetA The detection is sensitive, stable and specific.

[0126] Depend on Fig. 20 It can be seen that at a wavelength of 520nm, the absolute fluorescence change intensity (FF 0 ) and qPCR detection tetA There was a positive linear correlation between the concentrations, which verified the applicability of this system for the detection of resistance genes in environmental samples.

[0127] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. An isothermal enzyme-free cascade amplification system based on HCR-DNAzyme technology, characterized in that: The system includes: an identification module, an amplification module, a feedback loop module, and a detection module; The recognition module is connected to the auxiliary hairpin structure H h Specifically recognize target genes and trigger cascade reactions; The amplification module is composed of a hairpin structure H1, a hairpin structure H2, a hairpin structure H3, a hairpin structure H4 and a substrate-blocking complex, and realizes exponential amplification of the signal through the synergistic effect of HCR and DNAzyme; The feedback loop module is that DNAzyme cuts the substrate-blocking complex, releasing a new initiator chain (I), further activating HCR, and forming an autocatalytic feedback loop; The detection module is a fluorescence spectrometer, which is used to detect changes in fluorescence signals and quantify the concentration of target genes.

2. The system according to claim 1, characterized in that In the recognition module, the auxiliary hairpin structure H h The nucleotide sequence is shown in SEQ ID NO:

9.

3. The system according to claim 1 or 2, characterized in that: In the amplification module, the nucleotide sequence of the hairpin structure H1 is shown as SEQ ID NO:1, the nucleotide sequence of the hairpin structure H2 is shown as SEQ ID NO:2, the nucleotide sequence of the hairpin structure H3 is shown as SEQ ID NO:3, and the nucleotide sequence of the hairpin structure H4 is shown as SEQ ID NO:

4.

4. The system according to claim 1 or 2, characterized in that: In the amplification module, the hairpin structure H1 comprises an ab domain for binding to the initiator chain (I), and the nucleotide sequence of the initiator chain (I) is as shown in SEQ ID NO: 8; and / or, in the amplification module, the hairpin structure H2 comprises a c*-b domain for binding to the cb* sequence of the hairpin structure H1; and / or, in the amplification module, the hairpin structure H3 comprises a db domain for binding to the db sequence of the hairpin structure H2; And / or, in the amplification module, the hairpin structure H4 comprises an e*-b domain for binding to the eb* sequence of the hairpin structure H3, and is labeled with a fluorescent donor FAM at the 5' end and a fluorescent quencher TAMRA at the 3' end.

5. The system according to claim 1 or 2, characterized in that: In the amplification module, the substrate-blocking complex includes a substrate chain S and a blocking chain B, and the substrate chain S contains a ribonucleotide rA for cutting DNAzyme, and the blocking chain B is complementary to the substrate chain S to prevent signal leakage; and / or, in the substrate-blocking complex, the nucleotide sequence of the substrate strand S is as shown in SEQ ID NO:5, and the nucleotide sequence of the blocking strand B is as shown in SEQ ID NO:7; And / or, in the substrate-blocking complex, the molar ratio of the substrate strand S to the blocking strand B is 1:1.

2.

6. The system according to claim 1 or 2, characterized in that: In the feedback loop module, DNAzyme is self-assembled by hairpin structure H1, hairpin structure H2, hairpin structure H3, and hairpin structure H4, and is formed by Mg 2+ The catalytic action of the nucleotide sequence cleaves the ribonucleotide rA site in the substrate chain S, releasing a new initiator chain (I).

7. The system according to claim 1 or 2, characterized in that: In the detection module, the excitation wavelength of the fluorescence spectrometer is 490 nm, and the emission wavelength is 508-650 nm.

8. A method for detecting a tetA The method is characterized in that The method comprises the following steps: (1) Filter the environmental samples to remove particulate matter, and then take 200 mL of the environmental samples and place them in sterile containers, which are respectively recorded as sample S1 to sample S15; Extract DNA from the above sterile containers respectively, and confirm the quality and concentration of the extracted DNA by ultraviolet spectrophotometer respectively, randomly select 10 samples for establishing a linear correlation curve, record them as spare sample DNA S1 to spare sample DNA S10, and obtain information of each sample; the remaining 5 samples are used as prediction samples to evaluate the accuracy of the linear correlation curve, record them as spare sample DNA S11 to spare sample DNA S15, and obtain information of each sample; Then they were dissolved in 1×TAE-Mg 2+ Buffer and stored at -20°C; (2) respectively heating the standby sample DNA S1 to the standby sample DNA S15 to boiling, treating them in a boiling state for 15 minutes, and then cooling them rapidly at 0° C. for standby use, and recording them as sample DNA 1 to sample DNA 15 respectively; Prepare 15 portions of auxiliary hairpin structure H at a concentration of 400 nmol / L h , a hairpin structure H1 with a concentration of 400nmol / L, a hairpin structure H2 with a concentration of 400nmol / L, a hairpin structure H3 with a concentration of 400nmol / L, a hairpin structure H4 with a concentration of 400nmol / L, and a substrate-blocking complex (SB) with a concentration of 200nmol / L, and respectively mixed with the sample DNA1 to the sample DNA15, and incubated at 25°C for 30min, respectively, to start the HCR reaction, and measure the change of the fluorescence signal by a fluorescence spectrometer to obtain the absolute fluorescence change intensity, respectively, and monitor the fluorescence recovery during the reaction in real time; (3) Target genes in samples S1 to S15 in step (1) tetA The concentration is quantitatively analyzed, and the accuracy of the linear correlation curve is evaluated based on the linear correlation curve of the samples S1 to S10 in step (1) and the samples S11 to S15 in step (1).

9. The method according to claim 8, characterized in that In step (2), the absolute fluorescence change intensity is calculated using the following formula: ΔF = F_sample- F_blank, where "F_sample" represents the absolute fluorescence intensity of the sample and "F_blank" represents the absolute fluorescence intensity of the blank control sample.

10. The method according to claim 8 or 9, characterized in that: In step (3), the target gene in the samples S1 to S15 in step (1) is tetA The concentration is quantitatively analyzed, and the quantitative analysis includes: substituting the absolute fluorescence change intensity obtained in step (2) into the linear correlation curve described in step (1) to obtain the target gene tetA concentration.

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