Simple, rapid and cost-effective DNA extraction techniques for detection of economically important fruit flies in...

Fruit flies (Diptera: Tephritidae) include nearly 200 economically important species, many of which are quarantine pests. Accurate identification is vital for pest management, but morphological diagnosis is often difficult for non-specialists due to similarities in diagnostic traits. DNA barcoding has become the gold standard, providing precise species identification using molecular markers. However, conventional DNA extraction methods, though reliable, are time-consuming and require advanced laboratory facilities, while commercial kits are costly and unsuitable for large-scale use and resource-limited settings. To address this, we established four new rapid and inexpensive DNA extraction methods using Tween 20 + NaOH solution (RDI) buffer, Phosphate Buffer Saline (PBS), Tris-EDTA (TE) buffer and Chelex + Proteinase K solution (Chelex buffer). These methods consistently yielded DNA of sufficient quality and concentration across five major tephritid pests: Zeugodacus cucurbitae, Z. tau, Bactrocera dorsalis, B. divenderi and B. zonata. DNA integrity was confirmed through fluorometric and spectrophotometric analysis and successful amplification of the mitochondrial COI gene.•Here, we developed rapid and inexpensive DNA extraction protocols capable of producing DNA from five major fruit fly pests..•Requires only 20-45 min, without special equipment and produces DNA of sufficient quality for PCR-based barcoding.•Provides a practical alternative for resource-poor laboratories.