TY - JOUR
AU - Dasch, Gregory A.
AB - Abstract To meet the need for high-throughput sample testing, DNA extraction kits based on the 96-well plate format have been developed for use with blood and tissue samples. These methods have not been applied to DNA extractions from ticks. To meet this need, we developed a high-throughput method for DNA extraction and polymerase chain reaction (PCR) testing of tick samples. A liquid-handling robot was used to extract DNA in a 96-well binding column plate with vacuum manifold. The quantity, purity, and quality of DNA recovered from Ixodes scapularis Say, 1821 nymphs with this method were reproducible and comparable with existing manual DNA extraction techniques. The DNA yield from pools of five nymphal ticks averaged 0.432 ± 0.04 μg (95% CI). The robot also prepared real-time PCR reactions in 96-well plates, directly from the extracted DNA. A modification of the existing P20 tool resulted in accurate pipetting of 1- to 2-μl volumes with a reproducibility of ±0.038 μl when dispensing 1.0 μl. By using this process, 96 samples can be extracted and tested while reducing human labor to ≈30 min. DNA extraction, polymerase chain reaction, robotic, automated, ticks Population screening of ticks for vector-borne pathogens requires the collection, DNA extraction, and polymerase chain reaction (PCR) testing of hundreds or thousands of ticks (Fang et al. 2002, Levin et al. 2002). DNA can be manually extracted from ticks in individual tubes (Hill and Gutierrez 2003). These methods produce high-quality DNA (Schwartz et al. 1997), but they have a limited throughput capability. To meet the growing need for high-throughput sample testing, laboratory automation was developed for use in molecular biology applications, such as DNA extraction and PCR (Clarke 2002, Smith et al. 2003). DNA extraction kits based on the 96-well plate format are commercially available; these kits allow for higher-throughput sample processing with reduced human labor (Read 2001, Harris et al. 2002). Although some DNA extraction kits were evaluated for use with whole blood (Smit et al. 2000, Smith et al. 2003), few have been tested with arthropods. A high-throughput method of RNA extraction from mosquitoes was described by Shi et al. (2001), but DNA extractions differ in protocol. Allender et al. (2004) described the application of a plate based DNA extraction kit to fleas. This method used bead milling to mechanically disrupt fleas, and the extractions are performed using the 96-well DNeasy tissue kit (QIAGEN, Valencia, CA). This method requires a laboratory technician to manually pipette all reagents, and the total time to process 96 samples was estimated to be 2 to 3 h. We report here the development of an automated, high-throughput method of DNA extraction and PCR testing of arthropods. The method was developed using Ixodes scapularis Say, 1821 nymphs, and the Promega Wizard SV96 genomic DNA purification system (Promega, Madison, WI). This commercially available 96-well DNA extraction kit was chosen based on its compatibility with the Biomek 2000 liquid handling robot (Beckman Coulter, Fullerton, CA) and previously documented yield and quality of extracted DNA (Smith et al. 2003). Our automated process reduces human labor to 30 min for 96 samples, increasing sample processing capacity with no loss in DNA yield or quality. Materials and Methods Sample Preparation. Uninfected, laboratory-reared I. scapularis nymphs were obtained from the Medical Entomology Laboratory, Centers for Disease Control and Prevention, Atlanta, GA. Ticks were separated into pools of five nymphs and placed in 1.7-ml conical microcentrifuge tubes. IsoQuick DNA Extractions. Manual DNA extractions were performed using the IsoQuick DNA extraction kit (ORCA Research, Bothell, WA). Twenty pools of five ticks were frozen in liquid nitrogen and ground into a fine powder by using sterile Teflon pestles (Kontes, Vineland, NJ). DNA was extracted as described previously (Schwartz et al. 1997). Each DNA sample was resuspended in 50 μl of nuclease free water. Robotic DNA Extractions. Twenty-five pools of five ticks were manually disrupted as described above. The ground ticks were suspended in a tick lysis buffer consisting of 160 μl of nuclei lysis solution per sample (Promega), 40 μl of 0.5 M EDTA, pH 8.0 (Promega), and 20 μl of Proteinase K (20 mg/ml, Roche Diagnostics, Indianapolis, IN). The mixture was incubated at 55°C for 2 h before DNA extraction. To assess the effect of bead milling, other pools of ticks were disrupted using a Mixer Mill MM 301 (Reutsch, Haan, Germany) for 10 min at 3,000 strokes/min. Each well contained one pool of five nymphs, three 3-mm steel beads, tick lysis buffer, and 20 or 40 μl of Antifoam-A (Fluka, Seelze, Germany). These samples were incubated at 55°C for 2 h, and steel beads were removed using a magnet. For DNA extraction, the tubes containing the milled ticks were placed into 24-Place tube racks with 11-mm inserts (Beckman Coulter) and loaded onto the robot. The remaining steps of the DNA extraction were automated using the Biomek 2000 automated liquid handling robot (Beckman Coulter). A P1000L robotic pipette tool (Beckman Coutler) was used to individually mix and transfer samples to a 96-well DNA binding plate that rested on the vacuum manifold collar and vacuum manifold. Tick samples were mixed with 250 μl of SV lysis buffer (Promega). After all 96 samples were added to the plate, the plate was subjected to a vacuum (15–20 mmHg) for 1 min to load the DNA samples onto the membranes. Wells were washed three times with 750 μl of wash buffer (Promega), delivered by the eight-channel wash tool, with a 1-min vacuum after each of the three washes. The plate was subjected to a 5-min vacuum to completely remove the wash buffer. The 96-well DNA binding plate was blotted on a paper towel, and the 0.5-ml 96-well DNA elution plate was moved beneath the DNA binding plate on the vacuum manifold. The MP200 tool then delivered 100 μl of nuclease-free water (Promega) to each well of the DNA binding plate. A 4-min incubation was followed by a final 5-min vacuum to collect DNA from the plate. The plate was removed from the robotic workstation by a laboratory technician, sealed using nuclease-free foil sealing film (Beckman Coulter), and centrifuged to pool liquid in each well. DNA Analysis. Extracted DNA samples were tested for DNA yield, protein content, and presence of PCR inhibitors. To determine the amount of dsDNA content in each well, the PicoGreen dsDNA quantitation kit (Molecular Probes, Eugene, OR) was used, as per the manufacturer’s instructions. A Bradford assay (Bio-Rad, Hercules, CA) was performed to determine the amount of protein contamination in each sample using a standard curve of bovine serum albumin (Sigma, St. Louis, MO), such that the lower limit of detection was 0.8 μg protein/ml. The assays were run according to the manufacturers’ instructions. The presence of PCR inhibitors was determined using a previously described real-time PCR assay (Loftis et al. 2003). One thousand copies per microliter of plasmid encoding the 16S rRNA gene of Ehrlichia chaffeensis Anderson et al. 1992 was added to each extracted DNA sample and to three samples of PCR-grade water. Real-time PCR reactions were performed using 2.0 μl of DNA template. Reactions were performed using the iCycler (Bio-Rad), and threshold cycle (CT) values were determined. Significant inhibition of real-time PCR by the presence of extracted DNA was defined as a threshold cycle 1.5 standard deviations greater than the average of the three control samples. Calibration of the P20 Low-Level Tool. The P20 Low-Level tool is a special calibration to dispense 1–5 μl by using the P20 Single Tip tool. The slope and offset of this tool were altered as per the calibration recommendations from the manufacturer (Beckman Coulter), with the following modifications: fluorescent dye was used instead of colorimetric dye, and calibrations were performed using volumes of 1.0 and 5.0 μl rather than 5.0 and 20.0 μl. For calibration, 1 and 5 μl of 250 ng/ml fluorescein dye was delivered into 99 and 95 μl of phosphate-buffered saline (Invitrogen, Carlsbad, CA), respectively. Sixteen replicates of each dilution were performed. A standard curve generated by hand was used to determine the volume of liquid dispensed by the robot. The fluorescence of each well was read on a Fluoroskan Ascent FL (Thermo Electron Corporation, Waltham, MA). The resulting P20 Low-Level tool has a reproducibility of ±0.038 μl when dispensing 1.0 μl and ±0.077 μl when dispensing 5.0 μl (data not shown). Real-Time PCR The P20 Single-Tip tool was used to dispense 18 μl of Master Mix, containing PCR buffer, magnesium chloride, dNTPs, forward and reverse primers, fluorescent TaqMan probe, and Taq DNA polymerase (Stratagene Brilliant qPCR kit, Stratagene, La Jolla, CA) prepared by the laboratory technician and dispensed into two 1.7-ml conical microcentrifuge tubes of equal volume, to each well of the PCR plate. The P20 Low-Level tool was then used to transfer 2.0 μl of DNA template from the sample plate into the PCR plate. Real-time PCR reactions were set up in duplicate, with a new tip used for each well. To compare the accuracy and precision of the robot to that of a trained laboratory technician, duplicate real-time PCR reactions were prepared both by hand and by the robot. A previously described, TaqMan assay for the 17-kDa antigenic gene of Rickettsia spp. was used (Jiang et al. 2004). Ten-fold dilution series (from 10−1 to 10−5) of genomic R. rickettsii (Wolbach 1919) (ATCC VR891, bitterroot), R. sibirica Zdrodovskii 1948 (246CWPP), R. africae Kelly et al. 1996 (Eth SF2474–3), and R. prowazekii da Rocha-Lima 1916 (Madrid E parent) DNA in water were used as standards. Rickettsia DNAs were from reference stocks maintained by the WHO Reference Center for Rickettsial and Bartonella-Associated Diseases, at the Centers for Disease Control and Prevention in Atlanta. Real-time PCR reactions were run on the iCycler platform, as described above. The average CT for each sample and the absolute value of the difference between the CTs for each pair of duplicates (ΔCT) were calculated. Results DNA Extraction of Tick Samples. Pools of five I. scapularis nymphs were extracted using the robotic platform and 96-well plate based DNA extraction kit. The DNA obtained by this method was compared with DNA obtained from the same cohort of ticks by using a previously described manual extraction technique (Schwartz et al. 1997). An overview of the automated extraction process is provided in Fig. 1. For the first experiment, ticks were prepared using manual disruption in liquid nitrogen, as described previously (Schwartz et al. 1997), and extracted using the two different techniques. Twelve pools of ticks were prepared and extracted on the robotic platform by using an elution volume of 50 μl; however, the average recovered volume of eluted DNA was 6.25 μl/well (data not shown). These data were excluded from further analysis, (because of this poor recovery). Twenty-five samples were manually disrupted, extracted using the robotic platform, and eluted with 100 μl of nuclease-free water. The average recovered volume was 55 ± 1.85 μl/well (95% CI), and the average amount of DNA present in each sample was 0.432 ± 0.04 μg (95% CI). The DNA yield was comparable to the manual extraction method, by which 20 samples were extracted with a DNA yield of 0.378 ± 0.03 μg (95% CI; P = 0.064) (Table 1). Using a Bradford assay, with a lower limit of detection of 0.8 μg/ml, no protein contamination was detected in any tick DNA extracted. Real-time PCR was used as an assay for the presence of PCR inhibitors; inhibition was not detected in the tick DNAs extracted using the robotic extraction technique and in one of 20 samples extracted using a manual method (Table 1). Table 1 Comparison of the yield, purity, and quality of DNA obtained from manual and robotic extraction methods Open in new tab Table 1 Comparison of the yield, purity, and quality of DNA obtained from manual and robotic extraction methods Open in new tab Fig. 1 Open in new tabDownload slide Simplified flow chart of DNA extraction from arthropods. Sample preparation was done by hand and DNA extractions were executed on the liquid-handling robot. Fig. 1 Open in new tabDownload slide Simplified flow chart of DNA extraction from arthropods. Sample preparation was done by hand and DNA extractions were executed on the liquid-handling robot. For the second experiment, 20 additional pools of ticks were disrupted using a commercial bead beater (Mixer Mill MM 301) and extracted using the robotic platform; Antifoam-A was added to prevent foaming. Eight samples were disrupted in the presence of 40 μl of Antifoam-A, and 12 samples were disrupted in the presence of 20 μl of Antifoam-A. Bead disruption of the tick samples resulted in a significantly decreased DNA yield of 0.101 ± 0.01 μg (95% CI; P < 1 × 10−9) (Fig. 2). There was no statistical difference in DNA yield between the samples containing 20 or 40 μl of Antifoam-A (P < 1 × 10−12). Four pools of ticks were then disrupted manually in the presence of 40 μl of Antifoam-A, and DNA was extracted using the robotic platform. There was a significant decrease between the DNA yield from these samples (0.055 ± 0.011 μg) and from samples disrupted manually without Antifoam-A (P = 0.004) (Table 1). No protein contamination was detected in any of these 24 DNA extracts. Fig. 2 Open in new tabDownload slide Real-time PCR detection of a 10-fold dilution series of R. rickettsii genomic DNA. Reactions were prepared manually and using the P20 Low-Level tool on the Biomek 2000 liquid-handling robot. Average threshold cycle (CT) is shown on the y-axis and the dilution factor is shown on the x-axis. Error bars represent the standard deviation. Fig. 2 Open in new tabDownload slide Real-time PCR detection of a 10-fold dilution series of R. rickettsii genomic DNA. Reactions were prepared manually and using the P20 Low-Level tool on the Biomek 2000 liquid-handling robot. Average threshold cycle (CT) is shown on the y-axis and the dilution factor is shown on the x-axis. Error bars represent the standard deviation. PCR Setup Using Robotic Platform. Preparation of real-time PCR reactions by the robot was compared with a laboratory technician, by using 10-fold dilution series of four different standards. There was no significant difference between the average CT values obtained using both methods for any of these samples (Fig. 2). The precision for both pipetting methods was assessed by the difference in CT between duplicate wells (Fig. 3). The average variation between duplicate wells was 0.71 ± 0.25 (95% CI) for the reactions prepared by the robot and 0.56 ± 0.30 (95% CI; P = 0.436) for the reactions prepared manually. Fig. 3 Open in new tabDownload slide Precision of pipetting. Duplicate wells of DNA for a real-time PCR reaction were prepared manually and using the P20 Low-Level tool on the Biomek 2000 liquid handling robot. The y-axis represents the threshold cycle (CT) difference between each duplicate. Fig. 3 Open in new tabDownload slide Precision of pipetting. Duplicate wells of DNA for a real-time PCR reaction were prepared manually and using the P20 Low-Level tool on the Biomek 2000 liquid handling robot. The y-axis represents the threshold cycle (CT) difference between each duplicate. Discussion High-throughput extraction of DNA on an automated robotic platform was performed on I. scapularis nymphs. To evaluate the yield and quality of DNA obtained from the high-throughput extraction method, samples were collected on the same day under the same conditions, and DNA was extracted in parallel using two different methods: a manual DNA extraction kit (IsoQuick), and an automated, plate based DNA extraction kit (Promega Wizard SV96). Samples of DNA were tested to determine total DNA yield, protein contamination, and presence of PCR inhibitors. Using the PicoGreen assay for sensitive quantitation of double-stranded DNA (Ahn et al. 1996, Rengarajan et al. 2002), DNA yield from nymphs was not significantly different between the two extraction methods. Protein contamination was not detected in any arthropod DNA samples. Inhibition of PCR was assessed using a real-time PCR assay for the 16S rRNA gene of E. chaffeensis. This assay is very sensitive to the presence of PCR inhibitors but is not inhibited by mammalian or tick DNAs extracted using the manual extraction protocol (Loftis et al. 2003). There was no statistical difference between the presence of PCR inhibitors in DNA extracts produced using both methods: inhibition was detected in one of 25 tick DNA samples extracted using the automated method and in zero of 20 tick DNA samples by using the manual method. Mechanically disrupting the tick samples using the Mixer Mill MM 301 resulted in a significant loss of DNA compared with DNA extraction from ticks by using liquid nitrogen with manual disruption. The nuclei lysis buffer provided with the plate based DNA extraction kit contains a detergent that foams when used with the bead beater, requiring the addition of a chemical to prevent foaming (Antifoam-A). The loss of DNA might be attributed to the Antifoam-A, which reduced the DNA yield even when samples were disrupted manually. Antifoam-A contains a silicon polymer that might bind DNA and cause a significant reduction in DNA yield. This complication was not reported by Allender et al. (2004); however, the DNA yield from their technique was not compared with other extraction methods. Our attempts to improve DNA yield with the bead beater, by using alternate lysis buffers that do not require the addition of Antifoam-A, have been unsuccessful (data not shown). Preparation of PCR reactions on the robotic platform was achieved by creating a P20 Low-Level pipetting tool. The P20 Low-Level tool dispensed volumes with an accuracy comparable to manual pipettors and produced accurate and reproducible real-time PCR duplicates. The average time required to set up a real-time PCR plate, by using slow, accurate robotic pipetting, was ≈35–40 min, whereas the time needed to set up a less accurate, qualitative PCR plate was 15–20 min. We conclude that the volume, quantity, and quality of DNA recovered using the high-throughput, automated method of DNA extraction is comparable with the manual extraction method to which it was compared. Using the automated extraction method, 96 samples can be extracted using ≈30 min of human labor and 90 min of automated extraction time. This method was compatible with extraction of cells from in vitro culture and blood samples (data not shown). All three types of samples can be extracted in the same DNA binding plate at the same time; the only difference lies in the initial preparation of the samples. Upon completion of the automated DNA extraction, a simple change in the deck layout of the robot allows PCR setup from the 96-well plate containing the DNA samples. Preparation of PCR reactions requires an additional 15–40 min of robot time. To further streamline the automated process, a laboratory technician can prepare the reagents necessary for PCR while the robot is performing DNA extractions. PCR-based screening of tick samples, on a population scale, can be time-consuming. The use of laboratory automation capable of extracting DNA from ticks can reduce human labor, thereby allowing a larger number of samples to be processed and screened. In the past, high-throughput molecular biology methods have been applied to blood samples; the current study shows that arthropods also can be used for high-throughput applications. Acknowledgements We thank Michael L. Levin (Medical Entomology Laboratory, Centers for Disease Control and Prevention, Atlanta, GA) for providing uninfected I. scapularis nymphs and goat blood samples, and Marina E. Eremeeva (Centers for Disease Control and Prevention) for providing genomic Rickettsia DNA. References Cited Ahn S. J. Costa J. Emanuel J. R. . 1996 . 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Google Scholar Crossref Search ADS PubMed WorldCat © 2005 Entomological Society of America This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © 2005 Entomological Society of America
TI - High-Throughput Molecular Testing of Ticks Using a Liquid-Handling Robot
JF - Journal of Medical Entomology
DO - 10.1093/jmedent/42.6.1063
DA - 2005-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/high-throughput-molecular-testing-of-ticks-using-a-liquid-handling-kZ057cDHcC
SP - 1063
EP - 1067
VL - 42
IS - 6
DP - DeepDyve
ER -