Center for Disease Control and Prevention, USA
Title: Analytical validation of three alternative PCR master mixes for Bordetella species real-time PCR detection
Biography: Brunilís Burgos-Rivera
The Pertussis and Diphtheria Laboratory at the Centers for Disease Control and Prevention (CDC) developed a real-time PCR (rtPCR) assay for the detection of three Bordetella species. This assay is routinely used at the CDC, public health laboratories in the USA and internationally for pertussis diagnostics. It relies on one approved and validated PCR master mix, Applied Biosystems Taqman Gene Expression Master Mix (GE). In recent years PCR master mixes have been engineered to be resistant to PCR inhibitors, thus potentially providing more sensitive alternative master mixes to be used in this assay. Three commercially available alternative master mixes were identified: Quanta PerfeCTa SuperMix (QS), Quanta PerfeCTa ToughMix (QT) and Quanta PerfeCTa ToughMix with UNG (QTU). The performance of these master mixes was evaluated by comparing target-specific detection levels and consistency among three rtPCR instruments previously validated and currently used for routine pertussis diagnosis. The analytical sensitivity and specificity of these alternative PCR master mixes was assessed by the Bordetella species rtPCR multi-target assay. We report that QS produced Ct values comparable to GE, while QT and QTU produced lower Ct values (≥2 Ct value difference) and thus exhibited higher analytic sensitivity across all assays and instruments. Therefore, before selecting an alternative master mix or rtPCR platform, laboratories should validate their own diagnostic assay to ensure it is producing results with clinically relevant cut-offs, as those previously published.