Vancomycin-resistant gene identification from live bacteria on an integrated microfluidic system by using low temperature lysis and loop-mediated isothermal amplification.

Vancomycin-resistant Enterococcus (VRE) is a kind of enterococci, which shows resistance toward antibiotics. It may last for a long period of time and meanwhile transmit the vancomycin-resistant gene (vanA) to other bacteria. In the United States alone, the resistant rate of Enterococcus to vancomycin increased from a mere 0.3% to a whopping 40% in the past two decades. Therefore, timely diagnosis and control of VRE is of great need so that clinicians can prevent patients from becoming infected. Nowadays, VRE is diagnosed by antibiotic susceptibility test or molecular diagnosis assays such as matrix-assisted laser desorption ionization/time-of-flight mass spectrometry and polymerase chain reaction. However, the existing diagnostic methods have some drawbacks, for example, time-consumption, no genetic information, or high false-positive rate. This study reports an integrated microfluidic system, which can automatically identify the vancomycin resistant gene (vanA) from live bacteria in clinical samples. A new approach using ethidium monoazide, nucleic acid specific probes, low temperature chemical lysis, and loop-mediated isothermal amplification (LAMP) has been presented. The experimental results showed that the developed system can detect the vanA gene from live Enterococcus in joint fluid samples with detection limit as low as 10 colony formation units/reaction within 1 h. This is the first time that an integrated microfluidic system has been demonstrated to detect vanA gene from live bacteria by using the LAMP approach. With its high sensitivity and accuracy, the proposed system may be useful to monitor antibiotic resistance genes from live bacteria in clinical samples in the near future.

Rapid isolation and diagnosis of live bacteria from human joint fluids by using an integrated microfluidic system.

Arthroplasty is a general approach for improving the life quality for patients with degenerative or injured joints. However, post-surgery complications including periprosthetic joint infection (PJI) poses a serious drawback to the procedure. Several methods are available for diagnosing PJI, but they are time-consuming or have poor sensitivity and specificity. Alternatively, reverse-transcription PCR can detect live bacteria and reduce false-positive results but cannot avoid the cumbersome RNA handling and human contamination issues. In response, an integrated microfluidic system capable of detecting live bacteria from clinical PJI samples within 55 minutes is developed in this study. This system employs an ethidium monoazide (EMA)-based assay and a PCR with universal bacterial primers and probes to isolate and detect only the live bacteria that commonly cause PJI. The experimental results indicated that the developed system can detect bacteria in human joint fluids with a detection limit of 10(4) colony formation unit mL(-1). Furthermore, nine clinical samples were analyzed using the microfluidic system. The results obtained from the microfluidic system were negative for all culture-negative cases, indicating that the proposed system can indeed reduce false-positive results. In addition, experimental results showed that the EMA sample pre-treatment process was crucial for successful detection of live bacteria. The culture-positive cases were diagnosed as positive by the proposed system only when the clinical samples were treated with EMA immediately after being sampled from patients. Based on these promising results, the developed microfluidic system can be a useful tool to detect PJI and potentially be applied in other clinical situations.

Detection of viruses directly from the fresh leaves of a Phalaenopsis orchid using a microfluidic system.

Early detection of pathogens is crucial for the effective surveillance of diseases. Many efforts have been made to explore methods which can detect these pathogens within a short period of time without requiring a tedious protocol. However, these developed methods have disadvantages such as they are relatively time-consuming or require specialized laboratory facilities. In this work, we have developed an integrated microfluidic system for rapid and automatic detection of viruses by direct analysis from fresh Phalaenopsis orchid leaves. The entire protocol, including ribonucleic acid (RNA) purification, reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) and optical detection by measuring changes in turbidity was performed on a single chip. This is the first time that an integrated microfluidic system for the detection of viruses infecting the Phalaenopsis orchid has been demonstrated. The sensitivity of the developed system was also explored in this study to validate its performance. FROM THE CLINICAL EDITOR: In this study, the authors report the development of an integrated microfluidic system for rapid and automatic detection of viruses by direct analysis of fresh Phalaenopsis orchid leaves, performing the 3-step protocol using a single chip. Similar methods may find clinical application for fast and accurate detection of viral infections.

Micro flow cytometry utilizing a magnetic bead-based immunoassay for rapid virus detection.

The current study presents a new miniature microfluidic flow cytometer integrated with several functional micro-devices capable of viral sample purification and detection by utilizing a magnetic bead-based immunoassay. The magnetic beads were conjugated with specific antibodies, which can recognize and capture target viruses. Another dye-labeled anti-virus antibody was then used to mark the bead-bound virus for the subsequent optical detection. Several essential components were integrated onto a single chip including a sample incubation module, a micro flow cytometry module and an optical detection module. The sample incubation module consisting of pneumatic micropumps and a membrane-type, active micromixer was used for purifying and enriching the target virus-bound magnetic beads with the aid of a permanent magnet. The micro flow cytometry module and the optical detection module were used to perform the functions of virus counting and collection. Experimental results showed that virus samples with a concentration of 10(3)PFU/ml can be automatically detected successfully by the developed system. In addition, the entire diagnosis procedure including sample incubation and virus detection took only about 40min. Consequently, the proposed micro flow cytometry may provide a powerful platform for rapid diagnosis and future biological applications.