[4S05Am] 21. Open Topics in Biotechnology - Part 2

10:20 AM - 10:35 AM JST(1:20 AM - 1:35 AM UTC)

[4Open-02] CRISPR/Cas12a-Based Lateral Flow Assay for Rapid Detection of Tobacco Curly Shoot Virus in Solanaceous Crops of Jharkhand, India

Snehlata Kujur¹, ○Dinesh Prasad¹ (1. Birla Institute of Technology, Mesra, Ranchi, India (India))

Keywords:

CRISPR/Cas12a,Phytopathogen,Lateral Flow Assay,TbCSV,Biosensing

[Purpose] The emergence of viral pathogens in economically important crops poses a significant threat to regional food security and agricultural sustainability. Conventional diagnostic approaches, although widely used, often lack the sensitivity, specificity, and rapid turnaround required for timely disease management. This study aimed to identify and molecularly characterize the viral pathogen infecting tomato and chilli crops in Jharkhand, India, and to establish a foundation for the development of rapid, field-deployable diagnostic tools. Initial symptomology resembled that of the Tomato Yellow Leaf Curl Virus (TYLCV); however, repeated failure of amplification using TYLCV-specific primers suggested the presence of a divergent or previously unreported viral species.

[Method] Whole-genome sequencing was performed using ONT (P2 Solo) for accurate pathogen identification. Field-level screening was conducted to evaluate the host range and cross-infectivity. A ~200 bp genomic region spanning the AV2 and the intergenic region between AV2 and AC1 was selected for PCR-based amplification. Serial dilution experiments were conducted to determine the limit of detection of conventional PCR. A crRNA was designed using the CHOPCHOP platform to ensure sequence-specific targeting. The functionality of the crRNA was validated through LbCas12a-mediated fluorophore–quencher (FQ) probe cleavage assays. Based on these results, a CRISPR/Cas12a-based lateral flow assay (LFA) is proposed, utilizing collateral cleavage of labelled ssDNA reporters, with optimization of probe design and strip configuration for field applicability.

[Results] Morphological characterization using TEM and whole-genome sequencing identified the pathogen as tobacco curly shoot virus (TbCSV), a gemini virus with ssDNA genome of size 2699 bases, representing its first report in Jharkhand. Field studies confirmed its cross-infectivity in tomato and chilli crops, indicating its potential as an emerging agricultural threat. A specific ~200 bp region of the viral genome was successfully amplified using PCR. Serial dilution-based sensitivity analysis demonstrated that conventional PCR could detect the virus at levels below 100 copies of the viral genome, corresponding to approximately 0.4 fg of target DNA.

[Consideration] This study highlights the risk of misdiagnosis when relying solely on symptom-based identification and geographically limited primer sets, as evidenced by the initial assumption of TYLCV infection. The demonstrated sensitivity of conventional PCR provides a reliable baseline; however, further enhancement is required for rapid on-site detection. The integration of CRISPR/Cas12a with lateral flow platforms offers a promising strategy for improving sensitivity, specificity, and ease of use under field conditions.

[Conclusion] This study reports the first identification of TbCSV infecting tomato and chilli crops in Jharkhand and establishes a molecular framework for its detection. These findings validate the effectiveness of combining nanopore sequencing with PCR and CRISPR-based approaches for accurate pathogen identification and diagnostics. The proposed CRISPR/Cas12a-based lateral flow assay is expected to provide a rapid, user-friendly, and field-deployable diagnostic tool, contributing to improved disease surveillance, early intervention, and sustainable crop protection strategies.