Multiplex-PCR differentiation of two Hyalomma and two Haemaphysalis species (Acari: Ixodidae)

Hyalomma and Haemaphysalis are two most important genera of ticks (Acari: Ixodidae). The rapid and accurate identification of field collected specimens is crucial in faunal works, laboratory assays, anti-tick vaccine experiments, etc. The present study was designed to introduce a rapid and more sensitive method for the differentiation of closely related Hyalomma and Haemaphysalis species namely the pairs Hy. anatolicum-Hy. asiaticum and Ha. punctata-Ha. sulcata, as the main vectors of different animal and human pathogenic agents. Tick specimens were collected from domestic animals in Ardabil, Hormozgan, Khuzestan, Kurdistan, Lorestan, and Mazandaran and identified according to the taxonomic keys. DNA was extracted by the Phenol-Chloroform method. Then, PCR was carried out in a single PCR reaction tube using three pairs designed primers (one forward and two reverse) adapted from the internal transcribe spacer 2 (ITS2) and cytochrome oxidase subunit 1 (COI) genes for Hyalomma and Haemaphysalis, respectively. In the present study, results of Multiplex-PCR revealed that the pairs Hy. anatolicum-H. asiaticum and Ha. punctata-Ha. sulcata could be well differentiated on gel electrophoresis. Morphological misidentification of Hy. anatolicum-Hy. asiaticum and Ha. punctata-Ha. sulcata could be reduced significantly after using Multiplex-PCR.