Visual detector based on OAS1-Cas13aREC fusion protein and application thereof

Abstract

The invention relates to the technical field of biology, and discloses a visual detector based on an OAS1-Cas13aREC fusion protein and application of the visual detector. A Cas13 protein in a CRISPR-Cas system and an OAS protein are recombined to construct a fusion protein, an REC structural domain in the Cas13 protein serves as a crRNA recognizer, a protruding stem-loop structure in a secondary structure of crRNA serves as a direct repetitive sequence to be combined with the REC structural domain in the Cas13 protein, meanwhile, a target RNA specific complementary sequence contained in the crRNA is combined with a target chain of a detection sample to form an RNA double-chain activated OAS protein part, and the Cas13 protein and the OAS protein are combined to construct the Cas13 protein. PPi generated by the OAS protein under the combination of short dsRNA and ATP can be subjected to color development determination, so that the vibrio cholerae in a food sample can be quantitatively and qualitatively determined. The detection method based on the fusion protein is easy and convenient to operate and has extremely high sensitivity and specificity, and an efficient new strategy is provided for detection of pathogenic bacteria in food.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a visualization detector based on OAS1-Cas13aREC fusion protein and its application. Background Art

[0002] Pathogens in food threaten the health and lives of humans and animals, impacting social stability and economic development. Early, accurate, and rapid detection of pathogens is crucial for monitoring and treating human and animal diseases. The most common foodborne pathogens in food include Escherichia coli (EC), Staphylococcus aureus (SA), Bacillus cereus (BC), Pseudomonas aeruginosa (PA), Vibrio alginolyticus (VA), Listeria monocytogenes (LM), Vibrio parahaemolyticus (VP), and Vibrio cholerae (VC). Multiple contaminations of food with different pathogens are common. Among them, Vibrio cholerae is a highly destructive pathogen that can cause the severe acute diarrheal illness known as cholera. This disease is highly contagious and is typically transmitted through the ingestion of contaminated water or food. Since 1817, cholera has caused seven global pandemics, posing a persistent threat to global public health. Therefore, rapid, highly sensitive, low-cost, and easy-to-use detection of multiple foodborne pathogens is crucial for food safety.

[0003] Traditional culture-based foodborne pathogen detection methods typically require one to two days to obtain results, making them difficult to meet the demand for rapid testing. Techniques such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and flow cytometry are faster than culture-based methods, but they are time-consuming, have low sensitivity, or are laboratory-dependent, making them insufficient for point-of-care (POC) testing. There is an urgent need to develop new rapid detection technologies that are faster, more stable, more affordable, and simpler, with high sensitivity and specificity. Summary of the Invention

[0004] In view of this, the object of the present application is to provide a detection composition for detecting Vibrio cholerae, which can be used in the detection of Vibrio cholerae to achieve rapid and accurate visual detection, quantitative / semi-quantitative detection, and excellent sensitivity and specificity;

[0005] Another purpose of the present application is to provide related detection applications and products for Vibrio cholerae based on the above-mentioned detection composition.

[0006] In order to solve the above technical problems / achieve the above objectives or at least partially solve the above technical problems / achieve the above objectives, as a first aspect of the present application, a detection composition for detecting Vibrio cholerae is provided, comprising a fusion protein and crRNA;

[0007] The fusion protein comprises OAS protein and Cas13 protein; the crRNA is a single-stranded RNA comprising a sequence capable of forming a stem-loop structure and a sequence complementary to the base of Vibrio cholerae 16S rRNA.

[0008] Optionally, the OAS protein includes an OAS1 protein, and the Cas13 protein includes a Cas13aREC protein. Further optionally, the sequence of the OAS1 protein is shown in SEQ ID No. 1, and the sequence of the Cas13aREC protein is shown in SEQ ID No. 2.

[0009] Optionally, the sequence capable of forming a stem-loop structure is shown as SEQ ID No. 3, and the sequence complementary to the Vibrio cholerae 16SrRNA base is shown as SEQ ID No. 4.

[0010] As a second aspect of the present application, provided is the use of the detection composition in detecting Vibrio cholerae for non-diagnostic purposes or in preparing a product for detecting Vibrio cholerae.

[0011] As a third aspect of the present application, a visualization detector based on the OAS1-Cas13aREC fusion protein is provided, comprising the detection composition described in the present application, and one or more components selected from ATP, PPi colorimetric reagent, buffer, and EDTA.

[0012] As a fourth aspect of the present application, a method for detecting Vibrio cholerae for non-diagnostic purposes is provided, comprising:

[0013] Obtaining rRNA of the sample to be tested;

[0014] The rRNA is incubated with the detection composition described in the present application and ATP in a buffer solution, and then the reaction is terminated. A PPi color development reagent is added for reaction, and the presence of Vibrio cholerae is visually and qualitatively determined based on the color.

[0015] Optionally, the method further comprises preparing a standard curve of Vibrio cholerae concentration-absorbance value for quantitative detection:

[0016] A series of Vibrio cholerae bacterial solutions of known concentrations were prepared and tested according to the method for detecting Vibrio cholerae in the present application. The absorption spectra of the bacterial solutions of different concentrations after reaction were recorded using an instrument. The Vibrio cholerae concentration and its corresponding absorbance value were used as coordinates to obtain a curve of absorbance versus Vibrio cholerae concentration.

[0017] Find the point with linear change law on the curve, perform linear fitting, and obtain the standard curve of Vibrio cholerae concentration-absorbance value;

[0018] The absorbance value obtained after rRNA detection of the sample to be tested is substituted into the standard curve to obtain the quantitative detection result.

[0019] Optionally, the method further comprises preparing a standard colorimetric card of Vibrio cholerae concentration-colorimetric value for semi-quantitative detection:

[0020] Prepare a series of Vibrio cholerae solutions with known concentrations, perform tests according to the method for detecting Vibrio cholerae described in this application, record different Vibrio parahaemolyticus concentrations and their corresponding colorimetric values, and prepare a standard colorimetric card;

[0021] After the rRNA of the sample to be tested is detected, the color presented is compared with the prepared standard colorimetric card to obtain a semi-quantitative test result.

[0022] Optionally, methods for obtaining rRNA of a sample to be tested include:

[0023] After grinding the tissue of the food sample, LB culture medium containing NaCl was added to mix into a homogenate, and the supernatant of the food sample was obtained by centrifugation for rRNA extraction.

[0024] This application recombines the Cas13 protein and OAS protein in the CRISPR-Cas system to construct a fusion protein, using the REC domain in the Cas13 protein as a crRNA recognizer, and the raised stem-loop structure in the secondary structure of the crRNA as a direct repeat sequence to bind to the REC domain in the Cas13 protein. At the same time, the target RNA-specific complementary sequence contained in the crRNA binds to the target chain of the test sample to form an RNA double-stranded activation OAS protein portion. The PPi generated by the OAS protein under the combination of short dsRNA and ATP can be measured by colorimetric analysis, thereby quantitatively and qualitatively determining Vibrio cholerae in food samples. The detection method based on fusion protein in this application is simple to operate, has extremely high sensitivity and specificity, and provides an efficient new strategy for the detection of pathogens in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The exemplary embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application.

[0026] Figure 1 Shown is a schematic diagram of the principle of a visualization detector based on the OAS1-Cas13aREC fusion protein of this application;

[0027] Figure 2 Shown are gel images of OAS1 protein and the OAS1-Cas13aREC fusion protein of the present application;

[0028] Figure 3Shown are the feasibility analysis results of the present application for detecting Vibrio cholerae RNA using a visual detector based on the OAS1-Cas13aREC fusion protein;

[0029] Figure 4 Shown are the buffer optimization results of the visual detector based on the OAS1-Cas13aREC fusion protein in this application; the control group was subjected to a control experiment by adding 500 nmol / L OAS1-Cas13aREC fusion protein and 500 nmol / L crRNA under the same buffer conditions;

[0030] Figure 5 Shown are the optimization results of the reaction system of the visualization detector based on the OAS1-Cas13aREC fusion protein of this application;

[0031] Figure 6 Shown is the sensitivity detection of Vibrio cholerae RNA by the visualization detector based on the OAS1-Cas13aREC fusion protein of the present application;

[0032] Figure 7 Shown is the specific detection of Vibrio cholerae by the visualization detector based on the OAS1-Cas13aREC fusion protein of the present application;

[0033] Figure 8 Shown is the visualization detector based on OAS1-Cas13aREC fusion protein of the present application for detecting Vibrio cholerae in simulated samples and RT-qPCR control;

[0034] A: Detection results of five simulated positive samples and three negative samples of tilapia gill (oreochromis gill), tilapia liver (oreochromis liver), and Chinese velvet crab gill (eriocheir sinensis gill);

[0035] B: Detection results of five simulated positive samples and three negative samples of whiteleg shrimp pancreas (Litopenaeus vannamei hepatopancreas), whiteleg shrimp meat (Litopenaeus vannamei meat), and clam meat (Meretrix meretrix);

[0036] C: RT-qPCR test results of 5 simulated positive samples and 3 negative samples of tilapia gill (oreochromis gill), tilapia liver (oreochromis liver) and Chinese velvet crab gill (eriocheir sinensis gill);

[0037] D: RT-qPCR test results of five simulated positive samples and three negative samples of whiteleg shrimp pancreas (Litopenaeus vannamei hepatopancreas), whiteleg shrimp meat (Litopenaeus vannamei meat), and clam meat (Meretrix meretrix). DETAILED DESCRIPTION

[0038] The present application discloses a visual detector based on OAS1-Cas13aREC fusion protein and its application. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The products, processes and applications described in this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of this application.

[0039] It should be noted that, in this document, if relational terms such as "first" and "second", "step 1" and "step 2", and "(1)" and "(2)" appear, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "including a..." do not exclude the presence of other identical elements in the process, method, article or device comprising the elements. At the same time, the embodiments in this application and the features in the embodiments can be combined with each other in the absence of conflict.

[0040] In a first aspect of the present application, a detection composition for detecting Vibrio cholerae is provided, comprising a fusion protein and crRNA;

[0041] The fusion protein comprises OAS protein and Cas13 protein; the crRNA is a single-stranded RNA comprising a sequence capable of forming a stem-loop structure and a sequence complementary to the base of Vibrio cholerae 16S rRNA.

[0042] OAS1 is a double-stranded RNA (dsRNA) sensor, and its role in the cell is to activate potential RNaseL by converting ATP into 2', 5'-oligoadenylic acid (2-5A), thereby degrading single-stranded RNA and inhibiting viral replication, while also producing a byproduct pyrophosphate molecule (ppi). Although OAS1 can interact with short dsRNA (>18 base pairs (bp)), it lacks the RNA binding sequence that binds to the target chain and cannot accurately identify and locate. This application uses the three-dimensional structure of the REC domain of the Cas13 protein in the CRISPR-Cas system to allow the REC domain to accurately identify the raised stem-loop structure in the crRNA by designing a crRNA that can form a secondary structure of a stem-loop. The secondary structure is bound to the central channel of the positively charged nuclease (NUC) leaf, and the crRNA also contains a sequence that can specifically identify Vibrio cholerae 16S rRNA, which can capture the target to form a short dsRNA. By fusing the Cas13 protein with the OAS protein, the purpose of OAS recognizing the target is indirectly achieved, thereby initiating the interaction between OAS and short dsRNA to produce pyrophosphate molecules (PPi), and then the PPi colorimetric reagent can be used to qualitatively and quantitatively detect Vibrio cholerae.

[0043] In certain embodiments of the present application, the OAS protein includes an OAS1 protein, and the CRISPR protein includes a Cas13aREC protein. In other embodiments of the present application, the sequence of the OAS1 protein is as follows:

[0044] MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYIKNP

[0045] DNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSE

[0046] EDISEYDLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSF

[0047] EENKANYQKINENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFD

[0048] KLYKKEDIEKLFFLIENSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQN

[0049] VNNIKELIEKIPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMS

[0050] QLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKY

[0051] NYYLQVGEI (shown in SEQ ID No.1);

[0052] The sequence of the Cas13aREC protein is as follows:

[0053] HMELRHTPARDLDKFIEDHLLPNTCFRTQVKEAIDIVCRFLKERCFQG

[0054] TADPVRVSKVVKGGSSGKGTTLRGRSDADLVVFLTKLTSFEDQLRRRGEF

[0055] IQEIRRQLEACQREQKFKVTFEVQSPRRENPRALSFVLSSPQLQQEVEFDVL

[0056] PAFDALGQWTPGYKPNPEIYVQLIKECKSRGKEGEFSTCFTELQRDFLRNR

[0057] PTKLKSLIRLVKHWYQTCKKTHGNKLPPQYALELLTVYAWEQGSRKTDF

[0058] STAQGFQTVLELVLKHQKLCIFWEAYYDFTNPVVGRCMLQQLKKPRPVIL

[0059] DPADPTGNVGGGDTHSWQRLAQEARVWLGYPCCKNLDGSLVGAWTML

[0060] QKI (shown in SEQ ID No.2);

[0061] In certain embodiments of the present application, the fusion protein is connected to the Cas13aREC protein and the OAS1 protein through a linker protein, such as a flexible linker protein GGGGS (shown in SEQ ID No. 5), and the flexible linker protein sequence can be a plurality of repeat sequences, such as a flexible linker protein of 1-5 GGGGS.

[0062] In certain embodiments of the present application, the fusion protein further comprises a protein sequence of a screening tag and a promoter, which is generally a protein sequence expressed by a gene assembly inserted into an expression vector for preparing a fusion protein, such as a histidine screening tag and a T7 promoter. In some other embodiments of the present application, the sequence of the fusion protein from 5'-3' is: Cas13aREC protein-flexible linker protein-OAS1 protein-histidine tag-T7 promoter, and the more specific sequence is as follows (shown in SEQ ID No. 6):

[0063] .

[0064] In certain embodiments of the present application, the sequence capable of forming a stem-loop structure is as follows:

[0065] GAUUUAGACUACCCCAAAAACGAAAGGGACUAAAC (shown in SEQ ID No. 3, according to the requirements for making sequence listings in WIPOST.26, base U in RNA is T);

[0066] The sequence of the Vibrio cholerae 16S rRNA base complement is as follows:

[0067] CUCCACCUCGCGGUAUCGCU (shown in SEQ ID No. 4).

[0068] In some other embodiments of the present application, the sequence of the crRNA is as follows:

[0069] GAUUUAGACUACCCCAAAAACGAAAGGGACUAAAC CUCCACCUCGCGGUAUCGCU(SEQ ID No.3+ SEQ ID No.4) , which includes a 35nt direct repeat sequence that binds to the Cas13aREC domain, which can form a stem-loop secondary structure, and a 20nt reverse complementary part to the target chain. The reverse complementary sequence is designed based on the 16SrRNA sequence specific to Vibrio cholerae.

[0070] In a second aspect of the present application, the use of the detection composition for non-diagnostic detection of Vibrio cholerae or for the preparation of a product for detecting Vibrio cholerae is provided. The non-diagnostic purpose refers to a method for detecting Vibrio cholerae solely for food safety, without directly assessing the health status of the sample being tested. The food sample can be a non-frozen or frozen sample, or the food sample's surrounding environment can be sampled for testing.

[0071] In certain embodiments of the present application, the food sample may be selected from fish, crustaceans, shellfish, and molluscs.

[0072] In the third aspect of the present application, a visualization detector based on the OAS1-Cas13aREC fusion protein of the present application is provided, comprising the detection composition described in the present application, and one or more components selected from ATP, PPi colorimetric reagent, buffer, and EDTA.

[0073] Conventional buffers include OAS1 and dsRNA reaction buffer and CRISPR-Cas13a and its crRNA assembly reaction buffer; wherein, the OAS1 and dsRNA reaction buffer components are 25mmol / LTris-HCl, 25mmol / L NaCl, 7mmol / L MgCl2, 1mmol / L DTT, 2mmol / L ATP; the CRISPR-Cas13a and its crRNA assembly reaction buffer components are 40mmol / L Tris-HCl, 60mmol / L NaCl, 6mmol / L MgCl2, 1mg / mL proline, pH 6.3; however, there is no buffer for OAS1-Cas13aREC fusion protein in the prior art. In certain embodiments of the present application, the present application provides an optimized buffer solution comprising 20 mmol / L±5 mmol / L HEPES, 8 mmol / L±2 mmol / LMgCl2, 1 mmol / L±0.2 mmol / L DTT, 1 mg / mL±0.5 mg / mL proline, and a pH of 6.5. The reaction buffer solution of the present application is optimized based on the reaction buffer system of the two proteins, and the reaction signal is increased by 3.33 times before and after optimization.

[0074] In certain embodiments of the present application, the PPi color development reagent includes 2.5% (w / v) ammonium molybdate, 2.5 mol / L H2SO4 and 0.5 M β-mercaptoethanol. Commercial PPi color development reagents can also be purchased.

[0075] In a fourth aspect of the present application, a method for detecting Vibrio cholerae for non-diagnostic purposes is provided, comprising:

[0076] Obtaining rRNA of the sample to be tested;

[0077] The rRNA is incubated with the detection composition described in the present application and ATP in a buffer solution, and then the reaction is terminated. A PPi color development reagent is added for reaction, and the presence of Vibrio cholerae is visually and qualitatively determined based on the color.

[0078] The reaction is terminated by adding EDTA. After the addition of the PPi colorimetric reagent, the color of the reaction solution gradually changes from yellow to dark blue as the concentration of Vibrio cholerae increases, allowing for direct visual assessment of the degree of contamination in food samples.

[0079] In certain embodiments of the present application, in order to further accurately detect the degree of contamination of food samples, a standard curve of Vibrio cholerae concentration-absorbance value is prepared for quantitative detection:

[0080] A series of Vibrio cholerae bacterial solutions of known concentrations were prepared and tested according to the method for detecting Vibrio cholerae in the present application. The absorption spectra of the bacterial solutions of different concentrations after reaction were recorded using an instrument. The absorbance intensity at 580 nm after PPi color development was typically measured using a UV-visible spectrophotometer. The Vibrio cholerae concentration and its corresponding absorbance were used as coordinates to generate a curve of absorbance versus Vibrio cholerae concentration.

[0081] Find the point with linear change law on the curve, perform linear fitting, and obtain the standard curve of Vibrio cholerae concentration-absorbance value;

[0082] The absorbance value obtained after rRNA detection of the sample to be tested is substituted into the standard curve to obtain the quantitative detection result.

[0083] In certain embodiments of the present application, a standard colorimetric card of Vibrio cholerae concentration-colorimetric value is prepared for semi-quantitative detection:

[0084] Prepare a series of Vibrio cholerae solutions with known concentrations, perform tests according to the method for detecting Vibrio cholerae described in this application, record different Vibrio parahaemolyticus concentrations and their corresponding colorimetric values, and prepare a standard colorimetric card;

[0085] After the rRNA of the sample to be tested is detected, the color presented is compared with the prepared standard colorimetric card to obtain a semi-quantitative test result.

[0086] The detection principle diagram of the method described in this application is shown in Figure 1 In the buffer, crRNA forms a stem-loop secondary structure and then binds to the OAS1-Cas13aREC fusion protein. If the Vibrio cholerae target strand is present in the rRNA of the test sample, the reverse complementary sequence in the crRNA binds to it to form a short dsRNA, activating the OAS1 moiety. The PPi generated by the OAS1 protein in response to the short dsRNA and ATP can be measured using colorimetric analysis, allowing for both quantitative and qualitative determination of the target RNA sequence added to the reaction system.

[0087] In certain embodiments of the present application, the method of obtaining rRNA of the sample to be tested includes:

[0088] After grinding the food sample tissue, add LB medium containing NaCl to form a homogenate. Centrifuge to obtain the food sample supernatant for rRNA extraction. rRNA extraction can be performed using a commercial kit or conventional rRNA extraction methods known in the art.

[0089] More specifically, it includes:

[0090] 0.5 g of food sample was taken in a mortar, ground, mixed with 4.5 mL of LB medium containing 3.5% NaCl and homogenized for 30 min to obtain a 1:10 homogenate. The food sample supernatant was obtained by centrifugation for rRNA extraction.

[0091] In certain embodiments of the present application, the food samples include but are not limited to heart, liver, kidney, pancreas, intestine, gills, skin mucus, gall bladder, spleen, etc. These food samples are obtained by processing and dissection, and then pre-processed to extract rRNA.

[0092] In the comparative experiments provided in this application, unless otherwise specified, other experimental conditions, materials, etc. are kept consistent except for the differences noted in each group, so as to provide comparability. In addition, all materials used in this application can be purchased from commercial sources. All solutions used in this application were prepared with DEPC water, and the sample (bacteria) RNA extraction kit model is Bacterial RNA Kit was purchased from OMEGA.

[0093] In the sequence information provided in this application, unless otherwise specified, the sequence is in 5'-3' order, that is, the default writing rules in the biological field are adopted.

[0094] The following further describes a visualization detector based on OAS1-Cas13aREC fusion protein and its application provided in this application.

[0095] Example 1:

[0096] 1. According to the OAS1-Cas13aREC fusion protein sequence, it was sent to the reagent company for synthesis and gel imaging was performed. The results are shown in Figure 2 ; Figure 2 The molecular weight of the fusion protein was 85KD, and that of the OAS1 protein was 41KD;

[0097] 2. Feasibility analysis

[0098] The frozen Vibrio cholerae (CICC23794) strain was inoculated into LB medium containing 3.5% NaCl and cultured at 37°C and 180 rpm for about 12 h until the OD value was 0.8, resulting in a concentration of about 1×10 8 CFU / mL of bacterial solution. Then, rRNA of Vibrio cholerae was extracted using a bacterial RNA extraction kit. The extracted RNA was verified by 1% agarose gel electrophoresis, and its concentration was measured and stored at -80°C.

[0099] Set up four samples for detection experiments:

[0100] Sample 1, including 1 μmol / L OAS1-Cas13aREC fusion protein and 2 mmol / L ATP;

[0101] Sample 2, including 1 μmol / L OAS1-Cas13aREC fusion protein, 2 mmol / L ATP, and 500 nmol / L crRNA;

[0102] Sample 3, including 1 μmol / L OAS1-Cas13aREC fusion protein, 2 mmol / L ATP, and 500 ng of Vibrio cholerae rRNA;

[0103] Sample 4, 1 μmol / L OAS1-Cas13aREC fusion protein, 2 mmol / L ATP, 500 nmol / L crRNA, and 500 ng Vibrio cholerae rRNA.

[0104] The experimental results are as follows Figure 3 According to the experimental results, sample 4 showed a high signal different from the previous three control samples, which can prove the feasibility of visual detection of the OAS1-Cas13aREC fusion protein.

[0105] Example 2:

[0106] 1. Buffer optimization

[0107] The buffer optimization experimental settings are shown in Table 1 below;

[0108] Table 1 OAS1-Cas13aREC fusion protein detector buffer optimization

[0109]

[0110]

[0111] The optimization was performed according to the parameters listed in Table 1. The results are shown in Figure 4 The control group shown in the figure was subjected to control experiments by adding 500nmol / L OAS1-Cas13aREC fusion protein and 500nmol / L crRNA under the same buffer conditions; the experimental group was subjected to reactions by adding 500nmol / L OAS1-Cas13aREC fusion protein, 500nmol / L crRNA and 500ng Vibrio cholerae rRNA under the buffer optimization conditions designed in the table. Figure 4The results show that when the buffer solution is 20mmol / L±5mmol / LHEPES, 8mmol / L±2mmol / L MgCl2, 1mmol / L±0.2mmol / L DTT, 1mg / mL±0.5mg / mL proline, and pH 6.5, a more obvious OD value difference can be obtained compared with other conditions, which is more conducive to the accuracy of the test results.

[0112] 2. Reaction system optimization

[0113] The experimental settings for reaction system optimization are shown in Table 2 below;

[0114] Table 2 Optimization of OAS1-Cas13aREC fusion protein detector reaction system

[0115]

[0116]

[0117] The optimization was performed according to the parameters listed in Table 2. The results are shown in Figure 5 ,according to Figure 5 The results show that when the amount of OAS1-Cas13aREC fusion protein added to the reaction system is 1 μmol / L±0.2 μmol / L, the amount of ATP added is 5 mmol / L±1 mmol / L, the optimal reaction temperature is 37°C±2°C, and the optimal reaction time is 12h±0.5h, a more obvious OD value can be obtained, which is more conducive to the accuracy of the detection results.

[0118] Example 3:

[0119] The frozen Vibrio cholerae (CICC23794) strain was inoculated into LB medium containing 3.5% NaCl and cultured at 37°C and 180 RPM for about 12 h until the OD value was 0.8, resulting in a concentration of about 1×10 8 CFU / mL of bacterial solution. Use a pipette to draw 100 μL of each and 900 μL of PBS buffer into a 1.5 mL centrifuge tube, and shake to mix, to obtain a 10-fold diluted bacterial solution. Use a pipette to draw 100 μL of the 10-fold diluted bacterial solution into 900 μL of PBS buffer into a 1.5 mL centrifuge tube, and shake to mix, to obtain a 10-fold diluted bacterial solution. 2 times of bacterial solution. By analogy, the concentration is 1×10 2 CFU / mL-1×10 8 CFU / mL bacterial solution, and the dilution process was completed in a clean bench.

[0120] Then, rRNA was extracted from different concentrations of Vibrio cholerae using a bacterial RNA extraction kit. The extracted RNA was verified by 1% agarose gel electrophoresis and its concentration was measured and stored at -80°C. The RNA extracted from different gradient concentrations of bacterial solution was added to the detection system for detection. The experimental results are shown in the attached figure. Figure 6 As shown in the experimental results, it can be seen that the color of the reaction solution gradually changes from yellow to dark blue with the increase of bacterial concentration, and the absorption intensity at 580nm gradually increases with the increase of bacterial concentration, and there is a good linear relationship between the two (R 2 =0.99), and its regression equation is -Lg Absorbance Trend(OD) =-0.19Lg v.cholerae(CFU / mL) The minimum detection concentration of the OAS1-Cas13aREC fusion protein visual detector for Vibrio cholerae was calculated to be 38.2 CFU / mL, demonstrating excellent sensitivity.

[0121] Example 4:

[0122] Six different bacterial species were cultured in a medium suitable for their growth to obtain a concentration of about 1×10 8 CFU / mL of bacterial solution, and then diluted to obtain 1×10 3 CFU / mL, 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 The bacterial solution of four gradients of CFU / mL was added to the visualization detection system of OAS1-Cas13aREC fusion protein for detection. The experimental results are shown in the attached Figure 7 As shown. The experimental results show that both the measured absorbance at 580 nm and visual observation of the reaction solution indicate that the detector produces no signal when detecting other bacterial species, while a clear signal characteristic is observed when detecting Vibrio cholerae. This demonstrates the specificity of the OAS1-Cas13aREC fusion protein visualization detector for Vibrio cholerae detection.

[0123] Example 5:

[0124] Clams, whiteleg shrimp, tilapia, and Chinese mitten crab were purchased from the market and processed and dissected to obtain tissue samples. The five samples included the pancreas of whiteleg shrimp (Litopenaeus vannamei hepatopancreas), the meat of whiteleg shrimp (Litopenaeus vannamei meat), the clam meat (Meretrix meretrix), the gills of tilapia (oreochromis gill), the liver of tilapia (oreochromis liver), and the gills of Chinese mitten crab (eriocheir sinensis gill). First, each tissue sample (0.5 g) was ground and mixed with 4.5 mL of LB medium containing 3.5% NaCl and homogenized for 30 minutes to obtain a 1:10 homogenate. Then, Vibrio cholerae was cultured in LB medium containing 3.5% NaCl to the logarithmic phase, and 0 to 200 μL of Vibrio cholerae solution was pipetted to achieve a final concentration range of 10 in each tube of sample. 5 CFU / mL to 0 CFU / mL; the liquid was cultured in a test tube in an incubator under the following conditions: 37°C 200 rpm for 10 h.

[0125] Each tissue sample was simulated to produce 5 positive groups and 3 negative groups. The cultured samples were centrifuged at 4°C and 500g for 5 minutes, and the supernatant (containing pathogens) was taken. 3 mL of the obtained bacterial supernatant was used to extract rRNA using a kit, and the extracted RNA was added to the OAS1-Cas13aREC fusion protein detection system for detection. At the same time, the extracted simulated sample RNA was subjected to RT-qPCR to verify the test results. The experimental results are shown in the attached figure. Figure 8 As shown, the visualization detector results of the OAS1-Cas13aREC fusion protein confirmed all 5 positive samples and 3 negative samples, which was consistent with the results obtained by RT-qPCR, proving that the detector has good accuracy in actual sample detection.

[0126] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. A detection composition for detecting Vibrio cholerae, characterized in that: Including fusion protein and crRNA; The fusion protein comprises OAS protein and Cas13 protein; the crRNA is a single-stranded RNA comprising a sequence capable of forming a stem-loop structure and a sequence complementary to the base of Vibrio cholerae 16S rRNA.

2. The detection composition according to claim 1, characterized in that The OAS protein includes OAS1 protein, and the Cas13 protein includes Cas13aREC protein.

3. The detection composition according to claim 2, characterized in that The sequence of the OAS1 protein is shown in SEQ ID No. 1, and the sequence of the Cas13aREC protein is shown in SEQ ID No.

2.

4. The detection composition according to claim 1, characterized in that The sequence capable of forming a stem-loop structure is shown in SEQ ID No.3, and the sequence complementary to the base of Vibrio cholerae 16S rRNA is shown in SEQ ID No.

4.

5. Use of the detection composition according to any one of claims 1 to 4 in detecting Vibrio cholerae for non-diagnostic purposes or in preparing a product for detecting Vibrio cholerae.

6. A visual detector based on OAS1-Cas13aREC fusion protein, characterized in that The invention comprises the detection composition according to any one of claims 1 to 4, and one or more components selected from ATP, PPi colorimetric reagent, buffer, and EDTA.

7. A method for detecting Vibrio cholerae for non-diagnostic purposes, characterized in that: include: Obtaining rRNA of the sample to be tested; The rRNA is incubated with the detection composition according to any one of claims 1 to 4 and ATP in a buffer, and then the reaction is terminated, a PPi colorimetric reagent is added for reaction, and the presence of Vibrio cholerae is intuitively and qualitatively determined based on the color.

8. The method according to claim 7, characterized in that: It also includes preparing a standard curve of Vibrio cholerae concentration-absorbance value for quantitative detection: A series of Vibrio cholerae bacterial solutions of known concentrations are prepared, and the detection is performed according to the method described in claim 7, and the absorption spectra of the bacterial solutions of different concentrations after the reaction are recorded using an instrument, and a curve of the change of absorbance value with the concentration of Vibrio cholerae is obtained by taking the concentration of Vibrio cholerae and its corresponding absorbance value as the coordinate; Find the point with linear variation law on the curve, perform linear fitting, and obtain the standard curve of Vibrio cholerae concentration-absorbance value; Substitute the absorbance value obtained after rRNA detection of the sample to be tested into the standard curve to obtain the quantitative detection result.

9. The method according to claim 7, characterized in that: It also includes the preparation of a standard colorimetric card of Vibrio cholerae concentration-colorimetric value for semi-quantitative detection: Prepare a series of Vibrio cholerae solutions with known concentrations, perform detection according to the method described in claim 7, record different Vibrio parahaemolyticus concentrations and their corresponding colorimetric values, and prepare a standard colorimetric card; After the rRNA of the sample to be tested is detected, the color presented is compared with the prepared standard colorimetric card to obtain a semi-quantitative test result.

10. The method according to claim 7, characterized in that: Methods for obtaining rRNA of the sample to be tested include: After grinding the tissue of the food sample, LB medium containing NaCl was added to mix into a homogenate, and the food sample supernatant was obtained by centrifugation for rRNA extraction.

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