Microbial ATP extraction and detection system

Abstract

The present invention is directed to compositions and methods for single-step extraction and detection of ATP levels from microbial cells. The disclosed compositions are formulated to efficiently elicit bioluminescent detection of ATP among a broad variety of different microorganisms using a common single-step reagent composition. Additional luminescence-based methods are provided for identifying other useful extracting agents or for screening compounds for their pharmaceutical or biological effects on microbial cells.

1 - 62 . (canceled) 
   
   
       63 . A method of detecting ATP in a sample containing or suspected of containing a microorganism comprising:
 (a) forming a composition comprising a luciferase enzyme, one or more ATP extracting agents collectively capable of disrupting the integrity of a bacterium or yeast to effect release of ATP therefrom, and one or more divalent cations, wherein the composition has a divalent cation concentration of less than about 5 mM;   (b) contacting the composition with the sample to form a mixture; and   (c) detecting luminescence.   
   
   
       64 . The method of  claim 63 , wherein the step of forming the composition comprises adding the one or more ATP extracting agents to a lyophilized source of luciferase enzyme in combination with lyophilized luciferin. 
   
   
       65 . The method of  claim 63 , wherein the one or more ATP extracting agents comprises a quaternary ammonium salt. 
   
   
       66 . The method of  claim 65 , wherein the one or more ATP extracting agents comprises cetyltrimethylammonium bromide. 
   
   
       67 . The method of  claim 63 , where the one or more one ATP extracting agents comprises chlorohexidine. 
   
   
       68 . The method of  claim 63 , where the one or more one ATP extracting agents comprises chlorohexidine and a non-ionic detergent. 
   
   
       69 . The method of  claim 63 , where the one or more ATP extracting agents comprises cetyltrimethylammonium bromide, chlorohexidine, and a non-ionic detergent. 
   
   
       70 . The method of  claim 63 , wherein the divalent cation concentration in the composition is less than about 2.5 mM. 
   
   
       71 . The method of  claim 63 , wherein the divalent cation concentration in the composition is less than about 1 mM. 
   
   
       72 . The method of  claim 63 , wherein the divalent cation concentration in the composition is less than about 0.5 mM. 
   
   
       73 . The method of  claim 63 , wherein one or more divalent cation chelators at a second concentration is added to the composition, wherein the difference between the first concentration and the second concentration is less than about 5.0 mM. 
   
   
       74 . The method of  claim 73 , where the difference between the first concentration and the second concentration is less than about 2.5 mM. 
   
   
       75 . The method of  claim 73 , where the difference between the first concentration and the second concentration is less than about 1.0 mM. 
   
   
       76 . The composition of  claim 63 , where the one or more ATP extracting agents comprises a compound or combination of compounds capable of disrupting the integrity of a gram negative bacterium. 
   
   
       77 . The method of  claim 76 , wherein the bacterium is  E. coli  or  P. aeruginosa.    
   
   
       78 . The method of  claim 63 , wherein the sample contains or is suspected to contain a heterogenous population of microbial cells. 
   
   
       79 . The method of  claim 63 , wherein the sample contains or is suspected of containing a pathogenic microbe. 
   
   
       80 . The method of  claim 63 , wherein a detectable luminescent signal is produced within 10 minutes after contacting the sample with the composition. 
   
   
       81 . The method of  claim 63 , wherein a detectable luminescent signal is produced within 5 minutes after contacting the sample with the composition. 
   
   
       82 . The method of  claim 63 , wherein the luminescence produces a luminescent signal with a half-life of at least 30 minutes.