A Novel Strategy for the Detection of Aedes albopictus kdr gene Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology

Abstract

Abstract Objectives The objective of this study was to construct a novel strategy for the mutation detection of knockdown resistance(kdr)gene in Aedes albopictus using multiplex PCR-mass spectrometry minisequencing technique (mPCR-MS minisequencing). Methods Based on the single-base mass probe extension of multiplex PCR amplification products in wild and mutant genotype, a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) method was established to detect 3 mutated sites in Ae. albopictus kdr gene: locus 1016,1532 and 1534. Results The detection of the three sites can be conducted simultaneously by double PCR amplification combined with MALDI-TOF MS, achieving a detection limit of 20fg/ul. This method is extensible and flexible, and can be used in a high-throughput manner, easily allowing the addition of new mutation sites as needed to identify and track new kdr gene as they emerge. Conclusions mPCR-MS minisequencing provides a new option for the detection of kdr gene in Ae. Albopictus.