Comparison of Methods for Economic and Efficient Tick and Borrelia DNA Purification

In this study, we compared factors that may have an influence on DNA extraction namely commercially available DNA extraction kits vs alkaline hydrolysis for DNA extraction. The methods were applied to questing Ixodes (I.) ricinus ticks and Borrelia cultures of defined cell concentrations. A total of 69 questing I. ricinus ticks were collected. From 34 ticks, total DNA was extracted using a commercial DNA extraction kit. Thirty-five ticks were treated with 1.25 % ammonium hydroxide (NH4OH). Six ticks from each batch were placed in 70 % ethanol (EtOH) for one week prior to DNA extraction to see the effect of EtOH preservation on total DNA yield. DNA yield was estimated in field-collected ticks using conventional PCR targeting the Ixodes Cytochrome C oxidase (coi) gene and in cultured Borrelia isolates using quantitative real-time PCR (qPCR) targeting the FlaB encoding gene of Borrelia.Column DNA extraction yielded slightly better results than NH4OH treatment when tested in a PCR targeting a tick-specific coi gene (96 % PCR-positive vs 86 % PCR-positive results, respectively). EtOH preservation had a slightly negative effect on DNA yield and – again – slightly stronger PCR products were observed by commercial kit extraction. A Shapiro-Wilk test conducted revealed a significance-level of 90 % for both the methods, indicating a normal distribution of the values generated by BioNumerics quantification. A two-sided t-test conducted revealed a significant (p < 0.01) mean ...
Source: Ticks and Tick borne Diseases - Category: Zoology Source Type: research