A Species-Specific COI PCR Approach for Discriminating Co-Occurring Thrips Species Using Crude DNA Extracts

Thrips are cosmopolitan agricultural pests and important vectors of plant viruses, and the increasing coexistence of multiple morphologically similar species has intensified the demand for species-specific molecular identification. However, traditional morphological identification and PCR assays using universal primers are often inadequate for mixed-species samples and field-adaptable application. In this study, we developed a species-specific molecular identification framework targeting a polymorphism-rich region of the mitochondrial cytochrome c oxidase subunit I (COI) gene, which is more time-efficient than sequencing-based COI DNA barcoding, for four economically important thrips species in southern China, including the globally invasive Frankliniella occidentalis. By aligning COI sequences, polymorphism-rich regions were identified and used to design four species-specific primer pairs, each containing a diagnostic 3'-terminal nucleotide. These primers were combined with a PBS-based DNA extraction workflow optimized for single-insect samples that minimizes dependence on column-based purification. The assay achieved a practical detection limit of 1 ng per reaction, demonstrated species-specific amplification, and maintained reproducible amplification at DNA inputs of ≥1 ng per reaction. Notably, PCR inhibition caused by crude extracts was effectively alleviated by fivefold dilution. Although the chemical identities of the inhibitors remain unknown, interspecific variation in inhibition strength was observed, with T. hawaiiensis exhibiting the strongest suppression, possibly due to differences in lysate composition. This integrated framework balances target specificity, operational simplicity, and dilution-mitigated inhibition, providing a field-adaptable tool for thrips species identification and invasive species monitoring. Moreover, it provides a species-specific molecular foundation for downstream integration with visual nucleic acid detection platforms, such as the CRISPR/Cas12a system, thereby facilitating the future development of portable molecular identification workflows for small agricultural pests.