Tolerance of Droplet-Digital PCR vs Real-Time Quantitative PCR to Inhibitory Substances

摘要

To the Editor:Real-time quantitative PCR (qPCR)1 is a rapid and sensitive method that forms the foundation for many clinical diagnostic tests. Droplet digital PCR (ddPCR) shares these qualities with qPCR, but owing to reaction partitioning, ddPCR is proposed to exhibit increased tolerance to interfering substances, making it an attractive alternative to qPCR for diagnostic applications (1, 2). The data to support this phenomenon and its mechanism, however, are currently lacking in the literature (3).Herein, we describe a series of experiments to compare the inhibition tolerance of laboratory-developed CMV qPCR and ddPCR (Bio-Rad Laboratories, QX-100) assays by introducing a panel of clinically relevant inhibitors (SDS, EDTA, and heparin) directly into the PCR reactions (4). Differences in the resulting inhibition curves and the half-maximal inhibitory concentrations (IC50) were then assessed. The laboratory-developed CMV qPCR is a double primer/probe Taqman assay that amplifies and detects the genes UL123 (IE)2 (enhances activation by IE2; interacts with basal transcriptional machinery and cellular transcription factor; disrupts ND10; involved in gene regulation [Human herpesvirus 5]) and UL55 (gB) (type 1 membrane protein; possible membrane fusogen; binds cell surface heparan sulphate; involved in cell entry; involved in cell-to-cell spread [Human herpesvirus 5]) with primers and probes previously described (5). The ddPCR assay uses the same primers and probes, with the dyes HEX replacing 6-FAM on the gB probe and BHQ-1 replacing TAMRA on both probes (Sigma-Aldrich).SDS, EDTA, and …