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

Human hepatocarcinoma (Huh7) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS; VWR Life Science Seradigm), 10 mM non-essential amino acids (NEAA; Life Technologies), and 5 mM L-glutamine (Life Technologies). Cells were passaged once every three days and maintained at 37°C with 5% CO 2 . Twentyfour hours prior to infection Huh7 cells were seeded into tissue culture plates. The following day, one plate was counted. The other two plates were used for mock-and ZIKV-infection, where cells were infected at a multiplicity of infection of one. The original Cambodia and Uganda (MR766) ZIKV stocks were a generous gift from Dr. Brett Lindenbach (Yale School of Medicine). RNA from mock-and ZIKV-infected cells was harvested by removing media from the cells was removed and then washing the cell monolayer washed once with ice-cold PBS. Hereafter, each tissue culture plate was lysed in 1 mL TRIzol (Invitrogen) and total RNA extracted per the manufacturer's instructions. For experiments using ZIKV infectious particles in PBS/urine, cell culture media from mock-and ZIKV-infected cells was collected and stored at -80 °C. The number of infectious particles was determined by plaques assay, as described previously. 38