Polymerase chain reaction detection efficiency of the human granulocytic ehrlichiosis agent (Rickettsiaceae: Ehrlichieae) in ticks (Acari: Ixodidae) is dependent on the DNA extraction method.

Abstract

Several methods of extracting DNA from ticks were examined to improve the efficiency of polymerase chain reaction (PCR) detection of the human granulocytic ehrlichiosis (HGE) agent. DNA was extracted from laboratory-reared uninfected and HGE-infected ticks using 3 separate methods. In one treatment, unfed nymphs and engorged larvae of Ixodes scapularis Say, either individually or in pools of 3, were homogenized in 40 microliters of 1x PCR buffer and boiled for 30 min. A 2nd group of ticks was extracted using the QiaAmp Tissue kit, a silica column separation method. A 3rd group was extracted with DNA-STAT, a guanidinium thiocynate method. Five microliters of each extract was used for PCR amplification. Pathogen-free tick DNA samples did not amplify a product. Laboratory-infected ticks extracted either with the QiaAmp kit or those homogenized and boiled in PCR buffer amplified product in 37.5% and 87.5% of the samples, respectively. Infected ticks extracted with DNA STAT-60 amplified a product in 100% of samples. No differences were observed in detection efficiency between ticks tested singly or in pools.