Design of qPCR Assays Including Standards and Controls for Pathogen Testing in Wastewater

Design of qPCR Assays Including Standards and Controls for Pathogen Testing in Wastewater

Abstract
Over the course of the COVID-19 pandemic, wastewater surveillance has emerged as a promising tool to monitor spread of the virus in a community. Wastewater is a complex matrix, so analytical methods like RT-qPCR needs to be designed with appropriate controls to quantitate and normalize viral levels for trend analysis. In this work we describe methods for designing and manufacture of quantification standards for SARS-CoV-2 and Pepper Mild Mottle Virus (PMMoV) RNA. PMMoV is a plant virus with a ssRNA genome and is a well-documented human fecal indicator. PMMoV RNA also serves as a useful process control and normalization tool for wastewater surveillance.
To generate quantitative standards, we first cloned the Envelope and Nucleocapsid (E/N) genes of SARS-CoV2 in pGEM3z vector. Likewise, a 362bp fragment of the PMMoV genome consisting the RT-qPCR target is also cloned in a pGEM3z vector. The two plasmids are then linearized by XbaI and in-vitro transcribed to generate RNA transcripts. Any remaining DNA in the reaction is eliminated by DNase treatment. RNA was then quantified with a fluorescent RNA dye (QuantiFluor RNA System) and by droplet digital PCR (ddPCR). The relationship of quantity of RNA (in pg) corresponding to copy number as determined by ddPCR is established. The RNA was then diluted at 4 million copies/ul corresponding to 20pg/ul and 7.5pg/ul for the SARS-CoV-2 (E/N) and PMMoV RNA respectively. Both the RNA was then tested with the primes/probe set prescribed by the US Centers for Disease Control (CDC) that target the nucleocapsid (N1 and N2) gene, or the envelope (E) gene of the SARS-CoV2 RNA and primers/probes for PMMoV for the PMMoV RNA. The quantitation standards were stored at -20°C and subjected to ten freeze-thaw cycles. No decrease in Ct values was observed implying that the quantification standards were stable. The standards were used to quantify SARS-CoV-2 and PMMoV RNA from three wastewater treatment plants in Dane county, WI. Normalization of SARS-CoV-2 RNA with PMMoV RNA resulted in better correlation with 7-moving average of new clinical cases a municipality served by a treatment plant