High-Throughput Extraction of Arthropod Vector and Pathogen DNA Using Bead Milling

BioTechniquesVol. 37, No. 5 BenchmarksOpen AccessHigh-throughput extraction of arthropod vector and pathogen DNA using bead millingChristopher J. Allender, W. Ryan Easterday, Matthew N. Van Ert, David M. Wagner & Paul KeimChristopher J. AllenderNorthern Arizona University, Flagstaff, W. Ryan EasterdayNorthern Arizona University, Flagstaff, Matthew N. Van ErtNorthern Arizona University, Flagstaff, David M. WagnerNorthern Arizona University, Flagstaff & Paul Keim*Address correspondence to: Paul Keim, Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011-5640, USA. e-mail: E-mail Address: Paul.Keim@nau.eduNorthern Arizona University, FlagstaffThe Translational Genomics Research Institute (TGen), Phoenix, AZ, USAPublished Online:6 Jun 2018https://doi.org/10.2144/04375BM03AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Many important human diseases require an arthropod vector to complete their transmission cycle. These vectors provide an efficient means to track and monitor vector-borne pathogens, including Yersinia pestis (plague) (1–3), West Nile Virus (West Nile encephalitis) (4,5), and Borrellia spp. (Lyme disease) (6). Here we present a rapid DNA extraction protocol to isolate pathogen and vector DNA from vectors. Our example involves fleas (Siphonaptera) and their role as vectors of plague, but the techniques are expected to be applicable to many vector-borne pathogens.As the most prevalent form of plague transmission is flea-mediated, a common method for determining the presence of the disease in an area involves collecting fleas, extracting DNA from those fleas, and screening the extracts for Y. pestis DNA using genetic techniques. Disruption of the flea cuticle is the most time-consuming aspect of this method, as it traditionally involves the use of micropestles and enzymes for cell lysis (7). As a result, for efficiency, fleas are usually pooled together for DNA extraction when using these techniques. In contrast, bead milling allows for rapid, high-throughput disruption of vector cuticles. Although bead-beating techniques have been described previously (8), here we present significant improvements in throughput over these previous techniques. These improvements make it cost- and time-effective to examine individual vectors, as opposed to vector pools, which facilitates analysis of detailed disease patterns, such as presence and quantity of pathogens within individual vectors.Our method for extracting DNA from vectors is novel as it couples automated 96-well bead mill mechanical disruption with a commercially available 96-well DNA purification kit. The mechanical disruption for this protocol uses a 2000 Geno/Grinder mill homogenizer (SPEX CertiPrep, Metuchen, NJ, USA) in conjunction with 2-mm very high-density zirconium oxide beads (Glen Mills, Clifton, NJ, USA). The 96-well DNA purification protocol is the 96-well DNeasy® Tissue Kit (Qiagen, Valencia, CA, USA) modified for use with the QIAvac vacuum filtration system. This system allows for complete processing of 96 samples in about 2 to 3 h, from loading samples to elution of DNA (100 µL).The process begins with adding beads, reagents, and samples into a deep-well 96-microtube plate (Qiagen). First, six beads are added to each 1.2-mL well of the microtube plate using our custom-designed bead transfer plate. The transfer plate was created by drilling 96, 12-mm-deep wells into a 10 × 14 × 1.3 cm piece of clear plexiglass. The wells of this plate are filled with beads by pouring beads across the surface of the plate until each well is full. As the beads are 2 mm in diameter, only six beads will be contained in each well. The beads are then added to the microtubule plate by placing it upside down on the loading plate and then flipping it over, transferring the beads. Second, 40 µL of a solution of proteinase K and buffer ATL (Qiagen) is prepared according to manufacturer's instructions and is added to each microtube along with 20 µL of Antifoam-A (Sigma, St. Louis, MO, USA). Finally, a single vector specimen is added to each microtube (Figure 1A). For biosafety, the bead homogenization is completed under secondary containment, which involves capping the microtubes and heat-sealing the 96 samples in a 9 × 12 inch Kapak bag (Kapak, Minneapolis, MN, USA). The secondarily contained plate is then placed into the Geno/Grinder mill, which is operated at 2000 strokes/min for 5 min. Following bead-beating (Figure 1B), the sealed plate is centrifuged at 2000× g for 1 min. The Kapak bag is then removed in a biosafety cabinet, and another 140 µL of the protein-ase K/buffer ATL working solution is added to each well, followed by 410 µL of buffer AL/E (Qiagen). These combined solutions are then transferred to the DNeasy 96-well plate. All remaining steps follow the DNeasy 96 tissue protocol for high-throughput DNA isolation from animal tissues, except we use a QIAvac 96 Vacuum Manifold for each centrifugation step.Figure 1. Flea pre- and post-milling.(A) Individual or multiple whole fleas are added to each well of the microtube plate, along with six beads (2 mm in diameter). (B) Following milling, there is significant disruption of the flea cuticle.To determine the amount of DNA obtained using the above methods, we extracted DNA from 30 individual fleas (Oropsylla hirsuta) and analyzed each sample with the PicoGreen® DNA quantification assay (Molecular Probes, Eugene, OR, USA) in conjunction with a TBS-380 fluorometer (Turner BioSystems, Sunnyvale, CA, USA). Each sample was tested in duplicate, and the mean concentration of DNA was computed. Variation among the 30 sample means was within one order of magnitude (mean = 2.47 µg/mL, se ± 0.19, range = 0.54–5.26), consistent with observed size differences among individual fleas (e.g., juveniles versus adults). To assess DNA quality of the 30 samples, we utilized a real-time PCR assay specific to a region of O. hirsuta ribosomal DNA. Each of the 30 samples was examined in triplicate using this assay, and the mean threshold cycles (Ct) were computed. All three replicates for all 30 samples were amplified, and the range of mean Ct values were consistent with measured DNA quantities (mean = 17.28 Ct, se ± 0.17, range = 15.97–19.82), indicating little or no PCR inhibition. Finally, to assess DNA shearing, we ran an agarose gel using extracts from 10 individual fleas. Estimated DNA fragment sizes for these samples ranged from approximately 7–20 kb (data not shown).This protocol is a significant improvement over previous DNA extraction methods for several reasons. First, we reduce the total time required for extracting DNA. Standard protocols, such as Qiagen and other kits that utilize lysis proteins, require long incubation steps (6–14 h) for complete lysis of tissues. In contrast, our system bypasses this incubation step by providing complete lysis of tissue in 5 min (Figure 1B). Second, the 96-well format of our technique allows for high-throughput DNA extraction, and this format carries through to most methods used for pathogen detection and characterization (e.g., real-time PCR). In addition, the high-throughput capability of this protocol affords an efficient means to extract pathogen DNA from individual vectors, providing the ability to address epidemiological questions involving individual vectors, such as pathogen loads (9). Third, bead-beating standardizes the quality of disruption by removing interoperator variability, which we have anecdotally observed with micropestle techniques. Finally, our use of zirconium oxide beads is advantageous because they do not rust when in storage with buffers and they lack heavy metals that may inhibit PCR. Other bead homogenization protocols used metallic beads (10), which may lead to these problems.Using this protocol, we have obtained high-quality genomic DNA from fleas and, when it is present in the fleas, Y. pestis. We have successfully used this DNA to support real-time PCR assays that identify the presence of Y. pestis DNA and identify flea specimens to species, as well as amplified fragment-length polymorphism (AFLP) analysis of fleas and multiple locus variable number tandem repeat analysis (MLVA) of Y. pestis (data not shown). In addition, this protocol also has been useful for obtaining DNA from individual fruit flies and parasitic leaf mites, and pending further studies, we anticipate it will be applicable to obtaining DNA from plant tissues when used in conjunction with plant DNA isolation kits.AcknowledgmentsC.J.A. and W.R.E. contributed equally to this work. This work was supported by grants from National institutes of Health (NIH), Department of Homeland Security (DHS), and the Cowden Endowment at NAU. We thank Stanley Goldberg at Glen Mills, as well as Christy Clark, Jeff Henrikson, and Scott Woolbright for their assistance with this study.Competing Interest StatementThe authors declare no conflicts of interest.References1. Engelthaler, D.M., K.L. Gage, J.A. Montenieri, M. Chu, and L.G. Carter. 1999. PCR detection of Yersinia pestis in fleas: comparison with mouse inoculation. J. Clin. Microbiol. 37:1980–1984.Crossref, Medline, CAS, Google Scholar2. Stevenson, H.L., Y. Bai, M.Y. Kosoy, J.A. 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This work was supported by grants from National institutes of Health (NIH), Department of Homeland Security (DHS), and the Cowden Endowment at NAU. We thank Stanley Goldberg at Glen Mills, as well as Christy Clark, Jeff Henrikson, and Scott Woolbright for their assistance with this study.Competing Interest StatementThe authors declare no conflicts of interest.PDF download