Kit and method for visually detecting cat parvovirus based on RAA-CRISPR / Cas12a system

Abstract

The invention discloses a kit and a method for visually detecting cat parvovirus based on a RAA-CRISPR / Cas12a system. The kit contains an RAA system substance and a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas12a system substance, the RAA system substance comprises an upstream primer and a downstream primer, and the upstream primer and the downstream primer comprise RAA-F1, RAA-R1, RAA-F2 and RAA-R2; the CRISPR / Cas12a system substance comprises Cas12a, a crRNA (Complementary Ribonucleic Acid) sequence and an ssDNA (Deoxyribose Nucleic Acid) fluorescent reporter probe; the crRNA sequence comprises crRNA01 and crRNA02, and the ssDNA fluorescence report probe comprises qPCR-F (quantitative polymerase chain reaction) and qPCR-R (quantitative polymerase chain reaction); the sequences of the upstream primer RAA-F1, the downstream primer RAA-R1, the downstream primer RAA-F2 and the downstream primer RAA-R2 are respectively as shown in SEQ ID NO. 1 to SEQ ID NO. 4; the sequence of the crRNA01 is as shown in SEQ ID NO.5, and the sequence of the crRNA02 is as shown in SEQ ID NO.6; the sequence of the qPCR-F is as shown in SEQ ID NO.7, and the sequence of the qPCR-R is as shown in SEQ ID NO.8.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal disease detection, and in particular relates to a kit and a method for visually detecting feline parvovirus based on a RAA-CRISPR / Cas12a system. Background Art

[0002] Feline parvovirus (FPV) seriously affects their health. FPV has a wide host range, including domestic and wild animals such as the cat family, raccoon family, mustelid family and fox family. In adult animals, it mainly infects lymphoid tissues and intestinal epithelial cells, causing symptoms such as severe leukopenia, severe diarrhea and fever, and even sudden death in acutely ill cats. Infected young animals develop cerebellar lesions and may lead to ataxia. FPV is not only transmitted through direct contact, but can also be transmitted indirectly through contaminated clothing, utensils and insects, and can remain infectious for months to years. In recent years, there have been more and more reports on the spread of epidemics caused by FPV at home and abroad. Therefore, in order to further prevent FPV epidemic infection, it is imperative to establish a simple, rapid and accurate point-of-care (POC) molecular diagnostic method for FPV in pets and wild animals.

[0003] At present, the preliminary diagnosis method of FPV can be judged based on the clinical symptoms and pathological changes of sick animals. Further diagnosis requires the help of conventional PCR and real-time fluorescence quantitative PCR (qPCR). Although it has the advantage of high sensitivity, FPV sample preparation and detection still require specific working conditions and professionals, which obviously cannot meet the deployment of instant detection programs in resource-limited areas. In contrast, isothermal amplification technologies with low instrument dependence and high nucleic acid amplification efficiency are more suitable for POC molecular diagnosis, such as strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), and recombinase polymerase amplification (RPA / RAA). Among them, RPA / RAA is considered to be the most effective and promising isothermal amplification technology due to its advantages such as rapidity, high sensitivity, and moderate reaction temperature. Obviously, combining RPA / RAA with agarose gel electrophoresis does not bring much improvement over PCR.

[0004] In recent years, the clustered regularly interspaced short palindromic repeats / CRISPR-associated (CRISPR / Cas) system has developed rapidly due to its high sensitivity, rapidity and ease of use, especially Cas12 and Cas13. These RNA-guided Class 2 Cas systems have shown broad prospects in various nucleic acid point-of-care diagnostic applications. At present, the detection method combining isothermal amplification technology with the CRISPR / Cas system has become a new trend in nucleic acid point-of-care detection because of its ultra-high sensitivity, and has been significantly developed in bacteria, viruses, fungi, parasites, toxins and other chemical pollutants. The development of FPV diagnostic methods based on the CRISPR / Cas system is relatively lagging. Therefore, there is an urgent need to establish a rapid, highly sensitive and specific diagnostic method for detecting FPV nucleic acid molecules in the market.

[0005] Wang et al. established an RPA-LFD method combining RPA with lateral flow chromatography (LFD) for FPV VP2 gene. The detection method can be completed within 15 min at 38 °C. The results showed that the minimum detection limit of FPV was 1.0 × 10 2 The amplified product has no cross-reaction with other feline-related viruses (including FHV, FECV, FeSFV, and FIV), and is 10 times more sensitive than conventional PCR methods, with high sensitivity and specificity. This method greatly reduces the need for professionals and precision instruments, and is more suitable for immediate on-site testing than conventional PCR and qPCR methods. The disadvantage of this method is that LFD cannot detect asymptomatic carriers with low virus titers and may produce false negative results. In addition, opening the test tube during LFD incubation can easily cause aerosol contamination of the RPA product.

[0006] Wang et al. established a two-tube method (RPA-Cas12a) for the FPV NS1 gene based on RPA and CRISPR / Cas12a technology. The detection method can be completed within 25 minutes at 37°C. The results show that the minimum detection limit for FPV is 1 copies / μL, and the amplified product has no cross-reaction with other viral genomes (including FIPV, FCV, FHV-1, MYC and CP), with higher sensitivity and specificity. This method can read the test results with a simple handheld ultraviolet lamp in resource-limited areas, significantly reducing the need for professionals and precision instruments, and is suitable for instant on-site detection, especially in resource-limited environments. The disadvantage of this method is that due to the high sensitivity of the method, the uncapping operation after RPA amplification is easily affected by small molecule nucleic acids accumulated in the environment and produces false positive results, so the test operation requirements are more stringent. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention aims to establish a rapid, highly sensitive and specific detection method for FPV, so as to solve the problems of the existing FPV detection method, such as long time consumption, low sensitivity, false positive results, and dependence on professionals, precision instruments and environment.

[0008] In order to achieve the above object, the technical solution provided by the present invention is:

[0009] The kit for visual detection of feline parvovirus based on the RAA-CRISPR / Cas12a system contains RAA system substances and CRISPR / Cas12a system substances; the RAA system substances include upstream and downstream primers, and the upstream and downstream primers include RAA-F1, RAA-R1, RAA-F2 and RAA-R2; the CRISPR / Cas12a system substances include Cas12a, crRNA sequences and ssDNA fluorescent reporter probes (oligonucleotides with FAM and BHQ modified at both ends, respectively); the crRNA sequence includes crRNA01 and crRNA02, and the ssDNA fluorescent reporter probe includes qPCR-F and qPCR-R; the sequences of the upstream and downstream primers RAA-F1, RAA-R1, RAA-F2 and RAA-R2 are shown in SEQ ID NO.1 to SEQ ID NO.4, respectively; the sequence of the crRNA01 is shown in SEQ ID NO.5, and the sequence of the crRNA02 is shown in SEQ ID NO.6; the qPCR-F sequence is shown in SEQ ID NO.7, and the qPCR-R sequence is shown in SEQ ID NO.8.

[0010] Preferably, the upstream and downstream primers are RAA-F1 and RAA-R1; and the crRNA sequence is crRNA02.

[0011] Preferably, the concentrations of the RAA-F1 and RAA-R1 are both 200-600 nM, the concentration of the crRNA02 is 50-150 nM, the concentration of the Cas12a is 50-150 nM, and the concentration of the ssDNA fluorescent reporter probe is 100-250 nM.

[0012] More preferably, the concentrations of RAA-F1 and RAA-R1 are both 500 nM, the concentration of crRNA02 is 150 nM, the concentration of Cas12a is 50 nM, and the concentration of the ssDNA fluorescent reporter probe is 200 nM.

[0013] Preferably, the kit also contains lyophilized powder (lyophilized powder is the protease and nucleotide system of RAA reaction, including recombinase, polymerase, helicase, four nucleotides, etc., which is a commercially available conventional well-known technology), buffer solution, and sterile enzyme-free water.

[0014] More preferably, the buffer is A buffer, B buffer, 1×Reaction Buffer 1 (A buffer is mainly a reconstitution reagent for stabilizing the protein / enzyme performance in the lyophilized powder, and is used in conjunction with the lyophilized powder in the kit; the main active ingredient of B buffer is magnesium ions that start the reaction; Buffer 1 includes DTT, NaCl, Tris-Tricine (pH 7.9), etc. that stabilize the protease system, and is a commercially available conventional well-known technology).

[0015] The present invention also provides a method for visually detecting feline parvovirus based on the above kit, which is a two-tube method and specifically comprises the following steps:

[0016] (1) Prepare 25 μL RAA reaction system: add 14.7 μL of A buffer completely melted and mixed with lyophilized powder, 1.25 μL of upstream primer, 1.25 μL of downstream primer, 1.3 μL of B buffer, 2.5 μL of the sample to be tested and 4 μL of sterile enzyme-free water into a 200 μL PCR tube to obtain 25 μL RAA reaction system;

[0017] (2) taking 12.5 μL of the RAA reaction system and placing it in a PCR tube at 36 to 40° C., preferably at 38° C., for amplification reaction for 20 to 40 minutes, preferably 30 minutes, to obtain an amplified product; after the reaction is completed, the RAA amplification product is obtained;

[0018] (3) The RAA amplification product was transferred into a PCR tube containing 27.5 μL of CRISPR / Cas12a system material and mixed thoroughly, followed by immediate real-time fluorescence detection.

[0019] The present invention also provides another method for visually detecting feline parvovirus based on the above kit, which is a one-tube method and specifically comprises the following steps:

[0020] (1) Prepare 25 μL RAA reaction system: add 14.7 μL of A buffer completely melted and mixed with lyophilized powder, 1.25 μL of upstream primer, 1.25 μL of downstream primer, 1.3 μL of B buffer, 2.5 μL of the sample to be tested and 4 μL of sterile enzyme-free water into a 200 μL PCR tube to obtain 25 μL RAA reaction system;

[0021] (2) Add 27.5 μL of CRISPR / Cas12a system material to the PCR tube cap, then add 12.5 μL of RAA reaction system to the bottom of the PCR tube, and then perform amplification reaction at 20 to 60° C., preferably 37° C. in a fluorescence monitor for 20 to 40 minutes, preferably 30 minutes. After the amplification reaction is completed, the mixture in the PCR tube is fully mixed, and then real-time fluorescence detection is immediately performed.

[0022] The present invention will be further described below:

[0023] The present invention proposes a two-tube method and a one-tube method for detecting FPV based on RAA combined with CRISPR / Cas12a system. The optimized RAA reaction is amplified at 38°C for 30 minutes, and the visualized fluorescence effect can be presented by a blue light instrument within 5 minutes at the fastest, and the fluorescence signal reaches saturation at about 10 minutes. The minimum detection limit of both methods is 4.277 copies / μL, which is basically equivalent to the detection limit of existing diagnostic methods (1 copy / μL), but is much more sensitive than the RPA-LFD method (100 copies / μL), proving that it has high sensitivity, and sensitivity has always been one of the important indicators for judging the performance of nucleic acid detection. High-sensitivity detection will be more conducive to the early detection and epidemic control of asymptomatic carriers or subclinical infections. At the same time, the specific primers designed for the conservative fragment of VP2 have no cross-reaction in the detection of cat pathogens such as FCV, FCoV, FHV-Ⅰ, and FIV, proving that it has good specificity. In addition, compared with the gold standard qPCR method for nucleic acid molecule detection, the one-tube method has a 100% consistency in detecting FPV clinical samples, and the two-tube method has a consistency of 97.22%. Both methods are shorter, easier to operate, and do not require complex equipment, which will help improve the efficiency of FPV detection. Compared with the two-tube method, the one-tube method is more convenient during operation, and effectively reduces the risk of aerosol contamination after the reaction starts. Finally, the development and application of the two-tube method and the one-tube method based on the RAA-CRISPR / Cas12a system in FPV detection were realized. The detection results of the two methods are visible to the naked eye, and the fluorescence results are presented as quickly as 5 minutes at 37°C under the blue light instrument. It has the advantages of high sensitivity, strong specificity, rapidity and accuracy. It is suitable for FPV detection in resource-limited and clinical environments, and will provide a method reference for early rapid diagnosis of FPV, epidemiological surveys, and disease prevention and control.

[0024] In summary, the present invention constructs a two-tube method and a one-tube method based on RAA combined with the CRISPR / Cas12a system, and optimizes and evaluates the reaction conditions and detection performance of the two methods for rapid and accurate detection of FPV. RAA amplification can quickly and accurately enrich the target DNA, and then mix it with the CRISPR / Cas12a system by an open-cap method or a closed-cap method, activating the trans-cutting activity of Cas12a to cut the fluorescent probe to generate a signal, and the fluorescent signal is observed by the naked eye in a short time. Therefore, the detection time is greatly shortened, the demand for professionals and precision instruments is significantly reduced, and a new solution is provided for clinical diagnosis and rapid screening in resource-limited areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 :Optimization of RAA reaction system: (A) Screening of RAA primers; (B) Optimization of RAA primer concentration; (C) Optimization of RAA reaction temperature; (D) Optimization of RAA reaction time;

[0026] Figure 2 : Optimization of CRISPR / Cas12a system: (A) Validation of crRNA sequence validity, NTC represents no template control; (B) Visualized fluorescence effects of 3 groups of crRNA; (C) Real-time fluorescence collection curves of 3 groups of crRNA; (D) Fluorescence intensity of fluorescence collection for 30 minutes at different temperatures; (E) Fluorescence intensity of 9 complexes at 30 minutes of fluorescence collection; (F) Visualized fluorescence effect of 9 complexes; (G) Real-time fluorescence collection curve of ssDNA fluorescent reporter probe at different concentrations; (H) Fluorescence intensity of ssDNA fluorescent reporter probe at different concentrations at 30 minutes of fluorescence collection; (I) Visualized fluorescence effect of ssDNA fluorescent reporter probe at different concentrations. Error bars represent the mean ± SD of n = 3 replicates;

[0027] Figure 3 :Specificity and sensitivity of the RAA-Cas12a system: (A) Real-time fluorescence acquisition curve of the two-tube method for specific analysis; (B) Fluorescence intensity of the two-tube method for specific analysis at 30 minutes of fluorescence acquisition; (C) Visualized fluorescence effect of the two-tube method for specific analysis; (D) Real-time fluorescence acquisition curve of the one-tube method for specific analysis; (E) Fluorescence intensity of the one-tube method for specific analysis at 30 minutes of fluorescence acquisition; (F) Visualized fluorescence effect of the one-tube method for specific analysis; (G) Real-time fluorescence acquisition curve of the two-tube method for sensitivity analysis; (H) Fluorescence intensity of the two-tube method for sensitivity analysis at 30 minutes of fluorescence acquisition; (I) Comparative analysis of the sensitivity of the two-tube method and the one-tube method; (J) Visualized fluorescence effect of the two-tube method for sensitivity analysis; (K) Visualized fluorescence effect of the one-tube method for sensitivity analysis. Error bars represent the mean ± SD of n = 3 replicates;

[0028] Figure 4 :RAA-Cas12a system clinical sample detection: (A) The heat map shows the detection of 18 blood samples by three methods; (B) The heat map shows the detection of 18 fecal samples by three methods;

[0029] Figure 5 : Fluorescence intensity after fluorescence collection for 30 min;

[0030] Figure 6 : Real-time fluorescence collection under different temperature reactions;

[0031] Figure 7 : PC and NTC groups can be clearly identified under blue light instrument irradiation;

[0032] Figure 8 : Real-time fluorescence acquisition of 9 groups of complexes;

[0033] Fig. 9 :Real-time fluorescence curves and visualized fluorescence results of clinical samples; In the figure: (A) Real-time fluorescence curve of blood samples detected by the two-tube method; (B) Real-time fluorescence curve of feces samples detected by the two-tube method; (C) Real-time fluorescence curve of blood samples detected by the one-tube method; (D) Real-time fluorescence curve of feces samples detected by the one-tube method; (E) Visualized fluorescence effect of blood and feces samples detected by the two-tube method; (F) Visualized fluorescence effect of blood and feces samples detected by the one-tube method. DETAILED DESCRIPTION

[0034] 1. Experimental Methods

[0035] 1.1 Experimental Materials

[0036] All primers and crRNA (sequences in Table 1) were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). CRISPR / Cas12a was purchased from MAGIGEN Biotech Co., Ltd (Guangzhou, China). ssDNA fluorescent reporter probe (Cas12a cleavage substrate) was purchased from EZassay Biotech Co., Ltd (Shenzhen, China). DNA isothermal rapid amplification kit was purchased from Amp-Future Biotech Co., Ltd (Changzhou, China). DH5α competent cells and rapid plasmid extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd (Beijing, China). 6× DNA loading buffer, 10000× YeaRed nucleic acid dye, and DL 2000Marker were purchased from Yisheng Biotechnology Co., Ltd (Shanghai, China).

[0037] Feline calicivirus (FCV), feline coronavirus (FCoV), feline herpesvirus type Ⅰ (FHV-Ⅰ), and feline human immunodeficiency virus (FIV) plasmid standards were provided by Yanyuan Biotechnology Co., Ltd. (Shenzhen, China). 36 FPV clinical samples (including 18 blood samples and 18 fecal samples) were provided by a local pet hospital (Shenzhen, China).

[0038] 1.2 Experimental instruments

[0039] G100 dry incubator was purchased from Youning Instrument Co., Ltd. (Hangzhou, China), GelVlew 5000Plus intelligent gel imaging system was purchased from Bolu Teng Biotechnology Co., Ltd. (Guangzhou, China), SLAN-96S fully automatic medical PCR analysis system was purchased from Hongshi Medical Technology Co., Ltd. (Shanghai, China), and Nano One ultra-micro-volume spectrophotometer was purchased from Youning Instrument Co., Ltd. (Hangzhou, China).

[0040] 1.3 Primer and crRNA design

[0041] According to the FPV VP2 gene sequence published in GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ) (accession number: KC473946.1), two pairs of RAA primers were designed using Primer Premier 5.0 software, and crRNA was designed by EZASSAY (https: / / ezassay.com / rna) and synthesized by Sangon Biotech (Shanghai) Co., Ltd., including one pair of qPCR primers. The sequence information of all the above gene fragments is provided in Table 1.

[0042] Table 1 RAA, qPCR primers and crRNA sequence information

[0043]

[0044] 1.4 Preparation of positive recombinant plasmid pUC57-VP2

[0045] The FPV standard plasmid template was designed based on the FPV VP2 gene sequence published in GenBank (accession number: KC473946.1), which was synthesized by Shanghai Sangon Biotechnology Co., Ltd. and inserted into the pUC57 vector to construct the recombinant plasmid pUC57-VP2. Next, pUC57-VP2 was transformed into DH5α competent cells, spread on LB solid medium containing Amp resistance, and incubated overnight at 37°C. The monoclonal bacteria were picked in LB liquid medium containing Amp resistance and cultured at 37°C. The obtained bacterial liquid was extracted with a rapid plasmid mini-extraction kit (spin column type) to obtain the FPV recombinant plasmid. Finally, the concentration of the recombinant plasmid was determined using a Nano One ultra-micro spectrophotometer (Youning Instrument Co., Ltd., Hangzhou, China). The copy number was calculated based on the plasmid concentration, and the number of plasmid copies per microliter (copies / μL) = plasmid concentration (g / μL) × NA (copies / mol) / molecular weight (g / mol). The calculated copy number of FPV plasmid was 4.277×10 11 The standard plasmid was diluted 10-fold in sequence, from 4.277×10 11 The copies / μL were diluted to 4.277×10 0 copies / μL, stored at -20℃ for future use.

[0046] 1.5 Prepare 25 μL RAA reaction system

[0047] In a 200 μL PCR tube, 14.7 μL of A buffer, 1.25 μL of upstream primer, 1.25 μL of downstream primer, 1.3 μL of B buffer, 2.5 μL of template (positive recombinant plasmid pUC57-VP2) and 4 μL of sterile enzyme-free water were added to obtain a 25 μL RAA reaction system. The RAA reaction system was placed in a G100 dry thermostat (Youning Instrument Co., Ltd., Hangzhou, China) for 30 min at 38 °C. After the reaction, in order to reduce the effect of protein in the reaction product on electrophoresis, the amplification product was extracted with an equal amount of Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1), and then verified by electrophoresis on a 2% agarose gel. Finally, the electrophoresis results were observed on a GelVlew 5000Plus intelligent gel imaging system (Bolu Teng Biotechnology Co., Ltd., Guangzhou, China) and photographed and recorded.

[0048] 1.6 Establishment of a two-tube method based on the RAA-Cas12a system

[0049] First, 12.5 μL RAA reaction system was placed in a PCR tube for amplification reaction for 30 minutes. After the amplification reaction, it was transferred to a PCR tube containing 27.5 μL CRISPR / Cas12a system and fully mixed, followed by real-time fluorescence detection. Among them, the 27.5 μL CRISPR / Cas12a system includes 50nM Cas12a, 150nM crRNA02, 200nM ssDNA fluorescent reporter probe, 1×Reaction Buffer 1, and sterile enzyme-free water. In addition, fluorescence collection was carried out in the SLAN-96S fully automatic medical PCR analysis system (Hongshi Medical Technology Co., Ltd., Shanghai, China), incubated at 37°C for 30 minutes, and fluorescence was collected every 1 minute. After the fluorescence collection was completed, the fluorescence effect was observed under blue light irradiation and photographed and recorded.

[0050] 1.7 Establishment of a one-tube method based on the RAA-Cas12a system

[0051] First, add 27.5μL of CRISPR / Cas12a system to the PCR tube cap. Next, add 12.5μL of RAA reaction system to the bottom of the tube, and then send it to the fluorescence monitor for amplification reaction for 30 minutes. After the amplification reaction, the mixture in the tube is fully mixed by centrifugation, vortexing, oscillation and other operations, and then immediately perform real-time fluorescence detection. The 27.5μL CRISPR / Cas12a system includes 50nM Cas12a, 150nM crRNA02, 200nM ssDNA fluorescent reporter probe, 1×Reaction Buffer 1, and sterile enzyme-free water. Similarly, incubate at 37°C for 30 minutes in the SLAN-96S PCR instrument, and collect fluorescence every 1 minute. After the fluorescence collection is completed, observe the fluorescence effect under blue light irradiation and take pictures to record.

[0052] 1.8 Evaluation of specificity and sensitivity of the RAA-Cas12a system

[0053] In order to evaluate the specificity of the one-tube and two-tube methods of the RAA-Cas12a system, 10 ng / uL of FPV, FCV, FCoV, FHV-Ⅰ, and FIV plasmid standards and Mix (samples of the above plasmid standards mixed in equal proportions) were used for detection, and sterile enzyme-free water was used as a blank control. Then, different concentrations of FPV standards were used to evaluate the sensitivity, with a concentration of 4.277×10 9 4.277×10 7 4.277×10 5 4.277×10 3 4.277×10 1 4.277×10 0copies / μL, and sterile enzyme-free water was used as a blank control. Each series of samples was performed in triplicate for three independent replicates.

[0054] 1.9 Clinical sample testing

[0055] FPV clinical samples were extracted by DNA extraction kit and then tested. Specifically, 200 μL of pre-treated sample, 20 μL of Proteinase K, 250 μL of lysis solution RLC and 250 μL of isopropanol were added to the centrifuge tube, vortexed and mixed, incubated at 56 ° C for 5 minutes and cooled to room temperature. Then transferred to the adsorption column and centrifuged at 12000 rpm for 1 minute, and the waste liquid in the collection tube was discarded. Then 700 μL of rinse solution PWT was added and centrifuged at 12000 rpm for 1 minute, the waste liquid in the collection tube was discarded, and repeated once. The adsorption column was centrifuged at 12000 rpm for 2 minutes and the waste liquid was discarded. The cover was opened at room temperature and placed in a new centrifuge tube for 1 minute, and then 100 μL of elution solution was added and the cover was covered and placed at room temperature for 2 minutes. Finally, the viral DNA obtained by centrifuging the adsorption column at 12000 rpm for 1 minute was used as a template for amplification and stored at -20 ° C for use.

[0056] Eighteen blood samples and 18 stool samples were tested using the two-tube method and one-tube method of the RAA-Cas12a system, and no-template control and positive control were set. The samples were operated in the SLAN-96S PCR instrument according to the one-tube method and two-tube method procedures, and the end-point fluorescence was observed on the blue light instrument and gel imaging system. At the same time, the SYBR GreenⅠ chimeric fluorescence method was used to perform qPCR detection on these clinical samples to verify the accuracy of the two methods of the RAA-Cas12a system. In brief, 10μL 2×Taq Pro Universal SYBR qPCR Master Mix, 0.4μL of 10μM upstream and downstream primers, 1.5μL sample, and 7.7μL sterile enzyme-free water were added to a 200μL PCR tube, for a total of 20μL qPCR reaction mixture. The reaction mixture was subjected to qPCR detection on a SLAN-96S instrument using a three-step qPCR protocol: first, pre-denaturation at 95°C for 30 s; then, denaturation at 95°C for 10 s, annealing at 54°C for 30 s, and extension at 72°C for 30 s for 40 cycles; finally, final extension at 72°C for 10 min.

[0057] 1.10 Statistical analysis

[0058] GraphPad Prism 9.0 was used to generate graphs. All data are expressed as SD ± mean from three independent experiments. Statistical analysis was performed using SPSS statistics (SPSS Inc., Chicago, IL, USA). Unpaired t-test was used to compare data between two groups, and Tukey test was used to compare data between multiple groups after one-way analysis of variance. The criteria for statistical significance were *p < 0.05, **p < 0.01, ***p < 0.001.

[0059] 1.11 Results

[0060] 1.11.1 Establishment and Optimization of RAA System

[0061] Two pairs of primers were used to amplify RAA, and 2% agarose gel electrophoresis showed that ( Figure 1 A), primer pairs F1 / R1 and F2 / R2 showed target bands at around 194 bp and 159 bp respectively, and the primer pair F1 / R1 had a brighter band, so primer pair F1 / R1 was selected for subsequent experiments. At the same time, the optimized primer concentration of F1 / R1 was 500 nM ( Figure 1 B). The optimized RAA system reaction temperature is 38°C and the reaction time is 30 min ( Figure 1 CD).

[0062] 1.11.2 Establishment and optimization of CRISPR / Cas12a system

[0063] Two crRNA sequences (crRNA01 and crRNA02) were designed and synthesized (Table 1), with the expression of 4.277×10 10 4.277×10 9 4.277×10 8 The effectiveness of fluorescence visualization was verified at a concentration of 100 copies / uL. Both crRNAs had good visualization fluorescence effects ( Figure 2 A). Next, we set up three groups of crRNAs, including crRNA01, crRNA02, and crRNA03 (mixed by crRNA01 and crRNA02 in equal proportions) to screen for crRNAs with better performance. 9 The real-time fluorescence acquisition was performed at a concentration of 100 copies / uL plasmid, and the PC group and NTC group could be clearly identified under the irradiation of the blue light instrument ( Figure 2 B), among which crRNA02 has a better fluorescence detection effect ( Figure 2 C), and compared the fluorescence intensity at 30 min of fluorescence acquisition ( Figure 5 ).

[0064] In order to improve the efficiency of the CRISPR / Cas12a system, this study also optimized the temperature, crRNA02 concentration, Cas12a concentration and ssDNA concentration of the CRISPR / Cas12a system. Real-time fluorescence acquisition was performed under different temperature reactions ( Figure 6 ), and showed good fluorescence intensity at 37°C ( Figure 2 D), PC and NTC groups can be clearly identified under blue light instrument irradiation ( Figure 7 ). The orthogonal design method was used to optimize the concentrations of crRNA02 and Cas12a (Table 2), and real-time fluorescence acquisition was performed on 9 groups of complexes ( Figure 8 ), the optimized concentrations of crRNA02 and Cas12a were 150nM and 50nM, respectively ( Figure 2 E), and the visualized fluorescence results after the reaction of the 9 groups of complexes are shown in Figure F. Finally, the experiment found that ssDNA had similar real-time fluorescence change curves when the final concentration was 200nM and 250nM ( Figure 2 G) and stronger fluorescence intensity ( Figure 2 H) and visualized fluorescence results ( Figure 2 I), considering the principle of reducing costs and increasing efficiency, 200 nM ssDNA was selected for subsequent experiments.

[0065] Table 2 Concentrations of crRNA02 and Cas12a incorporated into 9 different complexes

[0066]

[0067] 1.11.3 Specificity and sensitivity evaluation of RAA-Cas12a system

[0068] The specificity of the two-tube method and the one-tube method was evaluated. The real-time fluorescence curves and the fluorescence intensity at 30 min of the two methods can accurately detect FPV ( Figure 3 AB, DE), and at the same time, significant visual fluorescence results were shown under the blue light instrument ( Figure 3 C, F), and no cross-reaction with other common cat infectious viruses. To evaluate the sensitivity of the two-tube and one-tube methods of the RAA-Cas12a system, a concentration of 4.277×10 9 4.277×10 7 4.277×10 5 4.277×10 3 4.277×10 1 4.277×10 0 The plasmid dilution with 100 copies / uL was used as a template for real-time fluorescence acquisition and fluorescence intensity comparison after 30 minutes of reaction ( Figure 3GI), the minimum detection limit of the two-tube method and the one-tube method reached 4.277 copies / uL, and showed a clear visual fluorescence effect ( Figure 3 J).

[0069] 1.11.4 Clinical sample testing

[0070] The performance of the two methods, qPCR and RAA-Cas12a system, was compared using 36 clinical samples, including 18 blood samples and 18 stool samples. In the blood samples, 5 positive samples were detected by qPCR, two-tube method and one-tube method ( Figure 4 A). In the stool samples, 6 positive samples were detected by both the qPCR method and the one-tube method, and 5 positive samples were detected by the two-tube method ( Figure 4 B). As shown in Table 3, the consistency between the two-tube method and the qPCR method of the RAA-Cas12a system was 97.22%, while the consistency of the one-tube method reached 100%. Fig. 9 AF shows the real-time fluorescence curves and visualized fluorescence results of clinical samples.

[0071] Table 3 Comparison of two-tube method, one-tube method and qPCR method

[0072]

Claims

1. A kit for visual detection of feline parvovirus based on the RAA-CRISPR / Cas12a system, characterized in that: The kit contains RAA system substances and CRISPR / Cas12a system substances; the RAA system substances include upstream and downstream primers, and the upstream and downstream primers include RAA-F1, RAA-R1, RAA-F2 and RAA-R2; the CRISPR / Cas12a system substances include Cas12a, crRNA sequence and ssDNA fluorescent reporter probe; the crRNA sequence includes crRNA01 and crRNA02, and the ssDNA fluorescent reporter probe includes qPCR-F and qPCR-R; the sequences of the upstream and downstream primers RAA-F1, RAA-R1, RAA-F2 and RAA-R2 are shown in SEQ ID NO.1 to SEQ ID NO.4, respectively; the sequence of the crRNA01 is shown in SEQ ID NO.5, and the sequence of the crRNA02 is shown in SEQ ID NO.6; the qPCR-F sequence is shown in SEQ ID NO.7, and the qPCR-R sequence is shown in SEQ ID NO.

8.

2. The kit according to claim 1, characterized in that The upstream and downstream primers are RAA-F1 and RAA-R1; the crRNA sequence is crRNA02.

3. The kit according to claim 2, characterized in that The concentrations of the RAA-F1 and RAA-R1 are both 200-600 nM, the concentration of the crRNA02 is 50-150 nM, the concentration of the Cas12a is 50-150 nM, and the concentration of the ssDNA fluorescent reporter probe is 100-250 nM.

4. The kit according to claim 3, characterized in that The concentrations of the RAA-F1 and RAA-R1 are both 500 nM, the concentration of the crRNA02 is 150 nM, the concentration of the Cas12a is 50 nM, and the concentration of the ssDNA fluorescent reporter probe is 200 nM.

5. The kit according to claim 1, characterized in that The kit also contains lyophilized powder, buffer solution and sterile enzyme-free water.

6. The kit according to claim 5, characterized in that The buffers are A buffer, B buffer, and 1×Reaction Buffer 1.

7. A method for visually detecting feline parvovirus based on the kit according to claim 5 or 6, characterized in that: The method is a two-tube method, comprising the following steps: (1) Prepare 25 μL RAA reaction system: add 14.7 μL of A buffer that has been completely melted and mixed with lyophilized powder, 1.25 μL of upstream primer, 1.25 μL of downstream primer, 1.3 μL of B buffer, 2.5 μL of the sample to be tested, and 4 μL of sterile enzyme-free water into a 200 μL PCR tube to obtain 25 μL RAA reaction system; (2) Take 12.5 μL of RAA reaction system and place it in a PCR tube for amplification reaction at 36-40 °C for 20-40 min to obtain the amplification product; after the reaction is completed, the RAA amplification product is obtained; (3) Transfer the RAA amplification product into a PCR tube containing 27.5 μL of CRISPR / Cas12a system material and mix thoroughly, then immediately perform real-time fluorescence detection.

8. A method for visually detecting feline parvovirus based on the kit according to claim 5 or 6, characterized in that: The method is a one-tube method, comprising the following steps: (1) Prepare 25 μL RAA reaction system: add 14.7 μL of A buffer that has been completely melted and mixed with lyophilized powder, 1.25 μL of upstream primer, 1.25 μL of downstream primer, 1.3 μL of B buffer, 2.5 μL of the sample to be tested, and 4 μL of sterile enzyme-free water into a 200 μL PCR tube to obtain 25 μL RAA reaction system; (2) Add 27.5 μL of CRISPR / Cas12a system material to the PCR tube cap, then add 12.5 μL of RAA reaction system to the bottom of the PCR tube, and then perform amplification reaction at 20-60°C in a fluorescence monitor for 20-40 minutes. After the amplification reaction is completed, mix the mixture in the PCR tube thoroughly, and then immediately perform real-time fluorescence detection.

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