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

Plasmids containing the full-length ZIKV (Cambodia FSS13025 strain; pFLZIKV) and DENV-2 (strain 16681, pD2/IC-30P) cDNAs were gifts from Dr. Pei-Yong Shi (University of Texas Medical Branch) and Dr. Claire Huang (Centers for Disease Control), respectively. 30,63 pFLZIKV was linearized with ClaI (New England Biolabs, NEB), and pD2/IC-30P was linearized with XbaI (NEB). Digested plasmids were extracted with phenol:chloroform:isoamyl alcohol and then precipitated. Linearized plasmids were in vitro transcribed (Thermo Fisher Scientific) and the resulting viral RNA cleaned by MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific). We followed the protocols of these two kits except that we did not heat the purification column in the elution step of the viral RNA because we noticed that high temperature can result in degradation of the viral RNA. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.01.12.902452 doi: bioRxiv preprint Viral RNA fragmentation test Viral RNA was fragmented by using 10×Fragmentation buffer (NEB) and the recommended protocol. Briefly, the ZIKV RNA obtained from in vitro transcription was mixed with fragmentation buffer (1× final) and then incubated at 94°C in a thermal cycler for 1, 3, 6, or 9 min. Then RNA fragmentation analyzer (Agilent, model 5003) was used to quantify the length distribution of RNA fragments by using the DNF-471 Standard Sensitivity RNA Analysis Kit (Fig. 2C) .