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.

Amplicon-Based High-Throughput Sequencing Method for Genotypic Characterization of Norovirus in Oysters

“…AmpliTaq Gold has been widely applied in molecular virology ( 37 – 39 ), though there is limited literature evaluating its performance relative to other DNA polymerases. In this case, LunaScript combined with AmpliTaq Gold provided the highest-quality sequences; however, previous studies with other complex sample types provide conflicting results ( 27 , 29 , 40 – 43 ). Several factors may contribute to the performance of RT and DNA polymerases, such as low-input genomic material, RNA quality, matrix-specific factors, target-specific factors, and DNA synthesis speed.…”

A simple, effective and inexpensive method to isolate the nucleic acid (DNA/RNA) from a single tick for molecular detection of various pathogens

“…In another study, the comparison between different DNA isolation methods were done, but it was found that up to 2000 µg/mL DNA can be isolated with chloroform phenol method, it is not mentioned whether they used pooled samples, but it seems that this study used pooled ticks and used a cumbersome method to remove the parts of ticks, in spite of this, our mentioned method here is also in position to provide high amount of isolated nucleic acid from single tick [7].…”

“…Polymerase chain reaction (PCR) is one of the impor-tant detection methods to detect various pathogens like SARS CoV-2. Influenza virus, Hepatitis C virus, Coxiella burnetii and many other pathogens [1][2][3][4][5][6][7][8][9]. Moreover there are large number of RNA viruses in ticks, which are needed to be addressed [10].…”

Development of an amplicon-based high throughput sequencing method for genotypic characterisation of norovirus in oysters

“…however, previous studies in other complex sample types provide conflicting results (30,32,(43)(44)(45)(46). Several factors may contribute to the performance of RT and DNA polymerase enzymes, such as low input genomic material, RNA quality, matrixspecific factors, target-specific factors and DNA synthesis speed.…”