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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 × 10 3 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. Onestart, a ready-to-use system, can detect 21 pathogens in a fully integrated manner on a microchip within 1.5 h.

Timely and accurate molecular diagnostics at the point-of-care (POC) level is critical to global health. To this end, we propose a handheld convective-flow real-time polymerase chain reaction (PCR) system capable of direct sample-to-answer genetic analysis for the first time. Such a system mainly consists of a magnetic bead-assisted photothermolysis sample preparation, a closed-loop convective PCR reactor, and a wireless video camera-based real-time fluorescence detection. The sample preparation exploits the dual functionality of vancomycin-modified magnetic beads (VMBs) for bacteria enrichment and photothermal conversion, enabling cell pre-concentration and lysis to be finished in less than 3min. On the presented system, convective thermocycling is driven by a single-heater thermal gradient, and its amplification is monitored in real-time, with an analysis speed of less than 25min, a dynamic linear range from 10 6 to 10 1 copies/µL and a detection sensitivity of as little as 1 copies/µL. Additionally, the proposed PCR system is self-contained with a control electronics, pocket-size and battery-powered, providing a low-cost genetic analysis in a portable format. Therefore, we believe that this integrated system may become a potential candidate for fast, accurate and affordable POC molecular diagnostics., (Copyright © 2017 Elsevier B.V. All rights reserved.)

High-throughput and rapid identification of multiple foodborne bacterial pathogens is vital in global public health and food industry. To fulfill this need, we propose a segmented continuous-flow multiplex polymerase chain reaction (SCF-MPCR) on a spiral-channel microfluidic device. The device consists of a disposable polytetrafluoroethylene (PTFE) capillary microchannel coiled on three isothermal blocks. Within the channel, n segmented flow regimes are sequentially generated, and m-plex PCR is individually performed in each regime when each mixture is driven to pass three temperature zones, thus providing a rapid analysis throughput of m×n. To characterize the performance of the microfluidic device, continuous-flow multiplex PCR in a single segmented flow has been evaluated by investigating the effect of key reaction parameters, including annealing temperatures, flow rates, polymerase concentration and amount of input DNA. With the optimized parameters, the genomic DNAs from Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7 and Staphylococcus aureus could be amplified simultaneously in 19min, and the limit of detection was low, down to 10(2) copiesμL(-1). As proof of principle, the spiral-channel SCF-MPCR was applied to sequentially amplify four different bacterial pathogens from banana, milk, and sausage, displaying a throughput of 4×3 with no detectable cross-contamination., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A novel thermodynamic approach for integrating isothermal amplification with CRISPR detection in a closed assay vessel without the use of interior driving forces or human intervention was developed by harnessing internal air thermal expansion for autonomous liquid actuation by coupling a thermally responsive microfluidic cartridge with a simple two-step temperature profile. [Display omitted] • Seamless integration of CRISPR detection and target preamplification. • Automated actuation without external driving mechanism or human intervention. • Temperature-responsive architecture utilizes the internal air thermal expansion. • It shortens the sample-to-answer time without compromising sensitivity. The rising incidence of syphilis highlights the urgent need to develop molecular tests of Treponema pallidum at the point-of-care (POC). Traditional molecular approaches based on polymerase chain reaction (PCR) often involve expensive equipment, expert technicians, and lengthy turnaround time. Recently emerged approaches coupling CRISPR detection with isothermal amplification techniques hold great promise to advance the POC diagnostics, but commonly require multiple manual operations for postamplification reaction transfer or suffer from compromised performance in terms of sensitivity or assay time. To address these issues, we develop a thermometer-inspired microfluidic biosensing platform that seamlessly integrates CRISPR detection and isothermal amplification through temperature-programmed air expansion for intended fluidic manipulation. This platform not only allows for automated running of the canonical two-step recombinase polymerase amplification-CRISPR/Cas12a assay via a simple two-step temperature profile, but also provides a smartphone-enabled fluorescence readout with a limit of detection of a few DNA copies in 30 min. We detail the rational design of a thermally-responsive microfluidic cartridge and showcase how it harnesses the temperature difference in preamplification and CRISPR detection to implement the assay workflow without the need for an external driving mechanism or human intervention. Furthermore, we validate its applicability for rapid and sensitive detection of Treponema pallidum DNA in clinical samples (n = 30), exhibiting 100 % sensitivity and 100 % specificity compared to the quantitative PCR technique. The simplicity and effectiveness may lead this platform to be a powerful upgrade kit compatible with the existing CRISPR approaches, thus facilitating their widespread point-of-care diagnostic applications. [ABSTRACT FROM AUTHOR]

This paper describes the first demonstration of the combination of micropatterned paper devices (μPPDs) and polydiacetylene (PDA) colorimetric probes for double-stranded DNA (dsDNA) detection with high sensitivity and fast response. The device is rapidly fabricated on paper by wax-screen-printing. The amine-functionalized PDA vesicles are spotted to the paper chambers, followed by the introduction of the polymerase chain reaction (PCR)-amplified dsDNA targets. After the completion of the ionic interactions between the targets and the probes, a significantly different optical signal is produced, thereby facilitating the naked-eye detection. Additionally, the signals are readily documented through a smartphone for quantitative analysis. In this study, the PDA colorimetric assay in μPPDs is capable of discriminating the presence of the target down to 10 nM (the typical concentration of PCR amplicons is around 100 nM). At such concentration, the value of colorimetric response in paper is about 41% higher than that in tube, leading to an enhanced sensitivity. In addition, the colorimetric reaction in μPPDs is accomplished in 1 min, and the analytical speed is much faster than that observed in tube. These results show that the paper-based PDA colorimetry should have greatly enhanced applications as a technology for DNA diagnostics. [ABSTRACT FROM AUTHOR]