Application of iron-ruthenium nano-enzyme in preparation of nucleic acid detection product

Abstract

The invention discloses application of iron ruthenium nano-enzyme in preparation of nucleic acid detection products, and belongs to the technical field of biological detection and nano-materials. The RIN has stable peroxidase-like (POD) activity, and can catalyze a chromogenic reaction between 3, 3 ', 5, 5'-tetramethyl benzidine (TMB) and hydrogen peroxide (H2O2). The catalytic activity of the nano-enzyme can be selectively inhibited by pyrophosphate (PPi) generated in a nucleic acid amplification reaction, so that visual colorimetric analysis on whether target nucleic acid exists or not is realized. In combination with a polymerase chain reaction (PCR) system, the invention constructs a new nucleic acid detection strategy which does not need fluorescence labeling, is simple and convenient to operate and has high sensitivity. Through verification, the system can be widely applied to rapid on-site screening and visual interpretation of related nucleic acid of drug-resistant pathogens (such as ESKAPE flora) and the like, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application provides a kind of nucleic acid detection method based on iron ruthenium nanometer enzyme (RIN) and its application in the preparation of nucleic acid detection product.The RIN has significant peroxidase (POD) activity, can catalyze the color reaction of substrate 3, 3', 5, 5'-tetramethyl benzidine (TMB) under the condition of hydrogen peroxide (H2O2) exists.It is found that pyrophosphate ion (PPi) can specifically coordinate with RIN, thereby inhibiting its POD activity and inhibiting the color development process of TMB.Based on the characteristics of a large number of PPi produced during polymerase chain reaction (PCR) amplification, the present application constructs a kind of ESKAPE pathogen nucleic acid detection platform with RIN as signal response element.When target sequence exists, PCR reaction can be effectively amplified and a large number of PPi are generated, which further inhibits the catalytic color development of RIN.By monitoring the color development degree of the system, it can be judged whether the target nucleic acid sequence exists.Specifically, the specific gene sequence of ESKAPE seven drug-resistant pathogens is used as the detection target, and the accumulation of PPi in the system after PCR amplification can react with RIN to inhibit the color development of substrate TMB.By detecting the absorbance change, the specific recognition of pathogen nucleic acid can be realized, and the detection limit is as low as 100 copies.The detection method has the advantages of high sensitivity, strong specificity, simple operation, etc., and provides a new technical path for early warning and accurate diagnosis of clinical multi-drug resistant bacterial infection. BACKGROUND

[0002] With the rapid development of life science and molecular diagnosis technology, nucleic acid detection has become an indispensable technical means in the fields of modern medicine, public health and food safety, etc.Especially in response to infectious disease epidemic, screening genetic mutation, monitoring pathogenic microorganism, etc., real-time fluorescent quantitative PCR (qPCR), digital PCR (dPCR) and other methods have been widely used due to their high sensitivity and high specificity.However, these traditional detection methods generally rely on expensive equipment, require complex operation procedures and professional technical personnel, etc., which are difficult to meet the needs of point-of-care testing (POCT).Therefore, it has become a research hotspot to develop nucleic acid detection platform with simple operation, rapid response, visual detection and adaptation to multiple scene requirements.

[0003] In recent years, nanozymes have shown great potential in the fields of biosensing, tumor imaging, environmental monitoring, and so on, due to their high stability, low preparation cost, and easy mass production. Among them, POD-like nanozymes can simulate the natural peroxidase to catalyze the decomposition of H2O2 and promote the reaction of the chromogenic substrate, showing good prospects in the field of visual detection. Transition metal nanomaterials such as iron-based, ruthenium-based, and cobalt-based have been widely studied as representatives of POD-type nanozymes, but there are still some deficiencies in their catalytic efficiency, substrate selectivity, and detection system versatility.

[0004] Multi-metal cooperation is an effective strategy to improve the catalytic performance of nanozymes. Among them, iron-ruthenium bimetallic nanomaterials have become a hot spot in the research of POD-like nanozymes due to their excellent electronic coupling effect and structural stability. Iron has strong intrinsic enzyme-like activity, while ruthenium can provide excellent electron transfer ability. The synergistic effect of the two can not only enhance the chromogenic reaction rate, but also improve the recognition ability of the substrate. The RIN constructed in this invention shows significantly better POD-like catalytic activity than traditional single-metal nanozymes, laying a solid foundation for its application in visual molecular diagnostic platforms.

[0005] In the process of nucleic acid amplification detection, PPi is a typical byproduct of DNA synthesis reaction, and its generation amount can directly reflect the progress of the amplification reaction. PPi has strong metal ion chelating ability and can bind to certain metal catalytic sites, thereby regulating the catalytic activity of metal nanozymes. The invention found that PPi can specifically coordinate with the active site of RIN, inhibiting its chromogenic catalytic reaction on substrates such as TMB. Therefore, the PPi generated by PCR amplification can be used as a "molecular switch" to mediate the change of nanozyme activity, and the indirect detection of nucleic acids can be achieved through the change of chromogenic intensity. This mechanism does not require probe labeling or complex signal conversion devices, and is suitable for constructing a simple, rapid, and visual nucleic acid detection platform.

[0006] Current clinical and public health fields are facing the challenge of rapid screening of multiple drug-resistant pathogens such as ESKAPE. Due to the characteristics of multiple types, strong drug resistance, and similar early clinical symptoms of such pathogens, traditional detection methods are difficult to accurately distinguish in a short time. Based on the principle of "PCR amplification + PPi inhibition of RIN chromogenic" constructed in this invention, high-specificity detection of specific nucleic acid targets can be achieved, thereby judging whether specific pathogens exist. This detection platform does not rely on fluorescent probes or complex instruments, and the results can be judged by the naked eye, with high on-site operability and application universality, greatly improving the portability and on-site applicability of the detection system.

[0007] Although some studies have attempted to combine nano-enzyme and amplification by-products to achieve nucleic acid detection, there are still obvious deficiencies in response mechanism stability, detection sensitivity, background interference control and nanometer enzyme material universality. Especially in the aspect of constructing a visual detection system based on the synergistic effect of multi-metal nanometer enzyme and PPi, there is still a lack of systematic material design and mechanism verification. The present application provides a kind of nanometer enzyme RIN based on the synergistic design of iron-ruthenium bimetal, and constructs a high-sensitivity, low-cost, non-probe-labeled nucleic acid detection platform by using its response behavior to PPi. This scheme breaks through the dependence of traditional detection methods on complex instruments and labeled molecules, and provides a new path for the rapid construction of pathogen precise detection and molecular diagnosis platform, which has important theoretical significance and practical application value. SUMMARY

[0008] The present application aims at the deficiencies of existing nucleic acid detection technology in portability, cost and operation complexity, and proposes a visual nucleic acid detection method based on RIN, and studies its application in the identification of multi-drug resistant ESKAPE pathogens. First, the nanometer enzyme material RIN with uniform morphology and controllable particle size is synthesized by controlling the conditions. The material is spherical nanostructure, which exhibits significant catalytic ability of H2O2 oxidation TMB display. Further found that the PPi released in the PCR amplification process can specifically bind to the catalytic active site of RIN, significantly inhibit its POD-like activity, and then inhibit the color reaction. Based on this mechanism, a nucleic acid detection system without probe labeling and dependent on enzyme activity change response is constructed. Specifically, by taking the specific nucleic acid sequence of the target pathogen as the PCR amplification target, introducing Q5 polymerase system for thermal cycle amplification, the PPi in the amplification product acts as an "input signal" to regulate the color development catalytic ability of RIN, thereby realizing the visual judgment of the presence or absence of nucleic acid.

[0009] The technical scheme of the present application is as follows:

[0010] A kind of preparation of iron-ruthenium nanometer enzyme, its preparation method is as follows: by magnetic stirring, ultrasonic dispersion and other steps, by glycerol-isopropyl alcohol homogeneous dispersion metal salt (ruthenium salt, iron salt), then by hydrothermal synthesis, centrifugal washing, vacuum drying and program temperature calcination, iron-ruthenium nanometer enzyme RIN is prepared.

[0011] The application of iron-ruthenium nanometer enzyme in the preparation of nucleic acid detection product, its application method is as follows:

[0012] Step one: select the specific gene fragments of seven ESKAPE pathogens to design primers, and carry out PCR amplification reaction, without introducing fluorescent probe or dye in the amplification system.

[0013] Step two: PCR amplification products were reacted with RIN under acidic conditions (pH 4.0, NaAc / HAc buffer system), and the reaction system contained color developing substrate TMB and H2O2. After 5 min of catalytic reaction, the difference in light absorption value at 652 nm wavelength between the experimental group and the control group was detected synchronously by an enzyme marker, and the difference between the two groups was verified by statistical analysis. Simultaneously, agarose gel electrophoresis was performed to analyze the nucleic acid bands of the amplification products, and the PCR results were verified, forming a "color development-nucleic acid" double verification mechanism.

[0014] Step three: The detection of PCR products under different target concentrations was explored to determine the lower limit of detection.

[0015] Step four: RIN was used to detect simulated clinical samples of ESKAPE pathogen infection. First, the target gene RNaseP was amplified and detected, and then the target pathogen was amplified and detected to observe whether there was a visually distinguishable color change between the experimental group and the control group, thereby verifying the practical scene application feasibility of the constructed nucleic acid detection method.

[0016] The primer sequences of the seven ESKAPE pathogens in step one are shown in Table 1 below:

[0017] Table 1 Primer sequences

[0018] The control group in step two has a low level of target DNA template, and the catalytic activity of RIN is retained, and the color development reaction is obvious. If the experimental group amplifies successfully, a large amount of PPi is generated, thereby inhibiting the catalytic ability of RIN, and the color development intensity is significantly reduced. The catalytic reaction conditions are RIN final concentration 50 μg / mL, H2O2 final concentration 1 mM, TMB final concentration 832 μM, and the reaction is carried out in HAc / NaAc buffer solution at pH 4.0.

[0019] In step three, the detection results of PCR products of gram-negative bacteria Acinetobacter baumannii (gltA) and gram-positive bacteria Staphylococcus aureus (nuc) with copy numbers from 1 to 10000 were explored and the significant differences were analyzed.

[0020] In step four, the experimental group is a simulated clinical sample in which a solution containing a target pathogen plasmid is added to human serum samples, and the control group is a normal human serum sample without the addition of a target pathogen plasmid. The upstream primer sequence of the reference gene RNase P used is AGATTTGGACCTGCGAGC, and the downstream primer sequence is GAGCGGCTGTCTCCACAAGT.

[0021] Finally, through RIN detection, the visual identification of simulated ESKAPE infection clinical samples can be realized.

[0022] In the existing nucleic acid detection method, the present application has the following obvious advantages: taking RIN as the core reaction element, and by virtue of its high catalytic performance on the TMB / H2O2 system, the color reaction can be quickly triggered under weak acidic conditions; at the same time, the PPi released in the amplification reaction process has high affinity binding capacity to the active site of RIN, which can significantly inhibit the POD-like activity of RIN, so as to realize the label-free and fluorescence probe-free nucleic acid detection. The detection strategy driven by PCR takes PPi as the endogenous signal of the amplification product, and combines the RIN enzyme activity change for visual judgment, which significantly simplifies the composition of the detection system, reduces the complexity and cost of the reaction system. In addition, the RIN material is simple to synthesize, has high stability and good batch consistency, which provides a feasible basis for large-scale promotion and industrialization of the detection platform.

[0023] The visual nucleic acid detection strategy based on the PPi-RIN mechanism proposed in the present application has the advantages of simple operation, high sensitivity, no need for optical probes and precise instruments, and is especially suitable for resource-limited or on-site rapid screening scenarios, which provides new diagnostic technology support for rapid identification of pathogens. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the transmission electron microscopy image of the RIN nanoparticles prepared in the present application.

[0025] Figure 2 is the flow chart of the nucleic acid detection of ESKAPE pathogens in the present application.

[0026] Figure 3 is the agarose gel electrophoresis result of the experimental group and the control group of various ESKAPE pathogens.

[0027] Figure 4 is the feasibility verification result of the RIN prepared in the present application for identifying various ESKAPE pathogens, and *** represents the significance analysis result p<0.001.

[0028] Figure 5 is the PCR product detection result chart of Acinetobacter baumannii (gltA) and Staphylococcus aureus (nuc) at different target concentrations.

[0029] Figure 6 is the heat map analysis of the ΔA / A0 result of the simulated clinical sample.

[0030] Figure 7 is the heat map analysis of the ΔA / A0 result of the real clinical sample of Staphylococcus aureus infection. DETAILED DESCRIPTION

[0031] The application will be further described in conjunction with the specific embodiments, the examples given are only for illustrating the application, but not for limiting the scope of the application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application.

[0032] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0033] The equipment used in the following examples is shown in Table 2:

[0034] Table 2 Equipment used

[0035] The reagents used in the following examples are shown in Table 3:

[0036] Table 3 Reagents used

[0037] Example 1

[0038] Material preparation and morphology testing

[0039] 10 g of glycerol and 40 mL of isopropyl alcohol were weighed into a conical flask, and a magnetic stirrer was set to 800 rpm for continuous stirring for 1 hour until a homogeneous transparent solution was formed. 0.25 mmol of RuCl3 and 0.125 mmol of FeSO4·7H2O reagents were accurately weighed into a 5 mL centrifuge tube (Fe:Ru ratio = 1:2), and 1 mL of ultrapure water was added, followed by ultrasonic treatment to obtain a uniform metal salt dispersion. The uniform solution of metal salts was added dropwise to the mixed glycerol and isopropyl alcohol, and after stirring for 60 min, it was transferred to a 100 mL hydrothermal reactor, which was placed in an oven at 180°C for hydrothermal reaction for 6 h. After the reaction, the product was separated by centrifugation, the supernatant was discarded, and the product was washed twice with anhydrous ethanol by centrifugation, and then transferred to a vacuum drying oven for drying overnight. The dried sample was finely ground in an agate mortar and placed in a quartz boat in a tube furnace for programmed temperature calcination (5°C min -1 to 400°C for 2 h), and finally a powder product, iron-ruthenium nanoscale enzyme RIN, was obtained. The morphology of the iron-ruthenium nanoscale enzyme was determined by a Japanese Electronics JEM-1011 field emission transmission electron microscope (TEM), as shown in Figure 1 which showed that the RIN was regular spherical with a diameter of about 170 nm and a relatively flat surface.

[0040] Example 2

[0041] Take 10 g of glycerol and 40 mL of isopropyl alcohol into a conical flask, set the magnetic stirrer at 800 rpm for continuous stirring for 1 hour, until a homogeneous transparent solution is formed. Accurately weigh 0.1875 mmol of RuCl3 and 0.1875 mmol of FeSO4·7H2O reagents into a 5 mL centrifuge tube (Fe:Ru = 1:1), add 1 mL of ultrapure water, and then ultrasonic treatment to obtain a homogeneous metal salt dispersion. Add the homogeneous solution of metal salt to the mixed glycerol and isopropyl alcohol, stir for 60 min, then transfer to a 100 mL hydrothermal reactor, and hydrothermal reaction at 180°C for 6 h. After the reaction, centrifugal separation of the product, discard the supernatant, and then use anhydrous ethanol to centrifugal wash twice, and then transfer to a vacuum drying oven to dry overnight. After fine grinding of the dried sample in an agate mortar, place it in a quartz boat in a tube furnace for programmed temperature calcination (5°C min -1 to 400°C, and keep for 2 h), and finally obtain the powder product iron ruthenium nanoscale enzyme RIN.

[0042] Example 3

[0043] Take 10 g of glycerol and 40 mL of isopropyl alcohol into a conical flask, set the magnetic stirrer at 800 rpm for continuous stirring for 1 hour, until a homogeneous transparent solution is formed. Accurately weigh 0.1875 mmol of RuCl3 and 0.1875 mmol of FeSO4·7H2O reagents into a 5 mL centrifuge tube (Fe:Ru = 1:1), add 1 mL of ultrapure water, and then ultrasonic treatment to obtain a homogeneous metal salt dispersion. Add the homogeneous solution of metal salt to the mixed glycerol and isopropyl alcohol, stir for 60 min, then transfer to a 100 mL hydrothermal reactor, and hydrothermal reaction at 180°C for 6 h. After the reaction, centrifugal separation of the product, discard the supernatant, and then use anhydrous ethanol to centrifugal wash twice, and then transfer to a vacuum drying oven to dry overnight. After fine grinding of the dried sample in an agate mortar, place it in a quartz boat in a tube furnace for programmed temperature calcination (5°C min -1 to 400°C, and keep for 2 h), and finally obtain the powder product iron ruthenium nanoscale enzyme RIN.

[0044] Example 4

[0045] Take 10 g of glycerol and 40 mL of isopropyl alcohol into a conical flask, set the magnetic stirrer at 800 rpm for continuous stirring for 1 hour until a homogeneous transparent solution is formed. Respectively, accurately weigh 0.0625 mmol of RuCl3 and 0.3125 mmol of FeSO4·7H2O reagent into a 5 mL centrifuge tube (Fe:Ru ratio = 5:1), add 1 mL of ultrapure water, and then ultrasonic treatment to obtain a homogeneous metal salt dispersion. Add the homogeneous solution of metal salt to the mixed glycerol and isopropyl alcohol, stir for 60 min, and then transfer to a 100 mL hydrothermal reactor, and hydrothermal reaction at 180℃ for 6 h. After the reaction, centrifugal separation of the product, discarding the supernatant, and using anhydrous ethanol to centrifugal wash twice, and then transferring to a vacuum drying oven to dry overnight. After fine grinding of the dried sample in a corundum mortar, it is placed in a quartz boat in a tube furnace for programmed temperature calcination (5℃ min -1 to 400℃, and keep for 2 h), and finally obtain the powder product iron ruthenium nanoscale enzyme RIN.

[0046] Example 5

[0047] Feasibility analysis of ESKAPE pathogen nucleic acid detection

[0048] Based on the excellent response performance of RIN to PPi, an RIN analysis platform for ESKAPE pathogen nucleic acid detection was constructed. Considering that a large amount of PPi will be generated during the PCR amplification process, when the target nucleic acid sequence exists, the dNTP in the system will undergo chain reaction under the action of enzyme and primer, and the generated PPi can significantly inhibit the color development reaction of RIN catalyzing TMB, thereby realizing the recognition and visual judgment of target sequence, and the detection process is as shown in Figure 2 In this case, the specific genes (ddl, nuc, mdh, gltA, oprL, dnaJ, ampC) of seven ESKAPE pathogens are amplified by PCR, and no fluorescent probe or dye is introduced in the reaction.

[0049] 1. PCR reaction system configuration

[0050] The total amount of the PCR reaction system is 50 μL, and the composition is shown in Table 4:

[0051] Table 4 PCR reaction system Component Amount Final concentration 5x Q5 buffer 10 μL 1× 500 μM dNTP 3 μL 30 μM Upstream primer 2 μL 0.4 μM Downstream primer 2 μL 0.4 μM Plasmid DNA 1 μL 1 ng Q5 enzyme 0.5 μL 0.02 U / μL Enzyme-free water 31.5 μL -

[0052] 2. PCR reaction conditions

[0053] Initial denaturation: 98℃, 30s; denaturation: 98℃, 10s; annealing: according to the T mValue adjustment (Table 5), 30 s; extension: 72 °C, 60 s; cycle number: 35 cycles; final extension: 72 °C, 2 min; after reaction, keep at 4 °C. The annealing temperature of each gene is as follows: Table 5 Annealing temperature Bacteria Target gene Annealing temperature Enterococcus faecium ddl 54℃ Staphylococcus aureus nuc 56℃ Klebsiella pneumoniae mdh 56℃ Acinetobacter baumannii gltA 54℃ Pseudomonas aeruginosa oprL 59℃ Enterobacter cloacae dnaJ 60℃ Enterobacter aerogenes ampC 60℃

[0054] 3. PCR product detection

[0055] Seven ESKAPE pathogen-specific gene fragments (ddl, nuc, mdh, gltA, oprL, dnaJ, ampC) were amplified according to the reaction conditions in Table 4 and Table 5. After amplification, the PCR reaction products were separated by agarose gel electrophoresis, and the DNA bands were observed using nucleic acid dye. The dyed gel was placed under ultraviolet transilluminator to observe whether there were specific amplification bands, so as to verify whether the amplification was successful. As shown in Table 6, the experimental group (PS) containing target sequence produced clear specific bands after amplification, while the negative control group (NS) without target sequence had no amplification product, proving that the PCR system has high sequence specificity. Figure 3

[0056] 4. Color development reaction system configuration

[0057] After dilution, 10 μL of the diluted solution was mixed with 5 μL of RIN solution (1 mg / mL), 10 μL of TMB solution (2 mg / mL), 10 μL of H2O2 solution (10 mM), and 65 μL of HAc / NaAc buffer (pH 4.0, 0.1 M) to make the total volume of the reaction system 100 μL. After mixing, the reaction was carried out at room temperature in the dark for 5 minutes, and then colorimetric detection was immediately performed.

[0058] 5. Colorimetric detection and result determination

[0059] The change in absorbance at 652 nm wavelength was detected using an enzyme marker, and compared with the absorbance value without adding PCR product. If there was no significant change in absorbance at this wavelength, it indicated that the RIN catalytic activity was preserved, i.e. the reaction system had no specific target sequence. If the absorbance value decreased significantly, it indicated that PPi inhibited the activity of RIN, i.e. PCR amplification was successful, and the sample contained the target pathogen-specific gene fragment. As shown in Table 7, the color development of the PS group was significantly weakened, and the ΔA / A0 value was much lower than that of the NS group, with statistically significant difference (p < 0.001, one-way ANOVA). Figure 4

[0060] Example 6

[0061] Limit of detection of ESKAPE pathogen nucleic acid ​​

[0062] PCR product detection effect of specific gene fragments of gram-negative bacteria Acinetobacter baumannii (gltA) and gram-positive bacteria Staphylococcus aureus (nuc) under different plasmid copy numbers was tested. The PCR reaction system is shown in Table 4. The plasmid DNA of gltA and nuc was gradient diluted and the copy number was calculated to obtain the PCR product detection under the condition of 1 to 10000 copies. After PCR amplification, the samples were transferred to a well plate containing 1 mM H2O2, 832 μM TMB and 50 μg / mL RIN (0.1 M NaAc / HAc buffer, pH 4.0), and colorimetric detection was performed after catalytic reaction at room temperature for 5 min. As shown in Table 5, when the target nucleic acid concentration was reduced to 100 copies, the RIN detection system could still accurately identify and produce obvious POD-like activity inhibition signal. Figure 5

[0063] Example 7

[0064] RIN detection of simulated ESKAPE infection clinical samples

[0065] Three normal human serum samples were used for clinical sample simulation, and three groups of simulation samples were constructed for each ESKAPE pathogen. By spiking the solution containing the target pathogen plasmid into the human serum sample, seven kinds of simulated clinical samples of ESKAPE pathogen specific gene fragments (ddl, nuc, mdh, gltA, oprL, dnaJ, ampC) were constructed. The simulated samples were used to perform PCR amplification detection of the internal reference gene RNase P and the specific gene fragments of the target pathogen at the same time. The PCR reaction system is shown in Table 6: Table 6 PCR reaction system of simulated clinical samples Component Amount Final concentration 5x Q5 buffer 10 μL 1× 500 μM dNTP 3 μL 30 μM Upstream primer 2 μL 0.4 μM Downstream primer 2 μL 0.4 μM Plasmid DNA 1 μL 1 ng Q5 enzyme 0.5 μL 0.02 U / μL Serum 1 uL - Enzyme-free water 30.5 μL -

[0066] According to the annealing temperature in Table 5 and the PCR reaction system in Table 6, the nucleic acid amplification reaction (PS) was performed, and the amplification reaction was performed in the group without adding the corresponding plasmid DNA as the control group (NS). After amplification, the absorbance value of the nanoenzyme detection was analyzed by significant difference analysis. The nanoenzyme catalytic reaction conditions were consistent with the previous ones, i.e. the PCR samples were transferred to a test tube containing 1 mM H2O2, 832 μM TMB and 50 μg / mL RIN (0.1 M NaAc / HAc buffer, pH 4.0) for catalytic reaction at room temperature for 5 min, and colorimetric detection was performed immediately after the reaction. The distribution rule of ΔA / A0 value of three groups of samples was analyzed by heat map, and the results are shown in Table 7. Figure 6 ​As shown, the amplification products of the internal reference gene RNase P in all three groups of samples inhibited the enzyme-like activity of RIN, indicating that the three groups of serum samples are usable. Furthermore, the PS group with added plasmid DNA exhibited similar inhibitory characteristics to the RNase P-positive samples, indicating that RIN can be used to detect clinically simulated ESKAPE pathogens. The NS group showed no inhibitory effect, indicating that the relevant components in the serum do not affect the POD-like activity of RIN, and this inhibitory effect is indeed caused by PCR amplification.

[0067] Example 8

[0068] RIN testing of clinical samples with Staphylococcus aureus infection

[0069] Five clinical blood samples were collected from patients infected with Staphylococcus aureus (PS1–PS5) and non-infected individuals (NS1–NS5). Serum was extracted from the collected samples, and PCR amplification targeting the nuc gene (a Staphylococcus aureus-specific gene fragment) and the internal reference gene RNase P was performed. The primers targeting the nuc gene used were consistent with previous experiments. After amplification, the PCR products were transferred to a reaction system containing 1 mM H2O2, 832 μM TMB, and 50 μg / mL RIN (0.1 M NaAc / HAc buffer, pH 4.0), and the catalytic reaction was carried out at room temperature. The absorbance at 652 nm was measured to assess signal changes. The PCR reaction system is shown in Table 7: Table 7 PCR reaction system for clinical samples of Staphylococcus aureus infection Component Amount Final concentration 5x Q5 buffer 10 μL 1× 500 μM dNTP 3 μL 30 μM Upstream primer 2 μL 0.4 μM Downstream primer 2 μL 0.4 μM Q5 enzyme 0.5 μL 0.02 U / μL Clinical blood sample serum 1 uL - Enzyme-free water 31.5 μL -

[0070] Amplification was performed according to the PCR reaction system shown in Table 7. The detection principle is as follows: Since no nuc gene amplification occurred in the NS1–NS5 groups, no PPi was produced to inhibit the POD-like activity of RIN. In contrast, the PS1–PS5 groups showed successful amplification of the nuc gene, leading to the production of the byproduct PPi, thus exhibiting inhibition of RIN catalytic activity. The distribution pattern of ΔA / A0 values ​​of the ten groups was analyzed by heatmap, as shown below. Figure 7 As shown, the amplified products of the internal reference gene RNase P in all ten groups of samples inhibited the enzyme-like activity of RIN, indicating that the obtained clinical blood samples are usable. The nuc gene amplification results showed that the PS1–PS5 groups exhibited nuc amplification, demonstrating a significant inhibitory effect, while the NS1–NS5 groups did not amplify and did not show any inhibitory effect on the enzyme-like activity of RIN. These results indicate that the RIN-based detection platform has excellent specificity and sensitivity and can be effectively applied to the nucleic acid detection of ESKAPE pathogens in real clinical samples, showing promising translational potential.

Claims

1. A method for nucleic acid detection based on iron-ruthenium nanoszyme (RIN), characterized in that Comprising the following steps: (1) Synthesis of bimetallic oxide nanoscale enzyme: using self-template hydrothermal synthesis process, two kinds of metal salts of iron salt and ruthenium salt are introduced to form a precursor, and a bimetallic oxide nanoscale enzyme RIN with peroxidase-like (POD) activity is obtained by air calcination; The preparation of the bimetallic oxide nanoscale enzyme in step (1) : glycerol and isopropyl alcohol are mixed and stirred for 10-90 min, then two kinds of metal salts of iron salt and ruthenium salt are added and continue to stir, wherein the molar ratio of glycerol, isopropyl alcohol and metal salt is 1 : 3.5-6 : 2.5-4.5, and the molar ratio of the two kinds of metal salts is 1 : 0.5-5. The above solution is stirred for 20-90 min and then transferred to a hydrothermal reactor, which is hydrothermally reacted at 150-200℃ for 4-12 h, centrifuged, washed and dried to obtain the bimetallic nanoscale enzyme RIN; (2) PPi detection: in an acidic environment, the bimetallic oxide nanoscale enzyme catalyzes the color development reaction of hydrogen peroxide (H2O2) oxidizing 3,3',5,5'-tetramethylbenzidine (TMB), and the color development is weakened by the inhibition of enzyme activity caused by pyrophosphate (PPi) to realize PPi detection; (3) Nucleic acid detection: the target nucleic acid sample is subjected to polymerase chain reaction (PCR) amplification, and PPi byproduct is generated during the reaction; the amplification product is mixed with the bimetallic oxide nanoscale enzyme, TMB and H2O2, and reacted under acidic conditions and room temperature, and the existence of target nucleic acid is determined by the specific inhibition of PPi on the color development reaction of TMB catalyzed by the nanoscale enzyme.

2. The method of claim 1, wherein RIN catalytic color reagent comprises RIN, TMB and H2O2; the final concentration of RIN used in PPi detection and nucleic acid detection is 50 μg / mL, the final concentration of H2O2 is 1 mM, and the final concentration of TMB is 832 μM; the reaction is carried out in a pH 4.0 HAc / NaAc buffer solution; wherein the POD-like activity of RIN can be inhibited by the byproduct PPi generated during the PCR amplification reaction, and the colorimetric determination of the presence / absence of target pathogen nucleic acid is realized according to this characteristic.

3. The method of claim 1, wherein the amplification reaction targets ESKAPE pathogen- associated drug resistance genes using primers comprising gltA , ddl , mdh , nuc , oprL , dnaJ and ampC .

4. The method of claim 1, wherein when the sample does not contain the target pathogen nucleic acid, the PCR amplification condition is not met, and the process does not generate PPi, and RIN catalyzes TMB to produce blue color development reaction; and when the sample contains the target nucleic acid, the byproduct PPi is generated after PCR amplification, and the POD-like activity of RIN is inhibited by PPi, and the reaction system presents colorless or light blue color.

5. The method of claim 1, wherein the nucleic acid detection is read by the naked eye or a portable colorimeter.

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