# US 9,434,999 B1 · Specific detection of organisms derived from a sample > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,434,999 B1 - Document ID: US9434999B1 - Status: Granted - Kind: B1 - Application number: 14/883,918 - Filed: 2015-10-15 - Granted: 2016-09-06 - Earliest claimed: 2015-06-26 - Assignees: Great Basin Scientific - Applicants: Great Basin Scientific, Inc. - Inventors: Wanyuan Ao; Robert D. Jenison; Jamie Purcell - Web page: https://patentgrep.com/patent/US9434999B1 ## CPC - C12Q 1/689: for bacteria - C12Q 1/6844: Nucleic acid amplification reactions - C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction - C12Q 1/6851: Quantitative amplification - C12Q 2600/16: Primer sets for multiplex assays - 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 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The method of claim 1, wherein the amplification step comprises helicase dependent amplification. ### Claim 3 (depends on 1) The method of claim 1, wherein the amplification step comprises polymerase chain reaction. ### Claim 4 (depends on 1) The method of claim 1, wherein the detection step further comprises detecting precipitate from a reaction product catalyzed by horseradish peroxidase activity. ### Claim 5 (depends on 1) The method of claim 1, wherein the target cell comprises a Staphylococcal species, and the threshold amount is about 10,000 to about 100,000 colony forming units per milliliter. ### Claim 6 (depends on 1) The method of claim 1, wherein the target cell is a Staphylococcal species, and the threshold amount is about 500 colony forming units. ### Claim 7 (depends on 1) The method of claim 1, wherein the sample comprises human blood. ### Claim 8 (depends on 1) The method of claim 1, wherein the DNA primers amplify the target nucleic acid at an amplification rate equal to a rate of signal suppressor amplification; and wherein the quantity of signal suppressor required to achieve a desired threshold amount is the product of a number of primer molecules and a threshold amount divided by a lower limit of detection. ### Claim 9 (depends on 1) The method of claim 1, wherein the DNA primers amplify the target nucleic acid at a PCR efficiency equal to a PCR efficiency of the signal suppressor; and wherein the quantity of signal suppressor required to achieve a required threshold is the product of a number of primer molecules and a threshold amount divided by a lower limit of detection. ### Claim 10 (depends on 1) The method of claim 1, wherein the amplification step comprises polymerase chain reaction and the DNA primers amplify the target nucleic acid at a PCR efficiency different from the PCR efficiency of the signal suppressor; and wherein the quantity of signal suppressor used in the amplification step is at least the quantity S as defined in the equation: S = ( P × Ta × y n LLOD × x n ) wherein P is a number of primer molecules, Ta is a threshold amount, y is a quantity of an efficiency of amplification of the target nucleic acid, n is a number of cycles of polymerase chain reaction, LLOD is a lower limit of detection of the detection step, and x is an efficiency of the amplification of the signal suppressor raised to the nth power. ### Claim 11 (depends on 1) The method of claim 1, wherein the amplification step comprises helicase dependent amplification and the DNA primers amplify the target nucleic acid at amplification rate different from the amplification rate of the signal suppressor; wherein the quantity of signal suppressor nucleic acid used in the amplification step 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. ### Claim 12 (depends on 1) The method of claim 1, further comprising determining the lower limit of detection of a detection method utilized to analyze the product of the nucleic acid amplification. ### Claim 13 (depends on 1) The method of claim 1, further comprising determining an amplification rate of the target cell nucleic acid. ### Claim 14 (depends on 1) The method of claim 1, further comprising determining an amplification rate of the signal suppressor. ### Claim 15 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. ### Claim 16 (depends on 15) The method of claim 15, wherein the nucleic acid amplification reaction is a helicase-dependent amplification. ### Claim 17 (depends on 15) The method of claim 15, wherein the nucleic acid amplification reaction is a polymerase chain reaction. ### Claim 18 (depends on 15) The method of claim 15, wherein the at least one pair of DNA primers are biotinylated. ### Claim 19 (depends on 15) The method of claim 15, wherein the step of detecting a signal comprises hybridization of the product of the nucleic acid amplification reaction to immobilized complementary DNA strands to yield a hybridized sample. ### Claim 20 (depends on 15) The method of claim 15, wherein the at least one pair of DNA primers amplify a specific gene sequence chosen from the group consisting of a mecA-specific gene sequence, a tuf-specific gene sequence, and a nuc-specific gene sequence. ### Claim 21 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.