Oct 14, 2024
Overview of MultiPathogen Detection Workflow
- 1Imperial College London;
- 2Kwame Nkrumah University of Science and Technology;
- 3CMC Vellore
- GEMS - Genomic Environmental Microbial Surveillance
Protocol Citation: Shannon Fitz, Alex Shaw, michael Owusu, Dilip Abraham 2024. Overview of MultiPathogen Detection Workflow. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32jq7v25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 03, 2024
Last Modified: October 14, 2024
Protocol Integer ID: 109068
Keywords: multipathogen detection, hepatitis E, vibrio cholera, salmonella typhi, enterovirus
Funders Acknowledgement:
The Gates Foundation
Grant ID: INV-049092 PA4273
Abstract
This protocol offers a comprehensive overview of the sequential steps involved in the GEMS multipathogen detection workflow, spanning from sample concentration to sequencing. This multipathogen detection workflow targets detection of Hepatitis E, enteroviruses, Vibrio cholera, and Salmonella typhi from wastewater samples.
Concentration
Concentration
Invert collected samples several times prior to decanting to ensure even mixing.
Separate sample into appropriate volume per concentration method (PEG = 40 ml, Ceres = 35 ml), and follow the selected protocol:
Extraction
Extraction
Following concentration with the chosen method, carry out extraction using MagMAX Wastewater Ultra Nucleic Acid Isolation Kit (protocol included in the above listed concentration protocols)
PCR purification
PCR purification
After extraction, carry out the Zymo OneStep PCR Inhibitor Removal protocol.
PCR
PCR
Three distinct PCRs will be conducted, so samples will be tested separately at this next stage. Depending on timeline for testing, it may be beneficial to separate samples into different aliquots at the step prior to avoid multiple freeze-thaws.
Run EV/HEV, typhi, and cholera assays.
Sequencing
Sequencing
Lastly, amplicon sequencing for all samples will be carried out using the Oxford Nanopore Rapid Barcoding Kit.