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11 October 2023 Rapid assay using recombinase polymerase amplification and lateral flow dipstick for identifying Agrilus mali (Coleoptera: Buprestidae), a serious wood-boring beetle of the western Tianshan Mountains in China
Cheng-Jin Li, Hui-Quan Sun, Wen-Xia Zhao, Xiao-Yi Wang, Ruo-Zhu Lin, Yan-Xia Yao
Author Affiliations +
Abstract

Agrilus mali stands as a significant wood-boring pest prevalent in Northeast Asia. Identifying this pest beetle is often hindered by insufficient efficient, rapid, on-site discrimination methods beyond examining adult morphological features. As a result, an urgent need arises for developing and implementing a rapid and accurate molecular technique to distinguish and manage the beetle. This study presents a straightforward, swift, highly specific, and sensitive method built upon recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD). This method demonstrates the capability to promptly identify the beetle, even during its larval stage. RPA primers and probes were designed using the internal transcribed spacer 1 region. Through probe optimization, false-positive signals were successfully eliminated, with an accompanying discussion on the underlying causes of such signals. The RPA-LFD assays exhibited remarkable specificity and sensitivity, requiring as little as 10–3 ng of purified DNA. Furthermore, the extraction of crude DNA was achieved through immersion in sterile distilled water, thus streamlining the assay process. Achievable at temperatures ranging from 30 to 50 °C, the RPA-LFD assay can be executed manually without specialized equipment. By merging the RPA-LFD assay with DNA coarse extraction, A. mali can be detected within just 30 min. This current study effectively demonstrates the immense potential of RPA-LFD in quarantine and pest management. Additionally, it presents a universal technique for the rapid on-site diagnosis of insects, showcasing the wide applicability of this method.

Graphical Abstract

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Cheng-Jin Li, Hui-Quan Sun, Wen-Xia Zhao, Xiao-Yi Wang, Ruo-Zhu Lin, and Yan-Xia Yao "Rapid assay using recombinase polymerase amplification and lateral flow dipstick for identifying Agrilus mali (Coleoptera: Buprestidae), a serious wood-boring beetle of the western Tianshan Mountains in China," Journal of Economic Entomology 116(6), 1969-1981, (11 October 2023). https://doi.org/10.1093/jee/toad189
Received: 9 May 2023; Accepted: 27 September 2023; Published: 11 October 2023
JOURNAL ARTICLE
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KEYWORDS
false-positive signal
insect on-site identification
lateral flow dipstick
molecular distinction method
recombinase polymerase amplification
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