Rapid freezing probe fixed CRISPR (clustered regularly interspaced short palindromic repeats) cyclic cleavage mediated signal enhanced vibrio parahaemolyticus biosensor and application

Abstract

The invention discloses a quick freezing probe fixed CRISPR (clustered regularly interspaced short palindromic repeats) cyclic cleavage mediated signal enhanced vibrio parahaemolyticus biosensor and application thereof. The vibrio parahaemolyticus biosensor comprises an RPA (recombinase polymerase amplification) isothermal amplification primer pair, a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas12a system and an electrochemical three-electrode system, the RPA isothermal amplification primer pair comprises a tdh primer pair and a trh primer pair, the tdh primer pair comprises a tdh upstream primer and a tdh downstream primer, and the trh primer pair comprises a trh upstream primer and a trh downstream primer; the CRISPR / Cas12a system comprises tdh-crRNA (Ribonucleic Acid) aiming at a tdh gene, trh-crRNA aiming at a trh gene, and Cas12a protein. The vibrio parahaemolyticus detection method provided by the invention can be completed within 30 minutes, has the characteristics of simplicity, rapidness, strong anti-interference capability and the like, is suitable for the field of rapid detection of aquatic products, and opens up a new way in the aspect of promoting safety supervision of the aquatic products.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biological detection, and in particular relates to a CRISPR cycle cleavage-mediated signal enhancement Vibrio parahaemolyticus biosensor fixed with a rapid freezing probe and its application. Background Art

[0002] Vibrio parahemolyticus is listed as one of the most important bacteria causing foodborne diseases. It is widely found in seafood such as fish, shrimp, and shellfish. However, not all V. parahemolyticus are pathogenic. They are usually divided into two categories: (1) pathogenic strains that produce virulence factors, such as thermostable direct hemolysin (encoded by the tdh gene) or thermostable direct hemolysin-related hemolysin (encoded by the trh gene); (2) non-pathogenic strains that do not produce the above virulence factors and only produce non-thermostable hemolysin (encoded by the tlh gene).

[0003] Traditional detection methods for Vibrio parahaemolyticus mainly include separation and identification, immunoassay and nucleic acid detection. However, the isolation and identification method has its disadvantages of long detection cycle, large workload and high requirements for staff's experimental skills, which limits its scope of application. Although the immunoassay has strong specificity, its sensitivity is low. Nucleic acid detection PCR is not suitable for on-site detection because it needs to set different temperature stages to achieve amplification. Traditional detection methods for Vibrio parahaemolyticus cannot quickly identify whether the Vibrio parahaemolyticus to be tested is pathogenic. Therefore, it is urgent to establish a better detection method to detect the target Vibrio parahaemolyticus and determine whether it is pathogenic by detecting the target virulence factors. Summary of the invention

[0004] The main purpose of the present invention is to provide a CRISPR cycle cleavage-mediated signal enhancement biosensor for Vibrio parahaemolyticus fixed by rapid freezing probe and its application to overcome the shortcomings of the prior art.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0006] The embodiment of the present invention provides a CRISPR cycle cleavage-mediated signal enhancement biosensor for Vibrio parahaemolyticus fixed by rapid freezing probe, which includes: an RPA isothermal amplification primer pair, a CRISPR / Cas12a system and an electrochemical three-electrode system;

[0007] Among them, the RPA isothermal amplification primer pair includes a tdh primer pair and a trh primer pair, the tdh primer pair includes a tdh upstream primer and a tdh downstream primer, and the trh primer pair includes a trh upstream primer and a trh downstream primer; the CRISPR / Cas12a system includes tdh-crRNA for the tdh gene, trh-crRNA for the trh gene and Cas12a protein.

[0008] The embodiments of the present invention also provide the use of the aforementioned rapid freezing probe-fixed CRISPR cycle cleavage-mediated signal enhancement Vibrio parahaemolyticus biosensor in detecting Vibrio parahaemolyticus in aquatic food.

[0009] The embodiment of the present invention also provides a method for detecting Vibrio parahaemolyticus in aquatic food, which comprises:

[0010] Performing an RPA isothermal amplification reaction on a mixed reaction system comprising at least an RPA isothermal amplification primer pair and a sample to be tested to obtain an amplification product;

[0011] And, the amplified product is mixed with the CRISPR / Cas12a system and applied to the working electrode surface of the electrochemical three-electrode system for incubation, and then NaClO4 electrolyte is added to the electrochemical three-electrode system for detection, and whether the sample to be tested contains Vibrio parahaemolyticus is determined according to the change amplitude of the current value;

[0012] Among them, the RPA isothermal amplification primer pair includes a tdh primer pair and a trh primer pair, the tdh primer pair includes a tdh upstream primer and a tdh downstream primer, and the trh primer pair includes a trh upstream primer and a trh downstream primer; the CRISPR / Cas12a system includes tdh-crRNA for the tdh gene, trh-crRNA for the trh gene and Cas12a protein.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The present invention successfully established a Vibrio parahaemolyticus biosensor based on target gene amplicon activation CRISPR / Cas12a-mediated cyclic cleavage electrochemical probe to achieve signal amplification. The Vibrio parahaemolyticus biosensor can complete the entire operation within 30-60 minutes at 37°C when detecting Vibrio parahaemolyticus;

[0015] (2) The detection method provided by the present invention has the characteristics of simplicity, rapidity, and strong anti-interference ability. These characteristics make the method suitable for application in the field of rapid detection of aquatic products, opening up a new way to promote the safety supervision of aquatic products, which is crucial for the monitoring and control of Vibrio parahaemolyticus. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 It is a schematic diagram of a CRISPR / Cas12a-mediated cyclic signal amplification and electrochemical probe detection method in a typical embodiment of the present invention;

[0018] Figure 2A-2D It is a feasibility analysis diagram of a Vibrio parahaemolyticus biosensor in a typical embodiment of the present invention;

[0019] Figure 3A-3B This is a diagram showing the specific analysis results of the Vibrio parahaemolyticus biosensor in a typical embodiment of the present invention;

[0020] Figure 4A-4B The figure is an agarose gel electrophoresis diagram in a typical embodiment of the present invention. DETAILED DESCRIPTION

[0021] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Specifically, as one aspect of the technical solution of the present invention, a CRISPR cycle cleavage-mediated signal enhancement biosensor for Vibrio parahaemolyticus fixed with a rapid freezing probe comprises: an RPA isothermal amplification primer pair, a CRISPR / Cas12a system, and an electrochemical three-electrode system;

[0023] Among them, the RPA isothermal amplification primer pair includes a tdh primer pair and a trh primer pair, the tdh primer pair includes a tdh upstream primer and a tdh downstream primer, and the trh primer pair includes a trh upstream primer and a trh downstream primer; the CRISPR / Cas12a system includes tdh-crRNA for the tdh gene, trh-crRNA for the trh gene and Cas12a protein.

[0024] In some preferred embodiments, the tdh upstream primer has a sequence as shown in SEQ ID NO.1, and the tdh downstream primer has a sequence as shown in SEQ ID NO.2.

[0025] In some preferred embodiments, the trh upstream primer has a sequence as shown in SEQ ID NO.3, and the trh downstream primer has a sequence as shown in SEQ ID NO.4.

[0026] In some preferred embodiments, the tdh-crRNA has a sequence as shown in SEQ ID NO.5.

[0027] In some preferred embodiments, the trh-crRNA has a sequence as shown in SEQ ID NO.6.

[0028] In some preferred embodiments, the electrochemical three-electrode system includes: a gold electrode as a working electrode, a saturated calomel electrode as a reference electrode, and a platinum wire electrode as a counter electrode, and the surface of the gold electrode has an electrochemical probe modified by freezing.

[0029] Furthermore, the electrochemical probe has a sequence as shown in SEQ ID NO.7.

[0030] Furthermore, the 5' end of the electrochemical probe is modified with an MB signal molecule, and the 3' end of the electrochemical probe is modified with a thiol group.

[0031] Another aspect of an embodiment of the present invention further provides the use of the aforementioned rapid freezing probe-fixed CRISPR cycle cleavage-mediated signal enhancement Vibrio parahaemolyticus biosensor in detecting Vibrio parahaemolyticus in aquatic food.

[0032] Another aspect of the embodiments of the present invention further provides a method for detecting Vibrio parahaemolyticus in aquatic food, comprising:

[0033] Performing an RPA isothermal amplification reaction on a mixed reaction system comprising at least an RPA isothermal amplification primer pair and a sample to be tested to obtain an amplification product;

[0034] And, the amplified product is mixed with the CRISPR / Cas12a system and applied to the working electrode surface of the electrochemical three-electrode system for incubation, and then NaClO4 electrolyte is added to the electrochemical three-electrode system for detection, and whether the sample to be tested contains Vibrio parahaemolyticus is determined according to the change amplitude of the current value;

[0035] Among them, the RPA isothermal amplification primer pair includes a tdh primer pair and a trh primer pair, the tdh primer pair includes a tdh upstream primer and a tdh downstream primer, and the trh primer pair includes a trh upstream primer and a trh downstream primer; the CRISPR / Cas12a system includes tdh-crRNA for the tdh gene, trh-crRNA for the trh gene and Cas12a protein.

[0036] In the present invention, in the presence of the target virulence factor of Vibrio parahaemolyticus, the RPA isothermal amplification reaction is initiated in combination with the corresponding isothermal amplification primers, and a large number of tdh amplicons and trh amplicons are produced; the tdh-crRNA and trh-crRNA recognize the corresponding amplicons, and form a ternary complex with the Cas12a protein, which stimulates the trans-cleavage activity of the Cas12a protein, cuts the electrochemical probe pre-fixed on the electrode surface, and triggers a decrease in the electrochemical signal.

[0037] In some preferred embodiments, the CRISPR / Cas12a system also includes a Cas protein recognition buffer.

[0038] In some preferred embodiments, the tdh upstream primer has a sequence as shown in SEQ ID NO.1, and the tdh downstream primer has a sequence as shown in SEQ ID NO.2.

[0039] In some preferred embodiments, the tdh primer pair is used to obtain a tdh amplicon.

[0040] In some preferred embodiments, the trh upstream primer has a sequence as shown in SEQ ID NO.3, and the trh downstream primer has a sequence as shown in SEQ ID NO.4.

[0041] In some preferred embodiments, the trh primer pair is used to obtain a trh amplicon.

[0042] In some preferred embodiments, the tdh-crRNA has a sequence as shown in SEQ ID NO.5.

[0043] In some preferred embodiments, the trh-crRNA has a sequence as shown in SEQ ID NO.6.

[0044] In some preferred embodiments, the electrochemical three-electrode system includes: a gold electrode as a working electrode, a saturated calomel electrode as a reference electrode, and a platinum wire electrode as a counter electrode, and the surface of the gold electrode has an electrochemical probe modified by freezing.

[0045] Furthermore, the electrochemical probe has a sequence as shown in SEQ ID NO.7.

[0046] Furthermore, the 5' end of the electrochemical probe is modified with an MB signal molecule, and the 3' end of the electrochemical probe is modified with a thiol group.

[0047] In the present invention, the 5' end of the electrochemical probe is modified with an MB signal molecule for electrochemical signal output, and the 3' end is modified with a thiol group for coupling the probe on the surface of a gold electrode. The probe needs to be reduced first, and then fixed on the electrode surface. The modified electrochemically active probe is fixed on the electrode surface by freezing (based on the Au-S bond), thereby improving the fixation efficiency and stability of the electrochemically active probe. The traditional salt aging method requires 24 hours, or even longer, while the frozen coupling in the present invention only takes 15-30 minutes to complete the coupling.

[0048] Furthermore, the gold electrode needs to be soaked, polished, ultrasonicated, etc. to obtain a pretreated gold electrode. The pretreatment of the gold electrode includes: soaking the gold electrode in piranha solution for 30 minutes, then further polishing the gold electrode with alumina slurry to finally obtain a mirror-like flat electrode surface, ultrasonically treating the gold electrode in ethanol and double distilled water respectively, then electrochemically cleaning the electrode using cyclic voltammetry in 0.5M sulfuric acid, and finally rinsing with ultrapure water and drying with nitrogen to obtain a pretreated gold electrode.

[0049] Furthermore, the preparation method of the working electrode includes: mixing the electrochemical probe with tri(2-carboxyethyl)phosphine (TCEP) and incubating, then drop-coating on the surface of the gold electrode and sealing, and then freezing and incubating at -18 to -20°C for 15 to 30 minutes to obtain the working electrode.

[0050] Furthermore, the NaClO4 electrolyte includes NaClO4.

[0051] In some preferred embodiments, the detection method specifically comprises:

[0052] Performing an RPA isothermal amplification reaction on a mixed reaction system comprising an RPA isothermal amplification primer pair, a sample to be tested, an activator, and ddH2O to obtain an amplification product;

[0053] Also, the amplified product is mixed with the CRISPR / Cas12a system to form a mixed solution, and then the mixed solution is dropped on the surface of the working electrode for incubation, and then NaClO4 electrolyte is added to the electrochemical three-electrode system for detection, and the change in current value before and after the mixed solution is dropped on the surface of the working electrode is used to determine whether the sample to be tested contains Vibrio parahaemolyticus; when the change in current value is ≥8%, it indicates that the sample to be tested contains Vibrio parahaemolyticus; when the change in current value is less than 8%, it indicates that the sample to be tested does not contain Vibrio parahaemolyticus.

[0054] Furthermore, the current value change value ≥ 8% and the current value change value less than 8% described herein refer to the ratio of the absolute value of the current value difference before and after dripping the mixed liquid to the current value before dripping the mixed liquid.

[0055] Furthermore, the temperature of the sample to be tested was detected to be 37°C.

[0056] Furthermore, the activator includes a magnesium acetate activator, a magnesium sulfate activator, and the like.

[0057] In the present invention, whether the sample to be tested contains the virulence factors of thermostable direct hemolysin (tdh) and relatively thermostable direct hemolysin (trh) of Vibrio parahaemolyticus is used to determine whether there is a target Vibrio parahaemolyticus. Specifically, if the current value is greatly reduced, the sample to be tested contains the target virulence factor of Vibrio parahaemolyticus; if the current value does not change significantly, the sample to be tested does not contain the virulence factor of Vibrio parahaemolyticus.

[0058] In the present invention, RPA isothermal amplification technology is combined with CRISPR / Cas12a system, and two important virulence factors of Vibrio parahaemolyticus can be quickly and specifically effectively identified, thermostable direct hemolysin (tdh) and relatively thermostable direct hemolysin (trh): including the electrochemical probe is efficiently and stably fixed on the working electrode surface by adopting the co-freezing coupling method, and the traditional salt aging coupling method requires an extremely long coupling time (4°C, 10h), and the coupling time required for the co-freezing coupling method is extremely short (-20°C, 20-30min). When the target virulence factor exists, a large number of amplicons are obtained by RPA isothermal amplification technology, and the corresponding amplicons are respectively identified by the designed crRNA, and the trans-cutting ability of Cas12a protein is activated. On the one hand, the electrochemical probe on the surface of the electrode is cyclically cut to achieve the sensitization of the signal. Importantly, adding NaClO4 solution to the reaction electrolyte can significantly increase the redox current and improve the sensitivity of detection. The detection method can complete the entire operation and detection within 30-60 minutes at 37°C; at the same time, the detection method provided has the characteristics of simplicity, rapidity, and strong anti-interference ability. These characteristics make the method suitable for application in the field of rapid detection of aquatic products, opening up a new way to promote the safety supervision of aquatic products, which is crucial for the monitoring and control of Vibrio parahaemolyticus.

[0059] The method of the present invention is divided into four steps. The first step is to extract the bacterial template and perform RPA isothermal amplification to obtain a large number of amplicons; the second step is electrode pretreatment to obtain a pretreated gold electrode. The third step is to fix the electrochemical probe on the electrode surface based on the freezing principle, and then use MCH solution to complete the closure of the active site and measure the current value. The fourth step is to use the target gene amplicon to activate the CRISPR system to cut the electrochemical probe on the electrode surface. After the reaction is completed, the current value is measured again. By detecting whether the current value drops significantly, it is determined whether the sample to be tested contains the Vibrio parahaemolyticus virulence factor.

[0060] The schematic diagram of a typical embodiment of the present invention based on CRISPR / Cas12a-mediated cyclic signal amplification and electrochemical probe detection method is as follows: Figure 1 shown.

[0061] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0062] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0063] In the following examples, all sequences used were commissioned to be synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the sequence information is shown in Table 1.

[0064] Table 1

[0065]

[0066] Example 1 Detection system construction

[0067] Extract template:

[0068] The nucleic acid was extracted using a bacterial DNA extraction kit according to the instructions and stored in a -20°C refrigerator for later use;

[0069] Working electrode pretreatment:

[0070] The gold electrode was immersed in the piranha solution for 30 minutes, and then further polished with alumina slurry to obtain a mirror-like flat electrode surface; the gold electrode was ultrasonically treated in ethanol and double distilled water, and then electrochemically cleaned in 0.5M sulfuric acid using cyclic voltammetry, and finally rinsed with ultrapure water and dried with nitrogen to obtain a pretreated gold electrode;

[0071] Modified working electrode

[0072] Mix the probe and 10mM TCEP and incubate for 60 minutes. Then, drop the mixture on the electrode surface, seal the electrode with a plastic cap, and freeze and incubate at -20°C for 30 minutes to obtain a probe-modified gold electrode. Then, use a 1mM MCH solution to block the remaining active sites on the electrode surface. After blocking, rinse the remaining solution on the electrode surface with double distilled water and dry it with nitrogen to obtain a fully modified gold electrode. Then, measure the current value in an electrolytic solution with NaClO4 added;

[0073] RPA isothermal amplification

[0074] Using the total DNA of Vibrio parahaemolyticus as a template, using RPA isothermal amplification primers, 0.5 μL of 10 μM forward primer and 0.5 μL of 10 μM reverse primer, 1 μL template, 1 μL activator, 22 μL ddH2O, temperature 37°C, reaction time 30 min, RPA isothermal amplification reaction was performed to obtain RPA amplicon;

[0075] RPA amplicon activates CRISPR system to cleave electrochemical probe

[0076] Taking the RPA amplicon as the target, the CRISPR / Cas12a system (including Cas12a buffer, Cas12a protein, tdh-crRNA, trh-crRNA, ddH2O, etc.) was added and mixed, and then the mixture was dropwise applied to the surface of the gold electrode, incubated at 37°C for 30 minutes, and then the current value was measured in an electrolyte solution added with NaClO4;

[0077] Analyze the results

[0078] According to the change of the detected current value before and after, it is judged whether the sample to be tested contains the virulence factor of Vibrio parahaemolyticus. If the current value drops significantly (i.e., the change value of the current value is ≥8%), the sample to be tested contains the virulence factor of Vibrio parahaemolyticus; if the current value does not change significantly (i.e., the change value of the current value is less than 8%), the sample to be tested does not contain the virulence factor.

[0079] Example 2 Feasibility Analysis

[0080] Example 2 provides feasibility analysis verification. Figure 2A CV curves of different modified electrodes in 1 mMFe3(CN)6 / Fe4(CN)6, where: a bare AE, b AE / probe / MCH, c AE / probe / MCH / target DNA; Figure 2B EIS curves of different modified electrodes in 1mMFe3(CN)6 / Fe4(CN)6; Figure 2C 1 is a reaction system lacking Cas12a components; 2 is a reaction system lacking crRNA components; 3 is a reaction system lacking Target DNA components; 4 is a reaction system lacking buffer components; 5 is a reaction system with all components; Figure 2D This is a diagram showing the feasibility verification of the Vibrio parahaemolyticus biosensor.

[0081] Figure 2A and Figure 2B The results show that the bare electrode has the highest current and the lowest impedance ( Figure 2A and the black curve in Figure 2B). After modification of the signal probe and MCH blocking, the peak value of the oxidation current was significantly reduced, as shown in Figure 2A (red curve). This reduction indicates that the DNA molecule layer hinders the interfacial charge transfer. On the contrary, as the amplicon and CRISPR system are combined, the trans-cutting ability of Cas12a is activated. The peak value of the oxidation current increases significantly, as shown in Figure 2A (blue curve). In this case, the signal probe is cleaved and the MB is detached from the electrode surface, exposing the electroactive site. Figure 2BThe charge transfer resistance (Ret) results show that the bare gold electrode is a straight line (black curve), indicating that the bare gold electrode has a lower Ret. After modification with electrochemical probe and MCH, Ret increases significantly, such as Figure 2B (red curve). After the Cas12a protein is activated by the amplified product, it cuts the electrochemical probe, and Ret decreases ( Figure 2B The blue curve in the figure confirms that the Cas12a protein has the ability to trans-cleave after being activated by the RPA amplicon. In addition, differential pulse voltammetry (DPV) was used to verify the effect of different components on the trans-cleavage effect of the designed CRISPR system. Figure 2C The results clearly show that in the absence of Cas12a, crRNA, buffer or RPA amplicon ( Figure 1 In the case of 1-4) in E, the cutting efficiency of Cas12a protein is low, while when all components are involved, Cas12a protein has a very strong cutting efficiency. Figure 2D The DPV curves with and without target amplification products also prove that the amplicon can effectively activate the trans-cleavage effect of Cas12a.

[0082] Example 3 Specificity test

[0083] Determine whether the detection method established by the institute has cross-reactions with other common pathogens. Figure 3A and Figure 3B It indicates that the Ri value is significantly increased only when the virulence factors tdh and trh are present, or when other control strains are mixed with the virulence factors tdh and trh, indicating that the Vibrio parahaemolyticus biosensor has good specificity.

[0084] Example 4 Agarose gel electrophoresis

[0085] Take 2 μL of buffer and 5 μL of amplicon respectively, mix well and add into the prepared agarose gel. RPA product of Vibrio parahaemolyticus: Lane 1, negative control; Lane 2, RPA isothermal amplification product of Vibrio parahaemolyticus. Figure 4A : tdh amplicon and Figure 4B : trh amplicon.

[0086] In summary, the present invention successfully developed a Vibrio parahaemolyticus biosensor based on CRISPR / Cas12a-mediated cyclic signal amplification and electrochemical probe by combining RPA isothermal amplification technology with CRISPR / Cas12a system. This method can complete the entire operation within half an hour at about 37°C. It has the advantages of room temperature reaction, short time consumption, strong specificity, high sensitivity and good repeatability. It is suitable for detection in non-laboratory places with poor conditions and has good application prospects.

[0087] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0088] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical deformation made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the protection scope of the present invention.

Claims

1. A CRISPR cycle cleavage-mediated signal enhancement biosensor for Vibrio parahaemolyticus fixed by rapid freezing probe, characterized in that: include: RPA isothermal amplification primer pairs, CRISPR / Cas12a system and electrochemical three-electrode system; Among them, the RPA isothermal amplification primer pair includes a tdh primer pair and a trh primer pair, the tdh primer pair includes a tdh upstream primer and a tdh downstream primer, and the trh primer pair includes a trh upstream primer and a trh downstream primer; the CRISPR / Cas12a system includes tdh-crRNA for the tdh gene, trh-crRNA for the trh gene and Cas12a protein.

2. The Vibrio parahaemolyticus biosensor according to claim 1, characterized in that: The tdh upstream primer has a sequence as shown in SEQ ID NO.1, and the tdh downstream primer has a sequence as shown in SEQ ID NO.2; And / or, the trh upstream primer has a sequence as shown in SEQ ID NO.3, and the trh downstream primer has a sequence as shown in SEQ ID NO.4; And / or, the tdh-crRNA has the sequence shown in SEQ ID NO.5; And / or, the trh-crRNA has the sequence shown in SEQ ID NO.

6.

3. The Vibrio parahaemolyticus biosensor according to claim 1, characterized in that: The electrochemical three-electrode system comprises: a gold electrode as a working electrode, a saturated calomel electrode as a reference electrode, and a platinum wire electrode as a counter electrode. The surface of the gold electrode is provided with an electrochemical probe modified by freezing.

4. The Vibrio parahaemolyticus biosensor according to claim 3, characterized in that: The electrochemical probe has a sequence as shown in SEQ ID NO.7; And / or, the 5' end of the electrochemical probe is modified with an MB signal molecule, and the 3' end of the electrochemical probe is modified with a thiol group.

5. Use of the CRISPR cycle cleavage-mediated signal enhancement Vibrio parahaemolyticus biosensor fixed by rapid freezing probe according to any one of claims 1 to 4 in detecting Vibrio parahaemolyticus in aquatic food.

6. A method for detecting Vibrio parahaemolyticus in aquatic food, characterized in that: include: Performing an RPA isothermal amplification reaction on a mixed reaction system comprising at least an RPA isothermal amplification primer pair and a sample to be tested to obtain an amplification product; And, the amplified product is mixed with the CRISPR / Cas12a system and applied to the working electrode surface of the electrochemical three-electrode system for incubation, and then NaClO4 electrolyte is added to the electrochemical three-electrode system for detection, and whether the sample to be tested contains Vibrio parahaemolyticus is determined according to the change amplitude of the current value; Among them, the RPA isothermal amplification primer pair includes a tdh primer pair and a trh primer pair, the tdh primer pair includes a tdh upstream primer and a tdh downstream primer, and the trh primer pair includes a trh upstream primer and a trh downstream primer; the CRISPR / Cas12a system includes tdh-crRNA for the tdh gene, trh-crRNA for the trh gene and Cas12a protein.

7. The detection method according to claim 6, characterized in that: The tdh upstream primer has a sequence as shown in SEQ ID NO.1, and the tdh downstream primer has a sequence as shown in SEQ ID NO.2; And / or, the trh upstream primer has a sequence as shown in SEQ ID NO.3, and the trh downstream primer has a sequence as shown in SEQ ID NO.4; And / or, the tdh-crRNA has the sequence shown in SEQ ID NO.5; And / or, the trh-crRNA has the sequence shown in SEQ ID NO.

6.

8. The detection method according to claim 6, characterized in that: The electrochemical three-electrode system comprises: a gold electrode as a working electrode, a saturated calomel electrode as a reference electrode, and a platinum wire electrode as a counter electrode. The surface of the gold electrode is provided with an electrochemical probe modified by freezing.

9. The detection method according to claim 7, characterized in that: The electrochemical probe has a sequence as shown in SEQ ID NO.7; And / or, the 5' end of the electrochemical probe is modified with an MB signal molecule, and the 3' end of the electrochemical probe is modified with a thiol group; And / or, the preparation method of the working electrode comprises: mixing an electrochemical probe with tri(2-carboxyethyl)phosphine and incubating the mixture, then drop-coating the mixture on the surface of a gold electrode and sealing the mixture, and then freezing and incubating the mixture at -18 to -20°C for 15 to 30 minutes to obtain a working electrode; And / or, the NaClO4 electrolyte includes NaClO4.

10. The detection method according to claim 6, characterized in that: Specifically include: Performing an RPA isothermal amplification reaction on a mixed reaction system comprising an RPA isothermal amplification primer pair, a sample to be tested, an activator, and ddH2O to obtain an amplification product; Also, the amplified product is mixed with the CRISPR / Cas12a system to form a mixed solution, and then the mixed solution is dropped on the surface of the working electrode for incubation, and then NaClO4 electrolyte is added to the electrochemical three-electrode system for detection, and the change in current value before and after the mixed solution is dropped on the surface of the working electrode is used to determine whether the sample to be tested contains Vibrio parahaemolyticus; when the change in current value is ≥8%, it indicates that the sample to be tested contains Vibrio parahaemolyticus; when the change in current value is <8%, it indicates that the sample to be tested does not contain Vibrio parahaemolyticus.

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