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

## TL;DR

This paper introduces a new PCR method to identify four thrips species using a COI gene region, enabling efficient and accurate species discrimination in agricultural settings.

## Contribution

The study introduces a species-specific COI PCR framework optimized for crude DNA extracts, enabling rapid and field-adaptable identification of co-occurring thrips species.

## Key findings

- Species-specific amplification was achieved using COI gene polymorphisms in mixed-species samples.
- PCR inhibition from crude extracts was effectively mitigated by mild dilution.
- The method supports field-oriented workflows and potential integration with visual nucleic acid detection platforms.

## Abstract

Thrips are tiny insects that feed on many crops and can spread plant viruses. Different thrips species often co-occur on the same host plants but are morphologically similar, making species-level molecular identification challenging under field-oriented conditions. This is important because species differ in how much damage they cause; how they respond to insecticides; and whether they spread viruses. In this study, we developed a simple detection system that can quickly tell apart four important thrips species that attack crops in southern China; including one globally invasive species; the western flower thrips. The test uses species-diagnostic regions of the mitochondrial COI gene and a short-turnaround DNA extraction workflow for processing individual insects. Our results show that species-specific and reproducible amplification can be achieved under mixed-species backgrounds, and that PCR inhibition can be effectively alleviated by mild dilution of crude insect lysates. This framework supports laboratory- and field-oriented molecular identification workflows for greenhouse and open-field thrips surveillance and provides a methodological basis for potential integration with simplified visual nucleic acid readout platforms in future applications.

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.