# US 2014/0155283 A1 · Microarray for detecting viable organisms > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0155283 A1 - Document ID: US20140155283A1 - Status: Published application - Kind: A1 - Application number: 13/984,257 - Filed: 2012-02-10 - Published: 2014-06-05 - Earliest claimed: 2011-02-11 - Assignees: The Regents of the University of California - Inventors: Kasthuri Venkateswaran; Christina Dock; Gary Aandersen; Todd DeSantis - Web page: https://patentgrep.com/patent/US20140155283A1 ## CPC - C12Q 1/689: for bacteria - C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) The method of claim 1, wherein said nucleic acid is DNA. ### Claim 3 (depends on 1) 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. ### Claim 4 (depends on 3) The method of claim 3, wherein the agent that selectively modifies a nucleic acid of dead cells is a DNA intercalating agent. ### Claim 5 (depends on 4) The method of claim 4, wherein said DNA intercalating agent is propidium monoazide. ### Claim 6 (depends on 1) 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. ### Claim 7 (depends on 1) The method of claim 1, wherein said presence, absence, relative abundance, and/or quantity is detected with a confidence level greater than 95%. ### Claim 8 (depends on 1) The method of claim 1, further comprising quantifying the number of live cells in the sample. ### Claim 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. ### Claim 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. ### Claim 11 (depends on 9, 10) 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. ### Claim 12 (depends on 11) The method of claim 11, wherein the agent that selectively modifies a nucleic acid of dead cells is a DNA intercalating agent. ### Claim 13 (depends on 12) The method of claim 12, wherein said DNA intercalating agent is propidium monoazide. ### Claim 14 (depends on 9, 10) 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. ### Claim 15 (depends on 10) 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. ### Claim 16 (depends on 10) The method of claim 10, wherein each interrogation probe has 4 or more corresponding mismatch probes in the plurality of mismatch probes. ### Claim 17 (depends on 10) 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. ### Claim 18 (depends on 10) The method of claim 10, wherein said probes are attached to a substrate. ### Claim 19 (depends on 18) The method of claim 18, wherein said substrate comprises a bead or a microsphere. ### Claim 20 (depends on 18) The method of claim 18, wherein said substrate comprises glass, plastic, or silicon ### Claim 21 (depends on 9, 10) 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%. ### Claim 22 (depends on 9) The method of claim 9, further comprising quantifying the number of live cells in the sample. ### Claim 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. ### Claim 24 (depends on 23) The kit of claim 23, wherein the agent that selectively modifies a nucleic acid of dead cells is a DNA intercalating agent. ### Claim 25 (depends on 23) The kit of claim 23, wherein said DNA intercalating agent is propidium monoazide. ### Claim 26 (depends on 23) The kit of claim 23, wherein each interrogation probe has 4 or more corresponding mismatch probes in the plurality of mismatch probes. ### Claim 27 (depends on 23) 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.