Feb 09, 2026
Comparative evaluation of boil-based DNA extraction methods paired with multiplex qPCR for detection of Vibrio cholerae across multiple sample types.
This protocol is a draft, published without a DOI.
- KT Ayazika1,
- ET Baumgartner1,
- A Safir1,
- MO Islam2,
- KN Murt1,
- W Luo1,
- DA Sack1,
- J Liu3,
- M Taniuchi4,
- AK Debes1
- 1Department of International Health, Johns Hopkins University Bloomberg School of Public Health, Baltimore, United States;
- 2Department of Civil and Environmental Engineering, University of Virginia, Charlottesville, United States;
- 3School of Public Health, Qingdao University, Qingdao, Shandong, China;
- 4Division of Infectious Diseases & International Health, University of Virginia, Charlottesville, United States
Protocol Citation: KT Ayazika, ET Baumgartner, A Safir, MO Islam, KN Murt, W Luo, DA Sack, J Liu, M Taniuchi, AK Debes 2026. Comparative evaluation of boil-based DNA extraction methods paired with multiplex qPCR for detection of Vibrio cholerae across multiple sample types.. protocols.io https://protocols.io/view/comparative-evaluation-of-boil-based-dna-extractio-hqfzb5tp7
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: January 30, 2026
Last Modified: February 16, 2026
Protocol Integer ID: 241881
Keywords: vibrio cholerae across multiple sample type, vibrio cholerae across multiple enteric specimen type, cholera surveillance, vibrio cholerae, cholera treatment center, assessing dna extraction approach, based molecular diagnostic, multiplex qpcr for detection, dna extraction approach, molecular diagnostic, detection in enteric disease surveillance, dna extraction method, use of qpcr, based dna extraction method, multiplex quantitative polymerase chain reaction, multiplex qpcr, multiple enteric specimen type, comparative laboratory evaluation, enteric disease surveillance, qpcr, single patient specimen type, standardization of molecular workflow, evaluating alternative specimen type, selection of appropriate specimen type, laboratory setting, logistical workflow of specimen collection, alternative specimen types in combination, laboratory
Funders Acknowledgements:
Bill & Melinda Gates Foundation
Grant ID: INV-047157
National Institute of Allergy and Infectious Disease
Grant ID: R01 AI181283
Abstract
This protocol describes laboratory-based procedures for the comparative evaluation of boil-based DNA extraction methods paired with multiplex quantitative polymerase chain reaction (qPCR) assays for the detection of Vibrio cholerae across multiple enteric specimen types. The protocol was designed to support optimization and standardization of molecular workflows for cholera surveillance by systematically assessing DNA extraction approaches, specimen matrices, and qPCR assay performance under controlled conditions.
All parameters were optimized in a laboratory setting using independently prepared, spiked stool and alternative specimen types. These preparations do not reflect the chronological or logistical workflow of specimen collection and processing in a cholera treatment center. In field applications, a single patient specimen type would be collected, preserved according to standard practice, and extracted using the corresponding protocol described for that specimen. This comparative laboratory evaluation was conducted to inform selection of appropriate specimen types, DNA extraction methods, and qPCR assays for setting-specific surveillance needs.
The protocol was developed in response to the recent expanded use of qPCR-based molecular diagnostics in low-resource settings. By evaluating alternative specimen types in combination with simplified extraction methods and multiplex qPCR assays, this protocol aims to support informed methodological choices that maximize sensitivity and specificity for V. cholerae detection in enteric disease surveillance.
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