Methods and systems for detection of microorganisms

Abstract

Disclosed are methods and systems for the isolation and detection of microbes from a sample. The use of binding agents for isolation of a microbe of interest from a sample are described. In certain embodiments, the methods use ribosome-based and/or bacteriophage-based amplification of the signal in detection of bacteria and other microorganisms. For example, embodiments of the present invention can achieve total amplification of at least 10,000 from a single infected cell.

1 . A method for detecting a microorganism of interest comprising the steps of:
 isolating the microorganism from other components in the sample;   lysing the microorganism to release ribosomes present in the microorganism; and   detecting the ribosomes, or a constituent of the ribosomes, wherein detection of the ribosomes or a constituent of the ribosomes, indicates that the microorganism is present in the sample.   
     
     
         2 . The method of  claim 1 , wherein the microorganism comprises at least one of a bacterium, or a fungus, or yeast. 
     
     
         3 . The method of  claim 1 , wherein the step of isolating the microorganism comprises binding of the microorganism to a binding agent. 
     
     
         4 . The method of  claim 1 , wherein the step of isolating the microorganism comprises binding of the microorganism to a binding agent that is bound to a solid support. 
     
     
         5 . The method of  claim 4 , the binding agent is an antibody specific for the microorganism. 
     
     
         6 . The method of  claim 4 , wherein the microorganism is a bacterium, and the binding agent is a bacteriophage specific for the bacterium. 
     
     
         7 . The method of  claim 1 , wherein the detection of the ribosomes comprises the use of a primary antibody that recognizes the ribosomes, and at least one second primary antibody that also recognizes the ribosomes. 
     
     
         8 . The method of  claim 7 , wherein the second primary antibody is bound to a solid support. 
     
     
         9 . The method of  claim 8 , wherein solid support comprises a plurality of second primary antibodies. 
     
     
         10 . The method of  claim 1 , wherein the ribosomes are applied to a solid support comprising anti-ribosome antibodies and detected detected by flow of the ribosomes across the surface of the support. 
     
     
         11 . The method of  claim 9 , wherein the ribosomes bound to the solid support comprising anti-ribosome antibodies are visualized using at least one carbon black nano-string comprising a second population of anti-ribosome antibodies. 
     
     
         12 . A method for detecting a bacterium of interest comprising the steps of:
 isolating at least one bacterium from other components in the sample;   infecting the at least one bacterium with a plurality of a parental bacteriophage;   lysing the at least one infected bacterium to release progeny bacteriophage present in the bacterium; and   detecting the progeny bacteriophage, or a constituent of the progeny bacteriophage, wherein detection of the bacteriophage or a constituent of the bacteriophage, indicates that the bacterium is present in the sample.   
     
     
         13 . The method of  claim 12 , wherein the parental bacteriophage are separated from the progeny bacteriophage. 
     
     
         14 . The method of  claim 12 , wherein the progeny bacteriophage comprise an indicator moiety. 
     
     
         15 . The method of  claim 14 , wherein the indicator moiety comprises luciferase fused to the phage capsid protein. 
     
     
         16 . The method of  claim 14 , wherein the indicator moiety comprises soluble luciferase protein. 
     
     
         17 . The method of  claim 12 , further comprising the step of infecting indicator bacteria with the progeny bacteriophage, wherein the indicator bacteria comprise a protein that is released upon lysis of the indicator bacteria. 
     
     
         18 . The method of  claim 17 , wherein the protein comprises one or both a detectable moiety and a ribosomal subunit. 
     
     
         19 . (canceled) 
     
     
         20 . A kit comprising components for detecting a microorganism of interest comprising:
 a component for isolating the microorganism from other components in the sample;   a component for lysing the microorganism to release ribosomes present in the microorganism; and   a component for detecting the ribosomes, or a constituent of the ribosomes, wherein detection of the ribosomes or a constituent of the ribosomes, indicates that the microorganism is present in the sample.   
     
     
         21 . A kit comprising components for detecting a microorganism of interest comprising:
 a component for isolating at least one microorganism of interest from other components in the sample;   a component for infecting the at least one microorganism of interest with a plurality of a parental infectious agents;   a component for lysing the at least one infected microorganism to release progeny infectious agents present in the microorganism; and   a component for detecting the progeny infectious agent, or a constituent of the progeny infectious agent, wherein detection of the infectious agent or a constituent of the infectious agent, indicates that the microganism is present in the sample.