Digital PCR Validation for Characterization of Quantitative Reference Material of <i>Escherichia coli</i> O157<i>:H7</i> Genomic DNA
<i>Escherichia coli O157:H7</i>, a Shiga-toxin-producing <i>E. coli</i> (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. Althoug...
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2024-11-01
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| author | Claudia Patricia Tere-Peña Martha Nancy Calderon-Ozuna John Emerson Leguizamón Guerrero |
| author_facet | Claudia Patricia Tere-Peña Martha Nancy Calderon-Ozuna John Emerson Leguizamón Guerrero |
| author_sort | Claudia Patricia Tere-Peña |
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| description | <i>Escherichia coli O157:H7</i>, a Shiga-toxin-producing <i>E. coli</i> (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 bacterium, 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 <i>E. coli O157:H7</i> detection and quantification using seven specific gene sequences; this method quantified nucleic acids from different <i>E. coli</i> serotypes, with a detection range of 6.6 to 7900 copies/µL and a repeatability standard deviation over the concentration range of 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 <i>E. coli</i> O157:H7 genomic DNA were then produced and characterized for evaluation of copy number concentration with the validated ddPCR method, with assigned values of 164,770 ± 9251 and 172 ± 9 copies/μL. Thus, this study demonstrated the development of a validated method and reference material for dPCR and qPCR detection of <i>E. coli</i> O157:H7, a key STEC responsible for food poisoning. |
| format | Article |
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| institution | Kabale University |
| issn | 2409-9279 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | MDPI AG |
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| series | Methods and Protocols |
| spelling | doaj-art-cc0cd3659564409c9fea87e6cf1b849d2024-12-27T14:43:09ZengMDPI AGMethods and Protocols2409-92792024-11-01769410.3390/mps7060094Digital PCR Validation for Characterization of Quantitative Reference Material of <i>Escherichia coli</i> O157<i>:H7</i> Genomic DNAClaudia Patricia Tere-Peña0Martha Nancy Calderon-Ozuna1John Emerson Leguizamón Guerrero2Department of Chemistry, Faculty of Science, Universidad Nacional de Colombia, Bogotá 111321, ColombiaDepartment of Chemistry, Faculty of Science, Universidad Nacional de Colombia, Bogotá 111321, ColombiaDepartment of Chemistry, Faculty of Science, Universidad Nacional de Colombia, Bogotá 111321, Colombia<i>Escherichia coli O157:H7</i>, a Shiga-toxin-producing <i>E. coli</i> (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 bacterium, 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 <i>E. coli O157:H7</i> detection and quantification using seven specific gene sequences; this method quantified nucleic acids from different <i>E. coli</i> serotypes, with a detection range of 6.6 to 7900 copies/µL and a repeatability standard deviation over the concentration range of 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 <i>E. coli</i> O157:H7 genomic DNA were then produced and characterized for evaluation of copy number concentration with the validated ddPCR method, with assigned values of 164,770 ± 9251 and 172 ± 9 copies/μL. Thus, this study demonstrated the development of a validated method and reference material for dPCR and qPCR detection of <i>E. coli</i> O157:H7, a key STEC responsible for food poisoning.https://www.mdpi.com/2409-9279/7/6/94<i>E. coli</i> O157:H7reference materialmethod validationdigital PCR |
| spellingShingle | Claudia Patricia Tere-Peña Martha Nancy Calderon-Ozuna John Emerson Leguizamón Guerrero Digital PCR Validation for Characterization of Quantitative Reference Material of <i>Escherichia coli</i> O157<i>:H7</i> Genomic DNA Methods and Protocols <i>E. coli</i> O157:H7 reference material method validation digital PCR |
| title | Digital PCR Validation for Characterization of Quantitative Reference Material of <i>Escherichia coli</i> O157<i>:H7</i> Genomic DNA |
| title_full | Digital PCR Validation for Characterization of Quantitative Reference Material of <i>Escherichia coli</i> O157<i>:H7</i> Genomic DNA |
| title_fullStr | Digital PCR Validation for Characterization of Quantitative Reference Material of <i>Escherichia coli</i> O157<i>:H7</i> Genomic DNA |
| title_full_unstemmed | Digital PCR Validation for Characterization of Quantitative Reference Material of <i>Escherichia coli</i> O157<i>:H7</i> Genomic DNA |
| title_short | Digital PCR Validation for Characterization of Quantitative Reference Material of <i>Escherichia coli</i> O157<i>:H7</i> Genomic DNA |
| title_sort | digital pcr validation for characterization of quantitative reference material of i escherichia coli i o157 i h7 i genomic dna |
| topic | <i>E. coli</i> O157:H7 reference material method validation digital PCR |
| url | https://www.mdpi.com/2409-9279/7/6/94 |
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