Electrochemical phagemid assay for the specific detection of bacteria using Escherichia coli TG-1 and the M13KO7 phagemid in a model system.
We describe a reporter phagemid system for the specific amperometric detection of bacteria. We constructed a phagemid a bacteriophage containing a bacterial plasmid using the M13KO7 helper phage and a commercial plasmid, pFLAG-ATS-BAP, which contains a gene encoding for a reporter enzyme, alkaline phosphatase. In the bacteria, the enzyme reacts with the substrate, p-aminophenyl phosphate, in the periplamic space that separates the outer plasma membrane from the cell wall. Thus, the activity of the reporter enzyme can be measured directly in an electrochemical cell without further treatment. The product of the enzymatic activity, p-aminophenol, diffuses out and is oxidized at the working electrode with an applied potential of 220 mV vs the reference electrode Ag/AgCl. The lower detection limit was 1 cfu/mL E. coli TG1 in less than 3 h in a very specific manner. The use of plasmid alkaline phosphatase as the reporter increased the sensitivity by 10-fold over our earlier electrochemical lytic phage method. Such a system can be used for the rapid detection of any strain of bacteria using the appropriate bacteriophage and reporter gene.
Neufeld
Tova
T
Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat-Aviv, Israel 69978.
Mittelman
Adrian Schwartz
AS
Buchner
Virginia
V
Rishpon
Judith
J
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D001419:Bacteria / Q000302:isolation & purification*; D017104:Bacteriophage M13 / Q000235:genetics* / Q000302:isolation & purification; D001434:Bacteriophage Typing / Q000379:methods*; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics*
D000469:Alkaline Phosphatase