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. Author manuscript; available in PMC: 2011 Jan 1.
Published in final edited form as: J Med Entomol. 2010 Jan;47(1):89–94. doi: 10.1603/033.047.0112

Extraction of Total Nucleic Acids From Ticks for the Detection of Bacterial and Viral Pathogens

Chris D Crowder 1, Megan A Rounds 1, Curtis A Phillipson 1, John M Picuri 1, Heather E Matthews 1, Justina Halverson 1, Steven E Schutzer 2, David J Ecker 1, Mark W Eshoo 1
PMCID: PMC2837073  NIHMSID: NIHMS176800  PMID: 20180313

Abstract

Ticks harbor numerous bacterial, protozoal, and viral pathogens that can cause serious infections in humans and domestic animals. Active surveillance of the tick vector can provide insight into the frequency and distribution of important pathogens in the environment. Nucleic-acid based detection of tick-borne bacterial, protozoan, and viral pathogens requires the extraction of both DNA and RNA (total nucleic acids) from ticks. Traditional methods for nucleic acid extraction are limited to extraction of either DNA or the RNA from a sample. Here we present a simple bead-beating based protocol for extraction of DNA and RNA from a single tick and show detection of Borrelia burgdorferi and Powassan virus from individual, infected Ixodes scapularis ticks. We determined expected yields for total nucleic acids by this protocol for a variety of adult tick species. The method is applicable to a variety of arthropod vectors, including fleas and mosquitoes, and was partially automated on a liquid handling robot.

Keywords: Tick, Borrelia, Powassan virus, nucleic acid extraction, vector-borne disease

Vector-borne diseases caused over 148,000 deaths and >12.5 million disability-adjusted life years worldwide in 2002 (Beaglehole et al. 2004). The tick vector can transmit a variety of pathogens ranging from viruses and bacteria to protozoa (de la Fuente et al. 2008). The diseases caused by bacterial pathogens can vary from life threatening infections, such as tularemia and Rocky Mountain spotted fever, to potentially chronic infections, like Lyme disease (de la Fuente et al. 2008). Manifestations of protozoan infections such as Babesiosis can present from flu-like symptoms to severe recurring infections and death in humans (Vannier et al. 2008) and can also affect livestock and pets (Bock et al. 2004).

Tick-borne RNA viruses from a wide range of families can also cause serious illness and death. In North America, the Powassan virus and Deer Tick fever virus, both Flaviviruses, have been found in four species of Ixodes, as well as Dermacentor andersoni ticks (Romero and Simonsen 2008). Colorado tick fever virus, a Coltivirus from the Reoviridae family, is transmitted by D. andersoni and infects 200–400 people annually. In the United States alone, there were over 23,000 reported cases of tick-transmitted diseases in 2006 (McNabb and Jajosky 2008). In Europe and Asia, tick-borne encephalitis (TBE), another Flavivirus, affects more then 10,000 people each year (Lindquist and Vapalahti 2008). Additionally, the Crimean-Congo hemorrhagic fever virus, an RNA virus that belongs to the Bunyaviridae family, can be transmitted by ticks of the Hyalommai genus; cases have been found throughout Europe, Africa, and Asia (Ergonul 2006).

Most direct pathogen detection assays employ polymerase chain reaction (PCR) and require the efficient homogenization of the ticks, lysis of the pathogens, and extraction of the nucleic acids from inhibitors of PCR. A number of methods have been reported for extracting nucleic acids from ticks, such as crushing frozen ticks with a mortar and pestle, homogenizing the ticks with small beads in a bead-beater, or cutting apart the tick with a scalpel (Exner and Lewinski 2003, Hill and Gutierrez 2003, Halos et al. 2004, Moriarity et al. 2005). However, none of these methods have been reported to simultaneously extract both DNA and RNA from a single tick. Extraction of both DNA and RNA is vital for pathogen surveillance, as some tick-borne pathogens are RNA viruses (Romero and Simonsen 2008) and co-infections are common (Clay et al. 2008, Steiner et al. 2008). The method we report here employs very high density yttria stabilized zirconium oxide beads to homogenize the ticks and physically lyse bacteria and protozoa; proteases are used to lyse viruses. Asilica-gel column is then used to remove PCR inhibitors and cellular debris from nucleic acids. Here we demonstrate the extraction of total nucleic acids from a variety of ticks and show that a PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) method efficiently detected Borrelia burgdorferi and Powassan virus from infected Ixodes ticks collected in the field.

Materials and Methods

Tick Lysis and Nucleic Acid Extraction

Ticks were obtained from the Oklahoma State University tick-rearing facility (Stillwater, OK), CA State vector control departments, and field collections in Tennessee and New York. A modification of the Qiagen Virus MinElute kit (Qiagen, Valencia, CA) was used to extract RNA and DNA from the ticks. Specifically ticks were homogenized in 0.5 ml screw-cap tubes (Sarstedt, Newton, NC). The tubes were filled with 750 mg of 2.0 mm yttria stabilized zirconium oxide beads (ziconia/yttria), 150 mg of 0.1 mm zirconia/yttria beads (Glen Mills, Clifton, NJ), and 450 µl of lysis buffer consisting of 419 µl of Qiagen ATL buffer, 6 µl of a 1 mg/ml stock of sonicated poly A (Sigma-Aldrich, St. Louis, MO), and 25 µl proteinase K solution (Qiagen). The tubes were shaken in a BioSpec Mini Bead Beater 16 (BioSpec, Bartlesville, OK). The samples then were then centrifuged for 2 min at 6,000 g in a benchtop microcentrifuge. A 400 µl aliquot of the recoverable supernatant was transferred to a fresh microcentrifuge tube and 400 µl of AL buffer was added. The tubes were briefly mixed by vortexing for 30 s, pulse centrifuged, and incubated at 37°C for 10 min. Subsequently, 480 µl of 100% ethanol was added and samples were mixed by vortexing for 30 s and then centrifuged to remove liquid from the tube cap. The samples were then loaded onto the Qiagen MinElute column in two parts: first, 750 µl of sample was loaded and then centrifuged at 6,000 g for 1 min. The flow through was discarded and the remaining sample was loaded onto the column and the column was again centrifuged at 6,000 g for 1 min. The wash steps were carried out according to the kit instructions with the exception that a fresh 2.0 ml collection tube was used in each step. Nucleic acids were eluted by adding 100 µl of AVE elution buffer (Qiagen) to the column, incubating at room temperature for 5 min, and centrifuging for 1 min at 6,000 g. The quality of the nucleic acid extraction was measured by total nucleic acid yield and visual inspection of the DNA and ribosomal RNA bands in a gel. This method was also automated on the QiaCube automated nucleic acid extraction robot (Qiagen) following the bead-beating step and manual collection of the supernatant.

DNase and RNase Treatments

To examine the RNA yield using our extraction protocol, the DNA was digested with DNase I, RNase-free, enzyme (Roche, Indianapolis, IN), and the remaining nucleic acids visualized by gel electrophoresis. Briefly, a 17.5 µl aliquot of the nucleic acid extract was mixed with 2 µl of 10× DNase incubation buffer (Roche) and 0.5 µl of DNase I (5 U). The reaction was incubated at 37°C for 15 min with mixing and brief centrifugation every 5 min. To examine the DNA yield of the extraction, the RNA was digested with RNase, DNase-free enzyme (Roche). Specifically, 45 µl of the sample was mixed with 1 µl of RNase (0.5 µg) and incubated for 30 min at 37°C with mixing and brief centrifuging every 10 min. The resulting enzyme-treated extracts were visualized on a 1% agarose gel with 8 µl of the All Purpose Hi-Lo ladder (Bionexus, Oakland, CA) which ranges from 10,000 to 50 bp in size.

Total Nucleic Acid Quantitation

Total nucleic acid tick extracts (2 µl) were quantified by absorbance 260 measurement using a Nanodrop ND-1000 (Thermo Scientific, Wilmington, DE) using Qiagen AVE buffer as the blank. Water extractions were also quantitated to determine the contribution of the poly A carrier to the overall yield; this value was subtracted from the samples to determine the total nucleic acid yields from tick samples.

PCR

Detection of Borrelia DNA was performed using PCR primers targeting the flaB gene. The forward and reverse primer sequences used for Borrelia flaB gene were 5′-TGCTGAAGAGCTTGGAATGCA-3′ and 5′-TACAGCAATTGCTTCATCTTGATTTGC-3′, respectively. PCR was performed in a 40 µl reaction containing 1 µl nucleic acid extract in a reaction mix consisting of 1 U of Immolase Taq polymerase (Bioline USA, Taunton, MA), 20 mM Tris (pH 8.3), 75 mM KCl, 1.5 mM MgCl2, 0.4 M betaine, 200 µM dATP, 200 µM dCTP, 200 µM dTTP (each dNTP from Bioline USA), and 200 µM 13C-enriched dGTP (Cambridge Isotope Laboratories, Andover, MA), 20 mM sorbitol (Sigma-Aldrich), 2 µg/ml sonicated poly A RNA (Sigma-Aldrich), 500 µg/ml of ultrapure BSA (Invitrogen, Carlsbad, CA), and 250 nM of each primer. The following PCR cycling conditions were used on an MJ Dyad 96-well thermocycler (Bio-Rad, Hercules, CA): 95°C for 10 min, followed by eight cycles of 95°C for 30 s, 48°C for 30 s, and 72°C 30 s, with the initial 48°C annealing temperature increasing 0.9°C each cycle. PCR was then continued for 37 additional cycles of 95°C for 15 s, 56°C for 20 s, and 72°C for 20 s. The PCR ended with a final extension of 2 min at 72°C followed by a 4°C hold. The PCR product was visualized on a 4% agarose gel.

RT-PCR

One-step RT-PCR was performed in a reaction mix consisting of 1 U of Immolase Taq polymerase (Bioline USA), 20 mM Tris (pH 8.3), 75 mM KCl, 1.5 mM MgCl2, 0.4 M betaine, 200 µM dATP, 200 µM dCTP, 200 µM dTTP (each dNTP from Bioline USA, Randolph, MA), and 200 µM 13C-enriched dGTP (Cambridge Isotope Laboratories, Andover, MA), 10 mM dithiothreitol, 100 ng sonicated poly-A DNA (Sigma), 40 ng random primers (Invitrogen), 1.2 U Superasin (Ambion Corp., Austin TX), 400 ng T4 gene 32 protein (USB, Cleveland, OH), 2 U Superscript III (Invitrogen), 20 mM sorbitol (Sigma), and 250 nM of each primer. The following RT-PCR cycling conditions were used: 60°C for 5 min, 4°C for 10 min, 55°C for 45 min, 95°C for 10 min, followed by eight cycles of 95°C for 30 s, 48°C for 30 s, and 72°C 30 s, with the 48°C annealing temperature increasing 0.9°C each cycle. The PCR was then continued for 37 additional cycles of 95°C for 15 s, 56°C for 20 s, and 72°C for 20 s. The RT-PCR cycle ended with a final extension of 2 min at 72°C followed by a 4°C hold. Primer pair VIR2217 was used to detect the Flavivirues RdRp gene are 5′-TGTGTCTACAACATGATGGGAAAGAGAGA-3′ for the forward and 5′-TGCTCCCAGCCACATGTACCA-3′ for the reverse primer.

Mass Spectrometry and Base Composition Analysis

Mass spectrometry was performed on an Ibis T5000 Biosensor (Ibis Biosciences, Carlsbad, CA). After PCR amplification, 30 µl aliquots of each PCR reaction were desalted and purified using a weak anion exchange protocol described previously (Ecker et al. 2006). Accurate mass (±1 ppm), high-resolution (M/dM > 100,000 FWHM) mass spectra were acquired for each sample using high-throughput ESI-MS protocols described previously (Sampath et al. 2007). For each sample, ≈1.5 µl of analyte solution was consumed during the 74 s spectral acquisition. Raw mass spectra were postcalibrated with an internal mass standard and deconvolved to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides (Muddiman et al. 1997).

Results

DNA and RNA Extracted From Single Ticks

A mixture of 2.0 mm and 0.1 mm zirconia/yttria beads were used to homogenate either adult or nymphal ticks. We found the bead composition was an important factor in the final yield of nucleic acid as well as influencing downstream applications because of the presence of PCR inhibition with some bead types (data not shown). The 2.0 mm beads function to disrupt and homogenate the tough tick exoskeleton while the smaller 0.1 mm beads are used to physically lyse the tick tissues and any micro-organisms present. The addition of Proteinase K to the lysis buffer is to ensure degradation of any viral particle and release of viral nucleic acid. To determine the optimal bead beating time for nymphal or adult ticks we performed a series of extractions increasing the time bead beaten by 30 s intervals and examining the total nucleic acid for yield and quality. We observed optimal yields when nymphs and adults were bead-beaten for 1 and 2.5 min, respectively (data not shown). Increases in bead-beating times did not affect the total nucleic acid yield significantly but degradation of the ribosomal RNA bands was observed at longer times. Total nucleic acid was then isolated from a single flat adult D. variabilis tick to demonstrate the recovery of DNA and RNA. Figure 1A shows the gel analysis of total nucleic acids, DNA, and RNA, respectively, from an adult D. variabilis tick. We also obtained total nucleic acid, DNA, and RNA from an adult I. scapularis tick as shown in Fig. 1B.

Fig. 1.

Fig. 1

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Nucleic acids isolated from a single adult ticks. (A) Total nucleic acids, DNA, and RNA (left, center, and right, respectively) from an adult D. variabilis tick. Approximately 10% of the sample extract was used for each gel. The left lane in each panel is DNA size ladder. (B) Total nucleic acids, DNA, and RNA (left, center, and right, respectively) from an adult I. scapularis tick. Approximately 20% of the sample extract was used for each gel.

The total nucleic acid yields from various tick species were determined using laboratory-reared and field-collected adult ticks. Table 1 shows the mean, standard deviation, and 95% confidence intervals for the extraction of total nucleic acids from adult D. variabilis, D. occidentalis, Amblyomma americanum, and I. scapularis ticks. To account for the nucleic acid contribution from the poly A carrier, extractions were performed without the ticks and the 260 nm absorbance of the extract was determined on a Nanodrop-1000. The blank extractions were found to have a mean of 4.4 µg of poly A (n = 5, σ = 0.18). The amount of total nucleic acid from each tick was calculated by subtracting this amount from the measured values.

Table 1.

Total nucleic acid yields from adult ticks

Species n Total microgram nucleic acid per tick
Mean Standard
deviation		 95% confidence
interval
D. variabilis 7 18.4 3.9 2.9
D. occidentalis 11 9.0 2.5 1.0
A. americanum 4 8.8 1.6 2.4
I. scapularis 15 5.8 2.0 1.0
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Detection of a Tick-Borne Bacterial Pathogen From Single Ticks

Extraction of total nucleic acids was performed on individual field-collected ticks. The extracts were then analyzed by PCR with primers targeting the Borrelia flaB gene. Figure 2A demonstrates the detection of the flaB gene for Borrelia by PCR in an extract from an I. scapularis nymph collected in New York. PCR/ESI-MS was then used to analyze the PCR amplicon to identify the species of Borrelia. PCR/ESI-MS nucleotide base compositions allowed us to identify this Borrelia sample as B. burgdorferi (Figs. 2B).

Fig. 2.

Fig. 2

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Detection of pathogens from single I. scapularis ticks. (A) Gel analysis of the PCR amplification of the Borrelia flaB gene from a nymph. (B) Identification of the B. burgdorferi flaB gene by PCR/ESI-MS on the T5000 biosensor. One percent of the tick extract was used in the PCR reaction for both (A) and (B). (C) Detection of a Flavivirus by RT-PCR in a frozen adult I. scapularis tick. (D) Powassan virus identification by PCR/ESI-MS. One percent of the tick extract was used in the RT-PCR reaction for both (C) and (D).

Detection of a Tick-Borne Viral Pathogen From Single Ticks

Extracts from individual field-collected ticks were screened by broad-range RT-PCR for Flaviviruses. Figure 2C shows the broad-range RT-PCR detection of the Flavivirus RNA from a frozen field collected I. scapularis adult from New York. The base count composition as determined by PCR/ESI-MS identified the sample as Powassan virus, Fig. 2D.

Discussion

The ability to simultaneously extract DNA and RNA from a tick allows for the monitoring for known and emerging vector-borne pathogens. As a number of tick viral pathogens are RNA viruses, researchers must analyze both RNA and DNA to determine the viral pathogens, as well as bacterial and protozoan pathogens, present. The method we report is simple and has been used to isolate the nucleic acids from live, ethanol-preserved, or frozen ticks. The method developed in this study employs two different size beads of identical density. The large, 2.0 mm beads homogenize the ticks and the smaller 0.1 mm beads lyse bacteria and protozoans. Proteinase K serves to lyse the viruses. This approach has the added advantage of minimizing exposure to the tick and its pathogens. Because the tick sample is not split between two extraction protocols there less labor and no reduced sensitivity in downstream detection assays because of less starting material going into the assay.

The use of mortar and pestles has been used in the past to homogenize ticks but is laborious and can be expensive. Furthermore, it still requires an enzymatic cell lysis step, which is slow and may not be effective against hard to lyses microorganisms. The use of bead-beating with very high-density yttria stabilized zirconium oxide beads is very rapid and can result in higher quality nucleic acids than those obtain from protocols employing lengthy enzymatic incubation steps where nucleases have the opportunity to degrade the nucleic acids of interest. We found that higher density beads allowed for shorter bead-beating times and resulted in overall higher yields then the standard, and less dense, zirconium-silica beads. Additionally, we did not observe the PCR inhibition in downsteam applications with the yttria stabilized zirconium oxide beads compared with zirconium-silica or steel beads. Lysis of microorganisms by mechanical bead-beating is a very effective method to quickly lysis all bacteria including hard to lyse cell types (O’Connor and Zusman 1988).

Because ticks can harbor a number of pathogens and closely related micro-organisms, PCR/ESI-MS was used in addition to gel analysis to ensure that the detected amplicon was that of the pathogen and not a PCR product from a related organism. PCR/ESI-MS has been used to detect and identify a variety of bacteria and viruses (Eshoo et al. 2007, Sampath et al. 2007, Ecker et al. 2008, Baldwin et al. 2009, Eshoo et al. 2009) Additionally, the PCR/ESI-MS allows the researcher to identify when there are mixtures of amplicons from two micro-organisms (Ecker et al. 2005).

The method described here could also be used for vector surveillance of other arthropods, for instance fleas, mosquitoes, mites, and various flies. Many arthropod vectors can transmit a combination of viruses (RNA and DNA), bacteria, and protozoa (Kalluri et al. 2007). Mosquitoes transmit RNA viruses such as West Nile and Dengue, as well as the protozoan Plasmodium falciparum, the etiological agent of malaria. Fleas and mites have been shown to transmit bacteria that cause a number of diseases and sandflies transmit the protozoa that cause Leishmaniasis along with viruses that cause Sandfly fever. Other reports have described homogenization by bead-beating the vector, but only in context of DNA isolation and from multiple ticks (Moriarity et al. 2005) or fleas (Allender et al. 2004).

Because of the rapidity of homogenization, along with the extraction of both DNA and RNA, this method could also be used for isolation of nucleic acid from various types of tissues and clinical samples. Tail or skin tissues or tough organs such as heart and spleen could be quickly homogenized and nucleic acids extracted without lengthy overnight digestions or manual homogenization by mortar and pestle. We have found that total nucleic acids from skin biopsies are readily obtained by this method (data not shown).

Using this protocol, we obtained high-quality DNA and RNA from individual ticks and from the pathogens that resided in the tick. We have successfully used nucleic acids obtained from this protocol to show the presence of B. burgdorferi and Powassan virus in ticks by both PCR and PCR/ESI-MS. The protocol can also be automated on the Qiagen QiaCube to increase the throughput of tick pathogen surveillance. Using the Qiagen MiniElute Virus extraction kit the total retail cost for all materials is $5 a sample. Recent in-house studies show the Qiagen DNeasy columns can be substituted for the MiniElute Virus columns and reduce the cost per samples by ≈50% (data not shown). This protocol may also have uses in other vector surveillance studies and in analysis of tissue samples.

Acknowledgments

We thank Scott Campbell from the Suffolk County Department of Health Services for supplying us with ticks. Additionally we would like to acknowledge the Lyme Disease Association, the Tami Fund, National Institute of Allergy And Infectious Diseases Grant 1R43AI077156–01 and CDC contract 200-2008-M-27504 for financial support for this work. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy And Infectious Diseases or the National Institutes of Health.

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