Tseng-Ming Hsieh

A self-contained all-in-one cartridge for sample preparation and real-time PCR in rapid influenza diagnosis

Herein we present a fully automated system with pseudo-multiplexing capability for rapid infectious disease diagnosis. The all-in-one system was comprised of a polymer cartridge, a miniaturized thermal cycler, 1-color, 3-chamber fluorescence detectors for real-time reverse transcription polymerase chain reaction (RRT-PCR), and a pneumatic fluidic delivery unit consisting of two pinch-valve manifolds and two pneumatic pumps. The disposable, self-contained cartridge held all the necessary reagents for viral RNA purification and reverse transcription polymerase chain reaction (RT-PCR) detection, which took place all within the completely sealed cartridge. The operator only needed to pipette the patients sample with lysis buffer into the cartridge, and the system would automatically perform the entire sample preparation and diagnosis within 2.5 h. We have successfully employed this system for seasonal influenza A H1N1 typing and sub-typing, obtaining comparable sensitivity as the experiments conducted using manual RNA extraction and commercial thermal cycler. A minimum detectable virus loading of 100 copies per μl has been determined by serial dilution experiments. This all-in-one desktop system would be suitable for decentralized disease diagnosis at immigration check points and outpatient clinics, and would not require highly skilled operators.

A self-contained polymeric cartridge for automated biological sample preparation

Sample preparation is one of the most crucial processes for nucleic acids based disease diagnosis. Several steps are required for nucleic acids extraction, impurity washes, and DNA/RNA elution. Careful sample preparation is vital to the obtaining of reliable diagnosis, especially with low copies of pathogens and cells. This paper describes a low-cost, disposable lab cartridge for automatic sample preparation, which is capable of handling flexible sample volumes of 10 μl to 1 ml. This plastic cartridge contains all the necessary reagents for pathogen and cell lysis, DNA/RNA extraction, impurity washes, DNA/RNA elution and waste processing in a completely sealed cartridge. The entire sample preparation processes are automatically conducted within the cartridge on a desktop unit using a pneumatic fluid manipulation approach. Reagents transportation is achieved with a combination of push and pull forces (with compressed air and vacuum, respectively), which are connected to the pneumatic inlets at the bottom of the cartridge. These pneumatic forces are regulated by pinch valve manifold and two pneumatic syringe pumps within the desktop unit. The performance of this pneumatic reagent delivery method was examined. We have demonstrated the capability of the on-cartridge RNA extraction and cancer-specific gene amplification from 10 copies of MCF-7 breast cancer cells. The on-cartridge DNA recovery efficiency was 54-63%, which was comparable to or better than the conventional manual approach using silica spin column. The lab cartridge would be suitable for integration with lab-chip real-time polymerase chain reaction devices in providing a portable system for decentralized disease diagnosis.