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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 10 2  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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiaomei Yan declares competing financial interest as a cofounder of NanoFCM Inc., a company committed to commercializing the nanoflow cytometry (nFCM) technology., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sensitive and rapid detection of Escherichia coli O157:H7, one of the most notorious bacterial pathogens, is urgently needed for public health protection. Yet, the existing methods are either lack of speed or limited in discriminating viable and dead cells. Using a recombinant bacteriophage, here we report the development of a rapid and sensitive method for live E. coli O157:H7 detection. First, the wild-type PP01 phage was engineered with a tetracysteine (TC)-tag fused with the small outer capsid (SOC) protein. Then, this PP01-TC phage was used to inoculate bacterial sample for 30min. Specific infection and rapid replication of PP01-TC phage in viable E. coli O157:H7 host cell yields a large number of progeny phages with capsids displaying TC tags that can be fluorescently labeled by a membrane permeable biarsenical dye (FlAsH). The bright green fluorescence of single E. coli O157:H7 cells can be readily detected by flow cytometry (FCM) and fluorescence microscopy. High specificity of the assay was verified with seven other bacterial strains. Practical application in E. coli O157:H7 detection in drinks was successfully demonstrated with artificially contaminated 100% apple juice. In less than three hours (including sample preconcentration) and with 40mL of sample volume, as low as 1cfu/mL E. coli O157:H7 can be detected in the presence of large excess of other nontarget bacteria via fluorescence microscopic measurement. The as-developed TC-PP01-FlAsH approach shows a great potential in the safeguard of liquid food products by providing rapid, sensitive, and specific detection of live E. coli O157:H7., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Advanced methods are urgently needed to determine the identity and viability of trace amounts of pathogenic bacteria in a short time. Existing approaches either fall short in the accurate assessment of microbial viability or lack specificity in bacterial identification. Bacteriophages (or phages for short) are viruses that exclusively infect bacterial host cells with high specificity. As phages infect and replicate only in living bacterial hosts, here we exploit the strategy of using tetracysteine (TC)-tagged phage in combination with biarsenical dye to the discriminative detection of viable target bacteria from dead target cells and other viable but nontarget bacterial cells. Using recombinant M13KE-TC phage and Escherichia coli ER2738 as a model system, distinct differentiation between individual viable target cells from dead target cells was demonstrated by flow cytometry and fluorescence microscopy. As few as 1% viable E. coli ER2738 can be accurately quantified in a mix with dead E. coli ER2738 by flow cytometry. With fluorescence microscopic measurement, specific detection of as rare as 1 cfu/mL original viable target bacteria was achieved in the presence of a large excess of dead target cells and other viable but nontarget bacterial cells in 40 mL artificially contaminated drinking water sample in less than 3 h. This TC-phage-FlAsH approach is sensitive, specific, rapid, and simple, and thus shows great potential in water safety monitoring, health surveillance, and clinical diagnosis of which trace detection and identification of viable bacterial pathogens is highly demanded.

Rapid quantification of viruses is vital for basic research on viral diseases as well as biomedical application of virus‐based products. Here, we report the development of a high‐throughput single‐particle method to enumerate intact viral particles by ultrasensitive flow virometry, which detects single viruses as small as 27 nm in diameter. The nucleic acid dye SYTO 82 was used to stain the viral (or vector) genome, and a laboratory‐built nano‐flow cytometer (nFCM) was employed to simultaneously detect the side‐scatter and fluorescence signals of individual viral particles. Using the bacteriophage T7 as a model system, intact virions were completely discriminated from empty capsids and naked viral genomes. Successful measurement of the physical virus titer and purity was demonstrated for recombinant adenoviruses, which could be used for gene delivery, therapeutic products derived from phage cocktails, and infected cell supernatants for veterinary vaccine production. [ABSTRACT FROM AUTHOR]

Viruses are by far the most abundant biological entities on our planet, yet existing characterization methods are limited by either their speed or lack of resolution. By applying a laboratory-built high-sensitivity flow cytometer (HSFCM) to precisely quantify the extremely weak elastically scattered light from single viral particles, we herein report the label-free analysis of viruses with a resolution comparable to that of electron microscopy and the throughput of flow cytometry. The detection of single viruses with diameters down to 27 nm is described. T7 and lambda bacteriophages, which differ in size by as little as 4 nm, could be baseline-resolved. Moreover, subtle structural differences of the same viral particles can be discriminated. Using monodisperse silica nanoparticles as the size reference standards, the virus sizes measured by the HSFCM are in agreement with the equivalent particle diameters derived from their structural dimensions. The HSFCM opens a new avenue for virus characterization. [ABSTRACT FROM AUTHOR]