A disposable chemical heater and dry enzyme preparation for lysis and extraction of DNA and RNA from microorganisms.

Sample preparation, including bacterial lysis, remains a hurdle in the realization of complete point-of-care tests for many pathogens. Here, we developed a sample preparation methodology for enzymatic lysis and sample heating for low-resource, point-of-care applications. We show an instrument-free chemical heater system for rapid lysis of a gram-positive bacterium (Staphylococcus aureus) and an RNA virus (human respiratory syncytial virus) using a dried lysis enzyme mixture (achromopeptidase) for S. aureus. After a lysis step (<1 minute), lysis enzymes are heat deactivated (<5 minutes) using a simple disposable chemical heater. We demonstrated that both DNA and RNA in the heat-treated sample could be directly amplified without purification, even in the presence of a clinically-obtained human nasal sample. This simple approach to dry enzyme storage and sample heating is adaptable to many applications where samples need to be lysed, including use in low-resource laboratories and in single-use or cartridge-based point-of-care diagnostic devices.

Precision chemical heating for diagnostic devices.

Decoupling nucleic acid amplification assays from infrastructure requirements such as grid electricity is critical for providing effective diagnosis and treatment at the point of care in low-resource settings. Here, we outline a complete strategy for the design of electricity-free precision heaters compatible with medical diagnostic applications requiring isothermal conditions, including nucleic acid amplification and lysis. Low-cost, highly energy dense components with better end-of-life disposal options than conventional batteries are proposed as an alternative to conventional heating methods to satisfy the unique needs of point of care use.

One-step purification and concentration of DNA in porous membranes for point-of-care applications.

The emergence of rapid, user-friendly, point-of-care (POC) diagnostic systems is paving the way for better disease diagnosis and control. Lately, there has been a strong emphasis on developing molecular-based diagnostics due to their potential for greatly increased sensitivity and specificity. One of the most critical steps in developing practical diagnostic systems is the ability to perform sample preparation, especially the purification of nucleic acids (NA), at the POC. As such, we have developed a simple-to-use, inexpensive, and disposable sample preparation system for in-membrane purification and concentration of NAs. This system couples lateral flow in a porous membrane with chitosan, a linear polysaccharide that captures NAs via anion exchange chromatography. The system can also substantially concentrate the NAs. The combination of these capabilities can be used on a wide range of sample types, which are prepared for use in downstream processes, such as qPCR, without further purification.

Electromechanical cell lysis using a portable audio device: enabling challenging sample preparation at the point-of-care.

Audio sources are ubiquitously available on portable electronic devices, including cell phones. Here we demonstrate lysis of Mycobacterium marinum and Staphylococcus epidermidis bacteria utilizing a portable audio device coupled with a simple and inexpensive electromagnetic coil. The resulting alternating magnetic field rotates a magnet in a tube with the sample and glass beads, lysing the cells and enabling sample preparation for these bacteria anywhere there is a cell phone, mp3 player, laptop, or other device with a headphone jack.

Stool cultures for Shigella spp: improved specificity by using MacConkey agar with xylose.

A total of 678 stool specimens were cultured on four different agars: on xylose-lysine-desoxycholate agar (XLD), MacConkey agar (Mac), MacConkey agar supplemented with xylose (Mac-X), and Hektoen enteric agar (HE). Isolation rates for shigellae were 77% on HE, 86% on Mac and Mac-X, and 91% on XLD. The specificities of the media were 61% for Mac, 75% for HE, and 78% for XLD and Mac-X. After overnight incubation, Mac-X is much easier to read than XLD, which requires incubation for at least 22 hours. Based on these results and also on the practical aspect that incubation for 22-21 hours does not fit well in our schedule, we now use Mac-X whenever shigellae need to be looked for (i.e. mainly patients with recent travel to tropical countries). As compared to our previous procedure the workload in the laboratory could be reduced by about 20%.