1

2

PARTICIPANTS

ARGENTINA

o Dra Elsa Velazquez, Instituto Fatala Chaben, Cdad de Buenos Aires.

o Dr Horacio Lucero, Universidad del Nordeste. Instituto de Medicina Regional. Resistencia, Chaco.

o Dr Patricio Diosque. Instituto de Patología Experimental, Universidad de Salta, Salta.

o Dra María Mercedes Monje. Instituto de Patología Experimental, Universidad de Salta, Salta.

o Dr Alejandro Uncos. Instituto de Patología Experimental, Universidad de Salta, Salta.

o Dr Sergio Sosa Estani, CeNDIE-ANLIS, MSAL.

BÉLGIUM

o Dr Stijn Deborgraeve, Instituto de Medicina Tropical, Antwerpen.

o Dr Philipe Bucher, Instituto de Medicina Tropical, Antwerpen.

BOLIVIA

o Dra Tatiana Tellez, Centro Universitario de Medicina Tropical, Facultad de Medecina, Universidad Mayor de San Simon, Cochabamba.

BRASIL

o Dr Constança Britto, Departamento de Bioquímica e Biologia Molecular. Laboratório de Biologia Molecular e Doenças Endêmicas FIOCRUZ, Rio de Janeiro.

o Dr Lucia Galvao, Faculdade de Farmácia, Petrópolis, Natal, RN.

o Dr J. E. Levi, Hospital Sirio Libanés, Molecular Biology and Serology. Instituto de Hemoterapia Sirio Libanés, San Pablo.

o Dr Alejandro Luquetti, Hosp da Clinicas, Univ. Fed Goias, Goias.

3

CHILE

o Dra Maria Isabel Jercic (Jefe Sección Parasitología). Santiago de Chile.

o Dra Inés Zulantay, Laboratorio de Parasitología Básico-Clínico, Programa de Biología Celular y Molecular, Santiago de Chile.

COLOMBIA

o Dra Ana Mejía Jaramillo, Grupo Chagas, Medellín.

o Dr Omar Triana Chavez, Grupo Chagas, Medellin.

o Dr Juan David Ramirez Gonzalez, CIMPAT, Univ de los Andes, Bogotá.

o Dr. Felipe Guhl Nannetti, CIMPAT, Univ de los Andes, Bogotá.

ESPAÑA

o Dra María Flores Centro de Mahahonda, Centro Nacional de Microbiologia, Instituto de Salud Carlos III Madrid.

o Dra María Piron, Banc de Sang i Teixits; Universitat de Barcelona, Barcelona.

FRANCE

o Dr Assedine Assal, FRENCH BLOOD SERVICES, LA PLAINE SAINT DENIS Paris.

o Dr Frederic Auter, FRENCH BLOOD SERVICES, LA PLAINE SAINT DENIS Paris.

GUYANNE FRANCAISE

o Dra Christine Aznar, Laboratorio Hospitalario, Universidad de Parasitología. Cayene.

o Vincent Verón, Laboratorio Hospitalario, Universidad de Parasitología. Cayene.

4

PARAGUAY

o Dra Graciela Russomando, Instituto de Investigaciones en Ciencias de la Salud – Universidad Nacional de Asunción. Asunción del Paraguay.

o Zunilda Elizabeth Sanchez Leon, Instituto de Investigaciones en Ciencias de la Salud – Universidad Nacional de Asunción. Asunción del Paraguay.

PERÚ

o Dra Gisely Hijar, Instituto Nacional de Salud. Lima.

SWITZERLAND

o Dr Janis Lazdins, PDE/TDR/WHO, Coordinator Product Development & Evaluation, TDR / World Health Organization, Ginebra.

UNITED KINGDOM

o Dr Debbie Nolder London School of Tropical Medicine and Hygiene Department of Clinical Parasitology, Hospital for Tropical Diseases. London.

URUGUAY

o Dr Carlos Robello, Institut Pasteur de Montevideo. Montevideo.

o Dr Pilar Zorrilla Institut Pasteur de Montevideo. Montevideo.

5

COORDINATION TEAM

o Sonia Lafón Secretary LABMECH INGEBI-CONICET

o Lic Tomás Duffy, Ph D student, LabMECH, INGEBI-CONICET

o Bioq Margarita Bisio, Ph D student, LABMECH INGEBI-CONICET

o Bioq Carolina Cura, Ph D student. LABMECH INGEBI-CONICET

o Dra Mariela Sued, Ph D, Instituto de Cálculo, University of Buenos Aires

o Dra Liliana Orellana PhD, INstituto de Cálculo University of Buenos Aires

o Dr Alejandro Schijman, Ph D. LABMECH INGEBI-CONICET

6

ANALYSIS OF BLOOD SAMPLES

Each participant will receive 6 blood samples mixed 1:1 with GE buffer (Guanidine – HCl (6M) / EDTA (0.2M)) and boiled during 15 minutes. They are pooled samples from seropositive patients with different degrees of parasitic loads and from seronegative individuals.

PROTOCOLS

I. DNA EXTRACTION FROM PERIPHERAL BLOOD SAMPLES

a. Silica Membrane Columns (QIAamp DNA Mini Kit, QIAGEN, Germany) b. Phenol Extraction and isopropanol precipitation.

II. PCR

a. Conventional k DNA Hot Start PCR b. Conventional Satellite DNA Hot Start PCR c. Satellite DNA Real Time PCR d. Internal Standard Real Time PCR

Guanidine EDTA Blood Samplewith Internal Standard

Phenol DNA extraction Silica Membrane Column

K DNA SATELLITE DNA REAL TIME PCR K DNA SATELLITE DNA REAL TIME PCR

Guanidine EDTA Blood Samplewith Internal Standard

Phenol DNA extraction Silica Membrane Column

K DNA SATELLITE DNA REAL TIME PCR K DNA SATELLITE DNA REAL TIME PCR

7

I. a. PROTOCOL USING QIAMP COLUMNS 1. Add 5 ul of Internal Standard (40 pg/μl ) to 400 μl of GEB sample (200 μl

peripheral blood + 200 μl of 2x lysis buffer containing 6 M guanidine

hydrochloride and 200 mM EDTA, pH 8.0).

2. Add 200 μl ethanol (96–100%) to the GEB sample with IS (if the tip touches

the tube wall change the tip).

3. Mix by pulse-vortexing for 15 s.

4. After mixing, briefly spin down the 1.5

ml microcentrifuge tube to remove

drops from the inside of the lid.

5. Carefully apply the mixture from step

5 to the QIAamp Mini spin column (in

a 2 ml collection tube) without wetting

the rim. Close the cap, and centrifuge

at full speed (20,000xg; 13,000 rpm)

for 1 min. Place the QIAamp Mini spin column in a clean 2 ml collection

tube, and discard the tube containing the filtrate.

Close each spin column in order to avoid aerosol formation during

centrifugation.

If the lysate has not completely passed through the column after

centrifugation, centrifuge again at higher speed until the QIAamp Mini spin

column is empty.

6. Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW1

without wetting the rim. Close the cap and centrifuge at full speed (20,000 x

g;13,000 rpm) for 1 min. Place the QIAamp Mini spin column in a clean 2 ml

collection tube, and discard the collection tube containing the filtrate.

Flow-through contains Buffer AW1 and is therefore not compatible with bleach.

7. Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW2

without wetting the rim. Close the cap and centrifuge at full speed (20,000 x

g;13,000 rpm) for 3 min.

8. Place the QIAamp Mini spin column in a new 2 ml collection tube and discard

the old collection tube with the filtrate. Centrifuge at full speed for 1 min.

8

This step helps to eliminate the chance of possible Buffer AW2 carryover.

9. Place the QIAamp Mini spin column in a clean 1.5 ml microcentrifuge tube,

and discard the collection tube containing the filtrate. Carefully open the

QIAamp Mini spin column and add 200 μl water. Incubate at room

temperature (15–25°C) for 5 min, and then centrifuge at full speed (20,000

x g;13,000 rpm) for 1 min.

10. For long-term storage of DNA, eluting in Buffer AE and storing at –20°C

is recommended.

OBSERVATIONS DURING PROCEDURE

Complete this table during the procedure

Steps  Workshop Method

Comments/Incidents

Etanol 200 μl

Blood + IS 400 μl

Apply to the column

600 μl

Centrifuge 1 min 13000 Buffer AW1 500 μl Centrifuge 1 min 13000 Buffer AW2 500 μl Centrifuge 3 min 13000 Centrifuge 1 min 13000 Buffer AE 200 μl Incubate 5 min Centrifuge 3 min 13000

9

I. b. DNA EXTRACTION WITH PHENOL CHLOROFORM

1. Distribute 200 μl of phenol-chloroform-isoamylic alcohol (25:24:1) (phenol

Tris–EDTA pH 8, 1:1), close all tubes (Tube 1). Corroborate the right volume

of the aliquots.

2. Add 5 μl of IS (40 pg/μl) to 100ul of GEB in an Eppendorf tube, vortex (3

pulses of 1 sec), centrifuge for 3 minutes at 13000 rpm.

3. Add 150 μl of distilled water (Tube 1),

vortex (5 pulses of 1 second) and

centrifuge during 3 minutes at 13000

rpm.

4. Withdraw the aqueous phase, 180-200

μl (upper), without disturbing the

interphase (white), and pour it to a new

Eppendorf tube (Tube 2). Use a p200 or

p100 and take twice 100 μl. (If the

interphase is thick and the volume of

the upper aqueous phase is lower than

180 μl, centrifuge 3 minutes at 13000 rpm and repeat this step)

5. Add 200 μl of chloroform to the aqueous phase (TUBE 2), vortex (5 pulses of

2 seconds) and centrifuge during 3 minutes at 13000 rpm.

6. Add 2 μl of carrier glycogen (SIGMA, 20 mg/ml in sterile distilled water) to

TUBE 3

7. Withdraw the aqueous phase (180-200 μl) from TUBE 2 and pour it to TUBE

3. Mix using the same tip 3 times.

8. Precipitate DNA and glycogen with 200 μl of isopropanol and vortex (3

pulses of 1 second). Let precipitate 35 min a – 20ºC.

9. Centrifuge 15 minutes at 13000 rpm, the tip of the rids of the tubes must be

outward in the rotor. Discard the supernatant using a p1000, avoiding

touching the pellet.

10. Wash the pellet adding 500ul of ethanol 70%, do not vortex, centrifuge

10 min and discard the supernatant using a p1000. Spin down to collect

drops from the walls and discard the remaining supernatant using a p200.

10

11. Incubate the pellet 10 min at 37-50ºC

12. Resuspend in 50 μl of distilled water. To facilitate suspension incubate at

37-50ºC, during 10 min and vortex.

OBSERVATIONS DURING PROCEDURE Complete this table DURING the procedure

Steps Workshop Method

Comments/Incidents

Blood (volume) 100 μl

Phenol Phenol: chloroform: isoamylic alcohol

Centrifuge 3 min 13000

1st extraction No

Water (volumen) 150 μl

Centrifuge 3 min 13000

2nd extraction Recovery 180-200 ul

Chloroform 200 μl

Centrifuge 3 min 13000

Recovery 180-200 μl

Glycogen 20 μl

Precipitation 200 μl Isopropanol

Incubation 35 min – 20ºC

Centrifuge 15 min 13000

Ethanol 70 % 500 μl

Centrifuge 15 min 13000

Dry 15 min 37-50ºC

Resuspend 50 μl distilled water 10 min 37-50ºC and vortex

11

II. a. KDNA HOT START PCR

MASTER MIX T. cruzi k-DNA

Reagents Final

concentration x 1

Buffer Taq platinum 10X (Invitrogen) 1X 3 μl

MgCl2 50 mM 3 mM 1.8 μl

dNTPs 25 mM 0.25 mM 0.3 μl

Primer 121 50pmol/μl 2.5 μM 1.5 μl

Primer 122 50pmol/μl 2.5 μM 1.5 μl Taq DNA polymerase platinum 5 U/ μl (Invitrogen) 0.6 U 0.12 μl

H20 16.78 μl

Total volume 25 μl

Sample 5 μl

Final volume 30 μl

121: 5´-AAATAATGTACGGGKGAGATGCATGA -3´

122: 5´-GGTTCGATTGGGGTTGGTGTAATATA -3´

K = G o T

12

CYCLING CONDITIONS T. cruzi k-DNA:

Temperature Time

94 ºC 3 minutes 94 ºC 1 minute

68 ºC 1 minute 72 ºC 1 minute 94 ºC 45 second

64 ºC 45 second 72 ºC 45 second 72 ºC 10 minutes 4 ºC Hold

M.J.R RESEARCH HOT BONNET THERMOCYCLER

DETECTION

Load 12 μl of PCR tube + 3 μl of Loading Buffer 6x(1) in 2% agarose gel in TAE(1) and 0.5 μg/ml Ethidium Bromide. Electrophoresis run at 5 Volt/cm. UV visualization of 330 pb amplicon. (1)Molecular Cloning: A Laboratory Manual (Third Edition). Joseph Sambrook, Peter

MacCallum; David Russell. CSHL Press, New York , 2001

M1: Negative sample

M6: Positive sample

M2-5: Unknown sample

CN: PCR Negative control

35 cycles

5 cycles

13

II. b. SATELLITE HOT START PCR

MASTER MIX T. cruzi sat-DNA

Reagents Final concentration

x 1

Buffer Taq platinum 10X (Invitrogen)

1X 3 μl

MgCl2 50 mM 3 mM 1.8 μl

dNTPs 25 mM 0.25 mM 0.3 μl Primers cruzi 1 50pmol/μl 0.5 μM 0.3 μl Primers cruzi 2 50pmol/μl 0.5 μM 0.3 μl Taq DNA polymerase platinum 5 U/ μl (Invitrogen)

0.6 U 0.12 μl

Bi distilled or MQWater 19.18 μl Total volume 25 μl Sample 5 μl Final volume 30 μl

Cruzi 1: 5´- ASTCGGCTGATCGTTTTCGA - 3´

Cruzi 2: 5´- AATTCCTCCAAGCAGCGGATA - 3´

S = C o G

14

CYCLING CONDITIONS T. cruzi sat–DNA:

Temperature Time

94 ºC 5 minutes

94 ºC 1 minute

68 ºC 30 second 72 ºC 1 minute 4 ºC Hold

MJR RESEARCH HOT BONNET THERMOCYCLER

DETECTION

Load 12 μls of PCR tube + 3 μl of Loading Buffer 6x(1) in 2% agarose gel in TAE(1) and 0.5 μg/ml Ethidium Bromide. Electrophoresis run at 5 Volt/cm. UV visualization of 166 pb amplicon. (1) Molecular Cloning: A Laboratory Manual (Third Edition). Joseph Sambrook, Peter MacCallum; David

Russell. CSHL Press, New York, 2001.

M1: Negative sample

M6: Positive sample

M2-5: Unknown sample

CN: PCR Negative control

40 cycles

15

II. c. SATELLITE DNA REAL TIME PCR MASTER MIX T. cruzi sat-DNA

Reagents Final concentration

x 1

Buffer Taq platinum 10X (Invitrogen)

1X 2 μl

MgCl2 50 mM 3 mM 1.2 μl

dNTPs 25 mM 0.25 mM 0.2 μl Primers cruzi 1 50pmol/μl 0.5 μM 0.2 μl Primers cruzi 2 50pmol/μl 0.5 μM 0.2 μl SYBR Green 10X 0.5 X 1 μl Taq DNA polymerase platinum 5 U/ μl (Invitrogen)

0.5 U 0.1 μl

Distilled water 13.1 μl Total volume 18 μl Sample 2 μl Final volume 20 μl

CYCLING CONDITIONS T. cruzi sat–DNA:

Temperature Time

94 ºC 5 minutes

94 ºC 10 second

58 ºC 10 second 72 ºC 10 second 80 ºC Read

CORBETT LIFE SCIENCE’S ROTOR-GENE™ THERMOCYCLER

40 cycles

16

DETECTION

Expected melting temperature: aprox 82-84ºC

II. d. INTERNAL STANDARD DNA BASED REAL TIME PCR

MASTER MIX SI-DNA

Reagents Final concentration

x 1

Buffer Taq platinum 10X (Invitrogen)

1X 2 μl

MgCl2 50 mM 3 mM 1.2 μl

dNTPs 25 mM 0.25 mM 0.2 μl Primers cruzi 1 50pmol/μl 1 μM 0.4 μl Primers cruzi 2 50pmol/μl 1 μM 0.4 μl SYBR Green 10X 0.5 X 1 μl Taq DNA polymerase platinum 5 U/ μl (Invitrogen)

0.5 U 0.1 μl

Water 13.1 μl Total volume 18 μl Sample 2 μl Final volume 20 μl

IS Fw: 5´-AACCGTCATG GAACAGCACGTAC-3´ IS Rv: 5´-CTAGAACATTGGCTCCCGCAACG-3´

17

CYICLING CONDITIONS T. cruzi SI–DNA:

Temperature Time

94 ºC 5 minutes 94 ºC 10 second

58 ºC 10 second 72 ºC 10 second 72 ºC Read

CORBETT LIFE SCIENCE’S ROTOR-GENE™ THERMOCYCLER

DETECTION

Expected melting temperature: aprox 81ºC

40 cycles

18

APPENDIX

I. BLOOD SAMPLES

I.a. Volume of peripheral blood

2 mL for pediatric samples

5 mL for adult samples

Collect blood in polypropylene tubes (Falcon, Corning, Costar), mix

immediately with guanidine buffer or store at 4 ºC up to 24 hs before mixing

with guanidine buffer. Some laboratories freeze the blood sample because

other DNA extraction methods are used, which have not been compared in the

context of this workshop.

I.b Storage of blood samples

Add the blood to a tube containing one volume of a solution of Guanidine/ClH

6M 0,2 EDTA pH 8,0 (Ávila et al, 1990). This can be done up to 16 hs after

blood collection. This is a lysis solution that also allows DNA preservation

because DNAses are denaturated. The GEB mixture may be stored at room

temperature during three months or indefinitely at 4 ºC. The GEB mixture

must be let stand at room temperature between 12 hs (overnight) to one week

before the boiling procedure.

Boiling: Boil the GEB mixture during 15 min at 100oC and let stand at room

temperature at least 12 hs (overnight).

II. QUALITY CONTROLS

Quality Controls

Extraction Control: As a negative control one can use blood sample

without parasites and as a positive control blood sample with 1 parasite/mL.

Do on duplicate

PCR control.

Negative control use a tube without DNA

Strong Positive control (10-100 fg/μL T.cruzi DNA)

19

Weak Positive control (DNA input corresponding to the detection limit of

the PCR )

It is recommended that the T.cruzi DNA control belongs to the lineage

prevalent in the region or country where the procedure is performed (Example

T.cruzi IId or IIe in Argentina, Chile or Bolivia, T.cruzi I in Mexico, Colombia,

Venezuela).

III. RECOMENDATIONS FOR DNA EXTRACTION

Before starting work put paper towel on the bench. Throw it away after

finishing the extraction procedure (red bags).

Use always aerosol resistant tips, changing them each time you

manipulate a sample.

Make aliquots of every reagent. In case of suspicion of contamination

use a new one.

Use gloves and change it frequently during manipulation or each time

you have to quit the lab or get in contact with materials that can become

contaminated ( mobile, telephone, etc ).

Manipulate tubes avoiding contact among them.

Open Eppendorf tubes using the metallic tool, clean it frequently with

bleach 10%.

Keep tubes closed. Only open them one by one during the procedure.

When the same reagent must be added to a panel of tubes, take care

not to touch with the same tip the inner walls of tubes containing sample or

DNA. In case of contact, discard the tip and use a new one.

After vortexing and before opening tubes, make a spin down.

If pipettes or bench become contaminated with samples, clean them

with papertowel, use 10% bleach and then 70% ethanol.

20

IV. REPORTING PCR DATA

Report of PCR findings

Not detectable

Detectable

Indicate the detection limit of the PCR run corresponding to the reported sample.

Example:

Technician Sample kDNA Satellite Satellite

Real-Time IS Real-

Time F109 F110 F111 F112 F113 F114 C109 C110 C111 C112 C113

C114 Conventional kDNA and Satellite Results:

0 = not detectable; 1 = weak; 2 = average; 3 = strong Real-Time Results: Detectable: Nº of C(t); Not detectable: 99

V. CONTROL OF AMPLICON CARRY-OVER

CONTAMINATION IN LABORATORY WORKING AREAS

1) Wet filter papers with sterile water.

2) Put filter papers on different working places, equipment, benchs, pipets,

hands and gloves, centrifuge rotors, etc., where there is a suspicion of

amplicon carry over contamination.

3) The filters are then incubated into an eppendorf tube containing 1 ml of

sterile water for 30 min at 50ºC.

4) 10 ul of the filter eluate is tested by the amplification procedure most

frequently performed in the laboratory (example kDNA)

21

VI. PRE –PCR WORKING LABORATORY WORKING

VII. POST–PCR WORKING LABORATORY

22

VIII. GUANIDINE/CLH BUFFER

REAGENTS

Guanidine hydrochloride (M.W. 95.53)

EDTA 0,5 M pH 8.0 (Disodium EDTA Dihydrate M.W. 372.24)

H2O distilled

Sodium hydroxide (NaOH).

• Guanidine 6,0 M; EDTA 0,2 M Buffer (GEB)

Dissolve 500 g Guanidine in 100 ml distilled water.

Add 350 ml EDTA 0,5 M pH 8.

Bring the volume to 870 ml with distilled water.

Pour into labelled bottles and autoclave before use.

• EDTA 0,5 M pH 8

Dissolve 186,1 g EDTA.H2O in 900 ml distilled water.

Reaches pH 8 with NaOH. Stir, on a magnetic stirrer with a magnetic

stirring bar, and gradually add solid sodium hydroxide pellets until pH

8 (approximately 20 g). (Continue until the solution is clear and the

pH is stable)

Bring the volume to 1000 ml with distilled water

Pour into labelled bottles.

23

AKNOWLEDGEMENTS

Financial support

To WHO-TDR for leading role of financial support of PCR standardization project including Multicentric Study – Workshop and Symposium.

To UNU-BIOLAC for financial support of Symposium.

To Fundación Bunge & Born and Grupo Centralab for Post-symposium

Events

Venues and technical assistance

To INGEBI, Dir Dr Mirta Flawiá and LabMECh, Dir Dr Mariano J. Levin for facilitating the Workshop Venue.

To Museo Nacional de Ciencias Naturales Dir Ing. for Symposium Venue.

To Chagas Group and INGEBI personnel for collaboration in workshop activities.