RE: Cambridge lighthouse study - how they did it18 Jul 2021 11:02
Tests completed using genesig HT kit Larry ;)
Direct to PCR (D2PCR) validationInitial experiments were performed using Oropharyngeal/Nasopharyngeal (OP/NP) swab samples withknown SARS-CoV-2 status (i.e., samples tested using the approved CCTC standard laboratory process) todetermine suitable conditions for Heat Inactivation of potential viable SARS-Cov-2 and RT-qPCR set-up.Heat Inactivation can be performed at the stage of sample receipt, or by another suitable method thatachieves the required heat exposure for viral inactivation prior to PCR set-up outside of biologicalcontainment. For example, this can be performed using a calibrated and heat mapped 96-well microplateand thermal cycler combination, with a suitable thermal resistant plate seal.?In the assay described herein, 100 µl of each OP/NP swab sample was transferred to a Hard-Shell Low-Pro?le Skirted 96-Well PCR Plate (Biorad #HSP9601), sealed with an Aluminium Foil Seal (BeckmanCoulter #538619) and heated in a PCR Max AlphaCycler, set to 65 °C for 20 minutes.In the absence of an RNA extraction step, the Internal Extraction Control (IEC)?– a synthetic RNA controlprovided with the PCR kit -?was included to con?rm successful RT-PCR in every sample (highlighting anyunexpected sample-mediated reaction inhibition). RT-PCR reactions were prepared in White, 384-wellLightCycler 480 Multiwell Plates (Roche #04729749001) by sequential addition of 3.5 µl of Genesig®Real Time PCR COVID-19 High Throughput HT-CE kit V2.0 (Genesig #Z-Path-COVID-19-CE) PCR mastermix using a ThermoFisher Multidrop Combi, followed by 2.5 µl of a 50-fold dilution of IEC in nuclease-freewater using an Agilent Bravo (omitting positive and negative control wells where IEC was not included).IEC was added to PCR master mix in 384-well microplates in this way to avoid weak or variable IECsignals that are seen when IEC is added to samples themselves, likely a result of RNA degradation.
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