CRISPR-Cas13d-based visual detection method for Seneca valley virus

Abstract

The invention discloses a visual detection method of Seneca valley virus based on CRISPR-Cas13d (clustered regularly interspaced short palindromic repeats-associated protein 13d), and belongs to the technical field of molecular biology diagnosis. The method comprises the following steps: firstly, amplifying a target virus sequence by utilizing an isothermal amplification technology RAA, specifically identifying and amplifying signals by utilizing a CRISPR-Cas13d system, specifically identifying and amplifying signals by utilizing a crRNA sequence as shown in SEQ ID NO.13, and finally, visually observing a detection result by utilizing various visualization methods, according to the detection method provided by the invention, the SVA virus can be accurately and quickly detected in time on site, the detection capability of the SVA virus is remarkably improved, and the biological safety risk is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biological diagnosis, and in particular relates to a visual detection method of Seneca Valley virus based on CRISPR-Cas13d. Background Art

[0002] Senecavirus A (SVA) is also known as Seneca Valley virus (SVV). Its Chinese translations include Senecavirus A, Seneca Valley virus, Seneca virus and Seneca virus. SVA is a single-stranded positive-strand RNA virus belonging to the genus Senecavirus of the family MicroRNAviridae. Its genome is about 7.2kb long and has an icosahedral structure. Its genome consists of the L region and three major protein regions (P1, P2, and P3). The P1 polyprotein can be hydrolyzed into VP1, VP3, and VPO proteins, of which VPO is further hydrolyzed into VP2 and VP4 during the assembly of the SVA virus. These four proteins - VP1, VP2, VP3, and VP4 - together constitute the capsid of the virus. P2 and P3 encode non-structural proteins that participate in viral replication. The genome is flanked by 5'UTR and 3'UTR, the latter of which is connected to a poly(A) tail.

[0003] SVA was first discovered in human fetal retinal cell lines in the United States in 2002, but it has been traced back to 1988 and has existed in American pig herds. Since it was introduced to Guangdong in 2015, SVA has spread rapidly from south to north, causing outbreaks in many provinces. As an emerging animal infectious disease, its transmission and pathogenic mechanism are still unclear, there is a risk of large-scale outbreaks, and there is currently no commercial vaccine, and the prevention and control situation is severe. Therefore, rapid and sensitive clinical point-of-care testing is crucial for the prevention and control of SVA. At present, Rt-PCR and Rt-qPCR are standard and effective diagnostic methods for detecting PRRSV virus with high specificity and sensitivity. However, it requires professional equipment, experienced technicians, and the process from sampling to results is time-consuming. Therefore, Rt-PCR is difficult to meet the needs of large-scale on-site point-of-care testing (POCT) in large-scale production.

[0004] For POCT (point-of-care testing), it is crucial to ensure the accuracy and convenience of testing. At present, isothermal amplification technologies such as enzyme polymerase amplification (RPA), recombinase-assisted amplification (RAA) and loop-mediated isothermal amplification (LAMP), as well as colloidal gene immunochromatographic test strips, are widely used in clinical applications. However, each of these methods has certain limitations. Isothermal amplification technology can efficiently amplify target DNA under constant temperature conditions, and has efficient amplification capabilities and strong anti-inhibition properties, but nonspecific amplification may affect the accuracy of the results, becoming an obstacle to its widespread application. On the other hand, it is difficult for colloidal gene immunochromatographic test strips to achieve a balance between sensitivity and specificity.

[0005] The CRISPR / Cas system, a gene editing technology derived from bacteria, has become an indispensable tool in the field of molecular biology and genome editing, and is hailed as the core of the "next generation of molecular diagnostic technology". The main components of the system include: CRISPR sequence: This is a specific class of DNA sequences containing clustered regularly interspaced short palindromic repeats (CRISPR), in which exogenous DNA fragments from viruses, the so-called "spacer sequences" are embedded; Cas protein: CRISPR-associated proteins (Cas) are a class of nucleases responsible for recognizing and cutting specific DNA or RNA sequences. Different types of Cas proteins have different functions and characteristics. The Cas13 effector protein family includes four subtypes: Cas13a, Cas13b, Cas13c and Cas13d. Among them, Cas13d has attracted attention for its many advantages in RNA targeting. It has a compact structure, high targeting specificity and non-destructive editing ability, and has shown great potential in gene knockdown, cell and animal antiviral defense, especially in recent virus detection applications. However, using CRISPR / Cas system alone for virus detection has problems such as insufficient sensitivity and quantitative ability, complex sample processing, difficulty in quantitative detection, long reaction time, false positive results, high cost and equipment dependence, as well as storage and transportation. These shortcomings limit its application in rapid, low-cost and high-throughput detection, especially in resource-limited settings. Summary of the invention

[0006] Based on the shortcomings of the prior art, the present invention introduces a rapid nucleic acid extraction method and a diversified result display technology to simplify sample processing and the interpretation of test results. First, the target virus sequence is amplified using isothermal amplification technology-RAA, and then the CRISPR-Cas13d system is used for specific identification and signal amplification. Finally, a variety of visualization methods are used to visualize the test results. Based on the above principles, the present invention has developed a method that can accurately and quickly detect SVA viruses on site in a timely manner. This is to enhance the pig farming industry's ability to detect SVA viruses and reduce biosafety risks. The specific plan is as follows:

[0007] The present invention provides a crRNA for detecting Seneca Valley virus based on CRISPR-Cas13d, and the sequence of the crRNA molecule is shown in SEQ ID NO.13.

[0008] The present invention also provides a product for detecting Seneca Valley virus, wherein the product contains the above-mentioned crRNA molecule.

[0009] Preferably, the product is a reagent or a kit.

[0010] More preferably, the reagent or kit also contains Cas13d protein, RNA fluorescent reporter probe, and upstream primers and downstream primers for RAA amplification.

[0011] More preferably, the sequences of the upstream primer and the downstream primer are shown as SEQ ID NO. 3 and 6, respectively.

[0012] The present invention also provides the use of the above-mentioned crRNA molecule in the preparation of a product for detecting Seneca Valley virus.

[0013] Preferably, the product is a kit.

[0014] More preferably, the kit also contains Cas13d protein, RNA fluorescent reporter probe, and upstream primers and downstream primers for RAA amplification.

[0015] More preferably, the method for using the kit is as follows:

[0016] (1) Using the DNA of the sample to be tested as a template to amplify the product, and transcribe in vitro to obtain ssRNA;

[0017] (2) reacting the ssRNA, Cas13d protein, RNA fluorescent reporter probe, and crRNA molecule obtained in step (1) in a CRISPR-Cas13d reaction system;

[0018] (3) After the reaction in step (2) is completed, the reaction system is placed under blue light or ultraviolet light to observe the detection results.

[0019] Preferably, the amplification in step (1) adopts RAA amplification method.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention significantly shortens the diagnosis time and reduces the cost, and does not need to rely on expensive professional equipment. The designed primers and crRNA show extremely high specificity and sensitivity, ensuring the accuracy and efficiency of detection. This method is not only simple to operate and quick to respond, but also provides easy-to-understand visual output.

[0022] Therefore, this detection scheme based on RAA and CRISPR-Cas13d is particularly suitable for on-site monitoring of SVA, which helps to improve the pathogen monitoring capabilities in the pig farming industry and reduce biosafety risks. The promotion of this technology has important practical significance for strengthening disease prevention and control measures in farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the RAA primer screening result in Example 1.

[0024] Figure 2 This is the result of protein activity verification in Example 1.

[0025] Figure 3 It is the screening result of crRNA screening in Example 1.

[0026] Figure 4 These are the test results of 2 positive samples, 1 negative sample, and 1 negative control using nuclease-free water as a template in Example 1.

[0027] Figure 5 It is the visualization detection result of 2 positive samples, 1 negative sample, and 1 negative control with nuclease-free water as template in Example 1, wherein samples 1 and 3 are positive samples, sample 2 is a negative sample, and N is a negative control.

[0028] Figure 6-1 , 6-2 These are the specific detection results of the method in Example 1, where 1 is PCV2, 2 is PCV3, 3 is PRV, 4 is SVA, 5 is CSFV, 6 is JEV, 7 is PEDV, and 8 is PRRSV-2.

[0029] Figure 7 The results of the sensitivity test of the method in Example 1, wherein N is a negative control. DETAILED DESCRIPTION

[0030] Example 1

[0031] 1. Design of RAA primers and crRNA

[0032] The full gene sequences of multiple strains of SVA virus were downloaded from GenBank, and the downloaded full gene sequences were compared and analyzed using MEGA 7.0 software to find the conserved sequences. Three pairs of primers were designed based on the conserved regions of the SVA gene and the principle of base degeneration. The three pairs of primers were screened and tested using the RAA amplification kit to find the best primer combination for subsequent experiments. Three groups of CrRNA were designed in the region amplified by the best primer combination, and the corresponding gDNA was designed according to the CrRNA sequence for in vitro transcription of CrRNA in combination with the T7 promoter sequence. The sequences of primers, gDNA, T7 and fluorescent probe are shown in Table 1.

[0033] Table 1

[0034]

[0035]

[0036] 2. RAA Primer Screening and Positive Standard Plasmid Construction

[0037] The RAA amplification kit (Hangzhou Zhongce Biotechnology Co., Ltd., catalog number S003ZC) was selected, and the positive SVA nucleic acid was used as a template. The primers in Table 1 were used for amplification. The amplification system is shown in Table 2. The program was 37°C and 30 min. After the amplification, 5 μL of the amplified product was extracted for agarose gel electrophoresis. The results are shown in Table 2. Figure 1 The results showed that primers RAA-F3 / R3 had the best amplification effect, and this pair of primers was selected for subsequent experiments.

[0038] Table 2

[0039] Components Volume (μL) Abuffer 29.4 Upstream primer 2 Downstream primer 2 template 2.5 <![CDATA[RNase-freedH2O]]> 11.6 Bbuffer 2.5

[0040] According to the experimental requirements, 2× Rapid Taq Master Mix (Nanjing Novogene Biotech Co., Ltd., Catalog No. P222-01) and primers RAA-F3 / R3 were used with positive nucleic acid samples for PCR amplification according to the instructions. After amplification, the products were analyzed by agarose gel electrophoresis. Subsequently, the DNA fragments separated by electrophoresis were purified using the universal DNA purification and recovery kit (Catalog No. DP214-03) of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0041] Next, the purified DNA fragment was ligated to pMD TM19 (Simple) vector (Cat. No. 3271) and complete the ligation reaction according to the operating instructions. The ligation product was then transformed into DH5α competent cells and spread on LB solid medium containing ampicillin resistance and cultured overnight at 37°C. The next day, single colonies were selected for PCR verification to confirm the presence of the inserted fragment.

[0042] For the successfully verified colonies, LB liquid medium containing ampicillin resistance was used for expansion culture, and the plasmid was extracted using a plasmid extraction kit (Tiangen Biochemical Technology, catalog number DP103-03). Finally, the concentration of the standard positive plasmid (pMD-19T-SVA) was determined using a micro-UV-Vis spectrophotometer (catalog number 840-317500) from Thermo Fisher Scientific and stored for later use.

[0043] 3. T7 in vitro transcription

[0044] 3.1 In vitro transcription to generate crRNA

[0045] The three designed gDNAs were mixed with equal volumes of T7 promoter to a final concentration of 10 μM, and the mixture was placed in a metal bath (Biyuntian Biotechnology Co., Ltd., Catalog No. E1523) at 95 ° C for 10 minutes, and then cooled naturally at room temperature. According to the instructions, the product was transcribed into CrRNA in vitro using an in vitro transcription kit. The transcribed CrRNA was purified using a column RNA purification kit (Shanghai Sangon Biotechnology Co., Ltd., Catalog No. B518661-0100), and stored at -80 ° C after the concentration was determined.

[0046] Table 3

[0047]

[0048] 3.2 In vitro transcription to generate target RNA-ssRNA

[0049] A RAA amplification kit was used to amplify the positive nucleic acid as a template. After purification using a universal DNA purification and recovery kit, the product was transcribed into ssRNA in vitro using an in vitro transcription kit according to the instructions, and the concentration was measured and stored at -80°C.

[0050] 4. Expression of EsCas13d protein induced expression

[0051] According to the supplier's guide for EsCas13d plasmid (pET28a-MH6-EsCas13d), the plasmid powder was first reconstituted. Subsequently, the reconstituted plasmid solution was transformed into B21 competent cells and cultured at 37 ° C on LB solid medium plates containing kanamycin to screen positive clones. After the selected positive clones were verified for correctness by single colony PCR and sequencing, the successful clones were selected for expansion culture.

[0052] The confirmed positive bacterial solution was inoculated into 200 ml LB medium at a ratio of 1:100 and cultured in a 37°C constant temperature shaker until OD600 reached 0.4-0.6. At this time, IPTG with a final concentration of 1 mM was added to induce protein expression, and then the culture temperature was lowered to 18°C ​​and continued to be cultured at 200 rpm for 18 hours.

[0053] After the induction expression is completed, the bacterial solution is transferred to a 50 ml centrifuge tube and the cell precipitate is collected by centrifugation at 10000 rpm for 15 minutes at 4 ° C. The precipitate is resuspended with PBS buffer and centrifuged and washed three times. Finally, the washed bacteria are suspended in 30 ml buffer, and lysozyme and PMSF (protease inhibitor, at a ratio of 1:100) are added. The ultrasonic crusher (model FB50A220) provided by Thermo Fisher Scientific is set to 30% power and intermittent ultrasonic treatment (2 seconds each time, 4 seconds interval), and the total treatment time is 40 minutes.

[0054] Next, the His tag protein purification kit (Cat. No. HRD0156) of Herui Biotechnology Co., Ltd. was used to purify and concentrate the lysed cell samples. The molecular weight and expression of the purified protein were analyzed by SDS-PAGE experiment, and further verified by Western Blot using His tag antibody (GenScript Biotech, mouse monoclonal antibody, Cat. No. A00186) as the primary antibody and HRP-labeled rabbit anti-mouse IgG (Sangon Biotech (Shanghai) Co., Ltd., Cat. No. D110073-0500) as the secondary antibody. Finally, the purified protein samples were stored at -80°C for subsequent use.

[0055] 5. CrRNA screening and EsCas13d protein activity verification

[0056] 5.1Cas13d buffer configuration and system construction

[0057] First, 2×Cas13d buffer was prepared with the following formula: 80mM HEPES pH 7.1, 200mM KCl, 20mMMgCl2 and 10% glycerol. The system for CrRNA screening and EsCas13d protein activity verification was configured using existing CrRNA, RNA-ssRNA, EsCas13d protein RNase inhibitor (Shanghai Biotechnology Co., Ltd., Catalog No. B600478-0005) and other reagents as shown in Table 4.

[0058] Table 4

[0059]

[0060]

[0061] 5.2 Screening CrRNA and verifying protein activity

[0062] After the system was constructed, three experimental groups lacking Cas13d protein, crRNA and ssRNA templates were set up, and their volumes were replaced with RNase-free water. The protein activity was verified using a fluorescence quantifier (Thermo Fisher, Catalog No. A51693). The results are as follows: Figure 2 As shown. Three groups of systems were set up according to three different CrRNAs for CrRNA screening. The results are shown in Figure 3 The results showed that CrRNA1 had the best effect, and CrRNA1 was selected for subsequent experiments.

[0063] 6. Establishment of a Visual Detection Method for SVA Based on RAA and CRISPR-Cas13d

[0064] 6.1 Method development

[0065] According to the instructions for the Rapid Nucleic Acid Releaser (DNA Type)-II (Cat. No. WLR8203-ES) provided by Anpu Future Biotechnology Co., Ltd., we extracted nucleic acids from two positive samples and one negative sample. Subsequently, the extracted nucleic acid was used for RAA amplification reaction. The final product of the RAA reaction was directly used for in vitro transcription, and the transcription product was purified and concentrated using the column-type RNA rapid concentration and purification kit (Cat. No. B518688-0050) of Shanghai Shenggong Biotechnology Co., Ltd. Using the above-mentioned purified products, set up the experimental system according to the configuration requirements in Table 4. Place the configured reaction mixture in a qPCR instrument and incubate at 37°C for 30 minutes. Evaluate and determine the experimental results by monitoring changes in the fluorescence signal. See Figure 4 .

[0066] 6.2 Visualization of detection methods

[0067] The experimental product of 6.1 was reacted on the qPCR instrument for 30 minutes and then observed under ultraviolet light and other light sources to see if it emitted fluorescence. The results showed that after the reaction system reacted on the qPCR instrument for 30 minutes, fluorescence could be observed under ultraviolet light and other light sources, while negative results showed no fluorescence. Figure 5 .

[0068] 6.3 Method specificity detection

[0069] The established method was used to conduct experiments using nucleic acid from SVA-positive samples and DNA or cDNA samples of several other pathogens that have a great impact on the pig industry, such as porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), pseudorabies virus (PRV), porcine epidemic diarrhea virus PEDV, classical swine fever virus (CSFV), and porcine Japanese encephalitis virus (JEV) (provided by the Biotechnology Center of Sichuan Agricultural University) as templates and the corresponding nuclease-free water was added as a negative control group. The experimental results are shown in Figure 6-1 and 6-2 This method has high specificity. Only the SVA experimental group can see obvious fluorescence under different light sources, showing a positive reaction, while no obvious fluorescence was observed in the experimental results of other swine disease pathogens and the corresponding negative control groups, and their test results were all negative.

[0070] 6.4 Method sensitivity test

[0071] In this study, the SVA recombinant plasmid standard: pMD-19T-SVA was diluted 10 times in a row, and 10 0 Up to 10 8 The plasmids of 9 different concentration levels with 10 copies / μL were used as templates, sterile deionized water was used as negative control, and amplification was performed according to the experimental method system. The results showed that this experimental method showed high sensitivity, and its detection limit could reach 10 0 copies / μL, and the results can still be visualized, see Figure 7 .

[0072] 6.5 Clinical sample testing

[0073] Ten SVA-positive and four SVA-negative clinical samples were tested using the established method and the qPCR method. The results of the established method and the qPCR method were consistent.

[0074] 7. Summary

[0075] The present invention proposes an innovative on-site visual detection method that combines recombinase-assisted amplification (RAA) and CRISPR-Cas13d technology for rapid identification of SVA (Seneca Valley virus). By introducing RNA fluorescent reporter molecules, this method enables intuitive detection of SVA on site. Specifically, different light source transilluminators are used for detection, and the results are interpreted by observing whether fluorescence is generated to determine the status of the sample. In addition, the method is also compatible with qPCR instruments and can provide test results in about one hour.

[0076] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A crRNA for detecting Seneca Valley virus based on CRISPR-Cas13d, characterized in that: The sequence of the crRNA molecule is shown in SEQ ID NO.

13.

2. A product for detecting Seneca Valley virus, characterized in that: The product contains the crRNA molecule according to claim 1.

3. The product according to claim 2, characterized in that The product is a reagent or a kit.

4. The product according to claim 3, characterized in that The reagent or kit also contains Cas13d protein, RNA fluorescent reporter probe, and upstream primers and downstream primers for RAA amplification.

5. The product according to claim 4, characterized in that The sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 3 and 6 respectively.

6. Use of the crRNA molecule described in claim 1 in preparing a product for detecting Seneca Valley virus.

7. The use according to claim 6, characterized in that: The product is a test kit.

8. The use according to claim 7, characterized in that: The kit also contains Cas13d protein, RNA fluorescent reporter probe, and upstream primers and downstream primers for RAA amplification.

9. The use according to claim 8, characterized in that: The method of using the kit is as follows: (1) Using the DNA of the sample to be tested as a template to amplify the product, and transcribe in vitro to obtain ssRNA; (2) reacting the ssRNA, Cas13d protein, RNA fluorescent reporter probe, and crRNA molecule obtained in step (1) in a CRISPR-Cas13d reaction system; (3) After the reaction in step (2) is completed, the reaction system is placed under blue light or ultraviolet light to observe the detection results.

10. The use according to claim 9, characterized in that: The amplification in step (1) adopts the RAA amplification method.

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