Methods for measuring enzyme activity useful in determining cell viability in non-purified samples
Inventors
O'Hara, Shawn Mark • Zweitzig, Daniel
Assignees
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Publication Number
US-10876173-B2
Publication Date
2020-12-29
Expiration Date
Abstract
An assay kit of reagents including a nucleic acid capable of acting as substrate for polymerase microorganism activity useful in a method of detecting polymerase activity as an indicator of the presence of a micro-organism in a sample are disclosed. 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 relates to sensitive detection of polymerase activity as an indicator of the presence of a microorganism in a sample, including non-purified samples and crude microbial lysates. A DNA substrate is provided that generates a detectable nucleic acid extension product in the presence of polymerase activity. The DNA substrate includes a DNA sense strand and an immobilised DNA antisense strand, where the two strands overlap to form a double stranded region and a single stranded portion of the antisense strand acts as a template with the sense strand acting as a primer to create an extension product when polymerase activity is present.
The approach is further defined by an assay kit that includes separate forward and reverse primers having sequences substantially identical to parts of the sense and antisense strands, respectively, from the immobilised DNA substrate. In an embodiment, the antisense strand contains uracil. The presence of polymerase activity is thereby linked to the generation of an extension product from the nucleic-acid substrate in the assay context used for microorganism detection.
The described problem is false positives and background from polymerase activity in unpurified lysates that can interfere with detection, including interference by DNA polymerase background when measuring ligase-related approaches in crude lysates. The described solution includes assay design features where unreacted nucleic-acid substrate does not contribute to signal, and the disclosed use of specific substrate/enzyme handling strategies, including uracil-containing substrates and polymerase interference strategies, to improve specificity and sensitivity when assaying viable microorganisms, particularly bacteria, in non-purified samples.
Claims Coverage
The partial claim set provided includes two independent assay-kit claims. Each independent claim centers on a DNA-substrate architecture that produces an extension product in the presence of polymerase activity, combined with separate forward and reverse primers that are substantially identical to portions of the sense and antisense sequences.
DNA sense strand and immobilised antisense strand overlapping to form double stranded region and single stranded template for extension product creation
An assay kit for detecting polymerase activity as an indicator of the presence of a microorganism, comprising a DNA substrate consisting of a DNA sense strand and an immobilised DNA antisense strand, wherein the two strands overlap 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 polymerase activity.
Separate forward primer and reverse primer substantially identical to parts of antisense and sense strands
An assay kit for detecting polymerase activity as an indicator of the presence of a microorganism, comprising a reverse primer having a sequence substantially identical to part of the sequence of the antisense strand and a forward primer having a sequence substantially identical to part of the sequence of the sense strand, wherein the forward primer and the reverse primer are separate molecules from the DNA substrate.
Uracil-containing DNA antisense strand overlapping with sense strand to form double stranded region and single stranded template for extension product creation
An assay kit for detecting polymerase activity as an indicator of the presence of a microorganism, comprising a DNA substrate consisting of a DNA sense strand and a uracil containing DNA antisense strand, wherein the two strands overlap 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 polymerase activity.
Across the independent claims, coverage is directed to an assay kit architecture where a sense strand and an antisense strand overlap to define a double stranded region and a single stranded template portion to enable primer-initiated extension products in the presence of polymerase activity, while using separate forward and reverse primers substantially identical to parts of the sense and antisense sequences, including an embodiment with a uracil containing antisense strand.
Stated Advantages
Reduced false positives because unreacted nucleic-acid substrate does not contribute to signal.
Very high analytical sensitivity (femtogram/attogram levels).
Improved specificity versus prior ligase-based approaches that fail in unpurified lysates due to interfering DNA polymerase background.
Direct assay on gently lysed crude samples.
Documented Applications
Detecting polymerase activity as an indicator of the presence of a microorganism in non-purified samples, including crude microbial lysates.
Detection of viable microorganisms, including bacteria, in non-purified samples using enzyme activity with nucleic-acid substrates that generate a novel detectable nucleic acid product.
Assaying microbe-spiked blood culture lysates.
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