Species-specific primers in multiplex PCR for Bactrocera minax identification using an internal transcribed spacer

Bactrocera minax (Enderlein), commonly known as the Chinese citrus fly, is a citrus pest native to China and nearby countries. B. minax can cause substantial losses in citrus orchards. B. minax can be spread to countries free from it by the global trade of citrus and by travelers carrying citrus. Timely, convenient, and accurate identi-fication of B. minax is essential in preventing its spread. In the present study, the nuclear ribosomal internal transcribed spacer 2 (ITS2) amplicon was used to design species-specific primer pairs that enable B. minax to be distinguished from 11 other fruit fly species. Four forward and four reverse species-specific primers were designed, and out of all possible sets of species-specific primer pairs obtained after intermixing them, seven sets of species-specific primer pairs were able to accurately identify B. minax. For B. minax identification, specific fragments ranging from 83 to 431 base pairs in length were amplified. The validity of the specific band only in B. minax was determined by visually inspecting the gel profile of the PCR product. B. minax was correctly identified using the designed species-specific primer pairs, with no cross-amplification with the 11 other fruit fly species included in the experiments. In addition to saving DNA sequencing costs, the application of these species-specific primer pairs facilitates rapid identification of B. minax, with identification in border scenarios being completable within 2-3 h.