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.

Digital PCR (dPCR) is a PCR method for the detection and quantitation of nucleic acids [14, 18, 24] . dPCR offers several advantages over real time methods [21, 25] . Digital PCR differs from real-time methods in the way the sample target is measured, by dividing up the reaction into multiple, smaller endpoint reactions which allows direct absolute quantification of the target [17, 26, 27] . dPCR methods do not depend on calibration curves [19, 25, 28] and are less subject to enzyme inhibition as they employ endpoint amplification [26, 29, 30] . They are more robust in the presence of viral sequence heterogeneity, showing less susceptibility to mismatches between primers-probe patient viral sequence. Such miss-matches are more likely to cause false-negative results or under-quantitation using qPRC, compared to dPCR [11, 31, 32] . This is particularly important for RNA viruses which have high mutation rates and need only a few weeks to escape immune response or to produce drug-resistant mutants [31, [33] [34] [35] [36] . Finally, clinical results generated by dPCR are more precise, reliable and reproducible than those by real-time PCR, thereby highlighting its potential diagnostic, prognostic, and predictive utility. Although dPCR has been previously used for quantitative detection of systemic viruses [17, 37, 38] , its use has been more limited for localized viral infections such as those in the respiratory tract.