Abstract
Bacteria detection through the polymerase chain reaction (PCR) often requires extraction and purification of the analyte DNA sequence, with the total time for analysis that is typically 5 to 8 h. In this contribution, we demonstrate that this workflow can be greatly simplified, reducing the total time to detection to 17 min. The on-electrode (oE) PCR technique conducts a surface-based 5-cycle PCR amplification by covalent attachment of a right primer to a pseudo-graphite fiber electrode. This foregoes electrophoretic separation steps. The target is a 225-base sequence of the 16S rDNA gene of Nitrobacter genomic DNA (3.4 million base pairs) and extracellular environmental (e) DNA. The oE-PCR technique overcomes Taq polymerase inhibition associated with untreated matrices of raw sewage and chicken juice. These are challenging media for all other variations of PCR. The detection of surface-based amplicons is through the introduction of the electrochemically active cobalt(III) tris-1,10-phenanthroline intercalator, followed by square-wave voltammetric (SWV) analysis. Detection was positive relative to a blank and no-template control (NTC). The total time required for the oE-PCR process is 17 min with a limit of detection of less than 20 aM. The LOD of this method surpasses the nM required for the detection of Nitrobacter DNA in wastewater matrices. While qPCR offers quantitative results, it is time-consuming. However, oE-PCR may act as a rapid screening tool for qPCR. The ability to detect rapidly Nitrobacter in such a complex matrix, from sample to result, may have important applications in medical, environmental, and forensic sciences.