Protocol
Optimization of DNA Extraction from Individual
Sand Flies for PCR Amplification
Lorena G. Caligiuri 1,2, Adolfo E. Sandoval 3, Jose C. Miranda 4, Felipe A. Pessoa 5,
María S. Santini 6, Oscar D. Salomón 7, Nagila F. C. Secundino 8 and Christina B. McCarthy 1,2,*
1 Centro Regional de Estudios Genómicos, Facultad de Ciencias Exactas, Universidad Nacional de La Plata,
La Plata 1900, Argentina; lgcaligiuri@gmail.com
2 Departamento de Informática y Tecnología, Universidad Nacional del Noroeste de la Provincia
de Buenos Aires, Pergamino, Buenos Aires 2700, Argentina
3 Laboratorio de Vectores, Secretaría de Calidad de Vida, Municipalidad de Posadas, Posadas, Misiones 3300,
Argentina; sandoval@inti.gob.ar
4 Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia 40296-710, Brasil;
jmiranda@bahia.fiocruz.br
5 Instituto Leonidas e Maria Deane, Fundação Oswaldo Cruz, Manaus, Amazônia 69057-070, Brasil;
favpessoa@gmail.com
6 Centro Nacional de Diagnóstico e Investigación en Endemoepidemias, Administración Nacional
de Laboratorios e Institutos de Salud, Ministerio de Salud, Buenos Aires 1063, Argentina;
mariasoledadsantini@gmail.com
7 Instituto Nacional de Medicina Tropical, Ministerio de Salud de la Nación, Puerto Iguazú, Misiones 3370,
Argentina; odanielsalomon@gmail.com
8 Laboratory of Medical Entomology, René Rachou Research Institute, Fundação Oswaldo Cruz,
Minas Gerais 30190-002, Brazil; secundinon@gmail.com
* Correspondence: mccarthychristina@gmail.com; Tel.: +54-221-423-6332
Received: 13 March 2019; Accepted: 3 May 2019; Published: 7 May 2019


Abstract: Numerous protocols have been published for extracting DNA from phlebotomines.
Nevertheless, their small size is generally an issue in terms of yield, e ciency, and purity, for large-scale
individual sand fly DNA extractions when using traditional methods. Even though this can be
circumvented with commercial kits, these are generally cost-prohibitive for developing countries.
We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain
reaction (PCR) and, for this reason, we evaluated various modifications on a previously published
protocol, the most significant of which was a di erent lysis bu er that contained Ca2+ (bu er TESCa).
This ion protects proteinase K against autolysis, increases its thermal stability, and could have
a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was
confirmed by amplification reactions using internal control primers that amplify a fragment of the
cacophony gene. To the best of our knowledge, this is the first time a lysis bu er containing Ca2+ has
been reported for the extraction of DNA from sand flies.
Keywords: sand fly; DNA extraction; calcium; PCR; lysis bu er; Lutzomyia
1. Introduction
Various protocols have been published for the extraction of DNA from phlebotomines,
including methods that eliminate DNA-associated proteins by using detergents and salts [1–3], or with
proteinase K and detergents [4], and others that also add extraction steps with phenol-chloroform
and precipitation with alcohol [5,6]; commercial DNA extraction kits [7,8]; and the use of Chelex-100
resin [9,10]. Nevertheless, the small size of the sand flies (around 3 mm long) can be an issue,
Methods Protoc. 2019, 2, 36; doi:10.3390/mps2020036 www.mdpi.com/journal/mps

Methods Protoc. 2019, 2, 36 2 of 15
especially in studies that require analysis on an individual basis, such as parasite infection, variability,
and population genetics. In particular, these large-scale individual DNA extractions using traditional
methods usually yield poor results in terms of e ciency, quantity and purity, which in turn a ect
PCR success and DNA conservation. This can be circumvented by the use of commercial kits [3],
particularly in terms of purity. Notwithstanding, in developing countries, an extensive use of the latter
is cost-prohibitive and, consequently, traditional protocols become indispensable.
In our studies we were interested in detecting parasite infection in Lutzomyia spp. sand flies by
PCR amplification [11]. Nevertheless, as parasite DNA is not necessarily expected, we first needed
to confirm that the DNA extraction had been successful, to ensure that negative results were not
due to a poor extraction. In our studies this was done with internal control primers that amplify
a fragment of the constitutive Lutzomyia cacophony gene [12,13]. We previously used a traditional
DNA extraction method with pools of 5 and 10 field-captured L. longipalpis sand flies. The protocol,
which we here refer to as pAC, uses detergent (SDS), proteinase K and phenol-chloroform extraction
([14] and Acardi personal communication). The DNA extracted from these pools of sand flies yielded
the expected results consistently when subjected to the internal control PCR. Nevertheless, when we
used pAC to process individual sand flies, we found that amplification was poor and inconsistent
(i.e., internal control PCR results were variable). For this reason, we decided to evaluate various
modifications and, as this method eliminates DNA-associated proteins with proteinase K, we focused
on this first crucial step. A number of researchers have reported that calcium ions activate proteinase
K and that they are required for the enzymatic action of the protein [15–17]. Even though another
study disputes the reduction in proteolytic activity of proteinase K when calcium is absent, it concedes
that calcium-free proteinase K precipitates irreversibly in the presence of EDTA, leading to a reduced
e ective concentration [18]. Because of this, we decided to add calcium to the lysis bu er (here referred
to as bu er TESCa). DNA extracted from individual sand flies using this and other variations we
implemented, produced consistent and successful results in the amplification reactions. To the best of
our knowledge, this is the first time a lysis bu er containing Ca2+ has been reported for the extraction
of DNA from sand flies.
2. Experimental Design
In the following scheme (Scheme 1) we show the main variations that were assayed to optimize
DNA extractions from one sand fly (for details see “Section 5”, Appendices B–E, and Table 1):

Methods Protoc. 2019, 2, 36 3 of 15
Methods Protoc. 2018, 1, x FOR PEER REVIEW 3 of 14


Scheme 1. This scheme summarizes the different variations that were analyzed for the DNA
extraction protocol. The assayed modifications are indicated by yellow boxes and bold text (for details
see “Section 5”, Appendices B–E, and Table 1). Figures are schematic (i.e., not an exact representation)
and are not drawn to scale. (*) In the third extraction with solvents, add 700 µl C:IAA. min, minutes;
hs, hours; pK, proteinase K; C:IAA, chloroform:isoamyl alcohol (24:1); , centrifuge; s/n, supernatant;
V, volumes; 14K rpm, 14,000 revolutions per minute; ddH2O, double-distilled water.
Table 1. Comparison of the different variations that were assayed for the DNA extraction protocol
(for details see “Section 5” and Appendices B–E). Conditions that improved results are highlighted in
bold. pK: proteinase K; C:IAA: chloroform:isoamyl alcohol; RT: room temperature; ON: overnight;
ddH2O: double-distilled water.
Step Variation A 1 Variation B 2 Variation C 3 Variation D 4
Homogenization
Grind with
micropestle for
8 min in 50 µL
buffer.
Grind with
micropestle for
8 min in 50 µL
buffer.
Grind with
micropestle for
8 min in 50 µL
buffer.
Grind with
micropestle for
8 min in 50 µL
buffer.
Lysis and protein
digestion
Buffer pAC;
Incubation with pK
at 58 °C for 0.5, 2, 3,
4 and 8 h.
Buffer pAC;
Incubation with
pK at 58 °C for
8 h.
Buffers pAC, TES
and TESCa;
Incubation with pK
at 50 °C for 8 h.
Buffer TESCa
Incubation with
pK at 50 °C for
1, 2, 3, 4 and 8 h.
Extraction with
solvents
Gentle mixing by
inversion with
C:IAA.
Gentle and
vigorous mixing
by inversion
with C:IAA.
Vigorous mixing by
inversion with
C:IAA.
Vigorous mixing
by inversion
with C:IAA.
DNA precipitation
Incubation with
alcohol: ON at
–20 °C, and no
incubation
Incubation with
alcohol at –20 °C
5
Incubation with
alcohol at –20 °C 5
Incubation with
alcohol: ON at
–20 °C and no
incubation
Final resuspension 20 µL ddH2O 20 µL ddH2O 10 µL ddH2O 10 µL ddH2O
1 See Figure A1; 2 See Figure A2; 3 See Figures 1 and A3; 4 See Figure A4; 5 The protocol was paused in
this step due to the length of the previous stages.
Scheme 1. This scheme summarizes the di erent variations that were analyzed for the DNA extraction
protocol. The assayed modifications are indicated by yellow boxes and bold text (for details see
“Section 5”, Appendices B–E, and Table 1). Figures are schematic (i.e., not an exact representation) and
are not drawn to scale. (*) In the third extraction with solvents, add 700 L C:IAA. min, minutes; hs,
hours; pK, proteinase K; C:IAA, chloroform:isoamyl alcohol (24:1); , centrifuge; s/n, supernatant; V,
volumes; 14K rpm, 14,000 revolutions per minute; ddH2O, double-distilled water.
Methods Protoc. 2018, 1, x FOR PEER REVIEW 7 of 14

ON at −20 °C). Amplification was variable for the samples processed with buffer TES (results were
positive for only 2 of the 10 samples; Figure 1), whereas amplification was successful for all the
samples treated with buffer TESCa (Figure 1). Chi-square analysis indicated that the association
between both tr atments (buffers TES and TESCa) and their outcomes was very significant (two-
tailed p value = 0.0019).

Figure 1. Analysis of samples processed with buffers TES and TESCa. DNA extractions were
evaluated with an internal control PCR (using primers 44F/44R) and were diluted (1:5). 1: Molecular
weight (MW) (Lambda/HindIII); 2 and 15: positive control; 3–12: lysis buffer TES; 16–25: lysis buffer
TESCa; 13 and 26: negative control.
Having determined that buffer TESCa and the previous modifications (incubation with pK at 50
°C, and vigorous mixing during the extraction with solvents), consistently improved DNA
extractions, we also analyzed if we could reduce the incubation periods with proteinase K in this new
lysis buffer, and eliminate the ON incubation at −20 °C. As we had found previously, longer
incubation periods with pK improved PCR amplification, and incubation at −20 °C seemed to have
little effect (Appendix E; Table 1).
5.2. Conclusions
To summarize, the main modifications for the final optimized DNA extraction protocol
consisted of:
(1) an 8-h incubation with proteinase K in buffer TESCa at 50 °C;
(2) vigorous mixing by inversion during the extraction with solvents step; and
(3) precipitation with alcohol with no ON incubation at –20 °C (Scheme 2). Pellets were
resuspended in 10 µL ddH2O and a 1:5 dilution was used for the PCR reactions. The
complete and detailed optimized protocol is described in “Section 3”.
Figure 1. Analysis of samples processed with bu ers TES and TESCa. DNA extractions were evaluated
with an internal control PCR (using primers 44F/44R) and were diluted (1:5). 1: Molecular weight (MW)
(Lambda/HindIII); 2 and 15: positive control; 3–12: lysis bu er TES; 16–25: lysis bu er TESCa; 13 and
26: negative control.

Methods Protoc. 2019, 2, 36 4 of 15
Table 1. Comparison of the di erent variations that were assayed for the DNA extraction protocol (for
details see “Section 5” and Appendices B–E). Conditions that improved results are highlighted in bold.
pK: proteinase K; C:IAA: chloroform:isoamyl alcohol; RT: room temperature; ON: overnight; ddH2O:
double-distilled water.
Step Variation A 1 Variation B 2 Variation C 3 Variation D 4
Homogenization
Grind with micropestle
for 8 min in 50 L
bu er.
Grind with micropestle
for 8 min in 50 L
bu er.
Grind with micropestle
for 8 min in 50 L bu er.
Grind with micropestle for
8 min in 50 L bu er.
Lysis and protein
digestion
Bu er pAC; Incubation
with pK at 58 C for
0.5, 2, 3, 4 and 8 h.
Bu er pAC; Incubation
with pK at 58 C for 8
h.
Bu ers pAC, TES and
TESCa; Incubation with
pK at 50 C for 8 h.
Bu er TESCa Incubation
with pK at 50 C for 1, 2, 3,
4 and 8 h.
Extraction with solvents
Gentle mixing by
inversion with C:IAA.
Gentle and vigorous
mixing by inversion
with C:IAA.
Vigorous mixing by
inversion with C:IAA.
Vigorous mixing by
inversion with C:IAA.
DNA precipitation
Incubation with
alcohol: ON at 20 C,
and no incubation
Incubation with
alcohol at 20 C 5
Incubation with alcohol
at 20 C 5
Incubation with alcohol: ON
at 20 C and no incubation
Final resuspension 20 L ddH2O 20 L ddH2O 10 L ddH2O 10 L ddH2O
1 See Figure A1; 2 See Figure A2; 3 See Figures 1 and A3; 4 See Figure A4; 5 The protocol was paused in this step due
to the length of the previous stages.
3. Final Procedure
See also Scheme 2 in Section 5.2.
3.1. Lysis and Elimination of Proteins. Time for Completion: ~8 h, 8 min
3.1.1. Homogenization of Sand Fly
1. Aliquot su cient volume of bu er TESCa (30 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS, 5 mM
CaCl2; see “Section 4.3”), i.e., 500 L per sample, and add proteinase K (pK) (to the aliquot) to
a final concentration of (0.42 g/ L).
2. Place one adult sand fly in a 1.5 mL microcentrifuge tube, and add 50 L of bu er TESCa + pK.
3.
Methods Protoc. 2018, 1, x FOR PEER REVIEW 4 of 14

3. Final Procedure
See also Scheme 2 in Section 5.2.
3.1. Lysis and Elimination of Proteins. Time for Completion: ~8 h, 8 min
3.1.1. Homogenization of Sand Fly
1. Aliquot sufficient volume of buffer TESCa (30 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS,
5 mM CaCl2; see “Section 4.3”), i.e., 500 µL per sample, and add proteinase K (pK) (to the
aliquot) to a final concentration of (0.42 µg/µL).
2. Place one adult sand fly in a 1.5 mL microcentrifuge tube, and add 50 µL of buffer TESCa +
pK.
3. CRITICAL STEP: Grind the sand fly thoroughly for 8 min with a Teflon micropestle.
To avoid cross-contamination between samples, the micropestle must be cleaned and
autoclaved after each grinding (i.e., one micropestle per sample).
3.1.2. Cell Lysis, and Protein Denaturation a d Digestion:
4. CRITICAL STEP: Add 450 µl buffer TESCa + pK (to a final volume of 500 µL), vortex
for 1 min, and incubate at 50 °C for 8 h, vortexing for 1 min every 30 min.
3.2. Extraction with Solvents. Time for Completion: ~25 min
3.2.1. First Extraction
5. CRITICAL STEP: Add 500 µL chloroform:isoamyl alcohol (C:IAA) (24:1 v/v) and mix
vigorously by inversion for 2 min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer
the supernatant (~480 µL) to a new 1.5 mL microcentrifuge tube.
3.2.2. Second Extraction
6. CRITICAL STEP: Add 500 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2
min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~460 µL) to
a new 1.5 mL microcentrifuge tube.
3.2.3. Third Extraction
7. CRITICAL STEP: Add 700 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2
min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~400 µL) to
a new 1.5 mL microcentrifuge tube.
3.3. DNA Precipitation. Time for Completion: ~35 min
3.3.1. Addition of Salt and Alcohol
8. Add 0.1 volumes (~40 µL) 3 M Sodium Acetate (NaOAc) pH 5.2 and 2.5 volumes (~1 mL)
100% ethanol, and gently mix by inversion for 1 min.
PAUSE STEP and OPTIONAL STEP: We found that after adding NaOAc and ethanol,
results improved when the sample was immediately centrifuged (i.e., was not incubated at
all). Nevertheless, due to the length of the previous stages (~9 h), the protocol can be paused
here and the sample stored overnight (ON) at –20 °C.
3.3.2. Centrifugation
9. Centrifuge at 14,000 rpm for 20 min and discard the supernatant by inversion.
CRITICAL STEP: Grind the sand fly thoroughly for 8 min with a Teflon micropestle. To avoid
cross-contamination between samples, the micropestle must be cleaned and autoclaved after each
grinding (i.e., one micropestle per sample).
3.1.2. Cell Lysis, and Protein Denaturation and Digestion
4.
Methods Protoc. 2018, 1, x FOR PEER REVIEW 4 of 14

3. Final Procedure
See also Scheme 2 i Section 5.2.
3.1. Lysis and Elimination of Proteins. Time for Completion: ~8 h, 8 min
3.1.1. Homogenization of Sand Fly
1. Aliquot suffici nt volume of buff r TESCa (30 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS,
5 mM CaCl2; see “Section 4.3”), i.e., 500 µL per sample, and add proteinase K (pK) (to the
aliquot) to a final concentration of (0.42 µg/µL).
2. Place one adult sand fly in a 1.5 mL microcentrifuge tube, and add 50 µL of buffer TESCa +
pK.
3. CRITICAL STEP: Grind the sand fly thoroughly for 8 min with a Teflon micropestle.
T avoid cross-contaminati n between samples, the micropestle must be cleaned and
autoclaved after each grinding (i.e., one micropestle per sample).
3.1.2. Cell Lysis, and Protein Denaturation and Digestion:
4. CRITICAL STEP: Add 450 µl buffer TESCa + pK (to a final volume f 500 µL), vortex
for 1 min, and incubate at 50 °C for 8 h, vortexing for 1 min every 30 min.
3.2. Extraction with Solvents. Time for Completion: ~25 min
3.2.1. First Extraction
5. CRITICAL STEP: Add 500 µL chloroform:isoamyl alcohol (C:IAA) (24:1 v/v) and mix
vigorously by inversion for 2 min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer
the supernatant (~480 µL) to a new 1.5 mL microcentrifuge tube.
3.2.2. Second Extraction
6. CRITICAL STEP: Add 500 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2
min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~460 µL) to
a new 1.5 mL microcentrifuge tube.
.2.3. Third Extraction
7 CRITICAL STEP: Add 700 µL C:IA (24:1 v/v) and mix vigorously by inver ion for 2
min. Immediately centrifuge at 14,000 rpm 5 Transfer the supernatant (~400 µL) to
a new 1.5 mL microcentrifuge tube.
3.3. DNA Precipitation. Time for Completion: ~35 min
3.3.1. Addition of Salt and Alcohol
8. Add 0.1 volumes (~40 µL) 3 M Sodium Acetate (NaOAc) pH 5.2 and 2.5 volumes (~1 mL)
100% ethanol, and gently mix by inversion for 1 min.
PAUSE STEP and OPTIONAL STEP: We found that after adding NaOAc and ethanol,
results improved when the sample was immediately centrifuged (i.e., was not incubated at
all). Nevertheless, due to the length of the previous stages (~9 h), the protocol can be paused
here and the sample stored overnight (ON) at –20 °C.
3.3.2. Centrifugation
9. Centrifuge at 14,000 rpm for 20 min and discard the supernatant by inversion.
CRITICAL STEP: Add 450 L bu er TESCa + pK (to a final volume of 500 L), vortex for
1 min, and incubate at 50 C for 8 h, vortexing for 1 min every 30 min.
3.2. Extraction with Solvents. Time for Completion: ~25 min
3.2.1. First Extraction
5.
Methods Protoc. 2018, 1, x FOR PEER REVIEW 4 of 14

3. Final Procedure
See also Scheme 2 in Section 5.2.
3.1. Lysis and Elimination of Proteins. Time for Completion: ~8 h, 8 min
3.1.1. Homogenization of Sand Fly
1. Aliquot sufficient volume of buffer TESCa (30 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS,
5 mM CaCl2; see “Section 4.3”), i.e., 500 µL per sample, and add proteinase K (pK) (to the
aliquot) to a final concentration of (0.42 µg/µL).
2. Place one adult sand fly in a 1.5 mL microcentrifuge tube, and add 50 µL of buffer TESCa +
pK.
3. CRITICAL STEP: Grind the sand fly thoroughly for 8 min with a Teflon micropestle.
To avoid cross-contamination betw en samples, the micropestle must be cleaned and
autoclaved after each grinding (i.e., one micropestle per sample).
3.1.2. Cell Lysis, and Protein D aturation and Digestion:
4. CRITICAL STEP: Add 450 µl buffer TESCa + pK (to a final volume of 500 µL), vortex
for 1 min, and incubate at 50 °C for 8 h, vortexing for 1 min every 30 min.
3.2. Extractio with Solvents. Time for Completion: ~25 min
3.2.1. First Extraction
5. CRITICAL STEP: Add 500 µL chloroform:isoamyl alcohol (C:IAA) (24:1 v/v) and mix
vigorously by inversion for 2 min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer
the supernatant (~480 µL) to a new 1.5 mL microcentrifuge tube.
3.2.2. Second Extraction
6. CRITICAL STEP: Add 500 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2
min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~460 µL) to
a new 1.5 mL microcentrifuge tube.
3.2.3. Third Extraction
7. CRITICAL STEP: Add 700 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2
min. Immediately centrifuge at 14,000 rpm for 5 min. Tr nsfer the supernatant (~400 µL) to
a new 1.5 mL microcentrifuge tube.
3.3. DNA Precipitation. Time for Completion: ~35 min
3.3.1. Addition of Salt and Alcohol
8. Add 0.1 volumes (~40 µL) 3 M Sodium Acetate (NaOAc) pH 5.2 and 2.5 volumes (~1 mL)
100% ethanol, and gently mix by inversion for 1 min.
PAUSE STEP and OPTIONAL STEP: We found that after adding NaOAc and ethanol,
results improved when the sample was immediately centrifuged (i.e., was not incubated at
all). Nevertheless, due to the length of the previous stages (~9 h), the protocol can be paused
here and the sample stored overnight (ON) at –20 °C.
3.3.2. Centrifugation
9. Centrifuge at 14,000 rpm for 20 min and discard the supernatant by inversion.
CRITICAL STEP: Add 500 L chloroform:isoamyl alcohol (C:IAA) (24:1 v/v) and mix vigorously
by inversion for 2 min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant
(~480 L) o a new 1.5 mL microcentrifuge tube.
3.2.2. Second Extraction
6.
Methods Protoc. 2018, 1, x FOR PEER REVIEW 4 of 14

3. Final Procedure
See also Scheme 2 i Section 5.2.
3.1. Lysis and Elimination of Proteins. Time for Completion: ~8 h, 8 min
3.1.1. Homogenization of Sand Fly
1. Aliquot suffici nt volume of buff r TESCa (30 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS,
5 mM CaCl2; see “Section 4.3”), i.e., 500 µL per sample, and add proteinase K (pK) (to the
aliquot) to a final concentration of (0.42 µg/µL).
2. Place one adult sand fly in a 1.5 mL microcentrifuge tube, and add 50 µL of buffer TESCa +
pK.
3 Grind the sand fly thoroughly for 8 min with a Teflo micr pestle.
T avoid cross-contaminati n between samples, the micropestle must be cleaned and
utoclaved after each grinding (i.e., one micropestle per sample).
1 2. Cell Lys s, nd Protein D aturati n and Digestion:
4. CRITICAL STEP: Add 450 µl buffer TESCa + pK (to a final volume f 500 µL), vortex
for 1 min, and incubate at 50 °C for 8 h, vortexing for 1 min every 30 min.
3.2. Extractio wi Solvents. Time for Completion: ~25 min
3.2.1. First Extraction
5. CRITICAL STEP: Add 500 µL chloroform:isoamyl alcohol (C:IAA) (24:1 v/v) and mix
vigorously by inversion for 2 min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer
the supernatant (~480 µL) to a new 1.5 mL microcentrifuge tube.
3.2.2. Second Extraction
6. CRITICAL STEP: Add 500 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2
min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~460 µL) to
a new 1.5 mL microcentrifuge tube.
.2.3. Third Extraction
7 CRITICAL STEP: Add 700 µL C:IA (24:1 v/v) and mix vigorously by inver ion for 2
min. Immediately centrifuge at 14,000 rpm 5 Transfer the supernatant (~400 µL) to
a new 1.5 mL microcentrifuge tube.
3.3. DNA Precipitation. Time for Completion: ~35 min
3.3.1. Addition of Salt and Alcohol
8. Add 0.1 volumes (~40 µL) 3 M Sodium Acetate (NaOAc) pH 5.2 and 2.5 volumes (~1 mL)
100% ethanol, and gently mix by inversion for 1 min.
PAUSE STEP and OPTIONAL STEP: We found that after adding NaOAc and ethanol,
results improved when the sample was immediately centrifuged (i.e., was not incubated at
all). Nevertheless, due to the length of the previous stages (~9 h), the protocol can be paused
here and the sample stored overnight (ON) at –20 °C.
3.3.2. Centrifugation
9. Centrifuge at 14,000 rpm for 20 min and discard the supernatant by inversion.
CRITICAL STEP: Add 500 L C:IAA (24:1 v/v) and mix vigo ously by inversion for 2 min.
Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~460 L) to a new
1.5 mL microcentrifuge tube.

Methods Protoc. 2019, 2, 36 5 of 15
3.2.3. Third Extraction
7.
Methods Protoc. 2018, 1, x FOR PEER REVIEW 4 of 14

3. Final Procedure
See also Scheme 2 in Section 5.2.
3.1. Lysis and Elimination of Proteins. Time for Completion: ~8 h, 8 min
3.1.1. Homogenization of Sand Fly
1. Aliquot sufficient volume of buffer TESCa (30 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS,
5 mM CaCl2; see “Section 4.3”), i.e., 500 µL per sample, and add proteinase K (pK) (to the
aliquot) to a final concentration of (0.42 µg/µL).
2. Place one adult sand fly in a 1.5 mL microcentrifuge tube, and add 50 µL of buffer TESCa +
pK.
3. CRITICAL STEP: Grind the sand fly thoroughly for 8 min with a Teflon micropestle.
To avoid cross-contamination between samples, the micropestle must be cleaned and
autoclaved after each grinding (i.e., one micropestle per sample).
3.1.2. Cell Lysis, and Protein Denaturation and Digestion:
4. CRITICAL STEP: Add 450 µl buffer TESCa + pK (to a final volume of 500 µL), vortex
for 1 min, and incubate at 50 °C for 8 h, vortexing for 1 min every 30 min.
3.2. Extraction with Solvents. Time for Completion: ~25 min
3.2.1. First Extraction
5. CRITICAL STEP: Add 500 µL chloroform:isoamyl alcohol (C:IAA) (24:1 v/v) and mix
vigorously by inversion for 2 min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer
the supernatant (~480 µL) to a new 1.5 mL microcentrifuge tube.
3.2.2. Second Extraction
6. CRITICAL STEP: Add 500 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2
min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~460 µL) to
a new 1.5 mL microcentrifuge tube.
3.2.3. Third Extraction
7. CRITICAL STEP: Add 700 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2
min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~400 µL) to
a new 1.5 mL microcentrifuge tube.
3.3. DNA Precipitation. Time for Completion: ~35 min
3.3.1. Addition of Salt and Alcohol
8. Add 0.1 volumes (~40 µL) 3 M Sodium Acetate (NaOAc) pH 5.2 and 2.5 volumes (~1 mL)
100% ethanol, and gently mix by inversion for 1 min.
PAUSE STEP and OPTIONAL STEP: We found that after adding NaOAc and ethanol,
results improved when the sample was immediately centrifuged (i.e., was not incubated at
all). Nevertheless, due to the length of the previous stages (~9 h), the protocol can be paused
here and the sample stored overnight (ON) at –20 °C.
3.3.2. Centrifugation
9. Centrifuge at 14,000 rpm for 20 min and discard the supernatant by inversion.
CRITICAL STEP: Add 700 L C:IAA (24:1 v/v) and mix vigorously by inversion for 2 min.
Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~400 L) to a new
1.5 mL microcentrifuge tube.
3.3. DNA Precipitation. Time for Completion: ~35 min
3.3.1. Addition of Salt and Alcohol
8. Add 0.1 volumes (~40 L) 3 M Sodium Acetate (NaOAc) pH 5.2 and 2.5 volumes (~1 mL) 100%
ethanol, and gently mix by inversion for 1 min.
Methods Protoc. 2018, 1, x FOR PEER REVIEW 4 of 14

3. Final Procedure
See also Scheme 2 in Section 5.2.
3.1. Lysis and Elimination of Proteins. Time for Completion: ~8 h, 8 min
3.1.1. Homogenization of Sand Fly
1. Aliquot sufficient volume of buffer TESCa (30 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS,
5 mM CaCl2; see “Section 4.3”), i.e., 500 µL per sample, and add proteinase K (pK) (to the
aliquot) to a final concentration of (0.42 µg/µL).
2. Place one adult sand fly in a 1.5 mL microcentrifuge tube, and add 50 µL of buffer TESCa +
pK.
3. CRITICAL STEP: Grind the sand fly thoroughly for 8 min with a Teflon micropestle.
To avoid cross-contamination between samples, the micropestle must be cleaned and
autoclaved after each grinding (i.e., one micropestle per sample).
3.1.2. Cell Lysis, and Protein Denaturation and Digestion:
4. CRITICAL STEP: Add 450 µl buffer TESCa + pK (to a final volume of 500 µL), vortex
for 1 min, and incubate at 50 °C for 8 h, vortexing for 1 min every 30 min.
3.2. Extraction with Solvents. Time for Completion: ~25 min
3.2.1. First Extraction
5. CRITICAL STEP: Add 500 µL chloroform:isoamyl alcohol (C:IAA) (24:1 v/v) and mix
vigorously by inversion for 2 min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer
the supernatant (~480 µL) to a new 1.5 mL microcentrifuge tube.
3.2.2. Second Extraction
6. CRITICAL STEP: Add 500 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2
min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~460 µL) to
a new 1.5 mL microcentrifuge tube.
3.2.3. Third Extraction
7. CRITICAL STEP: Add 700 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2
min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~400 µL) to
a new 1.5 mL microcentrifuge tube.
3.3. DNA Precipitation. Time for Completion: ~35 min
3.3.1. Addition of Salt and Alcohol
8. Add 0.1 volumes (~40 µL) 3 M Sodium Acetate (NaOAc) pH 5.2 and 2.5 volumes (~1 mL)
100% ethanol, and gently mix by inversion for 1 min.
PAUSE STEP and OPTIONAL STEP: We found that after adding NaOAc and ethanol,
results improved when the sample was immediately centrifuged (i.e., was not incubated at
all). Nevertheless, due to the length of the previous stages (~9 h), the protocol can be paused
here and the sample stored overnight (ON) at –20 °C.
3.3.2. Centrifugation
9. Centrifuge at 14,000 rpm for 20 min and discard the supernatant by inversion.
PAUSE STEP and OPTIONAL STEP: We found that after adding NaOAc and ethanol,
results improved when the sample was immediately centrifuged (i.e., was not incubated at all).
Nevertheless, due to the length of the previous stages (~9 h), the protocol can be paused here and the
sample stored overnight (ON) at 20 C.
3.3.2. Centrifugation
9. Centrifuge at 14,000 rpm for 20 min and discard the supernatant by inversion.
10. Add 500 L 70% ethanol and centrifuge at 14,000 rpm for 5 min. Discard the supernatant by
inversion and dry the pellet at 50 C for 5 min. Resuspend the pellet in 10 L double-distilled water.
4. Materials, Equipment, and Reagents Setup
4.1. Materials
TRIS bu er (NH2(CH2OH)3, 121.14 g/mol) (Anedra, Tigre, Argentina; Cat. no.: AN00915709)
Hydrochloric acid (HCl, 36.46 g/mol) (Biopack, Buenos Aires, Argentina; Cat. no.: 9632.08)
Sodium Dodecyl Sulfate (SDS, C12H25NaO4S, 288.38 g/mol) (Anedra, Tigre, Argentina; Cat.
no.: AN219483180)
EDTA (C10H16N2O8, 292.24 g/mol) (Anedra, Tigre, Argentina; Cat. no.: AN00605609)
Calcium chloride dihydrate (CaCl2 2H2O, 147 g/mol) (Anedra, Tigre, Argentina; Cat. No.: AN6456)
Proteinase K (Fermentas-Thermo Fisher Scientific, Waltham, MA, USA; Cat. No.: #EO0491)
Double-distilled water (ddH2O)
Chloroform (CHCl3, 119.38 g/mol) (Cicarelli Laboratorios, San Lorenzo, Argentina;
Cat. no.: 1116110)
Isoamyl alcohol (Anedra, Tigre, Argentina; Cat. no.: AN00659925)
Sodium acetate (CH3COONa, 82.03 g/mol) (Anedra, Tigre, Argentina; Cat. No.: AN00651808)
Glacial acetic acid (CH3COOH, 60,05 g/mol) (Anedra, Tigre, Argentina; Cat. No.: AN6082)
Absolute ethanol (C2H6O, 46.07 g/mol) (Biopack, Buenos Aires, Argentina; Cat. no.: 1654.08)
4.2. Equipment
Teflon micropestle (Eppendorf-Fisher Scientific, Leicestershire, UK; Cat. no.: 10683001)
Vortex (Denville Scientific, Metuchen, NJ, USA; Cat. no.: Vortexer S7030)
Water bath (Jiangsu Jinyi Instrument Technology Company Limited, Shanghai, China; Cat.
no.: SHZ-88)
High-speed bench-top centrifuge (Heal Force, Shanghai, China; Cat. no.: Neofuge 15)
Micropipettes p1000, p200, p20 (Gilson, Middleton, WI, USA; Cat. nos.: F144566, F144565,
and F144563)

Methods Protoc. 2019, 2, 36 6 of 15
4.3. Reagents Setup
Bu er TESCa
Composition: 30 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS; 5 mM CaCl2.
Calculate the necessary volumes for each stock solution. Add and mix the Tris-HCl pH 8, EDTA,
and CaCl2, autoclave, and then add the SDS.
Below we give an example (Table 2):
Table 2. Example of how to prepare an adequate volume of Bu er TESCa.
Reagent Final Concentration Volume (Vf 1 = 12.5 mL)
1 M Tris-HCl pH 8 30 mM 375 L
0.5 M EDTA 10 mM 250 L
100 mM CaCl2 5 mM 625 L
dH2O 2 - 10.625 mL
20% SDS 1% 625 L
Mix and autoclave all the reagents except the SDS, and then add the SDS. 1 Final volume; 2 Distilled water.
5. Results
As previously mentioned, we found that internal control PCR results were variable for individual
sand flies processed with the pAC protocol (results not shown). For this reason, we assayed various
modifications to optimize DNA extractions from one sand fly (Scheme 1). The quality and quantity
of the DNA extracts were measured using an AmpliQuant AQ-07 Spectrophotometer, but we found
there was no correlation between the amount of DNA quantitated and the success of the PCR reactions.
Similarly, a previous study describing the optimization of a DNA extraction procedure from individual
human hairs (which poses similar di culties to those faced by the extraction of DNA from individual
sand flies), showed that there was no correlation between the amount of DNA quantitated and the
success of STR genotyping, i.e., some extracts were correctly genotyped when quantitation failed to
detect any DNA [19]. Hence, and as our main objective was to analyze the DNA extracts in PCR
reactions for field studies, success was determined by evaluating each sample in amplification reactions
using internal control primers that amplify the IVS6 domain of the Lutzomyia constitutive cacophony
gene (~225 bp) [12,13] (5Llcac and 3Llcac, here referred to as 44F/45R; see Appendix A for detailed PCR
conditions). The positive control we used was DNA extracted from a pool of 10 L. longipalpis adults
from Posadas (Argentina) using the pAC protocol; the negative control was ddH2O. The variations we
assayed and the e ects they produced are mentioned below (see also Scheme 1 and Table 1); for all
these extractions we processed field-captured male adult L. longipalpis (Posadas, Argentina).
5.1. Assayed Modifications
We first analyzed the e ect of minor modifications on the pAC protocol and found that longer
incubation times with pK and no incubation at 20 C (in the DNA precipitation step) in general yielded
better results (Appendix B; Table 1). We also found that results improved when mixing by inversion
was done vigorously in the extraction with solvents step (Appendix C; Table 1). Nevertheless, as the
previous modifications did not determine a consistent improvement, we decided to evaluate changes of
greater magnitude (yet including these minor modifications that had produced slight improvements).
We decreased the incubation temperature from 58 (original pAC) to 50 C (our modification) because
pK digestion is routinely performed at 50 C [20], and to move as far away as possible from its
inactivation temperature (65 C) (manufacturer’s recommendation). More importantly, we assayed
three di erent lysis bu ers: the original bu er pAC (as control; 10 mM Tris-HCl pH 8; 200 mM
NaCl; 5 mM EDTA; 0.2% SDS) (according to Acardi personal communication), another commonly
used lysis bu er, here referred to as bu er TES (30 mM Tris-HCl pH 8, 10 mM EDTA and 1% SDS),

Methods Protoc. 2019, 2, 36 7 of 15
and this same bu er TES to which we added Ca2+ (5 mM), here referred to as bu er TESCa (30 mM
Tris-HCl pH 8, 10 mM EDTA, 1% SDS, and 5 mM CaCl2; see “Section 4.3”). There were various
reasons for evaluating the addition of calcium to the lysis bu er (bu er TESCa). Di erent studies
have reported that calcium ions greatly a ect the enzymatic activity of pK [15–17] and that enzymatic
activity is significantly reduced when they are removed (up to 80%) [15]. Even though another study
suggested that calcium ions stabilize the native conformation of the enzyme but do not a ect proteolytic
activity [18], it showed that Ca2+-free pK precipitated irreversibly in the presence of EDTA leading to
a much reduced e ective concentration [18]. Furthermore, even though Ca2+ forms a complex with
EDTA in the bu er, it is still capable of interacting with the enzyme [15]. In addition, a previous study
found that activation of proteinase K by calcium improved the extraction of DNA from individual
human hairs [19]. This same study showed that pK su ered loss of activity when the lysis bu er
contained EDTA but no calcium [19].
To evaluate these modifications, we processed specimens with the di erent lysis bu ers (pAC, TES,
and TESCa) (Table 1) and found that amplification was only positive when the samples were processed
with bu ers TES and TESCa (Appendix D). Due to these results we decided to further evaluate bu ers
TES and TESCa and processed more specimens with these bu ers. All samples were incubated with
pK (0.42 g/ L) at 50 C for 8 h, mixing by inversion was done vigorously for the three extractions
with C:IAA, and pellets were resuspended in 10 L ddH2O. Due to the length of the first three stages
(~9 h), the protocol was paused in the fourth step (i.e., the sample was precipitated ON at 20 C).
Amplification was variable for the samples processed with bu er TES (results were positive for only
2 of the 10 samples; Figure 1), whereas amplification was successful for all the samples treated with
bu er TESCa (Figure 1). Chi-square analysis indicated that the association between both treatments
(bu ers TES and TESCa) and their outcomes was very significant (two-tailed p value = 0.0019).
Having determined that bu er TESCa and the previous modifications (incubation with pK at 50 C,
and vigorous mixing during the extraction with solvents), consistently improved DNA extractions,
we also analyzed if we could reduce the incubation periods with proteinase K in this new lysis
bu er, and eliminate the ON incubation at 20 C. As we had found previously, longer incubation
periods with pK improved PCR amplification, and incubation at 20 C seemed to have little e ect
(Appendix E; Table 1).
5.2. Conclusions
To summarize, the main modifications for the final optimized DNA extraction protocol consisted of:
(1) an 8-h incubation with proteinase K in bu er TESCa at 50 C;
(2) vigorous mixing by inversion during the extraction with solvents step; and
(3) precipitation with alcohol with no ON incubation at 20 C (Scheme 2). Pellets were resuspended
in 10 L ddH2O and a 1:5 dilution was used for the PCR reactions. The complete and detailed
optimized protocol is described in “Section 3”.

Methods Protoc. 2019, 2, 36 8 of 15
Methods Protoc. 2018, 1, x FOR PEER REVIEW 8 of 14


Scheme 2. Summary of the main experimental stages and steps involved in the optimized DNA
extraction protocol (see “Section 3” for details). The approximate time needed to complete every stage
is indicated. Figures are schematic (i.e., not an exact representation) and are not drawn to scale. (*) In
the third extraction with solvents, add 700 µL C:IAA. min, minutes; hs, hours; pK, proteinase K;
C:IAA, chloroform:isoamyl alcohol (24:1); , centrifuge; s/n, supernatant; V, volumes; 14K rpm,
14,000 revolutions per minute; ddH2O, double-distilled water.
DNA obtained using our method is suitable for long-term conservation, since individual sand
fly DNA extracts were stored at –20 °C and used as a template as much as 6 years later in PCR
reactions which yielded positive results.
In conclusion, the above-mentioned changes (the most significant of which was the addition of
calcium ions to the lysis buffer) optimized DNA extraction from individual sand flies when compared
to the original pAC protocol, and enabled us to consistently obtain positive amplification results with
the internal control primers. Moreover, we used this optimized protocol to extract DNA from
individual field-captured Lutzomyia spp. from Brazil and Argentina, and the internal control
amplifications were successful (Appendix F).
Our results also suggest that pK activity is reduced when the lysis buffer contains EDTA but no
calcium (Figure 1), in accordance with a previous study that explored this same solution for
optimizing the extraction of DNA from individual human hairs [19], and supporting previous
evidence on the dependence of pK activity on the presence of calcium ions [15–17]. Furthermore, pK
digestion is routinely performed at 50 °C [20], and it has been reported that pK’s activity can increase
several fold within the 50 °C to 60 °C range [21]. In this context, buffers pAC, TES and TESCa were
tested within pK´s optimal temperature range (50–60 °C), and we only obtained consistently
successful results with the lysis buffer that contained calcium (buffer TESCa). Similarly, McNevin et
Schem . Summar f th mai experimental stage an step involve i th optimize DN
extractio rotocol (see “Section 3” for details). The approximate time needed to complete every
stage is indicated. Figures are schematic (i.e., not an exact representation) and are not drawn to scale.
(*) In the third extraction with solvents, add 700 L C:IAA. min, minutes; hs, hours; pK, proteinase
K; C:IAA, chloroform:isoamyl alcohol (24:1); , centrifuge; s/n, supernatant; V, volumes; 14 rpm,
14,000 revolutions per minute; dd 2 , double-distille water.
DNA obtained using our method is suitable for long-term conservation, since individual sand fly
DNA extracts were stored at 20 C and used as a template as much as 6 years later in PCR reactions
which yielded positive results.
In conclusion, the above-mentioned changes (the most significant of which was the addition of
calcium ions to the lysis bu er) optimized DNA extraction from individual sand flies when compared to
the original pAC protocol, and enabled us to consistently obtain positive amplification results with the
internal control primers. Moreover, we used this optimized protocol to extract DNA from individual
field-captured Lutzomyia spp. from Brazil and Argentina, and the internal control amplifications were
successful (Appendix F).
Our results also suggest that pK activity is reduced when the lysis bu er contains EDTA but no
calcium (Figure 1), in accordance with a previous study that explored this same solution for optimizing
the extraction of DNA from individual human hairs [19], and supporting previous evidence on the
dependence of pK activity on the presence of calcium ions [15–17]. Furthermore, pK digestion is
routinely performed at 50 C [20], and it has been reported that pK’s activity can increase several fold
within the 50 C to 60 C range [21]. In this context, bu ers pAC, TES and TESCa were tested within

Methods Protoc. 2019, 2, 36 9 of 15
pK´s optimal temperature range (50–60 C), and we only obtained consistently successful results with
the lysis bu er that contained calcium (bu er TESCa). Similarly, McNevin et al. [19] used a di erent
lysis bu er (at 56 C) and also found that DNA extraction only improved when calcium was added
to the bu er [19]. This would suggest that, when working within the enzyme´s optimal temperature
range, it is the addition of Ca2+ to the lysis bu er that improves DNA extraction.
Author Contributions: Conceptualization, C.B.M.; methodology, C.B.M. and L.G.C.; investigation, L.G.C. and
C.B.M.; validation, L.G.C. and C.B.M.; formal analysis, L.G.C. and C.B.M.; resources, C.B.M., A.E.S., J.C.M., F.A.P.,
N.F.C.S., M.S.S. and O.D.S.; writing—original draft preparation, C.B.M.; writing—review and editing, C.B.M.,
L.G.C., N.F.C.S., O.D.S., M.S.S. and J.C.M.; visualization, C.B.M.; supervision, C.B.M.; project administration,
C.B.M.; funding acquisition, C.B.M.
Funding: This research was funded by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (PICT
PRH 112 and PICT CABBIO 3632), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (PIP
0294), and Universidad Nacional del Noroeste de la Provincia de Buenos Aires (SIB 2010 Exp. 1388/2010 and SIB
2013 Exp. 2581/2012), grants to C.B.M. NFCS, FAP and JCM are supported by FIOCRUZ.
Acknowledgments: We gratefully acknowledge Lilian Tartaglino from the Posadas Municipality Quality of Life
Department and members of the Vector Laboratory (Secretaría de Calidad de Vida, Municipalidad de Posadas)
for their support, logistics and interest to abate the problems of the region; the neighbors of the city of Posadas
for opening their houses; and the Research Network for Leishmaniasis in Argentina (Red de Investigación de
Leishmaniasis en Argentina, REDILA), of which some of us are members. At the time this study was carried out
LGC was a recipient of an ANPCyT fellowship and AES was a member of the Vector Laboratory (Secretaría de
Calidad de Vida, Municipalidad de Posadas). CBM, MSS and ODS are members of the CONICET research career.
NFCS, FAP and JCM are researchers from FIOCRUZ Brazil.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A Internal PCR Control Conditions
An internal control PCR was implemented to confirm the e ciency and quality of the DNA
extractions using published primers 5Llcac and 3Llcac (here referred to as 44F and 45R), that amplify
a ~225 bp fragment of the Lutzomyia constitutive cacophony gene which includes the IVS6 domain [12,13].
Amplifications were completed in a GeneMax Thermal Cycler (Bioer Corporation). The reaction
mixture contained 1 PCR bu er (200 mM Tris-HCl pH 8.4; 500 mM KCl); 2.5 mM MgCl2; 0.125 mM
dNTPs; 0.3 U Taq Pegasusfi DNA polymerase (Productos Bio-Lógicos, Argentina); 0.5 M of each
primer (44F and 45R); 0.1 mg/mL bovine serum albumin (BSA), and 1 L template, in a final volume
of 10 L. DNA extractions were diluted (1:5), the positive control was a (1:25) dilution of a previous
DNA extraction (using the pAC protocol) from a pool of 10 L. longipalpis adults from Posadas
(Argentina), and the negative control was ddH2O. The following profile was adapted from [12,13]:
initial denaturation cycle at 95 C for 30 s; followed by 35 cycles with denaturation at 94 C for 30 s,
annealing at 53 C for 30 s and extension at 72 C for 30 s; and a final extension cycle at 72 C for 7 min.
PCR products were visualized on 1.5% (Figure 1) and 1% agarose gels (Figures A1–A7).
Appendix B Assaying Di erent Incubation Periods with pK in Bu er pAC, and ON or No
Incubation at 20 C (in the DNA Precipitation Step)
We simultaneously evaluated the e ect of di erent incubation periods (0.5, 2, 3, 4 and 8 h) with pK
(0.42 g/ L) in the original lysis bu er (pAC); and, in the DNA precipitation step, we assayed the e ect
of ON incubation at 20 C (original protocol) or no incubation (our variation) (Figure A1; Scheme 1).
The overall results indicated that, as expected, longer incubation times with pK (4 and 8 h) in general
yielded better results, as did no incubation with 100% ethanol, which was unexpected (Figure A1;
Table 1). They also suggested that, when precipitating with 100% ethanol at 20 C, incubation with
pK should only occur for 2–4 h and, when precipitating with 100% ethanol at room temperature (no
incubation), incubation with pK should occur for more than 3 h (Figure A1; Table 1).

Methods Protoc. 2019, 2, 36 10 of 15
Methods Protoc. 2018, 1, x FOR PEER REVIEW 10 of 14


Figure A1. Effects of different incubation periods with pK in buffer pAC, and ON or no incubation at
−20 °C (in the DNA precipitation step). DNA extractions were evaluated with an internal control PCR
(using primers 44F/44R), and were undiluted. The blue bracket indicates the samples that were
subjected to ON incubation at −20 °C (lanes 3–7), and the yellow bracket indicates the samples that
were not incubated before centrifugation (lanes 8–12); in both cases, the numbers below the brackets
indicate the hour/s of incubation with pK. 1: MW (pZero2/HaeII); 2: positive control; 3: 0.5 h (30 min)
pK + ON @−20 °C; 4: 2 h pK + ON @−20 °C; 5: 3 h pK + ON @−20 °C; 6: 4 hs pK + ON @20 °C; 7: 8 h pK
+ ON @−20 °C; 8: 0.5 h (30 min) pK + no incubation; 9: 2 h pK + no incubation; 10: 3 h pK + no
incubation; 11: 4 h pK + no incubation; 12: 8 h pK + no incubation; 13: negative control. MW: molecular
weight; ON: overnight; RT: room temperature.
Appendix C. Assaying the Intensity when Mixing by Inversion in the Extraction with Solvents
Step
Samples were incubated with pK in buffer pAC for 8 h and, in the extraction with solvents step,
mixing by inversion was done gently (standard protocol) and vigorously (our modification)
(Figure A2; Scheme 1). Due to the length of the first three stages (~9 h), the protocol was paused in
the fourth step (i.e., samples were incubated ON at −20 °C). Results showed there was an
improvement when the samples were mixed vigorously (Figure A2; Table 1), and we implemented
this minor modification to the protocol.

Figure A2. Effect of mixing intensity in the extraction with solvents step. DNA extractions were
evaluated with an internal control PCR (using primers 44F/44R). Gentle mixing by inversion (blue
bracket, sample G): lanes 3 (undiluted) and 4 (1:10 dilution); vigorous mixing by inversion (yellow
bracket, sample V): lanes 5 (undiluted) and 6 (1:10 dilution); lane 1: MW (pZero2/HaeII); lane 2:
positive control; lane 7: negative control. G: gentle; V: vigorous; MW: molecular weight.
Appendix D. Assaying different lysis buffers
As indicated in “Section 5”, we processed specimens with the three lysis buffers (pAC, TES, and
TESCa). Samples were incubated with pK (0.42 µg/µL) at 50 °C for 8 h, mixing by inversion was done
Figure A1. E ects of di erent incubation periods with pK in bu er pAC, and ON or no incubation
at 20 C (in the DNA precipitation step). DNA extractions were evaluated with an internal control
PCR (using primers 44F/44R), and were undiluted. The blue bracket indicates the samples that were
subjected to ON incubation at 20 C (lanes 3–7), and the yellow bracket indicates the samples that
were not incubated before centrifugation (lanes 8–12); in both cases, the numbers below the brackets
indicate the hour/s of incubation with pK. 1: MW (pZero2/HaeII); 2: positive control; 3: 0.5 h (30 min)
pK + ON @ 20 C; 4: 2 h pK + ON @ 20 C; 5: 3 h pK + ON @ 20 C; 6: 4 hs pK + ON @20 C; 7: 8 h
pK + ON @ 20 C; 8: 0.5 h (30 min) pK + no incubation; 9: 2 h pK + no incubation; 10: 3 h pK + no
incubation; 11: 4 h pK + no incubation; 12: 8 h pK + no incubation; 13: negative control. MW: molecular
weight; ON: overnight; RT: room temperature.
Appendix C Assaying the Intensity When Mixing by Inversion in the Extraction with
Solvents Step
Samples were incubated with pK in bu er pAC for 8 h and, in the extraction with solvents step,
mixing by inversion was done gently (standard protocol) and vigorously (our modification) (Figure A2;
Scheme 1). Due to the length of the first three stages (~9 h), the protocol was paused in the fourth step
(i.e., samples were incubated ON at 20 C). Results showed there was an improvement when the
samples were mixed vigorously (Figure A2; Table 1), and we implemented this minor modification to
the protocol.
Methods Protoc. 2018, 1, x FOR PEER REVIEW 10 of 14


Figure A1. Effects of different incubation periods with pK in buffer pAC, and ON or no incubation at
−20 °C (in the DNA precipitation step). DNA extractions were evaluated with an internal control PCR
(using primers 44F/44R), and were undiluted. The blue bracket indicates the samples that were
subjected to ON incubation at −20 °C (lanes 3–7), and the yellow bracket indicates the samples that
were not incubated before centrifugation (lanes 8–12); in both cases, the numbers below the brackets
indicate the hour/s of incubation with pK. 1: M (pZero2/HaeII); 2: positive control; 3: 0.5 h (30 min)
pK + ON @−20 °C; 4: 2 h pK + ON @−20 °C; 5: 3 h pK + ON @−20 °C; 6: 4 hs pK + ON @20 °C; 7: 8 h pK
+ ON @−20 °C; 8: 0.5 h (30 min) pK + no incubation; 9: 2 h pK + no incubation; 10: 3 h pK + no
incubation; 11: 4 h pK + no incubation; 12: 8 h pK + no incubation; 13: negative control. : molecular
weight; ON: overnight; RT: room temperature.
Appendix C. Assaying the Intensity when Mixing by Inversion in the Extraction with Solvents
Step
Samples were incubated with pK in buffer pAC for 8 h and, in the extraction with solvents step,
mixing by inversion was done gently (standard protocol) and vigorously (our modification)
(Figure A2; Scheme 1). Due to the length of the first three stages (~9 h), the protocol was paused in
the fourth step (i.e., samples were incubated ON at −20 °C). Results showed there was an
improvement when the samples were mixed vigorously (Figure A2; Table 1), and we implemented
this minor modification to the protocol.

Figure A2. Effect of mixing intensity in the extraction with solvents step. DNA extractions were
evaluated with an internal control PCR (using primers 44F/44R). Gentle mixing by inversion (blue
bracket, sample G): lanes 3 (undiluted) and 4 (1:10 dilution); vigorous mixing by inversion (yellow
bracket, sample V): lanes 5 (undiluted) and 6 (1:10 dilution); lane 1: MW (pZero2/HaeII); lane 2:
positive control; lane 7: negative control. G: gentle; V: vigorous; MW: molecular weight.
Appendix D. Assaying different lysis buffers
As indicated in “Section 5”, we processed specimens with the three lysis buffers (pAC, TES, and
TESCa). Samples were incubated with pK (0.42 µg/µL) at 50 °C for 8 h, mixing by inversion was done
Figure A2. ect of mixing intensity in the extraction with solvents step. DN extractions were
evaluated with an internal control PCR (using primers 44F/44R). Gentle mixing by inversio (blue bracket,
sampl G): lanes 3 (undiluted) and 4 (1:10 dilution); vigorous mixing by inversion (yellow bracket,
sampl V): lanes 5 (undiluted) and 6 (1:10 dilution); lane 1: MW (pZero2/HaeII); lane 2: positive control;
lane 7: negative control. G: gentle; V: vigorous; MW: molecular weight.

Methods Protoc. 2019, 2, 36 11 of 15
Appendix D Assaying Di erent Lysis Bu ers
As indicated in “Section 5”, we processed specimens with the three lysis bu ers (pAC, TES,
and TESCa). Samples were incubated with pK (0.42 g/ L) at 50 C for 8 h, mixing by inversion was
done vigorously for the three extractions with C:IAA, and pellets were resuspended in 10 L ddH2O
(Scheme 1, Table 1). Due to the length of the first three stages (~9 h), the protocol was paused in the
fourth step (i.e., the sample was precipitated ON at 20 C). We found that amplification was only
successful for the samples processed with bu ers TES and TESCa (Figure A3).
Methods Protoc. 2018, 1, x FOR PEER REVIEW 11 of 14

vigorously for the three extractions with C:IAA, and pellets were resuspended in 10 µ dd 2
(Scheme , able 1). Due t the lengt f the first three stages (~ h), the rotocol was pa se i the
fourt ste (i.e., the sample was recipitate at −20 °C). e foun that amplificatio was onl
successful for the samples rocesse wit ffers TES and TESCa (Figure A3).

Figure A3. Effect of different lysis buffers. DNA extractions were evaluated with an internal control
PCR (using primers 44F/44R), and were diluted (1:5). 1: MW (pZero2/HaeII); 2 and 3: positive control;
4: lysis buffer TES; 5: lysis buffer TESCa; 6: buffer pAC; 7: negative control. MW: molecular weight.
Appendix E. Assaying Different Incubation Periods with pK in Buffer TESCa, and ON or No
Incubation at −20 °C (in the DNA Precipitation Step)
To analyze if we could (1) reduce the incubation periods with proteinase K in the new lysis buffer
(TESCa), and (2) in the precipitation with alcohol step, eliminate the ON incubation at −20 °C, we
processed specimens which were incubated with pK at 50 °C during 1, 2, 3, 4, and 8 h (2 specimens
per condition). One sample of each incubation period was precipitated with alcohol ON at −20 °C,
and the other without (i.e., the sample was centrifuged immediately after adding NaOAc and alcohol)
(Scheme 1, Table 1). Results with the internal control primers indicated that: 1) Overall, and as we
had found before, for both treatments (with and without ON precipitation at −20 °C), band intensity
decreased as incubation time with proteinase K decreased (Figure A4), even though the faintly visible
PCR product in lane 9 (4 h incubation with pK) suggested that longer incubation times do not always
yield more DNA; 2) On the other hand, overall band intensity was greater for the samples that were
not precipitated at −20 °C, barring the aforementioned exception (lane 9, 4 h incubation with pK)
(Figure A4; Table 1).

Figure A4. Effects of different incubation periods with pK in buffer TESCa, and ON or no incubation
at −20 °C (in the DNA precipitation step). DNA extractions were evaluated with an internal control
PCR (using primers 44F/44R) and were diluted (1:5). The blue bracket indicates the samples that were
subjected to ON incubation at −20 °C (lanes 3–7), and the yellow bracket indicates the samples that
were not incubated before centrifugation (lanes 8–12); in both cases, the numbers below the brackets
indicate the hour/s of incubation with pK. 1: MW (pZero2/HaeII); 2: positive control; 13: negative
control.
Figur A3. ec f i erent lysis b ers. DN extraction evaluate wit interna cont
PCR (using primers 44F/44R), and were dilute (1:5). (pZe Ha II) an sitiv cont ol
: lysi TES; : lysi TESCa; : pAC; : negativ cont ol. : molecula weight.
Appendix E Assaying Di erent Incubation Periods with pK in Bu er TESCa, and ON or No
Incubation at 20 C (in the DNA Precipitation Step)
To analyze if we could (1) reduce the incubation periods with proteinase K in the new lysis
bu er (TESCa), and (2) in the precipitation with alcohol step, eliminate the ON incubation at 20 C,
we processed specimens which were incubated with pK at 50 C during 1, 2, 3, 4, and 8 h (2 specimens
per condition). One sample of each incubation period was precipitated with alcohol ON at 20 C,
and the other without (i.e., the sample was centrifuged immediately after adding NaOAc and alcohol)
(Scheme 1, Table 1). Results with the internal control primers indicated that: (1) Overall, and as we
had found before, for both treatments (with and without ON precipitation at 20 C), band intensity
decreased as incubation time with proteinase K decreased (Figure A4), even though the faintly visible
PCR product in lane 9 (4 h incubation with pK) suggested that longer incubation times do not always
yield more DNA; (2) On the other hand, overall band intensity was greater for the samples that were
not precipitated at 20 C, barring the aforementioned exception (lane 9, 4 h incubation with pK)
(Figure A4; Table 1).

Methods Protoc. 2019, 2, 36 12 of 15
Methods Protoc. 2018, 1, x FOR PEER REVIEW 11 of 14

vigorously for the three extractions with C:IAA, and pellets were resuspended in 10 µL ddH2O
(Scheme 1, Table 1). Due to the length of the first three stages (~9 h), the protocol was paused in the
fourth step (i.e., the sample was precipitated ON at −20 °C). We found that amplification was only
successful for the samples processed with buffers TES and TESCa (Figure A3).

Figure A3. Effect of different lysis buffers. DNA extractions were evaluated with an internal control
PCR (using primers 44F/44R), and were diluted (1:5). 1: MW (pZero2/HaeII); 2 and 3: positive control;
4: lysis buffer TES; 5: lysis buffer TESCa; 6: buffer pAC; 7: negative control. MW: molecular weight.
Appendix E. Assaying Different Incubation Periods with pK in Buffer TESCa, and ON or No
Incubation at −20 °C (in the DNA Precipitation Step)
To analyze if we could (1) reduce the incubation periods with proteinase K in the new lysis buffer
(TESCa), and (2) in the precipitation with alcohol step, eliminate the ON incubation at −20 °C, we
processed specimens which were incubated with pK at 50 °C during 1, 2, 3, 4, and 8 h (2 specimens
per condition). One sample of each incubation period was precipitated with alcohol ON at −20 °C,
and the other without (i.e., the sample was centrifuged immediately after adding NaOAc and alcohol)
(Scheme 1, Table 1). Results with the internal control primers indicated that: 1) Overall, and as we
had found before, for both treatments (with and without ON precipitation at −20 °C), band intensity
decreased as incubation time with proteinase K decreased (Figure A4), even though the faintly visible
PCR product in lane 9 (4 h incubation with pK) suggested that longer incubation times do not always
yield more DNA; 2) On the other hand, overall band intensity was greater for the samples that were
not precipitated at −20 °C, barring the aforementioned exception (lane 9, 4 h incubation with pK)
(Figure A4; Table 1).

Figure A4. Effects of different incubation periods with pK in buffer TESCa, and ON or no incubation
at −20 °C (in the DNA precipitation step). DNA extractions were evaluated with an internal control
PCR (using primers 44F/44R) and were diluted (1:5). The blue bracket indicates the samples that were
subjected to ON incubation at −20 °C (lanes 3–7), and the yellow bracket indicates the samples that
were not incubated before centrifugation (lanes 8–12); in both cases, the numbers below the brackets
indicate the hour/s of incubation with pK. 1: MW (pZero2/HaeII); 2: positive control; 13: negative
control.
Figure A4. E ects of di erent incubation periods with pK in bu er TESCa, and ON or no incubation
at 20 C (in the DN precipitation step). DN extractions were evaluated with an internal control
PCR (using primers 44F/44R) and were diluted (1:5). The blue bracket indicates the samples that were
subjected to ON incubation at 20 C (lanes 3–7), and the yellow bracket indicates the samples that were
not incubated before centrifugation (lanes 8–12); in both cases, the numbers below the brackets indicate
the hour/s of incubation with pK. 1: MW (pZero2/HaeII); 2: positive control; 13: negative control.
Appendix F Results Using the Optimized Protocol with Di erent Lutzomyia Spp.
As previously mentioned, we extracted DNA from various Lutzomyia spp. captured in di erent
regions of Brazil, and from L. longipalpis collected in Argentina (Table A1). DNA was extracted from
individual specimens using the protocol we optimized (Scheme 2) and, as we did for the optimization,
DNA extracts were analyzed by PCR using internal control primers (44F/45R) (Figures A5–A7).
Lins et al. [13] used these same primers, in conjunction with a set of degenerate primers, to analyze the
cacophony gene from all the species we analyzed, except for L. renei. Similar to what they reported,
our amplifications were successful for all species, including L. renei (which was not analyzed by [13]),
but not for L. migonei. As Lins et al. [13] did not specify which primers they used for each of the species
(44F/45R or the degenerate primers), it is possible that the cacophony fragment from L. migonei was
previously amplified using the degenerate primers (i.e., not 44F/45R). Below we show some of the
results we obtained for each of these species (Figures A5–A7).
Table A1. List of the Lutzomyia spp. that were analyzed. Color-coding for each species coincides with
the color-coding used in Figures A5–A7.
Species 1 City State/Province Country Figure
L. umbratilis
Presidente
Figueiredo
Amazonas Brazil A5, A6
L. migonei Baturite Ceara Brazil A5
L. renei Lagoa Santa Minas Gerais Brazil A6
L. intermedia Tancredo Neves Bahia Brazil A7
L. longipalpis
(cavunge strain)
Cavunge Bahia Brazil A5, A6
L. longipalpis
(jacobina strain)
Jacobina Bahia Brazil A5
L. longipalpis
(lapinha strain)
Lagoa Santa Minas Gerais Brazil A5
L. longipalpis Posadas Misiones Argentina A7
1 Total number of specimens that were analyzed individually = 136.

Methods Protoc. 2019, 2, 36 13 of 15
Methods Protoc. 2018, 1, x FOR PEER REVIEW 12 of 14

Appendix F. Results Using the Optimized Protocol with Different Lutzomyia Spp.
As previously mentioned, we extracted DNA from various Lutzomyia spp. captured in different
regions of Brazil, and from L. longipalpis collected in Argentina (Table A1). DNA was extracted from
individual specimens using the protocol we optimized (Scheme 2) and, as we did for the
optimization, DNA extracts were analyzed by PCR using internal control primers (44F/45R)
(Figures A5–A7). Lins et al. [13] used these same primers, in conjunction with a set of degenerate
primers, to analyze the cacophony gene from all the species we analyzed, except for L. renei. Similar to
what they reported, our amplifications were successful for all species, including L. renei (which was
not analyzed by [13]), but not for L. migonei. As Lins et al. [13] did not specify which primers they
used for each of the species (44F/45R or the degenerate primers), it is possible that the cacophony
fragment from L. migonei was previously amplified using the degenerate primers (i.e., not 44F/45R).
Below we show some of the results we obtained for each of these species (Figures A5–A7).
Table A1. List of the Lutzomyia spp. that were analyzed. Color-coding for each species coincides with
the color-coding used in Figures A5–A7.
Species 1 City State/Province Country Figure
L. umbratilis Presidente Figueiredo Amazonas
Brazil A5, A6
L. migonei Baturite Ceara Brazil A5
L. renei Lagoa Santa Minas Gerais Brazil A6
L. intermedia Tancredo Neves Bahia Brazil A7
L. longipalpis (cavunge strain) Cavunge Bahia Brazil A5, A6
L. longipalpis (jacobina strain) Jacobina Bahia Brazil A5
L. longipalpis (lapinha strain) Lagoa Santa Minas Gerais Brazil A5
L. longipalpis Posadas Misiones Argentina A7
1 Total number of specimens that were analyzed individually = 136.

Figure A5. PCR amplifications using DNA extracted with the optimized protocol from L. longipalpis
(cavunge, jacobina and lapinha strains), L. migonei, and L. umbratilis. DNA extracts were evaluated
with an internal control PCR (using primers 44F/44R) and were diluted (1:5). Color-coding coincides
with that used for Table A1. 1: MW (pZero2/HaeII); 2: positive control; 3–4: L. longipalpis cavunge
strain (Cavunge, Bahia, Brazil); 5–6: L. migonei (Baturite, Ceara, Brazil); 7–8: L. umbratilis (Presidente
Figueiredo, Amazonas, Brazil); 9–10: L. longipalpis jacobina strain (Jacobina, Bahia, Brazil); 11–12; L.
longipalpis lapinha strain (Lagoa Santa, Minas Gerais, Brazil); 13: negative control.
Figure A5. PCR amplifications using DNA extracted with the optimized protocol from L. longipalpis
(cavunge, jacobina and lapinha strains), L. migonei, and L. umbratilis. DNA extracts were evaluated
with an internal control PCR (using primers 44F/44R) and were diluted (1:5). Color-coding coincides
with that used for Table A1. 1: MW (pZero2/HaeII); 2: positive control; 3–4: L. longipalpis cavunge strain
(Cavunge, Bahia, Brazil); 5–6: L. migonei (Baturite, Ceara, Brazil); 7–8: L. umbratilis (Presidente Figueiredo,
Amazonas, Brazil); 9–10: L. longipalpis jacobina strain (Jacobina, Bahia, Brazil); 11–12; L. longipalpis
lapinha strain (Lagoa Santa, Minas Gerais, Brazil); 13: negative control.Methods Protoc. 2018, 1, x FOR PEER REVIEW 13 of 14


Figure A6. PCR amplifications using DNA extracted with the optimized protocol from L. longipalpis
(cavunge strain), L. umbratilis, and L. renei. DNA extracts were evaluated with an internal control PCR
(using primers 44F/44R) and were diluted (1:5). Color-coding coincides with that used for Table A1.
1: MW (pZero2/HaeII); 2: positive control; 3–5: L. longipalpis cavunge strain (Cavunge, Bahia, Brazil);
6–9: L. umbratilis (Presidente Figueiredo, Amazonas, Brazil); 10–13: L. renei lapinha (Lagoa Santa,
Minas Gerais, Brazil); 14: negative control.

Figure A7. PCR amplifications using DNA extracted with the optimized protocol from L. longipalpis
(Argentina) and L. intermedia. DNA extracts were evaluated with an internal control PCR (using
primers 44F/44R) and were diluted (1:5). Color-coding coincides with that used for Table A1. 1: MW
(pZero2/HaeII); 2: positive control; 3–4: L. longipalpis (Posadas, Misiones, Argentina); 5–6: L. intermedia
(Tancredo Neves, Bahia, Brazil); 7: negative control.
References
1. Ready, P.D.; Lainson, R.; Shaw, J.J.; Souza, A.A. DNA probes for distinguishing Psychodopygus wellcomei
from Psychodopygus complexus (Diptera: Psychodidae). Mem. Inst. Oswaldo Cruz 1991, 86, 41–49.
2. Parvizi, P.; Amirkhani, A. Mitochondrial DNA Characterization of Sergentomyia Sintoni Populations and
Finding Mammalian Leishmania Infections in This Sandfly by Using ITS-RDNA Gene. Iran. J. Vet. Res. 2008,
9, 9–18.
3. Golczer, G.; Arrivillaga, J. Modification of a Standard Protocol for DNA Extraction from Smaller Sandflies
(Phlebotominae: Lutzomyia). Rev. Colomb. Entomol. 2008, 34, 199–200.
4. Torgerson, D.G.; Velázquez, Y.; Woo, P.T.K.; Lampo, M. Genetic relationships among some species groups
within the genus lutzomyia (Diptera: Psychodidae). Am. J. Trop. Med. Hyg. 2003, 69, 484–493.
5. Michalsky, É.M.; Pimenta, P.F.; Secundino, N.F.; Fortes-Dias, C.L.; Dias, E.S. Assessment of PCR in the
detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera:
Psychodidae: Phlebotominae). Rev. Inst. Med. trop. S. Paulo 2002, 44, 255–259.
6. Kato, H.; Uezato, H.; Katakura, K.; Calvopiña, M.; Marco, J.D.; Barroso, P.A.; Gomez, E.A.; Mimori, T.;
Korenaga, M.; Iwata, H.; Nonaka, S.; Hashiguchi, Y. Detection and Identification of Leishmania Species
within Naturally Infected Sand Flies in the Andean Areas of Ecuador by a Polymerase Chain Reaction. Am.
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7. Beati, L.; Cáceres, A.G.; A Lee, J.; E Munstermann, L. Systematic relationships among Lutzomyia sand flies
(Diptera: Psychodidae) of Peru and Colombia based on the analysis of 12S and 28S ribosomal DNA
sequences. Int. J. Parasitol. 2004, 34, 225–234.
Figure A6. PCR amplifications using DN extracted with the optimized protocol fro L. longipalpis
(cavunge strain), L. umbratilis, and L. renei. DN extracts were evaluated with an internal control PCR
(using primers 44F/44R) and were diluted (1:5). Color-coding coincides with that used for Table A1.
1: (pZero2/HaeII); 2: positive control; 3–5: L. longipalpis cavunge strain (Cavunge, Bahia, Brazil);
6–9: L. umbratilis (Presidente Figueiredo, Amazonas, Brazil); 10–13: L. renei lapinha (Lagoa Santa,
Minas Gerais, Brazil); 14: negative control.
Methods Protoc. 2018, 1, x FOR PEER REVIEW 13 of 14


Figure A6. PCR amplifications using DNA extracted with the optimized protocol from L. longipalpis
(cavunge strain), L. umbratilis, and L. renei. DNA extracts were evaluated with an internal control PCR
(using primers 44F/44R) and were diluted (1:5). Color-coding coincides with that used for Table A1.
1: MW (pZero2/HaeII); 2: positive control; 3–5: L. longipalpis cavunge strain (Cavunge, Bahia, Brazil);
6–9: L. umbratilis (Presidente Figueiredo, Amazonas, Brazil); 10–13: L. renei lapinha (Lagoa Santa,
Minas Gerais, Brazil); 14: negative control.

Figure A7. PCR amplifications using DNA extracted with the optimized protocol from L. longipalpis
(Argentina) and L. intermedia. DNA extracts were evaluated with an internal control PCR (using
primers 44F/44R) and were diluted (1:5). Color-coding coincides with that used for Table A1. 1: MW
(pZero2/HaeII); 2: positive control; 3–4: L. longipalpis (Posadas, Misiones, Argentina); 5–6: L. intermedia
(Tancredo Neves, Bahia, Brazil); 7: negative control.
References
1. Ready, P.D.; Lainson, R.; Shaw, J.J.; Souza, A.A. DNA probes for distinguishing Psychodopygus wellcomei
from Psychodopygus complexus (Diptera: Psychodidae). Mem. Inst. Oswaldo Cruz 1991, 86, 41–49.
2. Parvizi, P.; Amirkhani, A. Mitochondrial DNA Characterization of Sergentomyia Sintoni Populations and
Finding Mammalian Leishmania Infections in This Sandfly by Using ITS-RDNA Gene. Iran. J. Vet. Res. 2008,
9, 9–18.
3. Golczer, G.; Arrivillaga, J. Modification of a Standard Protocol for DNA Extraction from Smaller Sandflies
(Phlebotominae: Lutzomyia). Rev. Colomb. Entomol. 2008, 34, 199–200.
4. Torgerson, D.G.; Velázquez, Y.; Woo, P.T.K.; Lampo, M. Genetic relationships among some species groups
within the genus lutzomyia (Diptera: Psychodidae). Am. J. Trop. Med. Hyg. 2003, 69, 484–493.
5. Michalsky, É.M.; Pimenta, P.F.; Secundino, N.F.; Fortes-Dias, C.L.; Dias, E.S. Assessment of PCR in the
detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera:
Psychodidae: Phlebotominae). Rev. Inst. Med. trop. S. Paulo 2002, 44, 255–259.
6. Kato, H.; Uezato, H.; Katakura, K.; Calvopiña, M.; Marco, J.D.; Barroso, P.A.; Gomez, E.A.; Mimori, T.;
Korenaga, M.; Iwata, H.; Nonaka, S.; Hashiguchi, Y. Detection and Identification of Leishmania Species
within Naturally Infected Sand Flies in the Andean Areas of Ecuador by a Polymerase Chain Reaction. Am.
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7. Beati, L.; Cáceres, A.G.; A Lee, J.; E Munstermann, L. Systematic relationships among Lutzomyia sand flies
(Diptera: Psychodidae) of Peru and Colombia based on the analysis of 12S and 28S ribosomal DNA
sequences. Int. J. Parasitol. 2004, 34, 225–234.
Figure A7. PCR amplifications using DNA extracted with the optimized protocol from L. longipalpis
(Argentina) and L. intermedia. DNA extracts wer evaluated with an internal control PCR (using
primers 44F/44R) and were diluted (1:5). Color-coding coincides with that used for Table A1. 1: MW
(pZero2/HaeII); 2: positive control; 3–4 L longipalpis (Posadas, Misiones, Argentina); 5–6: L. intermedia
Tancredo Neves, Bahia, Brazil); 7: negative control.

Methods Protoc. 2019, 2, 36 14 of 15
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2008, 9, 9–18.
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(Phlebotominae: Lutzomyia). Rev. Colomb. Entomol. 2008, 34, 199–200.
4. Torgerson, D.G.; Velázquez, Y.; Woo, P.T.K.; Lampo, M. Genetic relationships among some species groups
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' 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).