Second to mosquitos, ticks are considered the most dangerous arthropod vectors worldwide for the transmission of zoonotic infections [1]. Due to the blood meal processes in their life cycle, each tick is given numerous opportunities to pick up and spread new pathogens between their hosts. In addition to carrying numerous pathogens known to cause disease in humans and livestock, environmental factors are leading to expanding habitats of many tick species, resulting in previously unseen pathogens making their way into new environments via tick vectors [2].
This increasing burden of tick-borne disease has prompted numerous public health efforts for mitigation and epidemiologic surveillance of various disease-carrying ticks and the pathogens circulating within them. To fully evaluate the risk posed by a given tick population, their pathogen burden must be characterized and understood. The most sensitive and specific screening methods for pathogens seen in arthropod vectors currently rely on PCR-based testing; a tick may be screened by utilizing targeted primers for the presence of DNA from pathogens of interest. These screening measures can help medical and public health professionals identify, prepare for, and treat potential emerging infections in their communities.
These examination methods require DNA extraction from the tick, which can prove quite challenging due to their hard exoskeleton making complete lysis difficult to achieve. Conventional lysis methods typically involve manually bisecting the tick and allowing it to undergo enzymatic digestion for extended periods, ranging from a few hours to overnight incubations [3]. This extended digestion process prolongs the total time of the extraction for the DNA, delaying downstream analysis and significantly reducing throughput compared to other tissues processed similarly.
Mechanical disruption can be used to achieve lysis in less time when compared with enzymatic lysis, and the yield of DNA can be dramatically increased. One of the most effective forms of mechanical lysis is bead-beating homogenization. Bead-beating has been shown to produce equivalent results upon repeated trials across a wide range of tissue and sample types [4].
In addition to complete sample lysis, the quality of DNA extracted also plays a significant role in the effectiveness of downstream applications. Labs can also consider introducing automation in as many steps as possible. The chemagic™ 360 instrument is an automated nucleic acid extractor; using its patented magnetic bead technology, the chemagic 360 instrument allows for sample processing for DNA extraction in less time and with greater efficiency than manual DNA extraction methods [4].
Detection of pathogens via traditional PCR or bioinformatic methods can be costly and time-consuming to verify each pathogen that may be in the sample. Using multiplexed assays to target multiple pathogens per reaction can dramatically reduce processing time and allow deeper coverage of a tick’s actual pathogen burden. Herein, we utilize a multiplexed tick-borne pathogen (TBP) panel from ChromaCode to quantify the pathogen DNA of eight common tick pathogens relative to human diseases. ChromaCode leverages their proprietary High Definition PCR (HDPCR™) chemistry to detect nine of the most common tick borne pathogens in a single qPCR reaction.
The following study demonstrates a complete workflow incorporating the Omni Bead Ruptor Elite bead mill homogenizer, the chemagic 360 automated nucleic acid extractor and the HDPCR TBP Panel. Utilizing this workflow, we describe a method that can dramatically decrease the time required to screen a population of ticks and still detect low DNA copy numbers.
Table 1: Detection profile for nine tick-borne pathogens utilizing the ChromaCode HDPCR tick-borne pathogens kit on spiked samples. The spike columns represent how many replicates were detected at each dilution.