Integrated and universal ultrasonic microbial lysis and extraction

Quantitative lysis and extraction of high quality microbial nucleic acidsNAis a key to successful analysis of diverse samplesIn microbiome research and future diagnostics accurate enumerationidentificationand functional assessment of the members of microbial communities requires highly efficient extraction of NA without bias toward particular species or kinds of microorganismsSimilarlyefficient extraction of NA from blood samples suspected of infectionsepsiscoupled with sensitive molecular analysiscan directly impact the diagnosistreatmentand health outcomes of patients in profound waysAn ideal microbial sample preparation method would allow quantitative and unbiased NA extraction of samples with as few asorganisms mL with minimal damage due to NA shearing and heat degradation and a user friendly workflowBias in microbiome extraction due to differing enzymatic extraction efficiencies across organisms is alleviated to a degree by mechanical lysisbead beatinga relatively inefficient and difficult to automate method that can shear NA excessively and damage RNA through generated heatBead beating is also used in several sepsis detection systems operating with the highest sensitivityThe replacement of bead beating with a more efficient method will improve workflow and device manufacturability and yield greater sensitivityCovaris will further develop its beadless microbial lysis method is based on cavitation caused by high frequency ultrasound acting on engineered plastic substratesMicrobiome and sepsis samples will be processed using a centrifugal cartridgewith AFA inducing cavitation for lysiswhile centrifugal force and cartridge design will automate fluid handling and provide centrifugal separationsFor microbiome samplesmicrobes will be trappedand AFA will be applied to lyse them under the flow of buffereasy to lyse microbes will be lysed immediatelywhile refractory microbes will remain trapped for longer timesThis will result in a continuous extraction of DNA and will significantly improve on the benefits already seen using multiple rounds of AFAgreater extraction efficiency and higher measured microbial diversityFor sepsistwo workflows will be examinedFirstcentrifugal separation of the plasma fraction containingof microbes will be followed by lysis of trapped microbesas described for microbiome samplesseconda basic lysis solution will be used to lyse whole blood and inactivate human NA prior to buffer neutralization and AFA induced microbial lysisThese methods will be developed using mock fecal microbiomes and mock sepsis samples consisting of microbe spiked whole bloodwith analysis by qPCR for DNA extractionRT PCR for RNA extractionand fragment size analysis Quantitative lysis and extraction of high quality microbial nucleic acidsNAis a key to successful analysis of microbe containing samplessuch as human fecesfor microbiome researchand whole bloodfor blood infection or sepsis detectionThis project will develop a self containedultrasound enabled NA extraction system in a centrifugal cartridge to achieve lower limits of detection and more representative microbial genetic dataThis 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