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

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.

1 . A method for detecting polymerase activity as an indicator of the presence of a micro-organism in a sample, which method comprises:
 (a) contacting the sample with a nucleic acid molecule which acts as a substrate for polymerase activity in the sample;   (b) incubating the thus contacted sample under conditions suitable for polymerase activity; and   (c) specifically determining the presence (and/or the amount) of a nucleic acid molecule resulting from the action of the microorganism polymerase on the substrate nucleic acid molecule, thereby to indicate the presence of the microorganism.   
     
     
         2 . The method of  claim 1 , wherein the polymerase is either a DNA or RNA polymerase. 
     
     
         3 . The method of  claim 1 , wherein the microorganism detected is a viable microorganism in the sample. 
     
     
         4 . The method of  claim 1 , wherein the microorganism detected is an intact microorganism in the sample. 
     
     
         5 . The method of  claim 4 , wherein the intact microorganism detected is one in which the nucleic acid polymerase gene and its translated active protein polymerase is essential for viability of the microorganism. 
     
     
         6 . The method of  claim 1 , wherein the polymerase substrate is immobilized. 
     
     
         7 . The method of  claim 1 , wherein the sample in which the microorganism is detected is a normally sterile body fluid. 
     
     
         8 . The method of  claim 1 , wherein the sample is prepared using a differential cell lysis sample preparation method, thereby allowing only viable microorganism derived polymerase activity to modify the polymerase specific substrate. 
     
     
         9 . The method of  claim 1 , wherein the sample in which the microorganism is detected is prepared from crude cell lysates or purified cell fractions. 
     
     
         10 . The method of  claim 9 , wherein the method further comprises conducting a complete or partial microorganism genome and or transcriptome sequence analysis. 
     
     
         11 . The method of  claim 10 , wherein the complete or partial microorganism genome and or transcriptome sequence analysis can be performed simultaneously, in concert, or in parallel using a single sample preparation. 
     
     
         12 . The method of  claim 10 , wherein the complete or partial microorganism genome and or transcriptome sequence analysis of the microorganisms further comprises a method for the diagnostic measure and detection of agents with anti-microbial and or anti-polymerase activity useful in the management of patients. 
     
     
         13 . An assay kit comprised of reagents useful in the method of any of  claims 1  through  12 , for screening normally sterile body fluids for the presence of absence of microorganisms therein and to provide diagnostic, prognostic patient management information. 
     
     
         14 . A method of detecting an enzyme selected from the group consisting of NAD-dependent ligase or phosphatase or a mixture thereof as an indicator of the presence of a microorganism in a sample, which method comprises:
 (a) contacting the sample with a nucleic acid molecule which acts as a substrate for enzyme activity of the selected enzyme or mixture in the sample, while not allowing interfering signals from DNA polymerase;   (b) incubating the thus contacted sample under conditions suitable for enzyme activity; and   (c) specifically determining the presence (and/or the amount) of an enzyme modified nucleic acid molecule resulting from the action of the selected enzyme or mixture on the substrate nucleic acid molecule, thereby to indicate the presence of the microorganism.   
     
     
         15 . The method of  claim 14 , wherein the microorganism is one in which the selected enzyme or mixture is (or has been) expressed. 
     
     
         16 . The method of  claim 14  or  claim 15 , wherein the sample is prepared using a differential cell lysis sample preparation method, thereby allowing only viable microorganism derived ligase and/or phosphatase activity to modify the ligase specific substrate. 
     
     
         17 . An assay kit capable of performing an assay for detecting an enzyme selected from the group consisting of NAD-dependent ligase or phosphatase or a mixture thereof as an indicator of the presence of a microorganism in a sample, said kit comprising:
 (a) a substrate containing a nucleic acid molecule for activity of the selected enzyme or mixture in the sample, while not allowing interfering signals from DNA polymerase;   (b) Incubation means for incubating the sample and substrate under conditions suitable for enzyme activity; and   (c) Means for specifically determining the presence (and/or the amount) of a nucleic acid molecule resulting from the action of the selected enzyme or mixture on the substrate nucleic acid molecule, as an indication of the presence of the microorganism.