Methods for measuring enzyme activity useful in determining cell viability in non-purified samples
Inventors
O'Hara, Shawn Mark • Zweitzig, Daniel
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10870894-B2
Publication Date
2020-12-22
Expiration Date
Abstract
Methods for detecting microorganisms, in particular detection of bacteria and methods for measuring enzyme activity, such as Deoxyribonucleic acid (DNA) polymerase activity are disclosed. The aforesaid methods include, but are not limited to such methods performed on microbial crude lysates, useful for determining microbial enzyme activities, which can be linked to amplification signal generators such as real-time Polymerase Chain Reaction (PCR) techniques, thereby enabling determination of microbial pathogens in samples such as unpurified blood and other body fluids. Moreover, the disclosed embodiments also relate to reagents for use in such methods, and to test kits comprising such reagents for carrying out the methods.
Core Innovation
The invention provides a method for detecting the presence of a bacterium or a fungus in a sample by detecting DNA polymerase activity as an indicator of the presence of the bacterium or fungus. The method uses sample preparation that allows for only bacterium or fungi derived DNA polymerase activity to modify a DNA polymerase substrate. The workflow includes contacting the prepared sample with a nucleic acid molecule that acts as a substrate for DNA polymerase activity and incubating under conditions suitable for DNA polymerase activity to generate an extension product.
The nucleic acid substrate is a partially double-stranded DNA substrate consisting of a sense strand DNA oligonucleotide and an antisense strand DNA oligonucleotide. The strands overlap to form a double-stranded region and a single-stranded portion of the antisense strand acts as a template, while the sense strand of the double-stranded region acts as a primer to create an extension product in the presence of DNA polymerase activity. After generating the extension product, the method amplifies at least a portion of the extension product to generate an amplicon and detects the amplicon, thereby indicating the presence of the bacterium or fungus.
In blood or blood culture samples, the invention further includes recovering bacteria or fungi from the blood or blood culture sample before contacting the recovered bacteria or fungi with the partially double-stranded DNA substrate. The recovered organisms are contacted with the nucleic acid substrate, incubated to generate the extension product, and then an amplicon is generated by amplifying at least a portion of the extension product and detecting the amplicon. A further variant includes performing lysis of the recovered bacteria or fungi prior to contacting the DNA substrate.
Claims Coverage
Two independent claims are explicitly provided. They share a core inventive concept of detecting DNA polymerase activity using a partially double-stranded DNA substrate that yields an extension product followed by amplification and detection of an amplicon; they differ mainly in how the biological material is prepared.
DNA polymerase activity as indicator of bacterium or fungus presence
A method for detecting the presence of a bacterium or a fungus in a sample, wherein DNA polymerase activity is detected as an indicator of the presence of said bacterium or fungus in said sample.
Differential cell lysis enabling bacterium or fungi derived polymerase modification of the substrate
Preparing the sample using a differential cell lysis sample preparation method, thereby allowing for only bacterium or fungi derived DNA polymerase activity to modify a DNA polymerase substrate.
Partially double-stranded DNA substrate that uses template and primer regions to create an extension product
Contacting the prepared sample with a nucleic acid molecule which acts as a substrate for DNA polymerase activity, wherein the substrate is a partially double-stranded DNA substrate consisting of a sense strand DNA oligonucleotide and an antisense strand DNA oligonucleotide overlapping to form a double-stranded region and a single-stranded portion of the antisense strand acts as a template with the sense strand of the double-stranded region acting as a primer to create an extension product in the presence of DNA polymerase activity.
Amplifying and detecting an amplicon generated from the polymerase extension product
Incubating the contacted prepared sample and substrate under conditions suitable for DNA polymerase activity to generate the extension product, and amplifying at least a portion of the extension product to generate an amplicon and detecting the amplicon, thereby indicating the presence of the bacterium or fungus.
Recovering bacteria or fungi from blood or blood culture prior to polymerase substrate contact
Recovering bacteria or fungi from the blood or blood culture sample before contacting the recovered bacteria or fungi with a nucleic acid molecule that acts as a substrate for polymerase activity.
Using the partially double-stranded DNA substrate with recovered organisms to generate extension product, then amplicon detection
Contacting the recovered bacteria or fungi with a partially double-stranded DNA substrate consisting of a sense strand DNA oligonucleotide and an antisense strand DNA oligonucleotide overlapping to form a double-stranded region and a single-stranded portion of the antisense strand acting as a template with the sense strand acting as a primer to create an extension product in the presence of DNA polymerase activity, incubating to generate the extension product, amplifying at least a portion of the extension product to generate an amplicon, and detecting the amplicon thereby indicating the presence of the bacterium or fungus.
Across both independent claims, the inventive coverage centers on using DNA polymerase activity as an indicator of bacterium or fungus presence, generating a polymerase-dependent extension product from a partially double-stranded DNA substrate, and then detecting the resulting amplicon after amplification. The claim set differentiates preparation of the biological input by using differential cell lysis in the general sample method and by recovering bacteria or fungi specifically from blood or blood culture in the blood-focused method.
Stated Advantages
Detecting the presence of a bacterium or fungus by detecting DNA polymerase activity as an indicator of presence.
Reducing polymerase background/false positives by using differential cell lysis to allow only bacterium or fungi derived DNA polymerase activity to modify the DNA polymerase substrate.
Generating a detectable extension product that enables downstream amplification to generate and detect an amplicon.
Sensitivity down to femtogram/attogram levels is described.
Improved variants incorporating NAD-dependent DNA ligase activity and phosphatase activity are described as supporting viability indication and mitigating interfering DNA polymerase signals when targeting ligase/phosphatase.
Documented Applications
Detecting viable microorganisms (bacteria and fungus) in non-purified samples, including crude lysates and unpurified blood/blood cultures.
Detection in blood or blood culture samples using recovery of bacteria or fungi, polymerase substrate contact, amplification of extension product, and detection of the resulting amplicon.
Antimicrobial/anti-polymerase agent detection for managing patients, including diagnostic measurement and detection of agents having antimicrobial and/or anti-polymerase activity, tied to genome or transcriptome analysis.
Interested in licensing this patent?