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.

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.

The effect of immunization route on rat serum and tear antibody responses to Chlamydia trachomatis.

The effect of immunization route on the kinetics of serum and tear antibody responses to Chlamydia trachomatis was studied in a rat model. Rats received Chlamydia trachomatis serovar C/TW elementary bodies for two immunization cycles by ocular topical (OT) application or subconjunctival (SC) injection and in a second experiment for three immunization cycles by either OT application, gastrointestinal intubation (GI), or intraperitoneal injection (IP). Serum IgG and tear IgA antibodies to whole elementary bodies were measured sequentially following the secondary (2 degrees) and tertiary (3 degrees) immunization cycles. Serum IgG levels were minimal in the OT immunized group following 2 degrees and 3 degrees immunization. Serum IgG levels for the SC, GI, and IP immunized groups rose steadily following 2 degrees immunization reaching maximal levels between day (d)24 and d31. Levels of serum IgG antibodies were highest in the GI group following 3 degrees immunization. Tear IgA antibody responses were greatest in rats immunized by the OT route, the 2 degrees IgA response peaked by d9 and declined by d24. The 3 degrees OT tear IgA response peaked by d13 and was greater than the 2 degrees response. Tear IgA antibody levels in SC and GI immunized rats appeared by d3 following 2 degrees immunization but remained at low levels and were not noted until d20 in the GI group following 3 degrees immunization. Tear IgA antibody responses were not detectable in IP animals following 2 degrees or 3 degrees immunization. This study documents the immunogenicity of Chlamydia trachomatis serovar C/TW in the rat and shows that the OT route is most effective in eliciting IgA antibody responses in tears.

Induction of the cortical reaction in isolated sea urchin egg cortices: effects of Ca2+ and ionophore A23187.

The cortical reaction in isolated sea urchin (Strongylocentrotus purpuratus) egg cortices has been monitored with phase-contrast video microscopy. It was confirmed that the cortical reaction is induced by exposure to Ca2+. No induction was observed after exposure to the Ca2+-ionophore A23187, although the cortices remain sensitive to a subsequent exposure to Ca2+, and the cortical reaction in unfertilized eggs suspended in cortex isolation medium remains inducible by exposure to A23187. These results imply: (1) that A23187 does not induce the cortical reaction directly; (2) that the release of intracellular Ca2+, through which A23187 induces the cortical reaction, is not from storage sites localized entirely in the cortex; and (3) that intracellular storage sites for the Ca2+ involved in the cortical reaction are also present outside the cortex.