Qualitative Sybr Green real-time detection of single nucleotide polymorphisms responsible for target-site resistance in insect pests: the example of Myzus persicae and Musca domestica

Abstract

Chemical insecticides have been widely used to control insect pests, leading to the selection of resistant populations. To date, several single nucleotide polymorphisms (SNPs) have already been associated with insecticide resistance, causing reduced sensitivity to many classes of products. Monitoring and detection of target-site resistance is currently one of the most important factors for insect pest management strategies. Several methods are available for this purpose: automated and high-throughput techniques (i.e. TaqMan or pyrosequencing) are very costly; cheaper alternatives (i.e. RFLP or PASA-PCRs) are time-consuming and limited by the necessity of a final visualization step. This work presents a new approach (QSGG, Qualitative Sybr Green Genotyping) which combines the specificity of PASA-PCR with the rapidity of real-time PCR analysis. The specific real-time detection of Cq values of wild-type or mutant alleles (amplified used allele-specific primers) allows the calculation of ΔCqW-M values and the consequent identification of the genotypes of unknown samples, on the basis of ranges previously defined with reference clones. The methodology is applied here to characterize mutations described in Myzus persicae and Musca domestica and we demonstrate it represents a valid, rapid and cost-effective technique that can be adopted for monitoring target-site resistance in field populations of these and other insect species.

Transcriptome Analysis and Identification of Major Detoxification Gene Families and Insecticide Targets in Grapholita Molesta (Busck) (Lepidoptera: Tortricidae)

“…Target-site insensitivity was another insecticide resistance mechanism, which is often caused by point mutations in target protein ( Puggioni et al 2016 ). Unigenes with high sequence similarity to genes encoding insecticide targets or having functions in insecticide metabolism were also identified in G. molesta ( Table 6 ).…”

Structuration of multilocus genotypes associated with insecticide resistance of the peach potato aphid,Myzus persicae(Sulzer), in potato fields in southern Belgium

“…Its reproductive mode depends on lineages, climatic conditions and the availability of the primary host, Prunus persica, required for sexual reproduction. 17 Molecular methods to detect the main insecticide resistance mutations in M. persicae have been described 10,[18][19][20][21][22] and the use of neutral microsatellite markers has been shown to allow the assessment of clonal diversity and to infer the reproductive patterns. 15,21,[23][24][25] In combination, DNA diagnostic and genotyping methods are useful tools to detect the presence of resistances, to understand their evolution and to support reflection in pest management.…”

Target site mutations underlying insecticide resistance in Tunisian populations of Myzus persicae (Sulzer) on peach orchards and potato crops

“…The Qualitative Sybr Green real‐time for Genotyping (QSGG) 33 approach allowed the characterization of mutations linked to insecticide resistance by target‐site insensitivity to neonicotinoids, pyrethroids and carbamates in Tunisian populations of M. persicae .…”

“…PCR reactions were run on a CFX96™ Real-Time system (Bio-Rad, Hercules, CA, USA) according to the protocols by Puggioni and colleagues with some modification. 33 Each PCR reaction (10 μL) contained 3 μL of 1:10 diluted DNA extracted from a single aphid, 0.4 μM of each forward and reverse primers for all targets (0.5 μM for L1014F) and 5 μL of iTaq Universal SYBR® Green Supermix (Bio-Rad). DNA amplifications were performed using the following steps: 5 min of initial denaturation at 95°C followed by 40 cycles of denaturation at 95 °C for 5 s, 30 s at annealing temperature (depending on the target) (Table S2), and then 60 s of elongation at 72 °C.…”