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.
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.


CONCLUSIONS
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.
Sarkar
Manas
M
Medical Entomology Division, Defence Research Laboratory (DRDO), Tezpur, Assam, India. manas_sarkar54491@yahoo.com
Baruah
Indra
I
Srivastava
Ravi B
RB
Borkotoki
Aparajita
A
Bhattacharyya
Indra Kumar
IK
D000818:Animals; D003465:Culex / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D007305:Insecticide Resistance; D007306:Insecticides / Q000494:pharmacology; D015335:Molecular Probes / Q000235:genetics; D009154:Mutation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011722:Pyrethrins / Q000494:pharmacology*; D044366:Transition Temperature
D019476:Insect Proteins; D007306:Insecticides; D015335:Molecular Probes; D011722:Pyrethrins