Rapid potato Y virus detection method based on RPA-CRISPR / Cas12a-LFA

Abstract

The invention discloses a potato Y virus rapid detection method based on RPA-CRISPR / Cas12a-LFA, the RPA-CRISPR / Cas12a-LFA triple technology is adopted, the RPA-CRISPR / Cas12a-LFA triple technology comprises RPA isothermal amplification, Cas12a targeted cutting and LFA visual reading, the potato Y virus can be rapidly and efficiently detected, the sensitivity is high, the specificity is high, and the operation is simple and convenient.

Description

Technical Field

[0001] This invention belongs to the field of plant virus detection technology, and relates to a rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA. Background Technology

[0002] Potato virus Y (PVY) is also known as tobacco vein spot disease or tobacco vein spot virus disease. It has a wide host range, infecting various Solanaceae plants and causing severe mosaic or necrotic spots and streaks on potatoes. The virus particles are linear, measuring 11 nm × 680–900 nm, with an inactivation temperature of 52–62 °C, a dilution limit of 100–1000 times, and a survival time of 2–3 days in vitro.

[0003] Existing detection methods include multiplex PCR, traditional RPA, and CRISPR / Cas12a detection. Multiplex PCR requires a thermal cycler and gel electrophoresis equipment, is time-consuming, has limited sensitivity, and requires specialized laboratory support. While traditional RPA can amplify at isothermal temperatures (37-42℃), it relies on electrophoresis or fluorescence spectrometry for result interpretation, making it prone to false positives due to nonspecific amplification. CRISPR / Cas12a detection requires a pre-amplification step and largely depends on fluorescence spectrometry, making it difficult to apply on-site.

[0004] Currently, there is no "one-tube" PVY detection system that integrates RPA isothermal amplification, CRISPR / Cas12a high-specificity recognition, and LFA visual readout. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA. By using the RPA-CRISPR / Cas12a-LFA triple technology, potato virus Y can be detected rapidly and efficiently with high sensitivity, high specificity and simple operation.

[0006] The technical solution of the present invention: A rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA employs a triple technology of RPA-CRISPR / Cas12a-LFA, including RPA isothermal amplification, Cas12a targeted cleavage, and LFA visual readout. The method specifically includes the following steps: (1) Total RNA extraction and cDNA preparation: Take the potato sample tissue to be tested, extract total RNA using an RNA extraction kit, and obtain the sample cDNA template using a reverse transcription kit; (2) RPA isothermal amplification: Take the sample cDNA template, RPA amplification upstream primer, RPA amplification downstream primer and RPA premix solution, and perform isothermal amplification reaction to obtain RPA amplification product; The nucleotide sequence of the upstream primer for RPA amplification is: SEQ ID NO: 5: 5'-TCATGGCACATTTCTCAGATGTTGCAGAAGCG-3'; The nucleotide sequence of the downstream primer for RPA amplification is: SEQ ID NO: 6; 5'-GTGTGCCTCTCTGTGTTCTCCTCTTGTGTACT-3'; (3) CRISPR / Cas12a reaction: Take NE Buffer r2.1, 1µmol / L Lba Cas12a enzyme, 1µmol / L gRNA, ddH2O, 2µmol / L probe and RPA amplification product, mix them evenly, and react at a constant temperature to obtain CRISPR / Cas12a reaction solution. The nucleotide sequence of the gRNA is: SEQ ID NO: 9: 5'-UAAUUUCUACUAAGUGUAGAUAAUGUGUGCCUCUCUAGCCCUUA-3'; The nucleotide sequence of the probe is: SEQ ID NO:12: 5'-FAM-TTTTTTTATTTTTTT-Biotin-3'; (4) LFA detection: Take CRISPR / Cas12a reaction solution, add diluent, mix well, and drop onto the sample application area of ​​nucleic acid test strip. Read the result in 5-10 minutes.

[0007] In step (1) above, the amount of potato sample tissue taken is 0.05-0.15g, and the sampling site of the potato sample tissue is potato leaves, tubers or roots.

[0008] In step (2) above, the amount of sample cDNA template used is 6 μL, the amount of upstream primer for RPA amplification is 1 μL, the amount of downstream primer for RPA amplification is 1 μL, and the RPA premix consists of 10 μL of Rehydration Buffer and 2 μL of Starter.

[0009] In step (2) above, the isothermal amplification reaction conditions are 40-42℃ for 58-62 min.

[0010] Specifically, in step (2) above, the isothermal amplification reaction conditions are 41℃ for 60 min.

[0011] In step (2) above, the initial concentration of the upstream primer and the downstream primer for RPA amplification is 10 µmol / L.

[0012] In step (3) above, the CRISPR / Cas12a reaction is as follows: Take 3 μL of NE Buffer r2.1, 0.9 μL of 1 µmol / L LbaCas12a enzyme, 1 μL of 1 µmol / L gRNA, 19.4 μL of ddH2O, 0.6 μL of 2 µmol / L probe and 5 μL of RPA amplification product, mix them evenly, and react at a constant temperature of 36-38℃ for 29-31 min to obtain the CRISPR / Cas12a reaction solution.

[0013] Specifically, in the aforementioned step (3), the isothermal reaction condition is a constant temperature reaction at 37°C for 30 minutes; In step (4) above, the volume ratio of CRISPR / Cas12a reaction solution to diluent is 0.8-1.2:58-60.

[0014] In step (4) above, the volume ratio of CRISPR / Cas12a reaction solution to diluent is 1:59.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to integrate RPA, CRISPR / Cas12a and LFA into a potato virus Y detection system, breaking through the limitations of existing technologies that rely on equipment; 2. Fast and efficient: The entire process takes ≤1.5 hours; 3. High sensitivity: Detection limit down to 6.477 copies / μL; 4. Easy to operate: No thermal cycler, electrophoresis apparatus or fluorescence instrument is required, and LFA results are visible to the naked eye; 5. High specificity: Cas12a's gRNA guides targeted cleavage, avoiding cross-reactions (such as with similar viruses like PVX and PVS).

[0016] 6. Practicality: This method is easy to operate, highly accurate, and suitable for promotion at the grassroots level. Attached Figure Description

[0017] Figure 1 : Testing flowchart; Figure 2 : Gel images of amplification products of two sets of PVY RPA primers (M is DL2000 DNA marker, 1 is PVY-RPA-1 isothermal amplification product, 2 is PVY-RPA-2 isothermal amplification product). Figure 3RPA reaction results at different times (lanes 1-3: products amplified for 20 min, lanes 4-6: products amplified for 15 min, lane M: DL2000 DNA marker, lanes 7-8: products amplified for 25 min, lanes 10-12: products amplified for 30 min). Figure 4 RPA amplification products with different primer concentrations (M is the DL2000 DNA marker, lanes 1-7 represent RPA amplification products with final primer concentrations of 0.04 µmol / L, 0.08 µmol / L, 0.12 µmol / L, 0.16 µmol / L, 0.20 µmol / L, 0.24 µmol / L, and 0.28 µmol / L, respectively): Figure 5 Effects of different gRNAs on the fluorescence intensity of the CRISPR / cas12a reaction; Figure 6 Effect of gRNA concentration on fluorescence intensity of CRISPR / cas12a reaction (figure captions represent different initial concentrations, in µmol / L). Figure 7 Effect of Cas12a enzyme concentration on fluorescence intensity of CRISPR / Cas12a reaction (figure captions represent different initial concentrations, in µmol / L).

[0018] Figure 8 Effect of probe concentration on fluorescence intensity of CRISPR / Cas12a reaction (figure captions represent different initial concentrations, in µmol / L). Figure 9 The effect of the ratio of RPA amplification product to probe content on CRISPR / Cas12a cleavage efficiency; Figure 10 The effect of the ratio of RPA amplification product to probe content on CRISPR / Cas12a cleavage efficiency (0 represents negative control, B1~B8 represent groups with different product and probe contents; the two images above and below represent the test results of the test strips when the CRISPR reaction time is different; on the same test strip, the two red lines from top to bottom represent the control line and the test line respectively, two lines represent the negative sample, and only the control line represents the positive sample). Figure 11 Gel images of plasmid concentration gradient RPA products (in the left gel image, M is the DL2000 marker, lane 0 is the negative control, and A1-A8 are RPA products of different plasmid concentrations; in the middle gel image, M is the DL2000 marker, and A5-A8 are the corresponding group of plasmid RPA products; in the right gel image, M is the DL2000 marker, and A7 and A8 are the corresponding group of plasmid RPA products). Figure 12 Effect of low-concentration plasmid (A7, A8) RPA product to probe ratio on CRISPR / Cas12a (0-1: blank control with product:probe (v / v) ratio of 3:1; A7-1: experimental group of A7 with product:probe (v / v) ratio of 3:1; A8-1: experimental group of A8 with product:probe (v / v) ratio of 3:1; 0-2: blank control with product:probe (v / v) ratio of 5:0.8). A7-2 is the experimental group of A7 with a product:probe (v / v) ratio of 5:0.8; A8-2 is the experimental group of A8 with a product:probe (v / v) ratio of 5:0.8; 0-3 is the blank control with a product:probe (v / v) ratio of 5:0.6; A7-3 is the experimental group of A7 with a product:probe (v / v) ratio of 5:0.6; A8-3 is the experimental group of A8 with a product:probe (v / v) ratio of 5:0.6. Figure 13 : Gel images of RPA products with optimized plasmid concentration gradients and test strip detection results (in the left gel image, M is the DL2000 marker, and A1~A8 are RPA products of different plasmid concentrations; in the right gel image, test strip 0 is the negative control, and test strips of different plasmid concentrations A1~A8 are detection results). Figure 14 : RPA-CRISPR / Cas12a-LFA system sample detection results (M in the gel image is DL2000 DNA marker, and the corresponding test strip (the first test strip) is the negative control; lanes 1-20 are potato samples, and the corresponding test strips are the detection results of the Y virus RPA-CRISPR / Cas12a-LFA detection technology system in potato samples). Detailed Implementation

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0020] Example 1: Rapid Detection Method for Potato Virus Y (1) Total RNA extraction and cDNA preparation: Take 0.1g of potato leaf tissue to be tested, extract total RNA using an RNA extraction kit, and obtain sample cDNA template using a reverse transcription kit; (2) RPA isothermal amplification: Take 6 μL of sample cDNA template, 1 μL of RPA amplification upstream primer, 1 μL of RPA amplification downstream primer and 12 μL of RPA premix (RPA premix consists of 10 μL of Rehydration Buffer and 2 μL of Starter), and amplify at 41℃ for 60 min to obtain RPA amplification product; The nucleotide sequence of the upstream primer for RPA amplification is: SEQ ID NO: 5: 5'-TCATGGCACATTTCTCAGATGTTGCAGAAGCG-3'; The nucleotide sequence of the downstream primer for RPA amplification is: SEQ ID NO: 6; 5'-GTGTGCCTCTCTGTGTTCTCCTCTTGTGTACT-3'; (3) CRISPR / Cas12a reaction: Take 3 μL of NE Buffer r2.1, 0.9 μL of 1 µmol / L Lba Cas12a enzyme, 1 μL of 1 µmol / L gRNA, 19.4 μL of ddH2O, 0.6 μL of 2 µmol / L probe and 5 μL of RPA amplification product, mix them evenly, and react at 37℃ for 30 min to obtain CRISPR / Cas12a reaction solution; The nucleotide sequence of the gRNA is: SEQ ID NO: 9: 5'-UAAUUUCUACUAAGUGUAGAUAAUGUGUGCCUCUCUAGCCCUUA-3'; The nucleotide sequence of the probe is: SEQ ID NO:12: 5'-FAM-TTTTTTTATTTTTTT-Biotin-3'; (4) LFA detection: Take 1 μL of CRISPR / Cas12a reaction solution and add it to 59 μL of diluent. Mix well and drop it onto the sample application area of ​​the nucleic acid test strip. Read the result in 5-10 minutes.

[0021] Example 2: Rapid Detection Method for Potato Virus Y (1) Total RNA extraction and cDNA preparation: Take 0.15g of potato tuber tissue to be tested, extract total RNA using an RNA extraction kit, and obtain sample cDNA template using a reverse transcription kit; (2) RPA isothermal amplification: Take 6 μL of sample cDNA template, 1 μL of RPA amplification upstream primer, 1 μL of RPA amplification downstream primer and 12 μL of RPA premix (RPA premix consists of 10 μL of Rehydration Buffer and 2 μL of Starter), and amplify at 40℃ for 58 min to obtain RPA amplification product; The nucleotide sequence of the upstream primer for RPA amplification is: SEQ ID NO: 5: 5'-TCATGGCACATTTCTCAGATGTTGCAGAAGCG-3'; The nucleotide sequence of the downstream primer for RPA amplification is: SEQ ID NO: 6; 5'-GTGTGCCTCTCTGTGTTCTCCTCTTGTGTACT-3'; (3) CRISPR / Cas12a reaction: Take 3 μL of NE Buffer r2.1, 0.9 μL of 1 µmol / L Lba Cas12a enzyme, 1 μL of 1 µmol / L gRNA, 19.4 μL of ddH2O, 0.6 μL of 2 µmol / L probe and 5 μL of RPA amplification product, mix them evenly, and react at 36℃ for 29 min to obtain CRISPR / Cas12a reaction solution; The nucleotide sequence of the gRNA is: SEQ ID NO: 9: 5'-UAAUUUCUACUAAGUGUAGAUAAUGUGUGCCUCUCUAGCCCUUA-3'; The nucleotide sequence of the probe is: SEQ ID NO:12: 5'-FAM-TTTTTTTATTTTTTT-Biotin-3'; (4) LFA detection: Take 1.2 μL of CRISPR / Cas12a reaction solution and add 60 μL of diluent. Mix well and drop onto the sample application area of ​​the nucleic acid test strip. Read the result in 5-10 minutes.

[0022] Example 3: Rapid Detection Method for Potato Virus Y (1) Total RNA extraction and cDNA preparation: Take 0.05g of potato root tissue to be tested, extract total RNA using an RNA extraction kit, and obtain sample cDNA template using a reverse transcription kit; (2) RPA isothermal amplification: Take 6 μL of sample cDNA template, 1 μL of RPA amplification upstream primer, 1 μL of RPA amplification downstream primer and 12 μL of RPA premix (RPA premix consists of 10 μL of Rehydration Buffer and 2 μL of Starter), and amplify at 42℃ for 62 min to obtain RPA amplification product; The nucleotide sequence of the upstream primer for RPA amplification is: SEQ ID NO: 5: 5'-TCATGGCACATTTCTCAGATGTTGCAGAAGCG-3'; The nucleotide sequence of the downstream primer for RPA amplification is: SEQ ID NO: 6: 5'-GTGTGCCTCTCTGTGTTCTCCTCTTGTGTACT-3'; (3) CRISPR / Cas12a reaction: Take 3 μL of NE Buffer r2.1, 0.9 μL of 1 µmol / L Lba Cas12a enzyme, 1 μL of 1 µmol / L gRNA, 19.4 μL of ddH2O, 0.6 μL of 2 µmol / L probe and 5 μL of RPA amplification product, mix them evenly, and react at 38℃ for 31 min to obtain CRISPR / Cas12a reaction solution; The nucleotide sequence of the gRNA is: SEQ ID NO: 9: 5'-UAAUUUCUACUAAGUGUAGAUAAUGUGUGCCUCUCUAGCCCUUA-3'; The nucleotide sequence of the probe is: SEQ ID NO:12: 5'-FAM-TTTTTTTATTTTTTT-Biotin-3'; (4) LFA detection: Take 0.8 μL of CRISPR / Cas12a reaction solution and add 58 μL of diluent. Mix well and drop onto the sample application area of ​​the nucleic acid test strip. Read the result in 5-10 minutes.

[0023] This invention conducted corresponding experimental studies to verify the rapid detection method for Potato Virus Y. The results of this experimental study are as follows: 1. Materials and Methods 1.1 Materials and Reagents Potato tissues: Potato leaves, tubers, roots, and other tissues that need to be tested for Potato Y virus.

[0024] RNA extraction kit (FastPuerto University Plant Total RNA Isolation Kit), reverse transcription kit (HiScript Ⅲ 1st Stand cDNA Synthesis Kit), Green Taq Mix, DL2000 DNAmarker, FastPure Gel DNA Extraction Mini Kit, and FastPure Plasmid Mini Kit-BOX 2 were purchased from Nanjing Novizan Biotechnology Co., Ltd. RPA premix (containing 10 μL of Rehydration Buffer and 2 μL of Starter, purchased from Shenzhen Yizhi Biotechnology Co., Ltd.); DNA isothermal amplification (basic type) and nucleic acid test strips - line-strip erasure method (for CRISPR SHERLOCK) were both purchased from Shenzhen Yizhi Biotechnology Co., Ltd., with diluent provided with the nucleic acid test strips; NE Buffer r2.1 and Lba Cas12a (Cpf1) were purchased from New England Biotechnology (Beijing) Co., Ltd.; DH5α competent cells were purchased from Beijing Coolerbot Technology Co., Ltd. The nucleic acid primer sequences, gRNA nucleic acid sequences, and probes were all synthesized by Qingke Biotechnology Co., Ltd.

[0025] 1.2 Multiplex PCR method for detecting potato virus The potato PVY virus was detected by multiplex RT-PCR. The primer sequences used are shown in Table 1, and all were synthesized by Qingke Biotechnology Co., Ltd.

[0026] Table 1 Primer sequences for multiplex PCR detection of potato virus 1.3 Preparation of positive plasmids for PVY virus RPA products Potato nucleic acid samples that tested positive for PVY virus in multiplex PCR were used as templates for RPA amplification of PVY virus. The RPA amplification primers used are shown in Table 2 and were synthesized by Qingke Biotechnology Co., Ltd.

[0027] Table 2 PVY virus RPA sequence Positive RPA amplification products were recovered and purified, ligated into T vectors, and transformed into competent DH5α *E. coli*. Positive clones were screened using LB medium supplemented with ampicillin at a final concentration of 50 µg / mL. Three positive single colonies were picked and subjected to colony RPA amplification for preliminary verification. Plasmids from the positive single colonies were extracted and sequenced by Qingke Biotechnology Co., Ltd. The sequences were assembled using DNAMEN and compared with the NCBI database to confirm the positive colonies. Plasmids from the positive bacteria were extracted using a plasmid extraction kit for later use.

[0028] 1.4 Establishment of RPA-CRISPR / Cas12a-LFA detection technology 1.4.1 gRNA Design, Synthesis, and Activity Screening Based on the PVY core sequence in the positive plasmid of the PVY virus RPA product, a Cas12a enzyme-recognizable gRNA was designed using the CRISPR website (http: / / crispor.tefor.net / ) and synthesized by Qingke Biotechnology Co., Ltd. Details of the gRNA sequence used are shown in Table 3.

[0029] Table 3 PVY virus gRNA sequence Note: The bolded portion of the sequence represents the scaffold sequence of the gRNA.

[0030] Two probe sequences were used (Table 4), both synthesized by Qingke Biotechnology Co., Ltd.

[0031] Table 4 Two probe sequences 1.4.2 Screening of RPA amplification conditions The RPA amplification system followed the reagent instructions. The RPA reaction time gradients were 15 min, 20 min, 25 min, and 30 min; the final primer concentrations were 0.04 µmol / L, 0.08 µmol / L, 0.12 µmol / L, 0.16 µmol / L, 0.20 µmol / L, 0.24 µmol / L, and 0.28 µmol / L, respectively. All other conditions remained unchanged.

[0032] 1.4.3 CRISPR / Cas12a Conditional Screening The initial conditions for the Cas12a reaction are as per the reagent instructions. The initial concentration gradients (total volume 30 µL) for the Cas12a enzyme concentration screening assay are: 0.6 µmol / L, 0.7 µmol / L, 0.8 µmol / L, 0.9 µmol / L, 1.0 µmol / L, 1.1 µmol / L, 1.2 µmol / L, 1.3 µmol / L, 1.4 µmol / L, and 1.5 µmol / L. The initial concentration gradients (total volume 30 µL) for the gRNA concentration screening assay are: 0.6 µmol / L, 0.8 µmol / L, 1.0 µmol / L, 1.2 µmol / L, 1.4 µmol / L, and 1.6 µmol / L. The initial concentration gradients (total system size 30µL) in the probe concentration screening experiment were: 0.8µmol / L, 1.2µmol / L, 1.6µmol / L, 2.0µmol / L, 2.4µmol / L, 2.8µmol / L, and 3.2µmol / L.

[0033] The corresponding groups and screening conditions in the screening study of RPA amplification products and probe content ratio of PVY virus positive plasmids are shown in Tables 5 and 6.

[0034] Table 5 Screening based on the ratio of RPA amplification products to fluorescent probes Table 6 Screening of RPA amplification product to test strip probe content ratio 1.5 Data Analysis The data was analyzed using Excel software.

[0035] 2 Results and Analysis 2.2 Preliminary establishment of RPA-CRISPR / Cas12a-LFA detection system 2.2.1 RPA Primer Screening See Figure 2Two sets of RPA primers, PVY-RPA-1 and PVY-RPA-2, were used for isothermal amplification. Both primers showed bright bands near the target product. After purification, ligation, and transformation, the positive plasmid sequences were determined, and sequence alignment with the NCBI database confirmed that both primers had good amplification effects. Primer PVY-RPA-2 showed higher amplification specificity, and the longer amplification product was beneficial for subsequent gRNA site design. Therefore, primer PVY-RPA-2 and its amplification product were selected for subsequent research. In this study, the positive plasmid refers to the plasmid carrying the amplification product of primer PVY-RPA-2.

[0036] 2.2.2 Optimization of RPA reaction time and primer dosage When the required components are sufficient, the amount of RPA amplification product gradually increases with the extension of amplification time. Figure 3 A significant amount of amplification products can be obtained within 15 minutes. The positive plasmid concentration used in this study was relatively high. Compared to actual field virus detection, the amount of template in the virus sample may be significantly reduced. Sufficient viral nucleic acid copy number is a crucial factor affecting the Cas12a enzyme cleavage efficiency in the subsequent RPA-CRISPR / Cas12a-LFA detection system. Therefore, further adjustments will be needed in future studies based on the viral content in the sample.

[0037] The amount of primers used is also one of the factors affecting RPA amplification efficiency. The primer concentration gradient experiment results show that ( Figure 4 Under otherwise unchanged conditions, the amount of product generally increases with increasing primer content. When the final concentration reaches 0.20 µmol / L, the product amount remains basically stable, and then the primer dimer will gradually increase with increasing primer content. Therefore, 0.20 µmol / L was selected as the optimal final concentration of RPA primers for subsequent studies.

[0038] 2.2.3 CRISPR / Cas12a gRNA Screening The intensity of the fluorescence signal from the Cas12a enzyme cleavage probe can indirectly reflect the activity of the gRNA. The results of CRISPR mediated by four different gRNA sequences show that ( Figure 5Under the same conditions, gRNA3 mediated the highest enzyme cleavage activity, followed by gRNA4, with gRNA1 exhibiting the worst (sequence information is shown in Table 4). The difference between gRNA1 and gRNA3 is that the scaffold sequence lacks a uracil base, yet their activities differ significantly, indicating that the scaffold sequence of gRNA3 is more suitable for the Cas12a (Cpf1) enzyme. The difference between gRNA3 and gRNA4 is their guide sequences, suggesting that the guide sequence of gRNA3 is more suitable. In the RPA-CRISPR / Cas12a-LFA detection technology, stronger Cas12a enzyme cleavage activity is needed for more sensitive capture of the cleavage product; therefore, the most active gRNA, gRNA3, was selected for subsequent use.

[0039] 2.2.4 CRISPR / Cas12a enzyme and gRNA concentration screening The concentrations of Cas12a enzyme and gRNA are key factors affecting the CRISPR / Cas12a cleavage efficiency. The effects of different concentrations of Cas12a enzyme and gRNA on the CRISPR / Cas12a cleavage efficiency in the system were observed by recording changes in fluorescence intensity during the reaction using real-time quantitative PCR. Figure 6 , Figure 7 The results showed that when the initial concentration of Cas12a enzyme was fixed at 1 µmol / L, the fluorescence intensity was highest when the initial concentration of gRNA was also 1 µmol / L, indicating that the ratio of Cas12a enzyme to gRNA was optimal. However, when the initial concentration of gRNA was fixed at 1 µmol / L, the fluorescence intensity was highest when the initial concentration of Cas12a enzyme was 0.9 µmol / L. When gRNA was in excess, the excess free gRNA might form dimers through base pairing, thus reducing the efficiency of random probe cleavage during CRISPR / Cas12a cleavage of the target product. Conversely, when Cas12a was in excess, the excess free Cas12a enzyme might competitively bind to gRNA, leading to a decrease in the proportion of effective enzyme in the reaction system, thereby reducing the efficiency of CRISPR / Cas12a cleavage. In the CRISPR / Cas12a system, the optimal ratio of Cas12a enzyme to gRNA is 9:10. A ratio lower or higher than this will lead to a decrease in cleavage efficiency. Therefore, in the subsequent RPA-CRISPR / Cas12a-LFA detection system, a Cas12a enzyme to gRNA ratio of 9:10 was selected for further research.

[0040] 2.2.5 Effect of probe concentration on fluorescence intensity of CRISPR / Cas12a reaction When CRISPR / Cas12a cleaves the target product, it indiscriminately cleaves nearby probes. The broken probes emit fluorescent signals (fluorescent type), which are captured by the instrument, indirectly reflecting the cleavage activity of CRISPR / Cas12a. Therefore, the probe concentration in the system is also a key factor affecting the intensity of the fluorescent signal. Using different probe concentrations (initial concentrations) to conduct CRISPR / Cas12a reactions, the results showed that ( Figure 8 When the initial probe concentration is in the range of 0.8 µmol / L to 2.8 µmol / L, the fluorescence signal gradually increases with the increase of probe concentration, reaching the highest value at 2.8 µmol / L, and then decreases with the increase of probe concentration.

[0041] 2.2.6 Screening of RPA amplification product to probe content ratio in the RPA-CRISPR / Cas12a-LFA system Unlike fluorescent CRISPR / Cas12a systems, uncut probes in strip-based CRISPR / Cas12a systems can cause false negative results. Maintaining an appropriate ratio of RPA amplification products to probe content is a key parameter in the RPA-CRISPR / Cas12a-LFA detection system. Therefore, this study first explored the changing trend of the RPA amplification product to probe ratio using a fluorescent CRISPR / Cas12a system to provide a reference for subsequent use in the strip. The results showed that ( Figure 9 Before 70 minutes of CRISPR / Cas12a reaction, treatment group F8 (3µL product + 2µL probe) showed the strongest fluorescence signal. After 70 minutes, treatment group F6 (2µL product + 3µL probe) surpassed F8, reaching its highest value. High-efficiency cutting is essential for product detection on the test strips; therefore, product:probe (v / v) ratios of 3:2 and 2:3 were selected for subsequent test strip detection.

[0042] Based on the product-probe ratio derived from the fluorescence signal, the final product of RPA-CRISPR / Cas12a was detected using test strips. The results showed that ( Figure 10(REF _Ref194585193 \h\* MERGEFORMAT ), In experimental groups B1-B4 (product-to-probe volume ratio around 2:3, see Table 7), false negatives occurred in B1 and B4 after 30 min of CRISPR digestion, indicating that probe digestion was insufficient under these conditions. However, after extending the CRISPR reaction to 60 min, B1 and B4 showed positive results, indicating that the efficiency of the CRISPR reaction within 30 min was low in these two groups, but this could be compensated for by appropriately extending the reaction time. Experimental groups B5-B8 (product-to-probe volume ratio around 3:2) were all positive at both 30 and 60 min of CRISPR reaction, indicating that the CRISPR reaction efficiency was high under these four conditions, and the probe was completely digested within 30 min. Therefore, in CRISPR reactions detected by test strips, appropriately increasing the amount of product can effectively improve the efficiency of Cas12a enzyme digestion of the probe and reduce the probability of false negatives in test strip detection.

[0043] 2.2.7 Optimization of the RPA-CRISPR / cas12a-LFA system The positive plasmids were serially diluted and used to establish the RPA-CRISPR / cas12a-LFA system. The initial concentration of the positive plasmids (A1 group) was 2.81 ng / µL. The copy numbers of the diluted positive plasmids are shown in Table 7.

[0044] Table 7. Positive plasmid dilution factor and corresponding copy number First, RPA-CRISPR / cas12a-LFA detection was performed on each group of plasmids using previously optimized conditions. The RPA amplification products of each group showed ( Figure 11 (Left gel image) Under these RPA amplification conditions, the amount of target product in groups A5-A8 was relatively small. Therefore, by increasing the amount of template (positive plasmid) in the RPA amplification program from 1µL to 6µL and extending the amplification time from 15min to 40min, the amount of RPA product in groups A5 and A6 increased. Figure 11 Intermediate gel image); while groups A7 and A8, due to their low plasmid content, yielded more amplification products when the amplification time was extended to 60 minutes. Figure 11 (Right side of the glue image).

[0045] In the RPA-CRISPR / Cas12a-LFA detection system, the probe needs to be completely cleaved by Cas12a. Only when the test strip's detection line cannot capture the uncleaved probe will only a control line (a single line) be displayed. Therefore, the appropriate product-probe ratio is a key parameter for RPA-CRISPR / Cas12a-LFA detection. Low-concentration plasmid experimental groups, due to their lower template content, are prone to false negatives caused by incomplete Cas12a cleavage of the probe. Therefore, the A7 and A8 positive plasmid groups were mainly used to study the product-probe ratio. The results showed that ( Figure 12 When the product:probe (v / v) ratio was 3:1 (Figures 0-1, A7-1, A8-1), the Cas12a enzyme could not completely cleave the probe within 30 min of fixation, therefore, experimental groups A7-1 and A8-1 both showed false negatives; when the product:probe (v / v) ratio was increased to 5:0.8 (…), Figure 12 (0-2, A7-2, A8-2), the A8-2 experimental group was positive, while A7-2 was still a false negative; when the product:probe (v / v) ratio was increased to 5:0.6 ( Figure 12 The experimental groups (0-3, A7-3, A8-3) all showed positive results, while the negative control (0-3) was negative. This indicates that when the product:probe (v / v) ratio is increased to 5:0.6, the Cas12a enzyme can completely cleave the probe within a fixed 30 min, achieving the expected result.

[0046] Therefore, based on the optimized RPA-CRISPR / Cas12a-LFA detection system, virus detection was performed on positive plasmids of different concentrations (Table 8 and...). Figure 13 ).

[0047] Table 8 Positive plasmid groups and corresponding optimization conditions Under the optimized system conditions, different plasmid concentrations could all be detected. Figure 13 High-concentration plasmid groups require shorter detection times in the RPA-CRISPR / Cas12a-LFA detection system, while low-concentration groups require longer detection times due to limitations in nucleic acid copy number. For field samples in practical applications, the viral nucleic acid copy number extracted from tissues is relatively low, which may make low-concentration plasmid group detection systems more suitable.

[0048] 2.2.8 Sensitivity and Repeatability Determination of the RPA-CRISPR / cas12a-LFA System The positive plasmid was serially diluted and used to determine the detection limit of the RPA-CRISPR / cas12a-LFA system. The detection results showed that ( Figure 13The lowest copy number of the plasmid was 6.477 copies / µL, and the RPA-CRISPR / cas12a-LFA system test result was positive, indicating that the established system has a wide detection range and can detect target DNA with low copy numbers, thus having high sensitivity.

[0049] The RPA-CRISPR / cas12a-LFA system was used to test positive plasmid samples of different concentrations three times. The test strips in the experimental group all showed positive results, while the test strips in the control group all showed negative results. The results indicate that the RPA-CRISPR / cas12a-LFA system established in this study has good reproducibility.

[0050] 2.2.9 Comparison of RPA-CRISPR / cas12a-LFA system sample detection with multiplex PCR method Simultaneously, PVY virus detection was performed on 68 potato samples using the RPA-CRISPR / Cas12a-LFA technology system (low-concentration template copy number system) and multiplex PCR. Figure 14 The detection results of the RPA-CRISPR / Cas12a-LFA technology system are basically consistent with those of the multiplex PCR method. The RPA-CRISPR / Cas12a-LFA technology system can complete the detection process in only 1.5 hours, compared to approximately 2.5 hours for multiplex PCR, significantly shortening the detection cycle and improving efficiency. Furthermore, the RPA-CRISPR / Cas12a-LFA technology system requires no complex instruments; only simple isothermal amplification equipment and test strips are needed, making it more suitable for rapid on-site testing in grassroots laboratories.

[0051] Table 9 Comparison of RPA-CRISPR / Cas12a-LFA technology system and multiplex PCR method 3. Discussion and Analysis RPA-CRISPR / Cas12a-LFA technology has high applicability in other plant and animal fields, making significant breakthroughs in virus detection and disease diagnosis. It can also be used for the prevention and monitoring of viral diseases and their occurrence. Shin K et al. used the RPA-CRISPR / Cas12a-LFA system to detect citrus scab, completing the detection within 1 hour, demonstrating its rapid detection speed and high sensitivity. It also prevents cross-reactions with other common diseases, and the system is cost-effective. Paenkaew S et al., in their research on animals using a combination of RPA and Cas12a detection methods, demonstrated strong sensitivity, even detecting as low as 100 copies of *Ehrlich aspergillus* and *Anaplasma platyphylla*, indicating high sensitivity. Therefore, this detection system offers even higher sensitivity, meeting the demand for low-cost, efficient, and rapid potato virus detection, lowering the threshold for virus detection, and providing a feasible solution for smallholder farmers or remote areas.

[0052] In addition, this system has some shortcomings in virus detection. First, it requires the design of highly active gRNA to mediate Cas12a enzyme cleavage, and the verification of gRNA activity requires high precision. Second, although the RPA-CRISPR / Cas12a-LFA system requires less time for virus detection, the preliminary steps such as plant nucleic acid extraction still require a considerable amount of time. This study did not optimize the preliminary treatment, so further optimization can be aimed at addressing this issue.

Claims

1. A rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA, characterized in that: The RPA-CRISPR / Cas12a-LFA triple technology, including RPA isothermal amplification, Cas12a targeted cleavage, and LFA visual readout, specifically includes the following steps: (1) Total RNA extraction and cDNA preparation: Take the potato sample tissue to be tested, extract total RNA with RNA extraction kit, and obtain sample cDNA template with reverse transcription kit; (2) RPA isothermal amplification: Take the sample cDNA template, RPA amplification upstream primer, RPA amplification downstream primer and RPA premix solution, and perform isothermal amplification reaction to obtain RPA amplification product; The nucleotide sequence of the upstream primer for RPA amplification is: SEQ ID NO: 5: 5'-TCATGGCACATTTCTCAGATGTTGCAGAAGCG-3'; The nucleotide sequence of the downstream primer for RPA amplification is: SEQ ID NO: 6; 5'-GTGTGCCTCTCTGTGTTCTCCTCTTGTGTACT-3'; (3) CRISPR / Cas12a reaction: Take NE Buffer r2.1, 1µmol / L Lba Cas12a enzyme, 1µmol / L gRNA, ddH2O, 2µmol / L probe and RPA amplification product, mix them evenly, and react at a constant temperature to obtain CRISPR / Cas12a reaction solution. The nucleotide sequence of the gRNA is: SEQ ID NO: 9: 5'-UAAUUUCUACUAAGUGUAGAUAAUGUGUGCCUCUCUAGCCCUUA-3'; The nucleotide sequence of the probe is: SEQ ID NO:12: 5'-FAM-TTTTTTTATTTTTTT-Biotin-3'; (4) LFA detection: Take CRISPR / Cas12a reaction solution, add diluent, mix well, and drop onto the sample application area of ​​nucleic acid test strip. Read the result in 5-10 minutes.

2. The rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA according to claim 1, characterized in that: In step (1), the amount of potato sample tissue taken is 0.05-0.15g, and the sampling site of potato sample tissue is potato leaves, tubers or roots.

3. The rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA according to claim 1, characterized in that: In step (2), the amount of sample cDNA template used is 6 μL, the amount of upstream primer for RPA amplification is 1 μL, the amount of downstream primer for RPA amplification is 1 μL, and the RPA premix consists of 10 μL of Rehydration Buffer and 2 μL of Starter.

4. The rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA according to claim 1, characterized in that: In step (2), the isothermal amplification reaction conditions are 40-42℃ for 58-62 min.

5. The rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA according to claim 4, characterized in that: In step (2), the isothermal amplification reaction conditions are 41℃ for 60 min.

6. The rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA according to claim 1, characterized in that: In step (2), the initial concentrations of the upstream primer and the downstream primer for RPA amplification are both 10 µmol / L.

7. The rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA according to claim 1, characterized in that: In step (3), the CRISPR / Cas12a reaction is as follows: Take 3 μL of NE Buffer r2.1, 0.9 μL of 1 µmol / L LbaCas12a enzyme, 1 μL of 1 µmol / L gRNA, 19.4 μL of ddH2O, 0.6 μL of 2 µmol / L probe and 5 μL of RPA amplification product, mix them evenly, and react at a constant temperature of 36-38℃ for 29-31 min to obtain the CRISPR / Cas12a reaction solution.

8. The rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA according to claim 7, characterized in that: In step (3), the isothermal reaction condition is a constant temperature reaction at 37°C for 30 minutes.

9. The rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA according to claim 1, characterized in that: In step (4), the volume ratio of CRISPR / Cas12a reaction solution to diluent is 0.8-1.2:58-60.

10. The rapid detection method for potato virus Y based on RPA-CRISPR / Cas12a-LFA according to claim 1, characterized in that: In step (4), the volume ratio of the CRISPR / Cas12a reaction solution to the diluent is 1:59.

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