Method for identifying survival state of vibrio parahaemolyticus based on split crRNA and application thereof

Abstract

The application discloses a method for identifying survival state of Vibrio parahaemolyticus based on split crRNA and application. The method comprises the following steps: incubating a to-be-detected sample containing target Vibrio parahaemolyticus mRNA with a split crRNA scaffold chain, an ssDNA activator, a Cas12a protein and a reporter probe at room temperature to obtain a reaction product; wherein the target Vibrio parahaemolyticus mRNA, the split crRNA scaffold chain and the ssDNA activator form a ternary complex, and the ternary complex can activate the Cas12a protein to cut the reporter probe and generate a fluorescence signal; and detecting the fluorescence signal intensity of the reaction product, so as to identify the survival state of Vibrio parahaemolyticus according to the fluorescence signal intensity. The method has the advantages of high sensitivity, good specificity and no need for nucleic acid amplification, and is suitable for efficient and reliable identification of live Vibrio parahaemolyticus.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, specifically to a method and its application for identifying the survival status of Vibrio parahaemolyticus based on fission-type crRNA. Background Technology

[0002] The hazards posed by foodborne pathogens have become a major concern. Traditional methods such as colony counting, immunological detection, and nucleic acid detection (e.g., PCR) have significant limitations in distinguishing bacterial viability: the former is time-consuming, and the latter cannot differentiate between viable and non-viable bacteria due to residual DNA from dead bacteria, easily leading to false positives. Therefore, developing a detection technology that can rapidly, specifically, and sensitively distinguish the viability of foodborne pathogens is crucial.

[0003] CRISPR / Cas systems have been widely used in molecular diagnostics due to their strong specific recognition capabilities. However, traditional CRISPR detection methods typically rely on the pre-amplification of target nucleic acids (such as RPA and PCR), which not only increases operational complexity and the risk of aerosol contamination but also fails to address the interference from residual nucleic acids from dead bacteria. mRNA is a direct product of transcriptional activity in live bacteria and degrades rapidly after bacterial death. Therefore, targeting mRNA is an ideal approach for achieving specific detection of live bacteria. Summary of the Invention

[0004] The main objective of this invention is to provide a method and application for identifying the survival status of Vibrio parahaemolyticus based on fission-type crRNA, in order to overcome the shortcomings of the prior art.

[0005] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: This invention provides a method for identifying the viability of Vibrio parahaemolyticus based on fission-type crRNA, comprising: The test sample containing the target mRNA was incubated at room temperature with a splitting crRNA scaffold chain, an ssDNA activator, a Cas12a protein, and a reporter probe to obtain the reaction product. The target mRNA, the splitting crRNA scaffold chain, and the ssDNA activator formed a ternary complex, which could activate the Cas12a protein to cleave the reporter probe and generate a fluorescent signal. In addition, the fluorescence signal intensity of the reaction product is detected, and the survival status of Vibrio parahaemolyticus is identified based on the fluorescence signal intensity.

[0006] This invention also provides an amplification-free biosensor for identifying the viability of Vibrio parahaemolyticus, comprising: a splitting crRNA scaffold strand, an ssDNA activator, and a reporter probe; wherein the splitting crRNA scaffold strand has the sequence shown in SEQ ID NO. 1; the ssDNA activator has the sequence shown in SEQ ID NO. 2; and the reporter probe has the sequence shown in SEQ ID NO. 4.

[0007] The present invention also provides the application of the aforementioned amplification-free biosensor for identifying the viability of Vibrio parahaemolyticus in detecting the viability of Vibrio parahaemolyticus.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Compared with existing fluorescence detection methods, the present invention ensures the specificity of the analysis based on the complementary base pairing of the target Vibrio parahaemolyticus mRNA and ssDNA activator; (2) Compared with existing fluorescence detection methods, the present invention directly detects the RNA of Vibrio parahaemolyticus foodborne pathogens and identifies the survival status of Vibrio parahaemolyticus foodborne pathogens. No nucleic acid amplification step is required, thus avoiding the potential risk of aerosol contamination. (3) The present invention can complete the detection within 45 minutes and has the advantages of high sensitivity, good specificity and no need for nucleic acid amplification. It is suitable for efficient and reliable identification of live Vibrio parahaemolyticus. Attached Figure Description

[0009] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0010] Figure 1 This is a schematic diagram illustrating the principle of identifying the viability of Vibrio parahaemolyticus in a typical embodiment of the present invention; Figure 2 This is a standard curve obtained from the detection of Vibrio parahaemolyticus mRNA standard solution in Example 1 of the present invention; Figure 3 This is a graph showing the optimized concentration of Cas12a protein in Example 2 of the present invention; Figure 4 This is a diagram showing the optimized concentration of the splitting crRNA scaffold strand in Example 2 of the present invention; Figure 5 This is a diagram showing the optimized probe concentration in Example 2 of the present invention; Figure 6 This is the temperature optimization diagram in Embodiment 2 of the present invention; Figure 7 The fluorescence curves for surviving Vibrio parahaemolyticus and dead Vibrio parahaemolyticus were measured in Example 3 of this invention. Detailed Implementation

[0011] In view of the deficiencies of the prior art, the inventors of this case, through long-term research and extensive practice, have proposed the technical solution of this invention, which is mainly a fluorescence detection method based on the precise Cas12a amplification strategy assisted by splitting crRNA to detect the viable marker mRNA of Vibrio parahaemolyticus to identify the survival status of Vibrio parahaemolyticus foodborne pathogens. The operation steps of the fluorescence detection method are as follows: (1) assembly of the ternary complex; (2) activation of the trans-cleavage ability of Cas12a protein to cleave the reporter probe and generate a fluorescence signal; (3) optimization of reaction parameters; (4) detection of mRNA standard solution to establish a standard curve; (5) quantitative detection of viable bacteria in the sample containing mRNA.

[0012] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] Specifically, as one aspect of the technical solution of this invention, a method for identifying the survival status of Vibrio parahaemolyticus based on splitting crRNA includes: The test sample containing the target mRNA was incubated at room temperature with a splitting crRNA scaffold chain, an ssDNA activator, a Cas12a protein, and a reporter probe to obtain the reaction product. The target mRNA, the splitting crRNA scaffold chain, and the ssDNA activator formed a ternary complex, which could activate the Cas12a protein to cleave the reporter probe and generate a fluorescent signal. In addition, the fluorescence signal intensity of the reaction product is detected, and the survival status of Vibrio parahaemolyticus is identified based on the fluorescence signal intensity.

[0014] In some preferred embodiments, a schematic diagram illustrating the principle of identifying the viability of Vibrio parahaemolyticus in this invention is shown below. Figure 1 As shown.

[0015] In some preferred embodiments, the split crRNA scaffold strand has a sequence as shown in SEQ ID NO. 1.

[0016] In some preferred embodiments, the ssDNA activator has a sequence as shown in SEQ ID NO. 2.

[0017] In some preferred embodiments, the target mRNA has a sequence as shown in SEQ ID NO. 3.

[0018] In some preferred embodiments, the reporting probe has a sequence as shown in SEQ ID NO. 4.

[0019] In some preferred embodiments, the method specifically includes: (1) A series of target mRNA standard solutions of different concentrations were incubated together with split crRNA scaffold strands, ssDNA activator, Cas12a protein and reporter probe at room temperature to obtain a series of corresponding reaction products; (2) Measure the fluorescence intensity values ​​of a series of corresponding reaction products in the excitation light band to establish a standard curve of mRNA concentration-fluorescence intensity value; (3) The test sample containing the target mRNA is incubated together with the splitting crRNA scaffold chain, ssDNA activator, Cas12a protein and reporter probe at room temperature, and the fluorescence intensity value of the obtained reaction product in the excitation light band is measured and compared with the mRNA concentration-fluorescence intensity value standard curve to obtain the concentration of target mRNA in the test sample.

[0020] Furthermore, the method specifically includes: dissolving the standard target mRNA powder in ultrapure water to obtain a storage solution, and continuing to dilute it stepwise with ultrapure water to obtain a series of target mRNA standard solutions of different concentrations.

[0021] Furthermore, the method specifically includes: A series of target mRNA standard solutions of different concentrations were mixed with 1×Cas12a protein buffer, 10-100 nM of splitting crRNA scaffold strand, 10-200 nM of ssDNA activator, 200-1600 nM of reporter probe and enzyme-free water and incubated at 37-42℃ for 30-90 min to obtain a series of corresponding reaction products. A series of corresponding reaction products were measured at 520 nm to establish a standard curve of mRNA concentration-fluorescence intensity; the excitation wavelength used was 490 nm.

[0022] Further, the method specifically includes: mixing the test sample containing the target mRNA with 1×Cas12a protein buffer, 10-100 nM of splitting crRNA scaffold strand, 10-200 nM of ssDNA activator, 200-1600 nM of reporter probe and enzyme-free water, and incubating at 37-42℃ for 30-90 min, and measuring the fluorescence intensity value of the obtained reaction product at 520 nm, and comparing it with the mRNA concentration-fluorescence intensity value standard curve to obtain the concentration of target mRNA in the test sample.

[0023] Furthermore, the method specifically includes: the test sample containing the target mRNA includes a live bacterial sample.

[0024] In some preferred embodiments, the method for identifying the viability status of Vibrio parahaemolyticus based on splitting crRNA includes: In the presence of mRNA, it was incubated at room temperature with a splitting crRNA scaffold chain, an ssDNA activator, a Cas12a protein, and a reporter probe. The mRNA and ssDNA activator combined according to the base complementary pairing principle and assembled with the splitting crRNA scaffold chain to form a ternary complex. The ternary complex activated the trans-cleavage activity of the Cas12a protein, cleaved the reporter probe and generated a fluorescent signal, yielding the final reaction product. By detecting the fluorescence signal intensity of the final product, the concentration of the target mRNA can be obtained, thus enabling the identification of the viability status of Vibrio parahaemolyticus.

[0025] Specifically: (1) Mix 1×Cas12a protein buffer, 200 nM Cas12a protein, 100 nM splitting crRNA scaffold strand, 50 nM ssDNA activator, 400 nM reporter probe, enzyme-free water and target mRNA evenly, and react the mixture at 37℃ for 45 min to activate the trans-cleavage ability of Cas12a protein to cleave the reporter probe and obtain the final product of the reaction.

[0026] (2) According to step (1), a series of mRNA standard solutions of different concentrations are quantitatively detected, the fluorescence intensity value of the final product in the excitation light band is measured, and a standard curve of mRNA concentration-fluorescence intensity value is obtained. (3) According to step (1), the live bacterial sample containing mRNA is tested, the fluorescence intensity value of the product in the excitation light band is measured, and compared with the standard curve to obtain the concentration of mRNA in the live bacterial sample.

[0027] Furthermore, the sequence of the split crRNA scaffold chain is shown in SEQ ID NO. 1, specifically 5-UAAUUUCUACUAAGUGUAGAU-3.

[0028] Furthermore, the sequence of the ssDNA activator is shown in SEQ ID NO. 2, specifically 5-AGGTACAGGAGACAAGTAGAGTCTTAGTTTCATTAAATTGGTACTGGG-3.

[0029] Furthermore, the sequence of the reporting probe is shown in SEQ ID NO. 3, specifically FAM-TTATT-BHQ1, but is not limited thereto.

[0030] Furthermore, the Cas12a protein includes, but is not limited to, the LbCas12a protein.

[0031] In some more specific embodiments, the preparation method includes: in the presence of mRNA, incubating it together with a splitting crRNA scaffold chain, an ssDNA activator, a Cas12a protein and a reporter probe at room temperature, wherein the mRNA and the ssDNA activator combine according to the base complementary pairing principle and assemble with the splitting crRNA scaffold chain to form a ternary complex. This ternary complex activates the trans-cleavage ability of the Cas12a protein, cleaving the reporter probe and releasing a strong fluorescent signal. Finally, a fluorescence detection method for mRNA was established by observing the linear relationship between the intensity of the fluorescence signal and the mRNA concentration.

[0032] In some preferred embodiments, the present invention utilizes a Cas12a amplification strategy based on precise assistance of fission-type crRNA to detect viable bacterial marker mRNA in a fluorescence detection method for identifying the viability of Vibrio parahaemolyticus. The specific operation steps are as follows: (1) Mix 1×Cas12a protein buffer, 200 nM Cas12a protein, 2100 nM splitting crRNA scaffold strand, 50 nM ssDNA activator, 400 nM reporter probe, enzyme-free water and target mRNA evenly, and react the mixture at 37℃ for 45 min to activate the trans-cleavage ability of Cas12a protein to cleave the reporter probe and obtain the final product of the reaction.

[0033] (2) Detect the mRNA standard solution and establish a standard curve: ① Dissolve the purchased standard mRNA powder in DEPC-treated water to a 100 μM storage solution. Continue to dilute the storage solution with DEPC-treated water stepwise to obtain mRNA standard solutions of different concentrations; ② Quantitatively detect the mRNA standard solutions of different concentrations according to the methods described in steps (1) and (2), and measure the fluorescence intensity value of the product at 520 nm when the excitation light is 490 nm; ③ Establish a standard curve of mRNA concentration-fluorescence intensity value for mRNA detection with different concentrations of mRNA as the abscissa and the fluorescence intensity value at 520 nm as the ordinate. (3) Quantitative detection of actual biological samples containing mRNA: Take the unknown biological sample containing mRNA as the detection target, and perform detection according to the same processing method in steps (1) and (2). The obtained fluorescence intensity value is substituted into the standard curve of mRNA detection to calculate the concentration of mRNA.

[0034] Another aspect of the present invention provides an amplification-free biosensor for identifying the viability of Vibrio parahaemolyticus, comprising: a splitting crRNA scaffold chain, an ssDNA activator, and a reporter probe; wherein the splitting crRNA scaffold chain has the sequence shown in SEQ ID NO. 1; the ssDNA activator has the sequence shown in SEQ ID NO. 2; and the reporter probe has the sequence shown in SEQ ID NO. 4.

[0035] In some preferred embodiments, the amplification-free biosensor further includes the Cas12a protein.

[0036] Another aspect of the present invention provides the application of the aforementioned amplification-free biosensor for identifying the viability of Vibrio parahaemolyticus in detecting the viability of Vibrio parahaemolyticus.

[0037] In summary, the method for identifying the viability of Vibrio parahaemolyticus using a non-amplification biosensor provided by this invention has extremely high specificity and versatility, while eliminating the need for a nucleic acid amplification step and avoiding the potential risk of aerosol contamination.

[0038] The technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides detailed implementation methods and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0039] Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0040] In the following embodiments, all sequence information used is shown in Table 1: Table 1

[0041] Example 1 (1) Mix 1×Cas12a protein buffer, 200 nM Cas12a protein, 100 nM splitting crRNA scaffold strand, 50 nM ssDNA activator, 400 nM reporter probe, enzyme-free water and target mRNA evenly, and react the mixture at 40℃ for 30 min to activate the trans-cleavage ability of Cas12a protein to cleave the reporter probe and obtain the final product of the reaction.

[0042] (2) A series of mRNA standard solutions of different concentrations were quantitatively detected, and the fluorescence intensity of the final product in the excitation light band was measured to obtain a standard curve of mRNA concentration-fluorescence intensity value; ① The purchased standard mRNA powder was dissolved in DEPC-treated water to a 100 μM storage solution. The storage solution was further diluted stepwise with DEPC-treated water to obtain mRNA standard solutions of different concentrations; ② The mRNA standard solutions of different concentrations were quantitatively detected according to the methods described in (2) and (3), and the fluorescence intensity of the product at 520 nm when the excitation light was 490 nm was measured; ③ A standard curve for mRNA detection was established with different concentrations of mRNA as the abscissa and the fluorescence intensity at 520 nm as the ordinate, as shown in the figure. Figure 2 As shown.

[0043] (3) According to step (1), the live bacterial sample containing mRNA is tested, the fluorescence intensity value of the product in the excitation light band is measured, and compared with the standard curve to obtain the concentration of mRNA in the live bacterial sample.

[0044] Example 2 (1) Optimization of Cas12a protein concentration: The experimental concentrations were set at 50 nM, 100 nM, 200 nM, 300 nM, and 400 nM, from... Figure 3 It was found that when the Cas12a protein concentration reached 100 nM, the fluorescence intensity of the positive sample was much greater than that of the negative control, with a signal-to-noise ratio (SNR) of 21. As the protein concentration increased, the fluorescence intensity gradually increased, and the SNR gradually increased as well, reaching 64 when the protein concentration was 200 nM. To achieve a high SNR while conserving reagents, 200 nM was chosen as the optimal reaction condition.

[0045] (2) Optimization of crRNA scaffold chain concentration: The experimental concentrations were set at 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, and 100 nM, determined by... Figure 4 It can be seen that the fluorescence intensity gradually increases with the increase of concentration. When the concentration reaches 100 nM, the signal-to-noise ratio reaches its maximum of 56. Therefore, 100 nM is selected as the optimal reaction condition.

[0046] (3) Optimization of probe concentration: The experimental concentrations were set at 200 nM, 400 nM, 800 nM, 1000 nM, and 1600 nM, by... Figure 5 It can be seen that as the concentration of the reporter probe gradually increases, the fluorescence intensity also gradually increases. When the concentration is 400 nM, the signal-to-noise ratio is 59, which is already relatively high. In order to save reagent consumption, 400 nM is selected as the optimal reaction condition.

[0047] (4) Optimization of reaction temperature: The experimental temperatures were set at 37℃, 38℃, 39℃, 40℃, 41℃, and 42℃. Figure 6 It can be seen that the fluorescence intensity and signal-to-noise ratio are highest when the reaction temperature is 37℃, so 37℃ is chosen as the optimal reaction condition.

[0048] Example 3 (1) The suspected samples to be tested were divided into two groups: total RNA of viable Vibrio parahaemolyticus was extracted from the experimental group; total RNA of the same type of dead Vibrio parahaemolyticus was extracted from the control group after inactivation treatment (such as heating or disinfection). Both groups of samples were processed using the same commercial RNA extraction kit to obtain RNA template solution. The control group sample was used as an inactive target control for evaluating background signal.

[0049] (2) Mix 1×Cas12a protein buffer, 200 nM Cas12a protein, 100 nM splitting crRNA scaffold strand, 50 nM ssDNA activator, 400 nM reporter probe, enzyme-free water and the RNA nucleic acid sample prepared in step (1) evenly, and react the resulting mixture at 40℃ for 30 min to activate the trans-cleavage ability of Cas12a protein to cleave the reporter probe and obtain the final product of the reaction.

[0050] (3) Measure the fluorescence intensity of the product at 520 nm when the excitation light is 490 nm. Compare the fluorescence intensity of the experimental group (surviving Vibrio parahaemolyticus samples) with that of the control group (dead Vibrio parahaemolyticus samples). Determine whether there is a surviving target Vibrio parahaemolyticus in the sample by the signal difference between the two. Figure 7It can be seen that the fluorescence signal intensity of the experimental group (surviving Vibrio parahaemolyticus samples) is much greater than that of the control group (dead Vibrio parahaemolyticus samples), so the sensor can identify the survival status of Vibrio parahaemolyticus.

[0051] By means of the above technical solution, the method for identifying the viability of Vibrio parahaemolyticus without amplification biosensors provided by the present invention has a low background signal, a strong target signal, and a high signal-to-noise ratio (S / N=59), which far exceeds the existing fluorescence detection methods. At the same time, the method has good specificity and can be used for the direct detection of live bacteria. It can also be used for the sensitive detection of other foodborne pathogens through simple design, and is a universal nucleic acid detection platform.

[0052] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0053] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. A method for identifying the viability status of Vibrio parahaemolyticus based on fission-type crRNA, characterized in that, include: The test sample containing live Vibrio parahaemolyticus mRNA of the target bacteria was incubated at room temperature with a fissuring crRNA scaffold chain, an ssDNA activator, a Cas12a protein, and a reporter probe to obtain the reaction product. The target Vibrio parahaemolyticus mRNA formed a ternary complex with the fissuring crRNA scaffold chain and the ssDNA activator. The ternary complex could activate the Cas12a protein to cleave the reporter probe and generate a fluorescent signal. In addition, the fluorescence signal intensity of the reaction product is detected, and the survival status of Vibrio parahaemolyticus is identified based on the fluorescence signal intensity.

2. The method according to claim 1, characterized in that: The splitting crRNA scaffold strand has the sequence shown in SEQ ID NO. 1; And / or, the ssDNA activator has a sequence as shown in SEQ ID NO. 2; And / or, the target Vibrio parahaemolyticus mRNA has the sequence shown in SEQ ID NO. 3; And / or, the reporting probe has a sequence as shown in SEQ ID NO.

4.

3. The method according to claim 1, characterized in that, Specifically, it includes: (1) A series of target Vibrio parahaemolyticus mRNA standard solutions of different concentrations were incubated together with fission-type crRNA scaffold strands, ssDNA activator, Cas12a protein and reporter probe at room temperature to obtain a series of corresponding reaction products; (2) Measure the fluorescence intensity values ​​of a series of corresponding reaction products in the excitation light band, thereby establishing a standard curve of target Vibrio parahaemolyticus mRNA concentration-fluorescence intensity value; (3) The test sample containing the target Vibrio parahaemolyticus mRNA was incubated together with the splitting crRNA scaffold chain, ssDNA activator, Cas12a protein and reporter probe at room temperature, and the fluorescence intensity value of the obtained reaction product in the excitation light band was measured and compared with the standard curve of the target Vibrio parahaemolyticus mRNA concentration-fluorescence intensity value to obtain the concentration of target Vibrio parahaemolyticus mRNA in the test sample.

4. The method according to claim 3, characterized in that, Specifically, it includes: The standard target Vibrio parahaemolyticus mRNA powder was dissolved in ultrapure water to obtain a stock solution, and then further diluted stepwise with ultrapure water to obtain a series of standard solutions of target Vibrio parahaemolyticus mRNA with different concentrations.

5. The method according to claim 3, characterized in that, Specifically, it includes: A series of target Vibrio parahaemolyticus mRNA standard solutions of different concentrations were mixed with 1×Cas12a protein buffer, 10-100 nM of fission-type crRNA scaffold strand, 10-200 nM of ssDNA activator, 200-1600 nM of reporter probe and enzyme-free water and incubated at 37-42℃ for 30-90 min to obtain a series of corresponding reaction products. A series of corresponding reaction products were measured at 520 nm to establish a standard curve of Vibrio parahaemolyticus mRNA concentration-fluorescence intensity; the excitation wavelength used was 490 nm.

6. The method according to claim 3, characterized in that, Specifically, it includes: The test sample containing live Vibrio parahaemolyticus mRNA was mixed with 1×Cas12a protein buffer, 10–100 nM of fissile crRNA scaffold strand, 10–200 nM of ssDNA activator, 200–1600 nM of reporter probe, and enzyme-free water and incubated at 37–42 °C for 30–90 min. The fluorescence intensity of the obtained reaction product at 520 nm was measured and compared with the standard curve of live Vibrio parahaemolyticus mRNA concentration-fluorescence intensity value to obtain the concentration of live Vibrio parahaemolyticus mRNA in the test sample.

7. The method according to claim 6, characterized in that: The test samples containing target Vibrio parahaemolyticus mRNA include live bacterial samples.

8. An amplification-free biosensor for identifying the viability of Vibrio parahaemolyticus, characterized in that, include: A splitting crRNA scaffold chain, an ssDNA activator, and a reporter probe; wherein the splitting crRNA scaffold chain has the sequence shown in SEQ ID NO. 1; the ssDNA activator has the sequence shown in SEQ ID NO. 2; and the reporter probe has the sequence shown in SEQ ID NO.

4.

9. The amplification-free biosensor according to claim 8, characterized in that: The amplification-free biosensor also includes the Cas12a protein.

10. The application of the non-amplification biosensor for identifying the viability of Vibrio parahaemolyticus as described in claim 8 or 9 in detecting the viability of Vibrio parahaemolyticus.

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