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.

Validations for influenza and RSV included multiple lot numbers of all reagents and spanned several months allowing between run variability to be assessed. All samples had Qiagen IPC added prior to extraction to control for inhibition and ranged from 5,000-15,000 copies/mL. Cross reactivity for all three assays was tested using the Zeptometrix respiratory panel. Validation also included determination of limit of detection, positive cut-off value, linearity, within and between run variability, and limit of quantitation. The limit of blank (LOB) was defined by the 95% cutoff for all no template controls and was considered a baseline value to eliminate false positive results due background noise. LOD 95 was initially calculated by determining the lowest non-zero concentration for which 95% of the replicates gave a positive result. The LOQ was based on the percent CV for linearity runs (lowest level with a CV <10%). As calculated LOD 95 and LOQ were both lower than the LOB. Clinical samples used in the validation were run blinded.