Clinical correlation of influenza and respiratory syncytial virus load measured by digital PCR

Hijano, Diego R.; Brazelton de Cardenas, Jessica; Maron, Gabriela; Garner, Cherilyn D.; Ferrolino, Jose A.; Dallas, Ronald H.; Gu, Zhengming; Hayden, Randall T.

Remnant samples from nasopharyngeal washes obtained from 2014 to 2017 for routine care were used for this study. Washes were performed using normal saline and immediately transported to the Clinical Virology Laboratory on ice and stored at 4ËšC until testing (within 24 hours of collection). Sample remaining after clinical testing was stored for quality assurances at -80ËšC until used in this evaluation. Digital PCR (dPCR) assays for influenza were performed using the RainDrop digital PCR system (RainDance Technologies) and ViroReal assay kits (Ingenetix). Viral RNA stock generated by in vitro synthesis and purchased from Ingenetix was used for analytical validation of the assay, along with viral stock material generated by ATCC (quantified by ATCC using dPCR, plaque-forming units, and 50% tissue culture infective dose) and Zeptometrix. The assay for RSV was also designed for the RainDrop digital PCR system and primer/probes previously designed for qPCR [39] . Synthetic viral RNA stock for analytical validation of the RSV assay was purchased from Bio-Synthesis, Inc., along with additional viral stock material from ATCC and Zeptometrix.