DNA polymerase activity assays and methods enabling determination of viable microbes
Inventors
O'Hara, Shawn Mark • Zweitzig, Daniel
Assignees
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Publication Number
US-9708651-B2
Publication Date
2017-07-18
Expiration Date
Abstract
A method for performing a diagnostic assay for the detection of the presence or amount of a microorganism within a sample matrix containing active DNA polymerase, is disclosed. The method utilizes the measurement of DNA polymerase extension activity, wherein the assay comprises the steps of incubating DNA polymerase in the sample matrix with a selected suitable substrate, and performing PCR cycling and detection via the use of a selected suitable nucleic acid probe, thereby to detect endogenous DNA polymerase extension activity in the sample matrix as an indication of the presence or amount of said microorganism.
Core Innovation
The diagnostic assay detects the presence or amount of a microorganism within a sample matrix containing active DNA polymerase by measuring DNA polymerase extension activity. The assay incubates DNA polymerase in the sample matrix with a substrate comprising two preannealed oligonucleotides in which one oligonucleotide contains deoxyuridine nucleosides, and the extension activity is then detected as an indication of the presence or amount of the microorganism.
The method performs PCR cycling and includes detection via the use of a selected suitable-nucleic acid probe to detect endogenous DNA polymerase extension activity in the sample matrix. The approach is described as a diagnostic DPE-PCR / ETGA method that uses a pre-annealed oligonucleotide substrate with uracil and qPCR probe detection after PCR cycling.
The detected signal is extension-specific and correlates with biological viability, including evidence using ddCTP termination and dCTP rescue and comparisons involving exonuclease-free polymerase. A key aspect presented is that measuring extension activity provides a viability-linked microbial readout rather than relying on genomic DNA detection.
Claims Coverage
The independent claim recites three inventive feature groups: incubating DNA polymerase with a substrate of two preannealed oligonucleotides where one contains deoxyuridine nucleosides, performing PCR cycling, and detecting via a selected suitable-nucleic acid probe to detect endogenous DNA polymerase extension activity as an indication of microorganism presence or amount. Dependent claims further refine extension specificity, relate the measured activity to bacterial viability, constrain sample matrix forms, and add optional process elements such as a bead mill lysis tube and a blocking agent.
Diagnostic detection by measuring endogenous DNA polymerase extension activity
A method for performing a diagnostic assay for the detection of the presence or amount of a microorganism within a sample matrix containing active DNA polymerase by the measurement of DNA polymerase extension activity, thereby to detect endogenous DNA polymerase extension activity in the sample matrix as an indication of the presence or amount of said microorganism.
Two preannealed oligonucleotide substrate with deoxyuridine nucleosides
Incubating DNA polymerase in the sample matrix with a substrate comprising two preannealed oligonucleotides wherein one of said oligonucleotides contains deoxyuridine nucleosides.
PCR cycling followed by probe-based nucleic acid detection
Performing PCR cycling and detection via the use of a selected suitable-nucleic acid probe.
Extension-specific detection excluding signals from other enzymatic modifications
Specifically detect microbial DNA polymerase extension activity while ensuring the signal does not come from substrate modification by enzymatic activities other than DNA polymerase.
Use of DNA polymerase extension activity as an indicator of bacterial viability
Measuring DNA polymerase extension activity is used as an indicator of bacterial viability in the sample matrix.
Sample matrix selected from purified enzymes, microbial lysates, or crude microbial lysates
The sample matrix is chosen from purified enzymes, microbial lysates, or crude microbial lysates.
Bead mill lysis tube disruption prior to assay incubation
Including, before performing the assay, adding a microbial lysate or crude microbial lysate to a bead mill lysis tube to disrupt microorganism cells and transferring the disrupted cells into the assay incubation step.
Blocking agent added to the sample mixture
Adding a blocking agent to the sample mixture.
Overall claim coverage centers on a diagnostic assay that measures endogenous microbial DNA polymerase extension activity using an incubation step with a two preannealed oligonucleotide substrate, followed by PCR cycling and probe-based detection. Dependent claims add refinements for extension specificity, link the readout to bacterial viability, constrain sample matrix forms, and optionally incorporate a bead mill lysis tube disruption step and a blocking agent.
Stated Advantages
Detects endogenous DNA polymerase extension activity as an indication of the presence or amount of a microorganism.
Produces an extension-specific signal linked to DNA polymerase activity.
Enables direct microbial detection from crude bead-mill lysates.
Supports pan detection across multiple microbes.
DNA polymerase extension activity correlates with viability after heat treatment, while genomic DNA detection does not.
Documented Applications
Diagnostic detection of the presence or amount of a microorganism within a sample matrix containing active DNA polymerase.
Assessment of bacterial viability using DNA polymerase extension activity, including correlation with viability after heat treatment.
Broad microbial detection across multiple microbes tested, including gram-positive/gram-negative bacteria and Candida species.
Detection directly from crude bead-mill lysates derived from microorganisms.
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