A Technique for Magnetic Glass Particle-Mediated Nucleic Acid Extraction from Blood

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines

1. Blood Collection from Patients

CAUTION: Take appropriate laboratory safety measures when collecting whole blood from the patient. Wear gloves and glasses. If the patient is in isolation, please follow biosafety level 4 procedures.

1. To collect intravenous whole blood from the patient, use a butterfly needle with small-bore extension tubing and a blood collection vacuum tube containing a lysis/binding buffer. Rest the patient's arm in a downward position and position the collection tube lower than the butterfly needle. Insert the butterfly needle into the vein of the patient and attach the blood collection vacuum tube to the small-bore extension.
   NOTE: This will prevent back-flow.
2. After the blood collection, mix the contents of the tube by flipping the tube 5 – 10 times.
   NOTE: Due to the remaining vacuum in the blood collection tube containing 1.6 mL of lysis/binding buffer, the volume of the sample collected will automatically be 1.6 mL.
3. Disinfect the outside of the tube using 70% ethanol.
4. Incubate the tubes for 20 min at room temperature.
   NOTE: The protocol can be paused here and the full blood collection tubes can be stored at -20 °C, 5 °C, 25 °C or 37 °C for at least 1 month.
5. Continue directly to the simplified NA extraction method.

2. Simplified NA Extraction of the Whole Blood

1. To purify NA from the lysis/binding buffer-inactivated collected blood, mix the contents of the tube by flipping the vacuum tube by hand 5 – 10x.
2. Remove the lid of the tube carefully and discharge the lid.
3. Pour the prepared aliquot of MGPs (1 mL) directly into the blood collection tube.
4. Place a new lid from an unused blood collection tube on the tube containing the sample.
5. Place a finger on the lid to ensure that the tube is tightly closed and mix the contents of the tube by flipping the blood collection tube by hand 5 – 10 times.
6. Place the tube in the magnetic holder and keep a finger on the lid to ensure the tube is tightly closed.
7. Flip the magnetic holder with the tube a few times by hand to make sure that all the MGPs are collected at the side of the tube with the magnet.
   NOTE: The magnetic holder can be replaced with an elongated magnet and a rubber band.
8. Remove the lid of the tube and discard the contents of the tube either by using a disposable pipette or simply by pouring the contents into a 50 mL collection tube.
   NOTE: Avoid aerosols from the 50 mL collection tube by closing the tube with a lid.
9. Pour the prepared aliquot of wash buffer-1 (WB-1, 4 mL) directly into the blood collection tube.
10. Place the lid on the tube and place a finger on the lid to ensure the tube is tightly closed.
11. Remove the tube from the magnetic holder, keeping the lid securely tightened with a finger.
    NOTE: If using a magnet and a rubber band, simply remove the rubber band and magnet from the tube.
12. Resuspend the MGPs by flipping the blood collection tube by hand 5 – 10 times.
13. Repeat steps 2.6 – 2.8.
14. Pour the prepared aliquot of WB-2 (1.5 mL) directly into the blood collection tube.
15. Place the lid on the tube and remove the tube from the magnetic holder.
    NOTE: If using a magnet and a rubber band, simply remove the rubber band and magnet from the tube.
16. Place a finger on the lid to ensure the tube is tightly closed.
17. Resuspend the MGPs by flipping the blood collection tube for a few seconds by hand.
18. Repeat steps 2.6 – 2.8.
19. Pour the prepared aliquot of WB-3 (3 mL) directly into the blood collection tube.
20. Place the lid on the tube and remove the tube from the magnetic holder.
    NOTE: If using a magnet and a rubber band, simply remove the rubber band and magnet from the tube.
21. Place a finger on the lid to ensure the tube is tightly closed.
22. Resuspend the MGPs by flipping the blood collection tube by hand 5 – 10 times.
23. Repeat steps 2.6 – 2.8.
24. Pour the prepared aliquot of elution buffer (EB, 100 µL) directly into the blood collection tube.
25. Place the lid on the tube and remove the tube from the magnetic holder.
    NOTE: If using a magnet and a rubber band, simply remove the rubber band and magnet from the tube.
26. Resuspend the MGPs in the EB by tapping the blood collection tube 5 – 10 times with a finger.
    NOTE: The protocol can be paused here, and the tubes can be stored at -20 °C.
27. Transfer one droplet (5 – 8 µL) of the resuspended MGPs to the downstream NA amplification reaction mix using a 1.5 mL disposable pipette (any downstream diagnostic NA amplification assay such as loop-mediated isothermal amplification (LAMP)/ Reverse-transcription LAMP (RT-LAMP) or quantitative polymerase chain reaction (qPCR)/RT-qPCR assays can be used).
    NOTE: The NAs will stick to the MGPs, so be sure to use the MGPs in the downstream NA amplification reaction. Mix the MGP suspension before use. After mixing, the MGPs will collect at the bottom of the tube.