Method for detecting geminivirus in tomato plant and bemisia tabaci based on aMIRA-CRISPR / Cas14a system

Abstract

The invention belongs to the field of virus detection, and particularly relates to a method for detecting geminivirus in tomato plants and bemisia tabaci based on an aMIRA-CRISPR / Cas14a system. The invention discloses a method for detecting geminivirus in a tomato plant and bemisia tabaci based on an aMIRA-CRISPR / Cas14a system. The method comprises the following steps: 1) performing DNA extraction on a tomato plant sample and the bemisia tabaci; 2) specifically amplifying target genes of geminivirus in tomato plants and bemisia tabaci through an asymmetric multi-enzyme constant-temperature rapid amplification technology to obtain an aMIRA amplification product; and 3) a CRISPR / Cas14a system is adopted, and specific sgRNA is designed to recognize single-stranded DNA in an amplification product, so that non-specific cleavage activity of Cas14a is activated, a fluorescent probe is cleaved, release of a fluorescent signal and interpretation of a detection result are realized, and accurate detection of a target virus is realized. The invention develops an AI enhanced MaC14a platform, based on a portable centrifugal microfluidic system, an aMIRA amplification technology and a CRISPR / Cas14a recognition mechanism are integrated, and onsite visual rapid and sensitive detection of geminivirus in tomato and bemisia tabaci can be realized.

Description

Technical Field

[0001] This invention belongs to the field of virus detection, specifically relating to a method for detecting geminitroviruses in tomato plants and whiteflies based on the aMIRA-CRISPR / Cas14a system. Background Technology

[0002] Geminiviridae comprised of insect-borne, single-stranded DNA viruses that cause significant damage to economically important crops such as tomatoes, with symptoms including leaf curling and stunted growth. Due to their error-prone replication, these viruses exhibit high mutation rates, leading to rapid evolution of pathogenic strains and exacerbating global agricultural challenges. Notably, viruses such as Tomato Yellow Leaf Curl Virus (TYLCCNV), Tomato Yellow Leaf Curl Virus (TYLCV), Tomato Leaf Roll Virus (ToLCNDV) in New Delhi, and Tobacco Curled Stem Virus (TbCSV) are particularly detrimental to tomato cultivation. The increasing prevalence of co-infection with these viruses results in more severe symptoms and higher economic losses. The spread of these viruses is primarily mediated by the globally invasive pest, the whitefly, which facilitates rapid regional and terrestrial transmission. Consequently, many Geminiviridae species have been designated as critical quarantine targets. For example, ToLCNDV has been identified as a major concern in China and Japan, while TYLCV and ToLCNDV are considered quarantine threats in the European Union. These designations underscore the need for robust surveillance and control strategies to mitigate the risk of cross-border transmission and potential agricultural damage.

[0003] Traditional control methods focus on measures taken after symptoms appear, such as using pesticides and removing infected plants. However, these methods often fail due to the later accumulation of the virus and insect transmission. This urgently necessitates advanced diagnostic technologies capable of: 1) predicting infection risk before crop exposure, and 2) detecting latent infections during the critical treatment window.

[0004] The CRISPR / Cas system is an acquired immune system in prokaryotes used to resist the invasion of foreign genetic elements present in bacteriophages or plasmids. It is a defense mechanism present in most bacteria and all archaea to destroy foreign plasmid or bacteriophage DNA. It is now widely used in genetic engineering. The combination of the CRISPR / Cas system with isothermal amplification technology significantly improves the efficiency of nucleic acid detection by promoting sequence-specific recognition through programmable incidental cleavage activity. Notably, the Cas12a-coupled RPA system has become a promising rapid on-site detection platform. However, a major challenge lies in the incompatibility between the CRISPR / Cas system and isothermal amplification, which hinders their binding in a single reaction vessel. Specifically, the PAM-dependent Cas12a system may degrade the DNA template and initial product, thereby reducing double-stranded DNA yield and detection sensitivity. Previous methods, such as phase separation using spatial separation or viscous additives (e.g., glycerol / sucrose), aimed to separate the amplification and detection phases. Recent innovative strategies, including photoactivated crRNA systems and heparin-mediated Cas12 inhibition, introduce dynamic regulation for precise control. Despite these advances, current single-tube platforms remain limited by complexity and time constraints, hindering their application in real-time detection for rapid vector screening and early plant diagnosis.

[0005] On the one hand, in patent CN103045753B, a real-time fluorescence quantitative PCR primer, probe, and method for detecting TYLCV (Tomato Yellow Leaf Curl Virus), a TaqMan probe is designed targeting the conserved sequence of TYLCV to achieve highly sensitive quantitative detection. However, polymerase chain reaction (PCR) and quantitative PCR detection require specialized equipment and personnel, posing a significant challenge to field applications. In patent CN102321166B, a monoclonal antibody against the TYLCV coat protein and its application, a detection method for preparing highly specific antibodies for ELISA or test strip development is provided. While ELISA-based methods are suitable for large-scale screening, their sensitivity is insufficient for detecting low-titer infections. Furthermore, neither of these patents allows for ultra-early detection or individual vector screening. On the other hand, in patent CN110484696A, a rapid detection kit for TYLCV based on RPA technology, recombinase polymerase amplification (RPA) technology enables rapid detection without the need for PCR instruments. However, this method is susceptible to nonspecific amplification, which can easily lead to false positives and affect the reliability of the diagnostic results. Summary of the Invention

[0006] To overcome the problems of unpredictable infection risk, difficulty in detecting latent infections during the window period, susceptibility to non-specific factors in the detection process, and operational difficulties, this invention designs a novel microfluidic aMIRA-Cas14a detection platform (MaC14a). This platform ingeniously combines isothermal amplification technology with the single-stranded DNA (ssDNA) specific nuclease activity of Cas14a, providing a method for detecting geminitroviruses in tomato plants and whiteflies based on the aMIRA-CRISPR / Cas14a system.

[0007] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0008] This invention provides a method for detecting geminitroviruses in tomato plants and whiteflies based on the aMIRA-CRISPR / Cas14a system:

[0009] 1) DNA was extracted from tomato plant samples and whiteflies;

[0010] 2) The specific genes of geminiviruses in tomato plants and whiteflies were amplified using MIRA-specific primers to obtain aMIRA amplification products;

[0011] 3) Using the CRISPR / Cas14a system, specific SgRNA targets and recognizes the aMIRA amplification product, activating the non-specific cleavage activity of CRISPR / Cas14a. The detection results are interpreted by a reporter probe, enabling the detection of geminiviruses in tomato plants and whiteflies.

[0012] Furthermore, the specific genes of the geminitroviruses in the tomato plants and whiteflies include the V1 gene of TYLCV, the AV1 gene of TYLCCNV, the AV1 gene of ToLCNDV, and the AV1 gene of TbCSV.

[0013] Furthermore, the aMIRA-specific primers include MIRA-specific primers for TYLCCNV, whose nucleoside sequences are shown in SEQ ID NO. 4-7; aMIRA-specific primers for TYLCV, whose nucleoside sequences are shown in SEQ ID NO. 21-24; aMIRA-specific primers for ToLCNDV, whose nucleoside sequences are shown in SEQ ID NO. 39-42; and aMIRA-specific primers for TbCSV, whose nucleoside sequences are shown in SEQ ID NO. 56-59.

[0014] To achieve sequence-specific nucleic acid amplification, this invention designed multiple primer pairs targeting conserved viral genes. For initial primer screening, total genomic DNA extracted from virus-infected tomato leaves was used. A standard MIR A reaction mixture (50 μL) contained 29.4 μL of buffer A (enzyme reaction buffer containing dNTPs) to dissolve the lyophilized reagent (enzyme system); 2.5 μL of buffer B (enzyme activator); 5 μL of DNA amplification template; 2 μL of each forward and reverse primer; and finally, 50 μL of distilled water. The reaction was incubated at 37°C in a thermal cycler (Bi o-Rad Laboratories Co., Ltd., Shanghai, China) for 20 min. The amplified products were purified by chloroform extraction (1:1 v / v) and amplified on a 2% (v / v) agarose gel using GelGreen. TM Nucleic acid dyes were used for electrophoresis analysis at 200V for 20 minutes. Primer pairs with the best amplification efficiency were selected for subsequent development of an asymmetric MIRA system.

[0015] Furthermore, the specific SgRNA includes SgRNA-specific primers for TYLCCNV, whose nucleoside sequences are shown in SEQ ID NO. 8-11; SgRNA-specific primers for TYLCV, whose nucleoside sequences are shown in SEQ ID NO. 25-28; SgRNA-specific primers for ToLCNDV, whose nucleoside sequences are shown in SEQ ID NO. 43-46; and SgRNA-specific primers for TbCSV, whose nucleoside sequences are shown in SEQ ID NO. 60-63.

[0016] Furthermore, the sgRNAs are specific to each virus as follows: TYLCCNV uses sgRNA1, whose nucleotide sequence is shown in SEQ ID NO. 8; TYLCV uses sgRNA3, whose nucleotide sequence is shown in SEQ ID NO. 27; ToLCNDV uses sgRNA2, whose nucleotide sequence is shown in SEQ ID NO. 44; and TbCSV uses sgRNA1, whose nucleotide sequence is shown in SEQ ID NO. 60.

[0017] To identify functional sgRNAs and optimize reaction parameters, this invention systematically evaluated the ssDNA cleavage activity of Cas14a using real-time fluorescence monitoring. Four different sgRNA sequences were designed for each viral genome based on the target conserved viral genes. sgRNA synthesis was performed via in vitro transcription using PCR-generated template DNA, with the forward primer containing the T7 promoter sequence and the reverse primer containing specific complementary spacers.

[0018] Furthermore, the nucleoside sequences of the probes targeting TYLCCNV, TYLCV, ToLCNDV, and TbCSV are shown in SEQ ID NO. 70.

[0019] Furthermore, the detection method is divided into a two-step method and a one-pot method. The two-step method consists of single-stranded DNA amplification and Cas14a detection in the aMIRA system; the one-pot method performs amplification and detection simultaneously.

[0020] In this invention, aMIRA and Cas14a detection are performed in two separate steps during the two-step detection process. First, preliminary amplification is carried out in 50 μL of the aMIRA reaction system under optimized conditions (37°C, 20 min), followed by enzyme heat inactivation at 95°C for 1 min. For CRISPR detection, 10 μL of the aMIRA amplification product is mixed with 10 μL of the Cas14a1 detection system, which contains: 300 nM Cas14a1 nuclease, 600 nM target-specific sgRNA, 1.5 × Holmes buffer, and 250 nM FAM-BHQ1 probe. The final reaction volume is 20 μL, incubated at 37°C for 60 min, and real-time fluorescence monitoring (every 15 s) is performed using a QuantGene 9600 real-time PCR instrument.

[0021] In this invention, amplification and detection are performed simultaneously in a single test tube during the one-pot detection process. 4 μL of the aM IRA amplification product mixture is added to a 10 μL Cas14a1 detection system. Next, 5 μL of DNA solution and 1 μL of B buffer are added to initiate the amplification and detection reaction. The mixture is run at 37°C for 60 min, followed by 20 min of 2% agarose gel electrophoresis.

[0022] Furthermore, the reaction temperature for the CRISPR / Cas14a ssDNA detection is between 37°C and 46°C.

[0023] Furthermore, the CRISPR / Cas14a ssDNA detection reagent has a concentration of 300 nM Cas14a and 600 nM sgRNA.

[0024] Furthermore, the amplification employs asymmetric MIRA (aMIRA) technology based on isothermal amplification, and the optimal primer ratio for detection is 20:1.

[0025] This invention generates single-stranded DNA (ssDNA) for PAM-independent Cas14a activation by optimizing primer ratios. This system overcomes key technical obstacles, achieving ultrasensitive detection (10 fM) while eliminating cross-contamination through a single-tube reaction. The generation of single-stranded DNA (ssDNA) was validated by adjusting the primer ratios from 1:5 to 1:40. This was verified using GelDoc. TM The XR+ imaging system (Bio-Ray) analyzes electrophoresis to determine the yield of single-stranded DNA.

[0026] The beneficial effects of this invention are mainly reflected in:

[0027] 1) This invention relates to a novel microfluidic aMIRA-Cas14a detection platform, which ingeniously combines isothermal amplification technology with the single-stranded DNA (ssDNA) specific nuclease activity of Cas14a, enabling nucleic acid amplification without the need for complex equipment, and using isothermal amplification method, the amplification results are reliable;

[0028] 2) The CRISPR-Cas14a system of this invention is characterized by its compact structure and PAM sequence-independent ssDNA cleavage capability. After activation, it exhibits both cis and trans cleavage activities, which makes it highly effective for the detection of targeted ssDNA.

[0029] 3) The PAM-independent target recognition mechanism of the MaC14a platform of this invention eliminates sequence restrictions and seamlessly integrates with MIR A amplification in a single reaction vessel, unaffected by the interference of Cas14a on dsDNA or ssDNA, thereby ensuring higher detection sensitivity.

[0030] 4) This invention can detect several samples simultaneously, which is beneficial for its widespread implementation in the production process;

[0031] 5) This invention establishes a shift from passive response to proactive disease management by targeting and suppressing virus-carrying whitefly populations before crop planting and removing infected plants early before secondary transmission. It can detect infection in pre-symptom plants and vector insects. This technology has significant application potential in customs, field monitoring, and virus-free seedling detection, marking a new era in plant disease management and agricultural sustainability. Attached Figure Description

[0032] Figure 1 This diagram illustrates the nucleic acid detection mechanism mediated by aMIRA-Cas14a.

[0033] Figure 2Schematic diagram of the optimization of the CRISPR / Cas14a system. (A) Schematic diagram of sgRNA screening, four sgRNAs were designed to identify the best candidate sequence. (B) Fluorescence kinetics of Cas14a activity at different temperatures. (C) Optimization of Cas14a protein concentration in the reaction system. (D) Determination of the optimal sgRNA concentration in the Cas14a reaction system. (E) Optimization of Holmes buffer concentration in the Cas14a reaction system. (F) Comparative analysis of changes in Cas14a activity with and without RNase inhibitors. (G) Detection limit of the Cas14a assDNA detection system. (H) Detection of Cas14a in virus-infected plant samples.

[0034] Figure 3 This is a screening diagram for sgRNAs against four types of geminiviruses. (A) TYLCCNV (B) TYLCV, (C) TYLCV (D) TbCSV. Four candidate sgRNA sequences were designed and identified for each virus.

[0035] Figure 4 This is a limit of detection (LOD) plot for the Cas14a1 ssDNA detection system. ssDNA targets from four different viruses (TYLCCNV, TYLCV, ToLCNDV, and TbCSV) were tested at sequential dilutions to evaluate the sensitivity of the Cas14a1-based detection system. Real-time fluorescence curves for ssDNA targets of TYLCCNV (A), TYLCV (C), ToLCNDV (E), and TbCSV (G) are shown. Endpoint fluorescence analysis (bar graph) was performed for TYLCCNV (B), TYLCV (D), ToLCNDV (F), and TbCSV (H).

[0036] Figure 5 Figure A shows the aMIRA-Cas14a detection system. (A) Figure A illustrates the reaction mechanism of the aMIRA-Cas14a detection platform. (B) Gel electrophoresis analysis verified the specific cleavage activity of Cas14a for aMIRA products. (C) A comparative analysis of one-step and two-step MIRA-Cas14a detection methods was performed. Note: In the two-step reaction, aMIR amplification was performed first for 20 min, followed by the addition of the Cas14a detection system for fluorescence monitoring. Therefore, fluorescence signal collection began at 20 min. (D) and (E) optimization experiments were conducted to determine the optimal ratio of forward and reverse primers. (FI) The specificity of the aMIRA-Cas14a system was verified by detecting the specificity of four different viral targets (TYLCCNV, TYLCV, TOLCNDV, and TbCSV) in plant samples. Mixed samples (labeled Mix) were prepared by mixing equal volumes of each viral DNA preparation.

[0037] Figure 6 CRISPR-Cas14a1 phylogenetic diagram for aMIRA. (A) Primer selection for MIRA amplification. FP: forward primer; R: reverse primer. The asterisk indicates the target band. (B) Temperature optimization for MIRA reaction efficiency. (C) Cleavage efficiency of Cas14a1 on single-stranded DNA generated by aMIRA. The cleavage efficiency of single-stranded DNA is comparable in both one-step and two-step methods. (D) Effect of forward / reverse primer ratio on single-stranded DNA amplification. Amplification efficiency of single-stranded DNA generated by aMIRA under different primer ratios (F:R).

[0038] Figure 7 The graph shows the sensitivity test results of aMIRA-Cas14a1 using a qPCR instrument. (A) Fluorescence curves of the limit of detection (LOD) experiment using aMIRA-Cas14a1 against four target geminiviruses. (B) Bar graph showing the endpoint fluorescence intensity of the corresponding LOD experiment. (C) Linear regression analysis of the endpoint fluorescence intensity in the MaC14a LOD experiment.

[0039] Figure 8 The following are performance evaluation graphs for MaC14a. (A) Fluorescence intensity response of MaC14a in specific virus-detecting plant samples, data from a single chip detection chamber. (B) Imaging results of the sheet under a blue light projector after specific detection. (C) Detection performance of MaC14a on mixed samples of four target geminiviruses (1-2) and non-target virus plant samples (3-8). (D, F, H, J) Fluorescence curves of LOD experiments using MaC14a on four target geminiviruses (E, G, I, K). Bar graphs of the endpoint fluorescence intensity of the corresponding LOD experiments. The inset in the upper right corner shows the linear regression analysis of the endpoint fluorescence intensity in the MaC14a LOD experiment. The dashed lines in the bar graphs and curves represent the detection threshold.

[0040] Figure 9 This is a graph showing the detection stability of the MaC14a system. The detection stability of the MaC14a system was evaluated. When detecting positive samples of the same concentration, the endpoint fluorescence intensity variation of the 24 wells on the CMC (chip microarray chamber) was minimal, with a relative standard deviation (RSD) of approximately 5.08%.

[0041] Figure 10This is a graph showing the real-time fluorescence signal analysis based on LSTM. (A) Overview of the dataset preparation, LSTM model training, and evaluation process. Model performance was evaluated using accuracy, precision, recall, and a majority voting mechanism. (B) Evaluation metrics throughout the 60-minute recording period. Average accuracy, precision, and recall vary over time. (C) Evaluation metrics in the first 10 minutes of recording. Note: Accuracy is calculated as the sum of true positives and true negatives divided by the total number of samples, reflecting the overall correctness of the model's predictions. Precision is determined by the ratio of true positives to the sum of true positives and false positives, reflecting the proportion of correct identifications. Recall is calculated as the ratio of true positives to the sum of true positives and false negatives, indicating the model's ability to correctly identify positive cases.

[0042] Figure 11 Evaluation of the AI-enhanced MaC14a system for the detection of TYLCV in tomatoes. (A) Disease infection progression of TYLCV in tomatoes. Top: Comparison of whole-plant morphology between healthy control and infected samples at 14, 28, and 42 days post-infection. Leaf view: Macroscopic view of the spread of characteristic symptoms. (B) Fluorescence measurement results of the MaC14a system in tomatoes at different infection stages. (C) Microfluidic chip readout verification. Blue light transmission in the reaction chamber shows the specific activation (green fluorescence) of CRISPR-Cas14a in infected samples. (D) Schematic diagram of the MaC14a system used to detect whitefly virus. (E) Fluorescence intensity map of virus in whiteflies detected using the MaC14a system. (F) Results of the double-blind validation experiment. Twenty tomato leaf samples and 20 whiteflies were analyzed. Color gradient (blue for intensity) reflects the fluorescence signal intensity measured by the aMIRA-Cas14a platform after 60 min of incubation. The "+ / -" annotations in the boxes indicate the classification results generated by the AI-enhanced MaC14a system within 5 minutes. The dashed lines in Figures B and E represent thresholds.

[0043] Figure 12 This chart illustrates the field-ready multiplex detection capabilities of the MaC14a platform. (A) Endpoint fluorescence intensity measured from 20 suspected virus-infected tomato leaves using the single-step asymmetric aMIRA-Cas14 detection system. A negative control is a healthy tomato leaf. (B) The bar chart shows the endpoint fluorescence intensity generated by the MaC14a platform after detecting 20 whitefly samples.

[0044] Figure 13Box plots showing the detection of diseases in tomato plants and whiteflies using qPCR (TaqMan). (A) Box plot of Ct values ​​for four viruses detected by qPCR (TaqMan) from 20 tomato leaf samples. (B) Box plot of Ct values ​​for four viruses detected by qPCR (TaqMan) from 20 whitefly samples. In the box plots, the horizontal line in each box represents the median, and the white square represents the mean. The height of the box represents the mean ± standard deviation. Each group contains n = 3 replicates, and the coefficient of variation for each replicate is less than 10%. Detailed Implementation

[0045] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0046] Example 1: DNA Extraction

[0047] Rapid DNA extraction was performed on plant samples and whiteflies using the Rapid Nucleic Acid Release Kit (DNA Type-II) provided by AMP Future Biotechnology Co., Ltd. 0.1 g of leaf sample or a single whitefly was mixed with 200 μL of PBS buffer. After brief centrifugation, 100 μL of the supernatant was mixed with the release reagent at a ratio of sample:LD-1:LD-2 = 25:4:1. The mixture was incubated at 40 °C for 4 min, followed by centrifugation to collect 20 μL of the lysis supernatant, which was used as the amplification template for subsequent reactions.

[0048] Example 2: Optimization of the Cas14a Nucleic Acid Detection System

[0049] To identify functional sgRNAs and optimize reaction parameters, the ssDNA cleavage activity of Cas14a was systematically evaluated using real-time fluorescence monitoring. Four different sgRNA sequences were designed for each viral genome based on target conserved viral genes (see Table S1), including the V1 gene of TYLCV (GeneBank: MN432609.1), the AV1 gene of TYLCCNV (GeneBank: AJ319675.1), the AV1 gene of ToLCNDV (GeneBank: OR157979.1), and the AV1 gene of TbCSV (GeneBank: AJ420318), to screen for optimal cleavage efficiency. sgRNA synthesis was performed via in vitro transcription using PCR-generated template DNA, with the forward primer containing the T7 promoter sequence and the reverse primer containing specific complementary spacers (see Table S1).

[0050] A Cas14a-based single-stranded DNA detection system was constructed in a 20 μL reaction volume. Systematic optimization was achieved through the following parameter gradient optimization schemes: (1) Cas14a concentration gradient (100–400 nM), (2) sgRNA titration concentration (100–800 nM), (3) temperature gradient (37–46 °C), and (4) buffer composition optimization. The reaction was initiated by adding 2 μL of target single-stranded DNA and monitored using a real-time PCR system under isothermal conditions (37 °C, 150 cycles, 30 s per cycle). Fluorescence signals (excitation / emission: 485 / 520 nm) were recorded during the extension phase of each cycle. All experimental conditions were tested in three independent replicates to ensure reproducibility of the results.

[0051] Example 3: Establishment of the aMIRA System

[0052] To achieve sequence-specific nucleic acid amplification, several primer pairs targeting conserved viral genes were designed (see Table S1). Total genomic DNA extracted from virus-infected tomato leaves was used for preliminary primer screening. A standard MIRA reaction mixture (50 μL) contained 29.4 μL of buffer A (enzyme reaction buffer containing dNTPs) to dissolve the lyophilized reagent (enzyme system); 2.5 μL of buffer B (enzyme activator); 5 μL of DNA amplification template; 2 μL of each forward and reverse primer; and finally, 50 μL of distilled water. The reaction was incubated at 37 °C in a thermal cycler (Bio-Rad Laboratories Co., Ltd., Shanghai, China) for 20 min. The amplified products were purified by chloroform extraction (1:1 v / v) and amplified on a 2% (v / v) agarose gel using GelGreen. TMElectrophoresis analysis was performed using nucleic acid dyes (Tiangen Biotech Co., Ltd., Beijing, China) at 200V for 20 minutes. Primer pairs with optimal amplification efficiency were selected for subsequent development of the asymmetric MIRA system. Single-stranded DNA (ssDNA) was generated by adjusting the primer ratios, with the forward and reverse primer ratios ranging from 1:5 to 1:40 for validation. Results were obtained using GelDoc. TM The XR+ imaging system (Bio-Ray) analyzes electrophoresis to determine the yield of single-stranded DNA.

[0053] Example 4: DNA detection using aMIRA combined with Cas14a

[0054] In the two-step detection process (aMIRA and Cas14a detection were performed in two separate steps), initial amplification was first carried out in 50 μL of the aMIRA reaction system under optimized conditions (37℃, 20 min), followed by enzyme heat inactivation at 95℃ for 1 min. For CRISPR detection, 10 μL of aMIRA amplification product was mixed with 10 μL of the Cas14a1 detection system, which contained: 300 nM Cas14a1 nuclease, 600 nM target-specific sgRNA, 1.5×Holmes buffer, and 250 nM FAM-BHQ1 probe. The final reaction volume was 20 μL, incubated at 37℃ for 60 min, and real-time fluorescence monitoring was performed (every 15 s) using a QuantGene 9600 real-time PCR instrument (Hangzhou Boer Technology Co., Ltd.). For the one-step method, where amplification and detection were performed simultaneously in a single test tube, 4 μL of the aMIRA amplification product mixture was added to 10 μL of the Cas14a1 detection system. Next, 5 μL of DNA solution and 1 μL of B buffer were added to initiate the amplification and detection reaction. The mixture was run at 37°C for 60 min, followed by 20 min of 2% agarose gel electrophoresis.

[0055] The MaC14a detection platform integrates three synergistic components: (1) aMIRA for enriching single-stranded DNA, (2) the CRISPR / Cas14a system for recognizing single-stranded DNA and transmitting signals, and (3) a dual detection strategy including single-tube fluorescence signal output coupled to a centrifugal microfluidic chip using a qPCR instrument or portable analyzer. Figure 1In this procedure, plant or whitefly samples undergo rapid extraction, with the entire process completed within 5 minutes. The extracted nucleic acids are then introduced into the aMIRA reaction system, which dynamically transitions from initial double-stranded DNA synthesis to the later amplification stage of single-stranded DNA overproduction. Target recognition is achieved by activating the transcleavage activity of Cas14a through the sequence-specific binding of crRNA to the single-stranded DNA amplification product. This enzymatic reaction catalyzes the hydrolysis of the FAM / BHQ1 dual-labeled fluorescent reporter molecule, generating a quantitative, real-time fluorescence signal.

[0056] Optimization of key procedures addresses the inherent incompatibility between isothermal amplification and CRISPR detection in traditional RPA-Cas12a systems. Previous methods for overcoming Cas12 interference with isothermal amplification primarily relied on physical isolation techniques: (i) spatial isolation using microfluidic chambers, (ii) liquid-phase separation via glycerol / sucrose gradients, or (iii) time control via photoactivated crRNA. While these methods achieve functional separation, they lead to operational complexity, including multi-step workflows, reagent incompatibility risks, or the need for specialized equipment. Cas14a's PAM-independent single-stranded DNA recognition mechanism not only overcomes sequence design limitations but also achieves time synchronization with aMIRA through its inherent insensitivity to double-stranded DNA. This unique biochemical characteristic maintains the structural integrity of double-stranded DNA amplification intermediates by preventing premature enzymatic cleavage, thereby enabling homogeneous single-phase detection without physical separation.

[0057] 1. Optimization of the CRISPR / Cas14a ssDNA detection system

[0058] The Cas14a-sgRNA complex specifically recognizes single-stranded DNA targets and initiates trans-cleavage activity to cleave fluorescent probes, thus constructing a highly specific single-stranded DNA detection platform. System performance is influenced by key parameters such as sgRNA design, reaction temperature, and reagent concentration. Through systematic sgRNA screening targeting four geminiviruses, we observed that Cas14a exhibits sequence-dependent trans-cleavage activity (…). Figure 2 A). For each virus, we identified the optimal sgRNAs (TYLCCNV: sgRNA1, TYLCV: sgRNA3, ToLCNDV: sgRNA2, TbCSV: sgRNA1), which exhibited superior fluorescence signals compared to the second-best candidate sequences. Figure 3 ).

[0059] The Cas14a-sgRNA complex can precisely recognize single-stranded DNA (ssDNA) targets, initiate transfection and cleavage activity, and degrade the FAM-BHQ1 probe, thus constructing a highly specific ssDNA detection platform. The performance of this system is influenced by several key factors, including sgRNA design, reaction temperature, and reagent concentration. Four different sgRNAs were designed and systematically screened for each viral target to determine the most effective candidate sequences. For each viral target, the optimal sgRNA was determined: sgRNA1 for TYLCCNV, sgRNA3 for TYLCV, sgRNA2 for ToLCNDV, and sgRNA1 for TbCSV. Figure 2 , 3 These optimized sgRNAs significantly enhanced the fluorescence signal compared to the suboptimal sgRNAs. Figure 3 This highlights the crucial role of sgRNA design in improving the efficiency of Cas14a detection systems.

[0060] After determining the optimal sgRNA, we further optimized the reaction conditions and the concentrations of key components to further improve the performance of the Cas14a-based detection system. The system exhibited good thermal stability in the range of 37 to 46 °C. Figure 2 B). The optimal reagent concentrations are 300 nM Cas14a and 600 nM sgRNA. Figure 2 C, D). Buffer optimization shows that 1.5×HOLMES buffer ( Figure 2 E) plus 1 U / μL RNase inhibitor ( Figure 2 F) is the most effective formulation, likely because it helps stabilize the sgRNA-target complex. Sensitivity analysis showed that for synthesized single-stranded DNA targets, the system reached a detection limit of 1 pM within 60 min. Figure 2 G, Figure 4 Given that the genome of geminiviruses consists of single-stranded circular DNA, we tested whether this system could directly detect viral DNA from total DNA extracts from virus-infected plants. However, no detectable signal was observed. Figure 2 H). This highlights the challenge posed by the low viral copy number in these samples and underscores the need to incorporate a pre-amplification step to improve sensitivity in practical plant virus diagnosis.

[0061] 2. The CRISPR-Cas14a system, combined with aMIRA, enables one-pot nucleic acid detection.

[0062] To overcome the sensitivity limitations of amplified CRISPR-Cas14a detection in virally infected plant DNA, we first sought an ssDNA amplification method compatible with the CRISPR-Cas14a system. Inspired by asymmetric PCR, we attempted to develop an asymmetric MIRA (aMIRA) technique based on isothermal amplification. In our design, aMIRA rapidly generates target-specific ssDNA through asymmetric amplification using low concentrations of restriction primers and high concentrations of excess primers. Initially, both primers promote dsDNA synthesis, but as the restriction primers are depleted, the excess primers primarily amplify ssDNA. The enriched ssDNA then activates the Cas14a system, enabling efficient recognition and detection of the target DNA. Figure 5 A). To ensure efficient and specific amplification of the target sequence, MIRA primers were first designed and optimized through systematic screening. Figure 6 A). Temperature optimization shows that the MIRA response is optimal at 37°C. Figure 6 B). In the aMIRA experiment, using a 10:1 ratio of forward to reverse primers, we successfully generated virus-specific ssDNA amplicones. This was confirmed by clear electrophoretic bands corresponding to the expected molecular weight. Figure 5 B, lane 2), verified the specificity and efficiency of ssDNA generation.

[0063] To further verify whether the aMIRA product can activate the Cas14 detection system, we first attempted a two-step method, directly introducing the aMIRA amplification product into the Cas14 detection system. Experimental results showed that single-stranded DNA (ssDNA) was specifically cleaved by Cas14, while double-stranded DNA (dsDNA) remained unaffected, confirming the effectiveness of the Cas14 system. Based on this, we designed a comprehensive one-pot method that integrates the two systems into a unified reaction environment. This method, guided by target-specific sgRNA, achieves complete and sequence-specific degradation of ssDNA. Figure 5 (B Lane 6). In contrast, control systems lacking Cas14a nuclease or containing non-complementary sgRNA did not show detectable cleavage activity ( Figure 5 (B, lanes 3-5). To quantitatively verify the trans-cleavage activity of Cas14a, we employed a fluorescence-based detection system using FAM-BHQ1-labeled ssDNA probes. Analysis showed that the detection efficiency of the one-pot and two-step methods was comparable within 60 minutes. Figure 5 C Figure 6 C). In summary, these findings establish the operational compatibility and synergistic potential of aMIRA amplification and Cas14a-mediated detection in precise DNA target identification.

[0064] To optimize the efficiency of the one-pot detection system (hereinafter referred to as aMIRA-Cas14a), we systematically screened primer ratios. ssDNA generation was observed in all tested primer ratios (5:1 to 40:1), with a 1:1 ratio used as a negative control. Figure 5 D). When the primer ratio is 20:1, the fluorescence intensity reaches its highest level. Figure 5 D, E), therefore this ratio was used in all subsequent experiments to ensure optimal sensitivity. Further evaluation of the aMIRA-Cas14a system showed significant detection sensitivity, with a limit of detection (LOD) of 1 fM for all four test viruses targeting synthetic ssDNA. Figure 7 A, B). Notably, fluorescence intensity exhibits a linear correlation with target concentration across five orders of magnitude ( Figure 7 (C) This demonstrates its powerful quantitative capability. Compared to the traditional amplification-free CRISPR-Cas14a detection system, aMIRA-Cas14a exhibits a 1000-fold increase in sensitivity. Subsequently, validation using DNA extracts from virus-infected plants confirmed the system's high specificity; for the four target viruses, only positive fluorescent signals were detected in each detection system (C). Figure 5 (FI). The aMIRA-Cas14a platform operates in a fully closed tube format, requiring only a single reagent addition, significantly improving the convenience of detection and minimizing the risk of contamination. Combined with the simplicity of single-tube operation, high specificity, and physiological temperature operation, this system provides a robust and field-deployable diagnostic solution for plant virus detection.

[0065] Example 5: Limit of Detection (LOD) Validation

[0066] The analytical sensitivity of the MaC14a platform was systematically evaluated using synthetic single-stranded DNA targets (ssDNA) derived from four viruses: TYLCCNV, TYLCV, ToLCNDV, and TbCSV. These ssDNA targets were serially diluted in eight logarithmic concentrations from 100 pM to 100 aM to establish a detection gradient. aMIRA-Cas14a assays were performed in a portable analyzer at 37°C for 60 min, with fluorescence signals captured every 30 s using an integrated optical system.

[0067] Example 6: Specificity evaluation of the MaC14a platform

[0068] To evaluate the detection specificity of the MaC14a platform, DNA was extracted from tomato leaves infected with seven different viral pathogens: TYLCCNV, TYLCV, ToLCNDV, TbCSV, Solanaceae leafroll virus (NCTV), Tomato golden leaf virus (TGMV), and Tomato leafroll virus (ToLCV). The extracted DNA (20 μL) was mixed with 8 μL of buffer B and 132 μL of nuclease-free water and loaded into designated injection areas of the microfluidic chip. aMIRA-Cas14a detection was performed for 60 min at 37 °C in a portable analyzer, with fluorescence signals captured every 30 s using an integrated optical system. Negative controls included DNA from healthy tomato plants and nuclease-free water treated under the same conditions.

[0069] Using microfluidic chips and a portable analyzer, we systematically evaluated the specificity and sensitivity of the MaC14a platform. By analyzing plant leaf samples infected with the virus, we confirmed that the platform showed no cross-reactivity against four target viruses (TYLCCNV, TYLCV, ToLCNDV, and TbCSV). Figure 8 A, B). For mixed virus samples ( Figure 8 C(1)–(2)), the fluorescence intensity in all detection chambers showed that the platform could detect co-infection. Most importantly, the fluorescence reactions produced by healthy plant leaf samples and non-target viruses (NCTV, TGMV, ToLCV) were indistinguishable from the negative control. Figure 8 C(3)–(7)), which further confirms the system’s specificity.

[0070] Sensitivity assessment of MaC14a showed that the detection limit for all four virus-derived single-stranded DNA targets was 1 femtomolar (fM). Figure 8 DK), whose performance is comparable to the qPCR-based aMIRA-Cas14a system ( Figure 7 Linear regression analysis showed a significant correlation between the fluorescence intensity of the tested pathogen and the viral concentration, confirming the quantitative capability of the system. Figure 8 E, G, I, K). Systematic stability assessment using 10 pM mixed virus-derived single-stranded DNA showed an average fluorescence intensity exceeding 14,000 au in 24 compartments with low variability (RSD = 5.08%). Figure 9 These results collectively demonstrate that the MaC14a platform meets three key diagnostic performance criteria: femtomolar sensitivity (1 fM LOD), excellent specificity (no cross-reactivity in infected leaf samples), and operational stability (5.08% RSD), making it a promising field detection tool for agricultural pathogens.

[0071] Example 7: Real-time detection driven by artificial intelligence in MaC14a

[0072] To improve the detection efficiency of the MaC14a platform, we adopted a Long Short-Term Memory (LSTM) neural network, implemented in PyTorch and optimized using the Adam algorithm, while also introducing a majority voting mechanism. Figure 10 A) This study aimed to achieve real-time fluorescence signal analysis and early detection. Real-time fluorescence data covered synthetic single-stranded DNA targets ranging from 1 nM to 10 aM, as well as detection data from plant samples used for specificity testing. 103 original datasets were processed using a Savitzky-Golay filter for noise reduction, labeled as positive (n=52) or negative (n=51), and divided into training (70%), validation (17%), and test (13%) sets. The LSTM model was trained on 80% of the data and validated on 20%. The source code and software implementation of this study are available on GitHub (https: / / github.com / MaC14a1-2025 / MaC14a1) under an open-source license.

[0073] We systematically evaluated the changes in the model's mean accuracy, precision, and recall over time during a 1-hour recording period. Figure 10 B). The results showed that all three evaluation indicators reached 100% at 8 minutes and 30 seconds. Figure 10 C). Notably, the mean accuracy, precision, and recall reached 95% as early as 5 minutes and remained relatively stable thereafter (see Table 1). Within the current dataset, these findings demonstrate that our algorithm can effectively replace manual operation and significantly reduce detection time, decreasing it from 60 minutes for traditional methods to reach a stable state to 5-8 minutes based on machine interpretation. However, further blind testing is needed to verify the system's actual detection efficiency.

[0074] Table 1 Performance evaluation of AI recognition algorithms at different time points

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[0076]

[0077] Note: a. Accuracy is calculated as the sum of true positives and true negatives divided by the total number of samples.

[0078] b. Accuracy is determined by the ratio of true positives to the sum of true positives and false positives.

[0079] c. Recall rate is calculated as the ratio of true positives to the sum of true positives and false negatives, indicating the model's ability to correctly identify positive cases.

[0080] Example 8: Field verification of MaC14a diagnostic efficiency

[0081] To evaluate the performance of the AI-enhanced MaC14a system in detecting TYLCV infection in field samples, we first assessed its effectiveness in detecting TYLCV in tomato plants at different infection stages. In the experiment, an infectious clone of the virus was introduced into tomato plants using Agrobacterium-mediated inoculation technology. Figure 11 A). At 14 days post-inoculation (early stage), slight leaf deformation was observed, indistinguishable from leaf damage caused by abiotic stress. By 28 days post-inoculation (mid-stage), leaf curling and yellowing became more pronounced. At 42 days post-inoculation (late stage), severe yellowing, curling, and downward curling of the leaves were observed, typical characteristics of TYLCV infection. Leaf samples were collected at each stage and tested using the MaC14a system. The AI-driven analysis system demonstrated perfect diagnostic capability within 5 minutes, successfully classifying all infected samples correctly as positive while maintaining negative results in the uninfected control group. Post-amplification validation, performed by endpoint fluorescence measurement and transilluminator visualization 1 hour after testing, further confirmed the detection accuracy. Figure 11 (B, C). This diagnostic result, aligned with the qPCR benchmark, highlights the capabilities of the MaC14a system in detecting asymptomatic viruses.

[0082] Detecting the presence of viruses in insect vectors is crucial for early disease management and prevention of virus transmission in agricultural systems. To evaluate the ability of the MaC14a system to detect virus-carrying insect vectors, we conducted an acquisition experiment of TYLCV (Tobacco Mosaic Virus) using a whitefly population. Whiteflies were acquired from TYLCV-infected plants for 3 days before being treated with the MaC14a system. Figure 11 D). Subsequent analysis showed that 8 out of 10 tested whiteflies (80%) displayed viral signals. Figure 11 E). Importantly, all samples from the negative control group (which fed only on healthy plants) maintained baseline fluorescence levels. These results demonstrate that the MaC14a system can accurately identify virus-carrying whiteflies at the individual level, highlighting its potential for monitoring and controlling virus transmission in agricultural environments.

[0083] In the subsequent double-blind validation experiment, we tested 20 tomato leaf samples (numbered S1 to S20) collected from two greenhouse cultivation areas in Hangzhou, Zhejiang Province, and Nanning, Guangxi Zhuang Autonomous Region, China. Five tomato leaf samples exhibiting typical viral infection symptoms (such as yellowing, mosaic spots, and leaf curling) and five whiteflies (a total of 20) were provided from each area. The AI-enhanced MaC14a system produced results at 5 minutes, and these results were in perfect agreement with the aMIRA-Cas14a assay and qPCR platform results at 60 minutes, showing 100% concordance. Figure 11 F, 12, 13 (Table S2). Furthermore, the virus species detected in the plant samples were highly consistent with those identified in the vector insects from the same cultivation area. These results highlight the significant potential of AI-enhanced MaC14a in field plant virus diagnosis, achieving rapid detection from nucleic acid extraction to result reading in just 10 minutes, high accuracy consistent with qPCR results, and portability.

[0084] Table 2. Samples that passed qPCR validation in the double-blind validation.

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[0087] Note: "+" indicates a positive virus sample; blank indicates a negative result.

[0088] Table 3. DNA oligonucleotides used in this study.

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[0091]

Claims

1. A method for detecting geminitroviruses in tomato plants and whiteflies based on the aMIRA-CRISPR / Cas14a system, characterized in that, Includes the following steps: 1) DNA was extracted from tomato plant samples and whiteflies; 2) Amplify the specific genes of geminiviruses in tomato plants and whiteflies using aMIRA-specific primers to obtain aMIRA amplification products; 3) Using the CRISPR / Cas14a system, specific SgRNA targets and recognizes the aMIRA amplification product, activating the non-specific cleavage activity of CRISPR / Cas14a. The detection results are interpreted by a reporter probe, enabling the detection of geminiviruses in tomato plants and whiteflies.

2. The detection method as described in claim 1, characterized in that, The specific genes of the geminiviruses in the tomato plants and whiteflies include the V1 gene of TYLCV, the AV1 gene of TYLCCNV, the AV1 gene of ToLCNDV, and the AV1 gene of TbCSV.

3. The detection method as described in claim 1 or 2, characterized in that, The aMIRA-specific primers include a MIRA-specific primer for TYLCCNV, whose nucleoside sequences are shown in SEQ ID NO. 4-7; a MIRA-specific primer for TYLCV, whose nucleoside sequences are shown in SEQ ID NO. 21-24; a MIRA-specific primer for ToLCNDV, whose nucleoside sequences are shown in SEQ ID NO. 39-42; and a MIRA-specific primer for TbCSV, whose nucleoside sequences are shown in SEQ ID NO. 56-59.

4. The detection method as described in claim 1 or 2, characterized in that, The specific SgRNAs include SgRNA-specific primers for TYLCCNV, whose nucleoside sequences are shown in SEQ ID NO. 8-11; SgRNA-specific primers for TYLCV, whose nucleoside sequences are shown in SEQ ID NO. 25-28; SgRNA-specific primers for ToLCNDV, whose nucleoside sequences are shown in SEQ ID NO. 43-46; and SgRNA-specific primers for TbCSV, whose nucleoside sequences are shown in SEQ ID NO. 60-63.

5. The detection method as described in claim 4, characterized in that, The sgRNAs are specific to each virus: TYLCCNV uses sgRNA1, whose nucleotide sequence is shown in SEQ ID NO.8; TYLCV uses sgRNA3, whose nucleotide sequence is shown in SEQ ID NO.27; ToLCNDV uses sgRNA2, whose nucleotide sequence is shown in SEQ ID NO.44; and TbCSV uses sgRNA1, whose nucleotide sequence is shown in SEQ ID NO.

60.

6. The detection method as described in claim 1 or 2, characterized in that, The nucleoside sequence of the probe is shown in SEQ ID NO.

70.

7. The detection method according to any one of claims 1-6, characterized in that, The detection method is divided into a two-step method and a one-pot method. The two-step method consists of single-stranded DNA amplification and Cas14a detection in the aMIRA system; the one-pot method performs amplification and detection simultaneously.

8. The detection method as described in claim 7, characterized in that, The reaction temperature for CRISPR / Cas14assDNA detection in the two-step method is 37℃-46℃.

9. The detection method as described in claim 7, characterized in that, In the two-step method, the concentrations of the CRISPR / Cas14assDNA detection reagents were 300 nM Cas14a and 600 nM sgRNA.

10. The detection method as described in claim 7, characterized in that, In the one-pot method, amplification is performed using aMIRA technology, and the optimal primer ratio for detection is 20:1.

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