Comparison of DNA Extraction and Amplification Techniques for Use with Engorged Hard-Bodied Ticks

Abstract

Tick-borne infections are a serious threat to humans, livestock, and companion animals in many parts of the world, often leading to high morbidity and mortality rates, along with decreased production values and/or costly treatments. The prevalence of the microbes responsible for these infections is typically assessed by the molecular identification of pathogens within the tick vectors. Ticks sampled from animals are often engorged with animal blood, presenting difficulties in the amplification of nucleic acids due to the inhibitory effects of mammalian blood on the enzymes used in polymerase chain reactions (PCRs). This study tested two tick preparation methods, three methods of DNA extraction, and four commercially available DNA polymerases to determine the most reliable method of extracting and amplifying DNA from engorged ticks. Our study found that the phenol–chloroform extraction method yielded the highest concentration of DNA, yet DNA extracted by this method was amplified the least successfully. Thermo Scientific’s Phusion Plus PCR Master Mix was the best at amplifying the tick 16s rRNA gene, regardless of extraction method. Finally, our study identified that using the Qiagen DNeasy Blood & Tissues kit for DNA extraction coupled with either Phusion Plus PCR Master Mix or GoTaq DNA polymerase Master Mix is the best combination for the optimized amplification of DNA extracted from engorged ticks.