To beat or not to beat a tick: comparison

of DNA extraction methods for ticks

(Ixodes scapularis)
Alyssa D. Ammazzalorso1,* , Christine P. Zolnik1,2,* , Thomas J. Daniels2
and Sergios-Orestis Kolokotronis1
1 Department of Biological Sciences, Fordham University, Bronx, NY, USA
2 Vector Ecology Laboratory, Louis Calder Center–Biological Field Station, Fordham University,

Armonk, NY, USA

∗
These authors contributed equally to this work.

ABSTRACT
Background. Blacklegged ticks (Ixodes scapularis) are important disease vectors in
the United States, known to transmit a variety of pathogens to humans, including
bacteria, protozoa, and viruses. Their importance as a disease vector necessitates
reliable and comparable methods for extracting microbial DNA from ticks.
Furthermore, to explore the population genetics or genomics of this tick, appropriate
DNA extraction techniques are needed for both the vector and its microbes.
Although a few studies have investigated different methods of DNA isolation
from ticks, they are limited in the number and types of DNA extraction and lack
species-specific quantification of DNA yield.
Methods. Here we determined the most efficient and consistent method of DNA
extraction from two different developmental stages of I. scapularis—nymph and
adult—that are the most important for disease transmission. We used various
methods of physical disruption of the hard, chitinous exoskeleton, as well as
commercial and non-commercial DNA isolation kits. To gauge the effectiveness of
these methods, we quantified the DNA yield and confirmed the DNA quality via PCR
of both tick and microbial genetic material.
Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification Kit
Submitted 30 April 2015
Accepted 12 July 2015 resulted in the highest DNA yields and the most consistent PCR amplification when
Published 13 August 2015 combined with either cutting or bead beating with select matrices across life stages.
Corresponding author DNA isolation methods using ammonium hydroxide as well as the MoBio PowerSoil
Sergios-Orestis Kolokotronis, kit also produced strong and successful PCR amplification, but only for females.
[email protected]
Discussion. We contrasted a variety of readily available methods of DNA extraction
Academic editor from single individual blacklegged ticks and presented the results through a
Keith Crandall
quantitative and qualitative assessment.
Additional Information and
Declarations can be found on
page 11 Subjects Ecology, Entomology, Evolutionary Studies, Molecular Biology, Parasitology
DOI 10.7717/peerj.1147 Keywords Arthropod, Vector-borne, Blacklegged tick, Tick, DNA extraction, Nucleic acids,
DNA quantification
Copyright
2015 Ammazzalorso et al.

Distributed under INTRODUCTION

Creative Commons CC-BY 4.0
Blacklegged ticks (Ixodes scapularis Say, 1821) are hard-bodied, hematophagous arthropod
OPEN ACCESS (Arachnida, Ixodida) ectoparasites of vertebrates in North America. During its two-year

How to cite this article Ammazzalorso et al. (2015), To beat or not to beat a tick: comparison of DNA extraction methods for ticks
(Ixodes scapularis). PeerJ 3:e1147; DOI 10.7717/peerj.1147
 life cycle, the tick acquires a bloodmeal at each developmental stage (i.e., larva, nymph,
and adult) prior to molting or egg-laying, in the case of adult females. These ticks are of
great public health importance as pathogen vectors because they carry and transmit a
variety of human disease agents, such as Borrelia burdgorferi, the causative agent of Lyme
disease, Anaplasma phagocytophilum which causes human granulocytic anaplasmosis, and
Babesia microti, a protozoan responsible for the malaria-like illness, babesiosis (Speilman,
1976; Steere, Broderick & Malawista, 1978; Pancholi et al., 1995). Recently, I. scapularis has
been found to transmit Borrelia miyamotoi (Scoles et al., 2001) and Powassan virus lineage
2 (a.k.a., Deer Tick Virus) (Telford et al., 1997). Their importance as human pathogen
vectors necessitates research that involves successful isolation of genetic material needed in
investigations of both the vector itself and of the wide range of pathogens that they carry.
However, DNA isolation in ticks is challenging due to the hard chitinous exoskeleton and
the small amount of microbial nucleic acids present (Halos et al., 2004). Furthermore, tick
DNA is suseptible to degradation (Hubbard, Cann & Wright, 1995; Hill & Gutierrez, 2003;
Halos et al., 2004) and PCR can be challenged by inhibitors (Halos et al., 2004).
Although DNA extraction from ticks for both pathogen isolation and tick genetic and
genomic research is performed routinely by researchers, there is no consensus regarding
the most effective method of DNA isolation from any tick species. A few such studies and
reviews have been conducted (Hill & Gutierrez, 2003; Halos et al., 2004; Crowder et al.,
2010; Mtambo et al., 2006; Sparagano et al., 1999); however, they are limited to a handful of
extraction techniques, and quantitative data on DNA concentration is lacking. In this study
we aim to identify the optimal DNA isolation procedure for both tick and microbial DNA
from an important tick pathogen vector, the blacklegged tick.

MATERIALS & METHODS

Tick collection
Nymphal and adult female blacklegged ticks are important life stages in the transmission
of disease agents compared to larvae, which are rarely infected with human pathogens,
and adult males, whose brief feeding bouts minimize the risk of pathogen transmission
(Piesman et al., 1986; Falco & Fish, 1988; Falco et al., 1999). Thus, the ability to dependably
extract DNA, and in particular microbial DNA, from nymphal and adult female
blacklegged ticks is of importance to tick-borne disease research and constitutes the focus
of this study.
Unfed, host-seeking nymphal and adult female blacklegged ticks were collected by
dragging a 1 m2 flannel cloth along the forest floor or along low vegetation, respectively,
during each life stage’s peak activity period. A total of 258 ticks (129 nymphs and 129 adult
females) were collected from sites in Westchester County, Putnam County, and Orange
County in New York state in the summers and falls of 2009–2011, and were subsequently
stored in 70% v/v ethanol at room temperature until DNA isolation in the summer of
2014.

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 DNA isolation
We contrasted the efficiency of extracting DNA from ticks stored in 70% v/v ethanol
using five different DNA isolation procedures coupled with either cross-sectional division
or bead-based physical disruption of the tick body. These procedures included four
commercially available DNA extraction kits: DNeasy Blood & Tissue Kit (QIAGEN,
Valencia, California, USA), GeneJET Genomic DNA Purification Kit (Thermo Scientific,
Waltham, Massachusetts, USA), Tissue & Insect DNA MicroPrep (Zymo Research, Irvine,
California, USA), PowerSoil DNA Isolation Kit (MoBio, Carlsbad, California, USA); and
one noncommercial DNA extraction method using ammonium hydroxide (NH4 OH) and
heat, which has been primarily used for DNA isolation from the European sheep tick,
Ixodes ricinus (Guy & Stanek, 1991; Rijpkema et al., 1996; Pichon et al., 2003; Humair et al.,
2007; Pangrácová et al., 2013).
All commercial kits examined here use a silica-based column procedure and have either
been used in previous studies for DNA isolation in ticks or are marketed for efficient
microbial DNA recovery or insect DNA isolation (Table 1). All kits also include filtering
through a silica gel membrane and a variety of associated buffers. The Zymo kit and the
NH4 OH treatment did not include a protein digestion step. DNA extraction kits were used
according to the manufacturers’ recommended protocols with few exceptions (Table 1),
including a final elution completed following a 5-min room-temperature incubation
in 100 µl of deionized, sterilized, distilled water (sdH2 O) for consistency. Each tick
was air-dried to evaporate the ethanol prior to DNA extraction, as ethanol may inhibit
PCR (Hubbard, Cann & Wright, 1995; Bessetti, 2007; Schrader et al., 2012). Due to the
components similarity of the QIAGEN and Thermo kits, we used the same Proteinase-K
incubation duration (overnight).
The NH4 OH method included adding 150 µl of a 0.7-M NH4 OH solution to the tick
sample in a 1.5-ml snap cap tube, and heating to 100 ◦ C for 15 min. The solution was
briefly centrifuged to concentrate fluid at the bottom and then was evaporated to 70–100 µl
by opening the tubes and heating at 100 ◦ C for an additional 15 min. The solution was then
centrifuged for 10 min at 10,000 × g and the supernatant was collected and respun for 2
min at 10,000 × g. The total supernatant was collected and stored at −20 ◦ C.
To determine the most effective method of physical disruption of the hard, chitinous
exoskeleton of ticks prior to DNA extraction, we compared cross-sectionally dividing ticks
(bisection for nymphs and quadrisection for adult females) and crushing the entire tick in
a variety of bead matrices using the BeadBlaster 24 (Benchmark Scientific, Edison, New
Jersey, USA) (Table 2). The MoBio and Zymo kits included their own bead matrices
(Table 2). The beads from these two kits were used according to the manufacturers’
instructions with either whole or cut ticks. MoBio-processed samples were beaten on a
GeneMate vortex mixer (BioExpress, Kaysville, Utah, USA) at maximum speed (3,200
rpm) for 10 min, and Zymo-processed samples were beaten on the BeadBlaster 24
for 10 min at 4 m/s. For DNA extraction methods that did not include bead matrices
(i.e., QIAGEN, Thermo, and NH4 OH), we used six different MP Bio Lysing Matrices
(http://www.mpbio.com/index.php?cPath=2 77 425&country=223), which were either

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 Table 1 Methods of DNA isolation and physical disruption of tick samples. Only samples treated with the MoBio kit were processed on the
GeneMate vortex mixer (BioExpress), while all remaining bead beating took place on the BeadBlaster 24 (Benchmark Scientific).

DNA extraction method Alterations to manufacturer protocols Physical disruption Bead beating
—speed
and duration
Overnight incubation at 56 ◦ C in lysis Bisection (Nymphs), Quadrisection N/A
QIAGEN DNeasy Blood & Tissue Kit buffer/proteinase K (Females)
(cat. no. 69506) Elution in 100 µl dsH2 0 with 5 min MP Bio Lysing Matrices 4 m/s
room temperature incubation 1.5 & 4.0 min
Overnight incubation at 56 ◦ C in lysis Bisection (Nymphs), Quadrisection N/A
Thermo GeneJET Genomic DNA buffer/proteinase K (Females)
Purification Kit
Elution in 100 µl dsH2 0 with 5 min MP Bio Lysing Matrices 4 m/s
(cat. no. K0722)
room temperature incubation 1.5 & 4.0 min
Elution in 100 µl dsH2 0 with 5 min Bisection (Nymphs), Quadrisection 4 m/s
room temperature incubation (Females) followed by beating 10 min
Zymo Research Tissue & Insect DNA with Zymo beads
MicroPrep
Zymo beads 4 m/s
(cat. no. D6015)
10 min
Elution in 100 µl of dsH2 0 with 5 min Bisection (Nymphs), Quadrisection 3,200 rpm
room temperature incubation (Females) followed by vortexing with 10 min
MoBio PowerSoil DNA Isolation Kit MoBio garnet beads
(cat. no. 12888) MoBio provided beads 3,200 rpm
10 min
Initial volume of 150 µl NH4 OH Bisection (Nymphs), Quadrisection N/A
NH4 OH Final volume of 70–100 µl dsH20 (Females)
(Guy & Stanek, 1991; Pichon et al., 2003) Second centrifugation for 2 min at MP Bio Lysing Matrices 4 m/s
10,000 × g 1.5 & 4.0 min

Table 2 Bead matrices and their attributes. The composition, characteristics, and recommended uses for the different bead matrices tested are
adapted from the manufacturers’ websites. (MP Bio: http://www.mpbio.com/index.php?cPath=2 77 425&country=223, MoBio: http://www.mobio.
com/soil-dna-isolation/powersoil-dna-isolation-kit.html, Zymo: http://www.zymoresearch.com/dna/genomic-dna/solid-ffpe-tissue-dna/zr-tissue-
insect-dna-miniprep).

Matrix Manufacturer Material Suggested use

G MP Bio 1.6 mm silicon carbide particles Samples with tough, hard, or brittle cell
membranes
H MP Bio 2 mm glass beads & 2 mm zirconium oxide beads Tough, hard cells and organisms within dense
exterior matrices, e.g., whole insects and ticks
I MP Bio 2 mm zirconium beads & one 4 mm ceramic sphere Very tough, hard samples including chitin
exoskeletons, e.g., whole insects and ticks
M MP Bio Two 6.35 mm zirconium oxide-coated ceramic grinding Tough tissues, seeds, spores
spheres
S MP Bio 3.175 mm stainless steel beads Tough tissues, seeds, spores
Z MP Bio 2.0 mm yttria-stabilized zirconium oxide spheres Tough plant and animal samples
PowerBeads MoBio Garnet Environmental samples
BashingBead Lysis Zymo Ceramic Ticks, mosquitoes, bees, lice, and Drosophila
Matrix melanogaster

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 marketed for tough samples or, as in the case of Matrices H and I, were marketed
specifically for ticks (Table 2). We beat the ticks with the MP Bio Lysing Matrices for 1.5
and 4 min at 4 m/s (Table 1).
The variables that we maintained constant across DNA extraction methods were: bead
beating speed (4 m/s), water volume for final DNA elution of 100 µl, and a 5-min duration
of incubation in sdH2 O during DNA elution.
DNA was extracted from three nymphs and three females for all combinations of
DNA isolation and physical disruption procedures, including the different bead beating
durations and MP Bio Lysing Matrices. DNA was extracted using the MoBio and Zymo
kits from six nymphs and six females each. Half were bisected or quadrisected and half
remained whole prior to DNA extraction with one of these two kits. The QIAGEN,
Thermo, and NH4 OH methods were used to extract DNA from 39 nymphs and 39 females
each. For every one of these three procedures, three ticks from each life stage were bisected
or quadrisected and 36 ticks were bead-beaten. Among the 36 bead-beaten ticks processed
per method, six nymphs and six females were beaten with one of the six MP Bio Lysing
Matrices, half for 1.5 min and half for 4 min. Overall, DNA was isolated from 129 nymphs
and 129 females for a total of 258 tick DNA extractions.

DNA quantification
The resulting DNA yields were quantified via double-stranded DNA (dsDNA) fluoro-
metric quantitation on a Qubit 2.0 flurometer (Life Technologies, Norwalk, Connecticut,
USA) using 10 µl of extracted DNA template in 190 µl of the High Sensitivty (HS) dsDNA
assay.

PCR validation
The isolated DNA was validated using PCR amplification of tick mitochondrial and
nuclear loci. We amplified the tick DNA using (1) the cytochrome c oxidase subunit 1
(Cox1) DNA barcode region (∼650 bp) located on the mitochondrial genome with the
HCO/LCO primers (Folmer et al., 1994), and (2) a dinucleotide (CA)n microsatellite repeat
located on the nuclear genome with the bac7ea/bac7eb primer pair (139–197 bp) (Chan,
2012).
We targeted the genus Rickettsia using PCR to validate the successful extraction of
microbial DNA from inside the tick, which is necessary for studies involving PCR detection
of human pathogens transmitted by this tick species. Members of this genus are obligate
intracellular bacteria and are abundant in blacklegged ticks (Benson et al., 2004; Moreno
et al., 2006; Noda, Munderloh & Kurtti, 1997; Steiner et al., 2008). We targeted a 532-bp
fragment of the ompA gene using Rickettsia-specific primers (Vitorino et al., 2007).
All thermal cycling conditions were slightly modified from their published protocols
and are detailed below. The thermal cycling conditions for the Cox1 DNA barcode region
began with an initial denaturation at 95 ◦ C for 5 min, followed by 35 cycles of 95 ◦ C for
30 s, 50 ◦ C for 30 s, and 72 ◦ C for 30 s, and then a final extension at 72 ◦ C for 7 min (Folmer
et al., 1994). The microsatellite region was amplified using a touchdown PCR with an
initial denaturation at 95 ◦ C for 1 min, then 5 cycles of 95 ◦ C for 20 s, 60 ◦ C for 20 s, and

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 72 ◦ C for 30 s; 30 cycles of 95 ◦ C for 20 s, 50 ◦ C for 25 s, and 72 ◦ C for 30 s; and a final
extension for 5 min at 72 ◦ C (Chan, 2012). The Rickettsia ompA locus was amplified with
an initial denaturation at 95 ◦ C for 5 min, 35 cycles of 95 ◦ C for 30 s, 52 ◦ C for 30 s, and
72 ◦ C for 30 s, and a final extension for 7 min at 72 ◦ C (Vitorino et al., 2007). All PCR
reactions were performed using a Techne Prime Elite Thermal Cycler (Bibby Scientific,
Burlington, New Jersey, USA).
Each PCR was performed in a final 25-µl volume with 6.25 µl 2 × MyTaq HS Mix
(Bioline, Taunton, Massachusetts, USA) and 0.2 µM of each forward and reverse primer.
The PCRs targeting nuclear and mitochondrial tick DNA were caried out using 16.25
µl sdH2 O and 1.5 µl DNA template. To account for the lower DNA concentrations of
microbial DNA within this tick, and specifically for nymphs, the PCRs targeting the
Rickettsia ompA fragment were carried out with 15.75 µl sdH2 O and 2 µl DNA template
from nymphs, and 16.75 µl sdH2 O and 1 µl DNA template for adult females. PCRs were
confirmed by agarose gel electrophoresis on a 1.5% w/v gel based on amplicon length.

Statistical analysis
Given the substantial differences in size and DNA yield between nymphs and adult
females, their DNA concentrations were not comparable. Therefore, statistical analyses
of the DNA concentration data from the two developmental stages were performed
separately. Following a one-way ANOVA, Tukey’s HSD test was used for post-hoc analysis
of the average DNA quantification values resulting from nymph bisection or female
quadrisection across the five DNA isolation methods (QIAGEN, Thermo, MoBio, Zymo,
and NH4 OH). The Bonferroni correction was used to account for multiple comparisons.
The same procedures were also used to compare the DNA yields resulting from the
different MP Bio Lysing Matrices (G, H, I, M, S, and Z) and nymph bisection or female
quadrisection methods using data from the highest-yielding DNA extraction method. The
effect of bead beating duration was assessed through a two-tailed Student’s t-test.

RESULTS AND DISCUSSION

Comparison of DNA yield based on bisection and quadrisection
The comparison of DNA concentrations resulting from nymph bisection and female
quadrisection across the five DNA extraction methods yielded no apparent differences
between the QIAGEN DNEasy Blood & Tissue Kit and the Thermo GeneJET Genomic
DNA Purification Kit (Fig. 1). For both nymphs and adult females, the QIAGEN and
Thermo methods resulted in markedly higher DNA yields than all other methods (Fig. 1,
Table 3). The Zymo Research Tissue & Insect DNA MicroPrep Kit produced DNA yields
that were too low to be quantifiable by the Qubit fluorometer in the case of nymphs and
were extremely low in the case of adult females (average = 0.02 ng/µl, SD = 0.01).

Comparison of DNA yield based on bead beating

For the two highest-yielding DNA isolation methods (QIAGEN and Thermo), Thermo
had a significantly higher DNA yield than QIAGEN across all bead matrices in the case of
both nymphs (P = 2.7e–06) and females (P = 0.02). When cut ticks were included in this

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 Figure 1 DNA concentrations (ng/µl) resulting from the five DNA extraction methods following
nymph bisection and female quadrisection as determined using the Qubit HS dsDNA Assay. Each
sample set consisted of three individual tick DNA extractions. Note the difference in scale between the
life stages. (A) Nymphs (B) Adult Females.

comparison, Thermo had a higher DNA yield for both nymphs (P = 2.39e–05) and females
(P = 0.04). Therefore, in this section we report results based solely on the Thermo method.
Specifically, the use of bead matrices H, I, S, and Z for nymphs, and H, I, and S for females
resulted in higher DNA yield (P < 0.04). For both nymphs and adult females the M and G
matrices were the least efficient (Fig. 2). Nymphal bisection also produced DNA yields that
were significantly better than M and G, while adult female quadrisection outperformed
matrices M, G, and Z (P < 0.03) (Fig. 2). The duration of bead beating had no effect on
DNA yield in either nymphs or adult females (P > 0.6 and P > 0.1, respectively) MP Bio

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 Table 3 Average DNA concentration (ng/µl) of whole and cut nymphal and adult female blacklegged
ticks. Average and standard deviation of the DNA concentration values determined using the Qubit HS
dsDNA Assay. Unless otherwise indicated, samples were stored in 70% v/v ethanol. Three single-tick
measurements were included in each treatment. All values listed as <0.0005 ng/µl indicate a reading of
“too low” from the Qubit fluorometer.

Method Life stage Nymphs Adult females

Bisected Whole Quadrisected Whole
Average 1.70 9.41
QIAGEN Table S1 Table S1
SD 0.640 1.63
Average 1.68 9.77
Thermo Table S1 Table S1
SD 0.11 2.35
Average 0.070 0.0373 4.51 0.119
MoBio
SD 0.0585 0.0457 0.492 0.0133
Average <0.0005 <0.0005 3.14 0.0146
Zymo
SD 0.00 0.00 1.44 0.00341
Average 0.677 0.0240
NH4 OH Table S1 Table S1
SD 0.086 0.0143

lysing matrix bead beating results for whole nymphs and adult females treated with the
QIAGEN, Thermo and NH4 OH methods are detailled in Table S1.

PCR validation
The Thermo method exhibited the strongest and most consistent gel electrophoresis PCR
product bands across physical disruption methods in the case of both nymphs and females
for mtDNA (Cox1), nuDNA (microsatellite), and bacterial DNA (Rickettsia ompA). The
DNA extractions resulting from the NH4 OH protocol yielded strong and mostly consistent
amplification of the tick mitochondrial and nuclear loci, and the bacterial gene for adult
females. Although the NH4 OH method yielded lower DNA concentration results than the
QIAGEN and Thermo kits when using bead-beaten or quadrisected adult female ticks, this
did not affect PCR success. However, PCR amplification was consistently poor for nymphs
treated with the NH4 OH protocol. Consistent with the very low DNA yields we measured,
the DNA extracted using the Zymo kit did not result in any successful PCRs for ticks (either
nymphs or adults) and only produced amplicons for Rickettsia ompA from quadrisected
females.
DNA extraction from adult female ticks with the MoBio kit resulted in the consistent
amplification of the three targeted loci, with quadrisection substantially enhancing
the amplicon gel band quality in contrast to whole females (beaten with garnet on a
vortex). Those loci were not successfully amplified in the case of bisected and whole
nymphs—concordant with low DNA yields.
Among the MP Bio Lysing Matrices H, I, and S produced some of the strongest and most
consistent PCR results across all targeted loci following bead-beating for 1.5 min for both
females and nymphs with the Thermo kit and for females with the NH4 OH method. These
results partially reflected the MP Bio website’s recommendation for using matrices H and I
with ticks, while matrix S is not mentioned in that capacity.

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 Figure 2 DNA concentrations (ng/µl) resulting from the Thermo DNA extraction method following
the bead beating of whole ticks. Bead beating was carried out with each of the MP Bio lysing matrices (G,
H, I, M, S, and Z). Nymphs were bisected and adult females were quadrisected. The DNA concentration
was determined using the Qubit HS dsDNA Assay. Six nymphs and six adults were used in each bead
beating treatment, while three nymphs and three adults were used in each cutting treatment. (A) Nymphs
(B) Adult Females.

Nymph bisection and female quadrisection produced PCR products that were generally
just as strong (on agarose gel) and consistent as bead beating with the H, I, and S matrices
combined with the Thermo kit. In the case of NH4 OH only adult females (quadrisected
and bead-beaten with matrices H, I, and S) had successful PCR results. The QIAGEN kit
consistently yielded successful PCR amplification when ticks were cut, but when using
bead beating PCR results were mixed and inconsistent.

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 In terms of rapidly quantifying PCR success, we can report the proportion of positive
PCRs, while acknowledging that this is merely based on PCR product band presence on
an agarose gel, rather than accounting for band intensity. The latter characteristic can be
discussed on case-by-case basis, rather than being summarized in subjective fluorescence
categories, e.g., faint, medium, strong. In terms of DNA extraction methods, the Thermo
kit yielded the most “amplifiable” DNA (201 positive PCRs out of 234, i.e., 85.9% success
rate) with comparable performance across developmental stages (87.18% of adult females
vs. 84.62% of nymphs). The two other methods that used bead beating (QIAGEN and
NH4 OH) did not perform as well overall (61.11% and 58.55%, respectively) and further-
more exhibited a skewed behavior across developmental stage with PCRs from female adult
ticks working more consistently (72.65% of females vs. 49.57% of nymphs for QIAGEN;
85.47% of females vs. 31.62% of nymphs in NH4 OH). PCRs using the DNA extracted with
the MoBio kit worked well with female adults (88.89%) but not with nymphs (11.11%).
The Zymo kit was the most poorly performing in terms of PCR with only three successful
reactions out of 36 (8.33%) with none of the nymphal samples producing amplified PCR
products. Those DNA isolation and physical disruption methods that were most successful
for the nuclear and mitochondrial tick gene PCRs were also most effective for the Rickettsia
ompA gene amplification. All of this information is detailed in Table S2, yet we raise cau-
tion against the strict interpretation of those results, as part of a full assessment should be
the PCR amplicon yield (i.e., number of PCR fragments) that, during gel electrophoresis, is
manifested by fluorescence intensity. Quantitative PCR can be used for such purposes.

CONCLUSIONS
Successful DNA extraction from tick species is important for both genetic and genomic
studies of the tick vector itself, as well as for studies aimed at detecting pathogen presence
in these tick vectors. This study was designed to determine the most reliable and efficient
method of DNA extraction, including physical disruption of the tick exoskeleton.
Among the tested tick DNA extraction procedures, we recommend several different
procedures depending on budget, time, contamination concerns, and study goals. The
Thermo kit is recommended for its high DNA yields coupled with high-quality PCR
amplification with both bead beating and nymph bisection or adult quadrisection, as well
as for its lower cost in comparison with the QIAGEN kit—a similarly structured kit. The
QIAGEN kit may be used if already available when cutting ticks, but is not recommended if
bead beating is the chosen physical disruption strategy.
The NH4 OH extraction method is an inexpensive alternative to commercially available
kits and produced high-quality PCR products for adult females, although the DNA yield
was generally lower than that of commercial kits. However, this method was not useful for
DNA extraction from nymphs, resulting in low DNA yield and poor to non-existent PCR
amplification, despite the frequent use of this method in studies on nymphal Ixodes ricinus
in Europe (Guy & Stanek, 1991; Rijpkema et al., 1996; Pichon et al., 2003; Humair et al.,
2007; Pangrácová et al., 2013).

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 The MP Bio H and I matrices, which MP Bio recommends for ticks, as well as the S
matrix were best for bead beating in conjunction with the Thermo kit for nymphs and
females or NH4 OH for females. These results confirm MP Bio’s recommendation of the
H and I matrices for ticks, and we would add to that list matrix S. Benefits of bead beating
include less processing time and reduced direct sample handling, which may decrease the
likelihood of contamination. However, cutting ticks requires less expensive equipment
than bead beating.
The Zymo kit was a poor choice for extracting DNA from nymphs and adult female
ticks, although it is marketed for DNA extraction from stored arthropods, including ticks
(as per the manufacturer’s website http://www.zymoresearch.com/dna/genomic-dna/
solid-ffpe-tissue-dna/zr-tissue-insect-dna-kits/zr-tissue-insect-dna-microprep). The
MoBio kit also performed poorly with nymphs but showed suceess with adult females.
Admittedly, the sample size of three nymph and three female replicates for each
combination of DNA extraction variables is limited. Additional replicates may better
capture the potential variability in DNA concentration and quality resulting from each
extraction procedure. Future studies with larger sample sizes would be useful to further
assess the efficacy of these different methods with perhaps greater precision, although there
is no reason to expect that increasing the number of replicates would necessarily yield
globally different results. Our results clearly demonstrate that DNA yield quality varies
among different extraction kits and methods, which can have an important impact on the
success of PCR-based studies.
Our study expands on previous work that determined DNA extraction success from
ticks based on PCR amplification alone, without a DNA quantification assessment (Halos
et al., 2004). While a recent study (Crowder et al., 2010) quantified DNA yield, the reported
values were averaged across multiple tick species and focused only on one developmental
stage—adults. In order to test the efficiency of the DNA extraction techniques, we kept
certain variables constant, such as the long-term storage method, bead beating speed,
elution volume, and incubation time prior to elution. Alteration of these variables may
result in increased DNA yield and should be considered when DNA concentration is
important in downstream applications, such as high-throughput sequencing, pathogen
surveillance, and microbial community profiling.

ACKNOWLEDGEMENTS
We thank Rachel Engstrand for assistance with R. We thank the reviewers and the editor
for their insightful comments and suggestions that helped us shape the final version of this
paper.

ADDITIONAL INFORMATION AND DECLARATIONS

Funding
This study was partly funded by a Fordham University Summer Science Internship to ADA.
The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.

Ammazzalorso et al. (2015), PeerJ, DOI 10.7717/peerj.1147 11/14

 Grant Disclosures
The following grant information was disclosed by the authors:
Fordham University Summer Science Internship.

Competing Interests
The authors declare there are no competing interests.

Author Contributions
• Alyssa D. Ammazzalorso performed the experiments, analyzed the data, wrote the paper,
prepared figures and/or tables, reviewed drafts of the paper.
• Christine P. Zolnik conceived and designed the experiments, performed the experi-
ments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed
drafts of the paper.
• Thomas J. Daniels contributed reagents/materials/analysis tools, wrote the paper,
reviewed drafts of the paper.
• Sergios-Orestis Kolokotronis conceived and designed the experiments, analyzed the
data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures
and/or tables, reviewed drafts of the paper.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/
10.7717/peerj.1147#supplemental-information.

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