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High‐throughput approach to detection of knockdown resistance ( kdr ) mutation in mosquitoes, Culex quinquefasciatus , based on real‐time PCR using single‐labelled hybridisation probe/melting curve analysis
Author(s) -
Sarkar Manas,
Baruah Indra,
Srivastava Ravi B,
Borkotoki Aparajita,
Bhattacharyya Indra Kumar
Publication year - 2011
Publication title -
pest management science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.296
H-Index - 125
eISSN - 1526-4998
pISSN - 1526-498X
DOI - 10.1002/ps.2044
Subject(s) - knockdown resistance , genotyping , culex quinquefasciatus , biology , melting curve analysis , culex , genetics , gene knockdown , population , genotype , insecticide resistance , pesticide resistance , microbiology and biotechnology , mutation , polymerase chain reaction , toxicology , larva , pesticide , deltamethrin , gene , aedes aegypti , ecology , medicine , environmental health , cyfluthrin
BACKGROUND: Knockdown resistance ( kdr ) mutation (L1014F) is a well‐defined mechanism of resistance to pyrethroids and DDT in many insect species. Sensitive detection of the mutations associated with resistance is a prerequisite for resistance management strategies. The authors have developed a new real‐time molecular diagnostic assay based on SimpleProbe ® /melting curve analysis for large‐scale kdr genotyping in the wild population of Culex quinquefasciatus Say, the principal vector of bancroftian filariasis. Melting curve analysis is based on the thermal stability difference between matched and mismatched DNA duplexes. The application of SimpleProbe ® chemistry in insects described here is novel in entomology research. RESULTS: The mosquitoes homozygous for knockdown‐resistant and knockdown‐susceptible allele showed melting peaks at 60.45 °C ( ± 0.25) and 64.09 °C ( ± 0.24) respectively. The heterozygous mosquitoes yielded both peaks at approximately 60.5 °C ( ± 0.2) and 64.20 °C ( ± 0.23). Among the 92 samples genotyped, 16 were found to be homozygous resistant, 44 homozygous susceptible and 32 heterozygous. Comparative assessments were made of all the reported methods for kdr genotyping. CONCLUSION: The present method is cheaper, faster, more reliable and versatile than other alternatives proposed in detecting correct kdr genotypes in mosquitoes. This is the first report using a single‐labelled hybridisation probe to detect point mutations in insect populations. Copyright © 2010 Society of Chemical Industry