Viral DNA/RNA Extraction from Respiratory Samples

EZgene^TM Viral DNA/RNA Mini Kit

This mini kit provides an easy and reliable method for isolating total viral DNA/RNA from plasma, serum, nasopharyngeal or oropharyngeal aspirates or washes, nasopharyngeal or oropharyngeal swabs, broncheoalveolar lavage, tracheal aspirates, and sputum. This procedure has been tested for isolating nucleic acids from COVID-19, Hepatitis A, HBV, Hepatitis C, and HIV. The isolated RNA can be used for PCR, qRT-PCR and other downstream applications.

Acceptable Samples

• Respiratory specimens including: nasopharyngeal or oropharyngeal aspirates or washes, nasopharyngeal or oropharyngeal swabs, bronchoalveolar lavage, tracheal aspirates, and sputum. Swab specimens should be collected only on swabs with a synthetic tip with aluminum or plastic shafts. Swabs with calcium alginate or cotton tips with wooden shafts are not acceptable
• Serum, plasma, or other body fluid.

Viral DNA/RNA Isolation Protocol

The protocol is developed for processing 50-300 μL samples.

1. Pipet 10 µL Proteinase K, 4 µL L Solution, and 300 µL Buffer LYE to a 1.5 mL tube.
   Calculate the number of samples to be processed and make a master mix of proteinase K, L Solution, and Buffer LYE.
2. Pipet 300 µL nasopharyngeal or oropharyngeal aspirates or washes, nasopharyngeal or oropharyngeal swabs, broncheoalveolar lavage, tracheal aspirates, or plasma, serum, into the 1.5 mL tube from Step 1. Mix well by pulse-vortexing for 10 seconds. Incubate at room temperature for 2-5 minutes to lyse the cells and viruses.
3. Add 600 µL isopropanol and mix well by pulse-vortexing for 5 seconds.  Note: Maintain the ratio of (Sample+ Buffer LYE): Isopropanol= 1:1
4. Transfer 600 µL of the sample from step 3 into an RNA column and centrifuge at 10,000 rpm for 30 seconds. Discard the collection tube contains the flow-through and transfer the column to a new collection tube. Apply the remaining sample to the column and spin at 10,000 rpm for 30 seconds.
5. Discard the collection tube with the flow-through and transfer the column to a new collection tube. Add 500 µL Buffer RB to the column and centrifuge at 10,000 rm for 30 seconds. Discard the collection tube contains the flow-through and transfer the column to a new collection tube.
6. Add 500 µL RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 20 seconds. Discard the collection with the flow-through and transfer the column to a new collection tube.
7. Centrifuge the empty column at 14,000 rpm (maximum speed) for 2 min. It is critical to remove residual ethanol for optimal elution.
8. Transfer the RNA column to an RNase-free 5 mL tube, add 35-50 µL DEPC-treated water to the column, and centrifuge at 10,000 rpm for 30 seconds. The viral RNA is in the flow-through liquid.
9. Optional: Add the eluent back to the column for a second elution.

Note:

• The first elution normally yields 70% of the RNA while the second elution yields another 20-30% of the RNA bound to the column.
• The purified RNA should be put on ice for downstream application or store at -20°C.