RESUMO
Staphylococcus species are a major pathogen responsible for nosocomial infections and foodborne illnesses. We applied a laser-based BARDOT (bacterial rapid detection using optical scattering technology) for rapid colony screening and detection of Staphylococcus on an agar plate and differentiate these from non-Staphylococcus spp. Among the six growth media tested, phenol red mannitol agar (PRMA) was found most suitable for building the Staphylococcus species scatter image libraries. Scatter image library for Staphylococcus species gave a high positive predictive value (PPV 87.5-100%) when tested against known laboratory strains of Staphylococcus spp., while the PPV against non-Staphylococcus spp. was 0-38%. A total of nine naturally contaminated bovine raw milk and ready-to-eat chicken salad samples were tested, and BARDOT detected Staphylococcus including Staphylococcus aureus with 80-100% PPV. Forty-five BARDOT-identified bacterial isolates from naturally contaminated foods were further confirmed by tuf and nuc gene-specific PCR and 16S rRNA gene sequence. This label-free, non-invasive on-plate colony screening technology can be adopted by the food industries, biotechnology companies, and public health laboratories for Staphylococcus species detection including S. aureus from various samples for food safety and public health management. Graphical abstract.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Difusão Dinâmica da Luz/métodos , Contaminação de Alimentos/análise , Carne/análise , Leite/microbiologia , Staphylococcus/isolamento & purificação , Animais , Técnicas de Tipagem Bacteriana/economia , Bovinos , Galinhas/microbiologia , Difusão Dinâmica da Luz/economia , Microbiologia de Alimentos , Análise de Perigos e Pontos Críticos de Controle/métodos , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus/genética , Staphylococcus/crescimento & desenvolvimentoRESUMO
The SARS-CoV-2 pandemic, an ongoing global health crisis, has revealed the need for new technologies that integrate the sensitivity and specificity of RT-PCR tests with a faster time-to-detection. Here, an emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to allow for the compartmentalization of LAMP reactions, leading to faster changes in emulsion characteristics, and thus lowering time-to-detection. Within these droplets, ongoing LAMP reactions lead to adsorption of amplicons to the water-oil interface, causing a decrease in interfacial tension, resulting in smaller emulsion diameters. Changes in emulsion diameter allow for the monitoring of the reaction by use of angle-dependent light scatter (based off Mie scatter theory). Mie scatter simulations confirmed that light scatter intensity is diameter-dependent and smaller colloids have lower intensity values compared to larger colloids. Via spectrophotometers and fiber optic cables placed at 30° and 60°, light scatter intensity was monitored. Scatter intensities collected at 5 min, 30° could statistically differentiate 10, 103, and 105 copies/µL initial concentrations compared to NTC. Similarly, 5 min scatter intensities collected at 60° could statistically differentiate 105 copies/µL initial concentrations in comparison to NTC. The use of both angles during the eLAMP assay allows for distinction between high and low initial target concentrations. The efficacy of a smartphone-based platform was also tested and had a similar limit of detection and assay time of less than 10 min. Furthermore, fluorescence-labeled primers were used to validate target nucleic acid amplification. Compared to existing LAMP assays for SARS-CoV-2 detection, these times-to-detections are very rapid.
Assuntos
Teste de Ácido Nucleico para COVID-19/instrumentação , COVID-19/diagnóstico , Difusão Dinâmica da Luz/instrumentação , Emulsões/química , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , SARS-CoV-2/isolamento & purificação , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Teste de Ácido Nucleico para COVID-19/economia , Teste de Ácido Nucleico para COVID-19/métodos , Difusão Dinâmica da Luz/economia , Difusão Dinâmica da Luz/métodos , Desenho de Equipamento , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Smartphone , Fatores de TempoRESUMO
A simple, fast, costless, sensitive and selective method of resonance light scattering coupled with HPLC was established for the determination of 6-mercaptopurine in human urine sample. In a Britton-Robinson buffer solution of pH5.5, the formation of coordination complex between 6-mercaptopurine and metal palladium (II) led to enhance the RLS intensity of the system. The RLS signal was detected by fluorescence detector at λ(ex)=λ(em)=315 nm. The analytical parameters were provided by the coupled system, the linear of 6-mercaptopurine response from 0.0615 to 2.40 µg L(-1) and the limit of detection (S/N=3) was 0.05 µg L(-1). The presented method has been applied to determine 6-mercaptopurine in human urine samples which obtained satisfactory results. Moreover, the reaction mechanism and possible reasons for enhancement of RLS were fully discussed.