Single-tube real-time PCR assay for differentiation of Ixodes affinis and Ixodes scapularis

Ixodes affinis Neumann (1899) and Ixodes scapularis Say (1821) are tick vectors of the etiologic agent of Lyme disease, Borrelia burgdorferi sensu stricto. Ixodes affinis and I. scapularis are morphologically very similar, and as they are sympatric in the mid- and south-Atlantic U.S. coastal states, their accurate identification is crucial to studies of disease and vector ecology in this area. This work describes a rapid, single-tube SYBR® Green-based real-time PCR assay for differentiation of I. affinis and I. scapularis at all life stages. The assay employs 2 pairs of species-specific primers directed against the internal transcribed spacer 2 (ITS2) region of the nuclear rRNA operon. Amplification products for these primer pairs differ in size and may be differentiated with a melt curve analysis. This tool is intended as a supplement to morphological methods for accurate identification of these ticks.