This method can help when performing point of care diagnostics, of patients with signs of viral infections. The main advantage of this technique is that it can be performed without laboratory equipment and electricity, and hence it can be performed in a hospital ward. Demonstrating the blood collection procedure, will be Medical Doctor Anders Fomsgaard, and Scientist Morten Rasmussen, both from my laboratory.
Begin by preparing the blood collection tubes. Use a 25 gauge one inch needle attached to a 3ML syringe, to inject 1.6ML of a commercial lysis buffer into a 4ML EDTA vacuum tube. It is critical to maintain the vacuum in the blood collection tube for blood collection.
Hence, always use a needle to inject the lysis buffer into the blood collection tube. Next completely re-suspend a 960 micro-liter volume of magnetic glass particles, or MGPs, and transfer to a labeled 1.8ML tube. Next pipet 4ML of wash buffer one into a 4.5ML tube labeled WB-1.1.5ML of wash buffer two into a 3.6ML tube labeled WB-2, and 3ML of wash buffer three into a 4.5ML tube labeled WB-3.
Finally add 100 micro-liters of elution buffer to a 1.8ML tube labeled EB, and leave the tubes at room temperature until use. To collect intravenous whole blood from the patient, use a butterfly needle with a small bore extension tubing, and one of the previously prepared blood collection vacuum tubes containing lysis buffer. Rest the patient's arm in a downward position.
Insert the butterfly needle into the vein of the patient, and attach the blood collection vacuum tube to the small bore extension. After blood collection mix the contents of the tube, by flipping the tube five to ten times. Disinfect the outside of the tube using 70%ethanol.
Then incubate the tubes for 20 minutes at room temperature for virus inactivation. To purify nucleic acids from the collected blood, again mix the contents of the tube by flipping the vacuum tube by hand five to ten times. Pour the prepared aliquot of MGP's directly into the blood collection tube.
The place a new lid from an unused blood collection tube on the tube containing the sample. Place a finger on the lid to ensure that the tube is tightly closed, and mix the contents of the tube by flipping five to ten times. Place the tube in the magnetic holder and keep a finger on the lid, to ensure the tube is tightly closed.
Then flip the magnetic holder with the tube a few times to disperse the MGP's before they collect on the side of the tube with the magnet. Remove the lid of the tube, and discard the contents of the tube into a 50ML collection tube, using a disposable pipet. Pour the prepared aliquot of WB-1 directly into the blood collection tube and close the lid on the tube.
Mix the content by hand five to ten times. Then collect the beads at the side of the tube, using the magnet and remove the liquid as before. Re-suspend the beads in the prepared aliquot of WB-2, then discard the solution, and repeat the procedure with a prepared aliquot of WB-3.
Finally, remove the tube from the magnetic holder and pour the prepared aliquot of EB directly into the blood collection tube. Place the lid on the tube, and re-suspend the MGP's by tapping five to ten times with a finger. Finally, use a 1.5ML disposable pipet to transfer one droplet of the re-suspended MGP's to the downstream nucleic acid amplification reaction mix.
Be sure to fully re-suspend the MGP's before transferring only one droplet into the PCR reaction. To demonstrate proof of concept, negative whole blood was spiked with 10 fold dilutions of the Ebola virus standard, prepared from the recent outbreak in southern Guinea, or three different concentrations of Epstein Barr virus. The extracted nucleic acids were analyzed by in-house virus specific R2 qPCR or qPCR.
The lower the crossing threshold, or CT, the higher the viral genome copy number. Here, whole bloods spiked with different concentrations of Ebola virus was detected by an Ebola virus specific lamp. The final concentration of virus in the spiked whole blood sample is seen on the X axis, and the minutes to positive is seen on the Y axis.
Here, whole blood was spiked with 10 fold dilutions of a hepatitis C WHO standardized serum sample, with detection by HCV specific RT qPCR. Finally, this image shows the results of an Epstein Barr virus specific lamp reaction, on Epstein Barr virus positive whole blood. While attempting this procedure it's important to use the specified lysis binding buffer.
This is a crucial step, and a crucial re-agent for the method. This lysis binding buffer will inactivate virus in the sample. Following this technique you can perform different molecular detection methods like lamp and PCR, and hence you can perform a differential diagnosis.
