Volume 15, Issue 3 (Summer 2026)
aumj 2026, 15(3): 0-0 |
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Research code: 815 - 847 - 4003899
Ethics code: عدم نیاز به کد اخلاق
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Kiani A, Minaeian S, Bambai B, Zolfaghari Emameh R. Investigation of carbonic anhydrase-encoding genes and introduction of β-carbonic anhydrase as a molecular target for the detection of Escherichia coli. aumj 2026; 15 (3) : 8
URL: http://aums.abzums.ac.ir/article-1-1974-en.html
URL: http://aums.abzums.ac.ir/article-1-1974-en.html
1- Department of Energy and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
2- Antimicrobial Resistance Research Center, Institute of Immunology and Infectious Diseases, Iran University of Medical Sciences, Tehran, Iran
3- Department of Systems Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
4- Department of Energy and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran ,zolfaghari@nigeb.ac.ir
2- Antimicrobial Resistance Research Center, Institute of Immunology and Infectious Diseases, Iran University of Medical Sciences, Tehran, Iran
3- Department of Systems Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
4- Department of Energy and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran ,
Abstract: (27 Views)
Introduction: Escherichia coli is one of the most important bacterial indicators of fecal contamination and is frequently used as a microbiological indicator for assessing the sanitary quality and safety of water, particularly drinking water. Therefore, the development of rapid, reliable, accessible, and specific methods for detecting E. coli contamination in water sources is essential for effective microbiological quality control and the prevention of waterborne infections. Carbonic anhydrases (CAs) are metalloenzymes involved in diverse physiological processes and are classified into several distinct evolutionary families, including the α-, β-, and γ-CA classes. The genes encoding these enzymes exhibit considerable sequence diversity among bacterial genera and species, making them potentially valuable targets for species-specific molecular detection. Polymerase chain reaction (PCR) provides a rapid and sensitive molecular approach for detecting specific microbial genetic targets. This study aimed to identify and evaluate suitable carbonic anhydrase-encoding genes as potential molecular targets for the specific detection of E. coli in water samples.
Methods: Bioinformatic analyses demonstrated that E. coli possesses 19 distinct carbonic anhydrase enzymes, comprising 1 α-CA, 12 β-CAs, and 6 γ-CAs. The corresponding carbonic anhydrase-encoding genes were systematically evaluated to identify suitable targets for molecular detection. Among the identified genes, one α-CA, one β-CA, and one γ-CA gene were selected for experimental evaluation. Specific primers were designed for the selected target sequences, followed by extraction of E. coli genomic DNA and assessment of DNA concentration and purity using a NanoDrop spectrophotometer. PCR reaction mixtures were subsequently prepared, and amplification of the selected target sequences was performed. The resulting PCR products were visualized by agarose gel electrophoresis, and the amplified bands were subjected to sequencing to confirm the identity and specificity of the detected target sequences. Because the study did not involve human or animal subjects or samples, an institutional ethics approval code was not required. All experimental procedures involving E. coli were conducted under Biosafety Level 1 (BSL-1) conditions. The study was supported by Grants 815 and 847 from the National Institute of Genetic Engineering and Biotechnology (NIGEB) and Grant 4003899 from the Iran National Science Foundation (INSF). No artificial intelligence tools were used in the design, analysis, or preparation of this study.
Results: Among the three selected carbonic anhydrase gene targets representing the α-, β-, and γ-CA families, the β-CA-encoding sequence demonstrated successful and specific amplification for the detection of E. coli. The complete molecular detection procedure could be accomplished within approximately 2–3 hours, providing a considerably shorter turnaround time than conventional microbiological and biochemical identification procedures. These conventional approaches include a combination of biochemical assays, such as Triple Sugar Iron (TSI) agar, Voges–Proskauer (VP), indole production, citrate utilization, and urease tests, which generally require additional time for bacterial growth and phenotypic characterization. Sequencing of the amplified PCR product further confirmed the identity of the target sequence, supporting the suitability of the β-CA-encoding gene as a molecular target for E. coli detection.
Conclusion: The findings of this study suggest that the β-carbonic anhydrase (β-CA)-encoding sequence may serve as a novel, specific, and potentially reliable molecular target for the rapid identification of E. coli in water sources. The successful amplification and subsequent sequence confirmation of this target indicate its potential utility in PCR-based microbiological quality-control assays. Because β-carbonic anhydrase is absent or substantially restricted in vertebrates, targeting the corresponding bacterial gene may provide an additional level of biological specificity for E. coli detection and may reduce the likelihood of nonspecific or false-positive amplification. Further validation using a larger collection of environmental water samples and a broader range of bacterial species is warranted to establish the analytical sensitivity, specificity, and practical applicability of this molecular detection strategy.
Methods: Bioinformatic analyses demonstrated that E. coli possesses 19 distinct carbonic anhydrase enzymes, comprising 1 α-CA, 12 β-CAs, and 6 γ-CAs. The corresponding carbonic anhydrase-encoding genes were systematically evaluated to identify suitable targets for molecular detection. Among the identified genes, one α-CA, one β-CA, and one γ-CA gene were selected for experimental evaluation. Specific primers were designed for the selected target sequences, followed by extraction of E. coli genomic DNA and assessment of DNA concentration and purity using a NanoDrop spectrophotometer. PCR reaction mixtures were subsequently prepared, and amplification of the selected target sequences was performed. The resulting PCR products were visualized by agarose gel electrophoresis, and the amplified bands were subjected to sequencing to confirm the identity and specificity of the detected target sequences. Because the study did not involve human or animal subjects or samples, an institutional ethics approval code was not required. All experimental procedures involving E. coli were conducted under Biosafety Level 1 (BSL-1) conditions. The study was supported by Grants 815 and 847 from the National Institute of Genetic Engineering and Biotechnology (NIGEB) and Grant 4003899 from the Iran National Science Foundation (INSF). No artificial intelligence tools were used in the design, analysis, or preparation of this study.
Results: Among the three selected carbonic anhydrase gene targets representing the α-, β-, and γ-CA families, the β-CA-encoding sequence demonstrated successful and specific amplification for the detection of E. coli. The complete molecular detection procedure could be accomplished within approximately 2–3 hours, providing a considerably shorter turnaround time than conventional microbiological and biochemical identification procedures. These conventional approaches include a combination of biochemical assays, such as Triple Sugar Iron (TSI) agar, Voges–Proskauer (VP), indole production, citrate utilization, and urease tests, which generally require additional time for bacterial growth and phenotypic characterization. Sequencing of the amplified PCR product further confirmed the identity of the target sequence, supporting the suitability of the β-CA-encoding gene as a molecular target for E. coli detection.
Conclusion: The findings of this study suggest that the β-carbonic anhydrase (β-CA)-encoding sequence may serve as a novel, specific, and potentially reliable molecular target for the rapid identification of E. coli in water sources. The successful amplification and subsequent sequence confirmation of this target indicate its potential utility in PCR-based microbiological quality-control assays. Because β-carbonic anhydrase is absent or substantially restricted in vertebrates, targeting the corresponding bacterial gene may provide an additional level of biological specificity for E. coli detection and may reduce the likelihood of nonspecific or false-positive amplification. Further validation using a larger collection of environmental water samples and a broader range of bacterial species is warranted to establish the analytical sensitivity, specificity, and practical applicability of this molecular detection strategy.
Article number: 8
Keywords: Escherichia coli, Carbonic Anhydrase, Polymerase Chain Reaction, Molecular Detection, Water Quality
Type of Study: Research |
Subject:
Special
Received: 2025/11/19 | Accepted: 2026/08/26 | Published: 2026/09/22
Received: 2025/11/19 | Accepted: 2026/08/26 | Published: 2026/09/22
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