A novel real-time PCR assay for the speciation of medically important ticks.

The identification of ticks using morphological characters is a well-established practice, however specimens that are small or damaged are often difficult to speciate. A novel, rapid real-time PCR assay, which targets the second internal transcribed spacer (ITS2) region in the nuclear ribosomal DNA gene, was developed for identification of four tick species of utmost medical importance in the United States: Ixodes scapularis, I. pacificus, Dermacentor variabilis, and Amblyomma americanum. Computational analyses of public databases and DNA sequencing studies revealed regions that could be specifically targeted with oligonucleotides optimized for TaqMan chemistry. The oligonucleotide sets designed in this study are specific at both the genus and species levels, and are sensitive at 0.1-1 pg of total tick DNA.