Viability detection and quantification assay of waterborne pathogens by enrichment
AssigneePhigenics, LLC
InventorsLeah Wickenberg, Katherine Fisher, William F. McCoy
Life of this patent
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Abstract
A process for detecting viable waterborne pathogens in a water sample, includes enriching a concentrate of bacteria from the sample and taking a first DNA extract at time T0 and a second DNA extract at time T2 after an incubation period. Real-time polymerase chain reaction performed on the DNA extracts yields respective cycle threshold values Ct. The change in Ct provides an indication of the targeted pathogen in the sample. An order of magnitude quantification of the sample can also be performed using a serial dilution technique on the T0 DNA extract. The process has particular application for detecting Legionella, including viable but not culturable cells.
Claims25
A process for detecting presence of viable non-culturable targeted waterborne pathogens comprising:
- (A) obtaining a liquid sample;
- (B) collecting a concentrate of bacterial cells from the liquid sample;
- (C) enriching the concentrate of bacterial cells to produce an enriched concentrate of bacterial cells;
- (D) extracting a T0 DNA extract from the enriched concentrate of bacterial cells at time T0;
- (E) storing the T0 DNA extract;
- (F) incubating the enriched concentrate of bacterial cells;
- (G) extracting a T2 DNA extract from the incubated enriched concentrate of bacterial cells at time T2;
- (H) analyzing the T0 DNA extract with real-time Polymerase Chain Reaction (PCR) to determine a cycle threshold Ct value for the T0 DNA extract;
- (I) analyzing the T2 DNA extract with real-time Polymerase Chain Reaction (PCR) to determine a cycle threshold Ct value for the T2 DNA extract;
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- (J) analyzing a difference in Ct value (ΔCt) between the T0 DNA extract and the T2 DNA extract to determine a qualitative assessment of the presence of viable but not culturable cells of the targeted waterborne pathogens in the liquid sample.
Dependencies are inferred from the claim text.