US2010105093A1PendingUtilityA1

Assay method for the detection of viable microbial cells in a sample

54
Individually held — no corporate assignee on recordPriority: Aug 1, 2006Filed: Aug 1, 2007Published: Apr 29, 2010
Est. expiryAug 1, 2026(~0 yrs left)· nominal 20-yr term from priority
Inventors:Gerald J. BuglerCatherine M. RamsayWilliam J. SimpsonMark Driscoll
C12Q 1/66C12Q 1/06
54
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Claims

Abstract

The present invention discloses an assay method for the detection of viable microbial cells in a sample, the assay method comprising the steps of: i) adding an ATP degrading enzyme to a sample suspected of containing viable microbial cells to substantially degrade any extracellular ATP in the sample; ii) adding a phosphate containing compound to the sample to substantially halt action of the ATP degrading enzyme; and iii) subjecting the sample to a detection assay to establish the level of undegraded ATP in the sample to provide an indication of the level of viable microbial cells in the sample.

Claims

exact text as granted — not AI-modified
1 . An assay method for the detection of viable microbial cells in a sample, the assay method comprising the steps of:
 i) isolating a sample containing viable microbial cells having intracellular ATP and extraneous material containing extracellular ATP;   ii) adding an ATP degrading enzyme to the sample to substantially degrade extracellular ATP in the sample;   iii) adding a phosphate containing compound to the sample to substantially halt action of the ATP degrading enzyme; and   iv) subjecting the sample to a detection assay to establish the level of undegraded ATP in the sample to provide an indication of the level of viable microbial cells in the sample.   
   
   
       2 . An assay method according to  claim 1 , wherein an extractant is added to the sample prior to assaying the level of undegraded ATP to extract the undegraded ATP from the sample. 
   
   
       3 . An assay method according to  claim 1 , wherein the ATP degrading enzyme is an ectoATPase. 
   
   
       4 . An assay method according to  claim 3 , wherein the ectoATPase is an apyrase. 
   
   
       5 . An assay method according to  claim 4 , wherein the apyrase reagent is buffered to have a pH value in the range selected from 9 to 5, or 8 to 6, or 7.5 to 6.2. 
   
   
       6 . An assay method according to  claim 5 , wherein the apyrase is at a concentration of 1 ml apyrase to 10μ of 10 −7  M of ATP. 
   
   
       7 . An assay method according to  claim 1 , which is carried out at room temperature and a reaction time between 1-30 minutes. 
   
   
       8 . An assay method according to  claim 1 , wherein divalent cations are added with the ATP degrading enzyme. 
   
   
       9 . An assay method according to  claim 8 , wherein the divalent cations are calcium ions and/or magnesium ions. 
   
   
       10 . An assay method according to  claim 1 , wherein the phosphate containing compound is selected from one or more of sodium phosphate, potassium phosphate, sodium pyrophosphate, penta-sodium triphosphate or sodium polyphosphate. 
   
   
       11 . An assay method according to  claim 2 , wherein the extractant is a surface-active agent that is used to extract ATP from live cells. 
   
   
       12 . An assay method according to  claim 11 , wherein the surface-active agents include one or more of quaternary ammonium compounds, dialkyl ammonium salts and bis-biguanides. 
   
   
       13 . An assay method according to  claim 2 , wherein the extractant is selected from one or more or the group consisting of N—N-dimethyldodecan-1-amine (GENAMIN), chloroprozamine, liquid lysate (LL1), and dichlorogallium (III) β -diketonato derivatives GaCl 2 (BDK). 
   
   
       14 . An assay method according to  claim 2 , wherein the extractants are used at a concentration of between 0.05-50 g/l. 
   
   
       15 . An assay method according to  claim 14 , wherein the extractant is allowed to react for a period of between 3-10 minutes. 
   
   
       16 . An assay method according to  claim 1 , wherein neutralizing agents are added to the sample containing the extractant. 
   
   
       17 . An assay method according to  claim 1 , wherein the phosphate-containing materials are precipitated prior to performing the apyrase reaction. 
   
   
       18 . (canceled) 
   
   
       19 . An assay method according to  claim 1 , wherein a second reagent is added to the sample, said second reagent being able to enhance levels of ATP in the sample by either increasing intracellular ATP levels by manipulation of cellular metabolism, or by enhancing the activity of luciferase and luciferin to detect ATP. 
   
   
       20 - 21 . (canceled) 
   
   
       22 . An assay kit for the detection of viable microbial cells in a sample, the assay kit including an ATP degrading enzyme, a phosphate containing compound, and an extractant, the kit including combining means so that an assay method comprising the following steps can be carried out:
 i) isolating a sample containing viable microbial cells having intracellular ATP and extraneous material containing extracellular ATP;   ii) adding the ATP degrading enzyme to the sample to substantially degrade extracellular ATP in the sample;   iii) adding the phosphate containing compound to the sample to substantially halt action of the ATP degrading enzyme;   iv) adding the extractant to the sample to extract the undegraded ATP from the sample; and   v) subjecting the sample to an assay to establish the level of undegraded ATP, in the sample to provide an indication of the level of viable microbial cells in the sample.   
   
   
       23 . (canceled) 
   
   
       24 . An assay kit according to  claim 22 , wherein the ATP degrading enzyme is an ectoATPase. 
   
   
       25 - 27 . (canceled) 
   
   
       28 . An assay kit according to  claim 22 , wherein divalent cations are added with ATP degrading enzyme. 
   
   
       29 . (canceled) 
   
   
       30 . An assay kit according to  claim 22 , wherein the phosphate containing compound is selected from one or more of sodium phosphate, potassium phosphate, sodium pyrophosphate, penta-sodium triphosphate or sodium polyphosphate. 
   
   
       31 . An assay kit according to  claim 22 , wherein the extractant is a surface-active agent selected from one or more of the group comprising quaternary ammonium compounds, dialkyl ammonium salts and bis-biguanides. 
   
   
       32 . An assay kit according to  claim 22 , wherein the extractant is selected from one or more or the group consisting of N—N-dimethyldodecan-1-amine, chloroprozamine, liquid lysate (LL1), and dichlorogallium (III) β -diketonato derivatives GaCl 2  (BDK). 
   
   
       33 . (canceled) 
   
   
       34 . An assay kit according to  claim 22 , including at least one neutralizing agent to neutralise the activity of the extractant. 
   
   
       35 - 36 . (canceled)

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