An advanced approach for rapid visual identification of Liposcelis bostrychophila (Psocoptera: Liposcelididae) based on CRISPR/Cas12a combined with RPA

Wenxin Deng; Shiqian Feng; Vaclav Stejskal; George Opit; Zhihong Li

doi:10.1093/jee/toad139

An advanced approach for rapid visual identification of Liposcelis bostrychophila (Psocoptera: Liposcelididae) based on CRISPR/Cas12a combined with RPA

J Econ Entomol. 2023 Oct 10;116(5):1911-1921. doi: 10.1093/jee/toad139.

Authors

Wenxin Deng^{1

2}, Shiqian Feng³, Vaclav Stejskal^{4

5}, George Opit⁶, Zhihong Li^{1

2}

Affiliations

¹ Department of Plant Biosecurity, College of Plant Protection, China Agricultural University, Beijing 100193, China.
² Sanya Institute of China Agricultural University, Yazhou Bay Science and Technology City, Yazhou District, Sanya 572025, Hainan, China.
³ State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
⁴ Crop Research Institute, Drnovská 507, 161 06 Prague 6, Czech Republic.
⁵ Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamycka 129, 165 00 Prague, Czech Republic.
⁶ Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA.

PMID: 37463293
DOI: 10.1093/jee/toad139

Abstract

Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae) is a booklouse pest that is a threat to commodity storage security worldwide. Accurate and sensitive methods of L. bostrychophila on-site identification are essential prerequisites for its effective management. Evidence suggests that L. bostrychophila contains 3 intraspecific biotypes that are morphologically indistinguishable but can be discriminated at the level of mitochondrial genome organization and sequences. The traditional molecular identification methods, such as DNA barcoding and PCR-RFLP, are instrumentally demanding and time-consuming, limiting the application of the identification in the field. Therefore, this study developed a new CRISPR/Cas12a-based visual nucleic acid system based on the mitochondrial gene coding for NADH dehydrogenase subunit 2 (nad2), combined with recombinase polymerase amplification (RPA) to accurately identify L. bostrychophila from 4 other common stored-product booklice, and also differentiate 3 biotypes of this species at the same time. The entire identification process could be completed at 37 °C within 20 min with high sensitivity. The system could stably detect at least 1 ng/μl of DNA template. The green fluorescence signal produced by the trans-cleaving of the single-stranded DNA reporter could be observed by the naked eye under blue light. Additionally, the suggested system combined with the crude DNA extraction method to extract DNA rapidly, enabled identification of all developmental stages of L. bostrychophila. With crude DNA, this novel diagnostic system successfully identified an unknown booklouse by holding the reaction tubes in the hand, thus can be considered as an accurate, rapid, highly sensitive, and instrument-flexible method for on-site visual identification of L. bostrychophila.

Keywords: Liposcelis bostrychophila; CRISPR/Cas12a; naked-eye readout; on-site identification; recombinase polymerase amplification (RPA).