Back to results
US 9,434,999 B1Granted
Specific detection of organisms derived from a sample
AssigneeGreat Basin Scientific
InventorsWanyuan Ao, Robert D. Jenison, Jamie Purcell
Life of this patent
Estimating the term…
Abstract
Methods, materials, and kits for distinguishing a population of cells or organisms truly present in a clinical specimen from contaminating cells or organisms is disclosed. The methods and kits use a suppressor to avoid false positive detection of contaminants in nucleic acid amplification reactions.
Claims21
A method of determining whether a sample contains a threshold amount of a target cell population, comprising:
- identifying at least one type of target cell to detect from a sample;
- identifying a threshold amount above which amplification of a target nucleic acid from the target cell is noncontaminating;
- adding at least one signal suppressor nucleic acid for each type of target cell to the sample in a quantity sufficient to ensure that amplification of the target nucleic acid of each type of target cell will not proceed to detectable levels if the target cell of that type is present in a quantity below the threshold amount;
- amplifying the target nucleic acid in an amplification step with a DNA polymerase, a deoxyribonucleotide triphosphate mixture, and at least one pair of DNA primers, each of the pair of DNA primers having a sequence that anneals to a signal suppressor nucleic acid and the target nucleic acid;
- and
- detecting a signal from the product of the amplification step in a detection step, to determine whether the cell type is present in a noncontaminating quantity.
3 more elements
A method of determining whether a sample contains at least 500 colony forming units per milliliter of Staphylococcus comprising:
- providing a sample suspected of having Staphylococcus;
- adding at least one signal suppressor nucleic acid to the sample in a quantity sufficient to ensure that amplification of a nucleic acid sequence from Staphylococcus will not proceed to detectable levels unless there are at least 500 colony forming units present;
- subjecting the sample to a nucleic acid amplification reaction using an amplification mixture comprising a DNA polymerase, a deoxyribonucleotide triphosphate mixture, and at least one pair of DNA primers, each of the pair of DNA primers having a sequence that anneals to the signal suppressor nucleic acid and the nucleic acid sequence from Staphylococcus;
- and
- detecting a signal from a product of the nucleic acid amplification reaction to determine whether at least 500 colony forming units of Staphylococcus are present.
2 more elements
A method of determining whether a sample contains a threshold amount of a target cell population, comprising:
- identifying at least one type of target cell to detect from a sample;
- identifying a threshold amount above which amplification of a target nucleic acid from the target cell is noncontaminating;
- adding at least one signal suppressor nucleic acid for each type of target cell to the sample in a quantity sufficient to ensure that amplification of the target nucleic acid of each type of target cell will not proceed to detectable levels if the target cell is present in a quantity below the threshold amount;
- amplifying the nucleic acid of the target cell in an amplification step with a DNA polymerase, a deoxyribonucleotide triphosphate mixture, and at least one pair of DNA primers, each of the pair of DNA primers having a sequence that anneals to a signal suppressor nucleic acid and the target nucleic acid,
- wherein the amplification step comprises polymerase chain reaction and the DNA primers amplify the target nucleic acid with a PCR efficiency equal to a PCR efficiency of the signal suppressor nucleic acid;
- wherein the quantity of signal suppressor nucleic acid is at least the quantity S as defined in the equation:
- S
- =
- (
- P
- ×
- Ta
- ×
- ⅇ
- k
- 2
-
- t
- -
- k
- 1
-
- t
- )
- LLOD
- wherein P is the number of primer molecules, Ta is the threshold amount, k2 is the amplification rate of the target nucleic acid, k1 is the amplification rate of the signal suppressor, t is amplification time, and LLOD is the lower limit of detection;
- and
- detecting a signal from the product of the amplification step to determine whether the target cell is present in a noncontaminating quantity.
25 more elements
Dependencies are inferred from the claim text.