659101

research-article2016
VDIXXX10.1177/1040638716659101A quantitative PCR developed for Anaplasma platys detectionSilva et al.

Full Scientific Report

Journal of Veterinary Diagnostic Investigation

A new quantitative PCR method for the 2016, Vol. 28(5) 529­–535
© 2016 The Author(s)
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DOI: 10.1177/1040638716659101
jvdi.sagepub.com
on the citrate synthase gene

Claudia B. da Silva,1 Marcus S. Pires, Joice A. R. Vilela, Maristela Peckle,

Renata L. da Costa, Gabriela L. V. Vitari, Leandro A. Santos,
Huarrisson A. Santos, Carlos L. Massard

Abstract. Anaplasma platys is an obligate intracellular bacterium that primarily affects dogs, but it can also infect humans.
Our study aimed to standardize a quantitative real-time (q)PCR method using the citrate synthase gene (gltA) as a specific
target for A. platys detection in naturally infected dogs. Primers (gltA84F and gltA84R) and probe (PLATYSp) were designed
to amplify an 84-bp fragment based on the gltA gene sequences of A. platys available in GenBank. A total of 186 dog
blood samples originating from the Brazilian state of Rio de Janeiro were tested by qPCR. Additionally, the same samples
were tested by cytology and a nested (n)PCR that targeted the 16S ribosomal DNA to determine the performance of our
qPCR method compared to these existing techniques. Among the samples tested with qPCR, 17.2% were considered positive,
significantly more than detected by nPCR (14.0%). Under optical microscopy, inclusions were observed in platelets of 25.3%
of the samples, and among these samples, only 33.9% were identified as positive for A. platys using qPCR. The qPCR
technique proved to be more specific than cytology and to have superior sensitivity to nPCR for detecting A. platys in dogs.
The development of this new qPCR method contributes to the advancement of research involving A. platys. Furthermore, it
can be used to quantify the presence of this bacterium to evaluate the treatment of infected animals, or even as a more sensitive
and specific tool for situations indicating possible clinical disease but with negative cytology.

Key words: Anaplasma platys; citrate synthase; dogs; real-time PCR.

Introduction
Anaplasma platys is an obligate intracellular, gram-negative
bacterium and pleomorphic organism that affects dog plate-
lets, causing canine infectious cyclic thrombocytopenia. This
bacterium was once housed in the genus Ehrlichia, but it has
been recently reclassified based on a phylogenetic analysis of
the 16S ribosomal DNA (rDNA) gene.15 Although this agent is
important for small animal clinics, A. platys has a broad host
range, including cats,11 cows,12 camels,24 and humans.4,7,26
The most common diagnostic methods for canine infec-
tious cyclic thrombocytopenia are the direct identification of
the morulae in blood smears, serologic methods to detect
antibodies, and DNA amplification by PCR.17 The newer
diagnostic tools are typically based on PCR, with advantages
of greater sensitivity and specificity. Targets such as the heat-
shock operon, 23S rDNA, and the citrate synthase gene have
been used in diagnostic and phylogenetic studies, as well as
in the characterization of A. platys strains.1,12,24 As well, a
nested (n)PCR approach targeting the 16S rDNA gene has
been reported.1,27 The quantitative real-time (q)PCR tech-
nique has been used to detect A. platys infection,19 but this
assay was based on the p44 polynucleotide that encodes the
surface-exposed protein P44.25 However, it is important to
search for new targets that are less mutable between strains
and that result in the lowest number of false negatives. Our
study sought to standardize a qPCR technique targeting the
gltA gene of A. platys for the detection of this agent in natu-
rally infected dogs.
Materials and methods
Standardization of qPCR
Primers and probe were designed using commercial softwarea
to examine the sequences of the gltA gene from A. platys
available in GenBank (EU516387, AB058782, KC342665,
JN121381, DQ525688, DQ525687, DQ525686, AY077620,
Department of Animal Parasitology (Silva, Pires, Peckle, Costa, Vitari,
Massard), Department of Epidemiology and Public Health, Veterinary
Institute (HA Santos, Vilela), and Department of Soils, Agronomy Institute
(LA Santos), Federal Rural University of Rio de Janeiro, Rio de Janeiro,
Brazil.
1
Corresponding author: Claudia Bezerra da Silva, Department of
Animal Parasitology, Federal Rural University of Rio de Janeiro, BR 465,
Km 7, Rio de Janeiro, Seropédica 23890-000, Brazil.
claudia_ufrrj@yahoo.com.br
530
Silva et al.
AY530807, and AF478130). The characteristics of primers,
combined and separate, and the probe were tested using com-
mercial software,b and primer specificity was tested using
both a software toolc and sequence alignment. The following
selected primers amplified a 84-bp fragment of the gltA gene
of A. platys: gltA84F (5′-GACCTACGATCCGGGATTCA-3′)
and gltA84R (5′-TGGCGCAGTATACCCTTTTCTC-3′). The
probe was PLATYSp (5′-FAM-TCTACCGCGGCATG
CAGCTCTG-TAMRA-3′).
To optimize the primer and probe concentrations, concen-
tration curves were generated using the positive control DNA
as a template. The positive A. platys DNA sample (GenBank
FJ755157) was obtained from a symptomatic dog with micro-
scopic inclusions in platelets and confirmed by PCR targeting
the 16S rDNA gene. This positive control has been character-
ized and represents the strain of A. platys circulating in Brazil.
An existing assayd was used to determine the optimal primer
concentration and the minimum primer concentration neces-
sary to have the lowest quantification cycle (Cq) and maximum
fluorescence signal, with the baseline subtracted (ΔRn) in the
absence of nonspecific peaks for temperature of dissociation
compared to the positive control. A PCR was performed with 4
replicates of each of the 9 conditions established for primer
testing (300 nM/300 nM, 300 nM/600 nM, 300 nM/900 nM,
600 nM/300 nM, 600 nM/600 nM, 600 nM/900 nM, 900
nM/300 nM, 900 nM/600 nM, and 900 nM/900 nM) according
to a commercial protocol.e The reactions were run in a total
volume of 20 µL comprising 1× commercial PCR master mixf
and 3 µL (100 ng/µL) of A. platys DNA using the following
thermocycling conditions: 95°C for 10 min, 40 cycles of 95°C
for 20 s, and 60°C for 1 min. A curve of dissociation was gener-
ated to verify the specificity of the amplifications.
After the standardization of the optimal concentrations of
primers, the optimal probe concentration was determined.
Single-probe assays were run in 4 replicates for each concen-
tration (50 nM, 100 nM, 150 nM, 200 nM, and 250 nM). The
PCR to determine the optimal concentration of the probe was
performed in a final volume of 20 µL comprising 1× com-
mercial PCR master mix,g 600 nM of each primer, 3 µL (100
ng/µL) of A. platys DNA, and the respective probe concen-
tration. The selection of the optimal probe concentration for
qPCR was based on the result of the lowest Cq and the max-
imum ΔRn obtained in the target detection assay. DNA
extraction, mixture preparation, and sample pipetting were
performed in separate locations, and filter tips were used in
all steps to avoid contamination.
Analytical sensitivity and specificity of qPCR
The analytical sensitivity of qPCR was evaluated by using
serial decimal dilutions of the amplicon cloned into plasmid.h
Plasmid DNA concentration and purity were verified using a
spectrophotometer.i The plasmid copy number versus Cq
values were plotted to determine the analytical sensitivity of
the qPCR based on commercial technology.j The number of
copies ranged from 1 to 1 × 104 per µL, with 5 separate dilu-
tion series performed for each point of the curve in triplicate.
This assay was performed using an exogenous internal posi-
tive control reagent (termed E-IPC)k that contains a probe
marked with a reporter dyel and a quencher.m The linear
regression, along with the determination coefficient (R2)
formed after determination of each point of the curve, can be
used to evaluate whether the qPCR assay has been optimized.
Each reaction’s efficiency was determined considering the
slope of standard curve using the following formula: [Effi-
ciency = 10(−1/slope) – 1].43
The specificity of the assay was evaluated using DNA from
the following related organisms and other pathogens transmit-
ted by ticks: Anaplasma phagocytophilum, Neorickettsia risti-
cii, and Rickettsia rickettsii obtained from cellular cultures;
Anaplasma marginale obtained from a blood sample of a natu-
rally infected bovine; Babesia canis vogeli, Ehrlichia canis,
and Hepatozoon canis obtained from blood samples of natu-
rally infected dogs; and Theileria equi obtained from a blood
sample of a naturally infected equine. All blood samples were
obtained from animals with high parasitemia, in acute phase,
with infection detected by microscopy and confirmed by the
relevant specific molecular assay.13,18,23,28,30,34
Sampling
Between June 2011 and June 2012, 186 blood samples were
collected from domestic dogs in the state of Rio de Janeiro,
Brazil. Collection was performed by cephalic venipuncture,
and the collected blood was placed in sterile tubes containing
ethylenediaminetetra-acetic acid (EDTA). These samples
were used to determine the performance of the new qPCR
technique compared to both cytology (blood smear) and
nPCR, which targeted 16S rDNA. These procedures were
approved by the Ethics Committee on Research of the Federal
Rural University of Rio de Janeiro (protocol 363/2013, pro-
cess 23083.003990/2013-93) and the Ethics Committee on
Animal Use (protocol 001/2014) from the same university.
DNA extraction
Genomic DNA was extracted from 300 μL of each blood sam-
ple using a commercial DNA purification kit,n according to the
manufacturer’s recommendations. Next, the samples were
quantified using a spectrophotometeri and diluted in aliquots
of 100 ng/µL. As noted above, the positive blood control was
obtained from a symptomatic dog with platelet inclusions.
Nuclease-free watero was used as a negative control.
Evaluation of the qPCR performance
The E-IPC was added to the reaction to identify the presence
of inhibitors. The experiment performed was a standard curve
reaction carried out in duplicate using the same equipment
used for qPCR.p The final volume was 20 µL comprising the
 A quantitative PCR developed for Anaplasma platys detection
following components: 1× E-IPC mix,k 1× E-IPC DNA,k 1×
universal PCR master mix,k 600 nM of each primer (gltA84F
and gltA84R), 250 nM of probe (PLATYSp), and 3 µL (~300
ng) of total DNA extracted from whole blood. The thermocy-
cling conditions were as follows: 52°C for 2 min, 95°C for 10
min (denaturation), 40 cycles of 95°C for 15 s, and 60°C for
1 min. Samples were considered positive if they had a Cq of
<40 and if the same result was observed between replicates.
Nested PCR
To evaluate the performance of qPCR, the same DNA sam-
ples extracted from whole blood were also submitted to
nPCR using primers targeting the 16S rDNA gene that was
performed as previously described.27 The amplification
products were subjected to electrophoresis in a 1.5% agarose
gel. The electrophoresis was run for 60 min at 90 V (5 V/cm).
The gel was stained with ethidium bromide (0.4 mg/mL) and
visualized under ultraviolet light.q
Blood smear evaluation
Smears were prepared using the same whole blood samples
used for DNA extraction and qPCR. The laminas were fixed
in methanol, stained with Giemsa, and subjected to immer-
sion microscopy (1,000×) to enable visualization of morulae
in platelets. Approximately 100 fields were evaluated per
lamina.
Statistical analysis
The McNemar test at a 5% significance level was used to mea-
sure the proportions of disagreement between qPCR and the
other tests evaluated for the diagnosis of A. platys. All statisti-
cal analyzes were performed using BioEstat 5.0 software.5
Results
After designing and testing the probe and primers, it was
found that the sequences were specific to the target after
analysis in silico using a software tool as well as the in vitro
test using DNA from other members of family Anaplasmata-
ceae. The optimal concentration chosen for the forward and
reverse primers was 600 nM, which reached a Cq of 16.98 in
the positive control (Supplemental Table 1, available at
http://vdi.sagepub.com/content/by/supplemental-data). The
primer concentration of 900 nM/900 nM (forward/reverse)
achieved the best results in relation to the average value of Cq
obtained (16.68) and ΔRn (3.830). However, after the analyses
of the dissociation curve, a primer dimerization was observed,
which led to the exclusion of this concentration. The optimal
concentration of the probe was 250 nM, reaching an average Cq
value of 16.33 in the positive control (Supplemental Table 2,
available at http://vdi.sagepub.com/content/by/supplemental-data)
and a higher ΔRn with the minimum Cq.
531
Figure 1.  Standard curve plotted from serial decimal dilutions
of plasmid DNA, containing the Anaplasma platys gltA gene and
internal positive control reagents included in the reaction. The
quantification cycle (Cq) value obtained by hydrolysis probe
quantitative real-time PCR was plotted as a function of the initial
number of plasmid copies.
The detection limit of the qPCR was 1 copy of the plasmid
per microliter in both tested curves. The determination coef-
ficient of the 5 dilutions tested in the standard curve with the
E-IPC was 99%, with a Cq range of 23.66 ± 0.17 cycles in the
first dilution (1 × 104 plasmid copies) to 37.77 ± 0.35 cycles
in the last dilution (1 plasmid copy; Fig. 1). The amplification
curve with the E-IPC had the same layout during the expo-
nential phase, and the reaction efficiency was 96.06%. This
finding demonstrates that, even in reactions with few targets,
the reaction was a success. Without the E-IPC, the reaction
efficiency of the PCR was 101.80%. The determination coef-
ficient of the 5 dilutions tested in the standard curve with the
E-IPC was 99%, with a Cq range of 23.52 ± 0.10 cycles in the
first dilution (1 × 104 plasmid copies) to 36.88 ± 0.40 cycles
in the last dilution (1 plasmid copy; Fig. 2). As before, even in
reactions with few targets, the reaction was efficient.
Regarding qPCR specificity, only A. platys DNA was ampli-
fied when other organisms were tested in vitro. The A. platys
hydrolysis probe qPCR assay proved to be efficient in the
amplification of a 84-bp fragment from the gltA gene in clin-
ical samples. Among the samples tested using qPCR, 17.20%
(n = 32/186) were positive, whereas only 13.98%
(n = 26/186) amplified the target when using nPCR. The
average value of Cq observed in positive samples was 28.83
± 6.48, with a range of 18.79–38.58 cycles. In all reactions,
the E-IPC amplified properly, producing an average value of
Cq of 27.77 ± 0.06, with a range of 27.71–27.90 cycles.
According to the equation generated by the standard curve,
the detection of the number of A. platys copies ranged from
1 to 234,188, with Cq values of 38.58 and 18.79, respectively
(Table 1).
All positive nPCR samples were found to be positive
when using qPCR. However, nPCR did not detect the target
532
Silva et al.

Table 1.  Relationship between the quantification cycle (Cq)

and the Anaplasma platys gltA gene copy number obtained from
quantitative real-time PCR using samples from naturally infected
dogs.*

Positive No. of A. platys DNA

Cq sample copies based on Cq
18.79 96 234,188.30
18.91 185 216,173.62
19.37 112 160,051.47
19.52 183 145,143.93
20.45 154 79,260.74
21.09 84 51,933.87
21.31 179 44,948.81
24.04 20 7,562.98
24.75 27 4,743.92
Figure 2.  Standard curve plotted from serial decimal dilutions 24.96 164 4,141.09
of plasmid DNA, containing the Anaplasma platys gltA gene, 25.48 46 2,951.83
without the addition of internal positive control reagents. The 26.05 55 2,025.03
quantification cycle (Cq) value obtained by hydrolysis probe 26.15 94 1,898.49
quantitative real-time PCR was plotted as a function of the initial 26.83 36 1,214.79
number of plasmid copies. 26.85 178 1,199.11
27.16 50 981.56
fragment in 18.75% (n = 6/32) of the positive samples identi- 29.41 60 224.65
fied using qPCR (p = 0.0313; Table 2). Likewise, the results 29.81 184 173.34
of qPCR compared with the results of blood smears (Table 2) 30.25 181 130.11
showed that there was a significant difference (p = 0.0455) 31.56 155 54.98
between the discordant pairs. 31.97 47 42.07
33.70 95 13.56
In the blood smears evaluation, 25.27% (n = 47/186) of the
33.79 31 12.86
samples showed basophilic inclusion suggestive of parasitism
34.21 4 9.73
in platelets. However, among these samples only 33.91% (n =
34.53 114 7.90
15/47) were positive for A. platys by qPCR (Table 2).
35.36 25 4.60
37.16 122 1.42
Discussion 37.33 44 1.26
37.73 3 0.97
This study presented the development of a hydrolysis probe 37.76 5 0.96
qPCR assay targeting the A. platys gltA gene for diagnostic 37.83 48 0.91
and quantitative detection of A. platys in dog blood samples. 38.58 2 0.56
Typically, approaches to the molecular detection of A. platys
focus on the 16S rDNA gene as a target.1,14,27 This gene is * Volume of blood was 300 µL.
commonly used in molecular diagnostic methods because of
the high conservation of the gene and the multiple copies in Table 2.  Analysis of the disagreement between the results
the bacteria genome,33 which makes the molecular technique from the quantitative real-time (q)PCR and nested (n)PCR assays
and blood smear tests.
more sensitive and reliable. These characteristics were used
in the choice of the 16S rDNA gene as a comparative target nPCR Blood smear
to the gltA gene used in the qPCR. The gltA-based qPCR
  + – + –
developed in our study was more sensitive compared with
the nPCR targeting the 16S rDNA gene, even though the gltA qPCR
gene is a single-copy gene, as usually observed in rickettsial  + 26   6 15  17
agents.6 This fact demonstrates that the primers and probe  –  0 154 32 122
designed for our study had high sensitivity and specificity in p 0.0313* 0.0455*
the detection of A. platys in dog blood samples.
* p < 0.05 indicates significant disagreement between tests (McNemar).
The quantitative reverse transcription (qRT)-PCR targeting
16S rRNA has been used previously as a molecular diagnostic
tool.37,39,40 The qRT-PCR test is considered a robust diagnostic copy number, because of their role in the translation of genetic
method because ribosomes and rRNA are present in a higher code, than genes on the chromosome.37 However, a RNA-based
 A quantitative PCR developed for Anaplasma platys detection
technique is demanding because of the RNA degradation prob-
lem and the difficulty in handling RNA samples.38 The use of a
DNA-based method for the detection of hemoparasites, as used
in this and other studies,12,14,19,22 is a less demanding approach.
The qPCR technique can be used to monitor amplification prod-
ucts during the reaction and eliminate post-amplification pro-
cessing, thus decreasing the turnaround time for the assay.16 In
addition to the operational advantages, the qPCR technique has
a quantitative nature and is both sensitive and reproducible,
potentially replacing direct PCR assays in diagnostic routines.32
The primers and the probe used in our study were designed
to provide high specificity and sensitivity. At the end of the
probe, there is a purine (guanine), whereas in the E. canis
and E. chaffeensis sequences, there are 2 purines (adenine).
The pairing of these bases blocks the formation of hydrogen
bonds between the bases because of steric hindrance. The
bulky molecular structure of the purines45 interferes with the
hybridization of the DNA strand. We also sought the ideal
concentration for the primers and the appropriate conditions
to avoid the formation of primer dimers, and other factors
that may interfere with PCR.45
Our results showed that the potential inhibitors in the
reaction were absent because of adequate amplification of
E-IPC used in this assay. False-negative results in PCR may
be related to the presence of inhibitors, problems with assay
sensitivity, or unexpected pipetting errors.10 The use of an
internal positive control is common in reaction optimiza-
tions, and it is described as an appropriate tool because it
determines whether the absence of specific amplification is
actually a result of the absence of the target DNA.36
Marked thrombocytopenia and low parasitemia occur in
dogs during the initial period of A. platys infection. For this
reason, specific and sensitive tests are needed to identify this
etiologic agent.16 When the nPCR was analyzed in parallel
with the qPCR, it was clear that both could detect A. platys
DNA fragments in dog blood samples with high parasitemia.
However, the qPCR was more sensitive than the nPCR
because the qPCR was able to detect the target in samples
with low parasitemia. Although nPCR has been reported as a
technique with high sensitivity,27 our results demonstrate a
higher sensitivity with the newly developed qPCR technique.
Additionally, the risk of sample cross-contamination is low
in qPCR tests and high in nPCR assays.3
The adoption of a standard curve to evaluate qPCR effi-
ciency is considered a quality benchmark for the assay,43 and
it is recommended in guidelines for qPCR experiments.8 It
has been shown that the optimum efficiency for qPCR assays
range from 90% to 110%.44 The efficiency for the qPCR
developed in our study was 96.06%, which is considered
desirable for adequate target detection in a qPCR reaction.44
The curve without the E-IPC generated in this study had a
higher efficiency (101.80%). This demonstrates that the
presence of the E-IPC can interfere with the reaction because
of the occurrence of competition, and thus lower the amplifi-
cation efficiency of qPCR, a finding that has been previously
533
reported.21 However, this does not diminish the role of the
E-IPC, which in our study was useful in confirming the
absence of inhibitors in the reaction, maintaining the credi-
bility of qPCR.
One of the A. platys detection methods commonly used is
the search for morulae inside platelets in a blood smear,17
which is a rapid and inexpensive technique, but which offers
only low sensitivity.41 Other studies have shown a difference
in performance between a molecular method and blood
smears for detection of A. platys.35 In our study, the concor-
dance of qPCR with the blood smear was low, demonstrating
the accuracy of qPCR, as observed in the literature.31 Addi-
tionally, whereas blood smear and nPCR approaches can
detect A. platys, they cannot provide quantitative data,16
unlike the qPCR developed in our study.
The false-positive cytology results may be explained by
the occurrence of granulations in platelets a result of platelet
activation,17 dense granules, nuclear remnants of megakary-
ocytes,29 technical artifacts, or inclusions of E. canis in plate-
lets.2,29 We found some samples to be negative using the
blood smear technique and positive by qPCR. This finding
can be explained by the presence of thrombocytopenia dur-
ing infection, which interferes with the visualization of
microorganisms by microscopic observation17 because of the
cyclic parasitemia behavior of A. platys. Bacteremia is usu-
ally found to be low because of difficulty with detecting the
bacteria in blood smears where <1% of platelets may be
infected.20,27
Our study describes the development of a hydrolysis
probe qPCR assay targeting the gltA gene for the detection of
A. platys. Reliable and accurate diagnosis of canine infec-
tious cyclic thrombocytopenia is necessary to ensure correct
treatment for the animal and to monitor the treatment of
infected animals. Additionally, research has shown that
qPCR can be used as a suitable tool for monitoring parasite
load during hemoparasite investigations.9,42 As well, qPCR is
useful in the evaluation of therapy for diseases, and it is
capable of detecting and quantifying parasitism, even during
the chronic phase of infection.9 In our study, it was observed
that the developed qPCR method can detect this parasite,
even in samples with small copy numbers, thus demonstrat-
ing that this assay can be useful in monitoring the treatment
of both symptomatic and asymptomatic animals. The qPCR
method we have developed is more specific and more sensi-
tive than cytology and is more sensitive than the nPCR
approach used for comparison.
Authors’ contributions
CB da Silva, MS Pires, M Peckle, HA Santos, and CL Massard
contributed to conception and design of the study, and contributed
to acquisition, analysis, and interpretation of data. JAR Vilela con-
tributed to conception and design of the study, and contributed to
acquisition of data. RL da Costa and GLV Vitari contributed to
design of the study, and contributed to analysis and interpretation of
data. LA Santos contributed to design of the study, and contributed
534
Silva et al.
to analysis of data. All authors drafted the manuscript; critically
revised the manuscript; gave final approval; and agreed to be
accountable for all aspects of the work in ensuring that questions
relating to the accuracy or integrity of any part of the work are
appropriately investigated and resolved.
Sources and manufacturers
a. Primer Express 3.0, Thermo Fisher Scientific, Waltham, MA.
b. Oligo Explorer 1.2, Gene Link Inc., Hawthorne, NY.
c. primerBLAST, NCBI, Bethesda, MD.
d. SYBR Green assay, Applied Biosystems, Carlsbad, CA.
e. Getting Started Guide of Primer Express Software 3.0, Applied
Biosystems, Foster City, CA.
f. SYBR Green PCR master mix, Applied Biosystems, Carlsbad,
CA.
g. TaqMan universal PCR master mix, Applied Biosystems, Fos-
ter City, CA.
h. pGEM-T Easy vector system, Promega Corp., Madison, WI.
i. Spectrophotometer Nanodrop ND-2000, Thermo Fisher Scien-
tific, Wilmington, DE.
j. TaqMan Technology, Applied Biosystems, Foster City, CA.
k. TaqMan exogenous internal positive control reagents (VIC
probe), Applied Biosystems, Foster City, CA.
l. VIC, TaqMan Technology, Applied Biosystems, Foster City,
10. Conraths FJ, Schares G. Validation of molecular-diagnostic
CA
m. TAMRA (carboxytetramethylrhodamine), TaqMan Technol-
ogy, Applied Biosystems, Foster City, CA.
n. Wizard genomic DNA purification kit, Promega Corp., Madi-
son, WI.
o. Ambion nuclease-free water, Thermo Fisher Scientific, Wilm-
ington, DE.
p. StepOnePlus real-time PCR system, Applied Biosystems, Fos-
ter City, CA.
q. L-PIX Touch system, Loccus Biotecnologia, Cotia, São Paulo,
Brazil.
Declaration of conflicting interests
The author(s) declared that there is no conflict of interest with
respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article. The
National Council for Scientific and Technological Development
and the “Carlos Chagas Filho” Foundation for Research Support of
the State of Rio de Janeiro provided financial support.
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