Comparison of performance of multiplex and single RT-qPCR. CoviDetect™ is designed for use on open Real-Time PCR platforms by detection on FAM, HEX/VIC and Cy5 channels. For each sample, genotyping results were coded and identified based on exponential amplification curves in the CY5, ROX, FAM, and HEX channels in the above layout of four reaction tubes. A sample was considered negative if the … Samples were considered positive when a signal was detected at C t <40 for any gene. Figure 11.6A shows the raw data plot for two assays containing either a 6-FAM™ or a HEX™ (VIC ®) labeled probe. )|GAPDH, 18S, HSP, and H28S sequences were amplified either together in 4-plex PCR or in singleplex PCRs on the Rotor-Gene 3000 system using the QuantiTect Multiplex PCR NoROX Kit. The curves with the FAM probe are fine. C t from FAM (E gene), Cal Red 610 (RDRP gene), Quasar 670 (N gene) and HEX (internal control) were acquired. Although both assays show amplification, the HEX signal is approximately half of that of the FAM signal. A one-size fits all approach to designing qPCR and genotyping assays does not account for different experimental goals and probe sequence requirements such as percent GC content, secondary structure avoidance, target Tm values and more. (Data kindly provided by the University of Minnesota, Minneapolis, MN, USA. Review these recommendations for selecting dyes for multiplex qPCR that minimize background and Multiplex CE IVD kit for detection of SARS-CoV-2 virus is based on one-step RT-qPCR using fluorescently labelled probes (FAM and HEX channels) and exogenous positive control (Cy5 channel). If you are interested in our method, please contact us at info@pentabase.com It is frequently used when dealing with precious samples (e.g., clinical samples), or to enhance throughput (with fewer microtiter wells per sample there are more samples per plate). For example, FAM is a good dye choice to detect low copy transcripts because it has high uorescent signal intensity. Since this is an inherently weaker dye, this is a normal observation. The test has been developed to be used as a single one-step reverse transcription qPCR test for each patient sample, utilizing PCR amplification of the targets (N1, N2 and RNaseP) by FAM, HEX and Cy5 labelled probes. TaqMan probes labeled with FAM, HEX, or Cyanine 670 dye were used. TaqMan® probe is available with FAM, HEX, Yakima Yellow® and Cy5 according to customer requirements This accessory kit can be used with vDetect, rTEST and Multiplex rTEST Kit contains reagents for 400 rxns To confirm the sensitivity of the primer–probe sets (FAM, HEX, and Cy5 fluorophores) tested as single or multiplex reactions, as well as in comparison to the original single assay (FAM), we used nasopharyngeal swab and saliva samples from COVID-19 patients to detect SARS-CoV-2 RNA. Choosing a Probe. It is validated on BaseTyper™ (our PCR instrument), LightCycler 480 II, ABI 7500™ and ViiA7™.. I was thinking of changing the HEX probe to a Cy-5 probe. In multiplex with a FAM probe, I have been having a hard time getting consistent standard curves with the HEX probe. The assay was run as the AmpFire Multiplex HPV assay as described above. The reaction time is approximately 1 hour, it … The detection method is based on officially recommended WHO and Institut Pasteur designs for detection of SARS-CoV-2 and usage of our proprietary GEMINI™ probe technology. Multiplex PCR is economical—with fewer reactions, there is less reagent consumption. You can then use uorophores with lower signal intensities for more abundant transcripts (e.g., housekeeping genes). On BaseTyper™ ( our PCR instrument ), LightCycler 480 II, ABI and., FAM is a normal observation changing the HEX probe to a probe... 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