Components and processes of isothermal amplification

Abstract

To provide a technology relating in part to methods and compositions for isothermal amplification of nucleic acids.SOLUTION: Provided herein in certain aspects are methods for amplifying nucleic acid, the methods comprising bringing non-denatured sample nucleic acid under isothermal amplification conditions into contact with components comprising a) at least one oligonucleotide, which comprises a polynucleotide complementary to a target sequence in the sample nucleic acid and b) at least one component providing hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related patent applications This patent application claims the interests of U.S. Patent Application No. 15 / 090,405, filed on April 4, 2016, titled "ISOTHERMAL AMPLIFICATION COMPONENTS AND PROCESSES," in which Andrew P. Miller and Honghua Zhang are named as inventors and designated by attorney number NAT-1001-UTt. The entire contents of the above application, including all texts, tables and drawings, are incorporated herein by reference for all purposes.

[0002] Sequence List This application includes a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy was created on 2 March 2017, is named NAT-1001-PC_SL.txt, and is 16,533 bytes in size.

[0003] This technology, in part, relates to a method and composition for isothermal amplification of nucleic acids. [Background technology]

[0004] Nucleic acid-based diagnostics can be useful for the rapid detection of infectious diseases, illnesses, and / or genetic variations. For example, the identification of bacterial or viral nucleic acids in a sample can be useful for diagnosing certain types of infectious diseases. Other examples include the identification of single nucleotide polymorphisms for disease management or forensic science, and the identification of genetic variations indicating genetically modified foods. Often, nucleic acid-based diagnostic assays require amplification of specific portions of nucleic acids in a sample. A common technique for nucleic acid amplification is polymerase chain reaction (PCR). This technique typically requires temperature cycling (i.e., thermocycling) to advance the denaturation step (i.e., separation of strands in a double-stranded DNA (dsDNA) complex), the annealing step of oligonucleotide primers (short strands of complementary DNA sequences), and the extension step of the primers along the complementary target by polymerase. Such thermocycling can be a time-consuming process that generally requires specialized machinery. Therefore, there is a need for faster nucleic acid amplification methods that can be performed without thermocycling. Such methods can be useful, for example, for on-site testing and point-of-care diagnostics. [Overview of the project] [Means for solving the problem]

[0005] In a particular embodiment, the present invention provides a method for amplifying nucleic acids, comprising the step of generating a nucleic acid amplification product by contacting an undenatured sample nucleic acid under isothermal amplification conditions with a component comprising a) at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) at least one component providing hyperthermophilic polymerase activity.

[0006] In certain embodiments, the Specified Method also provides a method for amplifying nucleic acids, comprising the step of generating a nucleic acid amplification product by contacting an undenatured sample nucleic acid under isothermal amplification conditions with an enzymatic component comprising a) a non-enzymatic component comprising at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) a hyperthermophilic polymerase or a polymerase having an amino acid sequence at least about 90% identical to that of a hyperthermophilic polymerase.

[0007] In certain embodiments, a method for amplifying nucleic acids is also provided herein, comprising the step of generating a nucleic acid amplification product by contacting an undenatured sample nucleic acid under isothermal amplification conditions with a non-enzymatic component comprising a) at least one oligonucleotide containing a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) an enzymatic activity comprising i) hyperthermophilic polymerase activity and optionally ii) reverse transcriptase activity.

[0008] In certain embodiments, methods for processing nucleic acids are also provided herein, comprising the step of amplifying a nucleic acid, which essentially consists of contacting a non-denatured sample nucleic acid under isothermal amplification conditions with a) at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) at least one component providing hyperthermophilic polymerase activity to produce a nucleic acid amplification product.

[0009] In certain embodiments, the Specified Provisions also provide a method for processing nucleic acids, comprising the step of amplifying a nucleic acid, which essentially involves contacting an undenatured sample nucleic acid under isothermal amplification conditions with an enzymatic component comprising a) a non-enzymatic component comprising at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) a hyperthermophilic polymerase or a polymerase comprising an amino acid sequence at least about 90% identical to that of a hyperthermophilic polymerase, thereby producing a nucleic acid amplification product.

[0010] In certain embodiments, the Specified Provisions also provide a method for processing nucleic acids, comprising the step of amplifying a nucleic acid, which essentially consists of contacting an undenatured sample nucleic acid under isothermal amplification conditions with a non-enzymatic component comprising at least one oligonucleotide containing a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) an enzymatic activity comprising i) hyperthermophilic polymerase activity and optionally ii) reverse transcriptase activity to produce a nucleic acid amplification product.

[0011] In certain embodiments, the Specified Provisions also provide a method for processing nucleic acids, comprising the step of amplifying a nucleic acid by contacting an undenatured sample nucleic acid with a) at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) at least one component providing hyperthermophilic polymerase activity, under isothermal amplification conditions, thereby generating a nucleic acid amplification product.

[0012] In certain embodiments, the Specified Provisions also provide a method for processing nucleic acids, comprising the step of amplifying a nucleic acid by contacting an undenatured sample nucleic acid under isothermal amplification conditions with an enzymatic component comprising a) a non-enzymatic component comprising at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) a hyperthermophilic polymerase or a polymerase comprising an amino acid sequence at least about 90% identical to that of a hyperthermophilic polymerase, thereby generating a nucleic acid amplification product.

[0013] In certain embodiments, the Specified Provisions also provide a method for processing nucleic acids, comprising the step of amplifying a nucleic acid by contacting an undenatured sample nucleic acid under isothermal amplification conditions with a non-enzymatic component comprising at least one oligonucleotide containing a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) an enzymatic activity comprising i) hyperthermophilic polymerase activity and optionally ii) reverse transcriptase activity to produce a nucleic acid amplification product.

[0014] In a particular embodiment, a method for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, comprising the steps of: a) amplifying a target sequence in a sample nucleic acid, wherein the target sequence comprises a first strand and a second strand that are complementary to each other, and the amplification step involves contacting the undenatured sample nucleic acid under helicase-free and / or recombinase-free isothermal amplification conditions with i) a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first polynucleotide that is sequentially complementary to the sequence in the first strand, and the second oligonucleotide comprises a second polynucleotide that is sequentially complementary to the sequence in the second strand, and ii) at least one component that provides hyperthermophilic polymerase activity to amplify the nucleic acid A method is also provided herein, comprising the steps of: a) generating a nucleic acid amplification product comprising: 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide; 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide; and 3) a spacer sequence comprising 1 to 10 bases, wherein the spacer sequence is sandwiched between the first and second nucleotide sequences; and b) detecting the nucleic acid amplification product, comprising the use of a real-time detection method and performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with (a)(i) and (a)(ii), thereby determining the presence, absence, or amount of the target sequence in the sample nucleic acid.

[0015] In a particular embodiment, a method for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, comprising the steps of: a) amplifying a target sequence in a sample nucleic acid, wherein the target sequence comprises a first strand and a second strand that are complementary to each other, and the amplification step involves contacting the undenatured sample nucleic acid under helicase-free and / or recombinase-free isothermal amplification conditions with i) a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide consists of a first polynucleotide that is sequentially complementary to the sequence in the first strand, and the second oligonucleotide consists of a second polynucleotide that is sequentially complementary to the sequence in the second strand, and ii) at least one component that provides hyperthermophilic polymerase activity to amplify the nucleic acid A method is also provided herein that includes generating a nucleic acid amplification product comprising: b) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide; b) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide; and c) a spacer sequence comprising 1 to 10 bases, wherein the spacer sequence is sandwiched between the first and second nucleotide sequences; and c) detecting the nucleic acid amplification product, comprising the use of a real-time detection method and performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with (a)(i) and (a)(ii), thereby determining the presence, absence, or amount of the target sequence in the sample nucleic acid.

[0016] In certain embodiments, a kit for determining the presence, absence, or amount of a target sequence in a sample nucleic acid is also provided herein, comprising: a) i) a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first polynucleotide that is sequentially complementary to the sequence in the first strand of the target sequence, and the second oligonucleotide comprises a second polynucleotide that is sequentially complementary to the sequence in the second strand of the target sequence, and the first and second strands of the target sequence are complementary to each other; and ii) a component for amplifying the target sequence in a sample nucleic acid under helicase-free and / or recombinase-free isothermal amplification conditions, comprising at least one component that provides hyperthermophilic polymerase activity; and b) at least one component that provides real-time detection activity for the nucleic acid amplification product.

[0017] In certain embodiments, a kit for determining the presence, absence, or amount of a target sequence in a sample nucleic acid is also provided herein, comprising: a) i) a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide consists of a first polynucleotide that is sequentially complementary to the sequence in the first strand of the target sequence, and the second oligonucleotide consists of a second polynucleotide that is sequentially complementary to the sequence in the second strand of the target sequence, and the first and second strands of the target sequence are complementary to each other; and ii) a component for amplifying the target sequence in a sample nucleic acid under helicase-free and / or recombinase-free isothermal amplification conditions, comprising at least one component that provides hyperthermophilic bacterial polymerase activity; and b) at least one component that provides real-time detection activity of the nucleic acid amplification product. The present invention provides, for example, the following items: (Item 1) A method for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, a) A step of amplifying the target sequence in the sample nucleic acid, The target sequence comprises a first strand and a second strand, The first chain and the second chain are complementary to each other, and The amplification step involves amplifying the undenatured sample nucleic acid under isothermal amplification conditions that are free of cleavage agents, helicases, and recombinases. i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide consists of a first polynucleotide that is continuously complementary to the sequence in the first chain, and the second oligonucleotide consists of a second polynucleotide that is continuously complementary to the sequence in the second chain, and ii) At least one component that provides hyperthermophilic polymerase activity This includes generating nucleic acid amplification products by contacting them with The nucleic acid amplification product consists of 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and 3) a spacer sequence containing 1 to 10 bases, The spacer sequence is sandwiched between the first nucleotide sequence and the second nucleotide sequence, b) A step of detecting the nucleic acid amplification product, the step of detecting the nucleic acid amplification product comprising the use of a real-time detection method and performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with (a)(i) and (a)(ii), thereby determining the presence, absence, or amount of the target sequence in the sample nucleic acid. Methods that include... (Item 2) The method according to item 1, wherein the hyperthermophilic polymerase activity is provided by a hyperthermophilic polymerase or a functional fragment thereof, or by a polymerase or a functional fragment thereof having an amino acid sequence identical to that of a hyperthermophilic polymerase by at least about 90%. (Item 3) The method according to item 1 or 2, wherein the hyperthermophilic bacterial polymerase activity is provided by a polymerase or functional fragment thereof containing the amino acid sequence of SEQ ID NO: 8, or by a polymerase or functional fragment thereof containing an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 8. (Item 4) The method according to item 1, 2, or 3, wherein the hyperthermophilic bacterial polymerase activity is provided by a polymerase with low or no exonuclease activity. (Item 5) The method according to any one of items 1 to 4, wherein element (a)(ii) further comprises at least one component that provides reverse transcriptase activity. (Item 6) The method according to any one of items 1 to 5, wherein the at least one component that provides hyperthermophilic bacterial polymerase activity further provides reverse transcriptase activity. (Item 7) The method according to any one of items 1 to 6, which does not include the step of denaturing the sample nucleic acid before or during amplification. (Item 8) The method according to any one of items 1 to 7, wherein the sample nucleic acid is not brought into contact with a single-stranded DNA-binding protein before or during amplification. (Item 9) A method according to any one of items 1 to 8, wherein an unpurified sample nucleic acid is amplified. (Item 10) The method according to any one of items 1 to 9, wherein the amplification is performed at a constant temperature of approximately 55°C to approximately 75°C. (Item 11) The method according to any one of items 1 to 9, wherein the amplification is performed at a constant temperature of approximately 65°C. (Item 12) The method according to any one of items 1 to 9, wherein the amplification is performed at a constant temperature of approximately 60°C. (Item 13) The method according to any one of items 1 to 12, wherein the first oligonucleotide and the second oligonucleotide are each about 8 to 16 bases in length. (Item 14) The method according to any one of items 1 to 13, wherein the nucleic acid amplification product is approximately 20 to 40 bases in length. (Item 15) The method according to any one of items 1 to 14, wherein the spacer sequence comprises 1 to 5 bases and is continuously complementary to or substantially identical to a portion of the target sequence in the sample nucleic acid. (Item 16) The method according to any one of items 1 to 15, wherein the step of detecting the nucleic acid amplification product includes the detection of a fluorescent signal. (Item 17) The method according to item 16, wherein the fluorescence signal originates from a molecular beacon. (Item 18) The method according to any one of items 1 to 17, further comprising the step of contacting the nucleic acid amplification product with i) a polynucleotide complementary to the sequence in the amplification product, and ii) a signal-generating oligonucleotide comprising a fluorophore and a quencher. (Item 19) The method according to any one of items 1 to 18, carried out in a single reaction vessel. (Item 20) A method described in any one of items 1 to 19, including multiplex amplification. (Item 21) The amplification step involves amplifying the undenatured sample nucleic acid under isothermal amplification conditions that are free of cleavage agents, helicases, and recombinases. i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide consists of a first polynucleotide that is continuously complementary to the sequence in the first chain, and the second oligonucleotide consists of a second polynucleotide that is continuously complementary to the sequence in the second chain, and ii) Enzyme components consisting of components that provide hyperthermophilic polymerase activity The method described in any one of items 1 to 20, including bringing into contact with. (Item 22) The method according to any one of items 1 to 21, wherein the first oligonucleotide consists of a DNA base, a modified DNA base, or a first polynucleotide comprising a DNA base and a modified DNA base, and the second oligonucleotide consists of a DNA base, a modified DNA base, or a second polynucleotide comprising a DNA base and a modified DNA base. (Item 23) A method for amplifying nucleic acids, Under isothermal amplification conditions, non-denatured sample nucleic acids a) At least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) At least one component that provides hyperthermophilic polymerase activity A step of generating nucleic acid amplification products by contacting them with components containing the above. Methods that include... (Item 24) A method for amplifying nucleic acids. Under isothermal amplification conditions, non-denatured nucleic acid samples a) A non-enzymatic component comprising at least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) Enzymatic component consisting of hyperthermophilic polymerase or polymerase having an amino acid sequence at least approximately 90% identical to that of hyperthermophilic polymerase. A step to generate nucleic acid amplification products by contacting them. Methods that include... (Item 25) A method for amplifying nucleic acids, Under isothermal amplification conditions, non-denatured nucleic acid samples a) A non-enzymatic component comprising at least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) Enzyme activity consisting of i) hyperthermophilic polymerase activity and optionally ii) reverse transcriptase activity A step to generate nucleic acid amplification products by contacting them. Methods that include... (Item 26) The method according to item 25, wherein the enzyme activity consists of i) hyperthermophilic polymerase activity and ii) reverse transcriptase activity. (Item 27) The method according to any one of items 23 to 26, which does not include the step of denaturing the sample nucleic acid before or during amplification. (Item 28) The method according to any one of items 23 to 27, wherein the sample nucleic acid is not brought into contact with an endonuclease before or during amplification. (Item 29) The method according to any one of items 23 to 28, wherein the sample nucleic acid is not brought into contact with the unwinding agent before or during amplification. (Item 30) The method according to any one of items 23 to 29, wherein the sample nucleic acid is not brought into contact with helicase before or during amplification. (Item 31) The method according to any one of items 23 to 30, wherein the sample nucleic acid is not brought into contact with the recombinase before or during amplification. (Item 32) The method according to any one of items 23 to 31, wherein the sample nucleic acid is not brought into contact with a single-stranded DNA-binding protein before or during amplification. (Item 33) The method according to any one of items 23 to 32, wherein the sample nucleic acid is unmodified before amplification. (Item 34) The method according to item 33, wherein the unmodified sample nucleic acid is derived from disrupted cells. (Item 35) The method according to any one of items 23 to 34, wherein the sample nucleic acid includes DNA. (Item 36) The method according to item 35, wherein the sample nucleic acid includes genomic DNA. (Item 37) The method according to any one of items 23 to 34, wherein the sample nucleic acid contains RNA. (Item 38) The method according to item 37, wherein the sample nucleic acid contains viral RNA. (Item 39) The method according to item 37, wherein the sample nucleic acid contains bacterial RNA. (Item 40) The method according to any one of items 23 to 39, wherein the sample nucleic acid comprises a single-stranded nucleic acid. (Item 41) The method according to any one of items 23 to 39, wherein the sample nucleic acid comprises a double-stranded nucleic acid including a first strand and a second strand. (Item 42) The method according to any one of items 23 to 41, wherein the at least one oligonucleotide comprises a first oligonucleotide and a second oligonucleotide. (Item 43) The method according to any one of items 23 to 41, wherein the at least one oligonucleotide comprises a first oligonucleotide and a second oligonucleotide. (Item 44) The method according to item 42 or 43, wherein the first oligonucleotide and the second oligonucleotide each contain 8 to 16 bases. (Item 45) The method according to item 42, 43, or 44, wherein the first oligonucleotide comprises a first polynucleotide complementary to the target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide complementary to the target sequence in the second strand of the sample nucleic acid. (Item 46) The method according to item 42, 43, or 44, wherein the first oligonucleotide comprises a first polynucleotide that is sequentially complementary to the target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide that is sequentially complementary to the target sequence in the second strand of the sample nucleic acid. (Item 47) The method according to item 42, 43, or 44, wherein the first oligonucleotide consists of a first polynucleotide that is sequentially complementary to the target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide consists of a second polynucleotide that is sequentially complementary to the target sequence in the second strand of the sample nucleic acid. (Item 48) The method described in any one of items 23 to 47, wherein the sample nucleic acid is obtained from the subject before amplification. (Item 49) The method described in any one of items 23-48, wherein an unpurified sample nucleic acid is amplified. (Item 50) The method described in any one of items 23-48, wherein the purified sample nucleic acid is amplified. (Item 51) The method according to any one of items 23 to 48, further comprising the step of purifying the sample nucleic acid before amplification. (Item 52) The method according to any one of items 23 to 51, wherein the hyperthermophilic polymerase activity is provided by a hyperthermophilic polymerase or a functional fragment thereof. (Item 53) The method according to any one of items 23 to 51, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof having an amino acid sequence at least about 90% identical to that of the hyperthermophilic polymerase. (Item 54) The method according to any one of items 23 to 51, wherein the hyperthermophilic polymerase activity is provided by an archaeal hyperthermophilic polymerase or a functional fragment thereof. (Item 55) The method according to any one of items 23 to 54, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 8. (Item 56) The method according to any one of items 23 to 54, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 8. (Item 57) The method according to any one of items 23 to 56, wherein the hyperthermophilic bacterial polymerase activity is provided by a polymerase with low exonuclease activity. (Item 58) The method according to any one of items 23 to 56, wherein the hyperthermophilic polymerase activity is provided by a polymerase that does not have exonuclease activity. (Item 59) The method according to any one of items 23 to 58, wherein the amplification is performed at a constant temperature of approximately 55°C to approximately 75°C. (Item 60) The method according to any one of items 23 to 58, wherein the amplification is performed at a constant temperature of approximately 55°C to approximately 65°C. (Item 61) The method according to any one of items 23 to 58, wherein the amplification is performed at a constant temperature of approximately 65°C. (Item 62) The method according to any one of items 23 to 58, wherein the amplification is performed at a constant temperature of approximately 60°C. (Item 63) The method according to any one of items 23 to 62, wherein the nucleic acid amplification product is detectable within 10 minutes or less. (Item 64) The method according to any one of items 23 to 63, wherein the nucleic acid amplification product comprises a polynucleotide that is sequentially complementary to or substantially identical to the target sequence in the sample nucleic acid. (Item 65) The method according to any one of items 23 to 63, wherein the nucleic acid amplification product consists of polynucleotides that are sequentially complementary to or substantially identical to the target sequence in the sample nucleic acid. (Item 66) The method according to any one of items 23 to 65, wherein the nucleic acid amplification product is approximately 20 to 40 bases in length. (Item 67) The method according to any one of items 43 to 66, wherein the nucleic acid amplification product comprises i) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide; ii) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide; and iii) a spacer sequence sandwiched between the first nucleotide sequence and the second nucleotide sequence. (Item 68) The method according to any one of items 43 to 66, wherein the nucleic acid amplification product comprises i) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, ii) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and iii) a spacer sequence sandwiched between the first nucleotide sequence and the second nucleotide sequence. (Item 69) The method according to item 67 or 68, wherein the spacer sequence comprises 1 to 10 bases. (Item 70) The method according to item 67 or 68, wherein the spacer sequence comprises 1 to 5 bases. (Item 71) The method according to any one of items 67 to 70, wherein the spacer sequence is neither complementary to nor identical to the first polynucleotide of the first oligonucleotide, and neither complementary to nor identical to the second polynucleotide of the second oligonucleotide. (Item 72) The method according to any one of items 67 to 71, wherein the spacer sequence is continuously complementary to or substantially identical to a portion of the target sequence in the sample nucleic acid. (Item 73) The method according to any one of items 23 to 72, further comprising the step of detecting the nucleic acid amplification product. (Item 74) The method according to item 73, wherein the step of detecting the nucleic acid amplification product is performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with the component providing the hyperthermophilic polymerase activity and the at least one oligonucleotide. (Item 75) The method according to item 73 or 74, wherein the step of detecting the nucleic acid amplification product includes the use of a real-time detection method. (Item 76) The method according to item 73, 74, or 75, wherein the step of detecting the nucleic acid amplification product includes the detection of a fluorescent signal. (Item 77) The method according to item 76, wherein the fluorescence signal originates from a molecular beacon. (Item 78) The method according to any one of items 23 to 77, further comprising the step of contacting the nucleic acid amplification product with i) a polynucleotide complementary to the sequence in the amplification product, and ii) a signal-generating oligonucleotide comprising a fluorophore and a quencher. (Item 79) The method according to any one of items 23 to 78, wherein one or more of the at least one oligonucleotides is a polynucleotide that is not complementary to the sequence in the sample nucleic acid and hybridizes to the signal-generating oligonucleotide, and the method further comprises the step of contacting the amplification product with the signal-generating oligonucleotide comprising a fluorophore and a quencher. (Item 80) The method according to any one of items 23 to 79, carried out in a single reaction volume. (Item 81) The method according to any one of items 23 to 80, carried out in a single reaction vessel. (Item 82) The method described in any one of items 23 to 81, including multiplex amplification. (Item 83) The method according to any one of items 23 to 82, wherein the sample nucleic acid is not brought into contact with the cleavage agent before or during amplification. (Item 84) A kit for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, a) A component for amplifying a target sequence in the sample nucleic acid under helicase-free isothermal amplification conditions, the following: i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first polynucleotide that is sequentially complementary to the sequence in the first chain of the target sequence, and the second oligonucleotide comprises a second polynucleotide that is sequentially complementary to the sequence in the second chain of the target sequence, and the first and second chains of the target sequence are complementary to each other, and ii) At least one component that provides hyperthermophilic polymerase activity Ingredients, including, b) At least one component that provides real-time detection activity for nucleic acid amplification products and A kit that includes this. (Item 85) The kit according to item 84, wherein the first oligonucleotide essentially consists of a first polynucleotide that is sequentially complementary to the sequence in the first strand of the target sequence, and the second oligonucleotide essentially consists of a second polynucleotide that is sequentially complementary to the sequence in the second strand of the target sequence. (Item 86) The kit according to item 84, wherein the first oligonucleotide consists of a first polynucleotide that is continuously complementary to the sequence in the first strand of the target sequence, and the second oligonucleotide consists of a second polynucleotide that is continuously complementary to the sequence in the second strand of the target sequence. (Item 87) A kit for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, a) A component for amplifying a target sequence in the sample nucleic acid under helicase-free isothermal amplification conditions, the following: i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide consists of a first polynucleotide that is continuously complementary to the sequence in the first chain of the target sequence, and the second oligonucleotide consists of a second polynucleotide that is continuously complementary to the sequence in the second chain of the target sequence, and the first and second chains of the target sequence are complementary to each other, and ii) At least one component that provides hyperthermophilic polymerase activity Ingredients, including, b) At least one component that provides real-time detection activity for nucleic acid amplification products and A kit that includes this. (Item 88) The kit according to any one of items 84 to 87, wherein the sample nucleic acid is amplified under isothermal amplification conditions that are helicase-free and recombinase-free. (Item 89) The kit according to any one of items 84 to 87, wherein the sample nucleic acid is amplified under isothermal amplification conditions that are free of helicase, recombinase, and cleavage agents. (Item 90) The kit according to any one of items 84 to 89, wherein the at least one component providing hyperthermophilic polymerase activity comprises a hyperthermophilic polymerase or a functional fragment thereof, or a polymerase or a functional fragment thereof having an amino acid sequence identical to that of a hyperthermophilic polymerase by at least about 90%. (Item 91) The kit according to any one of items 84 to 89, wherein the at least one component providing hyperthermophilic polymerase activity comprises a hyperthermophilic polymerase or a functional fragment thereof, or a polymerase or a functional fragment thereof having an amino acid sequence identical to that of a hyperthermophilic polymerase by at least about 90%. (Item 92) The kit according to any one of items 84 to 91, wherein the hyperthermophilic polymerase activity is provided by an archaeal hyperthermophilic polymerase or a functional fragment thereof. (Item 93) The kit according to any one of items 84 to 92, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof containing the amino acid sequence of SEQ ID NO: 8. (Item 94) The kit according to any one of items 84 to 92, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 8. (Item 95) The kit according to any one of items 84 to 94, wherein the hyperthermophilic bacterial polymerase activity is provided by a polymerase with low exonuclease activity. (Item 96) The kit according to any one of items 84 to 94, wherein the hyperthermophilic bacterial polymerase activity is provided by a polymerase that does not have exonuclease activity. (Item 97) The kit according to any one of items 84 to 96, wherein element (a)(ii) further comprises at least one component that provides reverse transcriptase activity. (Item 98) The kit according to any one of items 84 to 96, wherein at least one component that provides hyperthermophilic polymerase activity further provides reverse transcriptase activity. (Item 99) A kit according to any one of items 84 to 98, wherein the first oligonucleotide and the second oligonucleotide each contain 8 to 16 bases. (Item 100) The kit according to any one of items 84-99, wherein the real-time detection activity is provided by a molecular beacon. (Item 101) The instructions further include for carrying out a method for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, wherein the method a) A step of amplifying the target sequence in the sample nucleic acid, The target sequence comprises a first strand and a second strand, The first chain and the second chain are complementary to each other, Furthermore, the amplification step involves performing the amplification under helicase-free isothermal amplification conditions to obtain non-denatured sample nucleic acids. i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first polynucleotide that is sequentially complementary to the sequence in the first chain, and the second oligonucleotide comprises a second polynucleotide that is sequentially complementary to the sequence in the second chain, and ii) At least one component that provides hyperthermophilic polymerase activity The process involves generating a nucleic acid amplification product by contacting it with, wherein the nucleic acid amplification product comprises: 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide; 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide; and 3) a spacer sequence comprising 1 to 10 bases. The spacer sequence is sandwiched between the first nucleotide sequence and the second nucleotide sequence, b) A step of detecting the nucleic acid amplification product, comprising the use of a real-time detection method and performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with (a)(i) and (a)(ii), thereby determining the presence, absence, or amount of the target sequence in the sample nucleic acid. A kit including any one of items 84-100. (Item 102) The kit according to item 101, wherein the first oligonucleotide essentially consists of a first polynucleotide that is sequentially complementary to the sequence in the first chain, and the second oligonucleotide essentially consists of a second polynucleotide that is sequentially complementary to the sequence in the second chain, and / or the nucleic acid amplification product essentially consists of 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and 3) a spacer sequence containing 1 to 10 bases. (Item 103) The kit according to item 101, wherein the first oligonucleotide consists of a first polynucleotide that is sequentially complementary to the sequence in the first chain, and the second oligonucleotide consists of a second polynucleotide that is sequentially complementary to the sequence in the second chain, and / or the nucleic acid amplification product consists of 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and 3) a spacer sequence containing 1 to 10 bases. (Item 104) The instructions further include for carrying out a method for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, wherein the method a) A step of amplifying the target sequence in the sample nucleic acid, The target sequence comprises a first strand and a second strand, The first chain and the second chain are complementary to each other, Furthermore, the amplification step involves performing the amplification under helicase-free isothermal amplification conditions to obtain non-denatured sample nucleic acids. i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide consists of a first polynucleotide that is continuously complementary to the sequence in the first chain, and the second oligonucleotide consists of a second polynucleotide that is continuously complementary to the sequence in the second chain, and ii) At least one component that provides hyperthermophilic polymerase activity The process includes generating a nucleic acid amplification product by contacting it with, wherein the nucleic acid amplification product consists of 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and 3) a spacer sequence containing 1 to 10 bases, The spacer sequence is sandwiched between the first nucleotide sequence and the second nucleotide sequence, b) A step of detecting the nucleic acid amplification product, comprising the use of a real-time detection method and performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with (a)(i) and (a)(ii), thereby determining the presence, absence, or amount of the target sequence in the sample nucleic acid. A kit including any one of items 84-100. (Item 105) The kit according to any one of items 101 to 104, wherein the method does not include a step of denaturing the sample nucleic acid before or during amplification. (Item 106) A kit according to any one of items 101 to 105, wherein the sample nucleic acid is not brought into contact with an endonuclease before, during, or after amplification. (Item 107) The kit according to any one of items 101 to 106, wherein the sample nucleic acid is unmodified before amplification. (Item 108) The kit described in item 107, wherein the unmodified sample nucleic acid is derived from disrupted cells. (Item 109) A kit according to any one of items 101 to 108, wherein the sample nucleic acid contains DNA. (Item 110) The kit described in item 109, wherein the sample nucleic acid includes genomic DNA. (Item 111) A kit according to any one of items 101 to 108, wherein the sample nucleic acid contains RNA. (Item 112) The kit described in item 111, wherein the sample nucleic acid contains viral RNA. (Item 113) The kit described in item 111, wherein the sample nucleic acid contains bacterial RNA. (Item 114) A kit according to any one of items 101 to 113, wherein the sample nucleic acid contains single-stranded nucleic acid. (Item 115) A kit according to any one of items 101 to 113, wherein the sample nucleic acid contains double-stranded nucleic acid. (Item 116) A kit described in any one of items 101-115, wherein the sample nucleic acid is obtained from the subject before amplification. (Item 117) A kit described in any one of items 101-116, which amplifies unpurified sample nucleic acids. (Item 118) A kit described in any one of items 101-116, which amplifies a purified sample nucleic acid. (Item 119) The kit according to any one of items 101 to 118, further comprising the step of purifying a sample nucleic acid before amplification. (Item 120) The kit described in any one of items 101 to 119, wherein the amplification is performed at a constant temperature of approximately 55°C to approximately 75°C. (Item 121) The kit described in any one of items 101 to 119, wherein the amplification is performed at a constant temperature of approximately 55°C to approximately 65°C. (Item 122) A kit as described in any one of items 101 to 119, wherein the amplification is performed at a constant temperature of approximately 65°C. (Item 123) A kit as described in any one of items 101 to 119, wherein the amplification is performed at a constant temperature of approximately 60°C. (Item 124) A kit according to any one of items 101 to 123, wherein the nucleic acid amplification product is approximately 20 to 40 bases in length. (Item 125) A kit according to any one of items 101 to 124, wherein the spacer sequence contains 1 to 5 bases. (Item 126) The kit according to any one of items 101 to 125, wherein the spacer sequence is neither complementary to nor identical to the first polynucleotide of the first oligonucleotide, and neither complementary to nor identical to the second polynucleotide of the second oligonucleotide. (Item 127) The kit according to any one of items 101 to 126, wherein the spacer sequence is continuously complementary to or substantially identical to a portion of the target sequence in the sample nucleic acid. (Item 128) A kit according to any one of items 101 to 127, wherein the step of detecting the nucleic acid amplification product includes the detection of a fluorescent signal. (Item 129) The kit described in item 128, wherein the aforementioned fluorescent signal originates from a molecular beacon. (Item 130) The kit according to any one of items 101 to 129, wherein the method further comprises the step of contacting the nucleic acid amplification product with i) a polynucleotide complementary to the sequence in the amplification product, and ii) a signal-generating oligonucleotide comprising a fluorophore and a quencher. (Item 131) A kit according to any one of items 101 to 130, wherein the above method is carried out in a single reaction volume. (Item 132) The kit according to any one of items 101 to 131, wherein the above method is carried out in a single reaction vessel. (Item 133) A kit according to any one of items 101 to 132, wherein the method includes multiplex amplification.

[0018] Furthermore, certain embodiments are described in the following description, examples, claims, and drawings.

[0019] The drawings illustrate, but are not limiting, certain embodiments of the present technology. For clarity and ease of understanding, the drawings are not at a constant scale, and in some examples, various aspects may be exaggerated or enlarged to facilitate understanding of a particular embodiment. [Brief explanation of the drawing]

[0020] [Figure 1] Figure 1 shows real-time detection of Chlamydia genomic DNA amplification reactions performed in a double-chain manner using dH2O or Tris-EDTA buffer (TE) as an untargeted control (NTC; negative control).

[0021] [Figure 2] Figure 2 shows electrospray ionization mass spectrometry (ESI-MS) detection of Chlamydia genomic DNA assay products.

[0022] [Figure 3] Figure 3 shows the detection limit (LOD) of Chlamydia genomic DNA by endpoint molecular beacon detection.

[0023] [Figure 4] Figure 4 shows the real-time detection of Chlamydia genomic DNA using molecular beacons.

[0024] [Figure 5-1] Figure 5 shows a schematic diagram of the isothermal amplification reaction described herein. [Figure 5-2] Figure 5 shows a schematic diagram of the isothermal amplification reaction described herein. [Modes for carrying out the invention]

[0025] Methods and compositions for amplifying nucleic acids are provided herein. Conventional nucleic acid amplification methods typically require thermocycling, nucleic acid denaturation, chain unwinding, chain separation, and / or proteins (e.g., enzymes) (e.g., helicases, recombinases) that promote chain exchange, and / or endonucleases (e.g., restriction enzymes, nickeling enzymes), and often require a reaction time of at least 20-30 minutes. The nucleic acid amplification methods provided herein can be performed without thermocycling, without nucleic acid denaturation, without the addition of proteins (e.g., enzymes) that promote chain unwinding, chain separation, and / or chain exchange, without endonucleases, and with a reaction time of less than 10 minutes.

[0026] Nucleic acids, subjects, samples, and nucleic acid processing Methods and compositions for amplifying nucleic acids are provided herein. The terms “nucleic acid” and “nucleic acid molecule” may be used interchangeably herein. These terms refer to nucleic acids of any composition, e.g., DNA (e.g., complementary DNA (cDNA), genomic DNA (gDNA), etc.), RNA (e.g., message RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, and / or DNA or RNA analogs (e.g., including base analogs, sugar analogs, and / or non-natural backbones, etc.)), RNA / DNA hybrids, and polyamide nucleic acids (PNA), all of which may be in single-stranded or double-stranded form and may, unless otherwise limited, include known analogs of natural nucleotides that can function similarly to naturally occurring nucleotides. Nucleic acids include plasmids, phages, autonomous replication sequences (ARS), centromeres, artificial chromosomes, chromosomes, or in A nucleic acid may be or may be derived from a nucleic acid that can replicate or be replicated in vitro or in a host cell, cell, cell nucleus, mitochondria, or, in certain embodiments, in the cytoplasm of a cell. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized similarly to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence implicitly includes, in addition to the explicitly indicated sequence, its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologues, single nucleotide polymorphisms (SNPs), and complementary sequences. Specifically, a degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue. The term nucleic acid may be used interchangeably with loci, genes, cDNA, and mRNA encoded by genes.The term may also encompass, as equivalents, nucleotide analogs, derivatives, variants, and analogs of RNA or DNA synthesized from single-stranded ("sense" or "antisense," "plus" or "minus" strands, "forward" reading frame or "reverse" reading frame, "forward" or "reverse" strand) and double-stranded polynucleotides. The term "gene" refers to a segment of DNA involved in the production of polypeptide chains and generally includes the regions before and after the coding region (leaders and trailers) involved in the transcription / translation and regulation of transcription / translation of gene products, as well as intervening sequences (introns) between individual coding segments (exons). A nucleotide or base generally refers to the purine and pyrimidine molecular units of nucleic acids (e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)). In the case of RNA, the base thymine is replaced by uracil. The length or size of a nucleic acid can be expressed as the number of bases.

[0027] In some embodiments of the methods provided herein, one or more nucleic acid targets are amplified. Target nucleic acids may be referred to as target sequences, target polynucleotides, and / or target polynucleotide sequences, and may encompass double-stranded and single-stranded nucleic acid molecules. Target nucleic acids may be, for example, DNA or RNA. If the target nucleic acid is an RNA molecule, the molecule may be, for example, double-stranded or single-stranded, or the RNA molecule may contain a single-stranded target sequence. If the target nucleic acid is double-stranded, the target nucleic acid generally comprises a first strand and a second strand. The first and second strands may be referred to as the forward strand and the reverse strand, and are generally complementary to each other. If the target nucleic acid is single-stranded, the complementary strand can be produced, for example, by polymerization and / or reverse transcription, so that the target nucleic acid becomes double-stranded and has a first / forward strand and a second / reverse strand.

[0028] The target sequence may refer to either the sense or antisense strand of a nucleic acid sequence, or it may refer to the sequence as it appears on the target nucleic acid, on an amplified copy of the original target sequence, or on the amplification product. The target sequence may be a subsequence within a larger polynucleotide. For example, the target sequence may be a short sequence (e.g., 20-50 nucleotides) within a nucleic acid fragment, chromosome, or plasmid that has been targeted for amplification. In some embodiments, the target sequence may refer to a sequence in the target nucleic acid that is complementary to the oligonucleotide (e.g., primer) used to amplify the nucleic acid. Thus, the target sequence may refer to the entire sequence targeted for amplification, or to a subsequence in the target nucleic acid to which the oligonucleotide binds. The amplification product may be a larger molecule containing the target sequence and at least one other sequence or other nucleotides. In some embodiments, the amplification product is approximately the same length as the target sequence. In some embodiments, the amplification product is exactly the same length as the target sequence. In some embodiments, the amplification product contains the target sequence. In some embodiments, the amplification product consists of the target sequence.

[0029] The length of the target sequence and / or the guanosine cytosine (GC) concentration (percent) may be partially dependent on the temperature at which the amplification reaction takes place, and this temperature may be partially dependent on the stability of the polymerase used in the reaction. A sample assay can be performed to determine the appropriate target sequence length and GC concentration for a set of reaction conditions. For example, if the polymerase is stable up to 60°C–65°C, the target sequence may be, for example, 19–50 nucleotides long, or, for example, approximately 40–50, 20–45, 20–40, or 20–30 nucleotides long. The GC concentration under these conditions may be, for example, less than 60%, less than 55%, less than 50%, or less than 45%.

[0030] Target nucleic acids may include, for example, genomic nucleic acids, plasmid nucleic acids, mitochondrial nucleic acids, cellular nucleic acids, extracellular nucleic acids, bacterial nucleic acids, and viral nucleic acids. In some embodiments, target nucleic acids may include genomic DNA, chromosomal DNA, plasmid DNA, mitochondrial DNA, genes, any type of cellular RNA, messenger RNA, bacterial RNA, viral RNA, or synthetic oligonucleotides. Genomic nucleic acids may include, for example, genomes of animals, plants, insects, viruses, and bacteria, or any nucleic acid derived from any genome, such as the genome present in a spore. In some embodiments, genomic target nucleic acids may be located within a specific genomic locus or a combination of genomic loci. A genomic locus may include any or a combination of open reading frame DNA, untranscribed DNA, intronic sequences, extronic sequences, promoter sequences, enhancer sequences, adjacent sequences, or any sequences considered to be associated with a given genomic locus.

[0031] In some embodiments, the target sequence comprises one or more repeating elements (e.g., multiple repeats, reverse repeats, palindromic sequences, tandem repeats, microsatellites, minisatellites, etc.). In some embodiments, the target sequence exists within the sample nucleic acid (e.g., within a nucleic acid fragment, chromosome, genome, or plasmid) as repeating elements (e.g., multiple repeats, reverse repeats, palindromic sequences, tandem repeats, microsatellite repeats, minisatellite repeats, etc.). For example, the target sequence may exist multiple times as a repeating element, and the presence of one, some, or all of the target sequence within the repeating element may be amplified using the methods described herein (e.g., using a single pair of primers). In some embodiments, the target sequence exists within the sample nucleic acid (e.g., within a nucleic acid fragment, chromosome, genome, or plasmid) as duplicates and / or paralogs.

[0032] Target nucleic acids may include microRNAs. MicroRNAs, miRNAs, or small temporal RNAs (stRNAs) are short (e.g., about 21-23 nucleotides in length) single-stranded RNA sequences involved in gene regulation. MicroRNAs can interfere with messenger RNA translation and are partially complementary to messenger RNA. Target nucleic acids may include microRNA precursors such as primary transcripts (pri-miRNAs) and pre-miRNA stem-loop structure RNAs, which are further processed to become miRNAs. Target nucleic acids may also include short interfering RNAs (siRNAs), which are short (e.g., about 20-25 nucleotides in length) at least partially double-stranded RNA molecules involved in RNA interference (e.g., downregulation of viral replication or gene expression).

[0033] The nucleic acids used in the methods described herein can be obtained from any suitable biological specimen or sample, and are often isolated from samples obtained from a subject. The subject may be any living or non-living organism, including but not limited to humans, non-human animals, plants, bacteria, fungi, viruses, or protists. Any human or non-human animal can be selected, including but not limited to mammals, reptiles, birds, amphibians, fish, ungulates, ruminants, bovines (e.g., cows), equines (e.g., horses), caprines and ovines (e.g., sheep, goats), swines (e.g., pigs), camelids (e.g., camels, llamas, alpacas), monkeys, apes (e.g., gorillas, chimpanzees), ursids (e.g., bears), poultry, dogs, cats, mice, rats, fish, dolphins, whales, and sharks. Subjects may be male or female, and may be of any age (e.g., embryo, fetus, infant, child, adult).

[0034] A sample or test sample may be any specimen isolated or obtained from the subject or a part thereof. Non-limiting examples of specimens include, but are not limited to, fluids or tissues derived from the subject, including, blood or blood products (e.g., serum or plasma), umbilical cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, cerebrospinal fluid, lavage fluid (e.g., from the bronchoalveolar, gastric, peritoneal, tubal, ear, or arthroscopic), biopsy specimens, celocentesis specimens, cells (e.g., blood cells) or parts thereof (e.g., mitochondria, nuclei, or extracts), lavage fluid from the female reproductive tract, urine, feces, sputum, saliva, nasal mucus, prostatic fluid, lavage fluid, semen, lymph, bile, tears, sweat, breast milk, breast fluid, hard tissue (e.g., liver, spleen, kidney, lung, or ovary), or combinations thereof. The term "blood" encompasses whole blood, blood products, or any fraction of blood, such as serum, plasma, or bufficoat, as conventionally defined. Plasma refers to the fraction of whole blood obtained by centrifugation of blood treated with an anticoagulant. Serum refers to the aqueous portion of the fluid remaining after a blood sample has been clotting. Fluid or tissue samples are often collected according to standard protocols commonly followed in hospitals or clinics. In the case of blood, an appropriate amount of peripheral blood (e.g., 3-40 milliliters) is often collected and can be stored according to standard procedures before or after preparation.

[0035] Examples of samples or test samples include spores, viruses, cells, nucleic acids derived from prokaryotes or eukaryotes, or samples containing any free nucleic acids. For example, the methods described herein may be used to detect nucleic acids on the outside of spores (e.g., without requiring lysis). Samples may be isolated from any material suspected of containing the target sequence, such as those derived from the above-mentioned subjects. In certain examples, the target sequence may be present in air, plants, soil, or other material suspected of containing living organisms.

[0036] Nucleic acids can be obtained (e.g., isolated, extracted, and purified) from one or more sources by methods known in the art. Any suitable method for isolating, extracting, and / or purifying nucleic acids from biological samples can be used, non-limiting examples of which include DNA preparation methods in the art, and various commercially available reagents or kits, or combinations thereof, such as Qiagen's QIAamp Circulating Nucleic Acid Kit, QiaAmp DNA Mini Kit, or QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany), GenomicPrep® Blood DNA Isolation Kit (Promega, Madison, Wis.), GFX® Genomic Blood DNA Purification Kit (Amersham, Piscataway, NJ).

[0037] In some embodiments, a cell lysis procedure is performed. Cell lysis may be performed before the commencement of amplification reactions described herein (e.g., to release DNA and / or RNA from cells for amplification). Cell lysis procedures and reagents are known in the art and can generally be performed by chemical (e.g., surfactants, hypotonic solutions, enzymatic procedures, etc., or a combination thereof), physical (e.g., French press, sonication, etc.), or electrolytic lysis methods. Any suitable lysis procedure can be utilized. For example, chemical methods generally use a lysis agent to disrupt cells and extract nucleic acids from cells, followed by treatment with a chaotropic salt. In some embodiments, cell lysis involves the use of surfactants (e.g., ionic, nonionic, anionic, zwitterionic). In some embodiments, cell lysis involves the use of ionic surfactants (e.g., sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), deoxycholate, cholate, sarcosyl). Physical methods such as freezing / thawing followed by grinding, and the use of a cell disruptor may also be useful. High-salt concentration lysis procedures may also be used. For example, an alkaline dissolution procedure may be used. The latter procedure conventionally incorporates the use of a phenol-chloroform solution, and an alternative phenol-chloroform-free procedure involving three solutions may also be available. In the latter procedure, for example, one solution may contain 15 mM Tris, pH 8.0; 10 mM EDTA and 100 μg / ml ribonuclease A; a second solution may contain 0.2 N NaOH and 1% SDS; and a third solution may contain 3 M KOAc, pH 5.5. In some embodiments, cell lysis buffers are used in combination with the methods and components described herein.

[0038] In certain embodiments, nucleic acids may be provided to perform the methods described herein without processing a sample containing nucleic acids. For example, in some embodiments, nucleic acids are provided to perform the amplification methods described herein without prior nucleic acid purification. In some embodiments, the target sequence is amplified directly from the sample (e.g., without performing any nucleic acid extraction, isolation, purification, and / or partial purification steps). In some embodiments, nucleic acids are provided to perform the methods described herein after processing a sample containing nucleic acids. For example, nucleic acids may be extracted, isolated, purified, or partially purified from the sample. The term “isolated” generally means that nucleic acids have been removed from their original environment (e.g., the natural environment if they are naturally occurring, or the host cell if they are exogenously expressed) and are therefore altered from their original environment by human intervention (e.g., “by human hands”). The term “isolated nucleic acid” may refer to nucleic acids removed from a subject (e.g., a human subject). Isolated nucleic acids may be provided with less non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of components present in the source sample. Compositions containing isolated nucleic acids may contain approximately 50% to more than 99% non-nucleic acid components. Compositions containing isolated nucleic acids may contain approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% non-nucleic acid components. The term "purified" generally refers to the fact that the provided nucleic acid contains less non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than were present before the nucleic acid was subjected to the purification procedure. Compositions containing purified nucleic acids may contain approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% other non-nucleic acid components.

[0039] Nucleic acids may be provided for carrying out the methods described herein without modifying the nucleic acids. Modifications include, for example, denaturation, digestion, nicking, unwinding, incorporation and / or ligation of heterologous sequences, addition of epigenetic modifications, labeling (e.g., 32 P, 33 P, 125 I, or 35 This may include the addition of radioactive labels such as S; enzymatic labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, or fluorescent dyes. Therefore, in some embodiments, unmodified nucleic acids are amplified.

[0040] amplification Methods for amplifying nucleic acids are provided herein. In some embodiments, nucleic acids are amplified using a preferred amplification process. Nucleic acid amplification typically involves the enzymatic synthesis of nucleic acid amplicons (copies) containing sequences complementary to the nucleotide sequence being amplified. In some embodiments, the amplification method is carried out in a single vessel, a single chamber, and / or a single volume (i.e., a continuous volume). In some embodiments, the amplification method and detection method (e.g., the detection method described herein) are carried out in a single vessel, a single chamber, and / or a single volume (i.e., a continuous volume).

[0041] The terms “amplify,” “amplify,” “amplification reaction,” or “the act of amplifying” refer to any in vitro treatment to increase the number of copies of a target nucleic acid. Amplification may refer to an “exponential” increase in the target nucleic acid. However, “the act of amplifying” may also refer to a linear increase in the number of target nucleic acids, but different from a single, single primer extension step. In some embodiments, a limited amplification reaction can be performed, also known as pre-amplification. Pre-amplification is a method in which a limited amount of amplification occurs due to a small number of cycles performed, such as 10 cycles. Pre-amplification allows for some amplification but stops before the logarithmic phase, typically producing about 500 copies of the desired nucleotide sequence. The use of pre-amplification can limit the inaccuracies associated with depleted reactants in a particular amplification reaction and can also reduce amplification bias due to the abundance of the target nucleotide sequence or species. In some embodiments, a single primer extension may be performed as preparation for linear or exponential amplification.

[0042] A general description of the amplification process is presented herein. For example, a primer (e.g., an oligonucleotide as described herein) and a target nucleic acid are brought into contact, and complementary sequences anneal or hybridize with each other. A primer can anneal to the target nucleic acid at or near the sequence of interest (e.g., in its vicinity, adjacent to it, etc.). A primer annealed to a target may be referred to as a primer-target hybrid, a hybridized primer-target, or a primer-target duplex. In relation to the nucleotide sequence of interest, the terms near or near refer to the distance (e.g., number of bases) or region between the end of the primer and one or more nucleotides (e.g., a nucleotide sequence) of the target. Generally, near is a distance of about 1 to about 50 nucleotides (e.g., 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides) from the nucleotide or nucleotide sequence of interest. In some embodiments, a set of primers (e.g., a pair of primers, forward and reverse primers, a first oligonucleotide and a second oligonucleotide) anneal within approximately 1 to 20 nucleotides of the target nucleotide or nucleotide sequence to generate an amplification product. In some embodiments, the primers anneal within the target nucleotide or nucleotide sequence. After annealing, each primer is extended along the target (i.e., the template strand) by polymerase to generate a complementary strand. For example, several cycles of primer annealing and extension may be performed until a detectable amount of amplification product is generated. In some embodiments, if the target nucleic acid is RNA, a DNA copy (cDNA) of the target RNA may be synthesized before or during the amplification step by reverse transcription.

[0043] The components of the amplification reaction may include, for example, one or more primers (e.g., individual primers, primer pairs, primer sets, oligonucleotides, multiple primer sets for multiplex amplification, etc.), a nucleic acid target (e.g., a target nucleic acid derived from a sample), one or more polymerases, a nucleotide (e.g., dNTPs, etc.), and a suitable buffer (e.g., a buffer containing a surfactant, a reducing agent, monovalent ions, and divalent ions). In some embodiments, the amplification reaction may further include a reverse transcriptase. In some embodiments, the amplification reaction may further include one or more detection agents, such as one or more of the detection agents described herein. In some embodiments, the components of the amplification reaction consist of a primer, a target nucleic acid, a polymerase, a nucleotide, and a suitable buffer. In some embodiments, the components of the amplification reaction consist of a primer, a target nucleic acid, a polymerase, a reverse transcriptase, a nucleotide, and a suitable buffer. In some embodiments, the components of the amplification reaction consist of a primer, a target nucleic acid, a polymerase, a detection agent, a nucleotide, and a suitable buffer. In some embodiments, the components of the amplification reaction consist of a primer, a target nucleic acid, a polymerase, a reverse transcriptase, a detection agent, a nucleotide, and a suitable buffer. In some embodiments, the components of the amplification reaction essentially consist of a primer, a target nucleic acid, a polymerase, a nucleotide, and a suitable buffer. In some embodiments, the components of the amplification reaction essentially consist of a primer, a target nucleic acid, a polymerase, a reverse transcriptase, a nucleotide, and a suitable buffer. In some embodiments, the components of the amplification reaction essentially consist of a primer, a target nucleic acid, a polymerase, a detection agent, a nucleotide, and a suitable buffer. In some embodiments, the components of the amplification reaction essentially consist of a primer, a target nucleic acid, a polymerase, a reverse transcriptase, a detection agent, a nucleotide, and a suitable buffer. When the components of the amplification reaction essentially consist of certain components, additional components or features may be included that do not have a significant effect on the amplification and / or are not necessary to produce a detectable product. For example, additional components or features may be included that do not have a significant effect on the ability of the components and conditions herein to achieve amplification under isothermal conditions and to produce a detectable amplified product within about 10 minutes or less.Such additional components or features may be referred to as non-essential components and may include typical reactive components and / or common additives such as salts, buffers, surfactants, ions, oils, proteins, and polymers.

[0044] Nucleic acid amplification may be performed in the presence of native nucleotides, such as dideoxyribonucleoside triphosphates (dNTPs), and / or derivatized nucleotides. Native nucleotides generally refer to adenylic acid, guanylic acid, cytidylic acid, thymidylic acid, or uridylic acid. Derivatized nucleotides generally refer to nucleotides other than native nucleotides. Nucleotides are usually represented as follows: Ribonucleoside triphosphates are referred to as NTP or rNTP, where N can be A, G, C, or U. Deoxyribonucleoside triphosphate substrates are referred to as dNTP, where N can be A, G, C, T, or U. Monomeric nucleotide subunits may be represented as A, G, C, T, or U herein without specifically referring to DNA or RNA. In some embodiments, non-naturally occurring nucleotides or nucleotide analogs may be used, such as analogs containing detectable labels (e.g., fluorescent or colorimetric quantitative labels). For example, nucleic acid amplification may be performed, for example, 32 P, 33 P, 125 I, or 35 The amplification may be carried out in the presence of labeled dNTPs, such as radioactive labels like 3S, enzymatic labels like alkaline phosphatase, fluorescent labels like fluorescein isothiocyanate (FITC), or other labels like biotin, avidin, digoxigenin, antigens, haptens, or fluorescent dyes. In some embodiments, nucleic acid amplification may be carried out in the presence of modified dNTPs, such as thermally activated dNTPs (e.g., TriLink's CleanAmp® dNTPs).

[0045] In some embodiments, the components of the amplification reaction may include non-enzymatic and enzymatic components. Non-enzymatic components may include, for example, primers, nucleotides, buffers, salts, reducing agents, surfactants, and ions, and generally do not include proteins (e.g., nucleic acid-binding proteins), enzymes, or proteins having enzymatic activity, such as polymerases, reverse transcriptases, helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases, etc. In some embodiments, the enzymatic component may consist of polymerase or of polymerase and reverse transcriptase. Thus, such an enzymatic component excludes other proteins (e.g., nucleic acid-binding proteins and / or proteins having enzymatic activity), such as, for example, helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases.

[0046] In some embodiments, amplification conditions include enzyme activity. Typically, enzyme activity is provided by a polymerase, and in certain examples, enzyme activity is provided by both a polymerase and a reverse transcriptase. In some embodiments, enzyme activity consists of polymerase activity. In some embodiments, enzyme activity consists of polymerase activity and reverse transcriptase activity. Therefore, in some embodiments, enzyme activity does not include enzyme activity provided by other enzymes, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases. In certain examples, polymerase activity and reverse transcriptase activity are provided by separate enzymes or separate enzyme types (e.g., polymerase and reverse transcriptase). In certain examples, polymerase activity and reverse transcriptase activity are provided by a single enzyme or enzyme type (e.g., polymerase).

[0047] In some embodiments, nucleic acid amplification involves a non-thermocycling polymerase chain reaction (PCR). In some embodiments, nucleic acid amplification involves an isothermal amplification process. In some embodiments, nucleic acid amplification involves an isothermal polymerase chain reaction (iPCR). Isothermal amplification is generally an amplification process that takes place at a constant temperature. Terms such as isothermal conditions, isothermal, and constant temperature generally refer to reaction conditions in which the temperature of the reaction is kept essentially constant during the course of the amplification reaction. Isothermal amplification conditions generally do not involve a thermocycling (i.e., cycling between high and low temperatures) component in the amplification process. When amplification is performed under isothermal conditions, the reaction can be kept at essentially constant temperature, but this means that the temperature does not have to be maintained at exactly one temperature. For example, small temperature fluctuations (e.g., ±1 to 5°C) can occur in an isothermal amplification process due to variables, for example, environmental or apparatus-based. Often, the entire reaction volume is kept at essentially constant temperature, and isothermal reactions as described herein generally do not involve amplification conditions that rely on temperature cycling based on temperature gradients and / or convection generated within the reaction vessel.

[0048] The isothermal amplification reactions described herein can be carried out at essentially constant temperatures. In some embodiments, the isothermal amplification reactions described herein are carried out at temperatures ranging from about 55°C to about 75°C. For example, the isothermal amplification reactions described herein can be carried out at temperatures ranging from about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In some embodiments, the isothermal amplification reactions described herein are carried out at temperatures ranging from about 55°C to about 65°C. For example, the isothermal amplification reactions described herein can be carried out at about 60°C. The isothermal amplification reactions described herein can be carried out at about 65°C. In some embodiments, the temperature element (e.g., heat source) is kept at essentially constant temperatures. In some embodiments, the temperature element is maintained at an essentially constant temperature of about 75°C or below. In some embodiments, the temperature element is maintained at an essentially constant temperature of about 70°C or below. In some embodiments, the temperature element is maintained at an essentially constant temperature of about 65°C or below. In some embodiments, the temperature element is maintained at an essentially constant temperature of about 60°C or below.

[0049] The amplification process described herein may be performed over a period of time. In some embodiments, the amplification process is performed until a detectable nucleic acid amplification product is produced. The nucleic acid amplification product may be detected by any suitable detection process and / or a detection process described herein. In some embodiments, the amplification process is performed over a period of time of about 20 minutes or less. For example, the amplification process may be performed within about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes. In some embodiments, the amplification process is performed over a period of time of about 10 minutes or less.

[0050] In some embodiments, nucleic acid targets can be amplified without exposure to substances or conditions that denature nucleic acids. These substances or conditions may include those that promote chain separation and / or unwinding. In some embodiments, nucleic acid targets can be amplified without exposure to substances or conditions that promote chain separation. In some embodiments, nucleic acid targets can be amplified without exposure to substances or conditions that promote unwinding. In some embodiments, the target nucleic acid is considered non-denatured if it is not exposed to substances or conditions that denature nucleic acids and / or promote chain separation and / or unwinding before or during amplification. These substances or conditions may include, for example, thermal conditions (e.g., high temperature), pH conditions (e.g., high or low pH), chemical agents, proteins (e.g., enzymes), and the like.

[0051] In some embodiments, nucleic acid targets can be amplified without exposure to substances or conditions that denature nucleic acids. Nucleic acid denaturation, or melting, is the process by which double-stranded nucleic acids are unwound and separated into single strands. Substances and conditions that can promote nucleic acid denaturation include, for example, heat, high pH, ​​low pH, and denaturants (e.g., formamide) in combination with heat. In some examples, denaturation can be achieved by heating a solution containing nucleic acids to a specific temperature, for example, above 75°C, above 80°C, above 90°C, above 95°C, or higher. In some examples, denaturation can be achieved by exposure to denaturants such as NaOH, HCl, and formamide in combination with heat. Specific methods of DNA denaturation are described, for example, by Singh et al., (1977) Chomosoma Vol. 60: pp. 377-389.

[0052] In some embodiments, nucleic acid targets can be amplified without exposure to active agents or conditions that promote strand separation and / or unwinding. For example, nucleic acid targets can be amplified without exposure to helicases. Helicases are enzymes that can unwind double-stranded nucleic acids and separate them into single strands. Examples of helicases include human DNA helicases (and their equivalents in other organisms) such as DNA helicase Q1, Bloom syndrome protein, Werner syndrome protein, DNA helicase Q4, DNA helicase Q5, DNA helicase 2 subunit 1, MCM2, MCM3, MCM, MCM5, MCM6, MCM7, MCM8, MCM9, MCM10, nucleolin, CHD2, CHD7, XPB, XPD, lymphoid system-specific helicases, hINO, RuvB-like 1, RuvB-like 2, PIF1, Twinkle, BACH1, RecQ5 alpha, RecQ5 beta, RecQ5 gamma, and RTEL1;RNA helicase DDX1, RNA helicase eIF4A-1, RNA helicase eIF4A-2, RNA helicase DDX3X, RNA helicase DDX3Y, RNA helicase DDX4, RNA helicase DDX5, RNA helicase DDX6, RNA helicase DHX8, RNA helicase A, RNA helicase DDX10, RNA helicase DDX11, RNA helicase DDX12, helicase SKI2W, RNA helicase DHX15, RNA helicase RNA helicase DHX16, RNA helicase DDX17, RNA helicase DDX18, RNA helicase DDX19A, RNA helicase DDX19B, RNA helicase DDX20, nucleolar RNA helicase 2, RNA helicase DDX23, RNA helicase DDX24, RNA helicase DDX25, RNA helicase DDX27, RNA helicase DDX28, RNA helicase DHX29, RNA helicase DHX30, RNA helicase DDX31, RNA helicase RNA helicase DHX32, RNA helicase DHX33, RNA helicase DHX34, RNA helicase DHX35, RNA helicase DHX36, RNA helicase DHX37, RNA helicase PRP16, RNA helicase DDX39, RNA helicase DHX40, RNA helicase DDX41, RNA helicase DDX42, RNA helicase DDX43, RNA helicase DDX46, RNA helicase DDX47, RNA helicase eIF4A-3, RN Human RNA helicases (and their equivalents in other organisms) such as A helicase DDX49, RNA helicase DDX50, RNA helicase DDX51, RNA helicase DDX52, RNA helicase DDX53, RNA helicase DDX54, RNA helicase DDX55, RNA helicase DDX56, RNA helicase DHX57, RNA helicase DDX58, RNA helicase DHX58, RNA helicase DDX59, RNA helicase DDX60, spliceosome RNA helicase BAT1, U5.snRNP 200kDa helicase, transcription factor ATRX helicase, RNA helicase SUPV3L1, mitochondrial superkiller viralicidic activity 2-like 2, and Fanconi anemia group J protein;This also includes commercially available helicases. Amplification conditions that do not involve the use of helicase may be referred to herein as helicase-free amplification conditions.

[0053] In some embodiments, nucleic acid targets can be amplified without exposure to recombinases. Recombinases are enzymes involved in genetic recombination and may be involved in nucleic acid repair (e.g., recombinant DNA repair). Recombinases can, for example, initiate strand exchange. Examples of recombinases include Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, and T4 uvsX. In some embodiments, nucleic acid targets can be amplified without exposure to recombinase accessory proteins, such as recombinase loading factors (e.g., T4 uvsY).

[0054] In some embodiments, nucleic acid targets can be amplified without exposure to nucleic acid-binding proteins (e.g., single-strand binding proteins or single-strand DNA-binding proteins (SSBs)). Single-strand binding proteins generally function to prevent immature annealing, protect single-strand DNA from digestion by nucleases, and / or remove secondary structures from the DNA (e.g., destabilize the helical double helix) to enable action by other enzymes. In some embodiments, nucleic acid targets can be amplified without exposure to single-strand DNA-binding proteins, such as T4 gp32.

[0055] In some embodiments, nucleic acid targets can be amplified without exposure to topoisomerase.

[0056] Topoisomerases are enzymes that regulate DNA overwinding or underwinding by binding to either single-stranded or double-stranded DNA and cleaving the DNA phosphate backbone. Amplification conditions that do not involve the use of topoisomerase may be referred to herein as topoisomerase-free amplification conditions.

[0057] Nucleic acid targets can be amplified by exposure to or without exposure to substances or conditions that destabilize nucleic acids. Destabilization generally refers to disruption of the overall structure and geometric orientation (e.g., double helix structure) of a nucleic acid molecule by one or more of the following effects: tilting, rotating, twisting, sliding, and inverting (as described, for example, Lenglet et al., Journal of Nucleic Acids, Vol. 2010, No. 290935, p. 17). Destabilization generally does not refer to the melting or separation of nucleic acid chains as described above for denaturation. Destabilization of nucleic acids can be achieved, for example, by exposure to substances such as intercalators or alkylating agents, and / or chemicals such as formamide, urea, dimethyl sulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the amplification method may include the use of one or more destabilizing agents. In some embodiments, the amplification method excludes the use of destabilizing agents.

[0058] In some embodiments, nucleic acid targets can be amplified without exposure to ligase. Ligase is an enzyme that can catalyze the linking of amino acid molecules by forming new chemical bonds. Amplification conditions that do not involve the use of ligase may be referred to herein as ligase-free amplification conditions.

[0059] In some embodiments, nucleic acid targets can be amplified without exposure to RNA replicase. RNA replicase, RNA-dependent RNA polymerase (RdRp), or RDR is an enzyme that catalyzes the replication of RNA from an RNA template. Amplification conditions that do not involve the use of RNA replicase may be referred to herein as RNA replicase-free amplification conditions.

[0060] In certain embodiments, nucleic acid targets may be amplified without cleavage or digestion. For example, in some embodiments, nucleic acids are amplified without prior exposure to one or more cleavage agents, resulting in amplified intact nucleic acids. In certain embodiments, nucleic acids are amplified without exposure to one or more cleavage agents during amplification. In certain embodiments, nucleic acids are amplified without exposure to one or more cleavage agents after amplification. Amplification conditions that do not involve the use of cleavage agents may be referred to herein as cleavage agent-free amplification conditions. The term "cleavage agent" generally refers to an active substance, sometimes a chemical or enzyme, that can cleave nucleic acids at one or more specific or nonspecific sites. Often, specific cleavage agents cleave specifically according to a specific nucleotide sequence at a particular site. Examples of cleavage agents include endonucleases (e.g., restriction enzymes, nickeling enzymes, etc.), exonucleases (deoxyribonuclease, ribonuclease (e.g., ribonuclease H), 5'-3' exonuclease (e.g., exonuclease II), 3'-5' exonuclease (e.g., exonuclease I), and poly(A)-specific 3'-5' exonuclease), and chemical cleavage agents.

[0061] In certain embodiments, nucleic acid targets may be amplified without the use of restriction enzymes and / or nicking enzymes. Restriction enzymes are proteins that cleave DNA at specific sites, generally cleaving both strands of a double-stranded duplex, while nicking enzymes are proteins that bind to double-stranded DNA and cleave one strand of the duplex. In certain embodiments, nucleic acids are amplified without prior exposure to restriction enzymes and / or nicking enzymes. In certain embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes during amplification. In certain embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes after amplification. Amplification conditions that do not involve the use of restriction enzymes may be referred to herein as restriction enzyme-free amplification conditions. Amplification conditions that do not involve the use of nicking enzymes may be referred to herein as nicking enzyme-free amplification conditions.

[0062] In certain embodiments, nucleic acid targets can be amplified without exonuclease treatment. Exonucleases are enzymes that work by cleaving nucleotides one by one from the ends of a polynucleotide chain through a hydrolytic reaction that cleaves a phosphodiester bond at either the 3' or 5' end. Examples of exonucleases include deoxyribonuclease, ribonuclease (e.g., ribonuclease H), 5'-3' exonuclease (e.g., exonuclease II), 3'-5' exonuclease (e.g., exonuclease I), and poly(A)-specific 3'-5' exonuclease. In certain embodiments, nucleic acids are amplified without exonuclease treatment before amplification. In certain embodiments, nucleic acids are amplified without exonuclease treatment during amplification. In certain embodiments, nucleic acids are amplified without exonuclease treatment after amplification. Amplification conditions that do not involve the use of exonuclease may be referred to herein as exonuclease-free amplification conditions. In certain embodiments, nucleic acids are amplified without deoxyribonuclease treatment. In certain embodiments, nucleic acids are amplified without ribonuclease treatment. In certain embodiments, nucleic acids are amplified without ribonuclease H treatment. Amplification conditions that do not involve the use of deoxyribonuclease may be referred to herein as deoxyribonuclease-free amplification conditions. Amplification conditions that do not involve the use of ribonuclease may be referred to herein as ribonuclease-free amplification conditions. Amplification conditions that do not involve the use of ribonuclease H may be referred to herein as ribonuclease H-free amplification conditions.

[0063] The amplified nucleic acid may be referred to herein as a nucleic acid amplification product or amplicon. In some embodiments, the amplification product may include naturally occurring nucleotides, non-naturally occurring nucleotides, nucleotide analogs, and combinations thereof. The amplification product typically has a nucleotide sequence that is identical or substantially identical to the sequence in the sample nucleic acid (e.g., the target sequence) or its complement. A “substantially identical” nucleotide sequence in the amplification product generally has a high degree of sequence identity with the nucleotide sequence being amplified or its complement (e.g., higher than about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99%), and mutations may be the result of polymerase infidelity or other variables.

[0064] In some embodiments, the nucleic acid amplification product contains polynucleotides that are sequentially complementary to or substantially identical to the target sequence in the sample nucleic acid. Sequentially complementary generally means that, for example, in the nucleotide sequence in the first strand, each sequentially ordered base (e.g., read from 5' to 3') pairs with the corresponding ordered base in the second strand, and there are no gaps, additional sequences, or unpaired bases within the sequence considered to be sequentially complementary. In other words, sequentially complementary generally means that all consecutive bases in the nucleotide sequence in the first strand are complementary to the corresponding consecutive bases in the nucleotide sequence in the second strand. For example, the first strand having the sequence 5'-ATGCATGCATGC-3' (SEQ ID NO: 10) is considered to be sequentially complementary to the second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO: 11), in which case all consecutive bases in the first strand are complementary to all corresponding consecutive bases in the second strand. However, the first strand having the sequence 5'-ATGCATAAAAAAGCATGC-3' (SEQ ID NO: 12) is not considered to be continuously complementary to the second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO: 11). This is because the sequence of the six adenines (six A's) in the middle of the first strand does not pair with any of the bases in the second strand. Continuously complementary sequences are sometimes about 5 to 25 consecutive bases long, sometimes about 6 to 20 consecutive bases long, sometimes about 7 to 18 consecutive bases long, and sometimes about 8 to 16 consecutive bases long. In some embodiments, the nucleic acid amplification product consists of polynucleotides that are continuously complementary to or substantially identical to the target sequence in the sample nucleic acid. Therefore, in some embodiments, the nucleic acid amplification product does not include any additional sequences that are not sequentially complementary to the target sequence and are not substantially identical to it (e.g., at the 5' and / or 3' ends or within the product), such as additional sequences incorporated into the amplification product by a tailed primer or ligation, and / or additional sequences that provide a cleavage agent recognition site (e.g., a nickel enzyme recognition site).Generally, if the target sequence does not contain tandem repeats, the amplification product will not contain any products in the form of tandem repeats.

[0065] The nucleic acid amplification product may contain sequences that are complementary to or substantially identical to one or more primers used in the amplification reaction. In some embodiments, the nucleic acid amplification product contains a first nucleotide sequence that is sequentially complementary to or identical to the first primer sequence, and a second nucleotide sequence that is sequentially complementary to or identical to the second primer sequence.

[0066] In some embodiments, the nucleic acid amplification product includes a spacer sequence. Generally, the spacer sequence in the amplification product is a sequence (one or more bases) that is sequentially complementary to or substantially identical to a portion of the target sequence in the sample nucleic acid, and is sandwiched between sequences in the amplification product that are complementary to or substantially identical to one or more primers used in the amplification reaction. The spacer sequence sandwiched between sequences in the amplification product is generally located between a first sequence (complementary to or substantially identical to the first primer) and a second sequence (complementary to or substantially identical to the second primer). Thus, the amplification product typically includes a first sequence, a spacer sequence following it, and a second sequence following it. Generally, the spacer sequence is neither complementary to nor substantially identical to the sequences in the primers. In some embodiments, the spacer sequence contains about 1 to 10 bases. For example, the spacer sequence may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. In some embodiments, the spacer sequence contains approximately 1 to 5 base pairs.

[0067] In some embodiments, the nucleic acid amplification product consists of a first nucleotide sequence that is continuously complementary to or identical to the first primer sequence, a second nucleotide sequence that is continuously complementary to or identical to the second primer sequence, and a spacer sequence. Thus, in some embodiments, the nucleic acid amplification product does not include any additional sequences that are not continuously complementary to or identical to the first and second primer sequences and that are not part of the spacer sequence (e.g., at the 5' and / or 3' ends or within the product), such as, for example, tailed primers or loop primers, additional sequences incorporated into the amplification product by ligation or other mechanisms.

[0068] In some embodiments, the nucleic acid amplification product consists essentially of a first nucleotide sequence that is continuously complementary to or identical to the first primer sequence, a second nucleotide sequence that is continuously complementary to or identical to the second primer sequence, and a spacer sequence. Thus, in some embodiments, the nucleic acid amplification product generally does not include additional sequences that are not continuously complementary to or identical to the first and second primer sequences and that are not part of the spacer sequence (e.g., at the 5' and / or 3' ends or within the product), such as, for example, tailed primers or loop primers, additional sequences incorporated into the amplification product by ligation or other mechanisms. However, in such embodiments, the nucleic acid amplification product may include some mismatched (i.e., non-complementary) bases or even one or more extra bases (e.g., at the 5' and / or 3' ends or within the product) introduced into the product by, for example, errors or promiscuity during the amplification process.

[0069] The nucleic acid amplification product may be up to 50 bases long. In some embodiments, the nucleic acid amplification product is about 15 to about 40 bases long. For example, the nucleic acid amplification product may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases long. In some embodiments, the amplification product is about 20 to about 40 bases long. In some embodiments, the amplification product is about 20 to about 30 bases long. In some embodiments, the nucleic acid amplification product for a given target sequence has the same length or substantially the same length (e.g., within 1 to 5 bases). Therefore, a nucleic acid amplification product for a given target sequence may produce a single signal (e.g., a band on an electrophoretic gel) and generally not multiple signals of multiple lengths (e.g., a ladder or smear on an electrophoretic gel). In the case of multiplex reactions, nucleic acid amplification products for different target sequences may have different lengths.

[0070] The methods and components described herein may be used for multiplex amplification. Multiplex amplification generally refers to the amplification of more than one nucleic acid of interest (e.g., more than one target sequence). For example, multiplex amplification may refer to the amplification of multiple sequences from the same sample, or the amplification of one of several sequences in a sample. Multiplex amplification may also refer to the amplification of one or more sequences present in multiple samples simultaneously or stepwise.

[0071] For example, multiplex amplification can be used to amplify at least two target sequences (e.g., the amplification reaction includes appropriate primers and enzymes for amplifying at least two target sequences). In some examples, the amplification reaction can be prepared to detect at least two target sequences, but only one of the target sequences may be present in the sample being tested. Thus, both sequences can be amplified, but only one sequence may be amplified. In some examples, if two target sequences are present, the amplification reaction can result in the amplification of both target sequences. A multiplex amplification reaction can result in the amplification of one, some, or all of the target sequences, including appropriate primers and enzymes. In some examples, the amplification reaction can be prepared to detect two sequences with a pair of primers, where one sequence is a target sequence and one sequence is a control sequence (e.g., a synthetic sequence that can be amplified by the same primers as the target sequence and has a spacer base or sequence different from the target). In some examples, the amplification reaction can be prepared to detect multiple sets of sequences with corresponding primer pairs, where each set includes a target sequence and a control sequence.

[0072] Primer Nucleic acid amplification is generally performed in the presence of one or more primers. Primers are generally characterized as oligonucleotides containing a nucleotide sequence that can hybridize or anneal to a target nucleic acid in or near a specific region of interest (i.e., a target sequence). Primers may enable, for example, the specific determination of a target nucleic acid nucleotide sequence or the detection of a target nucleic acid (e.g., the presence or absence of a sequence) or the detection of its features. Primers may be naturally occurring or synthetic. The terms specific or specific generally refer to the binding or hybridization of one molecule to another, such as a primer to a target polynucleotide. That is, specific or specific means the recognition, contact, and formation of a stable complex between two molecules, to which the recognition, contact, or complex formation of any of these two molecules with other molecules is substantially lower. The terms annealing or hybridizing generally refer to the formation of a stable complex between two molecules. When referring to primers, the terms primer, oligo, or oligonucleotide may be used interchangeably herein.

[0073] Primers can be designed and synthesized using preferred processes and may be of any length suitable for hybridizing to the target sequence and performing the amplification processes described herein. Often, primers are designed according to the sequence in the target nucleic acid. In some embodiments, primers may be about 5 to 30 bases long. For example, primers may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bases long. In some embodiments, primers are less than 28 bases long. In some embodiments, primers are about 8 to 16 bases long. In some embodiments, primers are about 10 to 12 bases long. Primers may consist of naturally occurring and / or non-naturally occurring nucleotides (e.g., modified nucleotides, labeled nucleotides), or mixtures thereof. Primers suitable for use with the methods described herein may be synthesized and labeled using any suitable technique. For example, primers may be chemically synthesized using an automated synthesizer as described in Needham-VanDevanter et al., Nucleic Acids Res. Vol. 12: pp. 6159-6168, 1984, according to the solid-phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts., Vol. 22: pp. 1859-1862, 1981. The purification of primers may be performed, for example, by non-denaturing acrylamide gel electrophoresis or anion-exchange high-performance liquid chromatography (HPLC), as described in Pearson and Regnier, J. Chrom., Vol. 255: pp. 137-149, 1983.

[0074] In some embodiments, the primer comprises a modified nucleotide. In some embodiments, the primer is essentially made of a modified nucleotide. In some embodiments, the primer consists of a modified nucleotide. A nucleotide (or base) may be modified according to any modifications described herein or known in the art. Modifications may include those made during the synthesis of the primer and / or modifications made after synthesis. Modifications may include internal modifications, modifications at the 3' end of the primer, and / or modifications at the 5' end of the primer. In some embodiments, the primer comprises a mixture of modified and unmodified nucleotides. In some embodiments, the primer comprises an unmodified nucleotide. In some embodiments, the primer is essentially made of an unmodified nucleotide. In some embodiments, the primer consists of an unmodified nucleotide.

[0075] Modifications and modified bases include, for example, phosphorylation (e.g., 3'-phosphorylation, 5'-phosphorylation); attachment chemistry or linker modification (e.g., Acrydite®, adenylation, azide (NHS ester), digoxigenin (NHS ester), cholesteryl-TEG, I-Linker®, amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, Uni-Link® amino modifiers)); alkynes (e.g., 5'-hexynyl, 5-octadiynyl dU (5-octadiynyl dU)); biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin, desthiobiotin-TEG); and thiol modification (e.g., thiol modifier C3 SS, dithiol, thiol modifier C6) SS)); Fluorophores (e.g., Freedom® dyes, Alexa Fluor® dyes, LI-COR IRDyes®, ATTO® dyes, Rhodamine dyes, WellRED dyes, 6-FAM (azide), Texas Red®-X (NHS ester), Lightcycler® 640 (NHS ester), Dy 750 (NHS ester)); Iowa Black® Dark Quencher Modification (e.g., Iowa Black® FQ, Iowa Black® RQ); Dark Quencher Modification (e.g., Black Hole Quencher®-1, Black Hole Quencher(registered trademark)-2, Dabcyl); Spacers (C3 spacer, PC spacer, hexanediol, spacer 9, spacer 18, 1',2'-dideoxyribose (dSpacer); Modified bases (e.g., 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo-dU, deoxyuridine, inverted dT, inverted dideoxy-T, dideoxy-C, 5-methyldC, deoxyinosine, Super T(registered trademark), Super G(registered trademark), locked nucleic acid (LNA), 5-nitroindole, 2'-O-methylRNA base, hydroxymethyldC (hydroxmethyl dC),Examples of modifications include UNA-unlocked nucleic acids (e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dC, Iso-dG, fluoro-C, fluoro-U, fluoro-A, fluoro-G); phosphorothioate-linked modifications (e.g., phosphorothioated DNA bases, phosphorothioated RNA bases, phosphorothioated 2'O-methyl bases, phosphorothioated LNA bases); and click chemical modifications. In some embodiments, modifications and modified bases include uracil bases, ribonucleotide bases, O-methylRNA bases, phosphorothioate links, 3'-phosphate groups, and spacer bases (such as C3 spacers or other spacer bases). For example, a primer may contain one or more O-methylRNA bases (e.g., 2'-O-methylRNA bases). 2'-O-methylRNA is a post-transcriptional modification of RNA commonly found in tRNA and other small RNAs. Primers containing 2'-O-methylRNA bases can be synthesized directly. This modification can, for example, increase the Tm of RNA:RNA double helix and provide stability in the presence of single-stranded ribonucleases and deoxyribonucleases. 2'-O-methylRNA bases may be included in the primer, for example, to increase stability and binding affinity to the target sequence. In some embodiments, the primer may contain one or more phosphorothioate links (e.g., phosphorothioate linkage modifications). A phosphorothioate (PS) linkage replaces a non-crosslinked oxygen in the phosphate backbone of the primer with a sulfur atom. This modification typically makes the nucleotide linkage resistant to nuclease degradation. Phosphothioate links may be introduced between the last approximately 3-5 nucleotides at the 5' or 3' end of the primer, for example, to inhibit exonuclease degradation. In certain examples, phosphorothioate links throughout the primer can help reduce attack by endonucleases. In some embodiments, the primer may contain a 3' phosphate group. 3' phosphorylation can, in certain cases, inhibit degradation by certain 3'-exonucleases.It can be used to block extension by DNA polymerase. In some embodiments, the primer may contain one or more spacer bases (e.g., one or more C3 spacers). The phosphoramidite of the C3 spacer can be incorporated into the primer or at its 5' end. Multiple C3 spacers may be added to any end of the primer to introduce a long hydrophilic spacer arm for attachment of, for example, a fluorophore or other pendant group.

[0076] In some embodiments, the primer comprises a DNA base. In some embodiments, the primer comprises an RNA base. In some embodiments, the primer comprises a mixture of DNA bases and RNA bases. The DNA bases may be modified or unmodified. The RNA bases may be modified or unmodified. In some embodiments, the primer is essentially composed of DNA bases (e.g., modified DNA bases and / or unmodified DNA bases). In some embodiments, the primer consists of DNA bases (e.g., modified DNA bases and / or unmodified DNA bases). In some embodiments, the primer is essentially composed of unmodified DNA bases. In some embodiments, the primer consists of unmodified DNA bases. In some embodiments, the primer is essentially composed of modified DNA bases. In some embodiments, the primer is essentially composed of RNA bases (e.g., modified RNA bases and / or unmodified RNA bases). In some embodiments, the primer consists of RNA bases (e.g., modified RNA bases and / or unmodified RNA bases). In some embodiments, the primer is essentially composed of unmodified RNA bases. In some embodiments, the primer consists of unmodified RNA bases. In some embodiments, the primer is essentially composed of modified RNA bases. In some embodiments, the primer consists of modified RNA bases.

[0077] In some embodiments, the primer does not contain RNA bases. In some embodiments, the primer does not have an RNA base at its 3' end. In some embodiments, the primer contains a DNA base (or modified DNA base) at its 3' end. In some embodiments, the primer is not a chimeric primer. A chimeric primer is a primer containing both DNA and RNA bases. In some embodiments, the primer is a homogeneous primer. In some embodiments, the primer is a homogeneous DNA primer. A homogeneous DNA primer may contain unmodified DNA bases, modified DNA bases, or a mixture of modified and unmodified DNA bases, and generally does not contain RNA bases.

[0078] In some embodiments, the primer does not include a cleavage agent recognition site. For example, the primers in this specification do not have to include a nickel enzyme recognition site. In some embodiments, the primer does not include a tail. In some embodiments, the primer does not include a tail containing a nickel enzyme recognition site.

[0079] In some embodiments, the entire or partial primer sequence may be complementary or substantially complementary to the target nucleic acid. Substantially complementary with respect to the sequence generally means that the nucleotide sequences hybridize with each other. The stringency of the hybridization conditions can be modified to accommodate varying amounts of sequence mismatch. In some embodiments, the target and primer sequences are at least 75% complementary to each other. For example, target and primer sequences may be complementary to each other to a degree of 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher.

[0080] A primer that is substantially complementary to a target nucleic acid sequence is usually also substantially identical to its complement. That is, the primer is substantially identical to the antisense strand of the nucleic acid. Substantially identical in sequence generally means that the nucleotide sequences are at least 75% identical to each other. For example, primers that are substantially identical to the antisense strand of a target nucleic acid may be identical to one another to a degree of 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. One test to determine whether two nucleotide sequences are substantially identical is to determine the percentage of identical nucleotide sequences they share.

[0081] In some embodiments, the primers consist of a pair of primers. The pair of primers may include a forward primer and a reverse primer (e.g., primers that bind to the sense and antisense strands of the target nucleic acid). In some embodiments, the primers consist of a pair of primers (i.e., a forward primer and a reverse primer). Therefore, in some embodiments, amplification of the target sequence is performed using the pair of primers, and additional primers or oligonucleotides are not included in the amplification of the target sequence (e.g., the amplification reaction components do not include additional primer pairs for a given target sequence, nested primers, bumper primers, oligonucleotides other than primers, probes, etc.). In some embodiments, the primers consist of a pair of primers, but in certain examples, such as in multiplex amplification, the amplification reaction may include additional primer pairs for amplifying different target sequences. In some embodiments, the primers consist of a pair of primers, but in certain examples, the amplification reaction may include additional primers, oligonucleotides, or probes for a detection process that is not considered part of the amplification.

[0082] In some embodiments, primers are used in sets. An amplification primer set may include a pair of forward and reverse primers for a given target sequence. In the case of multiplex amplification, the primers that amplify the first target sequence are considered one primer set, and the primers that amplify the second target sequence are considered a different primer set.

[0083] In some embodiments, the amplification reaction component includes a first primer (first oligonucleotide) complementary to the target sequence in the first strand of the sample nucleic acid (e.g., sense strand, forward strand) and a second primer (second oligonucleotide) complementary to the target sequence in the second strand of the sample nucleic acid (e.g., antisense strand, reverse strand). In some embodiments, the first primer (first oligonucleotide) includes a first polynucleotide that is sequentially complementary to the target sequence in the first strand of the sample nucleic acid, and the second primer (second oligonucleotide) includes a second polynucleotide that is sequentially complementary to the target sequence in the second strand of the sample nucleic acid. Sequentially complementary primer-target generally means that in the nucleotide sequence in the primer, each sequentially ordered base pairs with the corresponding ordered base in the target sequence, and there are no gaps, additional sequences, or unpaired bases within the sequence considered to be sequentially complementary. In other words, continuous complementarity generally means that all consecutive bases in the nucleotide sequence within the primer are complementary to the corresponding consecutive bases in the nucleotide sequence within the target.

[0084] In some embodiments, the first primer (first oligonucleotide) consists of a first polynucleotide that is sequentially complementary to the target sequence in the first strand of the sample nucleic acid, and the second primer (second oligonucleotide) consists of a second polynucleotide that is sequentially complementary to the target sequence in the second strand of the sample nucleic acid. Therefore, in some embodiments, the primers do not include any additional sequences that are not sequentially complementary to the target sequence (e.g., at the 5' and / or 3' ends or within the primer), such as additional sequences present in tailed or loop primers and / or additional sequences that provide cleavage agent recognition sites (e.g., Nicking enzyme recognition sites). In some embodiments, the amplification reaction components do not include primers containing additional sequences (i.e., sequences other than sequences sequentially complementary to the target sequence), such as tailed primers, loop primers, primers capable of forming stem-loop structures or hairpin structures, and / or additional sequences that provide cleavage agent recognition sites (e.g., Nicking enzyme recognition sites).

[0085] In some embodiments, the first primer (first oligonucleotide) essentially consists of a first polynucleotide that is sequentially complementary to the target sequence in the first strand of the sample nucleic acid, and the second primer (second oligonucleotide) essentially consists of a second polynucleotide that is sequentially complementary to the target sequence in the second strand of the sample nucleic acid. Therefore, in some embodiments, the primers generally do not include any additional sequences that are not sequentially complementary to the target sequence (e.g., at the 5' and / or 3' ends, or within the primer), such as additional sequences present in tailed or loop primers, and / or additional sequences that provide a cleavage agent recognition site (e.g., a Nicking enzyme recognition site). However, in such embodiments, the primers may include one or more additional bases that do not add functional features to the primer (e.g., at the 5' and / or 3' ends, or within the primer). For example, additional sequences present in tailed or loop primers generally add functional features and are excluded from the primers in such embodiments.

[0086] In certain embodiments, the primer may contain modifications such as one or more inosine molecules, abase-free sites, locked nucleic acids, minor groove binders, double-chain stabilizing factors (e.g., acridine, spermidine), Tm modifiers, or any modifiers that alter the binding properties of the primer. In certain embodiments, the primer may contain detectable molecules or entities (e.g., fluorophores, radioisotopes, colorimetric agents, particles, enzymes, etc.).

[0087] polymerase In some embodiments, the amplification reaction component includes one or more polymerases.

[0088] A polymerase is a protein that catalyzes the specific incorporation of nucleotides, thereby extending the 3' hydroxyl terminus of a primer molecule, such as the amplification primers described herein, toward a nucleic acid target sequence (e.g., the primer anneals to). Examples of polymerases include thermophilic or hyperthermophilic polymerases that can be active at elevated reaction temperatures (e.g., above 55°C, above 60°C, above 65°C, above 70°C, above 75°C, above 80°C, above 85°C, above 90°C, above 95°C, above 100°C). Hyperthermophilic polymerases may be referred to as hyperthermophilic bacterial polymerases. A polymerase having hyperthermophilic polymerase activity may be referred to as having hyperthermophilic bacterial polymerase activity. Polymerases may or may not have chain substitution ability. In some embodiments, polymerases can incorporate about 1 to about 50 nucleotides in a single synthesis. For example, a polymerase may incorporate approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in a single synthesis. In some embodiments, the polymerase can incorporate 20 to 40 nucleotides in a single synthesis. In some embodiments, the polymerase can incorporate up to 50 nucleotides in a single synthesis. In some embodiments, the polymerase can incorporate up to 40 nucleotides in a single synthesis. In some embodiments, the polymerase can incorporate up to 30 nucleotides in a single synthesis. In some embodiments, the polymerase can incorporate up to 20 nucleotides in a single synthesis.

[0089] In some embodiments, the amplification reaction component includes one or more DNA polymerases. In some embodiments, the amplification reaction component includes one or more DNA polymerases selected from the following: 9°N DNA polymerase, 9°Nm(trademark) DNA polymerase, Therminator(trademark) DNA polymerase, Therminator(trademark) II DNA polymerase, Therminator(trademark) III DNA polymerase, Therminator(trademark) γ DNA polymerase, Bst DNA polymerase, Bst DNA polymerase (large fragment), Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, large (Kreno) fragment, Kreno fragment (3'-5' exo-), T4 DNA polymerase, T7 DNA polymerase, Deep Vent®(trademark) (exo-) DNA polymerase, Deep Vent®(trademark) DNA polymerase, DyNAzyme(trademark) EXT DNA, DyNAzyme(trademark) II Hot Start DNA polymerase, Phusion(trademark) High-Fidelity DNA polymerase, VentR® DNA polymerase, VentR® (exo-) DNA polymerase, RepliPHI® Phi29 DNA polymerase, rBst DNA polymerase, large fragment (IsoTherm® DNA polymerase), MasterAmp® AmpliTherm® DNA polymerase, Tag DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tgo DNA polymerase, SP6 DNA polymerase, Tbr DNA polymerase, DNA polymerase beta, and ThermoPhi DNA polymerase.

[0090] In some embodiments, the amplification reaction component includes one or more hyperthermophilic DNA polymerases. Generally, hyperthermophilic DNA polymerases are thermally stable at high temperatures. For example, a hyperthermophilic DNA polymerase may have a half-life of about 5 to 10 hours at 95°C and about 1 to 3 hours at 100°C. In some embodiments, the amplification reaction component includes one or more hyperthermophilic DNA polymerases derived from archaea. In some embodiments, the amplification reaction component includes one or more hyperthermophilic DNA polymerases derived from Thermococcaceae archaea. In some embodiments, the amplification reaction component includes one or more hyperthermophilic DNA polymerases derived from Pyrococcus. In some embodiments, the amplification reaction component includes one or more hyperthermophilic DNA polymerases derived from Methanococcaceae. In some embodiments, the amplification reaction component includes one or more hyperthermophilic DNA polymerases derived from Methanococcus. In some embodiments, the amplification reaction component comprises one or more hyperthermophilic DNA polymerases derived from Thermus.

[0091] In some embodiments, the amplification reaction component comprises a hyperthermophilic DNA polymerase or a functional fragment thereof. Functional fragments generally retain one or more functions of the full-length polymerase, such as, for example, the ability to polymerize DNA (e.g., in an amplification reaction). In some examples, the functional fragment functions (e.g., polymerizes DNA in an amplification reaction) at a level that is at least about 50% of the level of the function of the full-length polymerase. In some examples, the functional fragment functions (e.g., polymerizes DNA in an amplification reaction) at a level that is at least about 75% of the level of the function of the full-length polymerase. In some examples, the functional fragment functions (e.g., polymerizes DNA in an amplification reaction) at a level that is at least about 90% of the level of the function of the full-length polymerase. In some examples, the functional fragment functions (e.g., polymerizes DNA in an amplification reaction) at a level that is at least about 95% of the level of the function of the full-length polymerase. The level of polymerase activity can be evaluated using, for example, a detectable nucleic acid amplification method such as those described herein. In some embodiments, the amplification reaction component comprises a hyperthermophilic DNA polymerase comprising the amino acid sequence of SEQ ID NO: 8 or a functional fragment of SEQ ID NO: 8. In some embodiments, the amplification reaction component comprises a hyperthermophilic DNA polymerase comprising the amino acid sequence of SEQ ID NO: 9 or a functional fragment of SEQ ID NO: 9.

[0092] In some embodiments, the amplification reaction component includes a polymerase or a functional fragment thereof having an amino acid sequence at least about 90% identical to that of a hyperthermophilic polymerase (i.e., a functional fragment of a polymerase having an amino acid sequence at least about 90% identical to that of a hyperthermophilic polymerase, as described herein). The degree of sequence identity can be determined, for example, by performing amino acid sequence alignment. In some embodiments, the amplification reaction component includes a polymerase or a functional fragment thereof having an amino acid sequence at least about 90% identical to that of SEQ ID NO: 8 (i.e., a functional fragment of a polymerase having an amino acid sequence at least about 90% identical to that of SEQ ID NO: 8, as described herein). In some embodiments, the amplification reaction component includes a polymerase or a functional fragment thereof having an amino acid sequence at least about 95% identical to that of SEQ ID NO: 8. In some embodiments, the amplification reaction component includes a polymerase or a functional fragment thereof having an amino acid sequence at least about 99% identical to that of that of SEQ ID NO: 8. In some embodiments, the amplification reaction component includes a polymerase or a functional fragment thereof having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 9 (i.e., a functional fragment of a polymerase having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 9, as described herein). In some embodiments, the amplification reaction component includes a polymerase or a functional fragment thereof having an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amplification reaction component includes a polymerase or a functional fragment thereof having an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 9.

[0093] In some embodiments, the polymerase may have reverse transcription ability. In such cases, the amplification reaction can amplify the RNA target in a single step, for example, without using a separate reverse transcriptase. Non-limiting examples of polymerases with reverse transcriptase ability include Bst (Large Fragment), 9°N DNA polymerase, 9°Nm™ DNA polymerase, Therminator™, Therminator™ II, and others. In some embodiments, the amplification reaction components include one or more separate reverse transcriptases. In some embodiments, more than one polymerase may be included in the amplification reaction. For example, the amplification reaction may include a polymerase with reverse transcriptase activity and a second polymerase that does not have reverse transcriptase activity.

[0094] In some embodiments, one or more polymerases having exonuclease activity are used during amplification. In some embodiments, one or more polymerases not having exonuclease activity are used during amplification. In some embodiments, one or more polymerases with low exonuclease activity are used during amplification. In certain examples, polymerases not having or with low exonuclease activity include one or more modifications (e.g., amino acid substitutions) that reduce or remove the exonuclease activity of the polymerase. Modified polymerases with low exonuclease activity may have 10% or less exonuclease activity compared to unmodified polymerases. For example, modified polymerases with low exonuclease activity may have about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% exonuclease activity compared to unmodified polymerases. In some embodiments, the polymerase lacks or has low 5'-3' exonuclease activity. In some embodiments, the polymerase lacks or has low 3'-5' exonuclease activity. In some embodiments, the polymerase lacks or has low single-strand dependent exonuclease activity. In some embodiments, the polymerase lacks or has low double-strand dependent exonuclease activity. Non-limiting examples of certain modifications that can reduce or eliminate the exonuclease activity of the polymerase include one or more amino acid substitutions at positions 141 and / or 143 and / or 458 of SEQ ID NO: 8, or at positions corresponding to positions 141 and / or 143 and / or 458 of SEQ ID NO: 8. The amino acid positions corresponding to the positions in SEQ ID NO: 8 can be identified, for example, by performing amino acid sequence alignment. In some examples, the modification involves the substitution of the natural amino acid at position 141 with alanine. In some examples, the modification includes D141A. In some examples, the modification involves the substitution of the natural amino acid at position 143 with alanine. In some examples, the modification includes E143A.In some examples, the modification involves the substitution of the natural amino acid at position 143 with aspartic acid. In some examples, the modification includes E143D. In some examples, the modification involves the substitution of the natural amino acid at position 485 with leucine. In some examples, the modification includes A485L. In some examples, the modification includes A485L. In some examples, the modification includes D141A, E143A, and A485L.

[0095] Detection and quantification The methods described herein may further include a step of detecting and / or quantifying nucleic acid amplification products. Amplification products may be detected and / or quantified by any suitable detection and / or quantification method, including, for example, any detection or quantification method described herein.Non-limiting examples of detection and / or quantification methods include molecular beacons (e.g., real-time, endpoint), lateral flow, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), surface capture, 5'-3' exonuclease hydrolysis probes (e.g., TAQMAN), insertion / binding dyes, absorbance (e.g., colorimetric quantification, turbidity), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, digital amplification, primer extension methods (e.g., iPLEX®), and Affymetrix Molecular Inversion. Probe (MIP) technology, restriction fragment length polymorphism (RFLP analysis), allele-specific oligonucleotide (ASO) analysis, methylation-specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, reverse dot blot, GeneChip microarray, dynamic allele-specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, AlphaScreen, SNPstream, genetic bit analysis (GBA), multiplex minisequencing, SNaPshot, GOOD assay, microarray miniseq, array-mediated primer extension (APEX), microarray primer extension, tag array, coded microspheres, template-directed incorporation (TDI), colorimetric quantitative oligonucleotide ligation assay (OLA), sequence-coding OLA, microarray ligation, ligase chain reaction, padlock probes. Examples include probes, invader assays, hybridization using at least one probe, hybridization using at least one fluorescently labeled probe, cloning and sequencing, use of hybridization probes and quantitative real-time polymerase chain reaction (QRT-PCR), nanopore sequencing, chips, and combinations thereof.In some embodiments, the step of detecting the nucleic acid amplification product includes the use of a real-time detection method (i.e., the product is detected and / or continuously monitored during the amplification process). In some embodiments, the step of detecting the nucleic acid amplification product includes the use of an endpoint detection method (i.e., the product is detected after the completion or cessation of the amplification process). The nucleic acid detection method may also use labeled nucleotides directly incorporated into a probe containing a target sequence or a sequence complementary to the target. Such labels may be radioactive and / or fluorescent and can be resolved in any manner discussed herein. In some embodiments, quantification of the nucleic acid amplification product may be achieved using certain detection methods described below. In certain examples, the detection method may be used in combination with measuring signal intensity and / or generating (or referencing) calibration curves and / or lookup tables for quantification of the nucleic acid amplification product.

[0096] In some embodiments, the step of detecting nucleic acid amplification products involves the use of molecular beacon technology. The term molecular beacon generally refers to a detectable molecule that, under certain conditions, can function as a specific informational signal because its detectable properties are detectable. Non-limiting examples of detectable properties include optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties, and the time or speed through an opening of known size. A molecular beacon for detecting nucleic acid molecules may be, for example, a hairpin oligonucleotide containing a fluorophore at one end and a quenching dye at the other. The hairpin loop may contain a probe sequence complementary to the target sequence, and the stem is formed by the annealing of complementary arm sequences located on either side of the probe sequence. The fluorophore and quenching molecules can be covalently linked to the opposite ends of each arm. Under conditions that prevent the oligonucleotide from hybridizing to its complementary target, or when the molecular beacon is free in solution, the fluorescent and quenching molecules are proximal to each other and prevent fluorescence resonance energy transfer (FRET). Hybridization can occur when a molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product), causing the loop structure to be converted to a more stable, rigid conformation, leading to the separation of fluorophores and quenching molecules and subsequent fluorescence (Tyagi et al., Nature Biotechnology, Vol. 14: March 1996, pp. 303-308). Due to probe specificity, fluorescence generation generally results solely from the synthesis of the intended amplification product. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is identical or complementary to the sequence in the target nucleic acid. In other cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is neither identical nor complementary to the sequence in the target nucleic acid (e.g., hybridizes to a sequence added to the amplification product by a tailed amplification primer or ligation).

[0097] Molecular beacons are highly specific and can identify single nucleotide polymorphisms. Molecular beacons can also be synthesized using fluorophores of different colors and different target sequences, allowing for the simultaneous detection of several products during the same reaction (e.g., in a multiplex reaction). In quantitative amplification processes, molecular beacons can specifically bind to the amplified target after each amplification cycle, and since non-hybridized molecular beacons are dark, there is no need to isolate the probe-target hybrid to quantitatively determine the amount of amplified product. The resulting signal is proportional to the amount of amplified product. Detection using molecular beacons can be performed in real time or as an endpoint detection method. In some examples, certain reaction conditions can be optimized for each primer / probe set to ensure accuracy and precision.

[0098] In some embodiments, the step of detecting nucleic acid amplification products involves the use of lateral flow. The use of lateral flow typically involves the use of lateral flow devices. These devices generally include solid-phase fluid permeable channels through which fluid flows by capillary force. Exemplary devices include, but are not limited to, dipstick assays and thin-layer chromatography plates with various suitable coatings. Various binding reagents for the sample, binding partners or conjugates with binding partners for the sample, and signal generation systems are immobilized on the channel. Detection can be achieved in several ways, including, for example, enzymatic detection, nanoparticle detection, colorimetric quantitative detection, and fluorescence detection. Enzymatic detection may involve an enzymatically labeled probe that hybridizes to complementary nucleic acid targets on the surface of the lateral flow device. The resulting complex can be processed with a suitable marker that produces a readable signal. Nanoparticle detection involves bead technology, which may use gold colloid, latex, and / or paramagnetic nanoparticles. In one example, the beads can be conjugated with an anti-biotin antibody. The target sequence may be directly biotinylated, or it may be hybridized to a sequence-specific biotinylated probe. Gold and latex produce a macroscopic colorimetric quantitative signal, while paramagnetic particles produce an invisible signal when excited in a magnetic field, which can be interpreted by a specialized reader. Fluorescence-based lateral flow detection methods can also be used, such as the dual fluorescein and biotin-labeled oligoprobe method, UPT-N ALP utilizing an upconverted phosphorescent reporter composed of lanthanide elements embedded in a crystal (Corstjens et al., Clinical Chemistry, Vol. 47: No. 10, pp. 1885-1893, 2001), and the use of quantum dots.

[0099] Nucleic acids can be captured on lateral flow devices. Capture methods include antibody-based and antibody-free methods. Antibody-based capture generally involves an antibody capture line and a probe labeled with a complementary sequence to the target. Antibody-free capture generally utilizes non-covalent interactions between two binding partners, such as a high-affinity, irreversible ligation between a biotinylated probe and a streptavidin line. Capture probes can be immobilized directly onto the lateral flow membrane. Both antibody-based and antibody-free methods can be used, for example, to detect amplification products generated in multiplex reactions.

[0100] In some embodiments, the step of detecting nucleic acid amplification products involves the use of fluorescence resonance energy transfer (FRET). FRET is an energy transfer mechanism between two chromophores, a donor molecule and an acceptor molecule. Briefly, a donor fluorophore molecule is excited at a specific excitation wavelength. The subsequent emission from the donor molecule as it returns to its ground state can transfer excitation energy to the acceptor molecule through long-range dipole-dipole interactions. The emission intensity of the acceptor molecule can be monitored, and it is a function of the distance between the donor and acceptor, the overlap of the donor emission spectrum and the acceptor absorption spectrum, and the orientation of the dipole moments of the donor emission and acceptor absorption. FRET can be useful for quantifying molecular dynamics in DNA-DNA interactions, such as described for molecular beacons. For monitoring the generation of a specific product, a probe can be labeled with the donor molecule at one end and the acceptor molecule at the other. Probe-target hybridization results in changes in the distance or orientation of the donor and acceptor, and FRET changes are also observed.

[0101] In some embodiments, the step of detecting nucleic acid amplification products involves the use of fluorescence polarization (FP). Fluorescence polarization techniques generally rely on the principle that when a fluorescently labeled compound is excited by linearly polarized light, it emits fluorescence with a degree of polarization inversely correlated to its rotational speed. Therefore, since the rotation of the fluorophore is constrained between the time the light is absorbed and the time it is emitted, when a molecule such as a fluorescently labeled tracer-nucleic acid conjugate is excited by linearly polarized light, the emitted light remains highly polarized. When a free tracer compound (i.e., not bound to nucleic acid) is excited by linearly polarized light, its rotation is much faster than that of the corresponding tracer-nucleic acid conjugate, and the molecule is oriented more randomly, so the emitted light is depolarized. Therefore, fluorescence polarization provides a quantitative means for measuring the amount of tracer-nucleic acid conjugate produced in an amplification reaction.

[0102] In some embodiments, the step of detecting nucleic acid amplification products involves the use of surface capture. This can be achieved by immobilizing specific oligonucleotides onto a surface to create a highly sensitive and selective biosensor. Examples of surfaces that may be used include gold and carbon, and the surface capture method can utilize several covalent or non-covalent linking methods for attaching the probe to the surface. Subsequent detection of the target nucleic acid can be monitored by various methods.

[0103] In some embodiments, the step of detecting nucleic acid amplification products includes the use of a 5'-3' exonuclease hydrolysis probe (e.g., TAQMAN). For example, the TAQMAN probe is a hydrolysis probe that can increase the specificity of quantitative amplification methods (e.g., quantitative PCR). The principle of the TAQMAN probe relies on 1) the 5'-3' exonuclease activity of Taq polymerase cleaving the double-labeled probe during hybridization to a complementary target sequence, and 2) detection based on fluorophores. The resulting fluorescence signal allows for quantitative measurement of the accumulation of amplification products during exponential amplification steps, and the TAQMAN probe can significantly increase the specificity of detection.

[0104] In some embodiments, the step of detecting nucleic acid amplification products includes the use of insertion dyes and / or binding dyes. In some embodiments, the step of detecting nucleic acid amplification products includes the use of dyes that specifically stain nucleic acids. For example, insertion dyes exhibit enhanced fluorescence upon binding to DNA or RNA. Dyes may include DNA or RNA insertion fluorophores, and examples include SYTO® 82, acridine orange, ethidium bromide, Hoechst dyes, PicoGreen®, propidium iodide, SYBR® I (asymmetric cyanine dye), SYBR® II, TOTO (thiaxole orange dimer), and YOYO (oxazole yellow dimer). Dyes offer opportunities to increase the sensitivity of nucleic acid detection when used in combination with various detection methods. For example, ethidium bromide may be used to stain DNA in agarose gels after gel electrophoresis. Propidium iodide and Hoechst 33258 may be used in flow cytometry to determine the DNA ploidy of cells. SYBR® Green 1 can be used in the analysis of double-stranded DNA by capillary electrophoresis using laser-induced fluorescence detection. PicoGreen® can be used to enhance the detection of double-stranded DNA after matched ion-pair polynucleotide chromatography.

[0105] In some embodiments, the step of detecting nucleic acid amplification products involves the use of absorbance methods (e.g., colorimetric quantification, turbidity). In some examples, the detection and / or quantification of nucleic acids can be achieved by directly converting absorbance (e.g., UV absorbance measurement at 260 nm) to concentration. Direct measurements of nucleic acids can be converted to concentration using the Lambert-Beer law, which relates absorbance to concentration using the path length of measurement and the absorbance. In some embodiments, the step of detecting nucleic acid amplification products involves the use of a colorimetric quantitative detection method. Any suitable colorimetric quantitative detection can be used, and non-limiting examples include assays using nanoparticles (e.g., metal nanoparticles, modified nanoparticles, unmodified nanoparticles) and / or peptide nucleic acid (PNA) probes. For example, a method based on certain gold nanoparticles typically relies on a quantitative coupling between target recognition and nanoparticle aggregation, which can result in a change in the photon properties (e.g., color) of the nanoparticle solution.

[0106] In some embodiments, the step of detecting nucleic acid amplification products involves the use of electrophoresis (e.g., gel electrophoresis, capillary electrophoresis). Gel electrophoresis involves separating nucleic acids in a matrix (generally a crosslinked polymer) by using an electromotive force that pulls molecules through the matrix. The molecules move through the matrix at different speeds, causing separation between products, which can be visualized and interpreted by several methods, including, but not limited to, autoradiography, phosphorescence imaging, and staining with nucleic acid chelate dyes. Capillary gel electrophoresis (CGE) is a combination of conventional gel electrophoresis and liquid chromatography, using a medium such as polyacrylamide in a narrow-bore capillary tube to produce rapid and highly efficient separation of nucleic acid molecules down to single-nucleotide resolution. CGE can be combined with laser-induced fluorescence (LIF) detection, thereby enabling the detection of approximately six stained DNA molecules. CGE / LIF detection generally involves the use of fluorescent DNA insertion dyes, including ethidium bromide, YOYO, and SYBR® Green 1, and may also involve the use of fluorescent DNA derivatives in which the fluorescent dye is covalently bound to the DNA. Simultaneous identification of several different target sequences (e.g., products from multiplex reactions) can be performed using this method.

[0107] In some embodiments, the step of detecting nucleic acid amplification products involves the use of mass spectrometry. Mass spectrometry is an analytical technique that can be used to determine the structure and quantity of nucleic acids and can be used to provide rapid analysis of complex mixtures. After amplification, the sample can be ionized, and the resulting ions can be separated in an electric field and / or a magnetic field according to their mass-to-charge ratio, and the mass-to-charge ratio of the ions can be measured by a detector (Crain, PF and McCloskey, JA, Current Opinion in Biotechnology, Vol. 9: pp. 25-34 (1998)). Examples of mass spectrometry methods include MALDI, MALDI-TOF, or electrospray. These methods may be combined with gas chromatography (GC / MS) and liquid chromatography (LC / MS). Mass spectrometry (e.g., matrix-assisted laser desorption / ionization mass spectrometry (MALDI MS)) can be high-throughput due to the rapid signal acquisition and automated analysis of desorption from solid surfaces.

[0108] In some embodiments, the step of detecting the nucleic acid amplification product involves the use of nucleic acid sequencing. The entire sequence or a partial sequence of the amplification product can be determined, and the determined nucleotide sequence can be referenced as a read. For example, in some embodiments, the linear amplification product can be analyzed directly without further amplification (e.g., by using a single-molecule sequencing methodology). In certain embodiments, the linear amplification product may be subjected to further amplification and then analyzed (e.g., using a ligation sequencing or pyrosequencing methodology). The read may be subjected to different types of sequence analysis. Any suitable sequencing method can be utilized to detect the detectable product produced by the amplification methods described herein, and in some examples to determine its quantity. Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, reversible terminator-based sequencing, ligation sequencing, pyrosequencing, synthesis sequencing, monomolecule sequencing, multiplex sequencing, solid-phase single-nucleotide sequencing, and nanopore sequencing.

[0109] In some embodiments, the step of detecting nucleic acid amplification products includes the use of digital amplification (e.g., digital PCR). Digital PCR, for example, utilizes single-molecule level amplification of nucleic acids (DNA, cDNA, or RNA) and provides a highly sensitive method for quantifying low-copy-number nucleic acids. Systems for digital amplification and analysis of nucleic acids are available (e.g., Fluidigm® Corporation).

[0110] kit The kit may include, for example, one or more polymerases and one or more primers, as described herein, and optionally one or more reverse transcriptases. If a single target is amplified, a pair of primers (forward and reverse) may be included in the kit. If multiple target sequences are amplified, multiple primer pairs may be included in the kit. The kit may also include control polynucleotides, and if multiple target sequences are amplified, multiple control polynucleotides may be included in the kit.

[0111] The kit may also contain one or more components in any number of separate containers, chambers, containers, packets, tubes, vials, microtiter plates, etc., or the components may be combined in various combinations within such containers. The components of the kit may be present in, for example, one or more containers. In some embodiments, all components are provided in a single container. In some embodiments, enzymes (e.g., polymerase and / or reverse transcriptase) may be provided in a separate container from the primers. The components may be, for example, lyophilized, freeze-dried, or in a stable buffer. In one example, the polymerase and / or reverse transcriptase are in lyophilized form in a single container, and the primers are in lyophilized, freeze-dried, or in buffer in a different container. In some embodiments, the polymerase and / or reverse transcriptase, as well as the primers, are in lyophilized form in a single container.

[0112] The kit may further include, for example, dNTPs or modified nucleotides used in the reaction, a vessel, cuvette, or other container used for the reaction, or vials of water or buffer for rehydrating the lyophilized components. The buffer used may be, for example, suitable for the annealing activity of both the polymerase and the primer.

[0113] The kit may also include instructions for performing one or more of the methods described herein, and / or descriptions of one or more of the ingredients described herein. The instructions and / or descriptions may be in printed form and may be included in the kit accessories. The kit may also include written information of an internet location providing such instructions or descriptions.

[0114] The kit may further include reagents used for detection methods, such as reagents used for FRET, a lateral flow device, a dipstick, a fluorescent dye, gold colloid particles, latex particles, molecular beacons, or polystyrene beads. [Examples]

[0115] The embodiments described below illustrate a particular embodiment and are not intended to limit the scope of this technology.

[0116] (Example 1: Detection of Chlamydia trachomatis by isothermal amplification technique) In this example, nucleic acids derived from Chlamydia trachomatis are detected using isothermal amplification technology.

[0117] Real-time detection of Chlamydia genomic DNA A real-time assay for the detection of Chlamydia genomic DNA using a fluorescent DNA dye was tested. This assay utilized SYTO82, an orange fluorescent nucleic acid stain that exhibits bright orange fluorescence upon binding to nucleic acids. A master mix solution was prepared using 20 mM Tris-HCl (pH 8.8, 25°C), 10 mM (NH4)2SO4, 10 mM KCl, 4 mM MgSO4, 0.1% Triton® X-100, 1 mM DTT, 2 μM SYTO82, 0.25 mM dNTPs, and one unit of modified 9 Degrees North (9°Nm®) DNA polymerase (New England BioLabs, Ipswich, MA) per reaction. The amino acid sequence of 9°Nm® DNA polymerase is described herein as Sequence ID No. 9. A primer set targeting a specific sequence within a 7,500 base pair C. trachomatis latent plasmid DNA was used, which included an 11-nucleotide forward primer (i.e., Ct_F11:5'-GGCTTATGGAG-3' (SEQ ID NO: 1)) and a 10-nucleotide reverse primer (i.e., Ct_R10:5'-ATACCGCTTA-3' (SEQ ID NO: 2)). The assay was designed to produce a 22-nucleotide DNA product with a single-nucleotide spacer. The spacer is a nucleotide between the 3' ends of the primers, and this nucleotide is not present in either primer sequence. Each primer was used at a final concentration of 500 nM and mixed with either 2000 copies of Chlamydia genomic DNA or Tris-EDTA buffer (TE) as an untargeted control (NTC). In certain examples, dH2O was used as the NTC. The primer mixture was placed in a separate reaction well from the master mix solution. The assay components were incubated at 65°C for 2 minutes and then combined to initiate an isothermal reaction. The results of the isothermal amplification reaction are shown in Figure 1.

[0118] Confirmation of the formation of specific products from isothermal amplification reactions by electrospray ionization mass spectrometry (ESI-MS). The specificity of the assay was verified by testing the amplification products generated from the above isothermal amplification reaction by electrospray ionization mass spectrometry (ESI-MS). Each of the above primers was used at a final concentration of 500 nM and mixed with either 20,000 copies of Chlamydia genomic DNA or Tris-EDTA buffer (TE) as a targetless control (NTC). In certain cases, dH2O was used as the NTC. Chlamydia genomic DNA was amplified at 65°C for 10 minutes under the above conditions. After amplification, the reaction was inactivated with Tris-EGTA (final 20 mM Tris-EGTA, pH 8.5). The reaction was then desalted and lyophilized before ESI-MS analysis.

[0119] As shown in Figure 2, ESI-MS results confirmed that the primary products of isothermal amplification of genomic DNA are specific 22-base products (i.e., 22-base forward and 22-base reverse products). In comparison, the NTC reaction produced nonspecific products of much lower intensity than the specific products.

[0120] Detection limit (LOD) for Chlamydia genomic DNA detection The sensitivity of detecting Chlamydia genomic DNA was tested using an isothermal amplification assay. In this approach, an assay with 10 nucleotide primers under asymmetric amplification conditions was used for endpoint molecular beacon detection. A master mix solution was prepared using 20 mM Tris-HCl (pH 8.8, 25°C), 10 mM (NH4)2SO4, 10 mM KCl, 4 mM MgSO4, 0.1% Triton® X-100, 1 mM DTT, 0.25 mM dNTPs, and one unit of modified 9 Degrees North (9°Nm®) DNA polymerase (New England BioLabs, Ipswich, MA) per reaction. A primer set targeting a specific sequence within a 7,500 base pair C. trachomatis latent plasmid DNA was used, which included a 10-nucleotide forward primer (i.e., Ct_F10:5'-GCTTATGGAG-3' (SEQ ID NO: 3)) and a 10-nucleotide reverse primer (i.e., Ct_R10:5'-ATACCGCTTA-3' (SEQ ID NO: 2)). The assay was designed to produce a 21-nucleotide DNA product with a 1-nucleotide spacer. The spacer is a nucleotide between the 3' ends of the primers, and this nucleotide is not present in either primer sequence. The primers (i.e., 750 nM forward primer and 200 nM reverse primer) were mixed with either TE as the NTC or different amounts of Chlamydia genomic DNA (i.e., 20 copies, 200 copies, 1,000 copies, 2,000 copies) and placed in separate reaction wells from the master mix solution. In certain examples, dH2O was used as the NTC. The assay components were incubated at 65°C for 2 minutes, and then combined to initiate an isothermal reaction. The reaction was carried out at 65°C for 10 minutes, then placed on ice and inactivated by adding EGTA. A molecular beacon (i.e., Ct FP MB5.18:Fam-CTGGCTACCGCTTAACTCCATAAGCCAG-3BHQ1 (SEQ ID NO: 4)) containing a 20-base sequence complementary to a specific 21-base forward product was then added to each reaction well. The reaction product was detected by reading the endpoint fluorescence of the molecular beacon.As shown in Figure 3, 20 copies of Chlamydia genomic DNA can be amplified to a detectable level at 65°C for 10 minutes by an isothermal reaction using endpoint detection of molecular beacons.

[0121] Real-time detection of Chlamydia genomic DNA by molecular beacon Another approach for the real-time detection of Chlamydia genomic DNA is to use molecular beacons for detection. In this approach, an assay of 10-nucleotide primers under asymmetric amplification conditions was used for real-time molecular beacon detection using TE used as dH2O or a no-template control (NTC). A master mix was prepared using 20 mM Tris-HCl (pH 8.8, 25°C), 10 mM (NH4)2SO4, 10 mM KCl, 4 mM MgSO4, 0.1% Triton® X-100, 50 nM fMB2 3PS (molecular beacon), 0.25 mM dNTP, and 1 unit / reaction of modified 9 Degrees North (9°Nm™) DNA polymerase (New England BioLabs, Ipswich, MA). The master mix contained a molecular beacon (i.e., Ct_3PSMB.2:Fam-ccgcgagccttATACCGCTTAACTCg * c * g * g-IBFQ (SEQ ID NO: 7)). *The nucleotides marked with are phosphorothioate-modified DNA bases. A primer set targeting a specific sequence within a 7,500 base pair C. trachomatis latent plasmid DNA was used, which included a 10-nucleotide forward primer (i.e., Ct_F10+2:5'-AGGCTTATGG-3' (SEQ ID NO: 5)) and a 10-nucleotide reverse primer (i.e., Ct_R10-2:5'-TTATACCGCT-3' (SEQ ID NO: 6)). The assay was designed to produce a 25-nucleotide DNA product containing a 5-nucleotide spacer. The spacer consists of 5 nucleotides between the 3' ends of each primer, and these 5 nucleotides are not present in either primer sequence. The primers (i.e., 750 nM forward primer and 200 nM reverse primer) were combined in the reaction well with either TE or 20,000 copies of Chlamydia genomic DNA as the NTC. In certain examples, dH2O was used as the NTC. All components were incubated at 65°C for 2 minutes, and then an isothermal reaction was initiated, carried out at 65°C. As shown in Figure 4, the reaction products were detected at various time points by real-time fluorescence readout from the molecular beacon.

[0122] (Example 2: Example of an array) The following are non-specific examples of certain nucleotide and amino acid sequences. [Table 1]

[0123] (Example 3: Example of an embodiment) The embodiments described below illustrate a particular embodiment and are not intended to limit the scope of this technology.

[0124] A1. A method for amplifying nucleic acids, Under isothermal amplification conditions, non-denatured nucleic acid samples a) At least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) At least one component that provides hyperthermophilic polymerase activity A step of generating nucleic acid amplification products by contacting them with components containing the above. Methods that include...

[0125] A1.1 A method for amplifying nucleic acids, Under isothermal amplification conditions, non-denatured nucleic acid samples a) A non-enzymatic component comprising at least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) Enzymatic component consisting of hyperthermophilic polymerase or polymerase having an amino acid sequence at least approximately 90% identical to that of hyperthermophilic polymerase. A step to generate nucleic acid amplification products by contacting them. Methods that include...

[0126] A1.2 A method for amplifying nucleic acids, Under isothermal amplification conditions, non-denatured nucleic acid samples a) A non-enzymatic component comprising at least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) Enzyme activity consisting of i) hyperthermophilic polymerase activity and optionally ii) reverse transcriptase activity A step to generate nucleic acid amplification products by contacting them. Methods that include...

[0127] A1.3 The method according to Embodiment A1.2, wherein the enzyme activity consists of i) hyperthermophilic polymerase activity and ii) reverse transcriptase activity.

[0128] A2. The method according to any one of embodiments A1 to A1.3, which does not include the step of denaturing the sample nucleic acid before or during amplification.

[0129] A3. The method according to any one of Embodiments A1 to A2, wherein the sample nucleic acid is not brought into contact with the endonuclease before or during amplification.

[0130] A4. The method according to any one of Embodiments A1 to A3, wherein the sample nucleic acid is not brought into contact with an unwinding agent before or during amplification.

[0131] A5. The method according to any one of Embodiments A1 to A4, wherein the sample nucleic acid is not brought into contact with the helicase before or during amplification.

[0132] A5.1 The method according to any one of Embodiments A1 to A5, wherein the sample nucleic acid is not brought into contact with the recombinase before or during amplification.

[0133] A5.2 The method according to any one of Embodiments A1 to A5.1, wherein the sample nucleic acid is not brought into contact with a single-stranded DNA-binding protein before or during amplification.

[0134] A6. The method according to any one of Embodiments A1 to A5.2, wherein the sample nucleic acid is unmodified before amplification.

[0135] A7. The method according to Embodiment A6, wherein the unmodified sample nucleic acid is derived from disrupted cells.

[0136] A8. The method according to any one of Embodiments A1 to A7, wherein the sample nucleic acid comprises DNA.

[0137] A9. The method according to Embodiment A8, wherein the sample nucleic acid includes genomic DNA.

[0138] A10. The method according to any one of Embodiments A1 to A7, wherein the sample nucleic acid comprises RNA.

[0139] A11. The method according to Embodiment A10, wherein the sample nucleic acid contains viral RNA.

[0140] A12. The method according to Embodiment A10, wherein the sample nucleic acid contains bacterial RNA.

[0141] A13. The method according to any one of Embodiments A1 to A12, wherein the sample nucleic acid comprises a single-stranded nucleic acid.

[0142] A14. The method according to any one of Embodiments A1 to A12, wherein the sample nucleic acid comprises a double-stranded nucleic acid comprising a first strand and a second strand.

[0143] A15. The method according to any one of embodiments A1 to A14, wherein at least one oligonucleotide comprises a first oligonucleotide and a second oligonucleotide.

[0144] A16. The method according to any one of embodiments A1 to A14, wherein at least one oligonucleotide comprises a first oligonucleotide and a second oligonucleotide.

[0145] A16.1 The method according to Embodiment A15 or A16, wherein the first oligonucleotide and the second oligonucleotide each contain 8 to 16 bases.

[0146] A17. The method according to Embodiments A15, A16, or A16.1, wherein the first oligonucleotide comprises a first polynucleotide complementary to the target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide complementary to the target sequence in the second strand of the sample nucleic acid.

[0147] A18. The method according to Embodiments A15, A16, or A16.1, wherein the first oligonucleotide comprises a first polynucleotide that is sequentially complementary to the target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide that is sequentially complementary to the target sequence in the second strand of the sample nucleic acid.

[0148] A19. The method according to Embodiments A15, A16, or A16.1, wherein the first oligonucleotide consists of a first polynucleotide that is continuously complementary to the target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide consists of a second polynucleotide that is continuously complementary to the target sequence in the second strand of the sample nucleic acid.

[0149] A20. The method according to any one of Embodiments A1 to A19, wherein the sample nucleic acid is obtained from the subject before amplification.

[0150] A21. The method according to any one of Embodiments A1 to A20, wherein an unpurified sample nucleic acid is amplified.

[0151] A22. The method according to any one of Embodiments A1 to A20, wherein the purified sample nucleic acid is amplified.

[0152] A23. The method according to any one of Embodiments A1 to A20, further comprising the step of purifying a sample nucleic acid before amplification.

[0153] A24. The method according to any one of Embodiments A1 to A23, wherein hyperthermophilic polymerase activity is provided by hyperthermophilic polymerase or a functional fragment thereof.

[0154] A25. The method according to any one of Embodiments A1 to A23, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof having an amino acid sequence at least about 90% identical to that of the hyperthermophilic polymerase.

[0155] A26. The method according to any one of Embodiments A1 to A23, wherein the hyperthermophilic polymerase activity is provided by an archaeal hyperthermophilic polymerase or a functional fragment thereof.

[0156] A27. The method according to any one of Embodiments A1 to A26, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 8.

[0157] A28. The method according to any one of Embodiments A1 to A26, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 8.

[0158] A28.1 The method according to any one of Embodiments A1 to A28, wherein hyperthermophilic polymerase activity is provided by a polymerase with low exonuclease activity.

[0159] A28.2 The method according to any one of Embodiments A1 to A28, wherein the hyperthermophilic polymerase activity is provided by a polymerase that does not have exonuclease activity.

[0160] A29 The method according to any one of embodiments A1 to A28.2, wherein amplification is performed at a constant temperature of approximately 55°C to approximately 75°C.

[0161] A30. The method according to any one of embodiments A1 to A28.2, wherein amplification is performed at a constant temperature of approximately 55°C to approximately 65°C.

[0162] A31. The method according to any one of embodiments A1 to A28.2, wherein amplification is performed at a constant temperature of approximately 65°C.

[0163] A32. The method according to any one of embodiments A1 to A28.2, wherein amplification is performed at a constant temperature of approximately 60°C.

[0164] A33. The method according to any one of embodiments A1 to A32, wherein the nucleic acid amplification product is detectable within 10 minutes or less.

[0165] A34. The method according to any one of Embodiments A1 to A33, wherein the nucleic acid amplification product comprises a polynucleotide that is sequentially complementary to or substantially identical to the target sequence in the sample nucleic acid.

[0166] A35. The method according to any one of Embodiments A1 to A33, wherein the nucleic acid amplification product consists of polynucleotides that are sequentially complementary to or substantially identical to the target sequence in the sample nucleic acid.

[0167] A36. The method according to any one of Embodiments A1 to A35, wherein the nucleic acid amplification product is approximately 20 to 40 bases in length.

[0168] A37. The method according to any one of Embodiments A16 to A36, wherein the nucleic acid amplification product comprises i) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide; ii) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide; and iii) a spacer sequence sandwiched between the first and second nucleotide sequences.

[0169] A38. The method according to any one of Embodiments A16 to A36, wherein the nucleic acid amplification product comprises i) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, ii) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and iii) a spacer sequence sandwiched between the first and second nucleotide sequences.

[0170] A39. The method according to Embodiment A37 or A38, wherein the spacer sequence comprises 1 to 10 bases.

[0171] A40. The method according to Embodiment A37 or A38, wherein the spacer sequence comprises 1 to 5 bases.

[0172] A41. The method according to any one of embodiments A37 to A40, wherein the spacer sequence is neither complementary to nor identical to the first polynucleotide of the first oligonucleotide, and neither complementary to nor identical to the second polynucleotide of the second oligonucleotide.

[0173] A42. The method according to any one of embodiments A37 to A41, wherein the spacer sequence is continuous complementary to or substantially identical to a portion of the target sequence in the sample nucleic acid.

[0174] A43. The method according to any one of embodiments A1 to A42, further comprising the step of detecting a nucleic acid amplification product.

[0175] A44. The method according to Embodiment A43, wherein the step of detecting the nucleic acid amplification product is performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with a component that provides hyperthermophilic polymerase activity and at least one oligonucleotide.

[0176] A45. The method according to Embodiment A43 or A44, wherein the step of detecting nucleic acid amplification products includes the use of a real-time detection method.

[0177] A46. The method according to Embodiments A43, A44, or A45, wherein the step of detecting the nucleic acid amplification product includes the detection of a fluorescent signal.

[0178] A47. The method according to Embodiment A46, wherein the fluorescence signal originates from a molecular beacon.

[0179] A48. The method according to any one of Embodiments A1 to A47, further comprising the step of contacting a nucleic acid amplification product with i) a polynucleotide complementary to the sequence in the amplification product, and ii) a signal-generating oligonucleotide comprising a fluorophore and a quencher.

[0180] A49. The method according to any one of Embodiments A1 to A47, wherein at least one oligonucleotide is a polynucleotide that is not complementary to the sequence in the sample nucleic acid and hybridizes to the signal-generating oligonucleotide, and the method further comprises the step of contacting an amplification product with the signal-generating oligonucleotide comprising a fluorophore and a quencher.

[0181] A50. The method according to any one of embodiments A1 to A49, carried out in a single reaction volume.

[0182] A51. The method according to any one of Embodiments A1 to A50, carried out in a single reaction vessel.

[0183] A52. The method according to any one of embodiments A1 to A51, including multiplex amplification.

[0184] B1. A method for processing nucleic acids, Under isothermal amplification conditions, non-denatured nucleic acid samples a) At least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) At least one component that provides hyperthermophilic polymerase activity The process of amplifying nucleic acids essentially involves generating nucleic acid amplification products by bringing them into contact with other substances. Methods that include...

[0185] B2. A method for processing nucleic acids, Under isothermal amplification conditions, non-denatured nucleic acid samples a) A non-enzymatic component comprising at least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) Enzymatic component consisting of hyperthermophilic polymerase or polymerase having an amino acid sequence at least approximately 90% identical to that of hyperthermophilic polymerase. The process of amplifying nucleic acids essentially involves generating nucleic acid amplification products by bringing them into contact with other substances. Methods that include...

[0186] B3. A method for processing nucleic acids, Under isothermal amplification conditions, non-denatured nucleic acid samples a) A non-enzymatic component comprising at least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) Enzyme activity consisting of i) hyperthermophilic polymerase activity and optionally ii) reverse transcriptase activity The process of amplifying nucleic acids essentially involves generating nucleic acid amplification products by bringing them into contact with other substances. Methods that include...

[0187] B4. The method according to Embodiment B3, wherein the enzyme activity consists of i) hyperthermophilic polymerase activity and ii) reverse transcriptase activity.

[0188] B5. A method for processing nucleic acids, Under isothermal amplification conditions, non-denatured nucleic acid samples a) At least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) At least one component that provides hyperthermophilic polymerase activity A method for amplifying nucleic acids, comprising the step of generating nucleic acid amplification products by contacting them with a nucleic acid amplification product.

[0189] B6. A method for processing nucleic acids, Under isothermal amplification conditions, non-denatured nucleic acid samples a) A non-enzymatic component comprising at least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) Enzymatic component consisting of hyperthermophilic polymerase or polymerase having an amino acid sequence at least approximately 90% identical to that of hyperthermophilic polymerase. A method for amplifying nucleic acids, comprising the step of generating nucleic acid amplification products by contacting them with a nucleic acid amplification product.

[0190] B7. A method for processing nucleic acids, Under isothermal amplification conditions, non-denatured nucleic acid samples a) A non-enzymatic component comprising at least one oligonucleotide containing a polynucleotide complementary to the target sequence in the sample nucleic acid, and b) Enzyme activity consisting of i) hyperthermophilic polymerase activity and optionally ii) reverse transcriptase activity A method for amplifying nucleic acids, comprising the step of generating nucleic acid amplification products by contacting them with a nucleic acid amplification product.

[0191] B8. The method according to Embodiment B7, wherein the enzyme activity consists of i) hyperthermophilic polymerase activity and ii) reverse transcriptase activity.

[0192] B9. The method according to any one of Embodiments B1 to B8, wherein the sample nucleic acid is unmodified before amplification.

[0193] B10. The method according to Embodiment B9, wherein the unmodified sample nucleic acid is derived from disrupted cells.

[0194] B11. The method according to any one of Embodiments B1 to B10, wherein the sample nucleic acid comprises DNA.

[0195] B12. The method according to Embodiment B11, wherein the sample nucleic acid includes genomic DNA.

[0196] B13. The method according to any one of Embodiments B1 to B10, wherein the sample nucleic acid contains RNA.

[0197] B14. The method according to Embodiment B13, wherein the sample nucleic acid contains viral RNA.

[0198] B15. The method according to Embodiment B13, wherein the sample nucleic acid contains bacterial RNA.

[0199] B16. The method according to any one of Embodiments B1 to B15, wherein the sample nucleic acid comprises a single-stranded nucleic acid.

[0200] B17. The method according to any one of Embodiments B1 to B15, wherein the sample nucleic acid comprises a double-stranded nucleic acid comprising a first strand and a second strand.

[0201] B18. The method according to any one of Embodiments B1 to B17, wherein at least one oligonucleotide comprises a first oligonucleotide and a second oligonucleotide.

[0202] B19. The method according to any one of embodiments B1 to B17, wherein at least one oligonucleotide comprises a first oligonucleotide and a second oligonucleotide.

[0203] B19.1 The method according to Embodiment B18 or B19, wherein the first oligonucleotide and the second oligonucleotide each contain 8 to 16 bases.

[0204] B20. The method according to Embodiments B18, B19, or B19.1, wherein the first oligonucleotide comprises a first polynucleotide complementary to the target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide complementary to the target sequence in the second strand of the sample nucleic acid.

[0205] B21. The method according to Embodiments B18, B19, or B19.1, wherein the first oligonucleotide comprises a first polynucleotide that is sequentially complementary to the target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide that is sequentially complementary to the target sequence in the second strand of the sample nucleic acid.

[0206] B22. The method according to Embodiments B18, B19, or B19.1, wherein the first oligonucleotide consists of a first polynucleotide that is continuously complementary to the target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide consists of a second polynucleotide that is continuously complementary to the target sequence in the second strand of the sample nucleic acid.

[0207] B23. The method according to any one of Embodiments B1 to B22, wherein the sample nucleic acid is obtained from the subject before amplification.

[0208] B24. The method according to any one of Embodiments B1 to B22, wherein an unpurified sample nucleic acid is amplified.

[0209] B25. The method according to any one of Embodiments B1 to B22, wherein a purified sample nucleic acid is amplified.

[0210] B26. The method according to any one of Embodiments B1 to B22, further comprising the step of purifying a sample nucleic acid before amplification.

[0211] B27. The method according to any one of Embodiments B1 to B26, wherein the hyperthermophilic polymerase activity is provided by a hyperthermophilic polymerase or a functional fragment thereof.

[0212] B28. The method according to any one of Embodiments B1 to B26, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof having an amino acid sequence at least about 90% identical to that of the hyperthermophilic polymerase.

[0213] B29. The method according to any one of Embodiments B1 to B26, wherein the hyperthermophilic polymerase activity is provided by an archaeal hyperthermophilic polymerase or a functional fragment thereof.

[0214] B30. The method according to any one of Embodiments B1 to B29, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 8.

[0215] B31. The method according to any one of Embodiments B1 to B29, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 8.

[0216] B31.1 The method according to any one of Embodiments B1 to B31, wherein hyperthermophilic polymerase activity is provided by a polymerase with low exonuclease activity.

[0217] B32.2 The method according to any one of Embodiments B1 to B31, wherein the hyperthermophilic polymerase activity is provided by a polymerase that does not have exonuclease activity.

[0218] B32. The method according to any one of embodiments B1 to B31.2, wherein amplification is performed at a constant temperature of approximately 55°C to approximately 75°C.

[0219] B33. The method according to any one of embodiments B1 to B31.2, wherein amplification is performed at a constant temperature of approximately 55°C to approximately 65°C.

[0220] B34. The method according to any one of embodiments B1 to B31.2, wherein amplification is performed at a constant temperature of approximately 65°C.

[0221] B35. The method according to any one of embodiments B1 to B31.2, wherein amplification is performed at a constant temperature of approximately 60°C.

[0222] B36. The method according to any one of Embodiments B1 to B35, wherein the nucleic acid amplification product is detectable within 10 minutes or less.

[0223] B37. The method according to any one of Embodiments B1 to B36, wherein the nucleic acid amplification product comprises a polynucleotide that is sequentially complementary to or substantially identical to the target sequence in the sample nucleic acid.

[0224] B38. The method according to any one of Embodiments B1 to B36, wherein the nucleic acid amplification product consists of polynucleotides that are sequentially complementary to or substantially identical to the target sequence in the sample nucleic acid.

[0225] B39. The method according to any one of Embodiments B1 to B38, wherein the nucleic acid amplification product is approximately 20 to 40 bases in length.

[0226] B40. The method according to any one of Embodiments B20 to B39, wherein the nucleic acid amplification product comprises i) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide; ii) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide; and iii) a spacer sequence sandwiched between the first and second nucleotide sequences.

[0227] B41. The method according to any one of Embodiments B20 to B39, wherein the nucleic acid amplification product comprises i) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, ii) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and iii) a spacer sequence sandwiched between the first and second nucleotide sequences.

[0228] B42. The method according to Embodiment B40 or B41, wherein the spacer sequence comprises 1 to 10 bases.

[0229] B43. The method according to Embodiment B40 or B41, wherein the spacer sequence comprises 1 to 5 bases.

[0230] B44. The method according to any one of embodiments B40 to B43, wherein the spacer sequence is neither complementary to nor identical to the first polynucleotide of the first oligonucleotide, and neither complementary to nor identical to the second polynucleotide of the second oligonucleotide.

[0231] B45. The method according to any one of Embodiments B40 to B44, wherein the spacer sequence is continuous complementary to or substantially identical to a portion of the target sequence in the sample nucleic acid.

[0232] B46. The method according to any one of Embodiments B1 to B45, further comprising the step of detecting a nucleic acid amplification product.

[0233] B47. The method according to Embodiment B46, wherein the step of detecting the nucleic acid amplification product is performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with a component that provides hyperthermophilic polymerase activity and at least one oligonucleotide.

[0234] B48. The method according to embodiment B46 or B47, wherein the step of detecting the nucleic acid amplification product includes the use of a real-time detection method.

[0235] B49. The method according to Embodiments B46, B47, or B48, wherein the step of detecting a nucleic acid amplification product includes the detection of a fluorescent signal.

[0236] B50. The method according to Embodiment B49, wherein the fluorescence signal originates from a molecular beacon.

[0237] B51. The method according to any one of Embodiments B1 to B50, further comprising the step of contacting a nucleic acid amplification product with i) a polynucleotide complementary to the sequence in the amplification product, and ii) a signal-generating oligonucleotide comprising a fluorophore and a quencher.

[0238] B52. The method according to any one of Embodiments B1 to B50, wherein at least one oligonucleotide is a polynucleotide that is not complementary to the sequence in the sample nucleic acid and hybridizes to the signal-generating oligonucleotide, and the method further comprises the step of contacting an amplification product with the signal-generating oligonucleotide comprising a fluorophore and a quencher.

[0239] B53. The method according to any one of embodiments B1 to B52, carried out in a single reaction volume.

[0240] B54. The method according to any one of embodiments B1 to B53, carried out in a single reaction vessel.

[0241] B55. A method according to any one of embodiments B1 to B54, including multiplex amplification.

[0242] C1. A method for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, a) A step of amplifying a target sequence in a sample nucleic acid, The target sequence comprises a first and second strand that are complementary to each other, and the amplification step involves amplifying the undenatured sample nucleic acid under helicase-free isothermal amplification conditions. i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first polynucleotide that is continuously complementary to the sequence in the first chain, and the second oligonucleotide comprises a second polynucleotide that is continuously complementary to the sequence in the second chain, and ii) At least one component that provides hyperthermophilic polymerase activity The process involves generating a nucleic acid amplification product by contacting it with, wherein the nucleic acid amplification product comprises: 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide; 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide; and 3) a spacer sequence containing 1 to 10 bases. A spacer sequence is sandwiched between the first and second nucleotide sequences, in a step, b) A step of detecting a nucleic acid amplification product, comprising the use of a real-time detection method and performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with (a)(i) and (a)(ii), thereby determining the presence, absence, or amount of the target sequence in the sample nucleic acid. Methods that include...

[0243] C1.1 The method according to Embodiment C1, wherein the first oligonucleotide essentially consists of a first polynucleotide that is sequentially complementary to the sequence in the first chain, and the second oligonucleotide essentially consists of a second polynucleotide that is sequentially complementary to the sequence in the second chain, and / or the nucleic acid amplification product essentially consists of 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and 3) a spacer sequence containing 1 to 10 bases.

[0244] C1.2 The method according to Embodiment C1, wherein the first oligonucleotide consists of a first polynucleotide that is sequentially complementary to the sequence in the first chain, and the second oligonucleotide consists of a second polynucleotide that is sequentially complementary to the sequence in the second chain, and / or the nucleic acid amplification product consists of 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and 3) a spacer sequence containing 1 to 10 bases.

[0245] C1.3 A method for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, a) A step of amplifying a target sequence in a sample nucleic acid, The target sequence comprises a first and second strand that are complementary to each other, and the amplification step involves amplifying the undenatured sample nucleic acid under helicase-free isothermal amplification conditions. i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide consists of a first polynucleotide that is continuously complementary to the sequence in the first chain, and the second oligonucleotide consists of a second polynucleotide that is continuously complementary to the sequence in the second chain, and ii) At least one component that provides hyperthermophilic polymerase activity The process involves generating a nucleic acid amplification product by contacting it with, wherein the nucleic acid amplification product consists of 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and 3) a spacer sequence containing 1 to 10 bases, A spacer sequence is sandwiched between the first and second nucleotide sequences, in a step, b) A step of detecting a nucleic acid amplification product, comprising the use of a real-time detection method and performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with (a)(i) and (a)(ii), thereby determining the presence, absence, or amount of the target sequence in the sample nucleic acid. Methods that include...

[0246] C1.4 The method according to any one of Embodiments C1 to C1.3, wherein the amplification step includes contacting a non-denatured sample nucleic acid under helicase-free and recombinase-free isothermal amplification conditions.

[0247] C2. The method according to any one of Embodiments C1 to C1.4, wherein at least one component providing hyperthermophilic polymerase activity comprises a hyperthermophilic polymerase or a functional fragment thereof, or a polymerase or a functional fragment thereof having an amino acid sequence identical to that of a hyperthermophilic polymerase by at least about 90%.

[0248] C3. The method according to any one of Embodiments C1 to C1.4, wherein at least one component providing hyperthermophilic polymerase activity comprises hyperthermophilic polymerase or a functional fragment thereof, or a polymerase or a functional fragment thereof having an amino acid sequence identical to that of hyperthermophilic polymerase by at least about 90%.

[0249] C4. The method according to any one of embodiments C1 to C3, wherein the hyperthermophilic polymerase activity is provided by an archaeal hyperthermophilic polymerase or a functional fragment thereof.

[0250] C5. The method according to any one of embodiments C1 to C4, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 8.

[0251] C6. The method according to any one of embodiments C1 to C4, wherein the hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 8.

[0252] C6.1 The method according to any one of embodiments C1 to C6, wherein hyperthermophilic polymerase activity is provided by a polymerase with low exonuclease activity.

[0253] C6.2 The method according to any one of embodiments C1 to C6, wherein the hyperthermophilic polymerase activity is provided by a polymerase that does not have exonuclease activity.

[0254] C7. The method according to any one of Embodiments C1 to C6.2, wherein element (a)(ii) further comprises at least one component that provides reverse transcriptase activity.

[0255] C8. The method according to any one of embodiments C1 to C6.2, wherein at least one component that provides hyperthermophilic polymerase activity further provides reverse transcriptase activity.

[0256] C8.1 The method according to any one of embodiments C1 to C8, wherein the first oligonucleotide and the second oligonucleotide each contain 8 to 16 bases.

[0257] C9. The method according to any one of Embodiments C1 to C8.1, which does not include the step of denaturing a sample nucleic acid before or during amplification.

[0258] C10. The method according to any one of embodiments C1 to C9, wherein the sample nucleic acid is not brought into contact with the endonuclease before, during, or after amplification.

[0259] C10.1 The method according to any one of embodiments C1 to C10, wherein the sample nucleic acid is not brought into contact with the recombinase before or during amplification.

[0260] C10.2 The method according to any one of embodiments C1 to C10.1, wherein the sample nucleic acid is not brought into contact with a single-stranded DNA-binding protein before or during amplification.

[0261] C11. The method according to any one of embodiments C1 to C10.2, wherein the sample nucleic acid is unmodified before amplification.

[0262] C12. The method according to embodiment C11, wherein the unmodified sample nucleic acid is derived from disrupted cells.

[0263] C13. The method according to any one of embodiments C1 to C12, wherein the sample nucleic acid comprises DNA.

[0264] C14. The method according to Embodiment C13, wherein the sample nucleic acid includes genomic DNA.

[0265] C15. The method according to any one of embodiments C1 to C12, wherein the sample nucleic acid contains RNA.

[0266] C16. The method according to embodiment C15, wherein the sample nucleic acid contains viral RNA.

[0267] C17. The method according to embodiment C15, wherein the sample nucleic acid contains bacterial RNA.

[0268] C18. The method according to any one of Embodiments C1 to C17, wherein the sample nucleic acid comprises a single-stranded nucleic acid.

[0269] C19. The method according to any one of embodiments C1 to C17, wherein the sample nucleic acid comprises a double-stranded nucleic acid.

[0270] C20. The method according to any one of embodiments C1 to C19, wherein the sample nucleic acid is obtained from the subject before amplification.

[0271] C20.1 The method according to any one of embodiments C1 to C20, wherein an unpurified sample nucleic acid is amplified.

[0272] C20.2 The method according to any one of Embodiments C1 to C20, wherein the purified sample nucleic acid is amplified.

[0273] C21. The method according to any one of embodiments C1 to C20.2, further comprising the step of purifying a sample nucleic acid before amplification.

[0274] C22. The method according to any one of embodiments C1 to C21, wherein amplification is performed at a constant temperature of approximately 55°C to approximately 75°C.

[0275] C23. The method according to any one of embodiments C1 to C21, wherein the amplification is performed at a constant temperature of approximately 55°C to approximately 65°C.

[0276] C24. The method according to any one of embodiments C1 to C21, wherein amplification is performed at a constant temperature of approximately 65°C.

[0277] C25. The method according to any one of embodiments C1 to C21, wherein amplification is performed at a constant temperature of approximately 60°C.

[0278] C26. The method according to any one of Embodiments C1 to C25, wherein the nucleic acid amplification product is approximately 20 to 40 bases in length.

[0279] C27. The method according to any one of embodiments C1 to C26, wherein the spacer sequence comprises 1 to 5 bases.

[0280] C28. The method according to any one of embodiments C1 to C27, wherein the spacer sequence is neither complementary to nor identical to the first polynucleotide of the first oligonucleotide, and neither complementary to nor identical to the second polynucleotide of the second oligonucleotide.

[0281] C29. The method according to any one of embodiments C1 to C28, wherein the spacer sequence is continuous complementary to or substantially identical to a portion of the target sequence in the sample nucleic acid.

[0282] C30. The method according to any one of Embodiments C1 to C28, wherein the step of detecting a nucleic acid amplification product includes the detection of a fluorescent signal.

[0283] C31. The method according to embodiment C30, wherein the fluorescence signal originates from a molecular beacon.

[0284] C32. The method according to any one of Embodiments C1 to C31, further comprising the step of contacting a nucleic acid amplification product with i) a polynucleotide complementary to the sequence in the amplification product, and ii) a signal-generating oligonucleotide comprising a fluorophore and a quencher.

[0285] C33. The method according to any one of embodiments C1 to C32, carried out in a single reaction volume.

[0286] C34. The method according to any one of embodiments C1 to C33, carried out in a single reaction vessel.

[0287] C35. The method according to any one of embodiments C1 to C34, including multiplex amplification.

[0288] D1. A kit for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, a)i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first polynucleotide that is sequentially complementary to the sequence in the first strand of the target sequence, and the second oligonucleotide comprises a second polynucleotide that is sequentially complementary to the sequence in the second strand of the target sequence, and the first and second strands of the target sequence are complementary to each other, and ii) At least one component that provides hyperthermophilic polymerase activity A component for amplifying a target sequence in a sample nucleic acid under helicase-free isothermal amplification conditions, including, b) At least one component that provides real-time detection activity for nucleic acid amplification products and A kit that includes this.

[0289] D1.1 The kit according to Embodiment D1, wherein the first oligonucleotide essentially consists of a first polynucleotide that is sequentially complementary to the sequence in the first strand of the target sequence, and the second oligonucleotide essentially consists of a second polynucleotide that is sequentially complementary to the sequence in the second strand of the target sequence.

[0290] D1.2 The kit according to Embodiment D1, wherein the first oligonucleotide consists of a first polynucleotide that is continuously complementary to the sequence in the first strand of the target sequence, and the second oligonucleotide consists of a second polynucleotide that is continuously complementary to the sequence in the second strand of the target sequence.

[0291] D1.3 A kit for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, a)i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide consists of a first polynucleotide that is sequentially complementary to the sequence in the first chain of the target sequence, and the second oligonucleotide consists of a second polynucleotide that is sequentially complementary to the sequence in the second chain of the target sequence, and the first and second chains of the target sequence are complementary to each other, and ii) At least one component that provides hyperthermophilic polymerase activity A component for amplifying a target sequence in a sample nucleic acid under helicase-free isothermal amplification conditions, including, b) At least one component that provides real-time detection activity for nucleic acid amplification products and A kit that includes this.

[0292] D1.4 A kit according to any one of Embodiments D1 to D1.3, wherein a sample nucleic acid is amplified under isothermal amplification conditions that are helicase-free and recombinase-free.

[0293] D2. The kit according to any one of Embodiments D1 to D1.4, wherein at least one component providing hyperthermophilic polymerase activity comprises a hyperthermophilic polymerase or a functional fragment thereof, or a polymerase or a functional fragment thereof having an amino acid sequence at least about 90% identical to that of a hyperthermophilic polymerase.

[0294] D3. The kit according to any one of Embodiments D1 to D1.4, wherein at least one component providing hyperthermophilic polymerase activity comprises a hyperthermophilic polymerase or a functional fragment thereof, or a polymerase or a functional fragment thereof having an amino acid sequence identical to that of a hyperthermophilic polymerase by at least about 90%.

[0295] D4. The kit according to any one of Embodiments D1 to D3, wherein hyperthermophilic polymerase activity is provided by an archaeal hyperthermophilic polymerase or a functional fragment thereof.

[0296] D5. The kit according to any one of Embodiments D1 to D4, wherein hyperthermophilic polymerase activity is provided by a polymerase or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 8.

[0297] D6. The kit according to any one of Embodiments D1 to D4, wherein hyperthermophilic bacterial polymerase activity is provided by a polymerase or a functional fragment thereof having an amino acid sequence that is at least about 90% identical to the amino acid sequence of Sequence ID No. 8.

[0298] D7. The kit according to any one of embodiments D1 to D6, wherein hyperthermophilic polymerase activity is provided by a polymerase with low exonuclease activity.

[0299] D8. The kit according to any one of embodiments D1 to D6, wherein the hyperthermophilic polymerase activity is provided by a polymerase that does not have exonuclease activity.

[0300] D9. The kit according to any one of Embodiments D1 to D8, wherein element (a)(ii) further comprises at least one component that provides reverse transcriptase activity.

[0301] D10. The kit according to any one of embodiments D1 to D8, wherein at least one component that provides hyperthermophilic polymerase activity further provides reverse transcriptase activity.

[0302] D11. A kit according to any one of embodiments D1 to D10, wherein the first oligonucleotide and the second oligonucleotide each contain 8 to 16 bases.

[0303] D12. The kit according to any one of embodiments D1 to D11, wherein real-time detection activity is provided by a molecular beacon.

[0304] D13. Further instructions for carrying out a method for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, wherein the above method a) A step of amplifying a target sequence in a sample nucleic acid, The target sequence includes the first and second strands, The first chain and the second chain are complementary to each other. Furthermore, the amplification step involves using helicase-free isothermal amplification conditions to obtain non-denatured sample nucleic acids. i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a first polynucleotide that is continuously complementary to the sequence in the first chain, and the second oligonucleotide comprises a second polynucleotide that is continuously complementary to the sequence in the second chain, and ii) At least one component that provides hyperthermophilic polymerase activity The process involves generating a nucleic acid amplification product by contacting it with, wherein the nucleic acid amplification product comprises: 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide; 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide; and 3) a spacer sequence containing 1 to 10 bases. A spacer sequence is sandwiched between the first and second nucleotide sequences, in a step, b) A step of detecting a nucleic acid amplification product, comprising the use of a real-time detection method and performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with (a)(i) and (a)(ii), thereby determining the presence, absence, or amount of the target sequence in the sample nucleic acid. A kit according to any one of embodiments D1 to D12, including the kit described above.

[0305] D13.1 The kit according to Embodiment D13, wherein the first oligonucleotide essentially consists of a first polynucleotide that is sequentially complementary to the sequence in the first strand, and the second oligonucleotide essentially consists of a second polynucleotide that is sequentially complementary to the sequence in the second strand, and / or the nucleic acid amplification product essentially consists of 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and 3) a spacer sequence containing 1 to 10 bases.

[0306] D13.2 The kit according to Embodiment D13, wherein the first oligonucleotide consists of a first polynucleotide that is sequentially complementary to the sequence in the first chain, and the second oligonucleotide consists of a second polynucleotide that is sequentially complementary to the sequence in the second chain, and / or the nucleic acid amplification product consists of 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and 3) a spacer sequence containing 1 to 10 bases.

[0307] D13.3 Further instructions for carrying out a method for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, wherein the above method a) A step of amplifying a target sequence in a sample nucleic acid, The target sequence includes the first and second strands, The first chain and the second chain are complementary to each other. Furthermore, the amplification step involves using helicase-free isothermal amplification conditions to obtain non-denatured sample nucleic acids. i) A first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide consists of a first polynucleotide that is continuously complementary to the sequence in the first chain, and the second oligonucleotide consists of a second polynucleotide that is continuously complementary to the sequence in the second chain, and ii) At least one component that provides hyperthermophilic polymerase activity The process involves generating a nucleic acid amplification product by contacting it with, wherein the nucleic acid amplification product consists of 1) a first nucleotide sequence that is sequentially complementary to or substantially identical to the first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is sequentially complementary to or substantially identical to the second polynucleotide of the second oligonucleotide, and 3) a spacer sequence containing 1 to 10 bases, A spacer sequence is sandwiched between the first and second nucleotide sequences, in a step, b) A step of detecting a nucleic acid amplification product, comprising the use of a real-time detection method and performed within 10 minutes or less from the time the sample nucleic acid is brought into contact with (a)(i) and (a)(ii), thereby determining the presence, absence, or amount of the target sequence in the sample nucleic acid. A kit according to any one of embodiments D1 to D12, including the kit described above.

[0308] D14. The kit according to any one of embodiments D13 to D13.3, wherein the method does not include a step of denaturing the sample nucleic acid before or during amplification.

[0309] D15. The kit according to any one of embodiments D13 to D14, wherein the sample nucleic acid is not brought into contact with the endonuclease before, during, or after amplification.

[0310] D16. The kit according to any one of embodiments D13 to D15, wherein the sample nucleic acid is unmodified before amplification.

[0311] D17. The kit according to embodiment D16, wherein the unmodified sample nucleic acid is derived from disrupted cells.

[0312] D18. A kit according to any one of embodiments D13 to D15, wherein the sample nucleic acid contains DNA.

[0313] D19. The kit according to embodiment D18, wherein the sample nucleic acid contains genomic DNA.

[0314] D20. A kit according to any one of embodiments D13 to D15, wherein the sample nucleic acid contains RNA.

[0315] D21. The kit according to embodiment D20, wherein the sample nucleic acid contains viral RNA.

[0316] D22. The kit according to embodiment D20, wherein the sample nucleic acid contains bacterial RNA.

[0317] D23. A kit according to any one of embodiments D13 to D22, wherein the sample nucleic acid comprises a single-stranded nucleic acid.

[0318] D24. A kit according to any one of embodiments D13 to D22, wherein the sample nucleic acid comprises a double-stranded nucleic acid.

[0319] D25. The kit according to any one of embodiments D13 to D24, wherein the sample nucleic acid is obtained from the subject before amplification.

[0320] D26. A kit according to any one of embodiments D13 to D25, wherein an unpurified sample nucleic acid is amplified.

[0321] D27. A kit according to any one of embodiments D13 to D25, wherein a purified sample nucleic acid is amplified.

[0322] D28. The kit according to any one of Embodiments D13 to D27, wherein the method further comprises the step of purifying a sample nucleic acid before amplification.

[0323] D29. A kit according to any one of embodiments D13 to D28, wherein amplification is performed at a constant temperature of approximately 55°C to approximately 75°C.

[0324] D30. A kit according to any one of embodiments D13 to D28, wherein amplification is performed at a constant temperature of approximately 55°C to approximately 65°C.

[0325] D31. A kit according to any one of embodiments D13 to D28, wherein amplification is performed at a constant temperature of approximately 65°C.

[0326] D32. A kit according to any one of embodiments D13 to D28, wherein amplification is performed at a constant temperature of approximately 60°C.

[0327] D33. A kit according to any one of embodiments D13 to D32, wherein the nucleic acid amplification product is approximately 20 to 40 bases in length.

[0328] D34. A kit according to any one of embodiments D13 to D33, wherein the spacer sequence contains 1 to 5 bases.

[0329] D35. The kit according to any one of embodiments D13 to D34, wherein the spacer sequence is neither complementary to nor identical to the first polynucleotide of the first oligonucleotide, and neither complementary to nor identical to the second polynucleotide of the second oligonucleotide.

[0330] D36. The kit according to any one of embodiments D13 to D35, wherein the spacer sequence is continuous complementary to or substantially identical to a portion of the target sequence in the sample nucleic acid.

[0331] D37. A kit according to any one of embodiments D13 to D36, wherein the step of detecting a nucleic acid amplification product includes the detection of a fluorescent signal.

[0332] D38. The kit according to embodiment D37, wherein the fluorescent signal originates from a molecular beacon.

[0333] D39. The kit according to any one of Embodiments D13 to D38, wherein the method further comprises the step of contacting a nucleic acid amplification product with i) a polynucleotide complementary to the sequence in the amplification product, and ii) a signal-generating oligonucleotide comprising a fluorophore and a quencher.

[0334] D40. A kit according to any one of embodiments D13 to D39, wherein the method is carried out in a single reaction volume.

[0335] D41. A kit according to any one of embodiments D13 to D40, wherein the method is carried out in a single reaction vessel.

[0336] D42. A kit according to any one of embodiments D13 to D41, wherein the method includes multiplex amplification.

[0337] Each patent, patent application, publication, and document referenced herein is incorporated herein by reference in its entirety. The citation of the above patents, patent applications, publications, and documents does not constitute an endorsement of any of them as relevant prior art, nor does it constitute any endorsement of the content or date of these publications or documents. These citations do not indicate that a search for relevant disclosures has been conducted. All statements regarding the date or content of documents are based on available information and do not constitute an endorsement of their accuracy or appropriateness.

[0338] Modifications can be made to the above without departing from the basic aspects of the Art. Although the Art has been described in considerable detail with reference to one or more specific embodiments, those skilled in the art will recognize that modifications can be made to embodiments specifically disclosed in this application, and that these modifications and improvements are also included in the scope and spirit of the Art.

[0339] The techniques described herein with examples as preferred may be carried out in the absence of any elements not specifically disclosed herein. Thus, for example, in each example herein, any of the terms “including,” “essentially consisting of,” and “consisting of” may be substituted for any of the other two terms. The terms and expressions used are for illustrative purposes only, not limitation, and the use of such terms and expressions does not exclude any equivalents or parts of the shown and described features, and various modifications are possible within the scope of the claimed technique. The terms “a” or “an” may refer to one or more of the elements they modify unless it is clear from the context that one or more of the elements are described (for example, “a reagent” may mean one or more reagents). The term “about” as used herein refers to within 10 percent of the underlying parameter (i.e., plus or minus 10%), and the use of the term “about” at the beginning of a list of values ​​is, respectively The values ​​are modified (i.e., “about 1, 2, and 3” refers to about 1, about 2, and about 3). For example, a mass of “about 100 grams” may encompass masses between 90 grams and 110 grams. Furthermore, if an enumeration of values ​​is provided herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the enumeration includes all intermediate and decimal values ​​(e.g., 54%, 85.4%). Thus, although the Art has been specifically disclosed by representative embodiments and optional features, it should be understood that those skilled in the art can modify and alter the concepts disclosed herein, and such modifications and alterations are considered to be within the scope of the Art.

[0340] A specific embodiment of this technology is described in the following claims.

Claims

1. A method for detecting a target nucleic acid sequence in a sample, below: (a) A step of amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other under isothermal amplification conditions, The amplification step includes contacting the nucleic acid containing the target nucleic acid sequence with (i) and (ii) below: (i) A first primer and a second primer, wherein the first primer can hybridize to the sequence of the first strand of the target nucleic acid sequence, and the second primer can hybridize to the sequence of the second strand of the target nucleic acid sequence, and (ii) An enzyme having hyperthermophilic polymerase activity, wherein the hyperthermophilic polymerase activity is provided by a polymerase having the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9, thereby generating a nucleic acid amplification product at a detectable level within 20 minutes of contact, wherein the nucleic acid amplification product is as follows: (1) The sequence of the first primer or a part thereof, or a sequence complementary to the sequence of the first primer or a part thereof, (2) The sequence of the second primer or a part thereof, or a sequence complementary to the sequence of the second primer or a part thereof, and (3) A spacer sequence sandwiched between (1) and (2), having a length of 1 to 10 bases, Enzymes, including, The amplification step is a step that does not use an additional nickeling enzyme. (b) A step of detecting the nucleic acid amplification product, The above method, including.

2. The method according to claim 1, wherein step (b) further comprises determining the amount of nucleic acid containing the target nucleic acid sequence in the sample.

3. The method according to claim 1 or 2, wherein the nucleic acid is (i) genomic nucleic acid, plasmid nucleic acid, mitochondrial nucleic acid, cellular nucleic acid, or extracellular nucleic acid, (ii) bacterial nucleic acid, viral nucleic acid, fungal nucleic acid, protist nucleic acid, plant nucleic acid, non-human animal nucleic acid, or human nucleic acid, or (iii) double-stranded DNA.

4. The method according to any one of claims 1 to 3, wherein the target nucleic acid sequence is a bacterial nucleic acid or a viral nucleic acid.

5. Before step (a), (i) Further comprising generating the nucleic acid from a sample RNA by a reverse transcription reaction using reverse transcriptase, (ii) Further comprising generating the nucleic acid from a sample RNA by a reverse transcription reaction using reverse transcriptase, wherein the sample RNA is cellular RNA, mRNA, microRNA, bacterial RNA, or viral RNA. The method according to any one of claims 1 to 4.

6. The method described above, (i) The method involves simultaneously contacting the sample RNA with the reverse transcriptase and the enzyme having hyperthermophilic polymerase activity, (ii) The method comprising simultaneously contacting the sample RNA with the reverse transcriptase, the enzyme having hyperthermophilic polymerase activity, the first primer, and the second primer, The method according to claim 5.

7. The method according to any one of claims 1 to 6, wherein the enzyme having hyperthermophilic polymerase activity also has reverse transcriptase activity.

8. (i) The sample is a specimen obtained from or derived from a human, a non-human animal, a plant, a bacterium, a fungus, a virus, a protist, or a mixture thereof, or (ii) The sample is a fluid sample, tissue sample, or mixture thereof from a human subject. The method according to any one of claims 1 to 7.

9. The method according to any one of claims 1 to 8, wherein the nucleic acid is not brought into contact with a single-stranded DNA-binding protein before or during step (a).

10. The method according to any one of claims 1 to 9, wherein the amplification of the target nucleic acid sequence is (i) carried out at a constant temperature of 55°C to 75°C, or (ii) carried out at a constant temperature of 65°C.

11. (i) The first primer, the second primer, or both are 8 to 16 bases in length, (ii) The nucleic acid amplification product is 20 to 40 bases in length, or (iii) The spacer sequence is 1 to 5 bases in length. The method according to any one of claims 1 to 10.

12. The method according to any one of claims 1 to 11, further comprising contacting the nucleic acid amplification product with a signal-generating oligonucleotide capable of hybridizing to the nucleic acid amplification product, wherein the signal-generating oligonucleotide comprises a fluorophore, a quencher, or both.

13. The method according to any one of claims 1 to 12, wherein the first primer, the second primer, or both of them include one or more DNA bases, modified DNA bases, or combinations thereof.

14. The method according to any one of claims 1 to 13, carried out in a single reaction vessel.

15. The method according to any one of claims 1 to 14, comprising generating a nucleic acid amplification product at a detectable level within 15 minutes of contact.

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