KIT AND METHOD FOR VISUAL IDENTIFICATION OF RHDV TYPE 1 AND TYPE 2 BY MEANS OF LAMP-CRISPR / CAS12a

Abstract

The present invention belongs to the technical field of pathogenic microorganism detection. Provided are a kit and a method for visual identification of RHDV type 1 and type 2 by means of LAMP-CRISPR / Cas12a. In the present invention, a LAMP-CRISPR / Cas12a rapid detection kit and method for RHDV1 and RHDV2 are established. A platform has high sensitivity and can be used to detect a nucleic acid sample at a concentration of 10 copies / μL. The platform has good specificity and can specifically identify RHDV1 and RHDV2 strains. Moreover, the platform has no cross reaction with other common rabbit pathogens. A result which is visible to naked eyes is obtained within 1.5 h by means of further utilizing a lateral flow chromatography test strip and visual fluorescence. The kit of the present invention is a portable platform; has the advantages of rapidity, high sensitivity, high specificity, visualization and low equipment requirements; and can be used clinically in remote rural areas and resource-limited areas.

Description

Kit and method for visual identification of RHDV type 1 and type 2 using LAMP-CRISPR / Cas12a

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on January 19, 2024, with application number 2024100781175 and invention name “Kit and method for visual identification of RHDV type 1 and type 2 by LAMP-CRISPR / Cas12a”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the technical field of pathogenic microorganism detection, and specifically relates to a kit and method for visually identifying RHDV type 1 and type 2 using LAMP-CRISPR / Cas12a. Background Art

[0003] Rabbit hemorrhagic disease is an acute, severe, and highly contagious disease caused by the rabbit hemorrhagic disease virus (RHDV). It carries a high mortality rate and poses a serious threat to the rabbit industry. Compared to the classic RHDV, known as rabbit hemorrhagic disease virus type 1 (RHDV1), the more virulent strain, rabbit hemorrhagic disease virus type 2 (RHDV2), has a wider host range, multiple transmission pathways, and is more resilient in natural environments, causing significant economic losses to the rabbit industry. Efficient, accurate diagnostic tools that can differentiate between the two are crucial for preventing and controlling rabbit hemorrhagic disease outbreaks.

[0004] Currently, detection methods include PCR, fluorescent quantitative PCR, hemagglutination assays, and ELISA. Immunological detection methods such as hemagglutination and ELISA have poor accuracy and are subject to limitations such as difficulty detecting during the window period. Pathogenic detection methods such as RT-PCR and fluorescent quantitative PCR require specialized instrumentation and are typically laboratory-based, failing to achieve rapid on-site testing and meeting the testing needs of grassroots farms. Currently, there are no reports on rapid visual identification of rabbit hemorrhagic disease virus using LAMP and CRISPR-Cas12a, nor on the application of this method to the rapid differentiation of rabbit hemorrhagic disease virus type 1 from rabbit hemorrhagic disease virus type 2.

[0005] Summary of the Invention

[0006] The present invention provides a kit and method for visually identifying RHDV type 1 and type 2 using LAMP-CRISPR / Cas12a. The kit and method can accurately identify RHDV1 and RHDV2 viruses and have the advantages of being rapid, highly sensitive, highly specific, visual, and requiring low equipment.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The present invention provides a kit for LAMP-CRISPR / Cas12a visual identification of RHDV type 1 and type 2, which includes a LAMP amplification system and a CRISPR / Cas12a detection system; the LAMP amplification system includes an outer primer pair F3 and B3, and an inner primer pair FIP and BIP; the nucleotide sequences of the outer primer pair F3 and B3 in the LAMP amplification system for RHDV1 are shown as SEQ ID Nos.13-14, and the nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID Nos.15-16; the nucleotide sequences of the outer primer pair F3 and B3 in the LAMP amplification system for RHDV2 are shown as SEQ ID Nos.24-25, and the nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID Nos.26-27; the CRISPR / Cas12a detection system includes gRNA; the nucleotide sequence of the gRNA in the CRISPR / Cas12a detection system for RHDV1 is shown as SEQ ID No.37; The nucleotide sequence of the gRNA in the CRISPR / Cas12a detection system for RHDV2 is shown in SEQ ID No.39.

[0009] Preferably, the LAMP amplification system further includes loop primers LF and / or LB; the nucleotide sequence of the loop primer LB in the LAMP amplification system for RHDV1 is shown as SEQ ID No.17; the nucleotide sequences of the loop primers LF and LB in the LAMP amplification system for RHDV2 are shown as SEQ ID No.28-29.

[0010] Preferably, the LAMP amplification system further comprises LAMP / RT-LAMP 2X premix, nucleic acid to be tested and enzyme-free water.

[0011] Preferably, the CRISPR / Cas12a detection system further comprises LAMP amplification product, RNase inhibitor, NEBuffer 2.1, Cas12a protein, ssDNA reporter molecule and enzyme-free water.

[0012] Preferably, the kit further comprises a lateral flow chromatography test strip or a blue light analyzer.

[0013] Preferably, when the kit contains a lateral flow chromatography test strip, the ssDNA reporter molecule is a biotin ssDNA reporter molecule; when the kit contains a blue light analyzer, the ssDNA reporter molecule is a fluorescence quenching ssDNA reporter molecule.

[0014] The present invention provides an application of the kit in the preparation of a product for identifying RHDV1 and RHDV2. The present invention provides a method for non-disease diagnosis purposes for visually identifying RHDV1 and RHDV2 based on LAMP-CRISPR / Cas12a, comprising the following steps: (1) extracting RNA from a sample to be tested; (2) performing LAMP amplification in a LAMP amplification system using the RNA extracted in step (1) as a template; (3) enzymatically cleaving the LAMP amplification product obtained in step (2) in a CRISPR / Cas12a detection system and performing fluorescence detection; or enzymatically cleaving the obtained LAMP amplification product in a CRISPR / Cas12a detection system and performing lateral flow chromatography test strip detection.

[0015] Preferably, the total volume of the LAMP amplification system is 25 μL, including 12.5 μL LAMP / RT-LAMP 2X premix, 2.5 μL primer mixture, 2 μL extracted RNA and 8 μL enzyme-free water.

[0016] Preferably, in the LAMP amplification system for RHDV1, the final concentrations of the outer primer pair F3 and B3 in the primer mixture are 200 nM, the final concentrations of the inner primer pair FIP and BIP are 1200 nM, and the final concentration of the loop primer LB is 600 nM; in the LAMP amplification system for RHDV2, the final concentrations of the outer primer pair F3 and B3 in the primer mixture are 200 nM, the final concentrations of the inner primer pair FIP and BIP are 800 nM, and the final concentrations of the loop primers LF and LB are 600 nM.

[0017] Preferably, when fluorescence detection is performed, the total volume of the CRISPR / Cas12a detection system is 20 μL, 2 μL LAMP amplification product, 1 μL RNase inhibitor (final concentration 0.5 U / μL), 2 μL gRNA (final concentration 500 nM), 2 μL NEBuffer 2.1, 1 μL Cas12a protein (final concentration 125 nM), 1 μL fluorescence quenching ssDNA reporter molecule (final concentration 250 nM) and 11 μL enzyme-free water.

[0018] Preferably, when performing lateral flow chromatography test strip detection, the total volume of the CRISPR / Cas12a detection is 20 μL, 2 μL LAMP amplification product, 1 μL RNase inhibitor (final concentration 0.5 U / μL), 2 μL gRNA (final concentration 500 nM), 2 μL NEBuffer 2.1, 1 μL Cas12a protein (final concentration 125 nM), 1 μL biotin ssDNA reporter molecule (final concentration 100 nM) and 11 μL enzyme-free water.

[0019] Preferably, the LAMP amplification condition is 65-69° C. for 25-35 minutes.

[0020] Preferably, the reaction conditions in the CRISPR / Cas12a detection system are incubation at 35-38° C. for 25-35 min.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] To rapidly, conveniently, and specifically detect RHDV1 and RHDV2 viruses, the present invention established a LAMP-CRISPR / Cas12a rapid detection method for RHDV1 and RHDV2. The platform exhibited high sensitivity, capable of detecting 10 copies / μL of nucleic acid samples, with good specificity, capable of specifically identifying RHDV1 and RHDV2 strains, and had no cross-reaction with other common pathogens in rabbits.

[0023] The present invention further utilizes lateral flow chromatography test strips and visualized fluorescence to obtain visible results within 1.5 hours. Furthermore, 74 clinical samples were tested using the detection system for RHDV1 and RHDV2, respectively, with concordance rates of 97.30% and 97.30% with quantitative fluorescence PCR, demonstrating higher sensitivity than quantitative fluorescence PCR. The kit and detection method prepared by the present invention offer the advantages of rapidity, high sensitivity, high specificity, visualization, and minimal equipment requirements, making them suitable for clinical use in remote rural areas and resource-constrained regions. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the LAMP-CRISPR / Cas12a detection method combined with LFS (lateral flow test strip) and blue light analyzer.

[0025] Figure 2 shows three primer sets for detecting and screening RHDV1 and RHDV2 using fluorescent LAMP; A: RHDV1 primer set Set 1, Set 2, Set 3; B: RHDV2 primer set Set 1, Set 2, Set 3.

[0026] Figure 3 Cross-validation results of LAMP amplification of RHDV1 and RHDV2; A: nucleic acid gel electrophoresis cross-validation results, B: endpoint fluorescence value of the cross-validation experiment using fluorescent LAMP, n = 3, ****P < 0.0001.

[0027] FIG4 shows the results of reaction temperature screening for RHDV1 and RHDV2 using fluorescent LAMP; A: RHDV1 screening results, B: RHDV2 screening results.

[0028] Figure 5 shows the results of the internal and external primer ratio screening for RHDV1 and RHDV2 using fluorescent LAMP; A: RHDV1 screening results, B: RHDV2 screening results.

[0029] FIG6 shows the results of screening the concentration of the detection loop primers of RHDV1 and RHDV2 using fluorescent LAMP; A: screening results of RHDV1, B: screening results of RHDV2.

[0030] Figure 7 visualizes the detection results of LAMP for RHDV1 and RHDV2 respectively.

[0031] Figure 8 Results of LAMP combined with CRISPR / Cas12a detection method; A: Fluorescence value change curve of the orthogonal experiment of gRNA screening in the CRISPR stage of the RHDV1 detection system and optimization of the Cas12a protein concentration, B: Fluorescence value change curve of the orthogonal experiment of gRNA screening in the CRISPR stage of the RHDV2 detection system and optimization of the Cas12a protein concentration; C: Comparison of fluorescence values ​​at the endpoint of the orthogonal experiment (three replicates); D: Verification of the trans-cleavage activity of Cas12a protein and cross-experiment of the RHDV1 / RHDV2 detection method in the CRISPR stage; E: Results of the trans-cleavage activity verification and cross-experiment under blue light.

[0032] Figure 9 Specificity evaluation results of the LAMP-CRISPR / Cas12a detection method; A and B: Specificity of the LAMP-CRISPR / Cas12a fluorescence detection platform evaluated by endpoint fluorescence values ​​(n=3, ****P<0.0001); C and D: Specificity of the LAMP-CRISPR / Cas12a fluorescence detection platform evaluated by imaging results under blue light; E and F: Specificity of the LAMP-CRISPR / Cas12a-based LFS detection platform. A, C, and E correspond to the RHDV1 detection system, and B, D, and F correspond to the RHDV2 detection system.

[0033] Figure 10: Sensitivity evaluation results of the LAMP-CRISPR / Cas12a detection method; A and B: Sensitivity of the LAMP-CRISPR / Cas12a fluorescence detection method evaluated by endpoint fluorescence values ​​(n=3, ****P<0.0001); C and D: Sensitivity of the LAMP-CRISPR / Cas12a fluorescence detection method evaluated by imaging results under blue light; E and F: Sensitivity results of the LAMP-CRISPR / Cas12a-based LFS detection method. A, C, and E correspond to the RHDV1 detection system, and B, D, and F correspond to the RHDV2 detection system.

[0034] Figure 11: Clinical practicality evaluation results of the LAMP-CRISPR / Cas12a detection method; A and B: Endpoint fluorescence values ​​of 74 clinical samples detected by the LAMP-CRISPR / Cas12a fluorescence detection method (cutoff values ​​are 55482 and 57905, respectively); C and D: Blue light irradiation results of 74 clinical samples detected by the LAMP-CRISPR / Cas12a fluorescence detection method; E and F: Results of 74 clinical samples detected by the LAMP-CRISPR / Cas12a-based LFS detection method; G and H: Results of 74 clinical samples detected by qPCR. Circled numbers indicate that the test results of the sample are inconsistent with the method established in this experiment. A, C, and E correspond to the RHDV1 detection system, and B, D, and F correspond to the RHDV2 detection system. DETAILED DESCRIPTION

[0035] The present invention provides a kit for visually identifying RHDV type 1 and type 2 based on LAMP-CRISPR / Cas12a. The kit comprises a LAMP amplification system and a CRISPR / Cas12a detection system. The LAMP amplification system comprises an outer primer pair F3 and B3, and an inner primer pair FIP and BIP. The nucleotide sequences of the outer primer pair F3 and B3 for RHDV1 are shown in SEQ ID Nos.13-14, and the nucleotide sequences of the inner primer pair FIP and BIP are shown in SEQ ID Nos.15-16. The nucleotide sequences of the outer primer pair F3 and B3 for RHDV2 are shown in SEQ ID Nos.24-25, and the nucleotide sequences of the inner primer pair FIP and BIP are shown in SEQ ID Nos.26-27. The CRISPR / Cas12a detection system comprises gRNA. The nucleotide sequence of the gRNA for RHDV1 is shown in SEQ ID No.37, and the nucleotide sequence of the gRNA for RHDV2 is shown in SEQ ID No.39. The LAMP amplification system of the kit of the present invention also includes loop primers LF and / or LB; the nucleotide sequence of the loop primer LB for RHDV1 is shown in SEQ ID No. 17; the nucleotide sequences of the loop primers LF and LB for RHDV2 are shown in SEQ ID Nos. 28-29.

[0036] In the present invention, multiple sets of primers were designed for the conserved VP60 fragment of RHDV1 (isolate WF2007, GenBank accession number: FJ794180) and RHDV2 (isolate SC2020 / 04, GenBank accession number: MT383749). Primers were then screened to select the most efficient primer set, ensuring no cross-reaction between the RHDV1 and RHDV2 systems. The loop primers described in the present invention were designed based on the exo-inner primers.

[0037] In the present invention, the total volume of the LAMP amplification system is 25 μL, which includes 12.5 μL of LAMP / RT-LAMP 2X premix, 2.5 μL of primer mix, 2 μL of extracted RNA, and 8 μL of enzyme-free water. The primer mix includes outer primer pair F3 and B3, inner primer pair FIP and BIP, and loop primers LF and / or LB.

[0038] In the present invention, the preparation of the primer mixture for RHDV1 includes the following steps: diluting each primer to 100 μM; adding 12 μL each of FIP / BIP, 2 μL each of F3 / B3, and 6 μL of LB, and then adding enzyme-free water to a total volume of 100 μL to obtain the primer mixture for RHDV1.

[0039] In the present invention, the preparation of the primer mixture for RHDV2 includes the following steps: diluting each primer to 100 μM; adding 8 μL each of FIP / BIP, 2 μL each of F3 / B3, and 6 μL each of LF / LB, and then adding enzyme-free water to a total volume of 100 μL to obtain the primer mixture for RHDV2.

[0040] In the present invention, in the LAMP amplification system for RHDV1, the final concentrations of the outer primer pair F3 and B3 are 200 nM, the final concentrations of the inner primer pair FIP and BIP are 1200 nM, and the final concentration of the loop primer LB is 600 nM; in the LAMP amplification system for RHDV2, the final concentrations of the outer primer pair F3 and B3 are 200 nM, the final concentrations of the inner primer pair FIP and BIP are 800 nM, and the final concentrations of the loop primers LF and LB are 600 nM.

[0041] In the present invention, the CRISPR / Cas12a detection system also includes a LAMP amplification product, an RNase inhibitor, NEBuffer 2.1, a Cas12a protein, and an ssDNA reporter molecule. The LAMP amplification product of the present invention is a product amplified by the RNA extracted from the sample in the LAMP amplification system, and the amount of the LAMP amplification product is 1 to 3 μL. The concentration of the RNase inhibitor of the present invention is 0.2 to 0.8 U / μL, and the amount is 0.5 to 1.5 μL. The gRNA concentration of the present invention is 450 to 550 nM, and the amount is 1.5 to 2.5 μL. The NEBuffer 2.1 of the present invention is used in an amount of 1 to 3 μL. The Cas12a protein of the present invention is used in an amount of 0.5 to 1.5 μL, and the concentration is 50 to 250 nM. The ssDNA reporter molecule of the present invention is used in an amount of 0.5 to 1.5 μL, and the concentration is 50 to 300 nM. The present invention designs gRNA (composed of a spacer sequence and a repeat sequence, with the spacer sequence generally being the 20-21 bases after the PAM sequence) and ssDNA probe (5'FAM-TTATT-BHQ13' or 5' 6-FAM-TTATT-Biotin 3') for the fragment amplified by LAMP. The present invention screens gRNA and optimizes Cas12a protein concentration by generating fluorescence through CRISPR / Cas12a cleavage experiments.

[0042] In the present invention, the kit further comprises a lateral flow chromatography test strip. The lateral flow chromatography test strip of the present invention is a test strip adapted for rabbit hemorrhagic disease virus detection. The test strip of the present invention is preferably a CRISPR Cas12 / 13 HybriDetect test strip (purchased from Wobo Biotech (Nanjing, China), JY0301), the sample pad of the test strip is added with colloidal gold-labeled mouse anti-FAM antibody, the C line is labeled with streptavidin, and the T line is labeled with anti-mouse IgG antibody. When the test strip is contained in the kit of the present invention, the ssDNA probe is a biotin ssDNA probe (5'6-FAM-TTATT-Biotin 3'). The principle of the present invention using the test strip to judge the result is: when the ssDNA reporter molecule is not cut, the streptavidin on the quality control line binds to the biotin on the ssDNA to capture it, and the C line is colored; when the ssDNA reporter molecule is cut due to the activated trans-cleavage ability of Cas12a, the disconnected FAM end continues to surge upward, and the anti-mouse IgG antibody on the detection line binds to the colloidal gold-labeled mouse antibody bound to FAM, capturing the FAM end with colloidal gold, and the T line is colored; when only the C line is colored, it is negative, and when only the T line is colored, it is positive.

[0043] The present invention also provides a method for non-disease diagnosis purposes of RHDV1 and RHDV2 based on LAMP-CRISPR / Cas12a visualization, comprising the following steps: (1) extracting RNA of a sample to be detected; (2) using the RNA extracted in step (1) as a template, performing LAMP amplification in a LAMP amplification system; (3) enzymatically cleaving the LAMP amplification product obtained in step (2) in a CRISPR / Cas12a detection system and performing fluorescence detection. The present invention combines the LAMP-CRISPR / Cas12a detection method with LFS to achieve visualization and construct a portable detection platform. The present invention can also perform blue light instrument detection on the CRISPR / Cas12a detection system after enzyme cleavage to achieve fluorescence visualization, as shown in Figure 1. The present invention sets RHDV type 1 and RHDV type 2 for detection in two different systems, namely, a RHDV type 1 detection system and a RHDV type 2 detection system.

[0044] In the present invention, the total volume of the LAMP amplification system is 25 μL, consisting of 12.5 μL of LAMP / RT-LAMP 2X premix, 2.5 μL of primer mix, 2 μL of extracted RNA, and 8 μL of enzyme-free water. The primer mix comprises an outer primer pair, an inner primer pair, and / or a loop primer. For RHDV1, the volume ratio of outer primer pair: inner primer pair: loop primer pair is 1:6:1.5 or 1:6:3; for RHDV2, the volume ratio of outer primer pair: inner primer pair is 1:4:3. The present invention targets RHDV1, and the final concentrations of the outer primer pair F3 and B3 are 200 nM, the final concentrations of the inner primer pair FIP and BIP are 1200 nM, and the final concentration of the loop primer LB is 600 nM. Targeting RHDV2, the final concentrations of the outer primer pair F3 and B3 are 200 nM, the final concentrations of the inner primer pair FIP and BIP are 800 nM, and the final concentrations of the loop primers LF and LB are 600 nM.

[0045] In the present invention, the LAMP amplification reaction conditions are 65-69° C. for 25-35 min, preferably 67° C. for 30 min.

[0046] In the present invention, the total volume of the CRISPR / Cas12a detection system is 20 μL, 2 μL LAMP amplification product, 1 μL RNase inhibitor (0.5 U / μL), 2 μL gRNA (500 nM), 2 μL NEBuffer 2.1, 1 μL Cas12a protein (125 nM), and 1 μL ssDNA reporter molecule (250 nM fluorescence quenching probe / 100 nM biotin probe).

[0047] In the present invention, the reaction conditions in the CRISPR / Cas12a detection system are incubation at 35-38°C for 25-35 minutes.

[0048] In the present invention, the LAMP, i.e., loop-mediated isothermal amplification technology, is the most widely used isothermal amplification technology. Amplification can be completed in a short time using a water bath, and amplification efficiency is 2 to 5 higher than that of ordinary PCR, and the detection result can be visualized by using a dye. The CRISPR / Cas12a technology can be used for gene editing, and can also be used for pathogen detection. It is the most common detection method coupled with isothermal amplification technology to increase specificity. Cas12a protein is further combined with the target sequence after being combined with gRNA, activating cutting activity, not only can specifically cut the target nucleic acid, but also can indiscriminately cut the nearby ssDNA probe, if fluorescein and quencher are added to ssDNA two ends, after being cut, fluorescent group emits light, and can point to a positive result. The present invention is associated with lateral flow test strips on the basis of LAMP in combination with CRISPR / Cas12a technology, and a more intuitive detection result.

[0049] The present invention also provides application of the kit and the detection method in identifying RHDV1 and RHDV2.

[0050] In the present invention, unless otherwise specified, all components or reagents are commercially available products well known to those skilled in the art.

[0051] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0052] Example 1

[0053] 1. Design of LAMP primers

[0054] LAMP primers include two outer primers, F3 and B3, two inner primers, FIP and BIP, and, if necessary, loop primers, LF and LB. Multiple primer sets were designed for the conserved VP60 fragment of RHDV1 (isolate WF2007, GenBank accession number: FJ794180) and RHDV2 (isolate SC2020 / 04, GenBank accession number: MT383749). Primer Set 1 (RHDV1), Primer Set 2 (Set 2), and Primer Set 3 (Set 3) were designed for RHDV1 and RHDV2, respectively, as shown in Table 1. Primer sequences were synthesized from GenScript Biotech (Nanjing, China).

[0055] Table 1 LAMP primer sequences

[0056] 2. Extraction of RNA template

[0057] RNA was extracted from RHDV1 and RHDV2 strains (stored in our laboratory) using RNA extraction kits (purchased from Novozymes Biotech, Nanjing, China). The concentration of RNA extracted from the RHDV1 strain was 218.6 ng / μL, and the concentration of RNA extracted from the RHDV2 strain was 158.8 ng / μL, which were used for subsequent experiments.

[0058] 3. Establish LAMP amplification method for RHDV1 and RHDV2 and optimize primers and reaction conditions

[0059] (1) LAMP amplification step: Using the RNA extracted in step 2 as a template, add the designed primers according to the reaction system in Table 2, mix well, and react in a constant temperature shaker at 67°C for 30 minutes. The preparation of the primer mixture for RHDV1 includes the following steps: dilute each primer to 100 μM; add 12 μL each of FIP / BIP, 2 μL each of F3 / B3, 6 μL each of LB / LF or 6 μL of LB, and then add enzyme-free water to a total volume of 100 μL to obtain the primer mixture for RHDV1. The preparation of the primer mixture for RHDV2 includes the following steps: dilute each primer to 100 μM; add 8 μL each of FIP / BIP, 2 μL each of F3 / B3, and 6 μL each of LF / LB, and then add enzyme-free water to a total volume of 100 μL to obtain the primer mixture for RHDV2. The LAMP amplification systems for RHDV1 and RHDV2, as well as the volume and final concentration of each primer, are shown in Tables 2 to 4.

[0060] Table 2 LAMP amplification system

[0061] Table 3 Primer volume and final concentration in the LAMP amplification system for RHDV1

[0062] Table 4 Primer volume and final concentration in the LAMP amplification system for RHDV2

[0063] (2) Primer screening: Based on the primers designed in step 1 and the LAMP amplification step in step 3 (1), the nucleic acid dye SYTO9 (final concentration 250 nM) was added to the LAMP system. The most efficient primer set was selected by monitoring the fluorescence value under the Quant Studio 1 system. It was also ensured that there was no cross-reaction between the RHDV1 and RHDV2 systems. Water was used as a negative control during the experiment. The results are shown in Figure 2.

[0064] As shown in FIG2 , the primers with the highest amplification efficiency are primer set 3 for RHDV1 and primer set 2 for RHDV2.

[0065] A crossover experiment was performed using primer set 3 targeting RHDV1 and primer set 2 targeting RHDV2 to amplify RHDV1 and RHDV2 nucleic acids, respectively. The amplified results were analyzed by gel electrophoresis and fluorescence value analysis. The results are shown in Figure 3.

[0066] As shown in Figure 3, gel electrophoresis was used to test the crossover experiment. When RHDV1 was amplified using primer set 3, which was selected for RHDV1, typical ladder-shaped LAMP bands were obtained, but no bands were obtained when amplifying RHDV2. When RHDV1 and RHDV2 nucleic acids were amplified using primer set 2, which was selected for RHDV2, no bands were obtained when amplifying RHDV1, but typical ladder-shaped LAMP bands were obtained when amplifying RHDV2. A crossover experiment using fluorescent LAMP revealed a significant increase in fluorescence only when the RHDV1 primer set was used to amplify RHDV1 nucleic acid, and only when the RHDV2 primer set was used to amplify RHDV2 nucleic acid. These results demonstrate that the two selected primer sets have good specificity.

[0067] (3) Screening of reaction temperature, inner and outer primer concentration ratio, and loop primer concentration

[0068] Based on the selected primer sets, the aforementioned amplification steps, and the LAMP amplification system, the optimal reaction conditions for reaction temperature, outer-to-inner primer concentration ratio, and loop primer concentration were selected based on fluorescence trends. The selected reaction temperatures were 61°C, 63°C, 65°C, 67°C, and 69°C; the selected outer-to-inner primer concentration ratios were 200nM:400nM, 200nM:800nM, 200nM:1200nM, 200nM:1600nM, and 200nM:2000nM; and the selected loop primer concentrations were 200nM, 400nM, 600nM, 800nM, and 1000nM, respectively. Water was used as a negative control. The results are shown in Figures 4-7.

[0069] As shown in Figures 4-7, the optimal reaction conditions for RHDV1 are 67°C, an outer primer:inner primer ratio of 1:6 (200 nM:1200 nM), and a loop primer concentration of 600 nM. The optimal reaction conditions for RHDV2 are 67°C, an outer primer:inner primer ratio of 1:4 (200 nM:800 nM), and a loop primer concentration of 600 nM. The addition of neutral red dye to the LAMP system allows visualization of the results, with a clear color difference between positive and negative results.

[0070] 4. Establish CRISPR / Cas12a detection methods for RHDV1 and RHDV2 and optimize the reaction system

[0071] (1) gRNA and ssDNA probe design

[0072] gRNAs were designed based on the target fragments of RHDV1 and RHDV2 amplified in the LAMP phase. Two gRNAs (gRNA1 and gRNA2) were designed (comprising a spacer sequence and a repeat sequence, with the spacer sequence typically being the 20-21 bases following the PAM sequence). A ssDNA probe (5'FAM-TTATT-BHQ13') was also designed. Both gRNA and ssDNA probe sequences were synthesized from GenScript Biotech (Nanjing, China) and are listed in Table 5.

[0073] Table 5 gRNA sequences

[0074] (2) CRISPR / Cas12a detection steps: Using the fragment amplified by the selected optimal LAMP amplification system and amplification conditions as a template, add the sample according to the reaction system in Table 6, mix well, and incubate at 37°C for 30 minutes to obtain the reactant solution.

[0075] Table 6 CRISPR / Cas12a detection system

[0076] (3) Screening gRNA and optimizing Cas12a protein concentration by the fluorescence generated by CRISPR / Cas12a cutting experiment: Based on the primer gRNA in step 4 (1) and the detection step in step 4 (2), use QuantStudio 1 to record the change of fluorescence value and blue light instrument irradiation, judge the result by the fluorescence curve and whether it is fluorescent, and ensure that there is no cross reaction between RHDV1 and RHDV2 systems. Verify that the generated fluorescence is generated by gRNA guiding Cas12a protein to bind to the target fragment and activate its trans-cutting activity, and non-specifically cut the surrounding ssDNA. Water is used as a negative control in the experimental process, and the product amplified by enzyme-free water as a template in the LAMP stage is LAMP-NC.

[0077] The gRNA1 and gRNA2 of RHDV1 and RHDV2 were optimized and screened respectively, and three Cas12a protein concentrations (50nM, 125nM, 250nM) were set for orthogonal experiments, and the results are shown in Figure 8. As shown in Figure 8, under three different Cas12a concentrations, in the gRNA designed for RHDV1, gRNA2 showed a higher efficiency of cutting to produce fluorescence, and in the gRNA designed for RHDV2, gRNA2 showed a higher efficiency of cutting to produce fluorescence; when Cas12a concentration was increased from 50nM to 125nM, the fluorescence value produced was significantly improved (P<0.05), and when Cas12a concentration was further increased to 250nM, the fluorescence value produced was not significantly different from that of 125nM (P>0.05), so the reaction concentration of Cas12a protein was set to 125nM.

[0078] The selected gRNA was used to perform a cross-experiment between the trans-cleavage activity of Cas12a protein and the RHDV1 reaction system and the RHDV2 reaction system. The products amplified using the selected optimal LAMP amplification system and amplification conditions were used as templates for the CRISPR / Cas12a cleavage experiments of the RHDV1 and RHDV2 systems, respectively. Enzyme-free water was used as a template as a negative control, and the fluorescence generated was detected using the QuantStudio 1 system. The results are shown in Figure 8. The results show that only when the template is the product of RHDV1 primer amplification of RHDV1 nucleic acid and RHDV2 primer amplification of RHDV2 nucleic acid, there is a significant increase in fluorescence value in the system, and the same result is observed under blue light. The results indicate that the fluorescence generated by the RHDV1 and RHDV2 systems in the CRISPR / Cas12a cleavage experimental stage is generated by the trans-cleavage activity stimulated by the binding of Cas12a protein to the target fragment under the guidance of the corresponding gRNA, and at this stage, there is no cross-reaction between the RHDV1 and RHDV2 systems.

[0079] Example 2 Kit 1 for visually identifying RHDV type 1 and type 2 based on LAMP-CRISPR / Cas12a

[0080] The kit includes a LAMP amplification system and a CRISPR / Cas12a detection system.

[0081] The total volume of the LAMP amplification system was 25 μL, containing 12.5 μL of LAMP / RT-LAMP 2X premix, 2.5 μL of primer mix, 2 μL of extracted RNA, and 8 μL of enzyme-free water. For the LAMP amplification system for RHDV1, the primer set used is shown in Table 1 (RHDV1 Primer Set 3); the final concentrations of the outer primer pair F3 and B3 were 200 nM, the final concentrations of the inner primer pair FIP and BIP were 1200 nM, and the final concentration of the loop primer LB was 600 nM. For the LAMP amplification system for RHDV2, the primer set used is shown in Table 1 (RHDV1 Primer Set 2); the final concentrations of the outer primer pair F3 and B3 were 200 nM, the final concentrations of the inner primer pair FIP and BIP were 800 nM, and the final concentrations of the loop primers LF and LB were 600 nM.

[0082] The total volume of the CRISPR / Cas12a detection system is 20 μL, containing 2 μL LAMP amplification product, 1 μL RNase inhibitor (final concentration 0.5 U / μL), 2 μL gRNA (final concentration 500 nM), 2 μL NEBuffer 2.1, 1 μL Cas12a protein (final concentration 125 nM), and 1 μL ssDNA reporter molecule (final concentration 250 nM fluorescence quenching probe). For RHDV1, the gRNA used is shown in Table 5 as RHDV1 gRNA2; for RHDV2, the gRNA used is shown in Table 5 as RHDV2 gRNA2.

[0083] Example 3 Kit 2 for visually identifying RHDV type 1 and type 2 based on LAMP-CRISPR / Cas12a

[0084] In addition to the system described in Example 2, the kit also contains lateral flow chromatography test strips. The lateral flow chromatography test strips are CRISPR Cas12 / 13 HybriDetect test strips (purchased from Wobo Biotech (Nanjing, China), JY0301). The LAMP amplification system and CRISPR / Cas12a detection system are the same as in Example 2, wherein the fluorescence quenching ssDNA probe is replaced with a biotin ssDNA probe (5'6-FAM-TTATT-Biotin3'), and its final concentration in the reaction system is 100nM.

[0085] Example 4

[0086] The method for visually distinguishing RHDV type 1 and type 2 based on LAMP-CRISPR / Cas12a includes the following steps:

[0087] (1) RNA extraction kit (purchased from Novozymes Biotech, Nanjing, China) was used to extract RNA from the sample to be tested.

[0088] (2) Add the extracted RNA solution to the LAMP amplification system described in Example 2 or 3, mix well, and react in a constant temperature shaker at 67°C for 30 minutes to obtain a LAMP amplification product;

[0089] (3) Add the obtained LAMP amplification product to the CRISPR / Cas12a detection system described in Example 2 or 3, mix evenly, and incubate at 37°C for 30 minutes to perform enzyme digestion reaction to obtain a digestion reaction solution;

[0090] (4) The reactant solution was irradiated with a blue light analyzer (LABGIC, Beijing, China). If the reactant solution showed fluorescence, it was positive; if the reactant solution showed no fluorescence, it was negative.

[0091] Alternatively, the lateral flow chromatography test strip described in Example 3 can be placed in the reactant solution according to the following procedure. A positive result is obtained when only the T line develops color, while a negative result is obtained when only the C line develops color. The procedure includes taking 5 μL of the reaction solution, adding 45 μL of enzyme-free water, and inserting the lateral flow chromatography test strip into the reactant solution for color development.

[0092] Example 5 Methodological evaluation of the established LAMP-CRISPR / Cas12a detection method

[0093] (1) Specificity experiment: The method described in Example 4 was used to extract nucleic acids of seven pathogens (preserved by this laboratory), including RHDV1, RHDV2, Escherichia coli (E. coli), Salmonella typhi (S. typhi), Klebsiella pneumoniae (K. pneumoniae), Pasteurella multocida (P. multocida), and rotavirus (RV), for LAMP-CRISPR / Cas12a detection to verify the specificity of the established detection method. Water was used as a negative control during the experiment. The results are shown in Figure 9. The results showed that when the detection method for RHDV1 was used for detection, only RHDV1 was positive and the other pathogens were negative; when the detection method for RHDV2 was used for detection, only RHDV2 was positive and the other pathogens were negative; the results seen under blue light were consistent with the results displayed on the test strips. This shows that the detection methods established for RHDV1 and RHDV2 are both highly specific.

[0094] (2) Sensitivity test: The target gene was cloned using the full-length primers of the VP60 gene of RHDV 1 isolate WF2007 and RHDV 2 isolate SC 2020 / 04. The amplified gene sequence was ligated into the pMD18-T vector. After screening and sequencing, the positive pMD18-T-WF2007-VP60 and pMD18-T-SC2020-VP60 recombinant plasmids were obtained. The concentration of the recombinant plasmid was determined by Nanodrop and the copy number was calculated. The recombinant plasmid was diluted to 1×1010 copies / μL as template standards. Use a 10-fold dilution (1×10 6 copies / μL-1×10 0 The full-length VP60 plasmids of RHDV1 and RHDV2 (copies / μL) were used as detection templates, and the method described in Example 4 was used (the extracted RNA solution was replaced with the recombinant plasmid). Enzyme-free water was used as a negative control template during the experiment. The results are shown in Figure 10. The results show that the established LAMP-CRISPR / Cas12a detection method for RHDV1 and RHDV2 can detect a minimum of 1×10 1 The results of dsDNA template observed under blue light were consistent with those displayed on the test strip.

[0095] Example 6 Clinical Sample Detection

[0096] 74 clinical samples from different regions and farms were tested using qPCR and LAMP-CRISPR / Cas12a detection methods to compare the consistency and sensitivity of the qPCR method and the established LAMP-CRISPR / Cas12a detection method. Water was used as a negative control (NC) with a concentration of 1×10 5 copies / μL standard positive plasmid (RHDV1 or RHDV2) as positive control (PC)

[0097] The LAMP-CRISPR / Cas12a detection method is the same as in Example 4.

[0098] The qPCR testing procedure includes: RNA extraction from 74 clinical samples according to the Vazyme FastPure Cell / Tissue Total RNA Isolation Kit V2 instructions, followed by qPCR amplification using qPCR primers. The absolute expression of viral RNA in each tissue sample was measured using a Quant Studio 1 fluorescence quantitative PCR instrument to determine the viral content in each tissue sample. Each sample was tested in triplicate. For the TaqMan probe real-time fluorescence quantitative PCR method, the positive control Ct value was ≤35 and the amplification curve showed a clear logarithmic growth phase. Simultaneously, if the negative control amplification curve lacked a logarithmic growth phase, the sample's Ct value was ≤35.0 and a standard S-shaped amplification curve was present, indicating the presence of RHDV1 and / or RHDV2 nucleic acid. If there was no Ct value or a Ct value greater than 38 and no standard amplification curve, the sample was considered negative for RHDV1 and / or RHDV2 nucleic acid. When the test sample has a Ct value of 35.0≤≤38 and the amplification curve shows a standard S-shaped curve, it is judged as suspicious and a repeated experiment is required. If the above result is still obtained after repetition, it is judged as positive, otherwise it is judged as negative.

[0099] The qPCR primer sequences were as follows: upstream primer RHDV-F1 of the VP60 gene sequence: 5′-TGGARMTWGGYTTRAGTGTDGAYG-3′ (SEQ ID No. 40); downstream primer: RHDV-R 1: 5′-CAGACATAAGAAAARCCATTGGYTG-3′ (SEQ ID No. 41); RHDV1 probe sequence: 5′-FAM-TGAYTGAACTCATTGAYGTACGCCC-BHQ1-3′ (5′-FAM-SEQ ID No. 42-BHQ1-3′); RHDV2 probe sequence: 5′-VIC-TGTCAGAMCTTGTTGACATCCGCC-BHQ2-3′ (5′-VIC-SEQ ID No. 43-BHQ2-3′); primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. In the above sequence, R represents A or G, M represents A or C, W represents A or T, Y represents C or T, and D represents G or A or T.

[0100] The qPCR amplification reaction system was as follows: 10 μL of 2× One Step RT-PCR Buffer III, 0.6 μL each of upstream and downstream primers (10 μmol / L), 0.4 μL of ROX Dye II (50×), 0.8 μL each of fluorescent probes (10 μmol / L), 2 μL of RNA product, and 4 μL of ddH2O. The qPCR amplification reaction program was as follows: 42°C for 5 min, 95°C for 10 s, 95°C for 5 s, 60°C for 34 s, for 40 cycles.

[0101] The fluorescence of the reactant solutions obtained by the two detection methods was monitored using QuantStudio 1. The reactant solutions were then illuminated with blue light and detected using lateral flow strips. The results are shown in Figure 11. The results showed that when using the RHDV1 detection system, the method developed in the present invention detected 19 RHDV1-positive samples, while the qPCR method detected 17 RHDV1-positive samples, with a concordance rate of 97.30%. When using the RHDV2 detection system, the method developed in the present invention detected 32 RHDV2-positive samples, while the qPCR method detected 30 RHDV2-positive samples, with a concordance rate of 97.30%. Direct observation of the fluorescence under blue light by the two detection systems and the lateral flow strips showed consistent results, mutually validating the two readout strategies. The concordance rate with the qPCR method also verified the stability and reliability of the detection method, indicating that the detection method constructed in the present invention can be considered a new diagnostic method for the detection and differentiation of RHDV1 and RHDV2 and is feasible in clinical practice.

[0102] All data in the present invention were statistically analyzed using GraphPad Prism 8.0, and one-way analysis of variance (ANOVA) was used between different groups. All experiments were repeated at least 3 times, and the data are presented as mean ± standard deviation. When the p value < 0.05, the difference was considered statistically significant.

[0103] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Kit for visually identifying RHDV type 1 and type 2 based on LAMP-CRISPR / Cas12a, characterized in that, The kit includes a LAMP amplification system and a CRISPR / Cas12a detection system; The LAMP amplification system includes outer primer pairs F3 and B3, and inner primer pairs FIP and BIP; in the LAMP amplification system for RHDV1, the nucleotide sequences of outer primer pairs F3 and B3 are shown in SEQ ID No.13-14, and the nucleotide sequences of inner primer pairs FIP and BIP are shown in SEQ ID No.15-16; in the LAMP amplification system for RHDV2, the nucleotide sequences of outer primer pairs F3 and B3 are shown in SEQ ID No.24-25, and the nucleotide sequences of inner primer pairs FIP and BIP are shown in SEQ ID No.26-27; The CRISPR / Cas12a detection system includes gRNA; in the CRISPR / Cas12a detection system for RHDV1, the nucleotide sequence of gRNA is shown in SEQ ID No.37; in the CRISPR / Cas12a detection system for RHDV2, the nucleotide sequence of gRNA is shown in SEQ ID No.

39.

2. The kit according to claim 1, characterized in that, The LAMP amplification system further includes loop primers LF and / or LB; in the LAMP amplification system for RHDV1, the nucleotide sequence of loop primer LB is shown in SEQ ID No.17; in the LAMP amplification system for RHDV2, the nucleotide sequences of loop primers LF and LB are shown in SEQ ID No.28-29.

3. The kit according to claim 1, wherein The LAMP amplification system further includes a LAMP / RT-LAMP 2X premix, nucleic acid to be detected, and nuclease-free water.

4. The kit according to claim 1, characterized in that The CRISPR / Cas12a detection system further includes the LAMP amplification product, RNase inhibitor, NEBuffer 2.1, Cas12a protein, ssDNA reporter molecule, and nuclease-free water.

5. The kit according to claim 1, wherein The kit further includes a lateral flow chromatographic test strip or a blue light instrument.

6. The kit according to claim 1, characterized in that, When the kit contains a lateral flow chromatographic test strip, the ssDNA reporter molecule is a biotinylated ssDNA reporter molecule; when the kit contains a blue light instrument, the ssDNA reporter molecule is a fluorescence quenched ssDNA reporter molecule.

7. Use of the kit according to any one of claims 1 to 6 in the preparation of a product for differentiating RHDV1 and RHDV2.

8. A method for visually differentiating between RHDV type 1 and type 2 for non-disease diagnosis purposes based on LAMP-CRISPR / Cas12a, characterized in that, Comprising the following steps: (1) Extract the RNA of the sample to be detected; (2) Using the RNA extracted in step (1) as a template, perform LAMP amplification in the LAMP amplification system; (3) Digest the LAMP amplification product obtained in step (2) in the CRISPR / Cas12a detection system and perform fluorescence detection; Or digest the LAMP amplification product obtained in step (2) in the CRISPR / Cas12a detection system and perform lateral flow chromatographic test strip detection.

9. The method according to claim 8, wherein The total volume of the LAMP amplification system is 25 μL, including 12.5 μL of LAMP / RT-LAMP 2X premix, 2.5 μL of primer mixture, 2 μL of extracted RNA, and 8 μL of nuclease-free water.

10. The method according to claim 9, wherein In the LAMP amplification system for RHDV1, the final concentrations of the outer primer pair F3 and B3 in the primer mixture are 200 nM respectively, the final concentrations of the inner primer pair FIP and BIP are 1200 nM respectively, and the final concentration of the loop primer LB is 600 nM; in the LAMP amplification system for RHDV2, the final concentrations of the outer primer pair F3 and B3 in the primer mixture are 200 nM respectively, the final concentrations of the inner primer pair FIP and BIP are 800 nM respectively, and the final concentrations of the loop primers LF and LB are 600 nM respectively.

11. The method according to claim 8, wherein When fluorescence detection is carried out, the total volume of the CRISPR / Cas12a detection system is 20 μL, including 2 μL of LAMP amplification product, 1 μL of RNase inhibitor with a final concentration of 0.5 U / μL, 2 μL of gRNA with a final concentration of 500 nM, 2 μL of NEBuffer 2.1, 1 μL of Cas12a protein with a final concentration of 125 nM, 1 μL of fluorescent quenching ssDNA reporter molecule with a final concentration of 250 nM, and 11 μL of enzyme-free water.

12. The method according to claim 8, wherein When lateral flow chromatographic strip detection is carried out, the total volume of the CRISPR / Cas12a detection system is 20 μL, including 2 μL of LAMP amplification product, 1 μL of RNase inhibitor with a final concentration of 0.5 U / μL, 2 μL of gRNA with a final concentration of 500 nM, 2 μL of NEBuffer 2.1, 1 μL of Cas12a protein with a final concentration of 125 nM, 1 μL of biotinylated ssDNA reporter molecule with a final concentration of 100 nM, and 11 μL of enzyme-free water.

13. The method according to claim 8, wherein The LAMP amplification conditions are 65 - 69 °C for 25 - 35 min.

14. The method according to claim 8, wherein The reaction conditions in the CRISPR / Cas12a detection system are incubation at 35 - 38 °C for 25 - 35 min.