Exponential nucleic acid amplification with a base greater than 2

Abstract

The present description relates to methods and compositions for providing efficient nucleic acid amplification. In some embodiments, this allows for an increase in the amplification product of greater than two-fold per amplification cycle, and thus improves sensitivity and speed relative to conventional PCR.

Description

[0001] This application is a divisional application of a patent application for "Exponential Nucleic Acid Amplification with a Base Greater than 2" with an application date of December 15, 2015, an application number of 201580076131.0 (International Application No. PCT / US2015 / 065890).

[0002] Cross - reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 092,102, filed on December 15, 2014, which is hereby incorporated by reference in its entirety.

[0004] Statement regarding rights to inventions made under federally - sponsored research and development

[0005] Not applicable. Technical field

[0006] The methods and compositions described herein generally relate to the field of nucleic acid amplification. In particular, methods and compositions for increasing amplification efficiency are described herein. Background art

[0007] A variety of nucleic acid amplification methods are available, and many have been used to achieve sensitive diagnostic assays based on nucleic acid detection. Polymerase chain reaction (PCR) remains the most widely used method for DNA amplification and quantification. Nested PCR and two - step PCR are used to increase the specificity and sensitivity of PCR (U.S. Patent No. 4,683,195). Nested primers for PCR amplification are oligonucleotides having sequences complementary to the region between the target sites of the reverse and forward primers on the target sequence. However, PCR generally has several limitations. PCR amplification can only achieve an increase of less than two - fold in the amount of the target sequence per cycle. This is still relatively low. In addition, the sensitivity of this method is usually limited, making it difficult to detect targets that are present as only a few molecules in a single reaction. Summary of the invention

[0008] Methods and compositions are described herein that are based on the use of new primers (e.g., new inner primers) that are designed to maintain the outer primer binding sites in the amplicons generated after amplification.

[0009] The present disclosure provides the following embodiments: Embodiment 1: A nucleic acid primer set for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and optionally a second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprises at least two oligonucleotides in the form of a first primer or capable of forming at least two first primers, the first primer being capable of hybridizing to the first template strand, wherein the at least two first primers comprise a first outer primer and a first inner primer, the first outer primer comprising a primer sequence a that specifically hybridizes to the first template strand sequence a'; and the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to the first template strand sequence b', wherein b' is adjacent to and 5' of a', and wherein the single-stranded primer sequence b is linked at its 5'-end to the first strand of a double-stranded primer sequence that comprises the primer sequence a, which is adjacent to and 5' of the single-stranded primer sequence b; and a clamp sequence c, which is adjacent to and 5' of the primer sequence a, wherein the clamp sequence c is not complementary to the first strand template sequence d', which is adjacent to and 3' of the first strand template sequence a'.

[0010] Embodiment 2: The primer set of Embodiment 1, wherein the primer set optionally comprises at least one second primer capable of specifically hybridizing to the second template strand.

[0011] Embodiment 3: A method for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and optionally a second template strand, wherein the second template strand is complementary to the first template strand, the method comprising:

[0012] (a) contacting the sample with:

[0013] (i) at least two first primers capable of hybridizing to the first template strand, wherein the at least two first primers comprise a first outer primer and a first inner primer, the first outer primer comprising a primer sequence a that specifically hybridizes to the first template strand sequence a'; and the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to the first template strand sequence b', wherein b' is adjacent to and 5' of a', and wherein the single-stranded primer sequence b is linked at its 5'-end to the first strand of a double-stranded primer sequence that comprises the primer sequence a, which is adjacent to and 5' of the single-stranded primer sequence b; and a clamp sequence c, which is adjacent to and 5' of the primer sequence a, wherein the clamp sequence c is not complementary to the first strand template sequence d', which is adjacent to and 3' of the first strand template sequence a'; and

[0014] (ii) at least one second primer capable of specifically hybridizing to the second template strand,

[0015] wherein, if present, the contacting is carried out under conditions in which the primers anneal to their template strands; and

[0016] (b) Under conditions where strand displacement occurs, if present, a DNA polymerase lacking 5'-3' exonuclease activity is used to amplify the target nucleic acid to produce an amplicon, the amplicon comprising a sequence extending from the template sequence a' to the second primer binding site.

[0017] Embodiment 4: The primer set or method of any of the foregoing embodiments, wherein the DNA polymerase comprises strand displacement activity.

[0018] Embodiment 5: The primer set or method of any of the foregoing embodiments, wherein the T of the double-stranded form of the binding sequence c-a m is greater than the T of the double-stranded form of the binding sequence a-b m .

[0019] Embodiment 6: The primer set or method of any of the foregoing embodiments, wherein the binding sequence c-a is more GC-rich and / or contains more stable bases than the binding sequence a-b.

[0020] Embodiment 7: The method of Embodiments 3-6, wherein the amplification amplifies the target nucleic acid at a rate of up to 3 循环数 during the exponential phase of PCR.

[0021] Embodiment 8: The method of Embodiments 3-7, wherein the amplification allows detection of single-copy nucleic acid in a biological sample within about 12% - 42% fewer amplification cycles than required for the detection using only a single forward primer and a single reverse primer.

[0022] Embodiment 9: The primer set of Embodiments 1, 2, 5 or 6 or the method of Embodiments 3-8, wherein the second primer comprises at least two second primer forms or oligonucleotides capable of forming at least two second primers, the second primers being capable of hybridizing to the second template strand, wherein the at least two second primers comprise a second outer primer and a second inner primer, the second outer primer comprising a primer sequence e that specifically hybridizes to the second template strand sequence e'; and the second inner primer comprising a single-stranded primer sequence f that specifically hybridizes to the second template strand sequence f', where f' is adjacent to and 5' of e', and wherein the single-stranded primer sequence f is linked at its 5' end to the first strand of a double-stranded primer sequence that comprises the primer sequence e, which is adjacent to and 5' of the single-stranded primer sequence f; and a clamp sequence g, which is adjacent to and 5' of the primer sequence e, wherein the clamp sequence g is not complementary to the second strand template sequence h', which is adjacent to and 3' of the second strand template sequence e'.

[0023] Embodiment 10: The primer set or method of Embodiment 9, wherein the T of the double-stranded form of the binding sequence g-e mT greater than the binding sequence e-f in double-stranded form m .

[0024] Embodiment 11: The primer set or method of Embodiment 9 or 10, wherein the binding sequence g-e is more GC-rich and / or contains more stable bases than the binding sequence e-f.

[0025] Embodiment 12: The primer set or method of Embodiments 9-11, wherein the amplification amplifies the target nucleic acid at a rate of up to 6 循环数 during the exponential phase of PCR.

[0026] Embodiment 13: The primer set or method of Embodiments 9-12, wherein the amplification allows detection of single-copy nucleic acids in a biological sample within about 36%-66% fewer amplification cycles than required for the detection using only a single forward primer and a single reverse primer.

[0027] Embodiment 14: The primer set or method of any of the foregoing embodiments, wherein the clamp sequences c and g, if present, cannot be replicated during the amplification process.

[0028] Embodiment 15: The primer set or method of Embodiment 14, wherein the clamp sequence c and / or g, if present, comprises 2'-O-methyl RNA.

[0029] Embodiment 16: The primer set or method of any of the foregoing embodiments, wherein the first inner primer and / or, if present, the second inner primer, does not contain a hairpin sequence in its double-stranded primer sequence.

[0030] Embodiment 17: The primer set or method of Embodiments 3-15, wherein the double-stranded primer sequence of the first inner primer contains a hairpin sequence in which the clamp sequence c is linked to the complementary sequence c', and / or the second inner primer, if present, contains a hairpin sequence in which the clamp sequence g is linked to the complementary sequence g'.

[0031] Embodiment 18: A nucleic acid primer set for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and an optional second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprises at least three oligonucleotides in the form of first primers or capable of forming at least three first primers, the first primers being capable of hybridizing with the first template strand, wherein the at least three first primers comprise a first outer primer, a first middle primer and a first inner primer, the first outer primer comprising a primer sequence d that specifically hybridizes with the first template strand sequence d'; the first middle primer comprising a primer sequence a that specifically hybridizes with the first template strand sequence a', wherein a' is adjacent to and 5' of d', and wherein the single-stranded primer sequence a is linked at its 5' end to the first strand of a double-stranded primer sequence that comprises: a primer sequence d, which is adjacent to and 5' of the single-stranded primer sequence a; and a clamp sequence c1, which is adjacent to and 5' of the primer sequence d, wherein the clamp sequence c1 is not complementary to the first strand template sequence i', which is adjacent to and 3' of the first strand template sequence d'; and the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes with the first template strand sequence b', wherein b' is adjacent to and 5' of a', and wherein the single-stranded primer sequence b is linked at its 5' end to the first strand of a double-stranded primer sequence that comprises: a primer sequence a, which is adjacent to and 5' of the single-stranded primer sequence b; a primer sequence d, which is adjacent to and 5' of the primer sequence a; and a clamp sequence c2, which is adjacent to and 5' of the primer sequence d, wherein the clamp sequence c2 is not complementary to the first strand template sequence i'.

[0032] Embodiment 19: The primer set of Embodiment 18, wherein the primer set further comprises at least one second primer capable of specifically hybridizing with the second template strand.

[0033] Embodiment 20: A method for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and an optional second template strand, wherein the second template strand is complementary to the first template strand, the method comprising:

[0034] (a) contacting the sample with:

[0035] (i) At least three first primers capable of hybridizing to the first template strand, wherein the at least three first primers include a first outer primer, a first middle primer, and a first inner primer, the first outer primer comprising a primer sequence d that specifically hybridizes to the first template strand sequence d'; the first middle primer comprising a primer sequence a that specifically hybridizes to the first template strand sequence a', where a' is adjacent to and 5' of d', and wherein the single-stranded primer sequence a is linked at its 5' end to the first strand of a double-stranded primer sequence that includes: a primer sequence d, which is adjacent to and 5' of the single-stranded primer sequence a; and a clamp sequence c1, which is adjacent to and 5' of the primer sequence d, where the clamp sequence c1 is not complementary to the first strand template sequence i', which is adjacent to and 3' of the first strand template sequence d'; and the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to the first template strand sequence b', where b' is adjacent to and 5' of a', and wherein the single-stranded primer sequence b is linked at its 5' end to the first strand of a double-stranded primer sequence that includes: a primer sequence a, which is adjacent to and 5' of the single-stranded primer sequence b; a primer sequence d, which is adjacent to and 5' of the primer sequence a; a clamp sequence c2, which is adjacent to and 5' of the primer sequence d, where the clamp sequence c2 is not complementary to the first strand template sequence i'; and

[0036] (ii) At least one second primer capable of specifically hybridizing to the second template strand,

[0037] wherein, if present, the contacting is carried out under conditions in which the primers anneal to their template strands; and

[0038] (b) Under conditions for strand displacement, if present, amplify the target nucleic acid using a DNA polymerase lacking 5'-3' exonuclease activity to produce an amplicon that includes the sequence extending from the template sequence a' to the second primer binding site.

[0039] Embodiment 21: The primer set or method of embodiments 18 - 20, wherein the DNA polymerase comprises strand displacement activity.

[0040] Embodiment 22: The primer set or method of embodiments 18 - 21, wherein g1 has a sequence different from g2.

[0041] Embodiment 23: The primer set or method of embodiments 18 - 22, wherein the T of the double-stranded form of the binding sequence c1-d m is greater than the T of the double-stranded form of the binding sequence d-a m , and the T of the double-stranded form of the binding sequence c2-d-a m is greater than the T of the double-stranded form of the binding sequence d-a-b m .

[0042] Embodiment 24: The primer set or method of Embodiments 18-23, wherein the binding sequence c1-d is more GC-rich and / or contains more stable bases than the binding sequence d-a, and the binding sequence c2-d-a is more GC-rich and / or contains more stable bases than the binding sequence d-a-b.

[0043] Embodiment 25: The method of Embodiments 20-24, wherein the amplification amplifies the target nucleic acid at a rate of up to 4 循环数 during the exponential phase of PCR.

[0044] Embodiment 26: The method of Embodiments 20-25, wherein the amplification allows for the detection of single-copy nucleic acids in a biological sample within about 25%-55% fewer amplification cycles than those required for the detection using only a single forward primer and a single reverse primer.

[0045] Embodiment 27: The primer set or method of Embodiments 18-26, wherein the second primer comprises at least three oligonucleotides of the second primer form or capable of forming at least three of the second primers, and the second primer is capable of hybridizing to the second template strand, wherein the at least three second primers include a second outer primer, a second middle primer, and a second inner primer. The second outer primer comprises a primer sequence h that specifically hybridizes to the second template strand sequence h'; the second middle primer comprises a single-stranded primer sequence e that specifically hybridizes to the second template strand sequence e', where e' is adjacent to and 5' of h', and where the single-stranded primer sequence e is linked at its 5' end to the first strand of a double-stranded primer sequence that comprises: a primer sequence h that is adjacent to and 5' of the single-stranded primer sequence e; and a clamp sequence g1 that is adjacent to and 5' of the primer sequence h, where the clamp sequence g1 is not complementary to the second strand template sequence j' that is adjacent to and 3' of the second strand template sequence h'; the second inner primer comprises a single-stranded primer sequence f that specifically hybridizes to the first template strand sequence f', where f' is adjacent to and 5' of e', and where the single-stranded primer sequence f is linked at its 5' end to the first strand of a double-stranded primer sequence that comprises: a primer sequence e that is adjacent to and 5' of the single-stranded primer sequence f; a primer sequence h that is adjacent to and 5' of the primer sequence e; and a clamp sequence g2 that is adjacent to and 5' of the primer sequence h, where the clamp sequence c2 is not complementary to the first strand template sequence j'.

[0046] Embodiment 28: The primer set or method of Embodiment 27, wherein the T of the double-stranded form of the binding sequence g1-h m is greater than the T of the double-stranded form of the binding sequence h-e m , and the T of the double-stranded form of the binding sequence g2-h-e mT greater than the binding sequence h-e-f in double-stranded form m 。

[0047] Embodiment 29: The primer set or method of Embodiment 27 or 28, wherein the binding sequence g1-h is more GC-rich and / or contains more stable bases than the binding sequence h-e, and the binding sequence g2-h-e is more GC-rich and / or contains more stable bases than the binding sequence h-e-f.

[0048] Embodiment 30: The method of Embodiments 27-29, wherein the amplification amplifies the target nucleic acid at a rate of up to 8 循环数 during the exponential phase of PCR.

[0049] Embodiment 31: The method of Embodiments 27-30, wherein the amplification allows the detection of single-copy nucleic acids in a biological sample within about 42%-72% fewer amplification cycles than the amplification cycles required for the detection using only a single forward primer and a single reverse primer.

[0050] Embodiment 32: The primer set or method of Embodiments 18-31, wherein the clamp sequences c1 and c2 and g1 and g2, if present, cannot be replicated during amplification.

[0051] Embodiment 33: The primer set or method of Embodiment 32, wherein the clamp sequences c1 and c2 and g1 and g2, if present, comprise 2'-O-methyl RNA.

[0052] Embodiment 34: The primer set or method of Embodiments 20-33, wherein the double-stranded primer sequences of the first inner primer and the first middle primer and / or, if present, the second inner primer and the second middle primer do not contain hairpin sequences.

[0053] Embodiment 35: The primer set or method of Embodiments 20-33, wherein: the double-stranded primer sequence of the first inner primer contains a hairpin sequence in which the clamp sequence c2 is linked to the complementary sequence c2'; and / or the double-stranded primer sequence of the first middle primer contains a hairpin sequence in which the clamp sequence c1 is linked to the complementary sequence c1'; and / or the double-stranded primer sequence of the second inner primer, if present, contains a hairpin sequence in which the clamp sequence g2 is linked to the complementary sequence g2'; and / or the double-stranded primer sequence of the second middle primer, if present, contains a hairpin sequence in which the clamp sequence g1 is linked to the complementary sequence g1'.

[0054] Embodiment 36: The method of Embodiments 3-17 or 20-35, wherein the amplification comprises PCR.

[0055] Embodiment 37: The method of Embodiment 3-17 or 20-36, wherein the DNA polymerase comprises strand displacement activity and is thermostable.

[0056] Embodiment 38: The method of Embodiment 3-17 or 20-37, wherein the method comprises detecting and optionally quantifying the target nucleic acid.

[0057] Embodiment 39: The method of Embodiment 3-17 or 20-38, wherein the sample consists of nucleic acids from a single cell.

[0058] Embodiment 40: The primer set or method of any one of Embodiments 1-6, wherein the binding sequence a-b contains more unstable bases than the binding sequence c-a.

[0059] Embodiment 41: The primer set or method of Embodiments 9-11, wherein the binding sequence e-f contains more unstable bases than the binding sequence g-e.

[0060] Embodiment 42: The primer set or method of Embodiments 18-24, wherein the binding sequence d-a contains more unstable bases than the binding sequence c1-d, and / or the binding sequence d-a-b contains more unstable bases than the binding sequence c2-d-a.

[0061] Embodiment 43: The primer set or method of Embodiments 27-29, wherein the binding sequence h-e contains more unstable bases than the binding sequence g1-h, and / or the binding sequence h-e-f contains more unstable bases than the binding sequence g2-h-e. Description of the Drawings

[0062] Figure 1 : Schematically shows a complete nested PCR performed on a double-stranded DNA template. Flanking primers are as Figure 2 and Figure 3 shown.

[0063] Figure 2 : Schematically shows an exemplary double primer set hybridizing to one end of a target nucleotide sequence. The set can be, for example, a forward primer set. Different fragments of the primer sequences are represented as (a, b, c); complementary sequences are represented as (a’, b’, c’). The template sequence 3’-5’ is represented as d’, a’ and b’. The outer primer (a) is single-stranded. The inner primer has a single-stranded part (b) and a double-stranded part (a-c).

[0064] Figure 3 : Schematically shows Figure 2Exemplary dual primer sets that hybridize to opposite ends of the target nucleotide sequence shown. The primer sets can be, for example, reverse primer sets. Different segments of the primer sequences are designated (e, f, g); complementary sequences are designated (e’, f’, g’). The template sequence 3’-5’ is designated h’, e’ and f’. The outer primer (e) is single-stranded. The inner primer has a single-stranded portion (f) and a double-stranded portion (a-g).

[0065] Figure 4 : Schematically shows an exemplary triple primer set that hybridizes to one end of the target nucleotide sequence. The primer set can be, for example, a forward primer set. Different segments of the primer sequences are designated (a, b, c1, c2, d); complementary sequences are designated (a’, b’, c1’, c2’, d’). The template sequence 3’-5’ is designated i’, d’, a’ and b’. The outer primer (d) is single-stranded. The middle primer has a single-stranded portion (a) and a double-stranded portion (d-c1). The inner primer has a single-stranded portion (b) and a double-stranded portion (a-d-c2).

[0066] Figure 5 : Schematically shows hybridization to Figure 4 Exemplary triple primer sets that hybridize to opposite ends of the target nucleotide sequence shown. The primer sets can be, for example, reverse primer sets. Different segments of the primer sequences are designated (e, f, g1, g2, h); complementary sequences are designated (e’, f’, g1’, g2’, h’). The template sequence 3’-5’ is designated j’, h’, e’ and f’. The outer primer (d) is single-stranded. The middle primer has a single-stranded portion (e) and a double-stranded portion (h-g1). The inner primer has a single-stranded portion (f) and a double-stranded portion (e-h-g2).

[0067] Figure 6A -B: Schematically shows two alternative structures of the primer when an illustrative primer with a clamp sequence hybridizes to the template. A fluorescence quencher (Q) is present at a position in the primer where it quenches the corresponding fluorescence label (F) in the template strand. In Example 1, experiments were conducted to measure the T of the primer and the complementary target sequence in the presence or absence of the clamp m . (A) Structure formed when the T m of the double-stranded binding sequence c-a is greater than the T m of the double-stranded binding sequence a-b. (B) Structure formed when the T m of the double-stranded binding sequence c-a is less than the T m of the double-stranded binding sequence a-b.

[0068] Figure 7A-B: (A) Schematically shows an exemplary pair of primers, where a fluorescence quencher (Q) is present at a position in the inner primer, at which position it quenches the corresponding fluorescence label (F) in the template strand. (B) In the primer extension reaction of Example 2, fluorescence intensity as a function of time. The three rising traces are independent reactions with slightly different clamps; the horizontal trace has no outer (flanking) displacement primer present. Detailed Description

[0069] Definitions

[0070] Unless otherwise specified, the terms used in the claims and the specification are defined as follows.

[0071] The term "nucleic acid" refers to a nucleotide polymer, which, unless otherwise defined, includes analogs of known natural nucleotides that are capable of functioning in a manner similar to natural nucleotides (e.g., hybridization).

[0072] The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is the DNA representation of mRNA and is typically obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced by synthesis or by amplification; mRNA; and non-coding RNA.

[0073] The term nucleic acid includes double-stranded or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double-stranded or triple-stranded nucleic acid complexes, the nucleic acid strands need not be co-extensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).

[0074] The term nucleic acid also includes any chemical modifications thereof, such as by methylation and / or by capping. Nucleic acid modifications can include the addition of chemical groups that introduce additional charge, polarity, hydrogen bonding, electrostatic interactions, and functionality to individual nucleic acid bases or to the nucleic acid as a whole. Such modifications can include base modifications, such as sugar modifications at the 2'-position, pyrimidine modifications at the 5-position, purine modifications at the 8-position, modifications at the exocyclic amine of cytosine, substitution of 5-bromo-uracil, sugar phosphate backbone modifications, rare methyl pairing combinations such as isobases isocytidine and isoguanine, etc.

[0075] More specifically, in some embodiments, a nucleic acid can include polydeoxyribonucleotides (comprising 2-deoxy-D-ribose), polyribonucleotides (comprising D-ribose), and nucleic acids that are N- or C-glycosides of any other type of purine or pyrimidine base, as well as other polymers that include non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acids (PNAs)) and polymorpholino polymers (commercially available from Anti-Virals, Inc. (Corvallis, Oregon) under the trade name Neugene), and other synthetic sequence-specific nucleic acid polymers, provided that the polymers contain nucleobases in configurations that allow base pairing and base stacking, such as those found in DNA and RNA. The term nucleic acid also includes locked nucleic acids (LNAs), which are described in U.S. Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, which are hereby incorporated by reference in their entireties for their disclosure of LNAs.

[0076] A nucleic acid can be obtained from a fully chemical synthesis process, such as solid-phase mediated chemical synthesis; can be obtained from biological sources, such as by isolation from any species that produces nucleic acids; or can be obtained from processes that involve manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription; or can be obtained from a combination of these methods.

[0077] As used herein, the term "complementary" refers to the ability of two nucleotides to pair precisely; that is, two nucleic acids are considered to be complementary to each other at a given position if the nucleotide at that position in one nucleic acid is capable of hydrogen bonding with the nucleotide of the other nucleic acid to form a standard base pair. Complementarity between two single-stranded nucleic acid molecules can be "partial", where only some nucleotides bind, or can be complete when there is full complementarity between the two single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between the nucleic acid strands.

[0078] "Specific hybridization" refers to the binding of a nucleic acid to a target nucleotide sequence under defined stringent conditions without substantial binding to other nucleotide sequences present in the hybridization mixture. Those skilled in the art understand that relaxing the stringency of the hybridization conditions will allow for sequence mismatches.

[0079] In some embodiments, hybridization is carried out under stringent hybridization conditions. The phrase "stringent hybridization conditions" generally refers to a temperature range that is about 5°C to about 20°C or 25°C below the melting temperature (T m ) of a particular sequence at a determined ionic strength and pH. As used herein, T m is the temperature at which half of a population of double-stranded nucleic acid molecules dissociates into single strands. Calculating the T mThe methods are well-known in the art (see, e.g., Berger and Kimmel (1987) METHODS IN ENZYMOLOGY, VOL. 152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory, both of which are incorporated herein by reference for their descriptions of stringent hybridization conditions). As described in standard references, when nucleic acids are in 1 M aqueous NaCl, the T m value can be simply estimated by the following equation: T m = 81.5 + 0.41(%G + C) (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The melting temperature of the hybrid (and thus the conditions for stringent hybridization) is affected by a variety of factors, such as the length and nature of the primer or probe (DNA, RNA, base composition) and the nature of the target nucleic acid (DNA, RNA, base composition, present in solution or immobilized, etc.), as well as the concentration of salts and other components (e.g., presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well-known and are discussed in standard references in the art. Exemplary stringent conditions suitable for achieving specific hybridization of most sequences are: a temperature of at least about 60 °C and a salt concentration of about 0.2 molar at pH 7. The T m of an oligonucleotide sequence can be calculated according to nearest neighbor thermodynamics, as described in “A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics” John SantaLucia, Jr., PNAS February 17, 1998 vol. 95 no. 4 1460-1465 (which is incorporated herein by reference for this description).

[0080] The term “oligonucleotide” is used to refer to relatively short nucleic acids, which are typically shorter than 200 nucleotides, more specifically, shorter than 100 nucleotides, and most specifically, shorter than 50 nucleotides. Generally, oligonucleotides are single-stranded DNA molecules.

[0081] The term "primer" refers to an oligonucleotide that, under suitable conditions (i.e., in the presence of four different nucleoside triphosphates and a polymerization reagent such as DNA or RNA polymerase or reverse transcriptase), in a suitable buffer and at a suitable temperature, is capable of hybridizing (also known as "annealing") to a nucleic acid and serving as a starting point for nucleotide (RNA or DNA) polymerization. The suitable length of a primer depends on its intended use, but the primer is typically at least 7 nucleotides in length, and in some embodiments, the primer length is 10 to 30 nucleotides, or in some embodiments, the primer length is 10 to 60 nucleotides. In some embodiments, the primer length can be, for example, 15 to 50 nucleotides. Short primer molecules generally require a lower temperature to form a sufficiently stable hybridization complex with the template. The primer does not need to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template.

[0082] A primer is said to anneal to another nucleic acid if the primer or a portion thereof hybridizes to a nucleotide sequence within the nucleic acid. The statement that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer hybridizes completely or exclusively to that nucleotide sequence. For example, in some embodiments, amplification primers used herein are said to "anneal to" or be "specific for" a nucleotide sequence. This description includes primers that anneal completely to a nucleotide sequence, as well as primers that anneal only in part to a nucleotide sequence.

[0083] The term "primer pair" refers to a set of primers that includes a 5' "upstream primer" or "forward primer" that hybridizes to a complementary sequence at the 5' end of the DNA sequence to be amplified, and a 3' "downstream primer" or "reverse primer" that hybridizes to the 3' end of the sequence to be amplified. Those skilled in the art will appreciate that the terms "upstream" and "downstream" or "forward" and "reverse" are not intended to be limiting, but rather provide exemplary directions in some embodiments.

[0084] A "probe" is a nucleic acid that is capable of binding to a target nucleic acid of a complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through the formation of hydrogen bonds, thereby forming a double-stranded structure. The probe can be labeled with a detectable label to facilitate detection of the probe, particularly once it has hybridized to its complementary target. Alternatively, however, the probe can be unlabeled but detectable by specific binding directly or indirectly to a labeled ligand. The size of the probe can vary significantly. Generally, the probe length is at least 7 to 15 nucleotides. Other probe lengths are at least 20, 30, or 40 nucleotides. Other probes are longer, with lengths of at least 50, 60, 70, 80, or 90 nucleotides. Still other probes are even longer, with lengths of at least 100, 150, 200, or more nucleotides. The probe can also be of any length within any range defined by any of the above values (e.g., a length of 15 - 20 nucleotides).

[0085] A primer or probe may be fully complementary or not fully complementary to a target nucleotide sequence. In some embodiments, the primer has at least 65% identity, in some embodiments at least 75% identity, at least 85% identity, at least 90% identity, or at least 95%, 96%, 97%, 98% or 99% identity with the complementary sequence of the target nucleotide sequence over a sequence of at least 7 nucleotides, more typically over a sequence of 10 - 30 nucleotides, and in some embodiments over a sequence of at least 14 - 25 nucleotides. It is understood that it is generally desired that certain bases (e.g., the 3' base of the primer) be fully complementary to the corresponding bases of the target nucleotide sequence. Under stringent hybridization conditions, primers and probes generally anneal to the target sequence.

[0086] As used herein with respect to a portion of a primer or a nucleotide sequence within a primer, the term "specific for" a nucleic acid refers to a primer or nucleotide sequence that is capable of specifically annealing to a target nucleic acid under appropriate annealing conditions.

[0087] The amplification taught by the present invention includes any method that generally replicates at least a portion of at least one target nucleic acid in a template-dependent manner, including but not limited to a variety of techniques for linearly or exponentially amplifying nucleic acid sequences. Exemplary methods for performing the amplification step include PCR, nucleic acid sequence-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), etc., including multiple versions and combinations thereof, such as but not limited to OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction - CCR), helicase-dependent amplification (HDA), etc. Descriptions of such techniques can be found in Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al., Curr Opin Biotechnol. 1993 Feb.; 4(1):41-7, U.S. Patent No. 6,027,998; U.S. Patent No. 6,605,451, Barany et al., PCT Publication No. WO 97 / 31256; Wenz et al., PCT Publication No. WO 01 / 92579; Day et al., Genomics, 29(1):152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al.,Infection 28:97-102(2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available at World Wide Web: promega.com / geneticidproc / ussymp6proc / blegrad.html); LCR Kit Instruction Manual, Cat.#200520, Rev.#050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93(1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951(1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii(1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66(2002); Barany and Gelfand, Gene 109:1-11(1991); Walker et al., Nucl. Acid Res. 20:1691-96(1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lage et al., Genome Res. 2003 Feb.; 13(2):294-307, and Landegren et al., Science 241:1077-80(1988), Demidov, V., Expert Rev Mol Diagn. 2002 Nov.; 2(6):542-8., Cook et al., J Microbiol Methods. 2003 May; 53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 Feb.; 12(1):21-7, U.S. Patent No. 5,830,711, U.S. Patent No. 6,027,889, U.S. Patent No. 5,686,243, PCT Publication No. WO0056927A3 and PCT Publication No. WO9803673A1, and other sources.

[0088] In some embodiments, amplification comprises the following consecutive steps for at least one cycle: annealing at least one primer to a complementary or substantially complementary sequence in at least one target nucleic acid; synthesizing at least one nucleotide strand in a template-dependent manner using a polymerase; and denaturing the newly formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated. Amplification may include thermal cycling or may be carried out isothermally.

[0089] “Nested amplification” refers to the use of more than two primers to amplify a target nucleic acid.

[0090] “Semi-nested amplification” refers to the use of more than one (e.g., two or three) primers that anneal to one end of the target nucleotide sequence.

[0091] “Full nested amplification” refers to the use of more than one primer that anneals to each end of the target nucleotide sequence.

[0092] With respect to nested amplification, multiple primers that anneal to one end of the replicon are distinguished by use of the terms “outer,” “inner,” and “intermediate.”

[0093] “Outer primer” refers to a primer that anneals to a sequence closer to the end of the target nucleotide sequence than another primer that anneals to the same end of the target nucleotide sequence. In some embodiments, the outer primer sequence defines the end of the replicon produced from the target nucleic acid. Herein, “outer primer” is also referred to as “flanking primer.”

[0094] “Inner primer” refers to a primer that anneals to a sequence closer to the middle of the target nucleotide sequence than another primer that anneals to the same end of the target nucleotide sequence.

[0095] With respect to nested amplification, the term “intermediate primer” is used herein in nested amplifications in which at least three primers that anneal to one end of the target nucleotide sequence are used. An intermediate primer is a primer that anneals to a sequence between the inner primer and the outer primer.

[0096] The term “proximal to” as used herein refers to a sequence that is close enough for the method of operation. In some embodiments, sequences that are proximal to each other are directly adjacent with no intervening nucleotides.

[0097] “Multiplex amplification reaction” is a reaction in which two or more nucleic acids distinguishable by sequence are amplified simultaneously.

[0098] The term “qPCR” is used herein to designate quantitative real-time polymerase chain reaction (PCR), which is also known as “real-time PCR” or “kinetic polymerase chain reaction”; all terms refer to PCR with real-time signal detection.

[0099] "Reagent" broadly refers to any reagent used in a reaction other than the analyte (e.g., nucleic acid being analyzed). Exemplary reagents for nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases, dNTPs, etc. Reagents for enzyme reactions include, for example, substrates, cofactors, buffers, metal ions, inhibitors, and activators.

[0100] As used herein, the term "label" refers to any atom or molecule that can be used to provide a detectable and / or quantifiable signal. Specifically, a label can be directly or indirectly linked to a nucleic acid or protein. Suitable labels that can be linked to a probe include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron-dense particles, magnetic particles, spin labels, chemiluminescent-emitting molecules, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0101] As used herein, the term "dye" generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation.

[0102] As used herein, the term "fluorescent dye" generally refers to any dye that emits electromagnetic radiation of a longer wavelength through a fluorescence mechanism after being irradiated by an electromagnetic radiation source (e.g., a lamp, a photodiode, a laser, or another fluorescent dye).

[0103] General methods for improving amplification efficiency

[0104] U.S. Patent No. 8,252,558 and Harris et al., BioTechniques 54:93-97 (February 2013) teach a nested PCR format, called "polymerase chain displacement reaction" (PCDR) (both of these documents are incorporated herein by reference for this description). In PCDR, when extension occurs from the outer primer, the outer primer displaces the extension strand generated from the inner primer because the reaction uses a polymerase with strand displacement activity. In theory, this results in an increase in the amplification product of each amplification cycle greater than 2-fold, and thus the sensitivity and speed are relatively improved compared to conventional PCR. In practice, each amplicon generated from the nested primers no longer contains the primer annealing site of the outer primer. Therefore, PCDR cannot maintain an increase greater than 2-fold in the amplification product of each amplification cycle over a very large number of cycles. For this reason, PCDR only provides a modest reduction in the number of amplification cycles (e.g., from about 23 to about 20) required to detect the target nucleic acid. In contrast, Table 1 below shows that quadrupling (4 循环数 ) of each cycle should halve the number of cycles required to double the amplification for the same amplification. A 6-fold replication of each cycle should be achieved in 15 cycles, which would require 40 cycles in normal PCR.

[0105] Table 1 - Degree of Amplification with Different "Bases"

[0106]

[0107] The key to maintaining an increase in the amplification product of greater than two-fold per amplification cycle is to design internal (nested) primers such that the extension product of the internal (nested) primer contains the external (flanking) primer sequence. Figure 1 A scheme for complete nested PCR using forward internal and external primers and reverse internal and external primers is shown. The "flap" formed when the internal primer anneals to the template contains the external primer sequence such that each of the four new strands generated from the two template strands extends from (and contains) any of the forward external primer sequences (or their complementary sequences) and through (and contains) the reverse external primer sequence. However, more than just adding the external primer sequence to the 5' end of the internal primer is required because when the internal primer anneals, the added sequence will also immediately anneal and prevent the external primer from annealing. The solution to this problem is to use additional 5' addends of the internal primer (i.e., sequences other than the external primer sequence) and oligonucleotides complementary to these two sequences, which are referred to herein as "clamps". For ease of discussion, the added sequences are referred to as "clamp sequences". The configuration is as Figure 2 shown.

[0108] The clamp sequence c is not homologous to the template (d' region). Here, c-a / c'-a' is more stable than a-b / a'-b'. In some embodiments, this can be achieved by using a sequence c that is longer than a, GC-rich (i.e., more GC-rich than a), or contains one or more stabilizing bases (when a does not contain such bases) or more stabilizing bases than a. In some embodiments, this can be achieved by using a sequence c that is longer than b, GC-rich (i.e., more GC-rich than b), or contains one or more stabilizing bases (when b does not contain such bases) or more stabilizing bases than b. In some embodiments, stabilizing bases can be included in the a region of c-a-b and in the a' region of c'-a' to increase the stability of c-a / c'-a' relative to a-b / a'-b'. Optionally or additionally, b can contain one or more destabilizing bases, such as inosine. The external primer retains sequence a. In this case, the sequence a' in the template remains available to the external primer. At higher temperatures, the c'-a' clamp and the 5' end of the internal primer will anneal faster than any other sequence, so the c'-a' clamp does not have to be ligated to the internal primer to form a hairpin structure. However, in some embodiments, using an internal primer with this type of hairpin structure can increase the reaction rate.

[0109] In some embodiments, the c sequence (highlighted in red) in the inner primer is preferably not replicated during PCR. If it is replicated during PCR, these new templates will have a c'-a' tail, which will be generated together with the inner primer c-a-b / c'-a'-b' duplex, and this duplex will "win" in the strand displacement competition with other possible structures and prevent the annealing of the flanking primer sequence a again. To prevent this replication, the red sequence can be prepared from RNA (or 2'-O-methyl RNA, which is relatively easy to prepare by synthesis) that DNA polymerase cannot replicate well. The red sequence can be prepared from any base that can perform base pairing but cannot be replicated.

[0110] In some embodiments, the clamp oligonucleotide (a'-c') is blocked from extension at the 3' end, for example due to the lack of a 3' hydroxyl group or the use of a chemical blocking group, which can improve the specificity of amplification.

[0111] Sample

[0112] Samples containing nucleic acids can be obtained and prepared from biological sources using conventional methods known in the art. In particular, the nucleic acids that can be used in the methods described herein can be obtained from any source, including single-celled organisms and higher organisms such as plants or non-human animals, such as dogs, cats, horses, primates, and other non-human mammals, as well as humans. In some embodiments, the sample can be obtained from an individual suspected or known to be infected with a pathogen, an individual suspected or known to have a disease (such as cancer), or a pregnant individual.

[0113] Nucleic acids can be obtained from cell, body fluid (such as blood, blood components, urine, etc.) or tissue samples by any of a variety of standard techniques. In some embodiments, the methods utilize samples of plasma, serum, cerebrospinal fluid, lymph fluid, peritoneal fluid, pleural fluid, oral fluid, and outer sections of the skin; samples from the respiratory tract, intestine, genital tract, or urinary tract; samples of tears, saliva, blood cells, stem cells, or tumors. The sample can be obtained from a living organism or a dead organism, or from an in vitro culture. Exemplary samples can include single cells, paraffin-embedded tissue samples, and needle biopsy tissues. In some embodiments, the nucleic acid to be analyzed is obtained from a single cell.

[0114] The nucleic acid of interest can be isolated using methods well known in the art. The sample nucleic acid does not need to be in a pure form, but is usually pure enough to allow the steps of the methods described herein to be carried out.

[0115] Target nucleic acid

[0116] Any target nucleic acid that can be detected by nucleic acid amplification can be detected using the methods described herein. In typical embodiments, at least some nucleotide sequence information of the target nucleic acid is known. For example, if the amplification reaction used is PCR, sufficient sequence information of each end of a given target nucleic acid is usually available to allow the design of suitable amplification primers.

[0117] Targets can include, for example, nucleic acids associated with pathogens (such as viruses, bacteria, protozoa or fungi); RNAs, such as those whose overexpression or underexpression indicates a disease, those expressed in a tissue-specific or development-specific manner; or those induced by a specific stimulant; genomic DNA, for which specific polymorphisms (such as SNPs), alleles or haplotypes can be analyzed, for example in genotyping. Of particular interest is genomic DNA that is altered (such as amplified, deleted and / or mutated) in genetic diseases or other pathologies; sequences associated with desired or undesired traits; and / or sequences that uniquely identify an individual (such as in forensics or paternity testing).

[0118] Primer design

[0119] Primers suitable for nucleic acid amplification are long enough to initiate the synthesis of an extension product in the presence of a suitable nucleic acid polymerase. The exact length and composition of the primer will depend on many factors, including, for example, the temperature of the annealing reaction, the source and composition of the primer, and, in the case of using a probe, the proximity of the probe annealing site to the primer annealing site, and the primer:probe concentration ratio. For example, depending on the complexity of the target nucleic acid sequence, oligonucleotide primers typically contain from about 10 to about 60 nucleotides, although it can contain more or fewer nucleotides. The primers should be sufficiently complementary to selectively anneal to their respective strands and form stable duplexes.

[0120] Generally, those skilled in the art know how to design suitable primers capable of amplifying a target nucleic acid of interest. For example, PCR primers can be designed by using any commercially available software or open-source software such as Primer3 (see, for example, Rozen and Skaletsky (2000) Meth. Mol. Biol., 132:365-386; www.broad.mit.edu / node / 1060, etc.) or by accessing the Roche UPL website. The amplicon sequence is input into the Primer3 program, where the UPL probe sequence is in parentheses to ensure that the Primer3 program will design primers on either side of the probe sequence in the parentheses.

[0121] Primers will be prepared by any suitable method, including, for example, direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22:1859-1862; the solid support method of U.S. Patent No. 4,458,066, etc., or primers may be available from commercial sources. Primers are purified by using a Sephadex column (Amersham Biosciences, Inc., Piscataway, NJ) or other methods known to those skilled in the art. Primer purification can improve the sensitivity of the methods described herein.

[0122] Outer primer

[0123] Figure 2 Illustrated how a pair of primers anneals to the first template strand at one end of the target nucleotide sequence. For ease of discussion, this pair of primers can be considered a "forward" pair of primers. The outer primer contains a sequence a that specifically hybridizes to the sequence a' of the first template strand. Figure 3 Illustrated how a pair of primers anneals to the second template strand at the opposite end of the target nucleotide sequence. For ease of discussion, this pair of primers can be considered a "reverse" pair of primers. Here, the outer primer contains a sequence e that specifically hybridizes to the sequence e' of the first template strand. Figure 4 and 5 Illustrated exemplary "forward" and "reverse" three-primer sets. In Figure 4 , the forward outer primer contains a sequence d that specifically hybridizes to the sequence d' of the first template strand. In Figure 5 , the forward outer primer contains a sequence d that specifically hybridizes to the sequence d' of the first template strand. Generally, the considerations for designing suitable outer primers are no different from those for designing outer primers for conventional nested PCR. It is noted that in some embodiments, the T of any primer sequence that is "outer" relative to another primer sequence (such as an inner primer sequence or an intermediate primer sequence) m is preferably lower than the T of the inner (or intermediate) primer sequence m . Thus, for example, during the temperature ramp-down in PCR, the inner primer can anneal and start extension before the outer primer; or premature extension of the outer primer will block the target site of the inner primer and prevent the inner primer from annealing. More specifically, in the embodiment shown in Figure 2 , the T of primer sequence a m is lower than the T of primer sequence b m . Similarly, in Figure 3In the illustrated embodiments, the T of primer sequence e m is lower than the T of primer sequence f m . In some embodiments, the T m difference is at least about 4 °C and generally ranges from about 4 °C to about 20 °C. In some embodiments, the T m difference ranges from about 4 °C to about 15 °C. However, the T of the outer primer m is generally high enough to maintain efficient PCR. For example, in some embodiments, the T of the outer primer m is at least 40 °C. The T can be adjusted by modulating the sequence length, G-C content, and / or by including stabilizing or destabilizing bases in the sequence m .

[0124] "Stabilizing bases" include, for example, peptide nucleic acid (PNA) stretches that can be incorporated into DNA oligonucleotides to increase duplex stability. Locked nucleic acids (LNAs) and unlocked nucleic acids (UNAs) are RNA analogs that can be readily incorporated into DNA oligonucleotides during solid-phase oligonucleotide synthesis and increase and decrease duplex stability, respectively. Suitable stabilizing bases also include modified DNA bases that enhance base pair (and thus duplex as a whole) stability. These modified bases can be incorporated into oligonucleotides during solid-phase synthesis and provide a more predictable method of increasing DNA duplex stability. Examples include AP-dC (G-clamp) and 2-aminoadenine as well as 5-methylcytosine and C(5)-propargyl cytosine (replacing cytosine) and C(5)-propargyl uracil (replacing thymine).

[0125] "Destabilizing bases" are those bases that destabilize double-stranded DNA due to the formation of base pairs that are less stable than typical A-T and / or G-C base pairs. Inosine (I) is a destabilizing base because it pairs with cytosine (C), but the I-C base pair is less stable than the G-C base pair. This lower stability is due to inosine being a purine that is only capable of forming two hydrogen bonds compared to the three hydrogen bonds of the G-C base pair. Other destabilizing bases are known or readily identifiable to those of skill in the art.

[0126] Inner primer of a pair of primers

[0127] For Figure 2, the inner primer in the forward dual primer set contains a single-stranded primer sequence b that specifically hybridizes with the first template strand sequence b', where b' is adjacent to and is the 5' of a', and where the single-stranded primer sequence b is linked at its 5' end to the first strand of the double-stranded primer sequence. This first strand contains: a primer sequence a that is adjacent to and is the 5' of the single-stranded primer sequence b; and a clamp sequence c that is adjacent to and is the 5' of the primer sequence a, where the clamp sequence c is not complementary to the first strand template sequence d', which is adjacent to and is the 3' of the first strand template sequence a'. In some embodiments, the T of the double-stranded form of the binding sequence c-a (represented herein by a hyphen to denote the binding nucleic acid sequence composed of sequences c and a) (i.e., c-a / c'-a') m is greater than the T of the double-stranded form of the binding sequence a-b (i.e., a-b / a'-b') m . This can be readily achieved, for example, by preparing a binding sequence c-a that is longer and / or more GC-rich than the binding sequence a-b, and / or by designing the binding sequence c-a to contain more stable bases than the binding sequence a-b ("more" in this context includes the case where the sequence a-b does not contain G-C base pairs and / or does not contain stable bases). Optionally or additionally, the binding sequence a-b can be designed to contain more unstable bases than the binding sequence c-a ("more" in this context includes the case where the sequence c-a does not contain unstable bases). In some embodiments, the extension of a'-c' is blocked at its 3' end.

[0128] The forward dual primer set can be used with a single conventional reverse primer for semi-nested amplification, or with a reverse dual primer set.

[0129] For Figure 3 , the inner primer in the reverse dual primer set contains a single-stranded primer sequence f that specifically hybridizes with the first template strand sequence f', where f' is adjacent to and is the 5' of e', and where the single-stranded primer sequence f is linked at its 5' end to the first strand of the double-stranded primer sequence. This first strand contains: a primer sequence e that is adjacent to and is the 5' of the single-stranded primer sequence f; and a clamp sequence g that is adjacent to and is the 5' of the primer sequence e, where the clamp sequence g is not complementary to the first strand template sequence h', which is adjacent to and is the 3' of the first strand template sequence e'. In some embodiments, the T of the double-stranded form of the binding sequence g-e (i.e., g-e / g'-e') m is greater than the T of the double-stranded form of the binding sequence e-f (i.e., e-f / e'-f') m . This can be readily achieved, for example, by preparing a binding sequence g-e that is longer and / or more GC-rich than the binding sequence e-f, and / or by designing (the T of the double-stranded form of the binding sequence g-e m is greater than the T of the double-stranded form of the binding sequence e-f mThe binding sequence g-e incorporates more stable bases than the binding sequence e-f (the requirement for "more" includes the case where the sequence e-f does not contain G-C base pairs and / or does not contain stable bases). Optionally or additionally, the binding sequence e-f can be designed to incorporate more unstable bases than the binding sequence g-e (the requirement for "more" includes the case where the sequence g-e does not contain unstable bases). In some embodiments, the extension of e'-g' is blocked at its 3' end.

[0130] In some embodiments, the clamp sequences c and g, if present, cannot be replicated during amplification. RNA or RNA analogs (such as hydrolysis-resistant RNA analogs) can be used to provide the required base pairing to form a double-stranded clamp sequence without being replicated by a DNA-dependent polymerase during amplification. The most common RNA analog is 2'-O-methyl-substituted RNA. Other nucleic acid analogs that can specifically base pair but cannot be replicated include locked nucleic acid (LNA) or BNA (bridged nucleic acid), morpholino, and peptide nucleic acid (PNA). Although these oligonucleotides have different backbone sugars, or in the case of PNA, amino acid residues that replace the phosphoribose, they still bind to RNA or DNA according to Watson and Crick pairing but are not affected by nuclease activity. They cannot be synthesized enzymatically and can only be obtained by synthesis using the phosphoramidite strategy, or for PNA, by peptide synthesis.

[0131] If desired, the clamp sequence c can be covalently linked to the complementary sequence c' such that a-c / a-c' is formed from the hairpin structure; however, this is not necessary for the efficient formation of the double-stranded clamp portion of the primer. Similarly, the clamp sequence g can but does not need to be covalently linked to the complementary sequence g' such that e-g / e'-g' is formed from the hairpin structure.

[0132] Primers of a triple primer set

[0133] In some embodiments, a third primer can be used at one or both ends of the target nucleic acid sequence to further increase the number of copies produced in each cycle of amplification. Figure 4 and Figure 5 Exemplary "forward" and "reverse" three-primer sets are shown. The three-primer set includes the outer primer as discussed above and an intermediate primer that is substantially identical in structure to the inner primer discussed above. The additional primer is an inner primer that is designed to hybridize to the template strand 5' of the intermediate primer.

[0134] The inner primer in the forward three-primer set contains a single-stranded primer sequence b that hybridizes specifically to the first template strand sequence b', where b' is adjacent to and 5' of a'. The single-stranded primer sequence b is linked at its 5' end to the first strand of a double-stranded primer sequence that contains: a primer sequence a, which is adjacent to and 5' of the single-stranded primer sequence b; a primer sequence d, which is adjacent to and 5' of the primer sequence a; and a clamp sequence c2, which is adjacent to and 5' of the primer sequence d, where the clamp sequence c2 is not complementary to the first strand template sequence i'. The clamp sequence c2 can be the same as or different from the clamp sequence (c1) used for the inner primer. In a preferred embodiment, c1 and c2 are different sequences. Similar considerations apply to the design of the inner primer in the three-primer set, as discussed above for the inner primer in the two-primer set. The inner primer in the reverse three-primer set ( Figure 5 shown) has the same structure as the inner primer in the forward three-primer set. Extension of one or more (or all) of the clamp oligonucleotides ( Figure 4 d'-c1' and a'-d'-c2' in Figure 5 and h'-g1' and e'-h'-g2' in

[0135] can be blocked at their 3' ends. The forward three-primer set can be used for semi-nested amplification with a single conventional reverse primer, with a reverse two-primer set, or with a reverse three-primer set. m In some embodiments, the order of primer annealing and extension is controlled according to the T Figure 4 of the primer sequences such that any primer that is "inner" relative to another primer anneals and begins extension before that other primer. Thus, for example, in a two-primer set, the inner primer anneals and begins extension before the outer primer, and in a three-primer set, the inner primer anneals and begins extension before the middle primer, and the middle primer anneals and begins extension before the outer primer. For example, in the embodiment shown in m , the T m of the primer sequences has the following relationship: T m of d < T m of a < T Figure 5 of b. In the embodiment shown in m , the T m of the primer sequences has the following relationship: T m of h < T m of e < T m of f. As noted above, T

[0136] is a function of sequence length, C-G content, and the optional presence of stabilizing and / or destabilizing bases.

[0137] Polymerase

[0138] The disclosed method uses a polymerase for amplification. In some embodiments, the polymerase is a DNA polymerase lacking 5' to 3' exonuclease activity. The polymerase is used under conditions such that the strand extended from the first primer can be displaced by polymerization of the nascent strand extended from the second primer, which is "outside" the first primer. Conveniently, the polymerase is capable of displacing a strand complementary to the template strand, a property known as "strand displacement". Strand displacement results in the synthesis of multiple copies of the target sequence per template molecule. In some embodiments, the DNA polymerase used in the disclosed method is highly processive. Exemplary DNA polymerases include variants of Taq DNA polymerase lacking 5' to 3' exonuclease activity, such as the Stoffel fragment of Taq DNA polymerase (ABI), SD polymerase (Bioron), mutant Taq lacking 5' to 3' exonuclease activity as described in U.S. Pat. No. 5,474,920, Bca polymerase (Takara), Pfx50 polymerase (Invitrogen), Tfu DNA polymerase (Qbiogene). If thermal cycling is to be performed (such as in PCR), the DNA polymerase is preferably a thermostable DNA polymerase. Table 2 below lists polymerases available from New England Biolabs that do not have 5' to 3' exonuclease activity but have strand displacement activity with concomitant thermostability.

[0139] Table 2 - Thermostable Strand Displacement Polymerases Lacking 5' to 3' Exonuclease Activity

[0140]

[0141]

[0142] In some embodiments, the DNA polymerase comprises a fusion between Taq polymerase and a portion of topoisomerase, such as TOPOTAQ TM (Fidelity Systems, Inc.).

[0143] Strand displacement can also be facilitated by using a strand displacement factor, such as a helicase. Any DNA polymerase capable of strand displacement in the presence of a strand displacement factor is suitable for the disclosed methods, even if the DNA polymerase does not perform strand displacement in the absence of such a factor. Strand displacement factors suitable for the methods described herein include the BMRF1 polymerase accessory subunit (Tsurumi et al., J. Virology 67(12):7648-7653(1993)), adenovirus DNA binding protein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164(1994)), herpes simplex virus protein ICP8 (Boehmer and Lehman, J. Virology 67(2):711-715(1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA 91(22):10665-10669(1994)), single-stranded DNA binding protein (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919(1995)), and calf thymus helicase (Siegel et al., J. Biol. Chem. 267:13629-13635(1992)). Thermostable forms of helicase and SSB are available and are thus suitable for PCR.

[0144] Amplification

[0145] If present, the above primer set is contacted with the sample nucleic acid under conditions where the primers anneal to their template strands. The desired nucleic acid amplification method is carried out using a DNA polymerase lacking 5'-3' exonuclease activity that is capable of strand displacement under the reaction conditions used. The amplification produces amplicons that contain the sequences of all the primers used in the amplification reaction. The primer set can be conveniently added to the amplification mixture in the form of individual oligonucleotides. For example, a dual primer set can consist of three oligonucleotides (assuming the internal primer does not contain a hairpin structure), and a triple primer set can consist of five oligonucleotides (assuming neither the internal primer nor the middle primer contains a hairpin structure).

[0146] For semi-nested amplification using a dual primer set as described above, up to 3 循环数The rate. Amplification using a semi-nested dual primer set can reduce the number of amplification cycles required to detect single-copy nucleic acids by about 12% to about 42% (e.g., by 37%). This helps to detect single-copy nucleic acids in a biological sample within about 23 - 27 amplification cycles (which may require 40 or more cycles using other methods). In some embodiments, semi-nested dual primer set PCR helps to detect single-copy nucleic acids in a biological sample in 23, 24, 25, 26, or 27 amplification cycles.

[0147] Table 3 below shows the number of cycles required to amplify single-copy nucleic acids to 10 12 copies using different embodiments described herein. For fully nested amplification using a dual primer set as described above, a rate of up to 6 循环数 can be achieved during the exponential phase of PCR. Amplification using a fully nested dual primer set can reduce the number of amplification cycles required to detect single-copy nucleic acids by about 36% to about 66% (e.g., by 61%). This helps to detect single-copy nucleic acids in a biological sample within about 13 - 17 amplification cycles. In some embodiments, fully nested dual primer set PCR helps to detect single-copy nucleic acids in a biological sample in 13, 14, 15, 16, or 17 amplification cycles.

[0148] Table 3 - Reduction in the number of cycles required for amplification as a function of the PCR base

[0149]

[0150] For semi-nested amplification using a triple primer set as described above, a rate of up to 4 循环数 can be achieved during the exponential phase of PCR. Amplification using a semi-nested triple primer set can reduce the number of amplification cycles required to detect single-copy nucleic acids by about 25% to about 55% (e.g., by 50%). This helps to detect single-copy nucleic acids in a biological sample within about 20 amplification cycles (which may require 40 or more cycles using other methods). In some embodiments, semi-nested triple primer set PCR helps to detect single-copy nucleic acids in a biological sample in 18, 19, 20, 21, or 22 amplification cycles.

[0151] For fully nested amplification using a triple primer set as described above, a rate of up to 8 循环数 can be achieved during the exponential phase of PCR. Amplification using a fully nested triple primer set can reduce the number of amplification cycles required to detect single-copy nucleic acids by about 42% to about 72% (e.g., by 67%). This helps to detect single-copy nucleic acids in a biological sample within about 11 - 15 amplification cycles. In some embodiments, fully nested triple primer set PCR helps to detect single-copy nucleic acids in a biological sample in 9, 10, 11, 12, or 13 amplification cycles.

[0152] In some embodiments, PCR is used for the amplification step. For running a real-time PCR reaction, the reaction mixture typically contains a suitable buffer, a source of magnesium ions (Mg 2+ ) in the range of about 1 to about 10 mM (e.g., in the range of about 2 to about 8 mM), nucleotides, and optionally a detergent and a buffer. An example of a suitable buffer is a TRIS buffer at a concentration of about 5 mM to about 85 mM, preferably at a concentration of 10 mM to 30 mM. In one embodiment, the TRIS buffer concentration is 20 mM in the double-strength (2X) form of the reaction mixture. The pH range of the reaction mixture can be from about 7.5 to about 9.0, with a typical pH range from about 8.0 to about 8.5. The concentration range of the nucleotides is from about 25 mM to about 1000 mM, with a typical concentration range from about 100 mM to about 800 mM. Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700, and 800 mM. Detergents such as Tween 20, Triton X 100, and Nonidet P40 can also be included in the reaction mixture. Stabilizers such as dithiothreitol (DTT, Cleland’s reagent) or mercaptoethanol can also be included. Additionally, the reaction mixture (master mix) can optionally contain dUTP and uracil DNA glycosylase (uracil-N-glycosylase, UNG). The reaction mixture is commercially available from Applied Biosystems, Foster City, CA, USA( Universal Master Mix, catalog numbers 4304437, 4318157, and 4326708).

[0153] Labeling strategy

[0154] Any suitable labeling strategy can be used in the methods described herein. When analyzing a single amplification product present in the reaction, a universal detection probe can be used in the amplification mixture. In certain embodiments, a universal qPCR probe is used for real-time PCR detection. Suitable universal qPCR probes include double-stranded DNA dyes such as SYBR Green, Pico Green (Molecular Probes, Inc., Eugene, OR), Eva Green (Biotinum), ethidium bromide, etc. (see Zhu et al., 1994, Anal. Chem. 66:1941-48).

[0155] In some embodiments, one or more target-specific qPCR probes (i.e., probes specific for the target nucleotide sequence to be detected) are used in the amplification mixture to detect the amplification products. By carefully selecting the markers, an assay can be performed in which different markers are excited and / or detected at different wavelengths in a single reaction ("multiplex detection"). See, e.g., Fluorescence Spectroscopy (Pesce et al., Eds.) Marcel Dekker, New York, (1971); White et al., Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York, (1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, New York, (1971); Griffiths, Colour and Constitution of Organic Molecules, Academic Press, New York, (1976); Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene (1992).

[0156] In some embodiments, it may be convenient to include a marker on one or more of the primers used in the amplification mixture.

[0157] Exemplary automation and systems

[0158] In some embodiments, an automated sample handling and / or analysis platform is used to detect the target nucleic acid. In some embodiments, a commercially available automated analysis platform is utilized. For example, in some embodiments, the System (Cepheid, Sunnyvale, CA) is utilized.

[0159] The GeneXpert System is used to illustrate the methods described herein. Exemplary sample preparation and analysis methods are described below. However, the invention is not limited to a particular detection method or analysis platform. Those skilled in the art will appreciate that any number of platforms and methods can be utilized.

[0160] Utilize an independent, single-use cartridge. Sample extraction, amplification, and detection can all be performed inside this independent "laboratory in a cartridge" (see, for example, U.S. Patents 5,958,349, 6,403,037, 6,440,725, 6,783,736, 6,818,185, each of which is hereby incorporated by reference in its entirety for this description).

[0161] The components of the cartridge include, but are not limited to, processing chambers containing reagents, filters, and capture processes that can be used for extracting, purifying, and amplifying target nucleic acids. Valves enable the transfer of liquids from one chamber to another and contain nucleic acid lysis and filtration components. Optical windows enable real-time optical detection. Reaction tubes enable very fast thermal cycling.

[0162] In some embodiments, the system includes multiple scalable modules. Each module includes multiple cartridges, as well as sample processing and analysis components.

[0163] After adding the sample to the cartridge, the sample contacts the lysis buffer, and the released nucleic acids bind to a nucleic acid binding substrate (such as a silica or glass substrate). Then the sample supernatant is removed and the nucleic acids are eluted in an elution buffer (such as Tris / EDTA buffer). The eluate can then be processed in the cartridge to detect the target genes described herein. In some embodiments, the eluate is used to reconstitute at least some of the reagents that are present as lyophilized particles in the cartridge.

[0164] In some embodiments, PCR is used to amplify and detect one or more target nucleic acids present. In some embodiments, PCR uses Taq polymerase with a hot start function, such as AptaTaq (Roche).

[0165] In some embodiments, off-line centrifugation is used to improve assay results for samples with low cellular inclusions. The sample, with or without buffer added, is centrifuged and the supernatant is removed. Then the pellet is resuspended in a smaller volume of supernatant, buffer, or other liquid. Then the resuspended pellet is added to the previously described cartridge.

[0166] Kit

[0167] Kits for carrying out the methods described herein are also contemplated. Such kits include one or more reagents that can be used to implement any of these methods. A kit typically includes a package having one or more containers for holding the reagents, which are one or more separate compositions or, optionally, a mixture if reagent compatibility permits. The kit may also include other materials that are desirable from the user's perspective, such as buffers, diluents, standards, and / or any other materials that can be used in any other steps of sample processing, washing, or performing the assay.

[0168] The kit preferably includes instructions for carrying out one or more of the screening methods described herein. The instructions included in the kit may be attached to the packaging material or may be included as a package insert. Although the instructions are typically in written or printed form, they are not limited thereto. Any medium capable of storing such instructions and disseminating them to the end user can be utilized. Such media include, but are not limited to, electronic storage media (e.g., disk, tape, cassette, chip), optical media (e.g., CD ROM), etc. The term "instructions" as used herein may include an Internet address providing the instructions.

[0169] Examples

[0170] Example 1: Determining the effect of "clamp" oligonucleotides on the primer / target structure

[0171] An experiment was conducted in which the primer oligonucleotide (although called a "primer", it is not used as such in this experiment) and the T of the complementary target sequence in the presence and absence of the "clamp" oligonucleotide were measured m . The synthetic target oligonucleotide has a 5' fluorescent tag (fluorescein), and the primer incorporates a fluorescent quencher group (see Figure 6). Red indicates the presence of a 2'-O-methyl backbone. The oligonucleotide sequences tested are listed in Table 4 below. The T of this double helix region was measured by tracking the increase in fluorescence as the temperature increased and as the fluorophore and quencher were separated by the melting of the rightmost double helix region shown in the structure of Figure 6 m .

[0172] If the region of the target to which the primer binds as Figure 6A shown is clamped to b / b' binding, the T of this region is predicted to be m much lower than Figure 6B the T in the case of ab / a'b' binding in m . The oligonucleotides used in this experiment and the following experiments are listed in Table 4 below. Table 5 shows the predicted and measured Ts of the primer and target oligonucleotides in the presence or absence of the clamp oligonucleotide m .

[0173] Table 4 - Oligonucleotides Used

[0174]

[0175] *Blocker = a group that blocks extension

[0176] Table 5 - T m Measurement

[0177]

[0178]

[0179] The conditions for all hybrid melt analyses were: 0.01 M tris-HCl, 0.05 M KCl, and 0.006 M MgCl2. All oligonucleotides were 1 μM. The oligonucleotide mixtures in Table 4 were heated to 95 °C and slowly cooled to 45 °C using a Cepheid SmartCycler TM Monitoring fluorescein fluorescence. The Tm was determined as the temperature at which the rate of fluorescence change was greatest.

[0180] The Tm of b / b’ and ab / a’b’ was predicted using software (www.idtdna.com / analyzer / Applications / OligoAnalyzer). The measured Tm was consistent with a structure in which, in the presence of the clamping oligonucleotide, the d’-a’ region in the target remained single-stranded and available for hybridization.

[0181] As Figure 7A shown, the flanking primers (16147 to 16149), also present at 1 μM, caused a small difference in the measured Tm.

[0182] Example II: Extensibility of outer (flanking) primers

[0183] Under the conditions shown in Table 6, the extendibility of the outer (flanking) primers schematically shown in Figure 7A was tested in a PCR reaction.

[0184] Table 6

[0185] Reaction Flanking primer 1 16147 2 16148 3 16149 4 None

[0186] All reactions contained 10 mM Tris-HCl, 0.125 mM each of dATP, dTTP, dCTP, and dGTP, 0.15 μM primer oligonucleotide 16140 from Table 1 above, 0.125 μM target oligonucleotide 16145 from Table 1, 0.125 μM clamp oligonucleotide 16142 from Table 1, 45 mM KCl, 3.5 mM MgCl2, 14 units of AmpliTaq CS, an enzyme having DNA polymerase activity but no 5'-to-3' and 3'-to-5' exonuclease activity, and 15 units of Taq polymerase antibody, which provided a temperature-activated "hot start" for the incorporation reaction.

[0187] Add 0.125 mM or no flanking primer as per Table 6 above; monitor the reaction over time using a SmartCycler, raising the temperature to 95 °C to separate the oligonucleotides and activate the polymerase, then lowering the temperature to 60 °C to anneal the oligonucleotides and allow any primer extension to occur. The results are shown in Figure 7B . The three upward traces are independent reactions with slightly different clamps; the horizontal trace is in the absence of an outer (flanking) displacement primer. These results indicate that strand displacement of the quencher occurs when an outer (flanking) primer is present. Sequence Listing <110> Cepheid Higuchi, Russell <120> Exponential Nucleic Acid Amplification with a Base Greater than 2 <130> CPHDP007WO <140> PCT / US2015 / 065890 <141> 2015-12-15 <150> 62 / 092,102 <151> 2014-12-15 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 49 <212> DNA <213> Artificial <220> <223> Primer <220> <221> Modified Base <222> (1)..(17) <223> 2'-O-Methyl <220> <221> Modified base <222> (6)..(6) <223> Uracil <220> <221> Modified base <222> (45)..(45) <223> Uracil labeled with dabcyl quencher <400> 1 ggcgctccgg accggcgtag gctggtaacc aaccgctgaa ggcatacgg 49 <210> 2 <211> 46 <212> DNA <213> Artificial <220> <223> Primer <220> <221> Modified base <222> (1)..(17) <223> 2'-O-methyl <220> <221> Modified base <222> (6)..(6) <223> Uracil <220> <221> Modified base <222> (45)..(45) <223> Uracil labeled with dabcyl quencher <400> 2 ggcgctccgg accggcgtag gctggtaacc aaccgctgaa ggcata 46 <210> 3 <211> 31 <212> DNA <213> Artificial <220> <223> Clip <400> 3 tggttaccag cctacgccgg tccggagcgc c 31 <210> 4 <211> 38 <212> DNA <213> artificial <220> <223> target <400> 4 ccgtatgcct tcagcggttg gttaccagcc tacgcatt 38 <210> 5 <211> 35 <212> DNA <213> artificial <220> <223> target <400> 5 tatgccttca gcggttggtt accagcctac gcatt 35 <210> 6 <211> 15 <212> DNA <213> artificial <220> <223> flanking primer <400> 6 cgtaggctgg taacc 15 <210> 7 <211> 16 <212> DNA <213> artificial <220> <223> flanking primer <400> 7 gcgtaggctg gtaacc 16 <210> 8 <211> 16 <212> DNA <213> artificial <220> <223> flanking primer <220> <221> modified base <222> (5)..(5) <223> 2-aminopurine <220> <221> modified base <222> (13)..(13) <223> 2-Aminopurine <400> 8 gcgtaggctg gtaacc 16

Claims

1. A nucleic acid primer set for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and optionally a second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprises at least two oligonucleotides in the form of a first primer or capable of forming at least two first primers, the first primer being capable of hybridizing to the first template strand, wherein the at least two first primers comprise a first outer primer and a first inner primer, the first outer primer is single-stranded and comprises a primer sequence a that specifically hybridizes to the first template strand sequence a'; and the first inner primer has a single-stranded portion and a double-stranded portion with base complementary pairing, the single-stranded portion being a single-stranded primer sequence b that specifically hybridizes to the first template strand sequence b', wherein b' is adjacent to and is 5' of a', and wherein the single-stranded primer sequence b is linked at its 5' end to the first strand of the double-stranded portion, the first strand comprising the primer sequence a, which is adjacent to and is 5' of the single-stranded primer sequence b; and a clamp sequence c, which is adjacent to and is 5' of the primer sequence a, wherein the clamp sequence c is not complementary to the first strand template sequence d', which is adjacent to and is 3' of the first strand template sequence a', and wherein the T of the double-stranded form of the binding sequence c and the a fragment in the first inner primer m is greater than the T of the double-stranded form of the binding sequence a and the b fragment m , the clamp sequence c is RNA, the binding sequence a' and c' fragments cannot be extended from their 3' ends, the binding sequence c and a fragment is complementary to the binding sequence c' and a' fragment, wherein the primer set additionally comprises at least one second primer, the second primer being capable of specifically hybridizing to the second template strand, and wherein the first primer and the at least one second primer are a forward primer and at least one reverse primer, respectively, or the first primer and the at least one second primer are a reverse primer and at least one forward primer, respectively.

2. The primer set of claim 1, wherein the RNA is a locked nucleic acid (LNA), bridged nucleic acid (BNA), or peptide nucleic acid (PNA) sequence.

3. The primer set of claim 1, wherein the binding sequence c and a fragment in the first inner primer are more GC-rich and / or contain more stable bases than the binding sequence a and b fragment.

4. The primer set of claim 1, wherein the binding sequence a and b fragment in the first inner primer contains more unstable bases than the binding sequence c and a fragment.

5. The primer set according to any one of claims 1 to 4, wherein the second primer comprises at least two second primer forms or oligonucleotides capable of forming at least two second primers, the second primer being capable of hybridizing with the second template strand, wherein the at least two second primers comprise a second outer primer and a second inner primer, the second outer primer being single-stranded and comprising a primer sequence e that specifically hybridizes with the second template strand sequence e'; and the second inner primer having a single-stranded portion and a double-stranded portion with base complementarity, the single-stranded portion being a single-stranded primer sequence f that specifically hybridizes with the second template strand sequence f', where f' is adjacent to and is the 5' of e', and wherein the single-stranded primer sequence f is linked at its 5' end to the first strand of the double-stranded portion, the first strand comprising the primer sequence e, which is adjacent to and is the 5' of the single-stranded primer sequence f; and a clamp sequence g, which is adjacent to and is the 5' of the primer sequence e, wherein the clamp sequence g is not complementary to the second strand template sequence h', the sequence h' being adjacent to and being the 3' of the second strand template sequence e', wherein the T of the double-stranded form of the binding sequences g and e in the second inner primer m is greater than the T of the double-stranded form of the binding sequences e and f m , the clamp sequence g is RNA, the binding sequences g' and e' fragments cannot be extended from their 3' ends, and the binding sequences g and e fragments are complementary to the binding sequences g' and e' fragments.

6. The primer set of claim 5, wherein the RNA is a locked nucleic acid (LNA), bridged nucleic acid (BNA), or peptide nucleic acid (PNA) sequence.

7. The primer set of claim 5, wherein the binding sequence g and e fragment in the second inner primer are more GC-rich and / or contain more stable bases than the binding sequence e and f fragment.

8. The primer set of claim 5, wherein the binding sequence e and f fragment in the second inner primer contains more unstable bases than the binding sequence g and e fragment.

9. The primer set of claim 5, wherein the amplification amplifies the target nucleic acid at a rate of up to 6 循环数 during the exponential phase of PCR.

10. The primer set of claim 5, wherein the amplification allows for the detection of single-copy nucleic acid in a biological sample within 36% - 66% fewer amplification cycles than required for detection using only a single forward primer and a single reverse primer.

11. The primer set of claim 5, wherein the clamp sequence c and / or g contains 2'-O-methyl RNA.

12. The primer set of claim 5, wherein the double-stranded portion of the first inner primer and / or the second inner primer does not contain a hairpin sequence.

13. The primer set of claim 5, wherein the double-stranded portion of the first inner primer contains a hairpin sequence in which the clamp sequence c is linked to the complementary sequence c', and / or the double-stranded portion of the second inner primer contains a hairpin sequence in which the clamp sequence g is linked to the complementary sequence g'.

14. A method for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and optionally a second template strand, wherein the second template strand is complementary to the first template strand, the method comprising: (a) contacting the sample with: (i) At least two first primers capable of hybridizing with the first template strand, wherein the at least two first primers include a first outer primer and a first inner primer, the first outer primer is single-stranded and contains a primer sequence a that specifically hybridizes with the first template strand sequence a'; and the first inner primer has a single-stranded portion and a double-stranded portion with base complementary pairing. The single-stranded portion is a single-stranded primer sequence b that specifically hybridizes with the first template strand sequence b', where b' is adjacent to and is the 5' of a', and wherein the single-stranded primer sequence b is linked at its 5' end to the first strand of the double-stranded portion, the first strand containing the primer sequence a, which is adjacent to and is the 5' of the single-stranded primer sequence b; and a clamp sequence c, which is adjacent to and is the 5' of the primer sequence a, wherein the clamp sequence c is not complementary to the first strand template sequence d', which is adjacent to and is the 3' of the first strand template sequence a'; and wherein the T in the double-stranded form of the binding sequence c and a fragment in the first inner primer m is greater than the T in the double-stranded form of the binding sequence a and b fragment m , the clamp sequence c is RNA, the binding sequence a' and c' fragment cannot be extended from its 3' end, and the binding sequence c and a fragment is complementary to the binding sequence c' and a' fragment; and (ii) at least one second primer capable of specifically hybridizing to the second template strand, wherein the contacting is carried out under conditions in which the primers anneal to their template strands; and (b) amplifying the target nucleic acid using a DNA polymerase lacking 5'-3' exonuclease activity but containing strand displacement activity to produce an amplicon, the amplicon comprising a sequence extending from the template sequence a' to the second primer binding site, wherein the first primer and the at least one second primer are a forward primer and at least one reverse primer, respectively, or the first primer and the at least one second primer are a reverse primer and at least one forward primer, respectively.

15. The method of claim 14, wherein the RNA is a locked nucleic acid (LNA), bridged nucleic acid (BNA), or peptide nucleic acid (PNA) sequence.

16. The method of claim 14, wherein the binding sequence c and a fragment in the first inner primer are more GC-rich and / or contain more stable bases than the binding sequence a and b fragment.

17. The method of claim 14, wherein the binding sequence a and b segments in the first inner primer contain more unstable bases than the binding sequence c and a segments.

18. The method of any one of claims 14 - 17, wherein said amplification amplifies the target nucleic acid at a rate of up to 3 循环数 during the exponential phase of PCR.

19. The method of any one of claims 14-17, wherein the amplification allows for the detection of single-copy nucleic acid in a biological sample within 12%-42% fewer amplification cycles than required for detection using only a single forward primer and a single reverse primer.

20. The method of any one of claims 14-17, wherein the second primer comprises oligonucleotides of at least two second primer forms or capable of forming at least two second primers, the second primer being capable of hybridizing with the second template strand, wherein the at least two second primers comprise a second outer primer and a second inner primer, the second outer primer being single-stranded and comprising a primer sequence e that specifically hybridizes with the second template strand sequence e'; and the second inner primer having a single-stranded portion and a double-stranded portion with base complementary pairing, the single-stranded portion being a single-stranded primer sequence f that specifically hybridizes with the second template strand sequence f', wherein f' is adjacent to and is 5' of e', and wherein the single-stranded primer sequence f is linked at its 5' end to the first strand of the double-stranded portion, the first strand comprising the primer sequence e, which is adjacent to and is 5' of the single-stranded primer sequence f; and a clamp sequence g, which is adjacent to and is 5' of the primer sequence e, wherein the clamp sequence g is not complementary to the second strand template sequence h', the sequence h' being adjacent to and being 3' of the second strand template sequence e', wherein the duplex form of T of the binding sequence g and the e fragment in the second inner primer m is greater than the duplex form of T of the binding sequence e and the f fragment m , the clamp sequence g is RNA, the binding sequence g' and the e' fragment cannot be extended from its 3' end, and the binding sequence g and the e fragment are complementary to the binding sequence g' and the e' fragment.

21. The method of claim 20, wherein the RNA is a locked nucleic acid (LNA), bridged nucleic acid (BNA), or peptide nucleic acid (PNA) sequence.

22. The method of claim 20, wherein the binding sequence g and e segments in the second inner primer are more GC-rich and / or contain more stable bases than the binding sequence e and f segments.

23. The method of claim 20, wherein the binding sequence e and f segments in the second inner primer contain more unstable bases than the binding sequence g and e segments.

24. The method of claim 20, wherein said amplification amplifies the target nucleic acid at a rate of up to 6 循环数 during the exponential phase of PCR.

25. The method of claim 20, wherein the amplification allows for the detection of single-copy nucleic acid in a biological sample within 36%-66% fewer amplification cycles than required for detection using only a single forward primer and a single reverse primer.

26. The method of claim 20, wherein the clamping sequence c and / or g contains 2'-O-methyl RNA.

27. The method of claim 20, wherein the double-stranded portion of the first inner primer and / or the second inner primer does not contain a hairpin sequence.

28. The method of claim 20, wherein the double-stranded portion of the first inner primer contains a hairpin sequence in which the clamping sequence c is linked to a complementary sequence c', and / or the double-stranded portion of the second inner primer contains a hairpin sequence in which the clamping sequence g is linked to a complementary sequence g'.

29. A nucleic acid primer set for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and an optional second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprises at least three oligonucleotides in the form of first primers or capable of forming at least three first primers, the first primers being capable of hybridizing to the first template strand, wherein the at least three first primers comprise a first outer primer, a first middle primer and a first inner primer, the first outer primer being single-stranded and comprising a primer sequence d that specifically hybridizes to the first template strand sequence d'; the first middle primer having a single-stranded portion and a double-stranded portion with base complementary pairing, the single-stranded portion being a primer sequence a that specifically hybridizes to the first template strand sequence a', wherein a' is adjacent to and is the 5' of d', and wherein the single-stranded primer sequence a is linked at its 5' end to the first strand of the double-stranded portion, which first strand comprises: a primer sequence d, which is adjacent to and is the 5' of the single-stranded primer sequence a; and a clamp sequence c1, which is adjacent to and is the 5' of the primer sequence d, wherein the clamp sequence c1 is not complementary to the first strand template sequence i', which template sequence i' is adjacent to and is the 3' of the first strand template sequence d'; and the first inner primer having a single-stranded portion and a double-stranded portion with base complementary pairing, the single-stranded portion being a single-stranded primer sequence b that specifically hybridizes to the first template strand sequence b', wherein b' is adjacent to and is the 5' of a', and wherein the single-stranded primer sequence b is linked at its 5' end to the first strand of the double-stranded portion, which first strand comprises: a primer sequence a, which is adjacent to and is the 5' of the single-stranded primer sequence b; a primer sequence d, which is adjacent to and is the 5' of the primer sequence a; and a clamp sequence c2, which is adjacent to and is the 5' of the primer sequence d, wherein the clamp sequence c2 is not complementary to the first strand template sequence i', and wherein the T of the double-stranded form of the binding sequence c1 and d fragment in the first middle primer m is greater than the T of the double-stranded form of the binding sequence d and a fragment m , and the T of the double-stranded form of the binding sequence c2, d and a fragment in the first inner primer m is greater than the T of the double-stranded form of the binding sequence d, a and b fragment m , the clamp sequences c1 and c2 are RNA, the binding sequences c1' and d' fragments cannot be extended from their 3' ends, the binding sequences c2', d' and a' fragments cannot be extended from their 3' ends, the binding sequences c1 and d fragments are complementary to the binding sequences c1' and d' fragments, the binding sequences c2, d and a fragments are complementary to the binding sequences c2', d' and a' fragments, wherein the primer set further comprises at least one second primer capable of specifically hybridizing to the second template strand, and wherein the first primer and the at least one second primer are a forward primer and at least one reverse primer respectively, or the first primer and the at least one second primer are a reverse primer and at least one forward primer respectively.

30. The primer set of claim 29, wherein the RNA is a locked nucleic acid (LNA), bridged nucleic acid (BNA), or peptide nucleic acid (PNA) sequence.

31. The primer set of claim 29, wherein c1 has a different sequence from c2.

32. The primer set of any one of claims 29-31, wherein the binding sequence c1 and d segments in the first intermediate primer are more GC-rich and / or contain more stable bases than the binding sequence d and a segments, and the binding sequence c2, d, and a segments in the first inner primer are more GC-rich and / or contain more stable bases than the binding sequence d, a, and b segments.

33. The primer set of any one of claims 29-31, wherein the binding sequence d and a segments in the first intermediate primer contain more unstable bases than the binding sequence c1 and d segments, and / or the binding sequence d, a, and b segments in the first inner primer contain more unstable bases than the binding sequence c2, d, and a segments.

34. A primer set according to any one of claims 29 - 31, wherein the second primer comprises at least three oligonucleotides of the second primer form or capable of forming at least three of the second primers, the second primer being capable of hybridizing with the second template strand, wherein the at least three second primers include a second outer primer, a second middle primer, and a second inner primer, the second outer primer being single-stranded and comprising a primer sequence h that specifically hybridizes with the second template strand sequence h'; the second middle primer having a single-stranded portion and a double-stranded portion with base complementarity, the single-stranded portion being a single-stranded primer sequence e that specifically hybridizes with the second template strand sequence e', where e' is adjacent to and is the 5' of h', and wherein the single-stranded primer sequence e is linked at its 5' end to the first strand of the double-stranded portion, the first strand comprising: a primer sequence h that is adjacent to and is the 5' of the single-stranded primer sequence e; and a clamp sequence g1 that is adjacent to and is the 5' of the primer sequence h, wherein the clamp sequence g1 is not complementary to the second strand template sequence j', which is adjacent to and is the 3' of the second strand template sequence h'; and the second inner primer having a single-stranded portion and a double-stranded portion with base complementarity, the single-stranded portion being a single-stranded primer sequence f that specifically hybridizes with the second template strand sequence f', where f' is adjacent to and is the 5' of e', and wherein the single-stranded primer sequence f is linked at its 5' end to the first strand of the double-stranded portion, the first strand comprising: a primer sequence e that is adjacent to and is the 5' of the single-stranded primer sequence f; a primer sequence h that is adjacent to and is the 5' of the primer sequence e; and a clamp sequence g2 that is adjacent to and is the 5' of the primer sequence h, wherein the clamp sequence g2 is not complementary to the second strand template sequence j', wherein the T of the double-stranded form of the binding sequence g1 and h fragment in the second middle primer m is greater than the T of the double-stranded form of the binding sequence h and e fragment m , and the T of the double-stranded form of the binding sequence g2, h, and e fragment in the second inner primer m is greater than the T of the double-stranded form of the binding sequence h, e, and f fragment m , the clamp sequences g1 and g2 are RNA, the binding sequence g1' and h' fragment cannot be extended from its 3' end, the binding sequence g2', h', and e' fragment cannot be extended from its 3' end, the binding sequence g1 and h fragment is complementary to the binding sequence g1' and h' fragment, and the binding sequence g2, h, and e fragment is complementary to the binding sequence g2', h', and e' fragment.

35. The primer set of claim 34, wherein the RNA is a locked nucleic acid (LNA), bridged nucleic acid (BNA), or peptide nucleic acid (PNA) sequence.

36. The primer set of claim 34, wherein in the second intermediate primer, the binding sequence g1 and the h fragment are more GC-rich and / or contain more stable bases than the binding sequence h and the e fragment, and in the second inner primer, the binding sequence g2, h and e fragments are more GC-rich and / or contain more stable bases than the binding sequence h, e and f fragments.

37. The primer set of claim 34, wherein in the second intermediate primer, the binding sequence h and the e fragment contain more unstable bases than the binding sequence g1 and the h fragment, and / or in the second inner primer, the binding sequence h, e and f fragments contain more unstable bases than the binding sequence g2, h and e fragments.

38. The primer set of claim 34, wherein the clamp sequences c1 and c2 and g1 and g2 contain 2'-O-methyl RNA. The primer set of claim 34, wherein: The double-stranded portion of the first inner primer and the first intermediate primer and / or the second inner primer and the second intermediate primer does not contain a hairpin sequence. The primer set of claim 34, wherein: The double-stranded portion of the first inner primer contains a hairpin sequence in which the clamp sequence c2 is linked to the complementary sequence c2'; and / or the double-stranded portion of the first intermediate primer contains a hairpin sequence in which the clamp sequence c1 is linked to the complementary sequence c1'; and / or the double-stranded portion of the second inner primer contains a hairpin sequence in which the clamp sequence g2 is linked to the complementary sequence g2'; and / or the double-stranded portion of the second intermediate primer contains a hairpin sequence in which the clamp sequence g1 is linked to the complementary sequence g1'.

41. A method for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and optionally a second template strand, wherein the second template strand is complementary to the first template strand, the method comprising: (a) contacting the sample with: (i) At least three first primers capable of hybridizing with the first template strand, wherein the at least three first primers include a first outer primer, a first middle primer, and a first inner primer. The first outer primer is single-stranded and contains a primer sequence d that specifically hybridizes with the first template strand sequence d'. The first middle primer has a single-stranded portion and a double-stranded portion with base complementary pairing. The single-stranded portion is a primer sequence a that specifically hybridizes with the first template strand sequence a', where a' is adjacent to and is the 5' of d', and wherein the single-stranded primer sequence a is linked at its 5' end to the first strand of the double-stranded portion, and this first strand contains: a primer sequence d, which is adjacent to and is the 5' of the single-stranded primer sequence a; and a clamp sequence c1, which is adjacent to and is the 5' of the primer sequence d, where the clamp sequence c1 is not complementary to the first strand template sequence i', and this sequence i' is adjacent to and is the 3' of the first strand template sequence d'. And the first inner primer has a single-stranded portion and a double-stranded portion with base complementary pairing. The single-stranded portion is a single-stranded primer sequence b that specifically hybridizes with the first template strand sequence b', where b' is adjacent to and is the 5' of a', and wherein the single-stranded primer sequence b is linked at its 5' end to the first strand of the double-stranded portion, and this first strand contains: a primer sequence a, which is adjacent to and is the 5' of the single-stranded primer sequence b; Primer sequence d, which is adjacent to and is 5' of primer sequence a; and clamp sequence c2, which is adjacent to and is 5' of primer sequence d, wherein clamp sequence c2 is not complementary to the first-strand template sequence i', and wherein the T of the double-stranded form of binding sequences c1 and d fragments in the first intermediate primer m is greater than the T of the double-stranded form of binding sequences d and a fragments m , and the T of the double-stranded form of binding sequences c2, d and a fragments in the first inner primer m is greater than the T of the double-stranded form of binding sequences d, a and b fragments m , clamp sequences c1 and c2 are RNA, binding sequences c1' and d' fragments cannot be extended from their 3' ends, binding sequences c2', d' and a' fragments cannot be extended from their 3' ends, binding sequences c1 and d fragments are complementary to binding sequences c1' and d' fragments, binding sequences c2, d and a fragments are complementary to binding sequences c2', d' and a' fragments; and (ii) At least one second primer capable of specifically hybridizing with the second template strand, wherein the contacting is carried out under conditions where the primers anneal to their template strands; and (b) Amplifying the target nucleic acid using a DNA polymerase lacking 5'-3' exonuclease activity but containing strand displacement activity to produce an amplicon, the amplicon containing a sequence extending from the template sequence a' to the second primer binding site, and wherein the first primer and the at least one second primer are a forward primer and at least one reverse primer respectively, or the first primer and the at least one second primer are a reverse primer and at least one forward primer respectively.

42. The method of claim 41, wherein the RNA is a locked nucleic acid (LNA), bridged nucleic acid (BNA), or peptide nucleic acid (PNA) sequence.

43. The method of claim 41, wherein c1 has a different sequence from c2.

44. The method according to any one of claims 41 - 43, wherein the binding sequence c1 and d fragment in the first middle primer are richer in GC and / or contain more stable bases than the binding sequence d and a fragment, and the binding sequence c2, d, and a fragment in the first inner primer are richer in GC and / or contain more stable bases than the binding sequence d, a, and b fragment.

45. The method according to any one of claims 41 - 43, wherein the binding sequence d and a fragment in the first middle primer contain more unstable bases than the binding sequence c1 and d fragment, and / or the binding sequence d, a, and b fragment in the first inner primer contain more unstable bases than the binding sequence c2, d, and a fragment.

46. The method of any one of claims 41-43, wherein said amplification amplifies the target nucleic acid at a rate of up to 4 循环数 during the exponential phase of PCR.

47. The method of any one of claims 41-43, wherein said amplification allows for the detection of single-copy nucleic acid in a biological sample within 25% - 55% fewer amplification cycles than required for detection using only a single forward primer and a single reverse primer.

48. The method according to any one of claims 41 - 43, wherein the second primer comprises at least three oligonucleotides of the second primer form or capable of forming at least three of the second primers, the second primer being capable of hybridizing to the second template strand, wherein the at least three second primers include a second outer primer, a second middle primer, and a second inner primer, the second outer primer being single-stranded and comprising a primer sequence h that specifically hybridizes to the second template strand sequence h'; the second middle primer having a single-stranded portion and a double-stranded portion with base complementary pairing, the single-stranded portion being a single-stranded primer sequence e that specifically hybridizes to the second template strand sequence e', where e' is adjacent to and 5' of h', and wherein the single-stranded primer sequence e is linked at its 5' end to the first strand of the double-stranded portion, the first strand comprising: a primer sequence h, which is adjacent to and 5' of the single-stranded primer sequence e; and a clamp sequence g1, which is adjacent to and 5' of the primer sequence h, wherein the clamp sequence g1 is not complementary to the second strand template sequence j', which is adjacent to and 3' of the second strand template sequence h'; and the second inner primer having a single-stranded portion and a double-stranded portion with base complementary pairing, the single-stranded portion being a single-stranded primer sequence f that specifically hybridizes to the second template strand sequence f', where f' is adjacent to and 5' of e', and wherein the single-stranded primer sequence f is linked at its 5' end to the first strand of the double-stranded portion, the first strand comprising: a primer sequence e, which is adjacent to and 5' of the single-stranded primer sequence f; a primer sequence h, which is adjacent to and 5' of the primer sequence e; and a clamp sequence g2, which is adjacent to and 5' of the primer sequence h, wherein the clamp sequence g2 is not complementary to the second strand template sequence j', wherein the T of the double-stranded form of the binding sequence g1 and the h fragment in the second middle primer m is greater than the T of the double-stranded form of the binding sequence h and the e fragment m , and the T of the double-stranded form of the binding sequence g2, h, and e fragments in the second inner primer m is greater than the T of the double-stranded form of the binding sequence h, e, and f fragments m , the clamp sequences g1 and g2 are RNA, the binding sequences g1' and h' fragments cannot be extended from their 3' ends, the binding sequences g2', h', and e' fragments cannot be extended from their 3' ends, the binding sequences g1 and h fragments are complementary to the binding sequences g1' and h' fragments, and the binding sequences g2, h, and e fragments are complementary to the binding sequences g2', h', and e' fragments.

49. The method of claim 48, wherein said RNA is a locked nucleic acid (LNA), bridged nucleic acid (BNA), or peptide nucleic acid (PNA) sequence.

50. The method of claim 48, wherein the binding sequences g1 and h segments in the second intermediate primer are more GC-rich and / or contain more stable bases than the binding sequences h and e segments, and the binding sequences g2, h, and e segments in the second inner primer are more GC-rich and / or contain more stable bases than the binding sequences h, e, and f segments.

51. The method of claim 48, wherein the binding sequences h and e segments in the second intermediate primer contain more unstable bases than the binding sequences g1 and h segments, and / or the binding sequences h, e, and f segments in the second inner primer contain more unstable bases than the binding sequences g2, h, and e segments.

52. The method of claim 48, wherein said amplification amplifies the target nucleic acid at a rate of up to 8 循环数 during the exponential phase of PCR.

53. The method of claim 48, wherein said amplification allows for the detection of single-copy nucleic acid in a biological sample within 42% - 72% fewer amplification cycles than required for detection using only a single forward primer and a single reverse primer.

54. The method of claim 48, wherein the clamp sequences c1 and c2 and g1 and g2 comprise 2'-O-methyl RNA. The method of claim 48, wherein: The double-stranded portions of the first inner primer and the first intermediate primer and / or the second inner primer and the second intermediate primer do not contain a hairpin sequence. The method of claim 48, wherein: The double-stranded portion of the first inner primer contains a hairpin sequence in which the clamp sequence c2 is linked to the complementary sequence c2'; and / or the double-stranded portion of the first intermediate primer contains a hairpin sequence in which the clamp sequence c1 is linked to the complementary sequence c1'; and / or the double-stranded portion of the second inner primer contains a hairpin sequence in which the clamp sequence g2 is linked to the complementary sequence g2'; and / or the double-stranded portion of the second intermediate primer contains a hairpin sequence in which the clamp sequence g1 is linked to the complementary sequence g1'.

57. The method of claim 14 or 41, wherein said amplification comprises PCR.

58. The method of claim 14 or 41, wherein the DNA polymerase comprises strand displacement activity and is thermostable.

59. The method of claim 14 or 41, wherein the method comprises detecting and optionally quantifying the target nucleic acid.

60. The method of claim 14 or 41, wherein the sample consists of nucleic acid from a single cell.

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