Version 1
: Received: 17 June 2024 / Approved: 18 June 2024 / Online: 18 June 2024 (11:03:54 CEST)
How to cite:
Tere-Peña, C. P.; Calderon-Ozuna, M. N.; Leguizamón, J. E. Digital PCR Validation for Characterization of Quantitative Reference Material of Escherichia coli O157:H7 Genomic DNA. Preprints2024, 2024061226. https://doi.org/10.20944/preprints202406.1226.v1
Tere-Peña, C. P.; Calderon-Ozuna, M. N.; Leguizamón, J. E. Digital PCR Validation for Characterization of Quantitative Reference Material of Escherichia coli O157:H7 Genomic DNA. Preprints 2024, 2024061226. https://doi.org/10.20944/preprints202406.1226.v1
Tere-Peña, C. P.; Calderon-Ozuna, M. N.; Leguizamón, J. E. Digital PCR Validation for Characterization of Quantitative Reference Material of Escherichia coli O157:H7 Genomic DNA. Preprints2024, 2024061226. https://doi.org/10.20944/preprints202406.1226.v1
APA Style
Tere-Peña, C. P., Calderon-Ozuna, M. N., & Leguizamón, J. E. (2024). Digital PCR Validation for Characterization of Quantitative Reference Material of Escherichia coli O157:H7 Genomic DNA. Preprints. https://doi.org/10.20944/preprints202406.1226.v1
Chicago/Turabian Style
Tere-Peña, C. P., Martha Nancy Calderon-Ozuna and John Emerson Leguizamón. 2024 "Digital PCR Validation for Characterization of Quantitative Reference Material of Escherichia coli O157:H7 Genomic DNA" Preprints. https://doi.org/10.20944/preprints202406.1226.v1
Abstract
Escherichia coli O157:H7, a Shiga toxin-producing E. coli (STEC) is an important pathogen related to foodborne disease that is responsible for a growing number of outbreaks worldwide and has been detected in processed meats, dairy, and fresh vegetables. Although culturing is the gold standard method for detection of this bacteria, molecular methods based on nucleic acid amplification techniques such as PCR are becoming more common because of their rapidity, sensitivity, and specificity. However, to ensure reliable results among the several alternative PCR protocols (e.g., commercial kits and reference methods), different measurement assurance tools, including validated methods, reference materials, and proficiency tests among others, are required. Herein, we present a digital PCR method validation for E. coli O157:H7 detection and quantification using seven gene sequences; this method quantified nucleic acids from different E. coli serotypes, with a detection range between 6.6 to 7,900 copies/µL and a repeatability standard deviation over the concentration range of between 1% to 13.6%. The relative standard uncertainty was 3.5%–14.6%, and the detection limit was 0.27 copies/µL. Subsequently two batches of a candidate reference material based on E. coli O157:H7 genomic DNA were then produced and characterized for evaluation of copy number concentration with the validated dPCR method, with assigned values of 164,770 ± 9,251 and 172 ± 9 copies/μL. Thus, this study demonstrated the development a validated method and reference material for dPCR and qPCR detection of E. coli O157:H7, a key STEC responsible for food poisoning.
Keywords
E. coli O157:H7; Reference material; Method validation; digital PCR
Subject
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
