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

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 minutes, without special equipment and produces DNA of sufficient quality for PCR-based barcoding. • Provides a practical alternative for resource-poor laboratories.