ABSTRACT
The COVID-19 pandemic, in addition to the co-occurrence of influenza virus and respiratory syncytial virus (RSV), has emphasized the requirement for efficient and reliable multiplex diagnostic methods for respiratory infections. While existing multiplex detection techniques are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) and extraction and purification kits, the need for complex instrumentation and elevated cost limit their scalability and availability. In this study, we have developed a point-of-care (POC) device based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that can simultaneously detect four respiratory viruses (SARS-CoV-2, Influenza A, Influenza B, and RSV) and perform two controls in less than 30 min, while avoiding the use of the RNA extraction kit. The system includes a disposable microfluidic cartridge with mechanical components that automate sample processing, with a low-cost and portable optical reader and a smartphone app to record and analyze fluorescent images. The application as a real point-of-care platform was validated using swabs spiked with virus particles in nasal fluid. Our portable diagnostic system accurately detects viral RNA specific to respiratory pathogens, enabling deconvolution of coinfection information. The detection limits for each virus were determined using virus particles spiked in chemical lysis buffer. Our POC device has the potential to be adapted for the detection of new pathogens and a wide range of viruses by modifying the primer sequences. This work highlights an alternative approach for multiple respiratory virus diagnostics that is well-suited for healthcare systems in resource-limited settings or at home.
Subject(s)
Nucleic Acid Amplification Techniques , Point-of-Care Systems , SARS-CoV-2 , Humans , SARS-CoV-2/isolation & purification , SARS-CoV-2/genetics , Nucleic Acid Amplification Techniques/methods , Influenza A virus/isolation & purification , Influenza A virus/genetics , COVID-19/diagnosis , COVID-19/virology , Influenza B virus/isolation & purification , Influenza B virus/genetics , RNA, Viral/analysis , RNA, Viral/isolation & purification , RNA, Viral/genetics , Molecular Diagnostic Techniques/methods , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/virology , Respiratory Syncytial Viruses/isolation & purification , Respiratory Syncytial Viruses/geneticsABSTRACT
Additive laser-induced forward transfer (LIFT) of metal bactericidal nanoparticles from a polymer substrate directly onto food bacterial biofilms has demonstrated its unprecedented efficiency in combating pathogenic microorganisms. Here, a comprehensive study of laser fluence, metal (gold, silver and copper) film thickness, and the transfer distance effects on the antibacterial activity regarding biofilms of Gram-negative and Gram-positive food bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Listeria monocytogenes, Salmonella spp.) indicated the optimal operation regimes of the versatile modality. LIFT-induced nanoparticle penetration into a biofilm was studied by energy-dispersion X-ray spectroscopy, which demonstrated that nanoparticles remained predominantly on the surface of the biofilm.