A kit and method for closed tube visual detection of vibrio parahaemolyticus

Abstract

This invention discloses a kit and method for closed-tube visualization detection of Vibrio parahaemolyticus. The kit comprises a mixed reaction system within a detection tube and thiol-modified gold nanoparticles on the tube cap. The mixed reaction system contains a mixture of RPA amplification and a protective CRISPR / Cas12a reaction system. The thiol-modified gold nanoparticle mixture on the tube cap can be added to the mixed reaction system while the tube is closed. This invention utilizes a DNA-crRNA complex design to place the RPA system, CRISPR system, and gold nanoparticles in the same tube, forming a closed-tube detection system to prevent cross-contamination. Compared to existing detection methods, this method offers advantages such as high sensitivity, strong specificity, rapid and efficient operation, ease of use, user-friendliness, and low cost, requiring no additional detection or observation instruments.

Description

Technical Field

[0001] This invention belongs to the fields of biology and chemistry, and specifically relates to a kit and method for closed-tube visualization detection of Vibrio parahaemolyticus. Background Technology

[0002] With the continuous development and impact of society and industrialization, food safety has become one of the most discussed social issues. Various food quality problems caused by foodborne pathogens are constantly emerging, increasing the risk of outbreaks of certain foodborne illnesses. Vibrio parahaemolyticus is a Gram-negative, rod-shaped, arc-shaped, or oval-shaped non-spore-forming bacterium belonging to the genus Vibrio, and is a common pathogen. It is a halophilic Gram-negative bacterium that can ferment glucose, does not produce gas, cannot utilize sucrose or lactose, does not produce hydrogen sulfide, and is positive for oxidase tests, lysine decondensase and ornithine decondensase tests, and negative for arginine decondensase tests. Its main habitat is seawater. Consuming seafood contaminated with this bacterium can cause food poisoning. Many seafood products can be eaten raw, and direct consumption without heating or processing greatly increases the likelihood of foodborne illnesses. Therefore, detecting Vibrio parahaemolyticus and other foodborne pathogens in seafood is a necessary means of preventing foodborne illnesses.

[0003] Currently, detection techniques for Vibrio parahaemolyticus (V. parahaemolyticus) mainly include plate culture, MPN counting, and PCR amplification. These methods are time-consuming, prone to contamination leading to false positives, and can introduce pathogens into the environment, posing a threat to human health. Immunological methods rely on the specific reaction between antigens and antibodies, such as enzyme-linked immunosorbent assay (ELISA). While these methods offer high specificity, they have low sensitivity, are time-consuming, costly, require professional operation, and depend on large instruments for result observation. Temperature-sensitive or isothermal nucleic acid amplification techniques, such as PCR amplification, polymerase chain reaction (RPA), multiplex RPA, quantitative real-time RPA, and loop-mediated isothermal amplification (LAMP), all require large instruments for result observation and cannot be directly observed. Therefore, developing a time-saving, labor-saving, low-cost, and accurate one-step detection method has become the main research direction.

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR) are an immune mechanism in many bacteria and archaea to combat invasion. In nature, a fierce competition for survival exists between bacteria / archaea and bacteriophages. To resist invasion by foreign bacteriophages, bacteria and archaea have evolved the CRISPR-Cas system to recognize and cleave invading nucleic acids, thus protecting themselves. Cas12a is a type 2 V Cas protein. When inactive, it has no cleavage activity. When it binds to specific crRNA, Cas12a undergoes a conformational change, forming a Cas12a-crRNA binary complex. This complex specifically recognizes target DNA and activates its endonuclease activity. After Cas12a sequentially cleaves the DNA double strand in a cis-cleavage manner, this active site remains exposed, exhibiting trans-cleavage activity and non-specifically cleaving the surrounding ssDNA. Scientists have designed various highly sensitive and specific nucleic acid detection platforms to leverage the characteristics of Cas12a, and are also continuously developing portable, low-cost, and rapid detection platforms that do not require large instruments. However, because the CRISPR-Cas12 enzyme is highly temperature-sensitive, the amplification step of strain-specific fragments is usually performed at different reaction temperatures than the CRISPR-Cas12 system, requiring multiple tube opening and sample loading processes, which can lead to aerosol contamination and false positives. Furthermore, the fluorescence generated by Cas12 protein cleaving DNA probes requires specialized instruments for observation, further increasing detection costs. Therefore, a closed-tube visualization detection system is needed. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a kit and method for closed-tube visualization detection of Vibrio parahaemolyticus. This invention utilizes a DNA-crRNA complex design to place the RPA system, CRISPR system, and gold nanoparticles in the same tube, forming a closed-tube detection system to prevent cross-contamination.

[0006] Therefore, the technical solution adopted by the present invention is as follows:

[0007] This invention first provides a kit for closed-tube visualization detection of Vibrio parahaemolyticus, which includes a mixed reaction system located inside the detection tube and a mixture of thiol DNA-modified gold nanoparticles located on the cap of the detection tube.

[0008] The mixed reaction system in the detection tube includes a mixed RPA amplification system and a CRISPR / Cas12a reaction system with protective DNA modification.

[0009] The RPA amplification system is used to amplify specific gene fragments in the genome of Vibrio parahaemolyticus using RPA, and includes primer RPA-F, primer RPA-R, Primer-Free-R buffer, DNA template, RNAase-free water, and magnesium acetate.

[0010] The CRISPR / Cas12a reaction system comprises: Cas12a, a DNA-crRNA complex, a Linker-ssDNA, buffer C, and ddH2O. The DNA-crRNA complex is formed by the pairing of a protective DNA strand with crRNA. It can unwind at 37°C and slowly release crRNA to bind to the target DNA on a specific gene fragment in the genome of *Vibrio parahaemolyticus*. The sequence of the protective DNA strand is shown in SEQ ID No. 3, and the protective DNA is designed with a stem-loop structure. The first 16 bases of the protective DNA pair complementaryly with the 16 bases of the crRNA-specific sequence, and the last 8 bases, after unwinding, can complement the first 8 bases of the protective DNA strand to form a stem-loop structure. When the system reacts at 37°C for 10 minutes, the DNA-crRNA complex gradually unwinds, releasing crRNA. The DNA strand itself forms a stem-loop structure, ensuring that it does not form a complex again after unwinding due to complementarity with crRNA.

[0011] The gold nanoparticles modified with thiol DNA on the detection tube cap include Au NPs-DNA1, Au NPs-DNA2, and a saturated sodium chloride solution; wherein Au NPs-DNA1 and Au NPs-DNA2 are formed by modifying the gold nanoparticles with two different thiol DNA sequences, wherein the two thiol DNA sequences are complementary to the Linker-ssDNA in the mixed reaction system;

[0012] The mixture of thiol-DNA-modified gold nanoparticles on the tube cap is disposed on the lower surface of the tube cap, allowing it to be added to the mixed reaction system while the detection tube is closed.

[0013] As a preferred embodiment of the present invention, the volume of the in-tube mixing reaction system is equal to that of the mixture of gold nanoparticles modified with thiol DNA;

[0014] The total volume of the in-tube mixing reaction system was 10 μL, the RPA amplification system was 2.5 μL, and the CRISPR / Cas12a reaction system after DNA protection modification was 7.5 μL. The concentrations of each component were as follows: In the 2.5 μL RPA amplification system, primers RPA-F and RPA-R were 4.8 μM, magnesium acetate was 14 mM, primer-free-R buffer was 1.475 μL, DNA template was 0.1 μL, and RNAase-free water was 0.56 μL; In the 7.5 μL CRISPR / Cas12a reaction system, Cas12a was 250 nM, DNA-crRNA complex was 75 nM, and linker-ssDNA was 1 μM; the volume of buffer C added was 1 μL.

[0015] The buffer C is composed of the following components: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM MTT; the pH of buffer C is 7.9.

[0016] As a preferred embodiment of the present invention, the total volume of the gold nanoparticle mixture modified with thiol DNA is 10 μL, wherein the concentration of Au NPs-DNA1 is 2.7 μM and the concentration of Au NPs-DNA2 is 2.7 μM.

[0017] As a preferred embodiment of the present invention, the sequence of RPA-F is shown in SEQ ID No. 1, and the sequence of RPA-R is shown in SEQ ID No. 2.

[0018] As a preferred embodiment of the present invention, the sequence of the crRNA is shown in SEQ ID No. 4.

[0019] As a preferred embodiment of the present invention, the sequence of the Linker-ssDNA is shown in SEQ ID No. 5.

[0020] As a preferred embodiment of the present invention, the sequence of NPs-DNA1 is shown in SEQ ID No. 6; the sequence of NPs-DNA2 is shown in SEQ ID No. 7.

[0021] The sequences designed in this invention correspond as follows:

[0022]

[0023] The present invention also provides an application of the above-mentioned kit in the detection of Vibrio parahaemolyticus.

[0024] This invention also provides a method for closed-tube visualization detection of Vibrio parahaemolyticus using the above-mentioned kit, which includes the following steps:

[0025] 1) Add the sample to be tested into the mixed reaction system in the tube, and perform RPA amplification of specific gene fragments in the genome of Vibrio parahaemolyticus using amplification primers in the reaction system at 37°C;

[0026] Simultaneously, the DNA-crRNA complex in the reaction system unwinds at 37°C and slowly releases crRNA to bind to the target DNA on the specific gene fragment; under the mediation of crRNA, Cas12a specifically recognizes the amplified fragment of the specific gene fragment and activates nuclease activity to cleave the Linker-ssDNA that complements the two thiol DNAs in the reaction system.

[0027] 2) After the reaction in step 1) is complete, add the thiol-DNA modified gold nanoparticle mixture to the system in the tube and observe the color change of the system with the naked eye. When the sample contains Vibrio parahaemolyticus, the gold nanoparticles do not aggregate after Linker-ssDNA is cleaved, and the system is red. When the sample does not contain Vibrio parahaemolyticus, the gold nanoparticles aggregate, and the red color of the system fades. This achieves closed-tube detection of Vibrio parahaemolyticus.

[0028] As a preferred embodiment of the present invention, in step 2), after adding the mixture of gold nanoparticles modified with thiol DNA, the mixture is reacted for 5 minutes, then centrifuged, and the color change is observed.

[0029] Compared with existing technologies, the method of this invention can detect Vibrio parahaemolyticus (V. parahaemolyticus) under closed-tube conditions without the need for any detection or observation instruments. This invention utilizes a DNA strand protection design. The first 16 bases of the protected DNA strand are designed to pair complementaryly with the 16 bases of the crRNA-specific sequence. Under specific conditions, this complementary pairing forms a DNA-crRNA complex, thereby inhibiting the CRISPR reaction. The last 8 bases of the protected DNA strand are designed to pair complementaryly with the first 8 bases at the 5' end after unwinding, forming a stem-loop structure and preventing the DNA strand from rebinding to crRNA. The presence of the DNA-crRNA complex ensures that the RPA system reacts completely independently within the overall system. In this invention, the RPA and CRISPR systems are mixed and placed at the bottom of a tube, with gold nanoparticles placed on the tube cap. After reacting the system at 37°C, the DNA-crRNA complex gradually unwinds, releasing crRNA. The DNA strand transforms into a stem-loop structure, preventing further complementary formation of the complex with crRNA after unwinding. After the system at the bottom of the tube has reacted at 37°C for a certain period, the Au-DNA1 / Au-DNA2 from the tube cap is dropped and mixed with the RPA-CRISPR system. The color reaction change is observed, thus forming a closed-tube detection system to prevent cross-contamination. This invention provides a simple, rapid, sensitive, specific, and portable single-tube detection method for Vibrio parahaemolyticus. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the working principle of the present invention.

[0031] Figure 2 This figure shows the results of the closed-tube detection sensitivity experiment of Vibrio parahaemolyticuss in this invention.

[0032] Figure 3 This figure shows the results of the sensitivity detection of Vibrio parahaemolyticus in food samples using closed-tube methods according to the present invention.

[0033] Figure 4 This figure shows the results of the detection specificity experiment of Vibrio parahaemolyticuss in this invention. Detailed Implementation

[0034] The present invention will be further described and illustrated below with reference to specific embodiments. The embodiments described are merely examples of the content of this disclosure and do not limit the scope of the invention. The technical features of each embodiment in the present invention can be combined accordingly, provided that there is no mutual conflict.

[0035] I. Visual Detection of Vibrio parahaemolyticus using CRISPR / Cas12a System with DNA Protective Strains and Nanomaterials

[0036] Reference Figure 1 The procedure involved pretreating the actual samples using thermal lysis. Under aseptic conditions, 1 mL of the homogenized sample was transferred to a sterile centrifuge tube and centrifuged at 8000×g for 2 min, discarding the supernatant. The sample was resuspended in 200 μL of sterile deionized water, centrifuged at 8000×g for 2 min, and the supernatant was discarded. 100 μL of sterile deionized water was added, and the sample was boiled at 100℃ for 10 min and then cooled to serve as an RPA template for amplification. A protective DNA strand was designed, and the optimal fragment length of the protective DNA strand (i.e., SEQ ID No. 3) was selected by PAGE gel electrophoresis. The DNA strand was reacted with crRNA (i.e., SEQ ID No. 4) at 85℃ for 5 min, and the temperature was slowly reduced to 4℃ at a rate of 0.1℃ / s to obtain the DNA-crRNA complex.

[0037] The 2.5 μL RPA amplification system was constructed as follows: Primer RPA-F (SEQ ID No. 1) and primer RPA-R (SEQ ID No. 2) were at 4.8 μM each; magnesium acetate was at 14 mM; primer-free-R buffer was at 1.475 μL; DNA template was at 0.1 μL; and RNAase-free water was at 0.56 μL. A 7.5 μL CRISPR / Cas12a reaction system was constructed, including 250 nM Cas12a, 75 nM DNA-crRNA, 1 μM Linker-ssDNA (SEQ ID No. 4), 2.5 μL amplification product, and 1 μL buffer C (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH...). 7.9) Add RNase-free water to a final volume of 10 μL, and thoroughly mix the RPA and CRISPR systems at the bottom of the tube. Place 10 μL of Au-DNA1 / Au-DNA2 and an appropriate amount of saturated sodium chloride on the tube cap (Au NPs-DNA1 concentration is 2.7 μM, Au NPs-DNA2 concentration is 2.7 μM). Add the sample to be tested to the mixed reaction system in the tube and incubate at 37 °C for 40 min. After 40 min, shake off the gold nanoparticle mixture from the tube cap and mix it with the reaction system, then observe the colorimetric results.

[0038] II. Sensitivity Characterization of the Method of the Invention for Detecting Vibrio parahaemolyticus

[0039] Genomic DNA from foodborne pathogens was extracted using a bacterial genomic DNA extraction kit. To investigate the detection sensitivity of this invention against different concentrations of Vibrio parahaemolyticus in DNA, DNA samples with different concentrations of Vibrio parahaemolyticus (46 pg / μL, 4.6 pg / μL, 460 fg / μL, 46 fg / μL, 4.6 fg / μL, 460 ag / μL, 46 ag / μL, and 4.6 ag / μL) were added to a mixture containing RPA and CRISPR. The specific gene fragment system was analyzed using the closed-tube visualization detection method of this invention. Figure 2 In Figure A, we show the effect of different DNA contents of Vibrio parahaemolyticus on the detection and the colorimetric results. Figure 2 In section B, we demonstrate the use of a UV spectrophotometer and microplate reader to detect the sensitivity of Vibrio parahaemolyticus (V. parahaemolyticuss) at different DNA concentrations, plotting UV absorption peaks and error bar charts. V. parahaemolyticuss at concentrations of 46 pg / μL, 4.6 pg / μL, 460 fg / μL, 46 fg / μL, 4.6 fg / μL, 460 ag / μL, 46 ag / μL, and 4.6 ag / μL were subjected to RPA isothermal amplification. Analysis was performed using the CRISPR / Cas12a visualization detection system. Visual observation revealed the detection of V. parahaemolyticuss at concentrations as low as 4.6 ag / μL.

[0040] III. The method of this invention for detecting Vibrio parahaemolyticus in various food samples

[0041] The sensitivity of Vibrio parahaemolyticus serovar V. parahaemolyticuss at different bacterial concentrations in shrimp meat was detected using the closed-tube visualization detection method based on the CRISPR / Cas12a system. Figure 3 Figure A shows the effect of different Vibrio parahaemolyticus content in shrimp samples on colorimetric assays based on gold nanoparticle probes. The results can be directly observed with the naked eye down to the detection limit of 1×10⁻⁶. 0 CFU / mL. Figure 3 Figure B demonstrates the sensitivity detection of Vibrio parahaemolyticus (V. parahaemolyticuss) at different bacterial concentrations in shrimp meat using a UV spectrophotometer and ELISA reader, plotting UV absorption peaks and error bar charts. This invention uses concentrations of 1×10⁻⁶... 6 CFU / mL, 1×10 5CFU / mL, 1×10 4 CFU / mL, 1×10 3 CFU / mL, 1×10 2 CFU / mL, 1×10 1 CFU / mL, 1×10 0 CFU / mL of Vibrio parahaemolyticus was added to shrimp meat samples for pretreatment and followed by RPA isothermal amplification. Analysis was performed using a closed-tube visualization detection system based on the CRISPR / Cas12a protocol. Colorimetric results observed with the naked eye showed identification as low as 1×10⁻⁶. 0 CFU / mL of Vibrio parahaemolyticus.

[0042] IV. Characterization of the Detection Specificity of the Method of the Invention

[0043] To characterize the specificity of this CRISPR / Cas12a system for detecting Vibrio parahaemolyticus using a DNA protected strand and nanomaterial closed-tube visualization detection method, clinical isolates of Escherichia coli O157:H7, as well as Vibrio parahaemolyticus (VP), Salmonella (Sal), Listeria monocytogenes (Lm), and Staphylococcus aureus (Staphylococcus) were used. Using clinical isolates of *V. parahaemolyticus* (SA) as test samples, shrimp meat samples were pretreated, amplified using RPA, and mixed with a CRISPR / Cas12a system in one tube. An equal volume of gold nanoprobe was added to the tube cap for closed-tube visualization detection and analysis. Visual observation showed that the clinical isolate of *V. parahaemolyticus* was positive; visual observation of specific systems against *Salmonella*, *Listeria monocytogenes*, *Escherichia coli* O157:H7, and *Staphylococcus aureus* was negative. Results are as follows. Figure 4 As shown above, this demonstrates that the closed-tube visualization detection method of the CRISPR / Cas12a system has good accuracy and specificity.

[0044] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A closed-tube visual detection method for Vibrio parahaemolyticus Vibrio parahaemolyticus The reagent kit is characterized by This includes a mixed reaction system located inside the detection tube and a mixture of thiol-DNA-modified gold nanoparticles located on the cap of the detection tube; The mixed reaction system in the detection tube includes a mixed RPA amplification system and a CRISPR / Cas12a reaction system with protective DNA modification. The RPA amplification system is used for the detection of Vibrio parahaemolyticus. Vibrio parahaemolyticus RPA amplification of specific gene fragments in the genome includes primer RPA-F, primer RPA-R, Primer-Free-R buffer, DNA template, RNAase-free water, and magnesium acetate; the sequence of RPA-F is shown in SEQ ID No. 1, and the sequence of RPA-R is shown in SEQ ID No. 2; The CRISPR / Cas12a reaction system includes: Cas12a, a DNA-crRNA complex, a linker-ssDNA, buffer C, and ddH2O; wherein the DNA-crRNA complex is formed by the pairing of a protective DNA strand with crRNA, and can unwind at 37 °C to slowly release crRNA and Vibrio parahaemolyticus. Vibrio parahaemolyticus The DNA-crRNA complex binds to the target DNA on a specific gene fragment. The sequence of the protective DNA strand is shown in SEQ ID No. 3, and the protective DNA is designed with a stem-loop structure. The first 16 bases of the 3' end of the protective DNA pair complementaryly with the 16 bases of the crRNA-specific sequence. After unwinding, the last 8 bases can complement the first 8 bases of the protective DNA strand to form a stem-loop structure. When the system reacts at 37 °C for a certain period of time, the DNA-crRNA complex will gradually unwind and release crRNA. The DNA strand itself forms a stem-loop structure and ensures that it does not complement crRNA again to form a complex after unwinding. The buffer C consists of the following components: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM DTT; the pH of buffer C is 7.

9. The sequence of the crRNA is shown in SEQ ID No. 4; The gold nanoparticles modified with thiol DNA on the detection tube cap include Au NPs-DNA1, Au NPs-DNA2, and a saturated sodium chloride solution; wherein Au NPs-DNA1 and Au NPs-DNA2 are formed by modifying the gold nanoparticles with two different thiol DNA sequences, wherein the two thiol DNA sequences are complementary to the Linker-ssDNA in the mixed reaction system; The mixture of thiol-DNA-modified gold nanoparticles on the tube cap is disposed on the lower surface of the tube cap, allowing it to be added to the mixed reaction system while the detection tube is closed.

2. The reagent kit according to claim 1, characterized in that, The volume of the in-tube mixing reaction system is equal to that of the thiol DNA-modified gold nanoparticle mixture. The total volume of the in-tube mixing reaction system was 10 μL, the RPA amplification system was 2.5 μL, and the CRISPR / Cas12a reaction system after DNA protection modification was 7.5 μL. The concentrations of each component were as follows: In the 2.5 μL RPA amplification system, primers RPA-F and RPA-R were 4.8 μM, magnesium acetate was 14 mM, primer-free-R buffer was 1.475 μL, DNA template was 0.1 μL, and RNAase-free water was 0.56 μL; In the 7.5 μL CRISPR / Cas12a reaction system, Cas12a was 250 nM, DNA-crRNA complex was 75 nM, and linker-ssDNA was 1 μM; the volume of buffer C added was 1 μL.

3. The reagent kit according to claim 1, characterized in that, The total volume of the gold nanoparticle mixture modified with thiol DNA was 10 μL, wherein the concentration of Au NPs-DNA1 was 2.7 μM and the concentration of Au NPs-DNA2 was 2.7 μM.

4. The kit according to claim 1, characterized in that, The sequence of the Linker-ssDNA is shown in SEQ ID No.

5.

5. The kit according to claim 1, characterized in that, The sequence of NPs-DNA1 is shown in SEQ ID No. 6; the sequence of NPs-DNA2 is shown in SEQ ID No.

7.

6. The kit according to any one of claims 1-5 for detecting Vibrio parahaemolyticus in food samples. Vibrio parahaemolyticus Applications in [the field].

7. A closed-tube visualization method for detecting Vibrio parahaemolyticus in food samples based on the kit described in any one of claims 1-5. Vibrio parahaemolyticus The method is characterized by Includes the following steps: 1) Add the sample to be tested to the mixed reaction system in the tube. The amplification primers in the reaction system are for Vibrio parahaemolyticus. Vibrio parahaemolyticus Specific gene fragments in the genome were amplified by RPA at 37 ℃; Simultaneously, the DNA-crRNA complex in the reaction system unwinds at 37°C and slowly releases crRNA to bind to the target DNA on the specific gene fragment; under the mediation of crRNA, Cas12a specifically recognizes the amplified fragment of the specific gene fragment and activates nuclease activity to cleave the Linker-ssDNA that complements the two thiol DNAs in the reaction system. 2) After the reaction in step 1) is complete, add the thiol-DNA modified gold nanoparticle mixture to the system in the tube and observe the color change of the system with the naked eye; among them, when the sample contains Vibrio parahaemolyticus... Vibrio parahaemolyticus When Linker-ssDNA is cleaved, the gold nanoparticles do not aggregate, and the system turns red; when Vibrio parahaemolyticus is not present in the sample... Vibrio parahaemolyticus At this time, the gold nanoparticles aggregate, and the system loses its red color; thus achieving the effect of targeting Vibrio parahaemolyticus. Vibrio parahaemolyticus Closed-tube testing.

8. The method according to claim 7, characterized in that, In step 2), after adding the mixture of gold nanoparticles modified with thiol DNA, the mixture is reacted for 5 minutes, then centrifuged, and the color change is observed.

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