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:

1. Phosphate buffered saline solution, 0.01M, pH 7.2
2. EDTA solution, 0.5M, pH 8.0
3. Selected reagents from the FastDNA® kit available from MP Biochemicals 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)
4. 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.com) or similar product.
   

Procedure:

1. Select and label enough unused 1.5 ml siliconized tubes to perform the extractions.
2. Centrifuge an aliquot of 300 to 500 μl of each stool specimen at 14,000 × g at 4°C for 5 minutes.
3. 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.
4. Resuspend the pellet in PBS EDTA after the final centrifugation to obtain a total volume of approximately 300 µl of solubilized sample.
5. 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%.
6. Mix by vortexing. Close tubes tightly and place in the FP120 disrupter.
7. Run in the FP120 at 5.0-5.5 speed for 10 seconds.
8. Spin 5 minutes at 14,000 × g at room temperature in a centrifuge designed to hold 1.5 ml tubes.
9. Transfer 600 µl supernatant to new tube. Discard the tubes containing the debris and Multi Mix E Matrix.
10. Add 600 µl of Binding Matrix and mix gently by inverting the tubes.
11. Incubate 5 minutes at room temperature.
12. Spin at 14,000 × g for 1 minute at room temperature. Pour out the supernatant.
13. Resuspend the pellet in 500 µl of Sews-M. Resuspend it thoroughly by pipetting up and down.
14. Spin 1 minute at 14,000 × g at room temperature. Discard the supernatant.
15. Spin 10 seconds and remove residual liquid from the top of the matrix.
16. Resuspend the matrix in 100 µl of DES. Mix with the tip and pipette up and down as in step 13.
17. Incubate 2 to 3 minutes at room temperature.
18. Spin 2 minutes at 14,000 × g.
19. Transfer the supernatant to a clean, labeled tube and store the DNA sample at 4°C until PCR amplification.
20. 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.
21. 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.

Reference:

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.