RATIOMETRIC PRE-rRNA ANALYSIS

Abstract

Disclosed are compositions and methods for detecting the presence of viable cells in a sample. Included are compositions and methods for increasing the sensitivity of a nucleic acid amplification test for determining the presence of at least one target microorganism in a sample. Also disclosed are compositions and methods for detecting ribosomal RNA precursors (pre-rRNA) as dynamic indicators of viable microorganisms in a sample.

1 - 29 . (canceled) 
     
     
         30 . A method of detecting the viability of at least one target prokaryotic microorganism in a collected sample, the method comprising:
 quantifying the level of at least one target pre-rRNA of the at least one target prokaryotic microorganism in the collected sample;   nutritionally stimulating and incubating a portion of the collected sample for a time period of less than one generation time of the at least one target prokaryotic microorganism, thereby producing a nutritionally stimulated sample;   quantifying the level of the at least one target pre-rRNA of the at least one target prokaryotic microorganism in the nutritionally stimulated sample;   comparing the level of the at least one target pre-rRNA of the at least one prokaryotic microorganism in the nutritionally stimulated sample with the level of the at least one target pre-rRNA of the at least one target prokaryotic microorganism in the collected sample,   wherein when the ratio of the level of the at least one target pre-rRNA in the nutritionally stimulated sample to the level of the at least one target pre-rRNA in the collected sample is greater than 1, the at least one target prokaryotic microorganism in the collected sample is viable, and when the ratio is not greater than 1, no viable target prokaryotic microorganism is detected in the collected sample.   
     
     
         31 . The method of  claim 30 , wherein the percentage of the at least one target prokaryotic microorganism that is viable in the collected sample is at least one of approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. 
     
     
         32 . The method of  claim 30 , wherein at least one of the quantifying the level of the at least one target pre-rRNA in the collected sample and the quantifying the level of the at least one target pre-rRNA in the nutritionally stimulated sample comprises the use of a microfluidic device. 
     
     
         33 . The method of  claim 30 , further comprising an immunoseparation process wherein the collected sample is screened for the presence of a particular isolate of the at least one target prokaryotic microorganism. 
     
     
         34 . The method of  claim 30 , wherein the at least one target prokaryotic microorganism is a member of a genera of microorganisms selected from the group consisting of  Acinetobacter, Actinobacillus, Aeromonas, Arcobacter, Bacteroides, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Citrobacter, Cronobacter, Edwardsiella, Enterobacter, Escherichia, Eubacterium, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Moraxella, Morganella, Neisseria, Pasteurella, Plesiomonas, Porphyromonas, Prevotella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Stenotrophomonas, Treponema, Veillonella, Vibrio, Yersinia, Actinomyces, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Enterococcus, Lactobacillus, Listeria, Micrococcus, Mobiluncus, Mycobacterium, Nocardia, Peptostreptococcus, Propionibacterium, Rhodococcus, Staphylococcus, Streptococcus , and  Streptomyces.    
     
     
         35 . The method of  claim 30 , wherein the at least one target prokaryotic microorganism is at least one of  Chlamydia trachomatis, Legionella pneumonia, Listeria monocytogenes, Campylobacter jejuni, Clostridium difficile, Bacillus anthracis, Francisella tularensis, Rickettsia prowasekii, Rickettsia typhi , and  Helicobacter pylori.    
     
     
         36 . The method of  claim 33 , wherein the immunoseparation process comprises screening the collected sample for the presence of  E. coli  0157. 
     
     
         37 . The method of  claim 30 , wherein the at least one target prokaryotic microorganism is a  Mycobacterium  species. 
     
     
         38 . The method of  claim 30 , wherein the nutritionally stimulating and incubating a portion of the collected sample comprises enriching the collected sample with at least one nutrient to encourage an upshift in the production of the at least one target pre-rRNA of the at least one target prokaryotic microorganism. 
     
     
         39 . The method of  claim 30 , wherein the collected sample is derived from tissues or bodily fluids. 
     
     
         40 . The method of  claim 30 , wherein the collected sample is from the tissue, blood, or sputum of a human or animal subject. 
     
     
         41 . A method of detecting viable microorganisms in a sample, the method comprising:
 collecting a sample;   nutritionally stimulating a first aliquot of the sample;   maintaining a second aliquot of the sample under non-nutritionally stimulating control conditions;   incubating the first aliquot and the second aliquot;   comparing the level of at least one target pre-rRNA from at least one target microorganism from the first aliquot with the level of the at least one target pre-rRNA from at least one target microorganism in the second aliquot;   wherein the ratio of the level of the at least one target pre-rRNA in the first aliquot and the level of the at least one target pre-rRNA in the second aliquot is indicative of viable microorganisms in the sample.   
     
     
         42 . The method of  claim 41 , further comprising extracting RNA from the first aliquot and extracting RNA from the second aliquot; and
 quantifying the level of the at least one target pre-rRNA in the first aliquot and quantifying the level of the at least one target pre-rRNA the second aliquot.   
     
     
         43 . The method of  claim 41 , wherein the percentage of the at least one target microorganism that are viable in the sample is at least one of approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.5%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. 
     
     
         44 . The method of  claim 42 , wherein extracting the RNA from the first aliquot and the second aliquot and quantifying the at least one pre-rRNA from the first aliquot and the second aliquot comprises the use of a microfluidic device. 
     
     
         45 . The method of  claim 41 , further comprising an immunoseparation process wherein the sample is screened for the presence of a particular isolate of the at least one target microorganism in the sample. 
     
     
         46 . The method of  claim 41 , wherein the at least one target microorganism is a member of a genera of microorganisms selected from the group consisting of  Acinetobacter, Actinobacillus, Aeromonas, Arcobacter, Bacteroides, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Citrobacter, Cronobacter, Edwardsiella, Enterobacter, Escherichia, Eubacterium, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Moraxella, Morganella, Neisseria, Pasteurella, Plesiomonas, Porphyromonas, Prevotella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Stenotrophomonas, Treponema, Veillonella, Vibrio, Yersinia, Actinomyces, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Enterococcus, Lactobacillus, Listeria, Micrococcus, Mobiluncus, Mycobacterium, Nocardia, Peptostreptococcus, Propionibacterium, Rhodococcus, Staphylococcus, Streptococcus , and  Streptomyces.    
     
     
         47 . The method of  claim 41 , wherein the at least one target microorganism is at least one of  Chlamydia trachomatis, Legionella pneumonia, Listeria monocytogenes, Campylobacter jejuni, Clostridium difficile, Bacillus anthracis, Francisella tularensis, Rickettsia prowasekii, Rickettsia typhi , and  Helicobacter pylori.    
     
     
         48 . The method of  claim 41 , wherein the first aliquot is incubated for a time period that is less than the doubling time of the at least one target microorganism. 
     
     
         49 . The method of  claim 41 , wherein the collected sample is derived from tissues or bodily fluids.