Beyond SARS-CoV-2, application of Absolute Q digital PCR system for waterborne pathogens

Waterborne pathogens lead to deteriorating water quality and challenge global health and economy. The latest U.S. Environmental Protection Agency (EPA) Contaminant Candidate List (CCL4) contained 12 microbial contaminants, such as human adenoviruses, enteroviruses, noroviruses, Salmonella enterica, and Campylobacter jejuni, which are linked to enteric disease outbreaks. One of the challenges to detect enteric pathogens in environmental water samples is their low ambient concentrations, thus direct monitoring may not be possible. Various concentration methods are needed, and PCR inhibitors might be accumulated during sample concentration processes. The Absolute Q digital PCR system facilitates precisive quantification of waterborne pathogen nucleic acid targets using microfluidic array. Comparable to qPCR system, the Absolute Q dPCR Master Mix can be prepared within 20 minutes per plate, and the running time is around 90 minutes. The PCR reaction is divided into over 20,000 micro-chambers and micro-reactions, which enabled the absolute quantification of the waterborne pathogen without the development of standard curves. The limit of detection is as low as 0.34 gc/reaction and 1554 gc/L in environmental water and can be more resistant to PCR inhibitor compared to qPCR. Besides the use of digital PCR for the quantification of SARS-CoV-2 in wastewater, we have developed a rapid turnaround method and customized the primers and probe mix to quantify bacterial and viral pathogens on the CCL4 list using the Absolute Q digital PCR system. The digital PCR will facilitate the waterborne pathogen detection and quantification with improved accuracy, precision, and reproducibility.

 

Highlights:

 

1. Application of digital PCR in detecting waterborne pathogens (EPA CCL4 list) from environmental samples and highly sensitive absolute quantification without the need of standard curves

2. Rapid quantification of pathogen in response to public health events

3. PCR primers and probe assays are customized and prepared in Tulane and are validated. The primers and probe are not from ThermoFisher, which indicate the Absolute Q is compatible with reagents from other vendors.