Kit for detecting salmonella enteritidis and application thereof

Abstract

The invention discloses a kit for detecting salmonella enteritidis and application of the kit, and belongs to the technical field of biomolecular detection. The kit comprises the following components: an allosteric aptamer probe of which the nucleotide sequence is as shown in SEQ ID NO.1, a template chain of which the nucleotide sequence is as shown in SEQ ID NO.3 and a fluorescence report probe of which the nucleotide sequence is as shown in SEQ ID NO.4. According to the kit, salmonella enteritidis can be distinguished from other impurities, and the specificity is high; a visual fluorescence signal can be obtained and is easy to interpret; according to the invention, a template chain can be cyclically utilized in the reaction to generate a Trigger chain to guide the TtAgo protein to efficiently and specifically cut a fluorescent reporter probe, the output signal is increased, the detection time is short, the detection limit is low, the sensitivity is high, the specificity is good, and the method has practical popularization and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomolecule detection, and in particular relates to a kit for detecting enteritidis salmonella and an application thereof. Background Art

[0002] Salmonella is a common foodborne pathogen that is widely found in nature, especially in animal products such as meat and eggs. According to statistics, 70% to 80% of bacterial food poisoning in my country is caused by Salmonella. Once a person consumes a large amount of Salmonella (usually 10 5 ~10 6 / g) of animal products may cause bacterial infection, and then food poisoning occurs under the action of toxins, leading to diseases such as gastroenteritis, typhoid and paratyphoid. Therefore, the detection of Salmonella is an important means to prevent and control food poisoning and ensure public health safety. At present, the detection of Enteritidis Salmonella still mainly relies on traditional bacterial culture, ELISA, serotyping and mass spectrometry technology. Traditional bacterial culture, ELISA, and serotyping generally require a bacterial culture enrichment process, which takes a long time and has low sensitivity, and is not suitable for immediate diagnosis; the detection limit of mass spectrometry technology is 10 5 CFU / mL, and the accuracy rate is less than 90%. The instrument is expensive and not suitable for detection in primary hospitals or early screening of Enteritidis Salmonella. Restriction endonuclease analysis of 16sRNA gene amplified by PCR has high restrictions and requirements on laboratory environment and the professional level of operators. Therefore, this method is not suitable for primary hospitals or screening of large-scale outbreaks of Enteritidis Salmonella.

[0003] There are also detection kits for Enteritidis Salmonella in China. Wang Taihua et al. used the specific primer probe LAMP of Enteritidis to rapidly detect Enteritidis Salmonella, with a detection limit of 1×10 -3 Copies / μL, but this method requires sample nucleic acid extraction first, which is not suitable for some primary medical institutions. Secondly, Jiao Xin'an and others used a surface plasmon resonance technology assay kit to detect Salmonella. This kit mainly includes the preparation of a Salmonella detection chip; incubation of the bacteria to be tested with antibodies; separation of free antibodies; and detection of the response signal of free antibodies using surface plasmon resonance technology. The antibody is a monoclonal antibody against the Salmonella PagN protein, which is expensive, and the surface plasmon resonance technology detection and analysis is complex and has high requirements for instruments, so a new detection kit is needed to optimize the above defects.

[0004] Therefore, it is very necessary to design a detection method for Enteritidis Salmonella that is highly sensitive and specific, simple to operate, and rapid. Summary of the invention

[0005] To solve the above problems, the present invention aims to provide a kit for fluorescent detection of Salmonella enteritidis based on an allosteric aptamer isothermal amplification sensing method, a fluorescent biosensor and applications thereof.

[0006] The present invention provides an allosteric aptamer probe, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a kit for detecting Enteritidis Salmonella, which comprises the following components: an allosteric aptamer probe with a nucleotide sequence as shown in SEQ ID NO.1, a template strand with a nucleotide sequence as shown in SEQ ID NO.3, and a fluorescent reporter probe (Probe) with a nucleotide sequence as shown in SEQ ID NO.4.

[0008] Furthermore, the molar ratio of the allosteric aptamer probe, the template chain (Template), and the fluorescent reporter probe is 0.2-0.8: 0.02-0.08: 2.2-9: 10000-40000; preferably 0.4: 0.04: 4.5: 20000.

[0009] Furthermore, the kit also includes the following components: dNTP, 10×ThermoPol buffer, 10×NEBufferr3.1 buffer, single-stranded binding protein, Bst DNA polymerase, Nt.BstNBI nickase and TtAgo protein.

[0010] Furthermore, the ratio of the allosteric aptamer probe, dNTP, single-stranded binding protein, Bst DNA polymerase, Nt.BstNBI nickase, and TtAgo protein is 0.2-0.8pmol: 10-40nmol: 0.05-0.2μg: 0.5-2U: 10U: 0.62-2.5pmol, preferably 0.4pmol: 20nmol: 0.1μg: 1.04U: 10U: 1.25pmol.

[0011] Furthermore, the pH value of the 10×ThermoPol buffer is 8.8, and the components are: 200mM Tris-HCl, 100mM (NH4)2SO4, 100mM KCl, 20mM MgSO4, 1% X-100, and the rest is water.

[0012] Furthermore, the pH value of the 10×NEBufferr3.1 buffer is 7.9, and its components are: 1000 mM NaCl, 500 mM Tris-HCl, 100 mM MgCl2, 1000 μg / ml recombinant albumin, and the rest is water.

[0013] Furthermore, the recombinant albumin is bovine serum albumin.

[0014] Furthermore, the template chain and the fluorescent reporter probe chain are synthesized as dry powder and then diluted with DEPC-treated water; the allosteric aptamer probe is synthesized as dry powder and then diluted with PBS.

[0015] The present invention also provides use of the kit in preparing a reagent or device for detecting Salmonella enteritidis.

[0016] The present invention also provides a fluorescent biosensor for detecting Salmonella enteritidis. The fluorescent biosensor is prepared by using the above-mentioned kit according to the following steps:

[0017] 1) Preparation of reaction liquid A

[0018] Mix the template strand, dNTP, 10×ThermoPol buffer, and single-stranded binding protein on ice, and add DEPC-treated water to make up to volume to obtain reaction liquid A;

[0019] 2) Preparation of reaction liquid B

[0020] Mix Bst DNA polymerase, Nt.BstNBI nickase, and 10×NEBufferr3.1 buffer on ice, and add DEPC-treated water to make up to volume to obtain reaction liquid B;

[0021] 3) Preparation of reaction liquid C

[0022] Take TtAgo protein, fluorescent reporter probe, and 10×ThermoPol buffer, mix them on ice, and add DEPC-treated water to make up to volume to obtain reaction liquid C;

[0023] 4) Preparation of fluorescent biosensor

[0024] The sample solution to be tested and the allosteric aptamer probe are mixed and incubated to obtain a mixed solution; a portion of the mixed solution is mixed with reaction solution A and reaction solution B to react to obtain an amplification product; a portion of the amplification product is added to reaction solution C to continue the reaction to obtain a fluorescent biosensor for detecting Salmonella enteritidis.

[0025] Further, in step 1), the volume ratio of the 10×ThermoPol buffer to the reaction liquid A is 0.8:10; in step 2), the volume ratio of the 10×NEBufferr3.1 buffer to the reaction liquid B is 1:8; in step 3), the volume ratio of the 10×ThermoPol buffer to the reaction liquid C is 1.5:18.5; the volume ratio of the reaction liquid A, the reaction liquid B, and the reaction liquid C is 10:8:18.5;

[0026] And / or, in step 1), the concentration of the template strand is 50-200 nM, preferably 100 nM; the concentration of the dNTP is 10 mM; the concentration of the single-stranded binding protein is 500 μg / mL; in step 2), the concentration of the Bst DNA polymerase is 8 U / μL; the concentration of the Nt.BstNBI nickase is 10000 U / mL; in step 3), the concentration of the TtAgo protein is 1 μM; the concentration of the fluorescent reporter probe is 50-500 nM, preferably 225 nM;

[0027] And / or, in step 4), the volume ratio of the sample solution to be tested and the allosteric aptamer probe is 1:1, the volume of the partial mixed solution is one twentieth of the volume of the mixed solution, and the volume of the partial amplified product is three fortieths of the volume of the amplified product.

[0028] And / or, in step 4), the incubation temperature is 50-60°C, preferably 55°C, the time is 30-150min, preferably 60min; the reaction temperature is 50-60°C, preferably 55°C, the time is 4-5min, preferably 5min; the continued reaction temperature is 75-85°C, preferably 80°C, the time is 8-14min, preferably 10min;

[0029] Furthermore, the sample solution to be tested is milk, serum or feces suspension.

[0030] The present invention also provides a method for detecting Enteritidis Salmonella, which comprises the following steps: using an enzyme marker or an ultraviolet spectrophotometer to detect the above-mentioned fluorescent biosensor.

[0031] The present invention has achieved the following beneficial effects:

[0032] (1) Relying on molecular recognition elements - aptamers to capture Enteritidis Salmonella, the sequence of the aptamer in the fluorescent biosensor of the present invention is designed according to the characteristics of Enteritidis Salmonella, which can distinguish Enteritidis Salmonella from other impurities with strong specificity. The aptamer adopts the hairpin allosteric probe mode for blocking, which can effectively improve the blocking efficiency and detection signal-to-noise ratio compared with the traditional mode of adding additional blocking chains for blocking;

[0033] (2) Under the action of fluorescent reporter probes, a visualized fluorescent signal can be obtained, which is easy to interpret;

[0034] (3) The isothermal amplification involved in the fluorescent biosensor of the present invention can generate a large number of trigger chains at a constant temperature in a short period of time, so that the method can detect low-abundance targets in clinical specimens and significantly shorten the detection time;

[0035] (4) When using the fluorescent biosensor of the present invention for detection, there is no need for nucleic acid extraction or bacterial culture. The fluorescent sensor is cheap and portable and can be used in two modes: qualitative analysis by ultraviolet irradiation and naked eye analysis or quantitative analysis by instrument. It is suitable for use in environments with limited resources.

[0036] (5) The detection phase of the fluorescent biosensor of the present invention takes only 15 minutes, the detection speed is fast, the detection limit is 0.32 CFU / mL, the sensitivity is high, and the specificity is good.

[0037] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0038] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of the principle of isothermal amplification ultrasensitive detection of Enteritidis Salmonella based on TtAgo protein of the present invention.

[0040] Figure 2 The results of the feasibility analysis of the allosteric aptamer for recognizing Salmonella enteritidis are shown in the figure below. (a) The fluorescent aptamer probe verifies the ability of bacteria to open the allosteric aptamer; (b) Fluorescence confocal imaging verifies the binding ability of the aptamer to bacteria.

[0041] Figure 3 The PAGE electrophoresis results for the feasibility analysis and verification of isothermal amplification of the present invention are as follows: (a) PAGE electrophoresis verifies the feasibility of isothermal amplification triggered by the simulated chain; (b) PAGE electrophoresis verifies the feasibility of isothermal amplification triggered by bacteria.

[0042] Figure 4 The results of the feasibility analysis of the TtAgo protein cleavage in the present invention are as follows: (a) Fluorescence verification of the cleavage activity of different components of TtAgo protein; (b) PAGE electrophoresis verification of TtAgo cleavage of the reporter probe as the reaction time increases.

[0043] Figure 5 The results of the feasibility analysis of the overall detection system of the present invention are as follows: (a) Fluorescence results of the detection system of different components; (b) PAGE electrophoresis imaging of the detection system of different components.

[0044] Figure 6The results of the optimization of the action conditions in the incubation mixing stage of the sample solution and the allosteric aptamer probe and the isothermal amplification stage of the present invention are shown in FIG. (a) Optimization of the incubation temperature of the allosteric aptamer probe and the sample solution; (b) Optimization of the incubation time of the allosteric aptamer probe and the sample solution; (c) Optimization of the amount of the allosteric aptamer probe added; (d) Optimization of the concentration of the isothermal amplification polymerase; (e) Optimization of the concentration of the isothermal amplification nickase; (f) Optimization of the isothermal amplification action time.

[0045] Figure 7 The results of the optimization of the conditions for the TtAgo protein cleaving the reporter probe in the present invention are shown in Figure 1. (a) Optimization of TtAgo protein concentration; (b) Optimization of TtAgo protein action time; (c) Optimization of fluorescent reporter probe concentration.

[0046] Figure 8 The sensitivity analysis results of the detection method of the present invention are shown in Figure 1. (a) Fluorescence intensity response curve for different concentrations of Enteritidis Salmonella; (b) Fluorescence intensity summary curve for different concentrations of Enteritidis Salmonella.

[0047] Fig. 9 This is a calibration curve of the logarithm of the concentration of Salmonella enteritidis and the fluorescence intensity signal of the present invention. (a)

[0048] 10 0 ~10 3 Calibration curve of bacterial concentration; (b) 10 4 ~10 8 Calibration curve of bacterial concentration.

[0049] Fig.10 This is a graph showing the specificity analysis results of the detection method of the present invention. DETAILED DESCRIPTION

[0050] The reagents and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0051] The nucleotide sequence information involved in the specific implementation of the present invention is shown in Table 1, and the main materials and reagents information is shown in Table 2.

[0052] Table 1. Nucleotide sequence information

[0053]

[0054]

[0055] Table 2. Main materials and reagents

[0056]

[0057] Example 1 Kit for detecting Salmonella enteritidis of the present invention

[0058] 1. Kit Composition

[0059] Table 3. Kit composition

[0060]

[0061]

[0062] Among them, the nucleotide sequence of the allosteric aptamer probe is shown in SEQ ID NO.1, the nucleotide sequence of Template is shown in SEQ ID NO.3, and the nucleotide sequence of Probe is shown in SEQ ID NO.4.

[0063] The pH value of 10×ThermoPol buffer is 8.8, and the composition is: 200mM Tris-HCl, 100mM (NH4)2SO4, 100mM KCl, 20mM MgSO4, 1% X-100, and the rest is water.

[0064] The pH value of 10×NEBufferr3.1 buffer is 7.9, and the composition is: 1000mM NaCl, 500mM Tris-HCl, 100mM MgCl2, 1000μg / ml recombinant albumin, and the rest is water, wherein the recombinant albumin is bovine serum albumin.

[0065] 2. Use of the kit - Preparation of isothermal amplification fluorescent biosensor based on TtAgo protein

[0066] ① Allosteric aptamer probe-test sample incubation

[0067] Take 20 μL of 200 nM allosteric aptamer probe and an equal volume of the sample solution to be tested and incubate at 55°C for 60 min to obtain a mixed solution;

[0068] ②Prepare reaction solution A, reaction solution B, reaction solution C

[0069] Take 2 μL of 100 nM Template, 2 μL of 10 mM dNTP mixed solution, 0.8 μL of 10×ThermoPol buffer, and 0.2 μL of 500 μg / mL single-stranded binding protein, mix them on ice, and add DEPC-treated water (also known as DEPC-treated water) to a total volume of 10 μL to obtain reaction solution A;

[0070] Take 0.13 μL 8U / μL Bst DNA polymerase, 1 μL 10000U / mL Nt.BstNBI nickase, and 1 μL 10×NEBufferr3.1 buffer, mix them on ice, and add DEPC-treated water to 8 μL to obtain reaction solution B;

[0071] 1.25 μL of 1 μM TtAgo protein, 5 μL of 900 nM fluorescent reporter probe (referred to as Probe), and 1.5 μL of 10× ThermoPol buffer were mixed on ice, and DEPC-treated water was added to a total volume of 18.5 μL to obtain reaction solution C;

[0072] ③Isothermal amplification produces the trigger chain of the amplification product

[0073] After mixing reaction solution A and reaction solution B on ice, add 2 μL of the mixed solution incubated in step ①, and incubate the mixed solution at 55°C for 5 minutes to obtain the amplified product Trigger chain;

[0074] ④Preparation of fluorescent biosensor for detection of Salmonella enteritidis

[0075] Take 1.5 μL of the amplified product Trigger chain obtained in step ③ and add it to the reaction solution C. Incubate at 80°C for 10 min. Take 10 μL of the incubated liquid and detect the fluorescence signal in an ELISA instrument.

[0076] The result of combining steps ①②③④ is the isothermal amplification fluorescent biosensor based on TtAgo protein for detecting Enteritidis Salmonella.

[0077] Experimental Example 1 Ultrasensitive Detection of Salmonella Enteritidis by Isothermal Amplification Fluorescence Sensor Based on TtAgo Protein I. Detection Principle

[0078] The working principle of the isothermal amplification fluorescence sensor based on TtAgo protein is as follows:

[0079] When Salmonella enterica serovar Enteritidis is present in the sample solution, due to the effect of affinity, the allosteric aptamer probe will bind to Salmonella enterica serovar Enteritidis and undergo a conformational change, thereby opening its own hairpin structure, exposing the nucleic acid domain that can complementarily pair with the Template chain, and proceeding to the next reaction. After the allosteric aptamer probe binds to the bacteria, the exposed nucleic acid domain binds to the Template chain, and isothermally amplified under the action of Bst polymerase and Nt.BstNBI nickase, and continuously cycles in a short period of time to produce a large number of Trigger chains of specified lengths for the next reaction. The Trigger chain generated by isothermal amplification binds to the TtAgo protein and guides it to efficiently cut the substrate fluorescent reporter probe present in the system, so that the FAM fluorescent group blocked by BHQ releases fluorescence to generate a fluorescent signal, thereby achieving the purpose of detection. On the contrary, when Salmonella enterica serovar Enteritidis is not present in the system, the nucleic acid domain in the allosteric aptamer probe that complementarily pairs with the Template chain is blocked, and the next amplification cannot be performed, and there is no fluorescent signal output ( Figure 1 ).

[0080] 2. Research on biomolecular detection methods for Salmonella enteritidis

[0081] 1. Application research based on detection principles

[0082] 1.1. Verification of aptamer bacterial affinity

[0083] First, different concentrations of Salmonella enteritidis liquid (0, 10 6 , 10 8 CFU / mL), and 20μL of bacterial solution was taken for each portion. Subsequently, a fluorescent allosteric aptamer probe (SEQ ID NO.2) with a concentration of 1μM was prepared, incubated at 95℃ for 10min and 4℃ for 2h before use. After incubating equal volumes of bacterial solution and fluorescent allosteric aptamer probe at 50℃ for 1h, the differences in the three groups of fluorescence signals were detected using 494nm excitation wave and 522nm emission wave to verify the affinity of Enteritidis Salmonella with the aptamer and the feasibility of opening the allosteric aptamer probe. At the same time, laser confocal microscopy was used to verify the affinity and specificity of the aptamer to Enteritidis Salmonella ( Figure 2 ).

[0084] 1.2. Electrophoresis verification of isothermal amplification and TtAgo cleavage effects

[0085] First, prepare different concentrations of allosteric aptamer probe template binding domain simulation chain (SEQ ID NO.6) (hereinafter referred to as simulation chain) and different concentrations of enteritidis Salmonella liquid. Incubate the enteritidis Salmonella liquid and the allosteric aptamer probe at 50°C for 1 hour and set aside (hereinafter referred to as the mixed solution). Take the simulation chain and the mixed solution and add them to the amplification system composed of reaction solution A and reaction solution B. After incubation at 55°C for 5 minutes, the amplification product is obtained. The amplification product is verified by electrophoresis. The electrophoresis results are shown in Figure 3 .like Figure 3 a and Figure 3 As shown in b, in the amplified system, a new band identical to the target gDNA (SEQ ID NO.5, hereinafter referred to as Trigger) that guides TtAgo protein cleavage appeared in the lanes of the higher concentration bacterial solution and the simulated chain, indicating that the isothermal amplification system was successfully constructed. The amplified product Trigger was added to the reaction solution C with different component contents and incubated at 80°C for 10 minutes, and then the product fluorescence intensity was detected using 494nm excitation wave and 522nm emission wave, as shown in FIG. Figure 4 As shown in a, when TtAgo and Trigger (gDNA) are present at the same time, the substrate fluorescent reporter probe is well cleaved, which greatly increases the fluorescent signal. In addition, Trigger is added to the reaction solution C of all components and incubated at 80°C for different time periods, and the product is verified by electrophoresis, as shown in Figure 4As shown in b, with the increase of incubation time, the Trigger-Probe double strands decreased and the cleavage products increased, indicating that the Trigger chain guided the TtAgo protein to cleave the fluorescent reporter probe.

[0086] 2. Fluorescent biosensor validation

[0087] Simulate the reaction process of 1.2, fluorescence signal detection, and omit one or several components in the control group to study the effects of each component. Figure 5 .like Figure 5 As shown in curve a, when only the fluorescent reporter probe (hereinafter referred to as Probe) is present, the fluorescent signal is very low, indicating the presence of a weak background signal (curve a); when only TtAgo and Probe are present, the fluorescent signal is low, indicating that TtAgo will not non-specifically cut the Probe (curve b); when any enzyme is missing in the reaction system, the fluorescent signal is low, indicating the lack of any amplification or cutting link, and signal output cannot be achieved (curves c, d, e, f); when gDNA, TtAgo, and Probe are manually added, a fluorescent signal is generated, indicating the feasibility of cutting (curve h); when all components are present, the fluorescent signal is greatly enhanced, indicating the feasibility of the overall sensor and its signal amplification capability (curve g). At the same time, the overall reaction process of simulation 1.2 and fluorescence signal detection is verified by PAGE electrophoresis, as shown in Fig. Figure 5 As shown in b, when there is no Trigger generated by amplification in the system, no cutting effect can be produced (lane 5); only when all components are present in the system at the same time can the reporter probe be cut (lane 6); when any component of the polymerase, nickase, or TtAgo protein is missing, the fluorescent reporter probe cannot be cut (lanes 7, 8, and 9).

[0088] 3. Optimize experimental parameters

[0089] In order to obtain the best fluorescence signal, the experimental conditions of allosteric aptamer probe-bacteria recognition (corresponding to step ① in Example 1), isothermal amplification (corresponding to step ③ in Example 1), and TtAgo protein cleavage (corresponding to step ④ in Example 1) were optimized.

[0090] 3.1 Optimization of experimental conditions for allosteric aptamer probe-bacteria recognition

[0091] The method of Example 1 is referred to, except that the amount of aptamer added, incubation time and incubation temperature in step ① of Example 1 are changed.

[0092] like Figure 6As shown in Figure c, as the amount of aptamer added increases, the signal-to-noise ratio of the sensor system increases first and then decreases. When the amount of aptamer added is 10 nM, the signal-to-noise ratio of the sensor system is optimal. If the aptamer concentration is further increased, the fluorescence signal value will be increased, but the single background signal will also increase significantly. The present invention also compares the ability of bacteria to open allosteric aptamer probes at different temperatures and different incubation times, such as Figure 6 a and Figure 6 As shown in b, the best binding effect was achieved when the incubation time was 60 min and the incubation temperature was 55° C. Finally, the allosteric aptamer probe 10 nM, the bacteria-aptamer incubation time 60 min, and the incubation temperature 55° C. were selected for subsequent experiments.

[0093] 3.2 Optimization of experimental conditions for isothermal amplification

[0094] The method of Example 1 was referred to, except that the amount of Bst DNA polymerase added, the amount of Nt.BstNBI nickase added, and the isothermal amplification incubation time in step ③ of Example 1 were changed.

[0095] The reaction conditions of the isothermal amplification step will directly affect the trigger chain generated by the isothermal amplification. The present invention also optimizes the reaction conditions of the amplification step. Figure 6 As shown in Figure d, the fluorescence values ​​of the same target concentration experiments were detected when the Bst DNA polymerase addition amount was 0.25, 0.5, 0.75, 1, 1.25, 1.5, and 1.75 U. When the Bst DNA polymerase addition amount was 1 U, it had the highest signal-to-noise ratio. At the same time, the effect of the Nt.BstNBI nickase addition amount was compared, as shown in Figure d. Figure 6 As shown in Figure e, when the amount of Nt.BstNBI nickase added was 2, 4, 6, 8, 10, 12, 14, and 16 U, the signal-to-noise ratio curve first increased and then decreased. When the amount of Nt.BstNBI nickase added to the reaction system was 10 U, the best experimental effect was achieved. In addition, when the isothermal amplification incubation time was 5 min, the best amplification effect was achieved ( Figure 6 f).

[0096] 3.3 Optimization of experimental conditions for TtAgo protein cleavage

[0097] The method of Example 1 is referred to, except that the amount of TtAgo protein added, the action time, and the concentration of the substrate fluorescent reporter probe in step ④ of Example 1 are changed.

[0098] The TtAgo protein cleavage process is the signal conversion output part of the fluorescent sensor. The amount of TtAgo protein added, the amount of fluorescent reporter probe added, and the action time of TtAgo protein will have a profound impact on the signal output. Figure 7As shown in a, when the amount of TtAgo protein added was 0, 0.75, 1, 1.25, 1.5, 1.75, and 2 pmol, the fluorescent probe cleavage efficiency guided by the same concentration of target amplification product gradually increased, reaching a higher level at 1.25 pmol. From the perspective of economic benefit, 1.25 pmol was selected as the optimized concentration of TtAgo protein; in addition, the TtAgo cleavage time was optimized, as shown in Figure 7 As shown in b, TtAgo protein cuts the substrate fluorescent reporter probe under the guidance of the amplified product, and the best signal-to-noise ratio is achieved when the action time is 10 min; Figure 7 As shown in Figure c, the concentration of the substrate fluorescent reporter probe (referred to as Probe) was optimized, and 25, 75, 125, 175, 225, 275, and 325 nM fluorescent reporter probes were selected for the experiment. The signal-to-noise ratio curve showed a trend of first rising and then flattening, and finally 225 nM fluorescent reporter probe was selected as the optimal substrate level.

[0099] The best experimental conditions after optimization were obtained according to the above results (i.e. the experimental conditions of Example 1).

[0100] 4. Detection range and sensitivity

[0101] Under the above-mentioned optimal experimental conditions after optimization (i.e., the experimental conditions of Example 1), an isothermal amplification fluorescent biosensor based on TtAgo protein for the detection of Enteritidis Salmonella was prepared. Different concentrations of Enteritidis Salmonella liquid were prepared for testing, and an ELISA instrument was used to perform ultraviolet spectrum wavelength scanning (excitation wave of 490nm, emission wave range of 505-655nm) and fixed-point fluorescence signal detection (excitation wave of 494nm, emission wave of 522nm). The fluorescence signal response curve was drawn, as shown in FIG. Figure 8 As shown, in 10 0 ~10 7 CFU / mL, the fluorescence signal of the scanning spectrum increases with the increase of the concentration of Salmonella enteritidis. The calibration curve of the fluorescence signal and the logarithm of the target concentration is established by segment fitting. Fig. 9 The linear range of this sensor is shown as: 10 0 ~10 3 CFU / mL, linear regression equation: y = 216.44 + 114.78lgC, R 2 =0.95;10 4 ~10 8 CFU / mL, linear regression equation: y = -4865.72 + 1489.66lgC, R 2 =0.98. The detection limit was 0.32 CFU / mL.

[0102] 5. Detection specificity

[0103] Under the above optimized optimal experimental conditions (i.e., the experimental conditions of Example 1), an isothermal amplification fluorescent biosensor based on TtAgo protein for the detection of Enteritidis Salmonella was prepared. The fluorescent biosensor system was used to perform control analysis on common clinical bacteria including Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, Proteus, and Staphylococcus aureus to verify the specificity. The concentration of all bacterial solutions was unified to 10 7 CFU / mL, and measure the fluorescence signal of each group. Fig.10 As shown, there is a stronger fluorescence signal when Salmonella Enteritidis is present, and it is significantly higher than that of the control group, indicating that this reaction system has good specificity for Salmonella Enteritidis.

[0104] 6. Detection of Salmonella enteritidis in contaminated milk and clinical serum specimens

[0105] Use milk purchased from the market without bacterial contamination and serum samples from clinical examination as solvents. Dilute milk and serum tenfold to prepare 10 2 CFU / mL, 10 5 CFU / mL and 10 7 The recovery rates of the sensor were calculated after fluorescence detection of the Enteritidis Salmonella liquid with a concentration of CFU / mL. As shown in Table 4, the recovery rates of Enteritidis Salmonella at different concentrations in milk ranged from 95.11% to 105.88%; the recovery rates of Enteritidis Salmonella at different concentrations in serum ranged from 91.36% to 97.58%, indicating that the reaction system was not interfered by substances in milk and serum, and had excellent practical application value in the detection of Enteritidis Salmonella in clinical specimens.

[0106] Table 4. Recovery rate of sensors

[0107]

[0108] In summary, the present invention has developed an isothermal amplification fluorescent biosensor based on TtAgo protein, which can be used for ultrasensitive detection of enteritis Salmonella. When enteritis Salmonella is present, the allosteric aptamer probe preferentially binds to enteritis Salmonella, the allosteric aptamer probe is opened, exposing the Template chain binding nucleic acid domain, the allosteric aptamer probe binds to the Template chain and starts isothermal amplification, generating a large number of Trigger chains, and the Trigger chains generated by amplification guide TtAgo to specifically cut the substrate fluorescent reporter probe, thereby generating a fluorescent signal. The sensor of the present invention has a short detection time, a low detection limit, a high sensitivity, and a good specificity, and has a practical prospect for promotion and application.

Claims

1. An allosteric aptamer probe, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A kit for detecting Salmonella enteritidis, characterized in that: The kit comprises the following components: an allosteric aptamer probe having a nucleotide sequence as shown in SEQ ID NO.1, a template strand having a nucleotide sequence as shown in SEQ ID NO.3, and a fluorescent reporter probe having a nucleotide sequence as shown in SEQ ID NO.

4.

3. The kit according to claim 2, characterized in that: The molar ratio of the allosteric aptamer probe, the template chain, and the fluorescent reporter probe is 0.2-0.8: 0.01-0.04: 2.2-9: 10000-40000; preferably 0.4: 0.02: 4.5: 20000.

4. The kit according to any one of claims 2 to 3, characterized in that: The kit also includes the following components: dNTP, 10×ThermoPol buffer, 10×NEBufferr3.1 buffer, single-strand binding protein, Bst DNA polymerase, Nt.BstNBI nickase and TtAgo protein.

5. The kit according to claim 4, characterized in that: The ratio of the allosteric aptamer probe, dNTP, single-stranded binding protein, Bst DNA polymerase, Nt.BstNBI nickase, and TtAgo protein is 0.2-0.8pmol: 10-40nmol: 0.05-0.2μg: 0.5-2U: 10U: 0.62-2.5pmol, preferably 0.4pmol: 20nmol: 0.1μg: 1.04U: 10U: 1.25pmol.

6. Use of the kit according to any one of claims 2 to 5 in preparing a reagent or device for detecting Salmonella enteritidis.

7. A fluorescent biosensor for detecting Salmonella enteritidis, characterized in that: The fluorescent biosensor is prepared by using the kit according to any one of claims 4 to 5 according to the following steps: 1) Preparation of reaction liquid A Take the template strand, dNTP, 10×ThermoPol buffer, and single-stranded binding protein and mix them on ice, and add DEPC-treated water to make up to volume to obtain reaction liquid A; 2) Preparation of reaction liquid B Mix Bst DNA polymerase, Nt.BstNBI nickase, and 10×NEBufferr3.1 buffer on ice, and add DEPC-treated water to make up to volume to obtain reaction liquid B; 3) Preparation of reaction liquid C Take TtAgo protein, fluorescent reporter probe, and 10×ThermoPol buffer, mix them on ice, and add DEPC-treated water to make up to volume to obtain reaction liquid C; 4) Preparation of fluorescent biosensor The sample solution to be tested and the allosteric aptamer probe are mixed and incubated to obtain a mixed solution; a portion of the mixed solution is mixed with reaction solution A and reaction solution B to react to obtain an amplification product; a portion of the amplification product is added to reaction solution C to continue the reaction to obtain a fluorescent biosensor for detecting Salmonella enteritidis.

8. The fluorescent biosensor according to claim 7, characterized in that: In step 1), the volume ratio of the 10×ThermoPol buffer to the reaction liquid A is 0.8:10; in step 2), the volume ratio of the 10×NEBufferr3.1 buffer to the reaction liquid B is 1:8; in step 3), the volume ratio of the 10×ThermoPol buffer to the reaction liquid C is 1.5:18.5; the volume ratio of the reaction liquid A, the reaction liquid B, and the reaction liquid C is 10:8:18.5; And / or, in step 1), the concentration of the template strand is 50-200 nM, preferably 100 nM; the concentration of the dNTP is 10 mM; the concentration of the single-stranded binding protein is 500 μg / mL; in step 2), the concentration of the Bst DNA polymerase is 8 U / μL; the concentration of the Nt.BstNBI nickase is 10000 U / mL; in step 3), the concentration of the TtAgo protein is 1 μM; the concentration of the fluorescent reporter probe is 50-500 nM, preferably 225 nM; And / or, in step 4), the volume ratio of the sample solution to be tested and the allosteric aptamer probe is 1:1, the volume of the partial mixed solution is one twentieth of the volume of the mixed solution, and the volume of the partial amplified product is three fortieths of the volume of the amplified product. And / or, in step 4), the incubation temperature is 50-60°C, preferably 55°C, the time is 30-150min, preferably 60min; the reaction temperature is 50-60°C, preferably 55°C, the time is 4-5min, preferably 5min; the continued reaction temperature is 75-85°C, preferably 80°C, the time is 8-14min, preferably 10min; 9. The fluorescent biosensor according to claim 7 or 8, characterized in that: The sample solution to be tested is milk, serum or feces suspension.

10. A method for detecting Enteritidis Salmonella, characterized in that: The method comprises the following steps: using an ELISA reader or an ultraviolet spectrophotometer to detect the fluorescent biosensor according to any one of claims 7 to 9.

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