Frequency detection of pyrethroid resistance allele in Anopheles sinensis populations by real-time PCR amplification of specific allele (rtPASA)

To investigate the level of pyrethroid resistance in Anopheles sinensis Wiedemann 1828 (Diptera: Culicidae), a major malaria vector in Korea, we cloned and sequenced the IIS4-6 transmembrane segments of the sodium channel gene that encompass the most widely known kdr mutation sites. Sequence analysis revealed the presence of the major Leu-Phe mutation and a minor Leu-Cys mutation at the same position in permethrin-resistant field populations of An. sinensis. To establish a routine method for monitoring resistance, we developed a simple and accurate real-time PCR amplification of specific allele (rtPASA) protocol for the estimation of resistance allele frequencies on a population basis. The kdr allele frequency of a field population predicted by the rtPASA method (60.8%) agreed well with that determined by individual genotyping (61.7%), demonstrating the reliability and accuracy of rtPASA in predicting resistance allele frequency. Using the rtPASA method, the kdr allele frequencies in several field populations of An. sinensis were determined to range from 25.0 to 96.6%, suggestive of widespread pyrethroid resistance in Korea.