Kits for a selective assay for determining the identity of live microorganisms in a mixed culture

Abstract

The invention provides methods for determining the identity of an unknown live microorganism in a mixed culture. The microorganism can be a bacterium, fungus, virus, or protozoan. The invention further provides an assay for determining the ability of a selected microorganism in a mixed culture to replicate in the presence of a chemical agent. Kits for determining the identity of a live microorganism in a mixed culture and for determining the ability of a microorganism in a mixed culture to replicate are also provided.

What is claimed is:  
     
         1 . An assay for determining the identity of an unknown live microorganism in a mixed culture, said assay comprising the steps of: 
 i. culturing the microorganism in an aqueous medium, said medium able to culture one or more microorganisms other than the unknown microorganism, and comprising a nucleic acid analog which is recognized by an analog-specific binding member and which is incorporated into nucleic acids of replicating cells or virions of the unknown microorganism;    ii. lysing the microorganisms to release nucleic acids of the unknown microorganism, and other microorganisms which may be present;    iii. capturing nucleic acids incorporating the analog using the analog-specific binding member;    iv. separating the captured nucleic acids from any nucleic acids which have not been captured;    v. amplifying captured nucleic acids originating from the unknown microorganism; and,    vi. determining the identity of the microorganism from which the captured and amplified nucleic acids originate.    
     
     
         2 . An assay of  claim 1  wherein step (v) is a selective amplification of a subset of the captured nucleic acids.  
     
     
         3 . An assay of  claim 1  wherein the identity of the microorganism in step (vi) is determined by hybridization of the captured and amplified nucleic acids to a species-specific nucleic acid sequence.  
     
     
         4 . An assay of  claim 1  wherein the unknown microorganism is a bacterium.  
     
     
         5 . An assay of  claim 1  wherein the unknown microorganism is a pathogen.  
     
     
         6 . An assay of  claim 5  wherein the pathogen is a pathogen of a mammal.  
     
     
         7 . An assay of  claim 1  wherein the pathogen is a virus.  
     
     
         8 . An assay of  claim 1  wherein the pathogen is a fungus.  
     
     
         9 . An assay of  claim 1  wherein the pathogen is a protozoan.  
     
     
         10 . An assay of  claim 1  wherein the mixed culture is of two or more strains of the same species of microorganism.  
     
     
         11 . An assay of  claim 1  wherein the analog is bromodeoxyuridine.  
     
     
         12 . An assay of  claim 11  wherein the specific binding member is an antibody.  
     
     
         13 . An assay of  claim 1  wherein the aqueous medium further comprises an antibiotic.  
     
     
         14 . An assay for determining the ability of a selected microorganism in a mixed culture to replicate in the presence of a chemical agent comprising the steps of: 
 i. culturing the selected microorganism in an aqueous medium or cell culture, said medium or cell culture able to culture one or more microorganisms other than the selected microorganism, comprising a nucleic acid analog recognized by an analog-specific binding member and which is incorporated into nucleic acids of replicating cells or virions of the selected microorganism;    ii. lysing the microorganisms to release nucleic acids of the selected microorganism, and other microorganisms which may be present;    iii. capturing nucleic acids incorporating the analog using the analog-specific binding member;    iv. separating the captured nucleic acids from any nucleic acids which have not been captured;    v. amplifying captured nucleic acids originating from the selected microorganism; and,    vi. detecting the presence or absence of captured nucleic acids, wherein the series of steps i-vi is conducted at least twice, at least once with the series of steps inclusive of a chemical agent added to the culture media and at least once with the series of steps not having a chemical agent added to the culture media.    
     
     
         15 . An assay of  claim 14  wherein the amplification of step v is by polymerase chain reaction.  
     
     
         16 . An assay of  claim 14  wherein the analog binding member is attached to a solid support.  
     
     
         17 . A kit for determining the identity of a microorganism in a mixed culture comprising: 
 i. a DNA analog which is recognized by an analog-specific binding member and which is permissive of incorporation into nucleic acids of the microorganism;    ii. an analog-specific binding member for capturing nucleic acids incorporating the DNA analog;    iii. primers for amplifying nucleic acids from a microorganism whose identity is of interest, and,    iv. instructions for using components i, ii, and iii in an assay for determining the identity of the microorganism.    
     
     
         18 . A kit of  claim 17  wherein the primers are specific for amplifying microorganisms of a species or strain of interest.  
     
     
         19 . A kit of  claim 17 , further comprising species-specific nucleic acid sequences.  
     
     
         20 . A kit of  claim 17  wherein the DNA analog is bromodeoxyuridine.  
     
     
         21 . A kit of  claim 17  wherein the analog specific binding member is an antibody.  
     
     
         22 . A kit of  claim 17  which further comprises an antibiotic.  
     
     
         23 . A kit for determining the ability of a microorganism in a mixed culture to replicate comprising: 
 i. a DNA analog which is recognized by an analog-specific binding member and which is permissive of incorporation into nucleic acids of the microorganism;    ii. an analog-specific binding member for capturing nucleic acids incorporating the DNA analog;    iii. primers for amplifying nucleic acids from the microorganism whose ability to replicate is of interest, and,    iv. instructions for using components i, ii, and iii in an assay for determining the ability of the microorganism to replicate.    
     
     
         24 . A kit of  claim 23  wherein the primers are specific for amplifying microorganisms of a species or strain of interest.  
     
     
         25 . A kit of  claim 23 , further comprising species-specific nucleic acid sequences.  
     
     
         26 . A kit of  claim 23  wherein the DNA analog is bromodeoxyuridine.  
     
     
         27 . A kit of  claim 23  wherein the analog specific binding member is an antibody.  
     
     
         28 . A kit of  claim 23  which further comprises an antibiotic.