Development of a highly accurate and sensitive diagnostic tool for pyrethroid‐resistant chimeric P450 CYP337B3 of Helicoverpa armigera using loop‐mediated isothermal amplification

Abstract

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.

Development of a simple and accurate molecular tool forSpodoptera frugiperdaspecies identification using LAMP

“…As previously reported, the amount of DNA is generally not the limiting factor for the LAMP assay (Choi et al, 2018). Varying the incubation time can generally compensate for different amounts of DNA.…”

“…In another attempt to decrease the incubation time, we applied two other loop primers, loop forward (LF) between the F2 and F1 regions and loop backward (LB) between the B1 and B2 regions, which have been suggested to amplify additionally from the loops in the dumbbell forms (Choi et al, 2018). Finding suitable loop primers was difficult because of the high AT content, and the strategy of adding overhanging 'G's to the 5' end to increase the melting temperature was hard to use in the inner primers.…”

Development of a simple and accurate molecular tool for Spodoptera frugiperda species identification using LAMP

“…As previously reported, the amount of DNA is generally not the limiting factor in the LAMP assay. 20 Incubation temperature is more important for specificity. At temperatures below 59°C, M. separata produced false positives with four primers in this study (data not shown).…”

“…A simpler technique, loopmediated isothermal amplification (LAMP) is widely used for the rapid identification of pest species. [17][18][19][20] It is performed under isothermal conditions that require a set of four primers, a stranddisplacing DNA polymerase, and a water bath or heat block to maintain the temperature at about 60-65°C following one-time denaturation at 95°C or one-step incubation. 21,22 There are no additional steps.…”

Monitoring of four major lepidopteran pests in Korean cornfields and management of Helicoverpa armigera

“…In total, eight CYP337B3 alleles are known worldwide. Among them, two alleles (CYP337B3v2 and v7) were identified and we developed a highly accurate and sensitive diagnostic tool for pyrethroid-resistant chimeric P450 CYP337B3 of H. armigera using loop-mediated isothermal amplification (Choi et al 2018). Recently the genome of H. armigera was sequenced and annotated (Pearce et al 2017).…”