关键词: 杰克贝尔氏粉蚧, 重组酶聚合酶扩增, CRISPR/Cas12a检测, 荧光检测, 侧向流层析检测

Abstract: 【Aim】 This study aims to develop a rapid, sensitive, convenient and visual system based on fluorescent detection and lateral flow assay for Pseudococcus jackbeardsleyi using recombinase polymerase amplification (RPA)-CRISPR/Cas12a technology. This detection system has the potential to facilitate on-site detection in resource-limited settings, such as in the field and at ports. 【Methods】The specific primers and crRNAs for RPA were designed based on the 28S rDNA gene sequence of P. jackbeardsleyi. Subsequently, the RPA and CRISPR/Cas12a assay were conducted on P. jackbeardsleyi and other nine mealybug species (Paracoccus marginatus, Phenacoccus solani, Phenacoccus solenopsis, Planococcus citri, Planococcus lilacinus, Dysmicoccus neobrevipes, Planococcus minor, Pseudococcus cryptus and Pseudococcus odermatti) to validate the specificity of the system. The conditions of the RPA-CRISPR/Cas12a detection system were optimized, including the RPA reaction time, the concentration ratio of Cas12a to crRNA and their concentrations, the concentrations of  fluorescent reporter molecule FQ Reporter and the test strip reporter molecule LF Reporter, and the reaction time of CRISPR/Cas12a assay system to screen the optimal detection system. 【Results】The RPA system utilizing specific RPA primers could amplify a 201 bp 28S rDNA fragment of P. jackbeardsleyi. In conjunction with the CRISPR/Cas12a assay, P. jackbeardsleyi could be definitely identified from the other nine mealybug species with visual results. The optimal RPA-CRISPR/Cas12a detection system for P. jackbeardsleyi established by optimization of conditions included the reaction time of 20 min for RPA, a 1∶1 ratio of Cas12a to crRNA at the concentration of 50 nmol/L, the FQ Reporter concentration of 500 nmol/L, the LF Reporter concentration of 800 nmol/L, and the reaction time of the fluorescent detection system and the lateral flow assay system of 5 and 20 min, respectively. 【Conclusion】In this study, a RPA-CRISPR/Cas12a detection system for P. jackbeardsleyi was established, with the advantages of high specificity, rapid detection (25-40 min), instrument flexibility and visual results. This detection system offers an invaluable tool for the early identification and on-site detection of invasive mealybugs, and provides guidance for the development of suitable management strategies.

Key words: Pseudococcus jackbeardsleyi, recombinase polymerase amplification, CRISPR/Cas12a assay, fluorescent detection, lateral flow assay