Universally applicable lysis buffer and processing methods for the lysis of bodily samples
Inventors
Klein, Matthias • Lüdke, Gerd • Boos, Andreas
Assignees
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Publication Number
US-9598721-B2
Publication Date
2017-03-21
Expiration Date
Abstract
The present invention provides a universally applicable lysis buffer comprising a chaotropic 5 agent, a reducing agent, and a proteolytic enzyme suitable for processing a wide variety of different sample types, such as different types of bodily samples relevant for the diagnosis of a respiratory disease. Furthermore, the present invention provides the use of a chaotropic agent, a reducing agent, and a proteolytic enzyme for the lysis of a broad spectrum of bodily samples. Moreover, the present invention provides a method for processing bodily samples which is universally applicable to the lysis of a variety of different types of bodily samples. Furthermore, the present invention provides methods for analyzing a bodily sample or for detecting the presence of a pathogen in a bodily sample, preferably, for diagnosing a respiratory disease, such as pneumonia or tuberculosis. Preferably, these methods are universally applicable to a variety of sample types, are applicable as one-tube-processes, are 15 suitable for performance in a high-throughput setting, and are automatable.
Core Innovation
The invention describes a universally applicable lysis buffer and processing of a highly viscous bodily sample having a viscosity of at least 1×10^4 mPa·s. The highly viscous bodily sample is contacted with a combination comprising at least one chaotropic agent, at least one reducing agent, and at least one proteolytic enzyme, thereby forming a mixture of the bodily sample and the combination.
The mixture is incubated and subjected to a heating sequence that includes a first temperature and a second temperature higher than the first temperature. To liquefy the highly viscous bodily sample, bead milling is performed simultaneously to or during the heating steps, including during the first temperature step or the second temperature step, or simultaneously to or during the higher-temperature step.
The described approach is presented as a one-tube workflow compatible with nucleic-acid diagnostics and downstream nucleic acid isolation followed by nucleic acid amplification and analysis, including PCR. The document emphasizes automation, high-throughput processing, reduced contamination/infection risk, and preserving nucleic-acid diagnostics performance.
Claims Coverage
The document includes five independent claims that cover liquefaction of highly viscous bodily samples using chaotropic, reducing, and proteolytic enzyme combinations with staged heating and bead milling, plus downstream use for nucleic acid isolation/analysis and diagnosis of respiratory disease. Across the independent claims, the inventive feature set centers on a viscosity threshold, a three-component reagent combination, two-stage heating with the second temperature higher, and bead milling performed simultaneously to or during at least one heating step.
Liquefaction of highly viscous bodily sample using chaotropic, reducing, and proteolytic enzyme combination with staged heating and simultaneous bead milling
Contacting a highly viscous bodily sample (viscosity at least 1×10^4 mPa·s) with a combination comprising at least one chaotropic agent, at least one reducing agent, and at least one proteolytic enzyme, forming a mixture, heating to a first temperature and then to a second temperature higher than the first temperature, and bead milling the mixture performed simultaneously to or during steps (iii) or (iv), thereby liquefying the highly viscous bodily sample.
Liquefaction using three-component mixture with first and higher second temperature heating and simultaneous-to/during bead milling
Providing a mixture comprising the bodily sample (viscosity at least 1×10^4 mPa·s) with at least one chaotropic agent, at least one reducing agent, and at least one proteolytic enzyme, heating the mixture to a first temperature, heating the mixture to a second temperature higher than the first temperature, and bead milling the mixture with bead milling performed simultaneously to or during steps (ii) or (iii), thereby liquefying the highly viscous bodily sample.
Liquefaction by incubating, staged heating with higher second temperature, and bead milling during the second heating step
Providing a mixture comprising the bodily sample (viscosity at least 1×10^4 mPa·s) with at least one chaotropic agent, at least one reducing agent, and at least one proteolytic enzyme, incubating the mixture, heating the mixture to a first temperature and to a second temperature higher than the first temperature, and bead milling the mixture wherein step (v) is performed simultaneously to or during step (iv), thereby liquefying the highly viscous bodily sample.
Diagnosing a respiratory disease using liquefaction workflow and applying the mixture to a diagnosis method
Providing a mixture comprising a highly viscous bodily sample (viscosity at least 1×10^4 mPa·s) with at least one chaotropic agent, at least one reducing agent, and at least one proteolytic enzyme, incubating the mixture, heating to a first temperature and then to a second temperature higher than the first temperature, bead milling the mixture performed simultaneously to or during steps (iii) or (iv) to liquefy the highly viscous bodily sample, and applying the mixture to a method suitable for diagnosing the respiratory disease.
Across the independent claims, the core claimed coverage is a liquefaction workflow for highly viscous bodily samples defined by a viscosity of at least 1×10^4 mPa·s, using a combination of at least one chaotropic agent, at least one reducing agent, and at least one proteolytic enzyme, followed by staged heating with a higher second temperature and bead milling performed simultaneously to or during the defined heating steps. One independent claim further requires applying the liquefied mixture to a respiratory-disease diagnostic method.
Stated Advantages
Universally applicable lysis buffer for diverse highly viscous bodily samples, especially respiratory samples.
Compatibility with nucleic-acid diagnostics, including nucleic acid isolation and downstream PCR/analysis.
High-throughput and automation, reproducing manual performance.
Reduced contamination/infection risk.
Improved handling of highly viscous samples by liquefaction enabling nucleic-acid analysis.
Documented Applications
Processing highly viscous bodily samples for nucleic-acid diagnostics, including nucleic acid isolation and nucleic acid amplification/analysis (PCR).
Pathogen detection from processed highly viscous bodily samples compared with culture.
Diagnosing a respiratory disease in a subject using a processed mixture applied to a method suitable for diagnosing the respiratory disease.
Processing respiratory samples such as sputum and tracheal secretion, and other viscous bodily samples such as BAL, gastric juice, blood, pleural fluid/punctates, swabs/aspirates/lavages, and pus.
Application contexts mentioned for respiratory diseases including pneumonia, tuberculosis, bronchitis, COPD-related infections, and respiratory tumors.
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