Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection
A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity o...
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Published in | Biosensors & bioelectronics Vol. 23; no. 12; pp. 1805 - 1811 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Lausanne
Elsevier B.V
15.07.2008
Elsevier Science |
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Abstract | A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the
eaeA gene, related with the pathogenic activity of
Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5
ng
μl
−1 and 0.45
ng
μl
−1 of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes. |
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AbstractList | A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5 ng microl(-1) and 0.45 ng microl(-1) of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes. A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5ng mu l super(-) super(1) and 0.45ng mu l super(-) super(1) of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes. A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5 ng μl −1 and 0.45 ng μl −1 of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes. |
Author | Pividori, M.I. Lermo, A. Campoy, S. Delwiche, M. Barbé, J. Barak, J. Zacco, E. Alegret, S. |
Author_xml | – sequence: 1 givenname: A. surname: Lermo fullname: Lermo, A. organization: Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona. 08193 Bellaterra, Catalonia, Spain – sequence: 2 givenname: E. surname: Zacco fullname: Zacco, E. organization: Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona. 08193 Bellaterra, Catalonia, Spain – sequence: 3 givenname: J. surname: Barak fullname: Barak, J. organization: Produce Safety and Microbiology Research Unit. Albany, CA 94710, USA – sequence: 4 givenname: M. surname: Delwiche fullname: Delwiche, M. organization: Department of Biological and Agricultural, Engineering University of California. Davis, CA 95616, USA – sequence: 5 givenname: S. surname: Campoy fullname: Campoy, S. organization: Unitat de Microbiologia, Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona. 08193 Bellaterra, Catalonia, Spain – sequence: 6 givenname: J. surname: Barbé fullname: Barbé, J. organization: Unitat de Microbiologia, Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona. 08193 Bellaterra, Catalonia, Spain – sequence: 7 givenname: S. surname: Alegret fullname: Alegret, S. organization: Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona. 08193 Bellaterra, Catalonia, Spain – sequence: 8 givenname: M.I. surname: Pividori fullname: Pividori, M.I. email: Isabel.Pividori@uab.es organization: Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona. 08193 Bellaterra, Catalonia, Spain |
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Keywords | Magnetic beads Q-PCR Electrochemical genosensing Escherichia coli Electrochemical DNA biosensor (Strept)avidin Avidin Polymerase chain reaction Biosensor DNA Pathogenic Electrochemistry Bacteria Detection Enterobacteriaceae |
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Snippet | A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the... |
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SubjectTerms | (Strept)avidin Biological and medical sciences Biosensing Techniques - instrumentation Biosensing Techniques - methods Biosensors Biotechnology Colony Count, Microbial - instrumentation Colony Count, Microbial - methods Electrochemical DNA biosensor Electrochemical genosensing Electrochemistry - instrumentation Escherichia coli Escherichia coli O157 - genetics Escherichia coli O157 - isolation & purification Expressed Sequence Tags Fundamental and applied biological sciences. Psychology Magnetic beads Methods. Procedures. Technologies Polymerase Chain Reaction - instrumentation Polymerase Chain Reaction - methods Q-PCR Various methods and equipments |
Title | Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection |
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