Repositorio Institucional

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, efficiency, 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 different lysis buffer that contained Ca2+ (buffer 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 buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

Keywords: sand fly; DNA extraction; calcium; PCR; lysis buffer; 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,



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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 efficiency, quantity and purity, which in turn affect 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 effective concentration [18]. Because of this, we decided to add calcium to the lysis buffer (here referred to as buffer 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 buffer 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):



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3 of 15



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(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



Grind with



Grind with



Grind with



Grind with



Homogenization



micropestle for 8 min in 50 µL



micropestle for 8 min in 50 µL



micropestle for 8 min in 50 µL



micropestle for 8 min in 50 µL



buffer.



buffer.



buffer.



buffer.



Buffer pAC;



Buffer pAC;



Buffers pAC, TES



Buffer TESCa



Lysis and protein Incubation with pK Incubation with



and TESCa;



Incubation with



digestion



at 58 °C for 0.5, 2, 3, pK at 58 °C for Incubation with pK pK at 50 °C for



4 and 8 h.



8 h.



at 50 °C for 8 h.



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.



Figure 1. AFnigaulryesi1s. oAfnsaalymsips loefs sparmopcleesssepdrocwesistehdbwuifftherbsuTffEerSs aTnEdS TanEdSCTaE.SCDaN. DANeAxterxatcraticotinonsswweerreeevaluated with an inteevranlaulatceodnwtritohlaPnCinRte(runaslincognptrorilmPCeRrs(u4s4iFng/4p4rRim)earns d44wF/4e4rRe)dainldutweedre(1d:i5lu)t.e1d:(1M:5)o.l1e:cMuolalercuwlaeright (MW)

weight (MW) (Lambda/HindIII); 2 and 15: positive control; 3–12: lysis buffer TES; 16–25: lysis buffer

(Lambda/HTiEnSdCIaI;I)1;32anadn2d6:1n5e:gaptoivseitciovnetroclo. ntrol; 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



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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 Incubation with alcohol: ON



at −20 ◦C 5



at −20 ◦C and no incubation



Final resuspension



20 µL ddH2O



20 µL ddH2O



10 µL ddH2O



10 µL ddH2O



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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



3. Final Ptrooctehdeulreength of the previous stages.



See also Scheme 2 in Section 5.2.

3. Final Procedure

3.1. Lysis and Elimination of Proteins. Time for Completion: ~8 h, 8 min

See also Scheme 2 in Section 5.2.

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3.12..1.PHlacoemonoegadeunltizsaantdioflny inofa 1S.a5 nmdL mFilcyrocentrifuge tube, and add 50 µL of buffer TESCa +



3.1. Lysis

1. 3.



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10



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cleaned

4



and

of 14



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3.1.



2. Lysis



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micropestle. cleaned and



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33..1233..21.D..2FCN.i2eArls.ltmaPLSErnyiexenecst.cwirpsIao,mict1anat.mni5todidoemnndPE.LiraTxomtietmteilrcyienarfcoocDecrtneeCintnorotairmnftiuufpugrleeagtteiaioottnun1:b4~ae,3n0.50d0mDripingmesftoiorn5: min. Transfer the supernatant (~460 µL) to



5.



CRITICAL STEP: Add 500 µL chloroform:isoamyl alcohol (C:IAA) (24:1 v/v) and mix



3.236.31..4AT. hdidrvditigiEooCxCnrtRorRoauIfcTsIStlTiIayoClntIbACayLniAndSvTALeErlcsPSoio:hTnAoEldfodPr 24:5mA0iµndl.dIbmu5mff0eer0dTiaµEtSeLlCyaCc+e:npItAKrif(Autgoea(a2fti4n1:4a1,l0v0vo0/lrvupm)meafonfrd50mm0 iµniL.xT),rvvaoingrstfoeexrrously by inversion for 2 min.



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3.2.2E. xStercaoc1mtn0i.odi50n%E.mwIxemittrLhhmaacSemntoidoolvilnia,ectnaertnlsoyd. cTcgeeiemnnntetrtlifryfouirmfgCueioxgambtepy1le4tit,uni0ov0bne0:err~s.p2iom5nmfoinr 51 min. Transfer the supernatant (~400 µL) to



PaAnUeSwE1S.5TmEPL manicdroOcePnTtIrOifuNgAeLtuSbTe.EP: We found that after adding NaOAc and ethanol, 3.2.1.6F. irstreEsxuCtlrtaRscIitTmioIpnCrAovLeSdTwEhPe:nAtdhde s5a0m0 pµlLe Cw:aIAs iAm(m24e:d1iavt/evl)yacnedntmriifxugveigdo(rio.eu.,swlyabsynointvinercsuiboantefodra2t 33..33..1D.5AN. dAdvmahaPiltiernlgiier)one.oceCn.wiNrpaIRoominet1uIfvad.Tms5tSeliItaryoemhCtlndhtbeA.LieayasTlLnmateiiendmmsSilsvcyeATp,redfcolErloecesucPronieseo:ChtntnoArtootoirrmflfdeiuotfpddhugrleeeg2o5tle0viameo0etntnuir1gµn:nb4t~.Lihe,g3I0.m5cho0h0ftmmlt(rohOiepnredomNipfa)orftraeoemtvrlyi–5:oi2csum0eosna°isntCmrt.ai.yfTgulregaasenl(cs~aof9teh1rho4)tl,,h0(teC0h0s:eIurAppprAmeor)tnof(oa2crto4a:l51ncmtav(n/i~vnb4).6eTa0pnrµaadnuLmss)feteidoxr



33..23..32..8TC. henirtAtdhrideEfudxsgut0rap.at1eciortvinnooanltuamnte(s~(4~8400µµLL))t3o



a new 1.5 mL microcentrifuge tube. M Sodium Acetate (NaOAc) pH 5.2



and



2.5



volumes



(~1



mL)



3.2.2.79S.. eco1Cn0ed0n%CEtrxRieftIturhTagacIentCiaooAtln,L1a4Sn,0dT0E0gPern:pAtmlydfdmor7ix0200bymµLiinnvCae:nIrAsdiAodni(s2fco4ar:r1d1vmt/hvie)nas. undpemrnixatvanigtobryouinsvlyerbsyioinn.version for 2 PmAiUnS. IEmSmTeEdPiaatenldy cOenPtTriIfOugNeAaLt 1S4T,0E0P0 :rpWme ffooru5nmd itnh.aTtrafntsefreradthdeinsugpNeranOaAtacnta(n~d40e0thµaLn)otol,

6. raensueCwltRs1Ii.Tm5IpmCrALovLmeSdicTrwoEhcPe:nAttrdhifdeusg5ae0m0tupµblLe.Cw:aIAs iAm(m24e:d1iavt/evl)yacnedntmriifxugveigdo(rio.eu.,swlyabsynointvinercsuiboantefodra2t malli)n..NImevmeretdhiealteeslsy, dceunetrtoifuthgeelaetn1g4th,0o00f trhpemprfeovri5oumsisnt.aTgreasn(s~f9erh)t,htehseuppreortnoactoalncta(n~b46e0pµauLs)etdo

3.3. DNA ahPernerecewipaint1ad.5titomhne.LsTmaimmiceprfoloecresCtnootrrmeifpdulegotevioetnur:nb~ieg3.5htm(OinN) at –20 °C.



3.23.31. AThdidrditiEoxntroafcStiaolnt and Alcohol



3.1.1. Homogenization of Sand Fly



3.2. Ext1ra. ctioAnlwiqiuthotSosulvfefnictise.nTtimvoelfuomr Ceoomfpbluetfifoenr: T~2E5SCmain(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



3.2.1. First Eaxltirqaucotito)nto 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 +



5.



pCKR. ITICAL STEP: Add 500 µL chloroform:isoamyl alcohol (C:IAA) (24:1 v/v) and mix



Me3t.hvoidgsorPoCruoRstolIycT.bI2Cy0Ai1nL9v,eS2rTs,iE3oP6n:fGorri2nmd itnh.eImsamndedfilaytethlyorcoenutgrhifluygfeoart81m4,0in00wrpitmh afoTre5flmoninm. Ticrraonpsefesrtle. theTsoupaveornidatacnrot s(s~-4c8o0nµtaLm) itnoaatinonewb1e.t5wmeeLnmsaicmropcleens,tritfhuegemtiucbroep. estle must be cleaned and



5 of 15



3.2.2. SecondauEtxotcrlaacvtieodnafter each grinding (i.e., one micropestle per sample).

3.136.2...2C.3el.l LTCyhRsiIisTr, daICndEALPxrtSorTtaeEicnPt:DioAendndatu50ra0tiµoLn Can:IdADAig(2e4s:t1iovn/:v) and mix vigorously by inversion for 2



7.4. min. ICmCRmRIeTIdTIiCaItACeLlyAScTeLnEtPSri:fTAugEdedPa4:t51A04,µ0dl0d0burp7ffm0er0fToµEr S5LCmaCin+:.pITAKra(Antosfa(e2rfit4nh:ae1lsvuvop/levurm)naeataonnfdt5(0~m046µi0Lxµ),Lvv)oitgrotoexrously by inversion for 2 min. a nfIeomwr 1m1m.5eimnd,Liaanmdtiecirnloyccuecbnaettrneiftaurtgi5ef0utu°Cgbeef.ora8t h1,4v,o0rt0e0xinrgpfmor 1fmorin5evmeryin30. mTirna. nsfer the supernatant (~400 µL) to a new



3.23.3.2..TEhxitrrdac1Etix.o5tnramwctitiLohnSmolviecnrtso. cTeimnetfroirfCuogmeplettuiobn:e~.25 min



7.



CRITICAL STEP: Add 700 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2



3.23.1..3F.imrDsitnNE. xIAmtramcPteirdoeinactieplyitcaetnitornifu. gTeiamt 1e4,f0o0r0 rCpommfoprl5etmioinn.:Tr~an3s5femr thine supernatant (~400 µL) to



5. a newC1R.5ImTILCmAiLcrSoTceEnPt:riAfudgde5tu00beµ.L chloroform:isoamyl alcohol (C:IAA) (24:1 v/v) and mix



3.3. D3N.3A.1P.rvtehcAiigepodistruadotpuiioestnrliny.oaTbntiaymnioentffv(o~reS4rC8as0iolomtµnpLaflo)entrtiodo2nam:An~i3enlw5.cIomm1hi.mn5omeldLiamteilcyrocecenntrtirfiufuggeeattu1b4e,0.00 rpm for 5 min. Transfer



3.33.1.2.8.A2..dSdeictoiAonnddoEdfxSt0raal.tc1tainovdnoAlulcomhoels (~40 µL) 3 M Sodium Acetate (NaOAc) pH 5.2 and 2.5 volumes (~1 mL) 100%



8. 6.PP1AA0AdU0maed%tSUnihn0EeeC..Swa1tISRhmnEv1TaIo.oTnmE5loIPluSemCl,md,TaAaLaienannLEmstddedS(Pil~OcgyT4regcPoE0neaceTPtµneln:nIyntLOArdtt)mirNfld3iuyfidAxugMOegm5Lbe0yaSP0StitoiuxTT1nµdb4vEiLIbue,eP0O.mCry0:s0:iWNIoAiArnnpecAAmevfftooL(aer2futor4e1nrs:1(mSdi5NovTimtan/nhvO.Eia)nAtfPa.onaTc:)frdrtape1WmnrHsiamfxe5ed.rvd2itinifhganono.egrdusoNun2upa.s5deOlyrvnAobatclytuhaaminanntevtd(se~re(4as~t6hi1fo0atnmµneofLrLol)),rtao2dding NaOAc and ethanol,



3.2Nr.3e.esTvuhrhaeeillerlstrr)dus.etNlEhatisxneemtvidrmleaetprpchstthrreisooeo,svnlaevdemsdesup,dwdelehutwseeotnotohrtthethedheneesoalvlemteenhrpngnetligehgtwhoshftaat(smhOoiemfNpptm)rlhaeeetvedi–oiw2pau0tresa°lesCystva.cgiieomenstur(mi~sf9uesghtde)a,ditgah(ieet.eesp.,lr(wyo~tao9sccoenhlonc)tat,inrntichbfueuebpagpateeurdsdoeadtto(ic.eo.l,



was not incubated at can be paused here and



all). the



sa7m. ple sCtRoIrTeICdAoLvSeTrEnPi:gAhdtd (7O00NµL) aCt:IA−A20(24◦:1Cv./v) and mix vigorously by inversion for 2

3.3.2. Centrimfuigna.tIimonmediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~400 µL) to



39..3.2C.eanCtnreeifwnugt1er.5iafmtu1Lg4,m0a0ti0ciroropncmenftorirfu2g0emtuinbea.nd discard the supernatant by inversion.



3.3. DNA Precipitation. Time for Completion: ~35 min

9. Centrifuge at 14,000 rpm for 20 min and discard the supernatant by inversion.



3.31.10..AddAitidondof5S0a0lt aµnLd A7lc0o%holethanol and centrifuge at 14,000 rpm for 5 min. Discard the supernatant by 8. 1Ain0d0vd%e0er.1tshivaoonnloulma, anensdd(~gd4e0rnytµlyLth)m3eixMpbeySloilndevitueamrstioA5nc0efto◦arCte1 (fmNoianrO.5Amc)ipnH. 5R.2easnuds2p.5envodlutmhees p(~e1lmleLt)in 10 µL double-distilled water.



PAUSE STEP and OPTIONAL STEP: We found that after adding NaOAc and ethanol,

4. Maretesurlitsailms,prEoqveudiwphmenetnhet,saamnpdle RwaesaimgemnedtsiatSeleytcuenptrifuged (i.e., was not incubated at



all). Nevertheless, due to the length of the previous stages (~9 h), the protocol can be paused

4.1. Mhearteearniadltshe sample stored overnight (ON) at –20 °C.



3.3•.2. CenTtrRifuISgabtiounffer (NH2(CH2OH)3, 121.14 g/mol) (Anedra, Tigre, Argentina; Cat. no.: AN00915709) • 9. HCeyndtrrifougcehalto1r4i,c00a0crpidm (foHr 2C0lm, 3in6a.n4d6dgis/cmardolth)e(Bsuipoeprnaactakn,t bByuinevneorssioAn. ires, 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



Buffer 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 Buffer TESCa.



Reagent



Final Concentration Volume (Vf 1 = 12.5 mL)



1 M Tris-HCl pH 8 0.5 M EDTA

100 mM CaCl2 dH2O 2 20% SDS



30 mM 10 mM 5 mM

1%



375 µL 250 µL 625 µL 10.625 mL 625 µL



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 difficulties 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 effects 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 effect 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 different lysis buffers: the original buffer 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 buffer, here referred to as buffer TES (30 mM Tris-HCl pH 8, 10 mM EDTA and 1% SDS),



Methods Protoc. 2019, 2, 36



7 of 15



and this same buffer TES to which we added Ca2+ (5 mM), here referred to as buffer 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 buffer (buffer TESCa). Different studies have reported that calcium ions greatly affect 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 affect proteolytic activity [18], it showed that Ca2+-free pK precipitated irreversibly in the presence of EDTA leading to a much reduced effective concentration [18]. Furthermore, even though Ca2+ forms a complex with EDTA in the buffer, 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 suffered loss of activity when the lysis buffer contained EDTA but no calcium [19].

To evaluate these modifications, we processed specimens with the different lysis buffers (pAC, TES, and TESCa) (Table 1) and found that amplification was only positive when the samples were processed with buffers TES and TESCa (Appendix D). Due to these results we decided to further evaluate buffers TES and TESCa and processed more specimens with these buffers. All samples were incubated with pK (0.42 µg/µL) 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 µL 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 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 treatments (buffers TES and TESCa) and their outcomes was very significant (two-tailed p value = 0.0019).

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”.



Methods Protoc. 2019, 2, 36 Methods Protoc. 2018, 1, x FOR PEER REVIEW



8 of 15 8 of 14



Scheme 22.. SSuummmmaarryy ooff thee main experimennttaall stageess and steps involved in the optimized DNA

extraction pprroottooccooll((sseeee““SSecetcitoionn3”3”fofrodredtaeitlasi)l.sT).heTahpeparpoxpirmoxaitme taitmeetinmeedneedetdoecdomtopcloetme pevleetreyesvtaegrye

sistaignedicsaitneddi.cFaitgedu.reFsigaureresschaeremsacthicem(i.aet.i,cn(oi.tea.,nneoxtaacnt reexparcetsreenptraetsioent)aatniodna) raenndoatrdernaowtndtroawscnalteo. s(*c)aIlen.

(t*h)eInthtihrde tehxirtrdacetxitornacwtioitnh wsoitlhvesnotlsv,eandtsd, a7d00d µ70L0Cµ:LIACA:I.AmAi.nm, mini,nmutiensu; thes;, hhso,uhros;urpsK; ,pKpr,optreointeaisneaKse; KC;:ICA:AIA, Ach, lcohrloofroorfmor:imso:iasmoayml yalcaolhcohl o(2l 4(2:14):;1); , c,ecnetnritfruifgueg; es;/ns/,ns, usupeprenrantaatnatn;t;VV, ,vvoolluummeess;; 1144KK rpm,

1144,,000000 rreevvoolluuttiioonnss ppeerrmmiinnuuttee;;ddddHH22O, double-distilled water..

DDNNAA oobbttaaiinneedduussininggoouurrmmeeththooddisissusiutaitbalbelefofrolronlogn-gte-rtmermcocnosenrsvearvtiaotnio, sni,nscienicnediinvdidivuiadlusaalnsdanfldy DflyNADNexAtraecxttsrawctesrewsetoreresdtoarte−d20at◦C–2a0n°dCuasendd aussaedtemaspalatteemaspmlatuechasasm6uyceharassla6teyreianrsPClaRterreianctPioCnRs wrehaiccthioyniselwdhedichpoysiietlidveedrepsouslittsi.ve results.

IInn ccoonncclluussiioonn,, tthhee aabboovvee--mmeennttiioonneedd cchhaannggeess ((tthhee mmoosstt ssiiggnniiffiiccaanntt ooff wwhhiicchh wwaass tthhee aaddddiittiioonn ooff ccaallcciiuumm iioonnssttootthheellyyssiissbbuufffeferr) )oopptitmimizizededDDNNAAexetxrtarcatciotinonfrformominidnidviivdiudaulaslasnadnfldifelsiews hwehnecnomcopmapreadretdo tthoethoeriogrinigailnpaAl pCApCroptrooctool,coanl,danednaebnlaedblueds tuosctooncsoisntseinsttelyntolbytoaibntapionspitoivsietiavme pamlifipclaiftiiocantiroensurletssuwltisthwtihthe itnhteerinnatel rcnoanltrcoolnptrrioml eprsri.mMeorsr.eoMveorr,ewoveeurs, ewd ethuisseodptitmhiiszeodpptirmotizoecdol ptoroetxotcraoclttDoNeAxtrfaroctmDinNdAivifdruomal fiinedldiv-cidaputaulrefdielLdu-tczaopmtuyiraedsppL.uftrzoommyBiarazspilpa.ndfroAmrgeBnrtainzail, aannddthAerignetnertinnaal,coanntdrolthaempinlitfiecrnatailoncsownterroel saumcpcelisfsifcualti(oAnpspweenrdeixsuFc)c.essful (Appendix F).

OOuurr rreessuullttss aallssoo ssuuggggeesstt tthhaatt ppKK aaccttiivviittyy iiss rreedduucceedd wwhheenn tthhee llyyssiiss bbuuffffeerr ccoonnttaaiinnss EEDDTTAA bbuutt nnoo ccaallcciiuumm(F(Figiguurere1)1, i)n, ainccoarcdcoanrdceanwciethwaipthrevaiopursevstiuoduys tshtautdeyxptlhoartedexthpilsosraemd etshoislutsiaomn eforsoolputtiimonizifnogr tohpetiemxtizraincgtiotnheofeDxtNraActfioronmoifnDdiNviAdufarlohmuminadnivhidaiurasl[1h9u],maannd shuapirpso[r1t9in],gapnrdevsiouupspeovrtiidnegncpereovniothues deveipdeenndceenocne tohfepdKepaecntidveintyceoonftphKe pacrteisveintyceonofthcealpciruesmenicoenosf[c1a5l–c1iu7]m. Fiounrsth[1e5rm–1o7r]e. ,FpuKrthdeirgmesotrieo,npiKs rdoiugteisnteiolynpiserrfoourtmineedlyapt e5r0fo◦Crm[2e0d],aat n5d0 °itCh[a2s0]b,eaenndreitphoarstebdeethnarteppKo’rsteadcttihvaittypcKa’ns iancctriveaitsyecsaenveinraclrefoalsde wseivtheirnalthfoeld50w◦iCthtion6t0he◦C50ra°nCgteo[6201]°.CInrathnigseco[2n1t]e.xItn, bthuiffsecrosnptAexCt,, bTuEfSfearnsdpTAECS,CTaEwS earnedteTsEteSdCawwitheirne 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



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 buffer that contained calcium (buffer TESCa). Similarly, McNevin et al. [19] used a different lysis buffer (at 56 ◦C) and also found that DNA extraction only improved when calcium was added to the buffer [19]. This would suggest that, when working within the enzyme´s optimal temperature range, it is the addition of Ca2+ to the lysis buffer 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 efficiency 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 buffer (200 mM Tris-HCl pH 8.4; 500 mM KCl); 2.5 mM MgCl2; 0.125 mM dNTPs; 0.3 U Taq Pegasus® DNA polymerase (Productos Bio-Lógicos, Argentina); 0.5 µM of each primer (44F and 45R); 0.1 mg/mL bovine serum albumin (BSA), and 1 µL template, in a final volume of 10 µL. 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 Different Incubation Periods with pK in Buffer pAC, and ON or No Incubation at −20 ◦C (in the DNA Precipitation Step)

We simultaneously evaluated the effect of different incubation periods (0.5, 2, 3, 4 and 8 h) with pK (0.42 µg/µL) in the original lysis buffer (pAC); and, in the DNA precipitation step, we assayed the effect 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).



MMeetthhooddssPPrroottoocc..22001198,,21,,3x6FOR PEER REVIEW Methods Protoc. 2018, 1, x FOR PEER REVIEW



1100 ooff 1154 10 of 14



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FFFiiiggguuurrreeeAAA222... EEEffffffeeecccttt ooofff mmmiiixxxiiinnnggg iiinnnttteeennnsssiiitttyyy iininntththheeeeeexxxtrttrraaacccttitoiioonnnwwwitiihtthhssosoolvllvveeennntsttsssstsetteeppp. .. DDDNNNAAA eeexxxtttrrraaaccctttiiiooonnnsss wwweeerrreee eeevvvaaallluuuaaattteeedddwwwitiihtthhanaaninntiiennrttneerarnnl caaollncctoornontltrrPooCll RPPC(CuRRsin((uugsspiinnriggmpperrisimm44eeFrrss/4444R4FF).//4G444eRRn))t..leGGmeennixttillneegmmbiyixxiiinnnvggebrbsyyioiinnnv(vbeelrrusseiioobnnra((cbbkllueutee, sbbarrmaaccpkkleett,,Gss)aa: mmlappnlleeesGG3))(::ullananndeeilssu33te((duu)nnaddniillduutt4eedd(1)):1aa0nndd i44lu((t11io::11n00);ddviilliuugttoiioroonnu));;svvmiiggiooxrirnooguussbmymiixnxiivnneggrsbbioyyniinn(yvveelrrlssoiiowonnb((ryyaeecllkllooewwt, sbbarrmaaccpkkleettV,, )ss:aalmmanppellsee5VV(u))::nlldaainnlueestsed55)((auunnnddd6iilluu(1tt:ee1dd0))daailnnuddtio66n()(1;1:l:1a10n0edd1ii:lluuMttiiWoonn))(;;pZllaaennreeo211/:H: MMaeIWWI); (l(appnZZeee2rroo: 22p//oHHsaiateeiIIvIIe));;clloaannnteero22l;:: lppaonosesiit7tii:vvneeeccgooanntttirrvooell;;clloaannnteero77l::. nnGee:gggaaettiinvvteelecc;ooVnn:ttvrrooigll..oGGro::uggsee;nnMttlleWe;; V:Vm:: vvoiilggeoocurroolauurss;w; MMeiWWgh::tmm. oolleeccuullaarr wweeiigghhtt..

AAppppeennddiixx DD.. AAssssaayyiinngg ddiiffffeerreenntt llyyssiiss bbuuffffeerrss TTEESSCCAAaass))..iiSSnnaaddmmiiccppaatltleeeesdsdwwiinneer“r“eeSSeieinnccctctiiuuoobbnnaa55ttee””dd,, wwwweeiittpphhrrpopocKcKeess((0s0se.e.44dd22ssµµppggee//ccµµiimmLL))eeaannttss55ww00ii°°ttCChh tftfhohorere88tthhhhrr,,eemmee liliyxyxsisinniissggbbbbuuyyffffiieennrrvvssee((rrppssAAiiooCCnn,,wwTTaEaEssSSd,d, aaoonnnnddee



Methods Protoc. 2019, 2, 36



11 of 15



Appendix D Assaying Different Lysis Buffers

MethoAdssPriontodc.ic2a01te8,d1,ixnFO“SRePcEtiEoRnRE5V”,IEwWe processed specimens with the three lysis buffers (pAC11, TofE1S4, and TESCa). Samples were incubated with pK (0.42 µg/µL) at 50 ◦C for 8 h, mixing by inversion was dvoignoerovuigsolyrofuosrlythfoertthhreeetherexetreaxcttrioanctsiownsithwiCth:ICA:AIA, Aan, danpdeplleeltlsetws wereerererseususpspenenddededinin1100 µµLL ddddHH22OO ((SScchheemmee 11,, TTaabbllee 11)).. DDuuee ttoo tthhee lleennggtthh ooff tthhee fifirrsstt tthhrreeee ssttaaggeess ((~~99 hh)),, tthhee pprroottooccooll wwaass ppaauusseedd iinn tthhee ffoouurrtthh sstteepp ((ii..ee..,, tthhee ssaammppllee wwaass pprreecciippiittaatteedd OONN aatt −−2200 °◦CC)).. WWee ffoouunndd tthhaatt aammpplliifificcaattiioonn wwaass oonnllyy ssuucccceessssffuull ffoorr tthhee ssaammpplleess pprroocceesssseedd wwiitthhbbuuffffeerrssTTEESSaannddTTEESSCCaa((FFiigguurreeAA33))..



Figurree AA33.. Effffeect ooff ddiiffffeerreennttllyyssiissbbuuffffeerrss.. DDNNAA eexxttrraaccttiioonns wweeree eevvaalluuaated wwith aann iinntteerrnal ccoontrol PCR (usinngg pprriimmeerrss 4444FF//4444RR)),,aannddwweerreeddiilluutteedd((11::55)).. 1: MW (pZero2/HaeII); 2 and 3: positive control; 4: lysis buffffer TES; 5: lysis buffffer TESCa; 6: buffffer pAC; 7: negative control. MW: molecular weight.

IAIAnnppccpupuebebnnaadtdtiiiiooxxnnEEaa.ttAA−−s22ss00saay°◦yCiCnin(g(iginnDDttihhffiefeefrDeDerNnNetnAAItnPPIcnrrueecbcuciaibpptaiiiotttaianottiinPooennPrieSSorttdieeosppd))ws iwthitphKpiKn BinuffBeurfTfeErSTCEaS, aCnad, aOnNd oOrNNoor No w(dhabPpdPh(app(STSneuaenCearCeecrcrEdcodffddchRhRprcrcSceeetfteeoferToCTphrophaomomasnonreousaer(uscsdoTeedoe)eeennooaa,ddidEidsdd1nntttd1tasuhihuSi,a,aaaeosonbcebCeTcslldpTsnntderyytreaaieai)f)izfzsinbwwn).no.cbn(ope,ecl2cirOlrOiiemaeluleuie)tteaiafhcnhn,bf1n,bnieni1wodonf)aemfeawne)oe.ouut.ets(sirste9Riret9t2oRhaoacnbw((b)en(o(memnceiis4o4soi..uohseetnuttpwpptuhihuilhi..hml,cdrm,lltlhtleeieihtthttdstnnehshi(rcerec1ooecwcwieeewhw(wp)fufaup1assieritbibweier)titaatemtrttmehaaaaahmhmedhreccttcteeetruiiiipihhppptndhoopeonnhcrrllntneuniciteiioeeeostnsnnucatiwwttwww(ccnecie(tehbwnuuwiuiitiiaaieaontttntetbbbisthsehhinhaitraeataanrhcenschsdptpnttwaeceeeeaiiaKiKaunlaoondwlniKcnKlntnnbt)t)occhrdrwicdasosduhdtiippofuhtfuanwieboeuiuwetnelggocthclacpgrgritrrgingrtoitirsoeKteepehooieeohhtdodpaladsKdeosloosanpesttpuiieepuetlermwmwar,tddipddrsti5toemiOamtame0O5(mtdet(lssFrhhiF0Npeee°sNmiiepaiaporCdd,g◦gwrtstrpdireCiiuspuneaaelldiilrsororctinratctieduemheneeneinitwdcpupclreldAggiApyyiiiiipriincneipetttrt4ic4aaiaaahrnharogi)ta)ffttttta,egtii,ttateeeen1epneteeeedtddi1Oi,cvrrcrtdivon,ohuo2ueatawNenwa2n,hettbnbddnseh,3iaaiaaOeddati3ittat,tttnnthhhth,iiiK:tiN4−nnoac:oo4−o(a,auggsn2uni,121ulailneb0a)c0)ncgNNgtntonaoitihOcOK◦h°dmhmatahdhuCCivvteOOtooobe8iheh))8eennlnls,,aAAseerrhhbeObtOdaadtccfiafwaaho(lal(aoNotNal2lan2nien,i,nnnl−nnnddssayaana2tdadotoppnnsltliit0teyttynieenadda−sw−−accalt°tlvvceb2ici2el2lCaamimwwioon0n0u0slsss,yhhissiafeeawb◦◦°wwbsifoionynyCCtCtelillyleyessseeessr)),,, (yn(ynFFioioeieigttglldudpuprrrmrmeeeecocAoAiirrpp4e4ei;i;tDtDTaaTaNttNaeebbAddAllee;a;a1(1t2t2))).−)−.OO2200nn°◦ttChChe,e, bbooatathhrrrereiirrnnhghgaattnnhhddee,, aaooffvvooeerrrreeaammlllleebbnnaattnniiooddnniieennddtteeeennxxsscciietteyypptwwtiiooaannss g(g(llrraaeennaaeettee99rr,,ff4oo4rrhhtthhiinneeccssuuaabbmmaappttiillooeennss ttwwhhaaiitttthhwwppeeKKrree))

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



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 MneothtodpsrPercoitpoci.t2a0t1e9d, 2a, 3t6−20 °C, barring the aforementioned exception (lane 9, 4 h incubation wi1th2 opf 1K5) (Figure A4; Table 1).



FFiigguurreeAA44. .EEffffeeccttssooffddiiffffeerreennttininccuubbaattioionnppeerrioioddsswwitithhppKKininbbuufffeferrTTEESSCCaa, ,aannddOONNoorrnnooininccuubbaattioionn aatt−−2200◦°CC ((iinntthheeDDNNAApprreecciippiittaattiioonnsstteepp))..DDNNAAeexxttrraaccttiioonnsswweerreeeevvaalluuaatteeddwwiitthhaanninintteerrnnaallccoonnttrrooll PPCCRR((uussininggpprrimimeerrss4444FF/4/444RR))aannddwweerreeddiliulutetedd(1(1:5:5).).TThheebblluueebbrraacckkeettiinnddiiccaatteesstthheessaammpplleesstthhaattwweerree ssuubbjejeccteteddtotoOONNinicnucbuabtiaotnioant a−t2−02◦0C°(Clan(leasn3e–s73),–a7n),datnhde ytheelloywellborwackberat cinkdeticiantdesictahteessatmheplseasmthpalet swtehraet nwoterinecnuobtaitnecdubbeaftoerde cbeenfotrriefucgeanttiroinfu(glaantieosn8(–l1a2n)e; sin8–b1o2th); cinasbeos,ththceansuesm, tbheersnbuemlobwertshbeeblroawcktehtes ibnrdaicckaetets tihnedhicoauter/sthoef ihnocuurb/astioofninwciuthbaptKio.n1:wMitWh p(pKZ. e1r:oM2/HWae(IpI)Z; e2r: op2o/HsitaievIeI);co2n: tprools;i1ti3v:enceognattrivoel; c1o3n: tnroelg. ative AppecnodnitxroFl. 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

L. umbratilis

L. migonei L. renei

L. intermedia L. longipalpis (cavunge strain) L. longipalpis (jacobina strain) L. longipalpis (lapinha strain) L. longipalpis



City Presidente Figueiredo

Baturite Lagoa Santa Tancredo Neves

Cavunge



State/Province

Amazonas

Ceara Minas Gerais

Bahia

Bahia



Country

Brazil

Brazil Brazil Brazil

Brazil



Jacobina



Bahia



Brazil



Lagoa Santa



Minas Gerais



Brazil



Posadas



Misiones



Argentina



1 Total number of specimens that were analyzed individually = 136.



Figure A5, A6

A5 A6 A7 A5, A6

A5

A5 A7



L. intermedia



Tancredo Neves



Bahia



Brazil



L. longipalpis (cavunge strain)



Cavunge



Bahia



Brazil



L. longipalpis (jacobina strain)



Jacobina



Bahia



Brazil



L. longipalpis (lapinha strain) Lagoa Santa



Minas Gerais



Brazil



L. longipalpis



Posadas



Misiones



Argentina



Methods Protoc. 2019, 2, 36



1 Total number of specimens that were analyzed individually = 136.



A7 A5, A6

A5 A5 A7

13 of 15



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MethodllasopnPigrnoiphtoaacl.ps2its0r1al8ain,p1i(,nLxhaFagOosRatrPaSiEannERt(aLR,aMEgVoinIaEaSWsaGntear,aMis,inBaraszGile);ra1i3s:, nBeragzaitli)v;e13c:onnetrgoalt.ive control.



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Figure A6. PCR amplifications using DNA extracted with the optimized protocol from L. longipalpis FF(ciiaggvuuurreengAAe66s..tPPraCCinRR),aaLmm. pupmlliififbirccaaatttiiilooisnn,ssanuudssiinnL.ggrDDenNNeiAA. DeeNxxttArraaeccxtteetrddacwwtsiittwhhettrhheeeeoovppattliiummaiitzzeeedddwppirtrhoottaoonccooinll tfferroronmmalLLc..ollnoonntrggoiiplpaPallCppiiRss 1(((1(6(1cuucu:::–aasssMM9Mvviii:nnnuuW WWgLggnn.gpgpp(((uepeprprrmiiiZsZZsmmmttbeeerrreeearrraaorroroiitnsssn222il)//4)4/4i,HH,Hs444LLFFaaFa(..eeP/e//uu4I4I4IrIImI4m44e)))R;R;R;sbb22i2r)))rd:a::aaaaettpppninninlloooiddditssssse,,iiiwwwttataFiiinvneveveidgrrerdeeeeeuccLcLdeddooo..iiinninrrrllleeeuuttutnrnrrdtttoooeeeeeoilillddd..;,;; DD333A(((–1–1–1NNm55::5:555::AA:))a)LL.L..zee..C.CCoxxlllnoooooottnrrnlnallooaaogsggrccr,riii-p-ttp-pcBscscaaaooolrwlwlppdpdadiiieeziisissnnrrnilceeccggg)aaa;eecvcvvcvv1oououu0aaiiinnnn–lnlnuug1ggcccaai3eieeiddttd:eessseeeLtddttssrrsr.aaawwwwwriiinneniiiiinttttthhh(h((ehCCCiaatttaaahlhnnhavvvaaapiiuuuttnntinnnunuttueegggshssrreeeeeaenn,d,d,daaBBB(llfLffaaaooccoahhhroorrgiiinnTaTaaoTtt,,,aaaarrBBbBobobSlrllrrlleeeaaaaPPznzzAAACCiiitlll1Ra1R1)));.,;.;. 66M––9i9n:: aLLs..Guumemrbabrriasa,ttiiBlliirssa(z(PPilrr)ee;ss1ii4dd:eennnettgeeaFFtiiivggeuueecioirrneedtdrooo,,l.AAmmaazzoonnaass,, BBrraazziill));; 1100––1133:: LL.. rreenneeii llaappiinnhhaa ((LLaaggooaa SSaannttaa,, MMiinnaass GGeerraaiiss,, BBrraazziill));; 1144:: nneeggaattiivvee ccoonnttrrooll..



FFiigguurree AA77.. PPCCRR aammpplliifificcaattiioonnss uussiinngg DDNNAA eexxttrraacctteedd wwiitthh tthhee ooppttiimmiizzeedd pprroottooccooll ffrroomm LL.. lloonnggiippaallppiiss (F(AAigrrgugereennttAiinn7aa.))PaaCnnRddaLLm.. piinnltitfeeircrmmateeidodiinaa.s. uDDsNNinAAg DeexxNttrrAaaccettsxstwrwaeecrtreedeevwvaailltuuhaatttheeeddowwpitittihmh iaaznnediinnpttereorrnntoaaclloccloofnnrottrrmoollLPP. CCloRRng((iuupsasilinpniggs p(pArriirmmgeenrrssti4n444aFF)//4a44n4RdR))Laa.nndidnwtwereemrreeedddiiail.luuDtteeNddA((11::e55x)).t.rCCaocotllsoorrw--cceoorddeiinneggvaccloouiianntcceiiddeewss wiwthiitthhantthhiaantttueurssneeaddlffocoorrnTTtaraboblleePAAC11R.. 11(:u: MMsinWWg (p(pprZZimeerreoor2s2//H4H4aaFeeI/II4I)4);;R22:): papnoodssiittwiivveeereccoodnnitltrurootlel;;d33–(–144::5LL).. Cllooonnlggoiiprp-aaclloppidissi(n(PPgoocssoaadidnaacssi,,dMMesiisswiioointnheesst,h, AAatrrggueesnentdtiinnfaoa)r);;T55–a–6b6:l:eLL.A. iinn1t.tee1rr:mmMeeddWiiaa ((TpTaZannecrcroree2dd/oHoNaNeeIeIvv)e;es2s,:,BpBaoahshitiaai,v,BeBrcraaozzniiltl)r);o;7l7:;:3nn–ee4gg:aaLtti.ivvloeenccgooinpnattrlrpooilsl..(Posadas, Misiones, Argentina); 5–6: L. intermedia (Tancredo Neves, Bahia, Brazil); 7: negative control.

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