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Updated: Nov 4, 2025
Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres
Published on: October 23, 2011
Multiplexed detection of bacterial pathogens based on a cocktail of dual-modified phages
Lina Wu1, Xinyi Hong1, Tian Luan1
1Department of Chemical Biology, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Key Laboratory for Chemical Biology of Fujian Province, Collaborative Innovation Center of Chemistry for Energy Materials, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, 361005, People's Republic of China.
Abstract:
Rapid, quantitative, and sensitive assays for the multiplexed detection of bacterial pathogens are urgently needed for public health. Here, we report the generation of dual-modified phage sensors for the simultaneous detection of multiple pathogenic bacteria. The M13KE phage was dual modified to display the targeting peptide on the minor coat protein pIII (∼5 copies) and the streptavidin-binding (StrB) peptide on the major coat protein pVIII (∼2700 copies). The targeting peptide specifically recognizes the target bacteria, and the StrB peptide acts as the efficient signal amplification and transduction unit upon binding with fluorescently tagged streptavidin. The bright fluorescence emitted from individual target bacteria can be clearly distinguished from the background via both the flow cytometry and fluorescence microscopy. Three different dual-modified phages targeting E. coli O157:H7, Salmonella Typhimurium, and Pseudomonas aeruginosa were constructed, and high specificity was verified via a large excess of other non-target bacteria. Using a 40 mL sample volume, the target bacteria detection limit was approximately 102 cells/mL via flow cytometry measurement in the presence of other non-target bacteria. By combining these three dual-modified phages into a cocktail, simultaneous detection and quantification of three target bacterial pathogens was demonstrated with good linearity. The strategy of constructing dual-modified phage represents a promising tool in the detection of bacterial pathogens.
Insights
Researchers developed dual-modified phage sensors for rapid, simultaneous detection of multiple bacterial pathogens. This innovative method enhances public health by providing sensitive and quantitative bacterial identification.
Area of Science:
- Biotechnology
- Microbiology
- Nanotechnology
Background:
- Current methods for detecting bacterial pathogens lack the speed, sensitivity, and multiplexing capabilities required for effective public health surveillance.
- There is a critical need for advanced diagnostic tools capable of simultaneously identifying multiple harmful bacteria.
Purpose of the Study:
- To engineer dual-modified phage sensors for the simultaneous, sensitive, and quantitative detection of multiple bacterial pathogens.
- To validate the specificity and detection limits of these novel phage-based biosensors.
Main Methods:
- M13KE bacteriophage was genetically modified to display a targeting peptide on pIII protein and a streptavidin-binding (StrB) peptide on pVIII protein.
- Fluorescently tagged streptavidin was used for signal amplification and transduction, enabling detection via flow cytometry and fluorescence microscopy.
- Dual-modified phages targeting E. coli O157:H7, Salmonella Typhimurium, and Pseudomonas aeruginosa were constructed and tested.
Main Results:
- High specificity was confirmed, with successful differentiation of target bacteria from a large excess of non-target species.
- A detection limit of approximately 10^2 cells/mL was achieved for target bacteria using flow cytometry with a 40 mL sample volume.
- Simultaneous detection and quantification of three target bacterial pathogens were successfully demonstrated using a cocktail of the dual-modified phages.
Conclusions:
- Dual-modified phage sensors offer a promising strategy for rapid, sensitive, and multiplexed detection of bacterial pathogens.
- This approach provides a valuable tool for enhancing public health diagnostics and surveillance of infectious diseases.
