Qvella Corporation

Aye Aye Khine

Samad Talebpour

Tino Alavie

Robert Maaskant

Stephen Wesley Leonard

Vilcy Parmar

Sukhdev Manku

Alaleh Samiei

Samad Talebour

Stephan W Leonard

Methods and compositions are provided for the selective lysis of eukaryotic cells and the separation of microbial cells. Blood cells and/or other eukaryotic cells in a sample, may be selectively lysed by adding, to the sample, a blood lysis reagent including saponin and an alkaline buffer, and optionally sodium polyanethole sulfonate and a non-ionic surfactant, thereby forming a mixture, and agitating the mixture. Microbial cells in the mixture may then be separated, for example, using a separation method such as centrifugation or filtration, and optionally detected or cultured in growth media. Blood lysis reagent compositions are provided that are suitable for preserving the intactness of microbial cells upon mixing with the sample. In example embodiments in which the sample is a blood sample, the blood lysis reagent composition may be selected to avoid or reduce the presence of visible blood debris upon centrifugation or filtration.

Methods and compositions for the selective lysis of blood cells and separation of microbial cells

Methods are provided for performing antibiotic susceptibility testing based on the detection of RNA, such as tmRNA, from microbial cells after exposure to antibiotics. In some embodiments, aliquots are obtained from a sample, one of which contains a selected antibiotic. The aliquots, which include growth media, are incubated under conditions suitable for microbial growth, and the microbial cells in each aliquot are removed and lysed, and the lysate is subjected to reverse transcription and amplification in infer the effect of the selected antibiotic on the microbial cells. In one embodiment, a sample containing microbial cells is incubated in the presence of a selected antibiotic and a stimulus is provided to induce the production of m RNA within the microbial cells. The microbial cells are subsequently lysed without substantial degradation of the m RNA within the lysate, and the m RNA is detected to determine the effect of the antibiotic on the microbial cells.

Methods of targeted antibiotic susceptibility testing

Devices and methods are provided for electrically lysing cells and releasing macromolecules from the cells. A microfluidic device is provided that includes a planar channel having a thickness on a submillimeter scale, and including electrodes on its upper and lower inner surfaces. After filling the channel with a liquid, such that the channel contains cells within the liquid, a series of voltage pulses of alternating polarity are applied between the channel electrodes, where the amplitude of the voltage pulses and a pulse width of the voltage pulses are effective for causing irreversible electroporation of the cells. The channel is configured to possess thermal properties such that the application of the voltage produces a rapid temperature rise as a result of Joule heating for releasing the macromolecules from the electroplated cells. The channel may also include an internal filter for capturing and concentrating the cells prior to electrical processing.

Methods and devices for electrical sample preparation

Methods and devices are provided for pretreatment of a sample containing microbial cells. In some embodiments, the pretreatment of the sample is performed via the initial selective lysis, within a sample pretreatment vessel, of non-microbial cells (such as blood cells) and the subsequent centrifugal separation of the sample to remove the resulting debris and concentrate the microbial cells. An immiscible and dense cushioning liquid may be included for collecting the microbial cells adjacent to the liquid interface formed by the cushioning liquid upon centrifugation of the pretreatment vessel. After removal of a substantial quantity of the supernatant, resuspension of the collected microbial cells, and re-establishment of the cushioning liquid interface, at least a portion of the remaining suspension may be removed without substantially removing the cushioning liquid. One or more intermediate wash cycles may be performed prior to extraction of the remaining suspension, which provides a 'pretreated' sample.

Method for pretreatment of microbial cells

The present invention provides a microfluidic devices and methods of use thereof for the concentration and capture of cells. A pulsed non Faradaic electric field is applied relative to a sample under laminar flow, which results to the concentration and capture of charged analyte. Advantageously, pulse timing is selected to avoid problems associated with ionic screening within the channel. At least one of the electrodes within the channel is coated with an insulating layer to prevent a Faradaic current from flowing in the channel. Under pulsed application of a unipolar voltage to the electrodes, charged analyte within the sample is moved towards one of the electrodes via a transient electrophoretic force.

Cell concentration, capture and lysis devices and methods of use thereof

Methods are provided for the stabilization and separation of nucleic acids from a sample via contact of the sample with a lysis and stabilization reagent that includes a cationic detergent. The cationic detergent lyses cells in the sample and stabilizes the released nucleic acids via the formation of nucleic acid-surfactant (NAS) complexes. The NAS complexes are centrifugally precipitated, washed, the resuspended in an aqueous resuspension liquid, forming a NAS complex suspension. The suspension is thermally processed to disintegrate the NAS complexes, thereby releasing the nucleic acids and forming a nucleic acid solution. In some example embodiments, the aqueous resuspension liquid is selected to be suitable for performing molecular amplification assays, such that the nucleic acid solution may be employed for performing a molecular amplification assay in the absence of further nucleic acid extraction. Examples are provided whereby the present methods are adapted for performing transcriptomic biomarker assays.

Methods of performing nucleic acid stabilization and separation

Methods and devices are provided for pre-treatment of a sample containing microbial cells. In some embodiments, the pre-treatment of the sample is performed via the initial selective lysis, within a sample pre-treatment vessel, of non-microbial cells, such as blood cells, and the subsequent centrifugal separation of the sample to remove the resulting debris and concentrate the microbial cells. An immiscible and dense cushioning liquid may be included for collecting the microbial cells adjacent to the liquid interface formed by the cushioning liquid upon centrifugation of the pre-treatment vessel. After removal of a substantial quantity of the supernatant, resuspension of the collected microbial cells, and re-establishment of the cushioning liquid interface, at least a portion of the remaining suspension may be removed without substantially removing the cushioning liquid. One or more intermediate wash cycles may be performed prior to extraction of the remaining suspension, which provides a 'pretreated' sample.

Apparatus and method for extracting microbial cells

Devices and methods are provided for electrically lysing cells and releasing macromolecules from the cells. A microfluidic device is provided that includes a planar channel having a thickness on a submillimeter scale, and including electrodes on its upper and lower inner surfaces. After filling the channel with a liquid, such that the channel contains cells within the liquid, a series of voltage pulses of alternating polarity are applied between the channel electrodes, where the amplitude of the voltage pulses and a pulsewidth of the voltage pulses are effective for causing irreversible electroporation of the cells. The channel is configured to possess thermal properties such that the application of the voltage produces a rapid temperature rise as a result of Joule heating for releasing the macromolecules from the electroplated cells. The channel may also include an internal filter for capturing and concentrating the cells prior to electrical processing.

Methods are provided for performing antibiotic susceptibility testing based on the detection of RNA, such as tmRNA, from microbial cells after exposure to antibiotics. In some embodiments, aliquots are obtained from a sample, one of which contains a selected antibiotic. The aliquots, which include growth media, are incubated under conditions suitable for microbial growth, and the microbial cells in each aliquot are removed and lysed, and the lysate is subjected to reverse transcription and amplification in infer the effect of the selected antibiotic on the microbial cells. In one embodiment, a sample containing microbial cells is incubated in the presence of a selected antibiotic and a stimulus is provided to induce the production on m RNA within the microbial cells. The microbial cells are subsequently lysed without substantial degradation of the m RNA within the lysate, and the m RNA is detected to determine the effect of the antibiotic on the microbial cells.

Method for pretreatment of microbial samples

The present invention provides a microfluidic devices and methods of use thereof for the concentration and capture of cells. A pulsed non-Faradaic electric field is applied relative to a sample under laminar flow, which results to the concentration and capture of charged analyte. Advantageously, pulse timing is selected to avoid problems associated with ionic screening within the channel. At least one of the electrodes within the channel is coated with an insulating layer to prevent a Faradaic current from flowing in the channel. Under pulsed application of a unipolar voltage to the electrodes, charged analyte within the sample is moved towards one of the electrodes via a transient electrophoretic force.

Methods and devices are provided for pretreatment of a sample containing microbial cells. In some embodiments, the pretreatment of the sample is performed via the initial selective lysis, within a sample pretreatment vessel, of non-microbial cells (such as blood cells) and the subsequent centrifugation of the sample to remove the resulting debris and concentrate the microbial cells. An immiscible and dense cushioning liquid may be included, for collecting the microbial cells adjacent to a liquid interface formed by the cushioning liquid, upon centrifugation of the pretreatment vessel. After removal of a substantial quantity of the supernatant, resuspension of the collected microbial cells, and re-establishment of the liquid interface, at least a portion of the remaining suspension may be removed without substantially removing the cushioning liquid. One or more intermediate wash cycles may be performed to prior to extraction of the pretreated sample.

Growing community of inventors

Thornhill, Canada

Aye Aye Khine