Optimization of Dipstick-Based DNA Extraction for Cassava Mosaic Disease Detection Using Response Surface Methodology

Abstract

Cassava mosaic disease (CMD), caused by cassava mosaic begomoviruses (CMBs), remains a major constraint on cassava production in Nigeria, resulting in significant yield losses. Management of CMD requires rapid, reliable diagnostics, but the complexity of current DNA extraction methods constrains field application. This study presents an optimized paper-based dipstick for DNA extraction from cassava leaves, developed to facilitate real-time disease detection. Five extraction parameters: sample size, number of binding dips, washing dips, washing steps, and elution dips were evaluated using Design Expert® software. DNA extracted with the dipstick method was compared with that obtained using the CTAB method and amplified through PCR using the ACMV21F/R primer pair. The number of washing dips, the number of washing steps, and the sample size significantly affected the yield and purity of the extracted DNA. The optimal conditions for DNA extraction using the dipstick were 7 washing dips, 1 washing step, and 50 mg of leaf tissue. Under optimal conditions, average DNA yields of 78.2 ± 14.01 ng/µL and 1727.65 ± 584.42 ng/µL were obtained from the dipstick and CTAB method, respectively. Similarly, DNA purity values ranged from 1.75 to 2.06 with the dipstick method, whereas 1.99 to 2.21 were obtained with the CTAB method. Overall, both methods produced DNA with adequate concentration and purity for PCR amplification. The dipstick DNA extraction method provides a rapid, easy, and cost-effective means for diagnosing cassava viruses and enables early detection in resource-limited settings.