Quantitative lysis and extraction of high-quality microbial nucleic acids (NA) is a key to successful analysis of diverse samples. In microbiome research?and future diagnostics?accurate enumeration, identification, and functional assessment of the members of microbial communities requires highly efficient extraction of NA without bias toward particular species or kinds of microorganisms. Similarly, efficient extraction of NA from blood samples suspected of infection (sepsis), coupled with sensitive molecular analysis, can directly impact the diagnosis, treatment, and health outcomes of patients in profound ways. An ideal microbial sample preparation method would allow quantitative and unbiased NA extraction of samples with as few as 1-10 organisms/mL with minimal damage due to NA shearing and heat degradation and a user-friendly workflow. Bias in microbiome extraction due to differing enzymatic extraction efficiencies across organisms is alleviated to a degree by mechanical lysis (bead beating), a relatively inefficient and difficult-to-automate method that can shear NA excessively and damage RNA through generated heat. Bead beating is also used in several sepsis detection systems operating with the highest sensitivity. The replacement of bead beating with a more efficient method will improve workflow and device manufacturability and yield greater sensitivity. Covaris will further develop its beadless microbial lysis method is based on cavitation caused by high frequency ultrasound acting on engineered plastic substrates. Microbiome and sepsis samples will be processed using a centrifugal cartridge, with AFA inducing cavitation for lysis, while centrifugal force and cartridge design will automate fluid-handling and provide centrifugal separations. For microbiome samples, microbes will be trapped, and AFA will be applied to lyse them under the flow of buffer; easy-to-lyse microbes will be lysed immediately, while refractory microbes will remain trapped for longer times. This will result in a continuous extraction of DNA and will significantly improve on the benefits already seen using multiple rounds of AFA: greater extraction efficiency and higher measured microbial diversity. For sepsis, two workflows will be examined: First, centrifugal separation of the plasma fraction containing ~50% of microbes will be followed by lysis of trapped microbes, as described for microbiome samples; second, a basic lysis solution will be used to lyse whole blood and inactivate human NA prior to buffer neutralization and AFA-induced microbial lysis. These methods will be developed using mock fecal microbiomes and mock sepsis samples consisting of microbe-spiked whole blood, with analysis by qPCR for DNA extraction, RT-PCR for RNA extraction, and fragment size analysis.
Quantitative lysis and extraction of high-quality microbial nucleic acids (NA) is a key to successful analysis of microbe-containing samples, such as human feces (for microbiome research) and whole blood (for blood infection or sepsis detection). This project will develop a self-contained, ultrasound-enabled NA extraction system in a centrifugal cartridge to achieve lower limits of detection and more representative microbial genetic data. This system should positively impact human health by providing simpler and more accurate tools for the detection of infection and understanding of microbial populations in the human microbiome.
