Rapid Identification of Tropical Important Mealybugs Based on a Multiplex PCR Assay

The mealybug can severely threaten agricultural and horticultural crops and has a widespread distribution in tropical regions, particularly in high-risk invasion areas such as Hainan, which is an important trade port with superior geographical conditions. Traditional morphological methods can no longer meet the requirements for the rapid and precise identification of different insect stages or debris. DNA barcoding has been used to establish efficient molecular identification tools. In this study, a multiplex polymerase chain reaction (mPCR) assay based on the cytochrome c oxidase subunit I (COI) gene was successfully constructed for the rapid identification of mealybugs. The 5′ end COI gene fragments of 12 mealybug species were amplified and sequenced. Furthermore, an mPCR assay was established to identify three common mealybug species in Hainan, namely Dysmicoccus neobrevipes, Maconellicoccus hirsutus, and Paracoccus marginatus. Condition optimization, sensitivity detection, and field sample testing results prove that the assay can identify the three target species through a single PCR amplification. A sample DNA concentration of as low as 0.1–1 ng/μL can be detected. Additionally, the assay in conjunction with barcode sequencing can identify mealybugs collected in the field, clarifying the distribution and host plants of 12 mealybug species commonly found in Hainan. Thus, the rapid identification of important mealybug species is realized. The establishment of this technology provides an economical and efficient molecular tool for the quarantine and monitoring of mealybugs in Hainan and other regions, which are essential for the detection, monitoring, and early warning of invasive organisms.