Stool Specimens - Extraction of Parasite DNA from Fecal Specimens Using FastDNA® Kit
Note 1: Divide fecal specimens into multiple aliquots and store at -80°C without preservatives. Optionally, samples can be preserved in potassium dichromate (1:1 dilution with 5% w/v) or in absolute ethanol (1:1 dilution) and stored at 4°C.
Note 2: This protocol is a summary of the procedure included in the FastDNA® kit manual provided by the manufacturer. Please refer to the manual for detailed product information and protocols.
Special equipment needed:
FastPrep FP120 Disrupter (available from Q-Biogene, Carlsbad, Calif.) or similar product.
List of reagents:
- Phosphate buffered saline solution, 0.01M, pH 7.2
- EDTA solution, 0.5M, pH 8.0
- Selected reagents from the FastDNA® kit available from MP BiochemicalsExternal or similar product:
CLS-VF (Cell Lysis/DNA Solubilizing Solution for Vegetation, Cat. No. 6540-402)
PPS (Protein Precipitation Solution, Cat. No. 6540-403)
Lysing Matrix Multi Mix E (Cat. No. 6914-050/100)
Binding Matrix (Cat. No. 6540-408)
SEWS-M (Salt/Ethanol Wash Solution) (Cat. No. 6540-405)
DES (DNA Elution Solution) (Cat. No. 6540-406)
- PVP (Polyvinylpyrrolidone – Cat. No. 85, 645-2, Aldrich Chemical Company, Inc., Milwaukee, WI) or equivalent product.
QIAquick PCR purification kit (Cat. No. 28106, Qiagen Inc., Valencia, CA, http://www.qiagen.comExternal) or similar product.
- Select and label enough unused 1.5 ml siliconized tubes to perform the extractions.
- Centrifuge an aliquot of 300 to 500 μl of each stool specimen at 14,000 × g at 4°C for 5 minutes.
- Suspend the pellet obtained in the previous centrifugation in 1 ml of PBS-EDTA. Repeat this procedure two more times using the same centrifugation conditions.
- Resuspend the pellet in PBS EDTA after the final centrifugation to obtain a total volume of approximately 300 µl of solubilized sample.
- To the tubes containing the lysing matrix Multi Mix E Matrix, add 300 µl of the washed stool sample, 400 µl of CLS-VF, 200 µl of PPS, and PVP to a final concentration ranging from 0.1% to 1%.
- Mix by vortexing. Close tubes tightly and place in the FP120 disrupter.
- Run in the FP120 at 5.0-5.5 speed for 10 seconds.
- Spin 5 minutes at 14,000 × g at room temperature in a centrifuge designed to hold 1.5 ml tubes.
- Transfer 600 µl supernatant to new tube. Discard the tubes containing the debris and Multi Mix E Matrix.
- Add 600 µl of Binding Matrix and mix gently by inverting the tubes.
- Incubate 5 minutes at room temperature.
- Spin at 14,000 × g for 1 minute at room temperature. Pour out the supernatant.
- Resuspend the pellet in 500 µl of Sews-M. Resuspend it thoroughly by pipetting up and down.
- Spin 1 minute at 14,000 × g at room temperature. Discard the supernatant.
- Spin 10 seconds and remove residual liquid from the top of the matrix.
- Resuspend the matrix in 100 µl of DES. Mix with the tip and pipette up and down as in step 13.
- Incubate 2 to 3 minutes at room temperature.
- Spin 2 minutes at 14,000 × g.
- Transfer the supernatant to a clean, labeled tube and store the DNA sample at 4°C until PCR amplification.
OPTIONAL STEP (necessary for food samples):
Further purification of the eluted DNA may be required in some samples because PCR inhibitors may not be completely removed using the described procedure. In this case, purify DNA using a QIAquick spin column, following the manufacturer’s instructions. Use high quality deionized water to elute the DNA from the QIAquick spin column.
- Store the purified DNA at 4°C until PCR amplification.
For additional information on molecular diagnosis using stool specimens, call the Division of Parasitic Diseases at (404) 718-4120.
da Silva AJ, Bornay-Llinares FJ, Moura INS, Slemenda SB, Tuttle TL, Pieniazek NJ. Fast and reliable extraction of protozoan parasite DNA from fecal specimens. Mol Diagn 1999;4:57-63.
DPDx is an educational resource designed for health professionals and laboratory scientists. For an overview including prevention, control, and treatment visit www.cdc.gov/parasites/.