Absolute and Simultaneous Quantification of Specific Pathogenic Strains and Total Bacterial Cells

Author: admin     Date: February 22, 2024

Fast, sensitive, and specific detection of pathogenic bacteria is of great significance for food safety, environmental monitoring, disease diagnosis, treatment, and prevention of bioterrorism. Traditional pathogenic microbial detection methods require the isolation, culture, and a series of biochemical reactions of bacteria, which are complex in operation, long in detection cycle, and cannot be applied to pathogenic bacteria that are difficult to culture. This is far from meeting the actual needs. Traditional flow cytometry still has quite limitations in the highly sensitive detection of pathogenic bacteria. Using Alexa Fluor 647-R-PE as a fluorescent probe for E. coli O157:H7 monoclonal antibody and fluorescent SYTO 9 dye to stain the nucleic acids of bacteria to achieve simultaneous detection of dual fluorescent channels using nanoflow cytometry. Pathogenic bacteria can produce fluorescence signals simultaneously in the green and red fluorescence detection channels, while non-pathogenic bacteria only produce signals in the green channel.

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The Flow NanoAnalyzer overcomes the limitations of traditional detection methods, showing  potential in the rapid identification and quantitative analysis of pathogenic bacteria in food, environment and disease-related samples.

Anal. Chem., 2010, 82(3), 1109-1116.

Rapid Quantification of Pathogenic SalmonellaTyphimurium and Total Bacteria in Eggs

Author: admin     Date: February 22, 2024

Rapid quantification of pathogenic Salmonella Typhimurium (S. typhimurium) and total bacteria in eggs is highly desired for food safety control. However, the complexity of the egg matrix presents a significant challenge for sensitive detection of bacteria. In this study, a sample pretreatment protocol, including dilution, fat dissolution, protein degradation, filtration, and washing, was developed to circumvent this challenge. The Flow NanoAnalyzer was employed to analyze individual bacteria with nucleic acid and immunofluorescent staining. Eggs spiked with pathogenic S. typhimurium and harmless Escherichia coli K12 (E. coli K12) were used as the model system to optimize the sample pretreatment protocol. S. typhimurium and total bacteria in eggs can be quantified without cultural enrichment, and the whole process of sample pretreatment, staining, and instrument analysis can be accomplished within 1.5 hours. The as-developed approach can specifically distinguish S. typhimurium from other bacteria, and successful application to bacterial detection in eggs freshly purchased from supermarkets and spoiled eggs upon inappropriate storage was demonstrated.

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The bacterial recovery rate upon sample pretreatment, detection limit, and dynamic range for S. typhimurium in eggs were 92%, 2 × 103 cells/mL, and from 2 × 103 to 4 × 108 cells/mL, respectively.

Talanta, 2020, 217, 121020.

Phage cocktail is used for multi-detection of bacterial pathogens

Author: admin     Date: February 22, 2024

The Public health sector has an urgent need for rapid, quantitative, and sensitive detection methods for the multi-detection of bacterial pathogens. In this study, the target peptide-specific recognition of pathogenic bacteria was demonstrated through the double modification of the M13KE phage pair. The target peptide was modified on the secondary capsid protein pIII, and the streptavidin-binding (StrB) was modified on the major capsid protein pVIII. The peptide-targeted peptide could specifically recognize the target bacteria, and after binding to the fluorescently labeled streptavidin, the StrB peptide could effectively amplify the signal. With the Flow NanoAnalyzer and fluorescence microscopy, bright fluorescence from individual pathogens can be clearly distinguished from the background.

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Bacteriophages targeting Escherichia coli O157:H7, Salmonella typhimurium, and Pseudomonas aeruginosa were constructed, and high specificity was verified by a large excess of other non-target bacteria. By analyzing using the Flow NanoAnalyzer, the detection limit of the target pathogen was about 10^2 cells/mL in the presence of other non-pathogenic bacteria with only a 40 mL sample. The combination of three dual-modified phages into a cocktail can also quantitatively and linearly detect three target bacterial pathogens simultaneously.

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Figure 1. Binding of different strains and bacteriophages O157S-M13KE-StrB

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Figure 2. Mixed analysis of E. coli O157:H7 in different proportions with other non-target bacteria

The Flow NanoAnalyzer easily distinguished the strong fluorescence signal in E. coli O157:H7 and can detect weak fluorescence signals from other non-pathogenic strains.

Analytica Chimica Acta, 2021, 1166, 338596.