Diagnosis of infectious diseases in primary care is predominantly based on medical history and physical examination, as conventional laboratory investigations are often associated with delays that are unacceptable in medical practice. Point-of-care testing, and especially lab-on-a-chip (LoC) systems, are expected to result in a considerable reduction in associated healthcare costs and lead to fast, but appropriate and effective, personalized therapy. Although appropriate sample preparation is essential for final detection, most microfluidic-based approaches start from samples prepared by conventional laboratory procedures, therefore continuing to restrict the use of these systems to a laboratory setting. The lack of integrated sample preparation, especially for sample volumes in the milliliter range, is a major drawback of existing LoC systems. LoC systems that start with real samples and perform a full protocol from sample to result are still rare. In this article, the most recent advances in on-chip sample preparation are reviewed for microfluidic-based diagnosis of infectious diseases.
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.
Point-of-care (PoC) testing followed by personalized efficient therapy of infectious diseases may result in a considerable reduction of associated health care costs. Lab-on-a-chip (LoC) systems represent a potentially high efficient class of PoC tools. Here, we present a LoC system for automated pathogen analysis of respiratory viruses from nasopharyngeal specimens. The device prepares total nucleic acids from extracted swab samples using magnetic silica beads. After reverse transcription the co-purified viral RNA is amplified in accordance with the QIAplex multiplex PCR technology. Hybridized to corresponding QIAGEN LiquiChip beads and labelled with streptavidin R-phycoerythrin, the amplified target sequences are finally detected using a QIAGEN LiquiChip200 workstation. All chemicals needed are either stored freeze-dried on the disposable chip or are provided in liquid form in a reagent cartridge for up to 24 runs. Magnetic stir bars for mixing as well as turning valves with metering structures are integrated into the injection-moulded disposable chip. The core of the controlling instrument is a rotating heating bar construction providing fixed temperatures for fast cycling. PCR times of about half an hour (for 30 cycles) could be achieved for 120 μl reactions, making this system the fastest currently available high-volume PCR chip. The functionality of the system was shown by comparing automatically processed nasopharyngeal samples to ones processed manually according to the QIAGEN "ResPlex™ II Panel v2.0" respiratory virus detection kit. A prototype of the present instrument revealed slightly weaker signal intensities with a similar sensitivity in comparison to the commercially available kit and automated nucleic acid preparation devices, even without protocol optimization.
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