Double-virus visual detection kit based on CRISPR / Cas9-LFD technology, detection method and application

Abstract

The invention belongs to the field of molecular biological detection, and relates to a kit, in particular to a double-virus visual detection kit based on a CRISPR / Cas9-LFD (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated protein 9-lateral FD) technology, a detection method and application. According to the invention, a CRISPR-Cas9 technology and a lateral flow test strip technology are combined, a dual visual detection system for PNRSV and ApNMV viruses based on a CRISPR / Cas9-LFD technology is developed, the developed detection method is evaluated, and an optimal detection system is selected. A rapid, visual and cheap simultaneous visual detection technology for the PNRSV and ApNMV viruses is finally established through system optimization, large instruments are not needed, the detection sensitivity is 4.8 * 10 < 2 > copy / reaction and is 10 times that of RT-PCR, the specificity is good, and simultaneous visual detection of the two viruses can be achieved within 40 min under the condition of the constant temperature of 37 DEG C.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, and specifically relates to a dual-virus visualization detection kit. Background Technology

[0002] apple( Malus domesica Apples hold an important position in my country's fruit production due to their high nutritional and economic value. However, apple cultivation has long been plagued by viral diseases, with apple mosaic virus (APNMV) being prevalent in major apple-producing areas of my country, severely reducing apple yield and quality and causing irreparable economic losses to fruit growers. APNMV and Prunus necrotic ring-spot virus (PNRSV) are important pathogens of APNMV, widely occurring and causing serious damage in my country's apple-producing regions. Furthermore, PNRSV has been listed as a quarantine pest and is included in the quarantine pest lists of the Plant Protection Organization (EPPO) in China, Europe, and the Mediterranean. Currently, no commercially available varieties are completely resistant to these two viruses. Therefore, timely and accurate monitoring and quarantine of pathogens during seedling transportation and field production are crucial for prevention, control, and reducing the spread of these viruses.

[0003] In recent years, in vitro amplification techniques based on polymerase chain reaction (PCR) have been widely used in nucleic acid detection, including in the detection of apple viruses. PCR detection offers advantages such as accurate results and high sensitivity; however, this method is very complex, relying on numerous expensive and complex instruments, making it difficult for the average person to master and impossible to perform visual on-site testing in the field. Recombinant polymerase amplification (RPA) is a nucleic acid amplification technique that does not rely on sophisticated instruments, can amplify the target gene at a constant temperature of 37°C for a short time. Lateral flow dipstick (LFD) technology is a technique combining immunochromatography for rapid DNA molecule detection, capable of rapidly detecting amplified products within 3–5 minutes without the need for any signal reading equipment; the results can be observed with the naked eye. Combining LFD with RPA technology overcomes the limitations of instruments and professional technicians, allowing for visual detection of amplified products in a shorter time. In recent years, nucleic acid detection systems based on CRISPR-Cas proteins (such as Cas9, Cas12, and Cas13) have shown great promise in the field of molecular diagnostics. These systems, by recognizing specific target sequences and triggering cis-cleavage, exhibit trans-cleavage characteristics on non-specific targets, making them particularly suitable for detecting specific genes in viruses, pathogens, or transgenic organisms. Patent CN201910886954.X discloses a visual detection system and method for apple stem groove virus (ASGV) based on CRISPR-Cas12a technology. Based on the conserved sequence of ASGV, crRNA was designed, and AuNP-DNA for colorimetric detection was prepared. The effects of in vitro cleavage reaction time of Cas12a protein and linker DNA concentration on the detection effect were investigated, and an optimal visual detection system and method for ASGV were established. This method uses isothermal reaction conditions and improves specificity by recognizing the virus through the CRISPR-Cas12a-crRNA complex. The linker DNA is universal, eliminating the need for expensive double-labeled probes. Multiple samples can be detected within 40 minutes using low-speed centrifugation, with a detection limit of 5.62 × 10⁻⁶. 2 The Cas12 / Cas13 copy / reaction has a sensitivity 100 times higher than RT-PCR. However, the trans-cleavage activity of Cas12 and Cas13 has detection limitations, only able to detect one pathogen target at a time. These drawbacks limit their application in plant virus detection.

[0004] Therefore, it is necessary to develop a simple, convenient, time-saving, low-cost, and visually observable dual detection technology for apple viruses to ensure the healthy development of the apple industry and to make it a promising technology for detecting apple viral diseases. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a dual-virus visualization detection kit, detection method, and application based on CRISPR / Cas9-LFD technology.

[0006] The technical solution of this invention is implemented as follows: On the one hand, this invention proposes a dual-virus visualization detection system based on CRISPR / Cas9-LFD technology: the detection system includes Cas9 protein, PRA primer pair, gRNA and DNA probe.

[0007] Preferably, the above-mentioned PRA primer pairs include forward primer ApNMV-RT-RPA-F and reverse primer ApNMV-RT-RPA-R, forward primer PNRSV-RT-RPA-F and reverse primer PNRSV-RT-RPA-R, wherein the nucleotide sequence of ApNMV-RT-RPA-F is shown in SEQ ID No. 1, and the 5' end is labeled with FITC fluorescent pigment; the nucleotide sequence of ApNMV-RT-RPA-R is shown in SEQ ID No. 2; the nucleotide sequence of PNRSV-RT-RPA-F is shown in SEQ ID No. 3, and the 5' end is labeled with FITC fluorescent pigment; the nucleotide sequence of PNRSV-RT-RPA-R is shown in SEQ ID No. 4. The above-mentioned gRNAs include gRNA-ApNMV and gRNA-PNRSV, wherein the nucleotide sequence of gRNA-ApNMV is shown in SEQ ID No. 5, and the 5' end is labeled with digoxigenin; the nucleotide sequence of gRNA-PNRSV is shown in SEQ ID No. 6, and the 5' end is labeled with biotin. The aforementioned DNA probes include DNA probe 1 and DNA probe 2, wherein the nucleotide sequence of DNA probe 1 is shown in SEQ ID No. 7, and the 5' end is labeled with digoxigenin; the nucleotide sequence of DNA probe 2 is shown in SEQ ID No. 8, and the 5' end is labeled with biotin.

[0008] Secondly, a dual-virus visualization detection kit based on CRISPR / Cas9-LFD technology includes the aforementioned dual-virus visualization detection system.

[0009] Preferably, the kit further includes a lateral flow test strip, wherein the sample pad is coated with gold nanoparticles with FITC antibody, the test line T1 is coated with a biotin receptor, and the test line T2 is coated with a digoxin antibody.

[0010] Thirdly, a dual-virus visualization detection method based on CRISPR / Cas9-LFD technology is described below: (1) Extract total RNA from the sample to be tested; (2) Using total RNA as a template, multiplex RT-RPA amplification was performed using the above-mentioned dual-virus visualization detection kit to obtain RT-RPA amplification products; (3) Using the above-mentioned dual-virus visualization detection kit, take the RT-RPA amplification product and perform a CRISPR / Cas9 reaction to obtain the reaction product; (4) Add 100 µL of LFD hybridization buffer to the reaction product for dilution, then insert the lateral flow test strip into the dilution solution, let it stand for 5 min and observe the color development of the C line, T1 line and T2 line of the test strip to determine the detection result.

[0011] Preferably, in step (2) above, the multiplex RT-RPA amplification reaction system is 50 µL, containing 4 µL total RNA, 20 µL solubilizer, 2.5 µL each of RPA primer pairs, 14 µL sterile water, and 2 µL activator; the reaction conditions are 37℃ for 20 min.

[0012] Preferably, in step (3) above, the CRISPR / Cas9 reaction system is 20 µL, containing 2 µL of 4 µM Cas9 protein, 1 µL of 2 µM gRNA, 2 µL of RT-RPA amplification product, 1 µL of 10 µM DNA probe, 2 µL of 10×NEBuffer 2.1 buffer, and 10 µL of sterile water; the reaction conditions are 37℃ for 5 min.

[0013] Preferably, the method for determination in step (4) above is as follows: ① If red bands appear on line C, T1, and T2 of the test strip, the sample is determined to be double positive for PNRSV and ApNMV; ② If both the C line and T1 line of the test strip show red bands, the sample is determined to be a single positive for PNRSV; ③ If both the C line and T2 line of the test strip show red bands, the sample is determined to be ApNMV single positive; ④ If only line C of the test strip shows a red band, the sample is considered a healthy sample.

[0014] Preferably, the sensitivity of the above detection method is 4.8 × 10⁻⁶. 2 Copy / reaction.

[0015] Fourthly, the application of the aforementioned dual-virus visualization detection kit in field dual-virus visualization detection.

[0016] Preferably, the aforementioned dual viruses are PNRSV and ApNMV.

[0017] The present invention has the following beneficial effects: 1. This invention combines CRISPR-Cas9 technology and lateral flow test strip technology to develop a reagent kit for the visual dual detection of PNRSV and ApNMV based on CRISPR / Cas9-LFD technology. Through optimization of the detection system, a fast, intuitive, and inexpensive technology for the simultaneous visual detection of PNRSV and ApNMV has been established, which does not require large instruments. Under constant temperature conditions of 37°C, the simultaneous visual detection of the two viruses can be achieved within 40 minutes.

[0018] 2. Using PNRSV and ApNMV viral plasmids as templates, RT-PCR and CRISPR / Cas9-LFD detection were performed. The results showed that the detection sensitivity of RT-PCR was 4.8 × 10⁻⁶. 3 Copy / reaction, while the sensitivity of Cas9-LFD is 4.8 × 10⁻⁶. 2 The copy / reaction rate is 10 times that of RT-PCR, with better sensitivity, and the entire Cas9-LFD detection process only takes 40 minutes. In the Cas9-LFD detection results, only three samples, PNRSV+ApNMV, PNRSV, and ApNMV, showed test bands, indicating good specificity. In field testing, the CRISPR / Cas9-LFD detection results are completely consistent with RT-PCR, demonstrating good practical applicability. It can be used for field testing, and the entire workflow takes less than 1 hour. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention 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 of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a flowchart of the dual detection process for PNRSV and ApNMV based on CRISPR / Cas9-LFD technology.

[0021] Figure 2The image shows a dual detection visualization of PNRSV and ApNMV based on CRISPR / Cas9-LFD; (A) is a gel image of multiplex RT-RPA amplification of PNRSV and ApNMV viruses, and (B) is a test strip for PNRSV and ApNMV detection based on CRISPR / Cas9-LFD.

[0022] Figure 3 The figure shows a comparison of the sensitivity and specificity of RT-PCR and Cas9-LFD for the detection of the two viruses; where (A) is a comparison of the detection sensitivity of RT-PCR and Cas9-LFD, and (B) is an analysis of the detection specificity.

[0023] Figure 4 Figure 1 shows the results of RT-PCR and CRISPR / Cas9-LFD assays on 20 field apple samples. Detailed Implementation

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0026] This application develops a CRISPR-Cas9-based dual detection system for the dual detection of PNRSV and APNMV viruses. Furthermore, it combines CRISPR-Cas9 technology with lateral flow test strip technology to develop a dual visualization detection system for PNRSV and APNMV viruses based on CRISPR / Cas9-LFD technology. The two developed detection methods were evaluated, and the optimal detection system was selected. Through system optimization, a rapid, intuitive, and inexpensive technology for the simultaneous visualization detection of both PNRSV and APNMV viruses was finally established. This technology requires no large instruments and can achieve simultaneous visualization detection of both viruses within 40 minutes under a constant temperature of 37℃. Example

[0027] 1. Detection methods and principles Studies have shown that after the Cas9-sgRNA complex binds to the target dsDNA, it does not separate from it for a short period of time, accompanied by the release of 20 nt of the non-target strand, exposing the other 13 bases of the non-target strand to the Cas9 surface. Therefore, utilizing this characteristic of the Cas9 protein in conjunction with lateral chromatography strip technology, a method can be established... Figure 1 The flowchart shown is a dual-visualization detection process based on CRISPR / Cas9-LFD technology.

[0028] Specific principles: (1) Two pairs of FITC marker primers were designed for the conserved sequences of ApNMV and PNRSV viruses respectively. The viral sequences were rapidly amplified by multiplex RT-RPA technology to obtain a large amount of FITC marker target dsDNA.

[0029] (2) The Cas9-sgRNA complex recognizes the target and forms a Cas9 / sgRNA / dsDNA-FITC ternary complex. The 13 non-target strand bases on the surface of Cas9 are exposed outside the Cas9 protein. By adding biotin probes and digoxigenin probes that target the non-target strands, the non-target strands will hybridize with the biotin probes and digoxigenin probes to form Cas9 / sgRNA / dsDNA-FITC / biotin probe complexes and Cas9 / sgRNA / dsDNA-FITC / dgoxigenin probe complexes.

[0030] (3) The mixed droplets are placed on the test strip and bind to the anti-FITC labeled gold nanoparticles on the sample pad. As they flow through test line 1 (T1) and test line 2 (T2), they are captured by the anti-digoxin antibody and biotin receptor, and the gold nanoparticles gradually accumulate, showing a red band. The visualization results can be divided into 4 types: ① A double-positive sample of PNRSV and ApNMV will show three red bands on the T1, T2 and C lines of the test strip; ② PNRSV single positive samples will be captured by biotin receptors when flowing through T1, and two red bands will appear on the T1 line and C line of the test strip; ③ApNMV single positive samples will be captured by anti-digoxigenin antibody when flowing through T2, and two red bands will appear on the T2 and C lines of the test strip; ④ Healthy samples do not contain the target sequence and will not be bound by the probe; they will only show one red band on the C line.

[0031] 2. Testing Process (1) Rapid extraction of RNA from leaves Total RNA was rapidly extracted from leaves using a modified alkaline polyethylene glycol (PEG) solution: A small amount of leaf tissue (20-50 mg) was placed in a 1.5 mL centrifuge tube, and 300 µL of extraction buffer (6% PEG 200 and 20 mM NaOH) was added. The tissue was then homogenized using a handheld electric tissue homogenizer. After incubating the homogenate at room temperature for 3 min, the crude extract was directly used for virus detection.

[0032] (2) Multiple RT-RPA reactions Multiplex RT-RPA reactions were performed using an RT-based basic nucleic acid amplification kit (Syndagene, KS102). The multiplex RT-RPA primer sequences are shown in Table 1. The reaction volume was 50 µL, containing 4 µL of crude total RNA extract, 20 µL of solvent, 2.5 µL each of forward and reverse RPA primers, 14 µL of sterile water, and 2 µL of activator. After brief shaking to mix, the reaction tubes were incubated in a thermostat (37℃) for 20 min. 2 µL of the RT-RPA product was then used for CRISPR / Cas9-LFD visualization detection.

[0033] Table 1 Multiplex RT-RPA primers (3) CRISPR / Cas9-LFD detection The CRISPR / Cas9-LFD detection primers and probes are shown in Table 2. The CRISPR / Cas9 reaction system was 20 µL, containing 2 µL of Cas9 protein (4 µM), 1 µL each of gRNA-ApNMV and gRNA-PNRSV (2 µM), 2 µL of RT-RPA amplification product, 1 µL each of biotin and digoxigenin probes (10 µM), 2 µL of 10×NEBuffer 2.1 buffer, and 10 µL of sterile water. After reacting at 37℃ for 5 min, 100 µL of LFD hybridization buffer was added. The HybriDetect 2T (Milenia Biotec GmbH, Germany) test strip was vertically inserted into the centrifuge tube liquid. After standing for 5 min, the color development of the C line, T1, and T2 lines of the test strip was observed.

[0034] Table 2 CRISPR / Cas9-LFD detection primers and probes 3. Results Analysis (1) Establish a visual dual detection system based on CRISPR / Cas9-LFD Six apple leaves infected with ApNMV and PNRSV and one leaf from a virus-free seedling were taken and subjected to double RT-RPA amplification to test the amplification effect. Figure 2 As shown in (A), the PNRSV amplified fragment is 420 bp, and the ApNMV amplified fragment is 200 bp. Lanes 1-6 represent six infected apple leaves, showing two bands at 200 bp and 420 bp. N is the negative control, with no band appearing. The amplified products were then detected using Cas9-LFD. Figure 2As shown in (B), the detection results are consistent with the RT-RPA results, with two bands appearing on test line 1 (PNRSV) and test line 2 (ApNMV). However, the negative control sterile vaccine did not show a positive band. Therefore, the method established in this study can be used for rapid dual-visualization detection of PNRSV and ApNMV.

[0035] (2) Sensitivity test and specificity detection Using PNRSV and ApNMV viral plasmids as templates, 4.8 × 10 8 The initial concentration was used for the copy / reaction, and the mixture was diluted sequentially in multiples of 10. A negative control was also set up for RT-PCR and CRISPR / Cas9-LFD detection reactions. Figure 3 (A) The results showed that the detection sensitivity of RT-PCR was 4.8 × 10⁻⁶. 3 Copy / reaction, while the sensitivity of Cas9-LFD is 4.8 × 10⁻⁶. 2 The copy / reaction time is 10 times that of RT-PCR, and the entire Cas9-LFD detection process only takes 40 minutes. Figure 3 (B) The Cas9-LFD detection results showed that only three samples, PNRSV+ApNMV, PNRSV and ApNMV, showed test bands, indicating good specificity.

[0036] Implementation Results Example Field testing application: In apple orchards with trees over 10 years old in Luoning, Henan Province, 20 apple tree leaf samples were randomly selected. Following the optimized conditions of this application's embodiments, CRISPR / Cas9-LFD and RT-PCR detection technologies were used to test the 20 field samples. A negative control was also included. The results were observed, and the differences in detection methods were compared and analyzed.

[0037] Figure 4 The test results showed that 8 samples were double positive for PNRSV and ApNMV, which was completely consistent with the RT-PCR test results and has good practical applicability, making it suitable for field testing. By calculating the time spent in the entire workflow, the CRISPR-Cas9 / LFD strategy allows for rapid (<1 hour) simultaneous detection of PNRSV and ApNMV.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A dual-virus visualization detection system based on CRISPR / Cas9-LFD technology, characterized by: The detection system comprises Cas9 protein, PRA primer pairs, gRNA, and DNA probes.

2. The dual-virus visualization detection system based on CRISPR / Cas9-LFD technology according to claim 1, characterized in that: The PRA primer pairs include forward primer ApNMV-RT-RPA-F and reverse primer ApNMV-RT-RPA-R, forward primer PNRSV-RT-RPA-F and reverse primer PNRSV-RT-RPA-R, wherein the nucleotide sequence of ApNMV-RT-RPA-F is shown in SEQ ID No. 1, and the 5' end is labeled with FITC fluorescein; the nucleotide sequence of ApNMV-RT-RPA-R is shown in SEQ ID No. 2; the nucleotide sequence of PNRSV-RT-RPA-F is shown in SEQ ID No. 3, and the 5' end is labeled with FITC fluorescein; the nucleotide sequence of PNRSV-RT-RPA-R is shown in SEQ ID No.

4.

3. The dual-virus visualization detection system based on CRISPR / Cas9-LFD technology according to claim 2, characterized in that: The gRNAs include gRNA-ApNMV and gRNA-PNRSV, wherein the nucleotide sequence of gRNA-ApNMV is shown in SEQ ID No. 5, and the 5' end is labeled with digoxigenin; the nucleotide sequence of gRNA-PNRSV is shown in SEQ ID No. 6, and the 5' end is labeled with biotin; the DNA probes include DNA probe 1 and DNA probe 2, wherein the nucleotide sequence of DNA probe 1 is shown in SEQ ID No. 7, and the 5' end is labeled with digoxigenin; the nucleotide sequence of DNA probe 2 is shown in SEQ ID No. 8, and the 5' end is labeled with biotin.

4. A dual-virus visualization detection kit based on CRISPR / Cas9-LFD technology, characterized in that: The kit comprises the dual-virus visualization detection system as described in any one of claims 1-3.

5. The dual-virus visualization detection kit based on CRISPR / Cas9-LFD technology according to claim 4, characterized in that: The kit also includes a lateral flow test strip with a sample pad coated with gold nanoparticles containing FITC antibodies, a biotin receptor coated on the T1 line, and a digoxin antibody coated on the T2 line.

6. A dual-virus visualization detection method based on CRISPR / Cas9-LFD technology, characterized in that, The steps are as follows: (1) Extract total RNA from the sample to be tested; (2) Using total RNA as a template, perform multiplex RT-RPA amplification using the dual-virus visualization detection kit described in claim 5 to obtain RT-RPA amplification products; (3) Using the dual-virus visualization detection kit described in claim 5, take the RT-RPA amplification product and perform a CRISPR / Cas9 reaction to obtain the reaction product; (4) Add 100 µL of LFD hybridization buffer to the reaction product for dilution, then insert the lateral flow test strip into the dilution solution, let it stand for 5 min and observe the color development of the C line, T1 line and T2 line of the test strip to determine the detection result.

7. The dual-virus visualization detection method according to claim 6, characterized in that: In step (2), the multiplex RT-RPA amplification reaction system is 50 µL, containing 4 µL total RNA, 20 µL solubilizer, 2.5 µL each of the RPA primer pairs, 14 µL sterile water, and 2 µL activator; the reaction conditions are 37℃ for 20 min.

8. The dual-virus visualization detection method according to claim 7, characterized in that: In step (3), the CRISPR / Cas9 reaction system is 20 µL, containing 2 µL of 4 µM Cas9 protein, 1 µL of 2 µM gRNA, 2 µL of RT-RPA amplification product, 1 µL of 10 µM DNA probe, 2 µL of 10×NEBuffer 2.1 buffer, and 10 µL of sterile water; the reaction conditions are 37℃ for 5 min.

9. The dual-virus visualization detection method according to claim 8, characterized in that, The determination method in step (4) is as follows: ① If red bands appear on line C, T1, and T2 of the test strip, the sample is determined to be double positive for PNRSV and ApNMV; ② If both the C line and T1 line of the test strip show red bands, the sample is determined to be a single positive for PNRSV; ③ If both the C line and T2 line of the test strip show red bands, the sample is determined to be ApNMV single positive; ④ If only line C of the test strip shows a red band, the sample is considered a healthy sample.

10. The application of the dual-virus visualization detection kit according to claim 5 in field dual-virus visualization detection, characterized in that: The two viruses are PNRSV and ApNMV.

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