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.

After thawing at room temperature, nucleic acids were purified from 200 μL each of the previously frozen samples using the Specific A protocol of the bioMérieux NUCLISENS easy-MAG nucleic acid extractor and stored at -20˚C. An internal control, intype IC-RNA (QIA-GEN, Inc), was added to and extracted with each sample and read using the VIC channel during dPCR. dPCR was conducted using the RainDrop Digital PCR system. ViroReal assay kits (Ingenetix) were used for influenza A and B assays, whereas RSV assay primer/probes were adapted from a previously designed, laboratory-developed qRT-PCR assay [39] . An assay volume of 25 μL was used for all virus samples. Standard qPCR protocols from Ingenetix and QuantaBio were used for the master mix and cycling conditions. After cycling, reactions were transferred to the RainDrop reader and data were analyzed using RainDrop Analyst II software (v1.0.0.520). Results of copies per reaction were converted to log 10 copies per milliliter of the original patient sample pre-extraction.