Probe-based quantitative PCR and RPA-Cas12a molecular diagnostics for detection of the tomato pest <i>Phthorimaea absoluta</i> (Lepidoptera: Gelechiidae)

Lewald, Kyle M; Song, Wenqi; Eweis-LaBolle, Daniel; Truong, Cindy; Godfrey, Kristine E; Chiu, Joanna C

doi:10.1093/jee/toad079

Cited by 1 publication

(1 citation statement)

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“…RPA technology has been applied for insect identification 46–48 . For the fruit fly, the first RPA‐based technology research report was published in 2022 49 .…”

Section: Discussionmentioning

confidence: 99%

Application of recombinase polymerase amplification with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick assay for Bactrocera correcta

Li,

Cai,

Chen

et al. 2024

Pest Management Science

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BACKGROUNDBactrocera correcta is a quarantine pest that negatively impacts the fruit and vegetable industry. Differentiating B. correcta from similar species, especially in non‐adult stages, remains challenging. Rapid molecular identification techniques, such as Recombinase Polymerase Amplification (RPA) combined with CRISPR/Cas12a and Multienzyme lsothermal Rapid Amplification with lateral flow dipstick (MIRA‐LFD), play a crucial role in early monitoring and safeguarding agricultural production. Our study introduces two methods for the rapid visual identification of B. correcta.RESULTSB. correcta specific RPA primers, crRNA, and the LFD probe were designed based on the cox1 genes. The RPA reaction conditions were optimized (at 37 °C for 8 min) for effective template DNA amplification. Two nucleic acid detection methods were established to visualize RPA amplification. In the RPA‐CRISPR/Cas12a system, the optimal LbCas12a: crRNA concentration ratio was 200:400 nM. Successful amplification was determined by the presence or absence of green fluorescence following 15 min incubation at 37 °C. The MIRA‐LFD system achieved precise identification of the target species within 4 min at 37 °C. Both methods exhibited high specificity and sensitivity, allowing for detection from 1.0 × 10−1 ng/μL of DNA. Combined with rapid DNA extraction, rapid identification of individual B. correcta at different developmental stages was achieved, enhancing the practicality and convenience of the established methods.CONCLUSIONOur research findings demonstrate that both the RPA‐CRISPR/Cas12a and MIRA‐LFD methods for B. correcta detection was accurate and rapid (within 30 min and 10 min, respectively), at 37°C. Our methods do not rely on expensive equipment, thus possess high practical value, providing improved identification solutions for port quarantine pests and field applications.This article is protected by copyright. All rights reserved.

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“…RPA technology has been applied for insect identification 46–48 . For the fruit fly, the first RPA‐based technology research report was published in 2022 49 .…”

Section: Discussionmentioning

confidence: 99%