Apr 22, 2026
DNA Extraction from Trichuris trichiura Worm Samples
- James Owen Delaluna1,
- Hyun Beom Song1,
- Taeksang Lee1
- 1Seoul National University
- Seoul National University
Protocol Citation: James Owen Delaluna, Hyun Beom Song, Taeksang Lee 2026. DNA Extraction from Trichuris trichiura Worm Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2djeqg1y/v1
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: Working
We use this protocol and it's working
Created: April 20, 2026
Last Modified: April 22, 2026
Protocol Integer ID: 315367
Keywords: Trichuris trichiura, DNA extraction, parasite genomics, nematode, bead homogenization, TissueLyser, PCR-ready DNA, β-tubulin, ITS2, molecular diagnostics, trichuris trichiura worm sample, trichuris trichiura worm samples this protocol, extraction of genomic dna, dna extraction, worm samples for downstream molecular analysis, tubulin gene, trichuris trichiura, worm sample, nematode tissue, genomic dna, phylogenetic analysis, resulting dna, extraction
Abstract
This protocol outlines the extraction of genomic DNA from Trichuris trichiura worm samples for downstream molecular analyses. The workflow includes removal of residual preservatives, mechanical homogenization using bead-based disruption, and DNA extraction optimized for nematode tissues. The resulting DNA is suitable for amplification of the full-length β-tubulin gene and the ITS2 region, supporting applications such as sequencing, phylogenetic analysis, and population genetics.
Guidelines
- Ensure thorough washing of samples to remove residual fixatives (formaldehyde/ethanol), as these may inhibit downstream enzymatic reactions.
- Avoid repeated freeze–thaw cycles of samples prior to extraction.
- Use appropriate PPE when handling biological specimens and chemical preservatives.
- Homogenization efficiency is critical due to the thick nematode cuticle—insufficient disruption may reduce DNA yield.
Materials
Reagents
- Phosphate-buffered saline (PBS), sterile
- Clear-S Quick DNA Extraction Kit (inVIRUStech)
- Nuclease-free water
- 70% ethanol (optional, for cleaning)
Equipements
- TissueLyser LT (QIAGEN) or equivalent bead homogenizer
- Stainless steel beads (3–5 mm)
- Microcentrifuge tubes (1.5 mL or 2.0 mL)
- Pipettes and sterile tips
- Microcentrifuge
- Nanodrop spectrophotometer (or equivalent)
- Liquid nitrogen storage system
Troubleshooting
Problem
Low DNA yield
Solution
Increase cycles or bead size
Problem
Poor PCR amplification
Solution
Repeat washing step
Problem
Low purity ratio
Solution
Extend lysis or improve purification
Problem
DNA degradation
Solution
Maintain cold chain
Safety warnings
- Risk of PCR inhibition: Residual preservatives (e.g., formaldehyde or ethanol) from sample storage can inhibit amplification. Ensure thorough washing during DNA extraction.
- Long-range amplification sensitivity: The β-tubulin target (~4.2 kb) is sensitive to DNA degradation. Fragmented DNA may fail to amplify even when shorter targets (e.g., ITS2) amplify successfully.
- Contamination risk: PCR is highly sensitive. Perform reaction setup in a clean area using sterile filter tips and include a no-template control (NTC) in every run.
- Non-specific amplification and artifacts: Excessive cycle numbers or suboptimal primer handling may result in non-specific bands or primer-dimers.
- Barcoding cross-contamination: The ITS2 reverse primer contains sample-specific barcodes. Mislabeling or cross-contamination can lead to incorrect sample assignment during downstream sequencing.
- Polymerase-specific conditions: This protocol uses a two-step cycling condition optimized for KOD One PCR Master Mix. Using a different polymerase may require re-optimization of annealing and extension conditions.
- Gel electrophoresis safety: Handle DNA stains and UV illumination equipment with appropriate precautions to avoid chemical exposure and UV damage.
Samples
Sample Adult Trichuris trichiura worms preserved in formaldehyde or ethanol during transport
Procedure
Step 1: Sample Washing and Preparation
- Transfer worm samples into a clean microcentrifuge tube.
- Add sterile PBS to fully immerse the sample.
- Gently invert or pipette to wash.
Remove PBS and repeat washing 2–3 times to remove residual formaldehyde or ethanol.
Step 2: Sample Storage (if not processed immediately)
After washing, snap-freeze samples in liquid nitrogen.
Step 3: Tissue Selection
For intact worms:
- Excise and use the anterior portion to ensure consistency.
For fragmented samples:
- Use all available tissue material.
Step 4: Mechanical Homogenization
Place the sample into a tube containing stainless steel beads (2-4 small beads)
Add appropriate lysis buffer from the Clear-S kit.
Homogenize using TissueLyser LT:
- Frequency: 50 Hz
- Duration: 1 minute per cycle
- Number of cycles: 2–3 cycles
Briefly centrifuge between cycles if needed.
Step 5: DNA Extraction
Proceed with DNA extraction using the Clear-S Quick DNA Extraction Kit following manufacturer’s instructions.
Apply an extended lysis step to improve DNA recovery:
- Extend incubation time as needed (e.g., +10–20 minutes beyond standard protocol).
Step 6: DNA Elution
Elute DNA in nuclease-free water or provided elution buffer.
Typical elution volume: 30–50 µL.
Step 7: DNA Quantification and Quality Assessment
Measure DNA concentration using a Nanodrop spectrophotometer.
Record:
- DNA concentration (ng/µL)
- Purity ratios (A260/A280)
Step 8: Storage of Extracted DNA
Store extracted DNA at:
- −20 °C (short-term)
- −80 °C (long-term)
Expected Results
High-quality genomic DNA suitable for:
- Long-range PCR (β-tubulin ~4.2 kb)
- ITS2 amplification (~481 bp)
DNA concentration sufficient for downstream applications (>10–20 ng/µL recommended)