Applicable Quality Control Components for Molecular Analysis

SARS CoV-2 Wastewater Testing - State of the Science
Oral Presentation

Prepared by D. Alvi1, S. Frantz2, C. Eskridge2, A. Godrej2
1 - Occoquan Lab/Virginia Tech, 9408 Prince William St, Manassas, Virginia, 20110, United States
2 - Occoquan Lab, 9408 Prince William St, Manassas, VIRGINIA, 20110, United States

Contact Information:; 572-265-7287


There has been growing demand of molecular analysis in water environment monitoring, such as detecting virus, bacteria, opportunistic pathogens, environmental DNA (eDNA), and most recently wastewater-based epidemiology (WBE) for SARS-CoV-2 surveillance. Although standard method and the NELAC institute (TNI) have well defined quality control procedures for most water and wastewater analysis, quality control (QC) elements for molecular testing basically presented in literature and journal articles until EPA’s Method 1696/1697 published March 2019. There is however still lack of standard protocols to ensure data quality and absence of bias for experiment and molecular analysis using advanced technology digital polymerase chain reaction (dPCR). In this study, an 18 months water quality monitoring was conducted at Potomac River at border of Washington DC and state of Virginia. We aim to provide users with quality control components of microbial source tracking (MST) data based on guidelines from EPA, standard method, and TNI.

The District's combined sewer system was built before 1900. During wet weather conditions, the system capacity may be exceeded. Consequently the combined sewer overflow (CSO) is discharged directly to the Anacostia River, Rock Creek, Potomac River or tributary waters. The monitoring effort is target at one of the major concern of elevated bacteria level in receiving waters by untreated CSO.

Culture-based E. coli by IDEXX colilert, and molecular-based bacteroides markers of human- (BacH) and ruminants- (BacR) using chip-based dPCR (cdPCR) were conducted for bi-weekly base flow as well storm events. Efficiency of DNA isolation was evaluated using sample processing control (SPC) Sketa22 adopted from EPA 1696/1697. Reference DNA was applied to monitor amplification proficiency. System performance measurements included extraneous DNA contamination assessments using no template control (NTC) and extraction blanks. Matrix inhibition was monitored through series dilutions, duplicate, and spikes. Single digit gene copy per ship-reading was achieved and applied for level of detection (LOD) determination.

Performance criteria such as high sensitivity, consistent resistance to matrix inhibition was observed in cdPCR system for BacH and BacR assay. Human related fecal bacteria contamination was detected in almost all wet weather samplings, whilst ruminant sources were prominent throughout base flow collections. Data of molecular-based assay and cultural-based E. coli generally follow a similar trend. Study output demonstrated that cdPCR could be a viable tool for MST monitoring projects, not only increasing ability to differentiate fecal contamination sources, also detect low levels of genetic makers.