LINEARLY-AMPLIFIED INTERNAL CONTROL FOR NUCLEIC ACID AMPLIFICATION REACTION
The present disclosure provides, among other things, systems, methods, and kits include internal amplification controls. Provided internal amplification controls are or include non-target sequences that are amplified during nucleic acid amplification. Provided internal amplification controls are linearly amplified. Provided internal amplification controls are useful for systems, methods and kits for nucleic acid amplification.
1 . A method of performing a nucleic acid amplification reaction, comprising steps of:
providing an internal amplification control, comprising a single oligonucleotide primer and a nucleic acid template;
contacting the internal amplification control with a nucleic acid amplification reagent in a reaction vessel; and
performing a nucleic acid amplification reaction,
wherein the nucleic acid amplification template is linearly amplified, and
wherein when a target nucleic acid sample is also present in the reaction vessel, the target nucleic acid sample is exponentially or linearly amplified.
2 . The method of any of the preceding claims, wherein a sequence of the nucleic acid template is or comprises the single oligonucleotide primer or a sequence complementary to the single oligonucleotide primer.
3 . The method of any of the preceding claims, wherein when the single oligonucleotide primer binds with a nucleic acid template, the single oligonucleotide primer is extended by a polymerase.
4 . The method of any of the preceding claims, wherein when the single oligonucleotide primer is extended by the polymerase, the probe activates.
5 . The method of any of the preceding claims, wherein the nucleic acid amplification reagent comprises target specific proteins.
6 . The method of any of the preceding claims, wherein the target nucleic acid sample is an environmental sample.
7 . The method of any of the preceding claims, wherein the nucleic acid amplification reagent comprises a DNA polymerase at a concentration of at least about 8.0 U/reaction and a target specific primer concentration of at least about 1.5 μM, and a single oligonucleotide primer concentration of at least about 5.0 μM.
8 . The method of any of the preceding claims, comprising a step of activating the probe.
9 . The method of any of the preceding claims, wherein the nucleic acid template is a plasmid that has more than one complementary sequence to the single oligonucleotide primer and the probe is a hydrolysis probe.
10 . The method of any of the preceding claims, wherein probe activation produces a fluorescence signal.
11 . The method of any of the preceding claims, comprising a step of quantifying the internal amplification control by its cycle threshold, slope, and/or end point fluorescence.
12 . The method of any of the preceding claims, comprising quantifying an internal amplification control in a target nucleic acid sample and a reference sample.
13 . The method of any of the preceding claims, wherein the nucleic acid template and the target nucleic acid sample are present in the reaction vessel at a relative quantity, wherein the relative quantity is a ratio of nucleic acid template copy number to nucleic acid sample genomic units, wherein said ratio is at least about 0.001, about 0.005, about 0.01, about 0.05, about 0.1, about 0.5, about 1.0, about 1.5; about 2.0, about 2.5, about 5.0, about 10.0, about 15.0, about 20.0, about 50.0, about 100.0, about 500.0, or about 1000.0.
14 . An internal amplification control for a nucleic acid amplification reaction, comprising:
a single oligonucleotide primer; and
a nucleic acid template,
wherein when present with a target nucleic acid sample and contacted with a nucleic acid amplification reagent in a reaction vessel, the nucleic acid template linearly amplifies and the target nucleic acid sample exponentially or linearly amplifies, and
wherein when the nucleic acid template is present at a higher concentration than the target nucleic acid sample, amplification of the nucleic acid template does not consume nucleic acid amplification reagents at a faster rate than amplification of the target nucleic acid sample.
15 . The internal amplification control of claim 14 , wherein when an internal amplification control is amplified its cycle threshold, slope or end point fluorescence is determined.
16 . The internal amplification control of claim 14 or 15 , comprising a reference sample.
17 . The internal amplification control as in any of claims 14 - 16 , wherein a sequence of the nucleic acid template is or comprises the single oligonucleotide primer or a sequence complementary to the single oligonucleotide primer.
18 . The internal amplification control as in any of claims 14 - 17 , comprising a probe.
19 . The internal amplification control as in any of claims 14 - 18 , wherein when the single oligonucleotide primer binds to a nucleic acid template, the single oligonucleotide primer is extended by a polymerase.
20 . The internal amplification control as in any of claims 14 - 18 , wherein when the single oligonucleotide primer is extended by the polymerase, a probe activates.
21 . The internal amplification control as in any of claims 14 - 20 , wherein the nucleic acid template is a plasmid that has more than one complementary sequence to the single oligonucleotide primer and a hydrolysis probe.
22 . The internal amplification control as in any of claims 14 - 21 , wherein activation of a probe produces a fluorescence signal.
23 . The internal amplification control as in any of claims 14 - 22 , wherein the nucleic acid amplification reagent comprises a DNA polymerase at a concentration of at least about 8.0 U/reaction and a target specific primer concentration of at least about 1.5 and a single oligonucleotide primer concentration of at least about 5.0 μM.
24 . The method of any of claims 14 - 23 , wherein the nucleic acid template and the target nucleic acid sample are present in the reaction vessel at a relative quantity, wherein the relative quantity is a ratio of nucleic acid template copy number to nucleic acid sample genomic units, wherein said ratio is at least about 0.001, about 0.005, about 0.01, about 0.05, about 0.1, about 0.5, about 1.0, about 1.5; about 2.0, about 2.5, about 5.0, about 10.0, about 15.0, about 20.0, about 50.0, about 100.0, about 500.0, or about 1000.0.