Aug 26, 2025

Public workspaceBlood DNA extraction protocol compatible with LAMP-PCR for T. cruzi detection.

Microbiology Spectrum
DOI
https://dx.doi.org/10.17504/protocols.io.rm7vz9mnrgx1/v1
  • Sneider Gutierrez1,
  • Jessy Condori2
  • 1Jons Hopkins University;
  • 2Universidad Peruana Cayetano Heredia
Sneider Gutierrez
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vz9mnrgx1/v1
External link: https://doi.org/10.1128/spectrum.00172-25
Protocol Citation: Sneider Gutierrez, Jessy Condori 2025. Blood DNA extraction protocol compatible with LAMP-PCR for T. cruzi detection.. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz9mnrgx1/v1
Manuscript citation:
Guarnizo SAG, Condori BJ, Basma L, Equilia S, Malaga E, Defazio S, Arteaga E, Velarde JK, Obregón M, Takyar A, Duque C, Hakim J, Tinajeros F, Gilman RH, Bowman N, Mugnier MR A specific, stable, and accessible LAMP assay targeting the HSP70 gene of Trypanosoma cruzi. Microbiology Spectrum 13(11). doi: 10.1128/spectrum.00172-25
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: June 28, 2025
Last Modified: August 26, 2025
Protocol Integer ID: 221226
Keywords: Blood DNA extraction, cost-effective method, T. cruzi detection, compatible with LAMP-PCR and qPCR, blood dna extraction protocol compatible with lamp, blood dna extraction protocol, isolated dna, quality nucleic acids from fresh peripheral blood, pcr, fresh peripheral blood, reliable results for downstream molecular application, ctab lysis buffer, qpcr
Abstract
This cost-effective protocol uses CTAB lysis buffer and isopropanol precipitation to purify high-quality nucleic acids from fresh peripheral blood collected in EDTA. The protocol is about four times cheaper when compared with commercial kits. The isolated DNA is compatible with both LAMP-PCR and qPCR assays, providing reliable results for downstream molecular applications.
Guidelines
Compared to commercial kits, this protocol yields a comparable average DNA amount but with moderately higher variability.
Materials

Reagents
ABC
ReagentCompanyCatalog number/SKU
2-propanol sigma aldhrich1096341011
Hexadecyltrimethylammonium bromide sigma aldhrichH5882-100G
Tris-HCl, 1M Solution, pH 8.0, Molecular Biology Grade, UltrapureFisher scienificAAJ22638AE
Ethylenediaminetetraacetic acid solutionMERKE8008-100ML
Absolute ethanol Thermo scientificT038181000
Nuclease free waterMilipore4.86505.1000
NaCl (5 M), RNase-freeMiliporeS9625-1Kg
Disposable materials
ABC
Disposable materialCompanyCatalog number/SKU
Conical Base 2.0 mL Microcentrifuge TubesUSA Scientific 14202700
1000 uL tips (TipOne‱ Pipette Tips in Racks)USA Scientific 1111-0806
200 uL tips (TipOne‱ Pipette Tips in Racks)USA Scientific 1111-0806

Permanent materials
AB
Permanent materials/equipmentSKU
Vortex mixerCLS6885DB
Centrifuge (capable of 12,000 × g) (Benchmark MC-12 High Speed Microcentrifuge)BMS:C1612
Heater (prewarmed to 70 °C) (Corning‱ LSE‱ digital dry bath heater)CLS6885DB
Plastic Test Tube Rack Cube76263-262
Troubleshooting
Safety warnings
It is recommended to receive training before processing important samples and to perform extractions in replicates when possible.
Before start
Collect peripheral blood in an EDTA vacutainer and proceed with the checklist section.
Check List (Ensure you have the following materials ready before running the experiment)
10m 30s
Equipment·
  • Vortex mixer
  • Centrifuge (capable of 12,000 × g)
  • Heater (prewarmed to 70 °C)
  • Timer
30s
Cold Storage·
  • A bucket with ice
2m
Reagents & Buffers
  • CTAB 2% lysis buffer (CTAB: 2% (w/v), Tris-HCl (pH 8.0): 100 mM, EDTA: 20 mM, NaCl: 1.4 M.
  • 5 M NaCl (cold/onice)
  • Isopropanol (cold/onice)
  • 70% ethanol
  • PCR-grade nuclease-free water (incubate at 70 °C)
30s
Consumables & Labware·
  • Rack for 2 mL tubes
  • 2 mL microcentrifuge tubes (Catalog 1420-2700 USA Scientific; two per sample) Do not use low-binding tubes.
  • 1 mL pipette + tips
  • 100 μL pipette + tips
  • Paper towel
  • Dampen a paper towel with 10% bleach to clean gloves between samples
2m
Labeling & Organization·
  • Markers for labeling
  • Label the tubes as follows: o Left side: Date and operator’s initials o Top: Sample order (e.g., 1, 2, 3) + “NA” =nucleic acids + full sample name o Right side: Leave blank for DNA concentration in case you have the equipment to quantify
5m
Are you running the LAMP-PCR right after? If so,...

  • Make sure you have enough master mix vials pre-stored at -20 °C.
  • Thaw the SYBR Green at 4 °C.
30s
Optional
Cell lysis
12m 25s
Add 300mL of fresh whole blood in an EDTA to 2mL microcentrifuge tube
10s
Centrifigation
Pipetting
Add 500 µL of CTAB lysis buffer to the whole blood.
10s
Pipetting
5 seconds Vortex thoroughly to mix.
5s
Mix
Incubate at 65°C for 10 minutes
10m 30s
Incubation
1 min on ice to improve protein precipitation
1m 30s
Temperature
Protein precipitation
3m 10s
Add 500 µL of 5 M NaCl to the lysed sample by pipetting three times.
10s
Pipetting
5 seconds Vortex
10s
Mix
Centrifuge at 12,000x g for 2 minutes.
2m 30s
Centrifigation
Carefully pipette 1 mL of the supernatant from top to bottom, ensuring that the pellet is not touched.
10s
Pipetting
Transfer the 1 mL supernatant to a new, pre-labeled 2 mL tube.
10s
Pipetting
Nucleic acids precipitation
7m 40s
Add 1mL of cold isopropanol to the recovered supernatant
10s
Pipetting
Critical
Gently invert to mix 20 times

Note: At this point, you should see debris floating in the solution.


Trick: If you are working with multiple samples, place the tubes in the rack. Use the palms of your hands to make a sandwich, and gently rotate from right to left to mix the isopropanol


1m 30s
Mix
Trick: If you did not see precipitation during the previous step, incubate the sample on ice or -20 °C for 10 minutes before proceeding.
Optional
Centrifuge at 12,000xg for 5 minutes
5m 30s
Centrifigation
Carefully remove the supernatant by pipetting, making sure not to disturb the pellet stuck to the side at the bottom of the tube. Since the DNA is concentrated in the pellet, you can safely discard any blood debris present in the upper phases.
30s
Nucleic acids clean up and DNA quantification
18m 43s
Add 1 mL of 70% ethanol to wash the pellet
10s
Vortex for 1 second.
3s
Centrifuge at 12,000x g for 2 minutes
2m 30s
Discard the ethanol by inverting the tube and tapping it repeatedly on a paper towel until any remaining ethanol drains down the side.

Note: It is recommended to invert the tube upside down only once. While inverting, gently rotate the tube to prevent the liquid from putting pressure on one side of the tube.
Add 1 mL of 100% ethanol to wash the pellet
10s
Discard the ethanol by inverting the tube and tapping it repeatedly on a paper towel until any remaining ethanol drains down the side.
20s
Dry the DNA for 3 minutes at 70 °C.
3m 10s
Resuspend the dried pellet in preheated 25-50 µL by pippeting 20 times.
30s
Incubate at 70 °C for 5 minutes to improve DNA dissolution
5m 10s
Vortex for 5 seconds
10s
Centrifuge at 12000 for 1 minute
1m 10s
Centrifigation
Quantify DNA by NanoDrop, taking DNA from the top of the solution.
5m
Sample storage
1m
Store at 4°C for short-term (< 1week) or -20°C for long-term storage (>1 week).
1m
Running LAMP- PCR
1h 22m
Take out the LAMP-PCR mix stored at -20°C and spin it down.
Note: include a negative and positive control.
1m
Optional
Add 2 µL of SYBR Green to the lid.
10s
Optional
Load 5 µL of pre-extracted DNA and pipette 5 times.
20s
Optional
Incubate for 1 hour at 65 °C.
1h 20m
Optional
Spin it down and record the result: brown = negative, green = positive.
30s
Optional