Abstract: Abstract: 【Aim】To apply the droplet digital PCR (ddPCR) technology to establish a multiplex and precise detection method for three quarantine mealybug species, Planococcus minor, Pl. lilacinus and Paracoccus marginatus intercepted at high frequency. 【Methods】Using common mealybug species Pl. minor, Pl. lilacinu, Pa. marginatus, Dysmicoccus neobrevipes, Pseudococcus jackbeardsleyi, Maconellicoccus hirsutus, Ps. comstocki and Pl. citri intercepted at ports and collected domestically as test insects, the COⅠ gene sequence was used as the target. Homology alignment and design of specific primers and probes were performed using the GenBank database, MEGA11 and Beacon Designer 8. Based on the single ddPCR detection system of the three mealybug species Pl. minor, Pl. lilacinus and Pa. marginatus, the reaction system was optimized by screening the optimal concentrations of primers and probes, thereby establishing a multiplex ddPCR method for the rapid and absolute quantitative detection of the genomic copy numbers of Pl. minor, Pl. lilacinus and Pa. marginatus in the same reaction system, and compared with TaqMan qPCR method to determine the sensitivity, quantification limit and accuracy of this method. 【Results】 All sets of primers and probes could amplify the genomes of target mealybugs in the multiplex ddPCR detection system without cross-reactivity, and the lowest DNA concentration detected was consistent with that of TaqMan qPCR. The lower limit of absolute quantification of this nultiplex ddPCR detection system for the genomic detection of Pl. minor was 0.44 copies/μL, that for the genomic detection of Pl. lilacinus was 0.43 copies/μL, and that for the genomic detection of Pa. marginatus was 0.44 copies/μL. The linear coefficients of determination R² for the quantitative assay of this nultiplex ddPCR detection system were all greater than 0.99 within the range of quantification, and the reproducibility and linear correlation were good. 【Conclusion】The multiplex ddPCR detection method established in this study can realize the rapid and precise identification of three quarantine mealybug species Pl. minor, Pl. lilacinus and Pa. marginatus intercepted at high frequency, and provide technical service and support for the prevention and control of foreign pests at ports and the protection of China’s ecological security.
Key words: Planococcus minor; Planococcus lilacinus; Paracoccus marginatus; molecular identification; molecular marker; droplet digital PCR

Key words:  Planococcus minor, Planococcus lilacinus, Paracoccus marginatus, molecular identification, molecular marker, droplet digital PCR