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Methods for measuring enzyme activity useful in determining cell viability in non-purified samples

Inactive Publication Date: 2013-08-01
MOMENTUM BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes how the present invention has been tested using polymerase enzymes and has shown that certain substrates are sensitive and specific in detecting nucleic acids in crude cell lysates. This technology can be used in various applications such as microbe detection and blood culture analysis. Its technical effect is to provide a reliable and reliable method for detecting nucleic acids in various crude cell lysates.

Problems solved by technology

Culture approaches can be used to detect small numbers of bacteria but such techniques require several days to complete, especially when attempting to detect small numbers of bacteria and also when detecting slower growing microorganisms.
However, a number of problems exist with respect to bioluminescence detection.
For example, detection of microbial ATP only, in the presence of ATP from non-microbial sources can be a problem.
Accordingly, it can be seen that a number of problems exist with respect to the conventional art of microbe detection.
However, in the experiments leading to the development of the present invention, it was found that the techniques and teachings described in this published patent application WO / 2009 / 007719 could not be applied to the determination of viable microorganisms in unpurified samples, such as crude microbial lysates, blood or blood cultures, thereby constituting a major drawback of the technology as described in this reference.
However, it has been discovered that these methodologies, too, have problems.

Method used

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  • Methods for measuring enzyme activity useful in determining cell viability in non-purified samples
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  • Methods for measuring enzyme activity useful in determining cell viability in non-purified samples

Examples

Experimental program
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Effect test

example 1

Discovery of a Ligase Independent Mechanism

[0055]Three different DNA substrates (A) were incubated with E. coli ligase or no ligase and subjected to PCR containing full length DNA ligase substrate specific PCR primers in the presence / absence of UNG. PCR was monitored via SYBR green (qPCR) and the resultant reactions were subjected to gel analysis (B). Three different DNA substrates (A) were incubated with E. coli ligase or no ligase and subjected to PCR containing S1-Extension detection primers in the presence / absence of UNG. PCR was monitored via the commercially—available Zeus-Probe (qPCR) methodology (Zeus Scientific, Inc., Raritan, N.J.) and the resultant reactions were subjected to gel analysis (C). Decreasing amounts of a non-ligatable DNA substrate (S1 / AS only) was incubated with three different commercially available DNA polymerases and subjected to Zeus-Probe qPCR analysis. The results of these experiments are illustrated graphically in FIG. 1.

example 2

Non-Ligate-Able, Polymerase Favorable Substrates were Found to be Sensitive and Specific in Microbe Derived Crude Cell Lysates

[0056]Decreasing amounts microbes were beadmill-lysed and incubated with a DNA substrate (S1 / AS only) in the presence of DNA polymerase buffer and dNTP's at 37° C. for 30 min. (A). The lysates were then subjected to Zeus-Probe qPCR containing S1-extension specific primers. The results are displayed graphically in FIG. 2.

example 3

Non-Ligate-Able, Polymerase Favorable Substrates were Found to be Sensitive and Specific in Microbe Spiked Blood Culture Derived Crude Cell Lysates

[0057]Decreasing amounts microbes were spiked into 10 ml of blood broth. The microbes were subsequently recovered, subjected to beadmill-lysis and incubated with a DNA substrate (S1 / AS only) in the presence of DNA polymerase buffer and dNTP's at 37° C. for 30 min. (A). The lysates were then subjected to Zeus-Probe qPCR containing S1-extension specific primers. The results are displayed graphically in FIG. 3.

[0058]Accordingly, in yet another aspect the present invention improves upon the invention described and claimed in WO / 2009 / 007719. In accordance with the present invention, it has been discovered that the putative DNA ligase specific substrate in accordance with the disclosure of said WO / 2009 / 007719 yields robust signals from either purified DNA polymerase or purified DNA ligase, such that the methods set forth therein are not rendere...

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Abstract

The present invention relates generally to the field of detection of microorganisms, in particular detection of bacteria, to methods for measuring enzyme activity, such as DNA polymerase activity, and particularly relates 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. This invention also relates to reagents for use in such methods, and to test kits comprising such reagents for carrying out the methods.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a non-provisional application, which is incorporated by reference herein and claims priority, in part, of U.S. Provisional Application No. 61 / 324,939, filed Apr. 16, 2010, U.S. Provisional Application No. 61 / 324,949, filed Apr. 16, 2010 and U.S. Provisional Application No. 61 / 325,413, filed Apr. 19, 2010.FIELD OF THE INVENTION[0002]The present invention relates generally to the field of detecting microorganisms, and more particularly to the detection of bacteria. Also provided by the invention are improved methods of detecting microorganisms that are highly sensitive, are applicable to non-purified samples, and have numerous applications, together with assay kits, which rely upon the presence of ligase and / or phosphatase as an indicator of bacterial viability.BACKGROUND OF THE INVENTION[0003]Measuring the presence and levels of certain molecules which are associated with cell viability is important in a number of conte...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/04C12Q1/25C12Q1/6888C12Q1/48C12Q1/42C12Q1/6834
Inventor O'HARA, SHAWN MARKZWEITZIG, DANIEL
Owner MOMENTUM BIOSCI
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