Unlocking Precision: Advancing Rapid Field Molecular Identification of Tuta absoluta Across its Life Cycle Using Locked Nucleic Acid Strategies

Rapid and accurate insect identification is crucial for effective plant quarantine procedures. This study introduced a reverse transcription recombinase-assisted amplification-lateral flow strip (RT RAA-LFS), specifically designed for on-site detection of the globally quarantined pest, Tuta absoluta, within a 25-minute timeframe. A pivotal strategy involved integrating three locked nucleic acid (LNA) modifications into a species-specific probe, enabling RT RAA to selectively and efficiently amplify the unique haplotype of T. absoluta's mitochondrial cytochrome c oxidase subunit I (COI) gene. After determining the amplification product dilution factor and the volumes of primers, magnesium acetate, and nucleic acid lysate in the amplification system, the finalized RT RAA-LFS system is capable of direct visual detection, complemented by handheld fluorescence detection for quantum dots (QDs) fluorescence on the lateral flow strip. This technique successfully identifies various developmental stages of T. absoluta in crude nucleic acid lysates without the need for genomic DNA extraction. Moreover, it detected the target gene in the genomic solution, reaching concentrations as low as 1.6pg or 51 copies per 50-µL reaction system, aligning with qPCR results. Field validation with 50 insect samples supports the correlation between RT RAA-LFS quantum dots' fluorescence intensity and LFS color intensity, affirming the system's reliability in estimating the target gene copy number (R=0.584, p < 0.01). This validation emphasizes the practical efficacy of the RT RAA-LFS system for T. absoluta detection, highlighting its potential as a rapid on-site tool in agricultural pest management.