ABSTRACT
In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI.
Subject(s)
Community-Acquired Infections/microbiology , Community-Acquired Infections/virology , DNA, Bacterial/isolation & purification , Microfluidic Analytical Techniques/methods , RNA, Viral/isolation & purification , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virology , Analytic Sample Preparation Methods , Automation , Bacteria/genetics , Bacterial Physiological Phenomena , DNA, Bacterial/analysis , Humans , Influenza A virus/genetics , Influenza A virus/physiology , Microfluidic Analytical Techniques/instrumentation , RNA, Viral/analysisABSTRACT
There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a 'sample-in-result-out' mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5â kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20â µL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5â pM and a linear detection range spanning three orders of magnitude.