RT-qPCR Detection of Process Controls (Murine noroviurs and crAssphage) from Wastewater using 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 the murine norovirus (MNV; extraction control) and crAssphage (human indicator) RT-qPCR assay developed for use on the AB 7500 platform using software version 2.0 or 2.3. The assay incorporates an internal amplification control (IC) to prevent the reporting of false negatives due to inhibition or failure of the RT-qPCR. This multiplexed detection assay was developed for the determination crAssphage extracted from wastewater, as an endogenous control, and MNV as an extraction control. The assay is designed to be used in conjunction with the SARS-CoV-2 RT-qPCR detection assay. Valid sample results for SARS-CoV-2 detection are contingent upon the detection of the MNV extraction control from the sample being tested.