Microarray for detecting viable organisms

Abstract

A methodology of microarray using the fluorescent DNA intercalating agent propidium monoazide (PMA) to selectively block DNA of dead cells from amplification and its application in detecting and enumerating viable microbes in complex microbial communities is described. A phylogenetic array is used in the preferred embodiment to enhance the sensitivity of the method. The PMA-Microarray assay is particularly applicable for monitoring samples from environments with extremely low microbial burden such as spacecraft surfaces.

What is claimed is: 
     
         1 . A method for detecting live cells in a sample comprising:
 selectively amplifying a nucleic acid from live cells from a sample comprising live and dead cells; and   detecting the presence, absence, relative abundance, and/or quantity of one or more operational taxon units (OTUs) in the sample based on hybridization of amplified nucleic acid to a plurality of probes complementary to 16s rRNA sequences, wherein said one or more OTUs consist essentially of live cells from said sample.   
     
     
         2 . The method of  claim 1 , wherein said nucleic acid is DNA. 
     
     
         3 . The method of  claim 1 , wherein said selectively amplifying comprises pre-treating the sample with an agent that selectively modifies a nucleic acid of dead cells. 
     
     
         4 . The method of  claim 3 , wherein the agent that selectively modifies a nucleic acid of dead cells is a DNA intercalating agent. 
     
     
         5 . The method of  claim 4 , wherein said DNA intercalating agent is propidium monoazide. 
     
     
         6 . The method of  claim 1 , wherein said probes are used to detect the presence, absence, relative abundance, and/or quantity of at least 10,000 different OTUs in a single assay. 
     
     
         7 . The method of  claim 1 , wherein said presence, absence, relative abundance, and/or quantity is detected with a confidence level greater than 95%. 
     
     
         8 . The method of  claim 1 , further comprising quantifying the number of live cells in the sample. 
     
     
         9 . A method for detecting live cells in a sample comprising:
 selectively amplifying a nucleic acid from live cells from a sample comprising live and dead cells; and   determining the presence, absence, relative abundance, and/or quantity of at least 1,000 different OTUs in a single assay, wherein said OTUs consist essentially of live cells from said sample.   
     
     
         10 . A method for detecting live cells in a sample comprising:
 (a) selectively amplifying a nucleic acid from live cells from a sample comprising live and dead cells;   (b) hybridizing amplified nucleic acid to a plurality of probes;   (c) determining hybridization signal strength distributions for a plurality of different interrogation probes, each of which is complementary to a section within one or more highly conserved polynucleotides in one or more target OTUs;   (d) determining hybridization signal strength distributions for a plurality of mismatch probes, wherein for each interrogation probe, one or more different corresponding mismatch probes comprising one or more nucleotide mismatches with said section within said one or more highly conserved polynucleotides are included in the plurality of mismatch probes; and   (e) using the hybridization signal strengths of the interrogation probes and mismatch probes to determine the probability that the hybridization signal for the different interrogation probes represents the presence, absence, relative abundance, and/or quantity of said one or more OTUs, wherein said one or more OTUs consist essentially of live cells from said sample.   
     
     
         11 . The method of  claim 9  or  10 , wherein said selectively amplifying comprises pre-treating the sample with an agent that selectively modifies a nucleic acid of dead cells. 
     
     
         12 . The method of  claim 11 , wherein the agent that selectively modifies a nucleic acid of dead cells is a DNA intercalating agent. 
     
     
         13 . The method of  claim 12 , wherein said DNA intercalating agent is propidium monoazide. 
     
     
         14 . The method of  claim 9  or  10 , wherein contacting said sample with an agent that selectively modifies a nucleic acid of dead cells in said sample is followed by exposure to visible light. 
     
     
         15 . The method of  claim 10 , wherein said highly conserved polynucleotides are selected from the group consisting of 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene, nif13 gene, RNA molecules derived therefrom, and a combination thereof. 
     
     
         16 . The method of  claim 10 , wherein each interrogation probe has 4 or more corresponding mismatch probes in the plurality of mismatch probes. 
     
     
         17 . The method of  claim 10 , wherein said probes are used to detect the presence, absence, relative abundance, and/or quantity of at least 10,000 different OTUs in a single assay. 
     
     
         18 . The method of  claim 10 , wherein said probes are attached to a substrate. 
     
     
         19 . The method of  claim 18 , wherein said substrate comprises a bead or a microsphere. 
     
     
         20 . The method of  claim 18 , wherein said substrate comprises glass, plastic, or silicon 
     
     
         21 . The method of  claim 9  or  10 , wherein said presence, absence, relative abundance, and/or quantity is detected with a confidence level greater than 95%. 
     
     
         22 . The method of  claim 9 , further comprising quantifying the number of live cells in the sample. 
     
     
         23 . A kit for detecting live cells in a sample comprising:
 (a) an agent that selectively modifies a nucleic acid of dead cells;   (b) a plurality of different interrogation probes, each of which is complementary to a section within one or more highly conserved polynucleotides in one or more target operational taxon units (OTUs); and,   (c) a plurality of mismatch probes, wherein for each interrogation probe, one or more different corresponding mismatch probes comprising one or more nucleotide mismatches with said section within said one or more highly conserved polynucleotides are included in the plurality of mismatch probes.   
     
     
         24 . The kit of  claim 23 , wherein the agent that selectively modifies a nucleic acid of dead cells is a DNA intercalating agent. 
     
     
         25 . The kit of  claim 23 , wherein said DNA intercalating agent is propidium monoazide. 
     
     
         26 . The kit of  claim 23 , wherein each interrogation probe has 4 or more corresponding mismatch probes in the plurality of mismatch probes. 
     
     
         27 . The kit of  claim 23 , wherein said highly conserved polynucleotides are selected from the group consisting of 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene, nif13 gene, RNA molecules derived therefrom, and a combination thereof.