Diagnostic Procedures [Last Modified: ]
Diagnostic Procedures for
Blood Specimens

Extraction of DNA from blood specimens

Whole blood
This method is used for whole blood collected in Vacutainer® EDTA tubes.  Use preferably the QIAamp Blood Kit (Cat. No. 51106; Qiagen Inc., Chatsworth, CA, [http://www.qiagen.com]).  Similar products that can yield the comparable concentration of genomic DNA from the same volume of blood can be used if QIAamp is not available.

Note: This protocol is a summary of the procedure included in the QIAamp Blood kit manual provided by the manufacturer.  Please refer to the manual for detailed product information and protocols.

Before you start, be sure to do the following:

  1. Equilibrate samples to room temperature.
  2. Heat two water baths, one to 56°C and another to 95°C.
  3. Equilibrate buffer AE or water to room temperature for elution in step 10.
  4. Ensure that buffer AW1, Buffer AW2, and QIAGEN protease have been prepared according to the instructions.
  5. All centrifugation steps should be carried out at room temperature.
  6. Use carrier DNA if the sample contains less than 10,000 genome equivalents.
  7. 200 µl of the whole blood yields 3-12 µg of DNA.  Preparation of buffy coat is recommended if a higher yield is required.

Procedure

  1. Pipet 200 µl whole blood, 20 µl QIAGEN Protease, and 200 µl Buffer AL into a 1.5 ml low binding microcentrifuge tube (e.g., Cat. No. T6050G, Marsh Biomedical Products, Rochester, NY).  Mix by vortexing.
  2. Incubate at 56°C for 10 minutes.
  3. Incubate at 95°C for 10-15 minutes.  This step will inactivate potentially infectious agents.  Do not incubate over 15 minutes, since this may degrade DNA.
  4. Spin down briefly to remove drops from the inside of the tube.
  5. Add 200 µl of 96-100% ethanol and mix by vortexing.
  6. Carefully apply the mixture from step above to a QIAamp spin column.  Centrifuge 1 minute at full speed (15,000 × g).  Discard the tube containing the filtrate.  The DNA will be bound to the filters in the spin columns.  Place column in a clean 2 ml collection tube.
  7. Open the QIAamp spin column and add 500 µl Buffer AW1 without hitting the rim.  Close the cap and centrifuge 1 minute at full speed.  Place QIAamp spin column in a clean 2 ml collection tube.
  8. Carefully open the QIAamp spin column and add 500 µl Buffer AW2.  Close the cap and centrifuge the QIAamp column for 3 minutes at maximum speed.
    8a.  Discard the 2 ml collection tube containing the filtrate, place the QIAamp spin column in a new collection tube and spin for 1 minute to remove residual buffer AW2.
  9. Place the spin column in a clean 1.5 ml low binding microcentrifuge tube.  Add 200 µl Buffer AE or distilled water to the spin column.  Incubate at room temperature for 5 minutes to elute the DNA.  Centrifuge 1 minute at full speed.  Use 1 µl of extracted DNA for a 50 µl PCR.
  10. Store at 4°C.

Extraction from filter containing blood
Use preferably the Schleicher and Schuell IsoCode® Stix (Cat. No. 10495015; Schleicher & Schuell, Keene, NH, [http://www.schleicher-schuell.com/bioscience]).  Spot papers directly from whole blood or finger stick.  Do not use blood collected in EDTA to prepare the IsoCode® Stix.  Refer to the IsoCode® Stix brochure provided by the manufacturer for detailed product information.

  1. Label one 1.5 ml microcentrifuge for each IsoCode® Stix to be extracted.  Do not add the IsoCode® Stix to the tubes yet!  Label one additional tube for water to be used in step 7.
  2. Add 500 µl of deionized sterile water to each tube that will receive the IsoCode® Stix.  Add enough deionized sterile water in the additional tube to provide 50 µl per sample to be extracted.  Place the tubes under ultraviolet light (UV) for 20 minutes.  Do not expose the IsoCode® Stix to UV light!
  3. At the end of this incubation, hold the stick with triangle of dried blood over the respective tube containing water that was exposed to UV.  While closing the cover, pull the scored end of the stick so that the triangle detaches and falls directly into the tube.  If the water turns even faintly pink, the IsoCode® Stix was not dry and should be discarded.
  4. Vortex the tubes containing the triangles at full power 3× for at least 5 seconds to wash.
  5. After vortexing, centrifuge the tubes for a few seconds and remove the water with a sterile pipette.
  6. Centrifuge the tubes again for 5 seconds, and pipette off the residual water.
  7. Add 50 µl of deionized sterile water that was exposed to UV (from the additional tube prepared in steps 1 and 2), to each tube, using a new tip for each addition.  Completely immerse the triangle by a brief centrifugation.
  8. Place each tube in a 95°C heat block for 30 minutes.  Caution should be taken to prevent the lids from opening during incubation.  Lid locks can be used for this purpose.
  9. After incubation, gently tap tube several times (around 20 times) as most of the fluid is on the tube sides and lid.  Spin down tubes for a few seconds to prevent leaking when opening.
  10. Remove the supernatant to a clean siliconized 1.5 ml microcentrifuge tube.  For PCR amplification, use 5 µl of the extracted DNA per 50 µl volume PCR mix.  Alternatively, 2 µl of the extracted DNA per 20 µl of PCR mix can be used.
  11. Store extracted DNA at 4°C.

For more information on molecular diagnosis for blood specimens, contact the Division of Parasitic Diseases at 770-488-4474.  For information about the technical aspects of PCR for molecular diagnosis of malaria and babesiosis, send an e-mail to dpdx@cdc.gov.
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