Microarray for detecting viable organisms
AssigneeThe Regents of the University of California
InventorsKasthuri Venkateswaran, Christina Dock, Gary Aandersen, Todd DeSantis
Life of this patent
Estimating the term…
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.
Claims27
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.
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.
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.
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.
Dependencies are inferred from the claim text.