Article Sharing丨An AI-enhanced CRISPR-Cas14a microfluidic platform for accurate on-site detection of geminiviruses in tomato plants and whiteflies

Geminiviruses pose a severe threat to global tomato production through rapid evolution and worldwide whiteflymediated transmission. Current detection methods primarily target symptomatic plants, often delivering diagnostic results only after viral loads have reached transmissible levels, rendering disease control ineffective. Early intervention hinges on identifying infections prior to symptom onset-or even detecting viral presence in insect vectors before they transmit viruses to plants. While recent advances in isothermal amplification and CRISPR-based diagnostics offer potential solutions, practical implementation faces inherent limitations: incompatibility between enzymatic amplification and CRISPR systems, contamination risks from multistep procedures, and inadequate field adaptability of detection devices. To address these challenges, we developed MaC14a-an AI-enhanced microfluidic platform integrating asymmetric multienzyme isothermal rapid amplification (aMIRA) with CRISPR-Cas14a. This system overcomes key technical barriers by optimizing primer stoichiometry to generate ssDNA for PAM-independent Cas14a activation, achieving ultrasensitive detection (10 fM) while eliminating cross-contamination via single-tube reactions. Coupled with a centrifugal microfluidic chip, portable optical detection, and machine learning-based signal interpretation, MaC14a enables multiplexed detection of four geminiviruses within 5 min, demonstrating 100 % diagnostic accuracy for both plant and whitefly samples. Its breakthroughs include: (i) presymptomatic infection detection in plants, and (ii) precise determination of viral carriage in individual whiteflies-filling a critical technological gap in pre-transmission surveillance. Beyond providing a novel tool for geminivirus management, this study establishes an "AI-CRISPR-microfluidics" paradigm for crop protection. By shifting focus from symptomatic plants to viruliferous vectors and asymptomatic infections, this technology offers a transformative solution to disrupt viral transmission cycles at their source.

Integrated workflow of the MaC14a system for multiplex geminivirus surveillance.

(A) Mechanism of aMIRA-Cas14a-mediated nucleic acid detection. Schematic illustrating aMIRA-driven ssDNA amplification coupled with Cas14a's PAM independent ssDNA recognition and collateral cleavage activity, enabling single-tube detection (compatible with real-time fluorescent PCR instruments) or on-site microfluidic analysis (via custom-developed centrifugal detection devices). (B) Microfluidic detection (MaC14a) workflow for field-deployable diagnostics.The time steps are indicated by arrows, showing the duration of each process within the portable analyzer. (C) Flowchart of the real-time detection algorithm based on Long Short-Term Memory (LSTM) networks. The optimized algorithm facilitates real-time processing of fluorescence signals, enabling result interpretation within 5–10 min.

The aMIRA-Cas14a detection system

(A) The schematic diagram illustrates the reaction mechanism of aMIRA-Cas14a detection platform. (B) The specific cleavage activity of Cas14a on aMIRA products was verified through gel electrophoresis analysis (C) Comparative analysis of one-step and two-step aMIRA-Cas14a detection methods. Note: In the two-step reaction protocol, the process begins with a 20-minute aMIR amplification, followed by the addition of the Cas14a detection system for fluorescence monitoring. Consequently, the collection of fluorescence signals initiates at the 20-minute time point. (D) and (E) Optimization experiments were conducted to determine the optimalforward and reverse primer ratios. (F–I) The specificity of the aMIRA-Cas14a system was validated through the detection of four distinct viral targets (TYLCCNV,TYLCV, TOLCNDV, and TbCSV) in plant samples. The mixed sample (designated as Mix) was prepared by combining equal volumes of each viral DNA preparation.

To achieve signal amplification for trace amounts of viral nucleic acids and provide sufficient substrates for Cas14a, we developed aMIRA (asymmetric multienzyme isothermal rapid amplification) based on MIRA. The resulting aMIRA-Cas14a system offers the following key advantages:

1. Optimized primer stoichiometry allows MIRA to preferentially overproduce ssDNA, which serves as the direct substrate for Cas14a. By adjusting the forward-to-reverse primer ratio to 20:1, the system generates sufficient ssDNA to activate the PAM-independent cleavage activity of Cas14a, which specifically recognizes and cleaves ssDNA.
2. One‑pot integration of MIRA and CRISPR-Cas14a eliminates cross‑contamination risks.
3. MIRA significantly enhances detection sensitivity, enabling the detection of ultra‑low‑abundance viral nucleic acids.
4. aMIRA-Cas14a maintains high specificity, avoiding false positives caused by non‑specific amplification.

Ultimately, the one‑pot integration of MIRA and CRISPR-Cas14a eliminates contamination risks and achieves perfect synergy between MIRA's strengths and the CRISPR-Cas14a system. This resolves the industry‑wide challenge of incompatibility between isothermal amplification and CRISPR systems, representing the core technological breakthrough of the MaC14a platform. The aMIRA-Cas14a system achieves a 1,000‑fold improvement in sensitivity while ensuring specific amplification. Combined with the sequence‑specific recognition of Cas14a, this provides dual‑layer specificity validation, with no cross‑reactivity against non‑target viruses or healthy samples-addressing another industry pain point: the tendency for false positives.

The MIRA multienzyme isothermal rapid amplification reagents used in this study were provided by Amp‑Future (Changzhou) Biotech Co., Ltd. Beyond excellent reagent performance, Amp‑future Biotech also offers a professional and rapidly responsive technical support team.

MIRA demonstrates strong compatibility with centrifuge-based microfluidic chips developed for research and diagnostic applications:

1.Miniaturized reaction system , suited for the micro-volume reaction chambers characteristic of microfluidic chips;
2.Multiplex detection capability in a single run;
3.Compatibility with portable devices,smooth workflows from sample to result.

The portable analyzer and chip architecture.

(A) Exploded view of the portable detection device, showing the major components. (B) Modular architecture of the centrifugal microfluidic chip. (C) Centrifugation driven fluidic control. Analysis of liquid transport trajectories under programmable centrifugal forces.

we engineered an integrated portable "sample-in, answer-out" point-of-care testing (POCT) device optimized for fielddeployment, as illustrated in Fig. 4A. The compact prototype (23 cm (L) × 21 cm (W) × 14 cm (H), 12.5 kg total mass) achieves exceptional portability. The system's core components include a high-precision servo motor for accurate rotational control and an air heating unit for temperature regulation. An integrated optical detection module, positioned beneath the chip, facilitates fluorescence excitation and measurement, ensuring high sensitivity and accuracy in detection. The portable device is equipped with an embedded Android operating system, offering a user-friendly interface where operators can select preprogrammed operation files containing detailed parameters such as reaction time and temperature. Results and conclusions are displayed in real-time on an LCD screen, enabling rapid information acquisition.Additionally, the system incorporates a wireless communication module that supports real-time data transmission to mobile devices or cloud servers, integrating with artificial intelligence algorithms for immediate interpretation and analysis of test results.

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To evaluate the MaC14a system's capacity for detecting viralcarriage in insect vectors, we conducted TYLCV acquisition assays using

Bemisia tabaci populations. Whiteflies were allowed a 3-day acquisition feeding period on TYLCV-infected plants and then processed through the

MaC14a system (Fig. D). Subsequent analysis revealed that 8 out of 10 tested whiteflies (80%) exhibited positive viral signals (Fig. E).Importantly, all specimens from the negative control cohort (fed exclusively 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 viral transmission in agricultural settings.

In a subsequent double-blind validation experiment, we tested 20 tomato leaf samples (S1–S20) collected from two greenhouse cultivation zones in Hangzhou, Zhejiang Province, China, and two greenhouse cultivation zones in Nanning, Guangxi Province, China. Each zone provided five tomato leaf samples exhibiting typical symptoms of viral infection (such as chlorosis, mosaic mottling, and leaf curling) and five whiteflies (20 in total). The AI-enhanced MaC14a system delivered results at 5th minute, which were fully consistent with those obtained from a 60-min aMIRA-Cas14a assay and qPCR platform, demonstrating100 % agreement (Figs. F, S6-S7, Table S3). Additionally, the virus species detected in the plant samples were highly consistent with those identified in the vector insects from the same cultivation zone. These results highlight the significant potential of AI-enhanced MaC14a in on-site plant virus diagnostics, achieving high speed (from nucleic acid extraction to result reading in just 10 min), high accuracy (consistent with qPCR results), and portability.

Article Sharing丨An AI-enhanced CRISPR-Cas14a Microfluidic Platform For Accurate On-site Detection Of Geminiviruses in Tomato Plants And Whiteflies