Development of a rapid isothermal-based assay for detection of whitefly MEAM1 and MED biotypes using recombinase...

Whiteflies Bemisia tabaci (Hemiptera: Aleyrodidae) are well-known agricultural pests responsible for transmitting plant viruses and causing significant crop damage. B. tabaci is a complex of different cryptic species for which molecular techniques are crucial for discriminating between variants like the MEAM1 (formerly B) and MED (formerly Q) types of B. tabaci due to morphological inconsistencies. This study presents the development of a rapid, isothermal-based assay using recombinase polymerase amplification (RPA) to detect Whitefly MEAM1 and MED biotypes. By targeting mtcox1-specific genomic regions unique to each biotype, this assay provides a sensitive and efficient method for identifying these economically important whitefly variants in under 90 min, without the need for nucleic acid extraction and PCR machine; direct whitefly samples serve as templates and a small heat block is sufficient for assay performance. The study demonstrates successful primer validation, ensuring the specificity and sensitivity of the assay. Specifically designed primers accurately identified target mtcox1 regions unique to each type, enhancing the assay's precision. The RPA assay exhibited high specificity, detecting only the target variants without cross-reactivity to related species or non-target organisms. Additionally, the assay demonstrated exceptional sensitivity, capable of detecting ten-copy targets in Artificial Plasmid Control plasmid control and 100 pg whitefly nucleic acid. These results highlight the potential of RPA as a valuable tool for rapid and accurate detection of Whitefly MEAM1 and MED biotypes, aiding in the development of efficient pest control strategies in agriculture.