Journal: Microbial Biotechnology
Article Title: Sequential development of several RT‐qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS‐CoV‐2 from influenza A and B
Figure Lengend Snippet: Optimization, analytical sensitivity and clinical performance of rTEST COVID‐19/FLU qPCR kit. A. Heatmaps illustrate combinatorial testing of two IAV primer/probe sets at either high or low viral input (10 000 versus 10 copies per reaction). B. Heatmaps show combinatorial testing of IBV primer/probe sets at low viral input (10 copies per reaction). In panels A, B, the best performing primer/probe combinations (highlighted by green rectangles) were selected based on C t (darker colours denote higher sensitivity), fluorescent intensity (∆ R , lighter colours correspond to higher intensity) and the number of replicates that amplified. C. Analytical sensitivity of the multiplexed SARS‐CoV‐2 E and RdRP (both labelled with FAM), IAV PB1 and IBV PA (both labelled with YY), and RNase P assay. The dotted line at C t = 40 serves as a threshold after which amplification is considered invalid. D. Assessment of competitive interference of 1000 copies of IAV per reaction (500× LoD) on the analytical sensitivity of the SARS‐CoV‐2 E and RdRP assays multiplexed together. E. Assessment of competitive interference of 1000 copies of SARS‐CoV‐2 per reaction (500× LoD) on the analytical sensitivity of the IAV PB1 assay. F. Clinical performance of the rTEST COVID‐19/FLU qPCR kit. The dotted line and shaded area indicate samples that were not detected by a particular assay. C t , cycle threshold; E, envelope gene; IAV, influenza A; IBV, influenza B; PA, polymerase acidic protein; PB1, polymerase basic 1 protein; ND, not detected within 45 cycles; NTC, no template control; RdRP, RNA‐dependent RNA polymerase; Δ R , normalized fluorescent intensity.
Article Snippet: KBur., AS and NV optimized reaction conditions using gel electrophoresis for vDetec v2 (Agilient Brilliant III master mix) and rTEST (Solis Biodyne SOLIScript® 1‐step CoV Kit).
Techniques: Real-time Polymerase Chain Reaction, Amplification