Development of a highly accurate and sensitive diagnostic tool for pyrethroid-resistant chimeric P450 CYP337B3 of...

Recent studies have shown that pyrethroid resistance in the cotton bollworm (CBW) Helicoverpa armigera is conferred by the generation of a chimeric CYP337B3 gene, which resulted from unequal crossing-over between the CYP337B1 and CYP337B2 genes. In this study, we developed a diagnostic protocol based on the loop-mediated isothermal amplification (LAMP) assay for the detection of chimeric CYP337B3. The CYP337B3 LAMP assay utilized six primers and generated strong fluorescence signals visible to the naked eye under normal or ultraviolet light. The primers were designed based on CYP337B3v1 (JQ995292), the major allele detected in Australia. The detection limit of this LAMP assay was 10 fg genomic DNA in a 25-µl reaction mixture. Compared with CYP337B2v1, the Korean CYP337B3v2 allele had two nucleotide mismatches within the amplifying regions of this LAMP assay; therefore, we confirmed that polymerase chain reaction-synthesized CYP337B3v2 was well amplified using this LAMP assay. In addition, we determined that the presence of CYP337B3 from H. armigera collected by pheromone traps from Korean fields could be confirmed using this LAMP assay. This assay could detect CYP337B3 even in heterozygotes, which is relevant because CYP337B3 is dominant, and heterozygotes are pyrethroid resistant. Therefore, the newly developed CYP337B3 LAMP assay could detect the presence of pyrethroid resistance in H. armigera that were captured by pheromone traps during the early season and provide information on whether pyrethroids could be used to control H. armigera.