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Rapid and accurate identification of respiratory tract infection pathogens is of utmost importance for clinical diagnosis and treatment, as well as prevention of pathogen transmission. To meet this demand, a microfluidic chip-based PCR-array system, Onestart, was developed. The Onestart system uses a microfluidic chip packaged with all the reagents required, and the waste liquid is also collected and stored on the chip. This ready-to-use system can complete the detection of 21 pathogens in a fully integrated manner, with sample lysis, nucleic acid extraction/purification, and real-time PCR sequentially implemented on the same chip. The entire analysis process is completed within 1.5 h, and the system automatically generates a test report. The lower limit-of-detection (LOD) of the Onestart assay was determined to be 1.0 × 103 copies·mL−1. The inter-batch variation of cycle threshold (Ct) values ranged from 0.08% to 0.69%, and the intra-batch variation ranged from 0.9% to 2.66%. Analytical results of the reference sample mix showed a 100% specificity of the Onestart assay. The analysis of batched clinical samples showed consistency of the Onestart assay with real-time PCR. With its ability to provide rapid, sensitive, and specific detection of respiratory tract infection pathogens, application of the Onestart system will facilitate timely clinical management of respiratory tract infections and effective prevention of pathogen transmission. [ABSTRACT FROM AUTHOR]

Highlights: [•] A high-throughput and rapid microfluidic method for pathogen detection is proposed. [•] The method offers a simple and convenient way toward high-throughput DNA analysis. [•] Important parameters of segmented continuous-flow multiplex PCR were investigated. [•] The proposed method is suitable for high-throughput biomedical monitoring. [Copyright &y& Elsevier]

Rapid screening of infectious pathogens at the point-of-care (POC) is ideally low-cost, portable, easy to use, and capable of multiplex detection with high sensitivity. However, satisfying all these features in a single device without compromise remains a challenging task. Here, we introduce an ultraportable, automated RNA amplification testing device that allows rapid screening of infectious pathogens from clinical samples. In this device, 3D-printed structural parts incorporated with off-the-shelf mechanic/electronic components are utilized to create an inexpensive and automated droplet manipulation platform. On this platform, a simple configuration that couples a linear displacement of the chip with a tunable magnet array allows parallel and versatile droplet operations, including mixing, splitting, transporting, and merging. By exploiting a multi-channel droplet array chip to preload necessary reagents in "water-in-oil" format, bacteria lysis, RNA extraction and amplification are seamlessly integrated and implemented by the combination of droplet operations. Furthermore, visual readout and geometrically-multiplexed quantitative detection are provided by an integrated wireless video camera-enabled wide-field fluorescence imaging. We demonstrated that this droplet-based device could have a shorter RNA extraction time (12 min) and lower detection limits for pathogenic RNA (approaching to 102 copies per reaction). We also verified its clinical applicability for the rapid screening of four sexually transmitted pathogens from urine specimens. Results show that the sample-to-answer assay could be completed in approximately 42 min, with 100% concordance with the laboratory-based molecular testing. The exhibiting features may render this microdevice an easily accessible POC molecular diagnostic platform for infectious disease, especially in resource-limited settings. A pocket-sized microfluidic system automates multiplexed point-of-care RNA amplification testing for rapid screening of infectious pathogens from urine samples. [Display omitted] • An inexpensive microdevice automates multiplexed point-of-care molecular diagnosis. • It is made of 3D-printed parts with off-the-shelf mechanic/electronic components. • Multiplexed real-time detection by an ultracompact camera-enabled wide-field imaging. • Highly sensitive screening of 4 pathogens directly from urine samples in 42 min. • It is promising for screening of infectious diseases in resource-limited settings. [ABSTRACT FROM AUTHOR]