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1) Product Images from "Sequential development of several RT‐qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS‐CoV‐2 from influenza A and B"
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
Journal: Microbial Biotechnology
Figure Legend 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.
Techniques Used: Real-time Polymerase Chain Reaction, Amplification
Figure Legend Snippet: Schematic illustrating SARS‐CoV‐2 genome and regions targeted by RT‐qPCR primers and probes. A. Schematic overview portrays the SARS‐CoV‐2 genome with RdRP and E gene regions magnified to show the locations of primers and probes of the original Charité protocol, vDetect (v1 and v2), and rTEST RT‐qPCR assays. F, forward primer; P, probe; R, reverse primer. The inset boxes (from left to right) illustrate a SARS‐CoV‐2 particle with labelled structural proteins and RNA, legend describing panel A and the primers and probes used in each test to detect RNase P subunit p30 (RPP30). B. Diagram compares the sequences of RdRP and E gene primers and probes for the original Charité protocol, vDetect (v.1) and vDetect v.2 and rTEST RT‐qPCR assays to the Wuhan reference sequence. The numbers written above the Wuhan reference sequence correspond to the start and end base positions of the sequence Reverse primer sequences are written in the reverse complement (rc). Magenta lines and letters represent mixed bases found in the primers and probes in the Charité protocol that were replaced with the correct bases in vDetect v1 (blue lines and letters). Red lines and letters signify LNA‐modified bases.
Techniques Used: Quantitative RT-PCR, Sequencing, Modification
Figure Legend Snippet: Optimization of room temperature stable kit and dual probes for rTEST COVID‐19 qPCR kit. (A, B) Graphs depict the effects of decoy nucleic acids (tRNA or salmon sperm DNA) or pure oligonucleotides (pure) on the stability of lyophilized positive control left at room temperature over a one‐month period as assessed by amplification of RdRP (A) and E (B) genes. (C) Performance of either fresh or an rTEST COVID‐19 qPCR kit left a t room temperature for 1‐month on amplification of SARS‐CoV‐2 E and RdRP genes and human RNase P. Comparison of analytical sensitivity (D, F) and fluorescent intensity (E, G) between single probes versus dual probes for both RdRP (D, E) and E (F, G) genes. Dotted line at C t = 40 serves as a threshold after which amplification is considered invalid. * P
Techniques Used: Real-time Polymerase Chain Reaction, Positive Control, Amplification