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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

Published online by Cambridge University Press:  22 July 2016

V. Puggioni ,
O. Chiesa ,
M. Panini  and
E. Mazzoni
V. Puggioni
Affiliation:
Department of Sustainable Crop Production, Section Sustainable Crop and Food Protection, Università Cattolica del Sacro Cuore, Via Emilia Parmense, 84, I-29122 Piacenza, Italy
O. Chiesa
Affiliation:
Department of Sustainable Crop Production, Section Sustainable Crop and Food Protection, Università Cattolica del Sacro Cuore, Via Emilia Parmense, 84, I-29122 Piacenza, Italy
M. Panini
Affiliation:
Department of Sustainable Crop Production, Section Sustainable Crop and Food Protection, Università Cattolica del Sacro Cuore, Via Emilia Parmense, 84, I-29122 Piacenza, Italy
E. Mazzoni*
Affiliation:
Department of Sustainable Crop Production, Section Sustainable Crop and Food Protection, Università Cattolica del Sacro Cuore, Via Emilia Parmense, 84, I-29122 Piacenza, Italy
*
*Address for correspondence Fax: +39 0523 599268 Phone: +39 0523 599237 E-mail: [email protected]
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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.

Keywords

SNPs genotypingqualitative real-time PCRM. persicaeM. domesticaR81Tkdrs-kdrkdr-hisM918LMACE
Type
Research Papers
Information
Bulletin of Entomological Research , Volume 107 , Issue 1 , February 2017 , pp. 96 - 105
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Copyright
Copyright © Cambridge University Press 2016 

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