Behind mosquitos, ticks are the most common vector for pathogens that not only affect humans but livestock as well (1). As climate change and deforestation occur, these vectors are expanding to new areas, and as a result introducing previously unseen pathogens (2). As such, it should come as no surprise that ticks are becoming a hot topic for public health researchers. Fully examining the pathogen burden in a tick, however, can be a challenging endeavor. Part of the reason is due to the difficulty of fully exposing the genetic material of the pathogens within due to the tick’s hard exoskeleton.
Typical methods of examining a tick’s pathogen burden involve a lengthy process of incubation, sectioning, and enzymatic lysis. While these methods are effective, they introduce a considerable time burden limiting the overall sample throughput the researcher can achieve. Bead mill homogenization is a method that can be used to decrease time of sample preparation required for extraction and detection while also being robust enough to handle the tough nature of tick samples. The Bead Ruptor EliteTM Homogenizer is one of the most powerful bead mills on the market that is not only capable of disrupting even the toughest samples, but also provides a time saving alternative compared to traditional lysing methods. In addition to OMNI scientists, others have seen the effectiveness of our bead mills. Full lysis of the tick and its pathogenic payload is an important enough topic that it recently has received ample attention to be granted funding by the Department of Defense (DoD), Defense Health Program. In a study published in the Journal of Medical Entomology, many common bead mills were evaluated alongside the Bead Ruptor EliteTM Homogenizer in efforts to detect common tick-borne pathogens. The ultimate consensus of this study showed that the Bead Ruptor EliteTM Homogenizer scored the highest when evaluating DNA yield, DNA quality, maceration of the tick, and detection of pathogenic nucleic acids (3). Kudos to the Department of Defense for rating the OMNI Bead Ruptor EliteTM Homogenizer the best bead mill for the task at hand!
Beyond lysis, another hurdle is met when examining a tick’s pathogen burden during the nucleic acid extraction itself. Typical commercial methods rely on spin column kits that can be very time consuming and limit throughput. Additionally, more manual steps required exponentially increases the chances for sample variance, incomplete extractions, and introduction of contaminants. These variables can be reduced, however, by introducing automation. In tick-surveillance labs, automated nucleic acid extraction allows for numerous benefits, like walk-away processing capabilities, reduced human error, and increased extraction efficiency. Generally, automated extraction platforms are easily attainable as there are several commercially available solutions on the market.
To address the needs for automation, OMNI’s scientists utilized the chemagic 360 Nucleic Acid Extractor. This machine uses magnetic bead technology and unique chemistry to purify nucleic acids efficiently and effectively from a lysed sample. Adding this automation step is not only time saving but allows the user to process up to 96 samples at once with consistency and speed not found in manual methods. A collaboration between the scientists at OMNI and chemagen has resulted in a reliable workflow for extracting tick DNA (and other tissues) for downstream applications.
Unfortunately, obtaining purified DNA from the tick is only half the challenge. To fully understand the disease burden found in a tick, the DNA must be screened for a variety of potential pathogens. This is traditionally performed via PCR targeting of a single pathogen of interest. This method is effective; however, in a single plex reaction it requires many additional PCR reactions to fully scan the tick’s entire pathogenic burden, not to mention consuming a high volume of the extracted nucleic acids. A solution to this problem has been presented by the scientists at ChromaCode. Their team has developed a method to screen for 9 of the most common pathogens found within a tick, using their HDPCR Tick-Borne Pathogen (TBP) panel. This is made possible by the company’s unique primers and utilizing HDPCR technology. By evaluating all 9 of these pathogens in a single reaction, the end user can save a tremendous amount of time and inputs and have higher confidence for a fully screened population.
To evaluate the effectiveness of introducing OMNI homogenization systems and automated DNA extraction platforms into the ChromaCode workflow, a series of ticks were processed through their panel to see what level of specificity can be achieved. Certified pathogen free ticks were homogenized utilizing the Bead Ruptor EliteTM Homogenizer and the resulting lysate was spiked with one of the pathogens of interest in the ChromaCode panel in a series of dilutions from 1000 copies/mL down to 50 copies/mL. After spiking, the lysate was extracted via the chemagic 360 instrument and the purified DNA was amplified using ChromaCode’s HDPCR TBP panel.
Results of the HDPCR were quite promising with all but two replicates detected at extremely low copy numbers. A literature review was performed to examine typically observed copy numbers found in wild born ticks. With few exceptions the OMNI method of homogenization combined with automated extraction allowed the detection of pathogens of interest at much lower levels than what is typically observed in wild born ticks. This gives the team confidence as to the effectiveness of our workflow and the ability for researchers to fully monitor the pathogen burden within ticks.
If you are interested in OMNI’s method for lysing ticks, or other tough samples, reach out to our team today to see how our solutions can benefit your workflow.
For research use only. Not for use in diagnostic procedures.
APN 202209 - Bead Mill Homogenization and chemagic™ 360 Automated DNA Extraction Workflow for High-Definition PCR Detection of Tick-Borne Pathogens
Citations:
1. Parola, P., & Raoult, D. (2001). Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clinical infectious diseases, 32(6), 897-928.
2. Menchaca, A. C., Visi, D. K., Strey, O. F., Teel, P. D., Kalinowski, K., Allen, M. S., & Williamson, P. C.(2013). Preliminary assessment of microbiome changes following blood-feeding and survivorshipin the Amblyomma americanum nymph-to-adult transition using semiconductor sequencing. PloS one, 8(6), e67129
3. Jones, A. M., Van de Wyngaerde, M. T., Machtinger, E. T., Rajotte, E. G., & Baker, T. C. (2020). Choice of laboratory tissue homogenizers matters when recovering nucleic acid from medically important ticks. Journal of medical entomology, 57(4), 1221-1227.
