Programmable low-cost DNA-based platform for viral RNA detection

Lifeng Zhou; Arun Richard Chandrasekaran; Jibin Abraham Punnoose; Gaston Bonenfant; Stephon Charles; Oksana Levchenko; Pheonah Badu; Cassandra Cavaliere; Cara T. Pager; Ken Halvorsen

For the ZIKV related NASBA experiments, first we spiked the ZIKV infectious particles (started at 1.96 fM of particles) into PBS or human urine. Then samples were diluted into different concentrations 1.49, 0.33, and 0.03 fM with 1xPBS or 10% human urine. Subsequently, blocking oligo (200 nM) was added and the viral RNA was released by heating the samples at 94 °C for 3 minutes. For the human urine samples, RNase Inhibitor (NEB, Murine) was also added at concentration of 2 U/µl before heating. After cooling down at room temperature, 0.5 µl RNase Inhibitor at 40 U/ul (NEB, Murine) was added to 10 µl human urine sample to protect the viral RNA. Total volume of each NASBA reaction was scaled down to 6 µl which contains 1.25 µl Enzyme COCKTAIL (NEC 1-24), 2 µl 3× buffer (NECB-24), 0.48 µl NTPs mix at 25 mM, 0.3 μl dNTPs mix at 20 mM, 0.2 µl two primers mix at 10 uM, 0.2 μl RNase Inhibitor with 40 U/ul (NEB, Murine) and 1.57 µl sample with viral RNA. The sample was incubated at 41 °C for 2 hours in the thermal cycler and followed by heating at 94 °C for 10 minutes to deactivate all enzymes. Then 1 µl of the NASBA sample was used in the following DNA nanoswitch detection assay in PCR tubes with 10 µl final volumes. The assay was finished by incubating at room temperature for two hours. After mixing with GelRed (Biotium Inc.) at 1×concentration and 2 µl 6× blue loading dye, the detection samples were loaded to the 25 ml 0.8% agarose gel which was run in 0.5×Tris-Borate-EDTA (TBE) buffer at 75 V at room temperature for 45 minutes.