RT-qPCR Detection of SARS-CoV-2 from Wastewater Using the AB 7500 v1
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This protocol describes triplex and duplex assays for the RT-qPCR detection of the nucleocapsid region of the SARS-CoV-2 genome. These assays, along with the murine norovirus (MNV; extraction control) and crAssphage (human indicator) RT-qPCR assay (RT-qPCR Detection of Process Controls (Murine noroviurs and crAssphage) from Wastewater (protocols.io)), were developed for use on the AB 7500 platform using software version 2.0 or 2.3. All assays incorporate an internal amplification control (IC) to prevent the reporting of false negatives due to inhibition or failure of the RT-qPCR. These multiplexed detection assays were developed for the qualitative determination SARS-CoV-2 nucleocapsid gene extracted from wastewater. Valid sample results are contingent upon the detection of the MNV extraction control from the sample being tested.