Method for amplifying and detecting target nucleic acids with extremely high specificity and sensitivity - Patents.com

Abstract

A method for detecting a nucleic acid target by nucleic acid amplification, comprising: providing at least one target nucleic acid and at least one non-extendable or extendable oligonucleotide labeled with at least one luminescent group / moiety adapted to emit excess light when hybridized to or incorporated into a nucleic acid molecule; providing at least one extendable oligonucleotide labeled with at least one converter or acceptor group / moiety adapted to convert the color of the luminescent group / moiety to a different color or to thermalise it, which is incorporated into the amplification product; hybridizing or incorporating the luminescent group / moiety of the luminescent group-labeled oligonucleotide to the target nucleic acid amplification product, whereby the luminescent group / moiety emits excess light, the excess light emission being a measure of target amplification. In the method, any non-specific amplification does not produce any detectable signal under controlled attenuation by selectively controlling the removal of only the excess light emission of the luminescent group.

Description

[Technical field]

[0001] The present invention relates to a process for nucleic acid amplification, more specifically, the process comprising Libra primer pairs and Libra probe-primer pairs amplifying and detecting target nucleic acids with extremely high specificity and sensitivity. [Background technology]

[0002] Deoxyribonucleic acid (DNA), the hereditary (genetic) material present in all living organisms and cells, is the blueprint of all their properties and capabilities. For this reason, genetic material has been envisaged for the identification of an organism or virus or any special properties thereof, or for the determination of genotypic variations, or for the molecular analysis of diseases or defects of an organism. Due to the low amount of nucleic acid in cells and in available samples of organs and tissues of living organisms, the detection or analysis of nucleic acid requires the amplification of its amount for detection and / or quantification. Many nucleic acid amplification techniques have been developed in the past 40 years. The polymerase chain reaction (PCR) is the most important and was the first amplification technique developed (U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188; Saiki et al., 1985, Science 230:1350-1354), followed by LDR and PCR (U.S. Pat. No. 6,797,470), isothermal amplification reactions or procedures using one or more RNA polymerases (WO 2006 / 081222), strand displacement amplification (U.S. Pat. No. RE39,007E), ligase chain reaction (LCR) (Wu, et al. Genomics 4: 560-569 (1990); Barany, et al. Proc. Natl. Acad. Sci. USA 88: 189 - 193 (1991)), and Qβ Particular mention may be made of the RNA replicase system (WO 1994 / 016108), rolling circle amplification (RCA) (U.S. Pat. No. 5,854,033), NASBA (U.S. Pat. No. 5,130.238), loop-mediated amplification (LAMP) (Nucleic Acids Research ---), recombinase polymerase amplification (RPA), helicase polymerase amplification (HPA), each of which is incorporated herein by reference in its entirety.

[0003] Many chemistries have also been developed to generate measurable / quantifiable signals, among which fluorescence-based chemistries are the chemistries of choice, most notably the Taqman Chemistry, which uses a hydrolysis probe dual-labeled with a fluorophore and a quencher (U.S. Pat. No. 5,538,848), the Scorpion primer method, which includes a dual-labeled stem structure probe and a PCR blocker element hexaethylene monomer in one of the primers (Whitcombe, et al Nat Biotech. 17: 804-807 (1999); U.S. Pat. No. 6,589,743; Solinas et al Nucleic Acids Res 29: E96, 2001), ethidium bromide, an intercalating dye that fluoresces more strongly when intercalated into double-stranded DNA than when bound to single-stranded DNA or RNA (U.S. Pat. Nos. 5,994,056; 6,171,785, and 6,814,934), the intercalating dye SYBR Green 1 [Ririe et al., Analytical Biochem. 245: 154 (1997)], molecular beacons (U.S. Pat. Nos. 5,925,517 and 6,103,476; 6,485,901; 6,355,421; and 6,593,091), LUX primers (Nazarenko et al. Nucleic Acids Res. 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2030, 2030, 2040, 2050, 2060, 2070, 2080, 2090, 2100, 2110, 2120, 2130, 2140, 2150, 2160, 2170, 2180, 2190, 2200, 2210, 2210, 2220, 2230, 2240, 2250, 2260, 2270, 2280, 2 30:e37, 2002), and linear primer pairs labeled with donor and acceptor fluorophores that generate a FRET signal (WO 03 / 102239 A2), each of which is incorporated herein by reference in its entirety.

[0004] In nucleic acid amplification, the range of amplification is very large, and a few to billions of copies of the target nucleic acid can be synthesized. However, a problem with nucleic acid amplification is the generation of non-specific amplification products along with specific amplification products. One type of non-specific amplification product that is frequently observed is the template-independent amplification artifact "primer dimer". Primer dimers are double-stranded amplification products whose length typically approximates the sum of the lengths of the two primers used in the amplification reaction. They are formed when one primer is extended on the other primer. The resulting concatenation forms an undesired template that is amplified more efficiently due to its short length and competes with the specific target amplification product. Similarly, when using two amplification primers plus a probe, a similar undesired concatenated amplification artifact "primer dimer-like" product is formed when one primer is extended on the probe, whose length typically equals the sum of the lengths of the two primers and the probe. These non-specific amplification products are more likely to form when the copy number of the target nucleic acid is low, when inhibitors exist in the sample, and when the quality of the reagents, i.e., primers and probes, is poor. The formation of non-specific amplification products is very difficult to avoid, and can be reduced but cannot be eliminated. The formation of these non-specific products reduces the specificity and sensitivity of the detection method.

[0005] Moreover, in all these signal generating chemical reactions, non-specific amplification products also generate signals together with target amplification products, affecting the sensitivity and specificity of target detection. To overcome this problem, suppressing the formation of non-specific amplification products is provided as a solution to the problem. In response, hot start amplification, specifically hot start PCR amplification, is adopted as a solution. Many hot start methods with stepwise improvements have been developed.

[0006] For example, among others, use of heat-sensitive substances such as wax to separate or sequester reagents (U.S. Pat. No. 5,411,876), thermally reversible inhibition of DNA polymerase with DNA polymerase-specific antibodies as described in U.S. Pat. Nos. 5,338,671, 5,677,152, enzymatic degradation of extension products formed prior to the start of the reaction using the methods described in U.S. Pat. No. 5,418,149, chemical modification of primer(s) at the 3' end (U.S. Pat. No. 6,001,611), reversible chemical modification of nucleic acid polymerases that reverse or dissociate at high temperatures (U.S. Pat. No. 5,773,258 and Moretti et al, Biotechniques 25: 716, 1998), which are incorporated by reference in their entireties. A recent U.S. patent application dated November 26, 2020, bearing the publication number U.S. Published Patent Application No. 2020 / 0370109, discloses a dual hot start solution that uses a combination of two or more existing hot start mechanisms.

[0007] Despite all the solutions, a perfect or very good solution to the non-specific amplification problem of nucleic acid amplification that increases the sensitivity and specificity of nucleic acid target amplification has yet to be found. There is no single process with both good sensitivity and specificity, and all existing processes have roughly the same specificity and sensitivity (approximately 95% and 70-80%, respectively). Non-specific amplification reduces sensitivity and specificity. Other than hot start PCR, there is no other reasonable solution close to hot start PCR. Hot start PCR can prevent the formation of non-specific products during reaction set-up and the initial denaturation step. Since the polymerase is then active at all temperatures in the subsequent thermal cycling or amplification cycles, the formation of primer dimer or primer dimer-like non-specific products during this cycling step cannot be avoided. Non-specific amplification products are relatively more efficient templates for amplification due to their small size. The formation of non-specific products is a stochastic phenomenon, and if non-specific products are formed in the early cycles of the amplification process, the non-specific products will outpace the amplification of the target sequence, resulting in failure of target amplification or a lower yield of target amplification products and a lower sensitivity of target detection. Therefore, there is still a need for a solution to this problem and an improved nucleic acid amplification process for detection of target nucleic acid with higher specificity and sensitivity as discussed in the present invention.As a result, this problem is a technical constraint on the development of PCR technology that has not been overcome in the past 30 years.As a result, PCR and qPCR are losing out to digital droplet PCR (dPCR), which is a new technology and requires very expensive equipment.Therefore, there is a need for a solution or improvement to solve the above problems in PCR and other amplification processes in a cost-effective manner.Therefore, there is a need for further improvements or better solutions to this problem. Summary of the Invention

[0008] Object of the invention It is therefore a basic object of the present invention to provide a nucleic acid amplification method for the detection and / or quantification of a target nucleic acid.

[0009] Another object of the present invention is to provide a method for nucleic acid amplification having higher sensitivity and higher specificity with no or little signal from primer dimers and primer dimer-like non-specific nucleic acid amplification products.

[0010] Another object of the present invention is to solve the problem with a solution to the problems of conventional PCR or quantitative PCR (qPCR) and other nucleic acid amplification methods caused by interference of primer dimers and primer dimer-like non-specific amplification products by providing a method with reduction or elimination of signals from such non-specific signals.

[0011] Another object is to provide a kit or kits for the amplification and detection and / or quantification of a target nucleic acid, the kits comprising either a labeled primer pair of the present invention separately labeled with a donor fluorophore or an acceptor fluorophore (radioactive quencher) / non-radioactive quencher, or a probe labeled with a donor fluorophore of the present invention, a primer labeled with an acceptor fluorophore (radioactive quencher) / non-radioactive quencher and an unlabeled primer or two primers labeled with an acceptor fluorophore (radioactive quencher) / non-radioactive quencher, the present invention may also include one or more amplification reagents, such as, without limitation, a nucleic acid polymerase or ligase, nucleoside triphosphates and appropriate buffers.

[0012] Yet another object is to achieve absolute quantification of target nucleic acids in a sample using donor fluorophore and acceptor fluorophore (radioactive quencher) / non-radioactive quencher labeled primers or donor fluorophore labeled probes and acceptor fluorophore (radioactive quencher) / non-radioactive quencher labeled primer(s) of the present invention.

[0013] Summary of the Invention Thus, according to a basic aspect of the present invention, there is provided a method for detection and / or quantification of a nucleic acid target by nucleic acid amplification, comprising the steps of: providing at least one target nucleic acid and at least one non-extendable or extendable oligonucleotide labeled with at least one luminescent group / moiety adapted to emit extra light upon hybridization to or incorporation into a nucleic acid molecule; providing at least one extendable oligonucleotide that is labeled with at least one converter or acceptor group / moiety adapted to convert the color of the luminescent group / moiety to a different color or to thermalise it, and that is incorporated into the amplification product; hybridizing or incorporating a luminescent group / moiety of a luminescent group-labeled oligonucleotide to a target nucleic acid amplification product, whereby the luminescent group / moiety emits excess light, the excess light emission being a measure of target amplification; A method is provided in which nucleic acid amplification is performed such that any non-specific amplification does not generate any detectable signal under controlled attenuation by selectively controlling the removal of only the excess luminescence of the luminescent group in the non-specific amplification products by converter or acceptor groups / moieties, by selectively placing the signaling moiety on the signaling oligonucleotide of a specific signaling moiety and attenuator or acceptor moiety pair, and selectively placing the attenuator or acceptor moiety on the attenuator or acceptor oligonucleotide, thereby resulting in no net signal enhancement and net signal from the non-specific products, no net signal attenuation and reduction of the target amplification signal.

[0014] Another aspect of the invention provides a method, wherein the degree of signal attenuation in the non-specific amplification products is equal or approximately equal to the degree of signal enhancement of the signaling moiety in the non-specific amplification products, resulting in no net signal enhancement and net signal from the non-specific products and no net signal attenuation and reduction of the target amplification signal.

[0015] Yet another aspect of the invention is to provide a method, wherein the distance of the signaling moiety-labeled base from the 3' end of the signaling moiety-labeled oligonucleotide plus the distance of the attenuator or acceptor moiety-labeled base from the 3' end of the receiver or acceptor moiety-labeled oligonucleotide, minus the distance of possible overlap between the 3' end of the signaling moiety-labeled oligonucleotide and the 3' end of the attenuator or acceptor moiety-labeled oligonucleotide, comprises a separation distance between the signaling moiety-labeled base and the attenuator or acceptor moiety-labeled base in the non-specific amplification products that results in a decrease in signal equal to or approximately equal to the degree of signal enhancement in the non-specific amplification products.

[0016] Yet another embodiment of the invention provides a method, wherein the number of bases separating the signaling moiety-labeled base from the 3'-end of the signaling moiety-labeled oligonucleotide plus the number of bases separating the attenuator or acceptor moiety-labeled base from the 3'-end of the attenuator or acceptor moiety-labeled oligonucleotide, minus the number of possible base overlaps between the 3'-end of the signaling moiety-labeled oligonucleotide and the 3'-end of the attenuator or acceptor moiety-labeled oligonucleotide, constitutes the number of bases separating the signaling moiety-labeled base and the attenuator or acceptor moiety-labeled base in the non-specific amplification products that results in a decrease in signal equal to or approximately equal to the degree of signal enhancement in the non-specific amplification products.

[0017] Yet another embodiment of the invention provides a method, wherein the distance of the signaling moiety-labeled base from the 3' end of the signaling moiety-labeled oligonucleotide plus the distance of the attenuator or acceptor moiety-labeled base from the 3' end of the receiver or acceptor moiety-labeled oligonucleotide, minus the distance of possible overlap between the 3' end of the signaling moiety-labeled oligonucleotide and the 3' end of the attenuator or acceptor moiety-labeled oligonucleotide, constitutes a separation distance between the signaling moiety-labeled base and the attenuator or acceptor moiety-labeled base in the non-specific amplification products that results in a decrease in signal equal or approximately equal to the degree of signal enhancement in the non-specific amplification products.

[0018] Another aspect of the invention provides a method, wherein the degree of signal enhancement and corresponding attenuation is selectively controlled such that (i) for about 1-100% signal enhancement, the corresponding attenuation of signal for balanced non-substantial signal from non-specific amplification products is maintained at about 1-50%, (ii) for about 20-80% signal enhancement, the corresponding attenuation of signal for balanced non-substantial signal from non-specific amplification products is maintained at about 17-45%, and (iii) for about 30-70% signal enhancement, the corresponding attenuation of signal for balanced non-substantial signal from non-specific amplification products is selectively controlled to be maintained at about 23-40%.

[0019] Yet another aspect of the invention provides a method in which the oligonucleotide labeled with a signaling moiety used is a probe for monitoring nucleic acid amplification that hybridizes to one strand of a target amplification product or target, and the oligonucleotide labeled with an attenuator or acceptor moiety used is one of the nucleic acid amplification primers that anneals either to the same strand to which the probe hybridizes or to the other strand of the target amplification product or target, and both amplification primers used are selectively labeled with an attenuator or acceptor moiety.

[0020] Another embodiment of the invention provides a method, wherein the signaling moiety is located either on the 3' end or at a base up to 30 bases away from the 3' end, excluding the 5' end, of the oligonucleotide labeled with the signaling moiety, and the attenuator or acceptor moiety is located on any base at least 2 bases away from the 3' end or any base up to 30 bases away from the 3' end of the oligonucleotide labeled with the attenuator or acceptor moiety.

[0021] Yet another embodiment of the invention provides a method, wherein the signaling moiety-labeled oligonucleotide and the attenuator or acceptor moiety-labeled oligonucleotide used are two nucleic acid amplification primers that anneal separately to the two strands of a target amplification product or target nucleic acid and are extended by a polymerase(s).

[0022] A further embodiment of the invention provides a method, wherein the signaling moiety is located on any base at least 2 nucleotides away from the 3' end or any base up to 30 bases away from the 3' end, excluding the 5' end, of the oligonucleotide labeled with the signaling moiety, and the attenuator or acceptor moiety is located on any base at least 2 nucleotides away from the 3' end or any base up to 30 bases away from the 3' end of the oligonucleotide labeled with the attenuator or acceptor moiety.

[0023] Yet another aspect of the invention provides a method, wherein the signaling moiety used is a donor fluorophore and the attenuator or acceptor moiety used is an acceptor fluorophore or a non-radioactive acceptor or quencher moiety that accepts energy but does not emit any energy or electromagnetic radiation, the signal is a fluorescent signal, and the signal attenuation is quenching of the fluorescent signal of the donor fluorophore, and the donor fluorophore and acceptor moiety are an energy transfer pair.

[0024] Another embodiment of the invention provides a method, wherein the separation distance between the signaling moiety labeled base and the attenuator or acceptor moiety labeled base in the non-specific amplification product is selected such that the degree of signal enhancement in the non-specific amplification product is equal or approximately equal to the signal decay between 2R0 and R0 distances, respectively, where 3.7 Angstroms is the inter-base distance and R0 is the Förster radius of the signaling moiety and acceptor moiety pair, more specifically the donor fluorophore and acceptor fluorophore or non-radioactive acceptor moiety pair (Förster radius R0 values ​​range 22 Å to 75 Å).

[0025] A further aspect of the invention provides a method, wherein the separation distance between the signaling moiety-labeled base and the attenuator or acceptor moiety-labeled base in the non-specific amplification products is selected to result in an attenuation of signal equal to or approximately equal to the degree of signal enhancement in the non-specific amplification products, optionally between 1.3025R0 distance and 1.034R0 distance, respectively, where 3.7 Angstroms is the inter-base distance and R0 is the Förster radius of the signaling moiety and acceptor moiety pair, more specifically the donor fluorophore and acceptor fluorophore or non-radioactive acceptor moiety pair (Förster radius R0 values ​​range 22 Å to 75 Å).

[0026] Yet another embodiment of the present invention provides such a method, wherein the separation distance between the signaling moiety labeled base and the attenuator or acceptor moiety labeled base in the non-specific amplification products used results in an attenuation of the signal equal or approximately equal to the degree of signal enhancement in the non-specific amplification products, which is between 1.224R0 distance and 1.0699R0 distance, respectively, where 3.7 Angstroms is the inter-base distance and R0 is the Förster radius of the signaling moiety and acceptor moiety pair, i.e., donor fluorophore and acceptor fluorophore or non-radioactive acceptor moiety pair (Förster radius R0 value range 22 Å to 75 Å).

[0027] Yet another embodiment of the invention provides a method, wherein the number of bases separating the signaling moiety labeled base and the attenuator or acceptor moiety labeled base in the non-specific amplification product is selected to provide a signal attenuation of about (1.3025R0 / 3.7) bases to (1.034R0 / 3.7) bases that is equal or approximately equal to the degree of signal enhancement in the non-specific amplification product corresponding to a distance of 1.3025(R0) to 1.034(R0), where 3.7 Angstroms is the inter-base distance and R0 is the Förster radius of the donor fluorophore moiety and acceptor moiety energy transfer pair expressed in Angstroms, corresponding to about 6.1 to 26.4 bases for the full range of fluorophore moiety and acceptor moiety energy transfer pairs (Förster radius R0 values ​​range 22 Å to 75 Å).

[0028] Yet another embodiment of the invention provides that the number of bases separating the signaling moiety labeled base and the attenuator or acceptor moiety labeled base in the non-specific amplification product is selected to provide a signal attenuation equal or approximately equal to the degree of signal enhancement in the non-specific amplification product corresponding to a distance of 1.3025(R0) to 1.034(R0) of about (1.3025R0 / 3.7) bases to (1.034R0 / 3.7) bases, where 3.7 Angstroms is the inter-base distance and R0 is the Förster radius of the donor fluorophore moiety and acceptor moiety energy transfer pair expressed in Angstroms, corresponding to about 6.1 to 26.4 bases for the full range of fluorophore moiety and acceptor moiety energy transfer pairs (Förster radius R0 values ​​range 22 Å to 75 Å).

[0029] Another aspect of the invention provides a method, wherein the number of bases separating the signaling moiety labeled base and the attenuator or acceptor moiety labeled base in the non-specific amplification product is selected to provide a signal attenuation of about (1.224R0 / 3.7) bases to (1.0699R0 / 3.7) bases that is equal or approximately equal to the degree of signal enhancement in the non-specific amplification product corresponding to a distance of 1.224(R0) to 1.0699(R0), where 3.7 Angstroms is the base-to-base distance and R0 is the Förster radius of the donor fluorophore moiety and acceptor moiety energy transfer pair expressed in Angstroms, corresponding to about 6.4 to 24.8 bases for the full range of fluorophore moiety and acceptor moiety energy transfer pairs (Förster radius R0 values ​​range 22 Å to 75 Å).

[0030] Yet another aspect of the invention provides a method wherein the signaling moiety-labeled oligonucleotide and the attenuator or acceptor moiety-labeled oligonucleotide used are linear.

[0031] Yet another embodiment of the invention provides a method in which an additional labeling moiety, such as biotin, is placed at the 3' and / or 5' ends of the labeled probe.

[0032] Another aspect of the invention provides a method, wherein the probe labeled with a signaling moiety or donor fluorophore as defined in claim 6 or the primer labeled with a signaling moiety or donor fluorophore moiety as defined in claim 8 further comprises an acceptor fluorophore or quencher, or a sequence of 5-8 bases at the 5' end that is sufficiently complementary to the sequence of the labeled primer or probe adjacent to the fluorophore labeled base to form a stem structure, with or without a quencher, and with or without an intervening spacer between the fluorophore labeled primer or probe and the 5-8 base sequence.

[0033] Yet another embodiment of the invention provides a method, wherein the number of bases separating the base labeled with the signaling moiety or donor fluorophore moiety and the 3' end of the donor fluorophore labeled primer or probe, plus the number of bases separating the base labeled with the acceptor or quencher moiety and the 3' end of the primer labeled with the acceptor or quencher moiety, is between 6 and 40 bases for the full range of energy transfer pairs between the donor fluorophore moiety and the acceptor or quencher moiety.

[0034] A further embodiment of the invention provides a method, wherein the number of bases separating the signaling moiety or fluorophore labeled base and the 3' end of the fluorophore labeled primer or probe, plus the number of bases separating the acceptor or quencher moiety labeled base and the 3' end of the primer labeled with the acceptor or quencher moiety, is between 10 and 30 bases for the full range of donor fluorophore and acceptor or quencher moiety energy transfer pairs.

[0035] Yet a further embodiment of the invention provides a method, wherein two or more signaling moieties or donor fluorophores are disposed on a signaling moiety-labeled probe or primer, and two or more acceptor moieties or quenchers are disposed on the acceptor moiety-labeled primer(s).

[0036] Another aspect of the invention provides a method in which a first primer pair amplifies a first segment of a target nucleic acid and a second primer pair amplifies a second segment of the first segment in a nested nucleic acid amplification, the second primer pair being a libra primer pair labeled with a signaling moiety or donor fluorophore moiety and labeled with an acceptor or quencher moiety, the second primer pair comprising two nucleic acid amplification primers selected from a second primer pair labeled with an acceptor or quencher moiety.

[0037] A further aspect of the invention provides a method, wherein the primer of the first primer pair is provided further suitably labelled with an acceptor or quencher moiety.

[0038] Yet another aspect of the invention provides a method for semi-nested nucleic acid amplification in which a first primer and a second primer together amplify a first segment of a target nucleic acid and a third primer combines with the first primer to amplify a second segment of the first segment, wherein the first and third primers are a libra primer pair labeled with a signaling moiety or fluorophore moiety and labeled with an acceptor or quencher moiety, and wherein the second primer is also provided further labeled with an acceptor fluorophore or quencher.

[0039] A further aspect of the invention provides a method, wherein in a nested nucleic acid amplification, a first primer pair amplifies a first segment of a target nucleic acid and a second primer pair amplifies a second segment of the first segment, and a probe is used which hybridizes to the second segment, the probe and second primer pair being, respectively, a probe labeled with a signaling moiety or donor fluorophore moiety and a primer(s) labeled with an acceptor or quencher moiety; further provided that the first primer pair is also suitably labeled with an acceptor or quencher moiety.

[0040] Yet another aspect of the invention provides a method, wherein in a semi-nested nucleic acid amplification, a first primer and a second primer together amplify a first segment of a target nucleic acid and a third primer together with the first primer amplify a second segment of the first segment and a probe hybridizing to the second segment is used, wherein the probe as well as the first primer and / or the third primer are a probe labeled with a signaling moiety or a donor fluorophore moiety and a primer(s) labeled with an acceptor or quencher moiety, respectively.

[0041] A further aspect of the present invention provides a method, wherein a semi-synthetic target nucleic acid sequence is generated by adding a first non-target sequence or a first non-target sequence and a second non-target sequence to one or both ends of a target nucleic acid sequence by any method known in the art (there are many methods and the skilled artisan will be aware of these methods), and amplification of the target nucleic acid is driven either by one target specific primer and a first non-target sequence specific primer or by a first and a second non-target sequence specific primer, respectively, wherein the amplification primers are primers labeled with a signaling moiety or a donor fluorophore moiety and a primer labeled with an acceptor or quencher moiety according to claim 8 or the present invention, optionally comprising SEQ ID NOs: 21 and 22, and wherein the first non-target sequence and the second non-target sequence are added to the target sequence by tail PCR, wherein the first non-target sequence and the second non-target sequence are added to the 5' ends of the two PCR primers.

[0042] A further aspect of the invention provides methods further comprising providing a probe, wherein the probe and primer are the labeled primer(s) and probe of the invention, the probe being labeled with a signaling moiety or donor fluorophore.

[0043] Yet another aspect of the invention provides a method, wherein a signaling moiety or donor fluorophore labeled primer or probe, selectively comprising SEQ ID NO:22, either comprises a quencher / acceptor fluorophore at the 5' end or has an additional 5-8 base sequence that hybridizes to a base adjacent to the donor fluorophore labeled base of the donor fluorophore labeled primer or probe to form a hairpin, with or without a quencher at the 5' end and with or without a linker connected.

[0044] A further aspect of the invention relates to a method for detecting a signaling or fluorophore moiety comprising the steps of: (a) detecting a signaling or fluorophore moiety that is capable of detecting a signaling or fluorophore moiety in a target amplification product; (b) detecting a signaling or fluorophore moiety that is capable of detecting a signaling or fluorophore moiety in a target amplification product; and (c) detecting a signaling or fluorophore moiety that is capable of detecting a signaling or fluorophore moiety in a target amplification product; and (d) detecting a signaling or fluorophore moiety that is capable of detecting a signaling or fluorophore moiety in a target amplification product; In one embodiment, the separation of the acceptor or quencher moiety from the acceptor or quencher moiety is 0.8327R0 to 0.7284R0, which corresponds to 0.8327 (R0 / 3.7) bases to 0.7284 (R0 / 3.7) bases, where 3.7 Angstroms is the base-to-base distance and R0 is the Förster radius of the energy transfer pair between the fluorophore moiety and the acceptor moiety in Angstroms, which corresponds to approximately 4 to 16 bases for the full range of energy transfer pairs between the fluorophore moiety and the acceptor moiety (Förster radius R0 values ​​range 22 Å to 75 Å).

[0045] Another embodiment of the invention provides a method, wherein the number of bases separating the base labeled with the signaling moiety or fluorophore and the 3' end of the fluorophore labeled primer or probe, plus the number of bases separating the base labeled with the acceptor or quencher moiety and the 3' end of the primer labeled with the acceptor or quencher moiety, is between 4 and 16 bases.

[0046] A further aspect of the invention relates to a method for nucleic acid amplification using a first primer labeled with a first signaling moiety or a first fluorophore moiety, a second primer labeled with an acceptor or quencher moiety, and a probe labeled with a second signaling moiety or a second fluorophore moiety, wherein the first signaling moiety or the first fluorophore moiety generates a first signal upon target amplification and the second signaling moiety or the second fluorophore moiety of the probe generates a second fluorescent signal upon hybridization of the probe to a target amplification product, and wherein the first primer labeled with a first signaling moiety or a first fluorophore and a probe labeled with an acceptor or quencher moiety generate a second fluorescent signal upon hybridization of the probe to a target amplification product. and wherein the second primer labeled with a signaling moiety or fluorophore moiety of the invention is a pair of a primer labeled with an acceptor or quencher moiety acceptor or quencher moiety, and the first primer labeled with a first signaling moiety or fluorophore moiety, the second primer labeled with an acceptor or quencher moiety acceptor or quencher moiety, and the probe labeled with a second signaling moiety or second fluorophore moiety are labeled primers and probes of the invention, and the first signaling moiety or first fluorophore moiety is an acceptor fluorophore or quencher for the second signaling moiety or second fluorophore moiety or vice versa.

[0047] A further aspect of the invention provides a method in which a primer labeled with a donor fluorophore and a primer labeled with an acceptor fluorophore are used to amplify a target nucleic acid sequence, where the donor fluorophore and acceptor fluorophore are positioned at least 2 nucleotides away from the 3' end of the primer and the donor and acceptor fluorophores are separated by 5-30 bases in the target amplification product, such that a FRET signal, more specifically emission from the acceptor fluorophore upon excitation of the donor fluorophore, is generated upon target amplification.

[0048] Another aspect of the invention provides a method, in which one of two amplification primers is provided labeled with an acceptor fluorophore, and a donor fluorophore-labeled probe that hybridizes to a target sequence is used instead for target nucleic acid amplification, where the acceptor fluorophore is positioned at least two nucleotides away from the 3' end of the labeled primer, the donor fluorophore-labeled probe is labeled with the donor fluorophore at its 3' end or on a base away from the 3' end excluding the 5' terminal base, and the donor and acceptor fluorophores are separated by 5 bases or more when the probe hybridizes to the target amplification product, such that a FRET signal, more specifically emission from the acceptor fluorophore upon excitation of the donor fluorophore, is generated upon target amplification.

[0049] A still further embodiment of the invention provides a method in which a probe is provided that is labeled with an acceptor fluorophore and a labeled primer is provided that is alternatively labeled with a donor fluorophore.

[0050] Yet another embodiment of the invention provides a method in which the donor and acceptor fluorophores in the target amplification are separated by 10-20 bases.

[0051] A further embodiment of the invention provides a method wherein the donor and acceptor fluorophores are separated in the target amplification product by 14-20 bases.

[0052] Another embodiment of the invention provides a method, wherein the distance or number of bases separating the donor fluorophore-labeled base from the 3' end of the donor fluorophore-labeled primer or probe plus the distance or number of bases separating the acceptor fluorophore-labeled base from the 3' end of the acceptor fluorophore-labeled primer or probe minus the overlap distance or number of overlapping bases between the 3' end of the donor fluorophore-labeled primer or probe and the 3' end of the acceptor fluorophore-labeled primer or probe is equal to the distance or equivalent base separation, respectively, for static quenching between the donor and acceptor fluorophores for non-substantial signal from non-specific amplification products.

[0053] Further embodiments of the invention provide methods where the static quenching distance is plus or minus 3 or plus or minus 2 or plus or minus 1 or 0, or where the labeled bases are positioned opposite each other.

[0054] Yet another aspect of the invention is a method for the preparation of a nucleic acid sequence comprising the steps of: using a first oligonucleotide primer carrying a target specific sequence or a polythymidine sequence or a polythymidine sequence with one or more non-thymine bases at its 3' end and a promoter sequence at its 5' end; or using a double-stranded adapter having a 5' overhang of a number of bases for attaching the adapter to the target sequence and a promoter sequence at the 5' end of the adapter; or using a first oligonucleotide primer having a stretch of target sequence and a promoter sequence at its 5' end; The linear amplification of the target sequence is carried out by sequential polymerase extension using a second target-specific primer and a first oligonucleotide primer carrying a promoter sequence using a DNA polymerase or reverse transcriptase and deoxynucleoside triphosphates in conjunction with RNA transcription using an RNA polymerase and ribonucleoside triphosphates, where the first primer carrying a promoter sequence and the second target-specific primer are labeled with a donor fluorophore and a quencher / acceptor fluorophore, as described in any one of claims 8 to 10 and 17 of the present invention, the signaling moiety-labeled oligonucleotide and the attenuator or acceptor moiety-labeled oligonucleotide used are two nucleic acid amplification primers that anneal separately to the two strands of the target amplification product or the target nucleic acid and are extended by the polymerase(s) (claim 8); the signaling moiety is a signaling moiety-labeled oligonucleotide. the attenuator or acceptor moiety is located on any base at least 2 nucleotides away from the 3' end or any base up to 30 bases away from the 3' end, excluding the 5' end, of the nucleotide, and the attenuator or acceptor moiety is located on any base at least 2 nucleotides away from the 3' end or any base up to 30 bases away from the 3' end of the oligonucleotide labeled with the attenuator or acceptor moiety (claim 9); the signaling moiety used is a donor fluorophore, the attenuator or acceptor moiety used is an acceptor fluorophore or a non-radioactive acceptor or quencher moiety that accepts energy but does not emit any energy or electromagnetic radiation (claim 10), and the signaling moiety labeled oligonucleotide and the attenuator or acceptor moiety labeled oligonucleotide used are linear (claim 17).

[0055] Another aspect of the invention is a donor fluorophore-labeled probe and acceptor fluorophore or quencher-labeled primer(s) of the invention, wherein either or both of the first primer and the second primer carrying a promoter sequence are labeled with a quencher or acceptor fluorophore, and a donor fluorophore-labeled target specific probe is used, the labeled probe and labeled primer(s) having a signaling moiety or donor fluorophore-labeled probe according to claim 6; wherein the signaling moiety is located either on the 3' end or at a position up to 30 bases away from the 3' end, excluding the 5' end, of the oligonucleotide labeled with the signaling moiety, and wherein an attenuator is used. or the acceptor moiety is located on any base at least 2 bases away from the 3' end of the oligonucleotide labeled with the attenuator or acceptor moiety or on any base up to 30 bases away from the 3' end (claim 7); the signaling moiety used is a donor fluorophore and the attenuator or acceptor moiety used is an acceptor fluorophore or a non-radioactive acceptor or quencher moiety that accepts energy but does not emit any energy or electromagnetic radiation (claim 10); the signaling moiety labeled oligonucleotide and the attenuator or acceptor moiety labeled oligonucleotide used are linear (claim 17).

[0056] A further embodiment of the invention provides a method in which a quencher is also attached to the 5' end of a primer or probe labeled with a donor fluorophore.

[0057] A still further aspect of the present invention provides a method, wherein the nucleic acid amplification reaction comprises a polymerase chain reaction (PCR), the polymerase chain reaction (PCR) being, but not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), allele or allele specific polymerase chain reaction (allele PCR), allele RT-PCR, tail PCR, droplet PCR, emulsion PCR, digital PCR, asymmetric PCR (wherein one of two primers is used at a very low concentration compared to the other primer, generating a single stranded target amplification product), nested PCR, semi-nested PCR, methylation status PCR, in-situ PCR, and wherein the size of the target amplification product is 35-400 base pairs, preferably 50-250 base pairs.

[0058] A further aspect of the present invention provides a method, wherein the nucleic acid amplification reaction comprises an isothermal nucleic acid amplification reaction including, but not limited to, loop-mediated isothermal nucleic acid amplification reaction (LAMP), recombinase polymerase amplification reaction (RPA), helicase polymerase amplification reaction (HPA), nucleic acid sequence-based amplification (NASBA), wherein a loop primer used in LAMP promotes DNA strand separation, and a strand separation enzyme such as recombinase, helicase, gyrase, topoisomerase, etc. is used in RPA and HPA for denaturation or strand separation in conjunction with single-stranded binding (SSB) protein, and the size of the target amplification product is 75-1000 base pairs, preferably 100-250 base pairs.

[0059] A further embodiment of the invention is that absolute quantification of a target nucleic acid is performed using a labeled primer-probe pair, in which the oligonucleotide labeled with a signaling moiety used is a probe for monitoring nucleic acid amplification that hybridizes to the target amplification product or to one strand of the target, and the oligonucleotide labeled with an attenuator or acceptor moiety used is one of the nucleic acid amplification primers (as described in claim 6); the signaling moiety is located either on the 3' end or at a base up to 30 bases away from the 3' end, excluding the 5' end, of the oligonucleotide labeled with the signaling moiety, and the attenuator or acceptor moiety is located on any base at least 2 bases away from the 3' end or at any base up to 30 bases away from the 3' end of the oligonucleotide labeled with the attenuator or acceptor moiety (as described in claim 7); A method, wherein the signaling moiety used is a donor fluorophore and the attenuator or acceptor moiety used is an acceptor fluorophore or a non-radioactive acceptor or quencher moiety that accepts energy but does not emit any energy or electromagnetic radiation, the signal is a fluorescent signal and the signal attenuation is the quenching of the fluorescent signal of the donor fluorophore, and the donor fluorophore and the acceptor moiety are an energy transfer pair (claim 10); and labeled primer pairs selectively comprising SEQ ID NOs: 21 and 22 and kit(s) therefor, the kit(s) comprising at least one donor fluorophore-labeled oligonucleotide probe and an acceptor fluorophore or quencher-labeled oligonucleotide primer(s); or a kit(s) comprising in one or more containers two nucleic acid amplification primers, oligonucleotide primers, at least a donor fluorophore labeled oligonucleotide primer and an acceptor fluorophore or quencher labeled oligonucleotide primer, wherein the signaling moiety labeled oligonucleotide and the attenuator or acceptor moiety labeled oligonucleotide used are separately annealed to the two strands of a target amplification product or target nucleic acid and extended by a polymerase(s); The kit(s) may further contain a reaction buffer, multiple deoxynucleoside triphosphates, polymerase enzyme(s), a positive control template and corresponding labeled primer pairs for amplification, as well as additional components.

[0060] Another aspect of the invention provides a method, wherein the oligonucleotide is optionally 10-50 bases in length, preferably 15-35 bases in length, more preferably 20-30 bases in length, is complementary to a target sequence, has the ability to hybridize or anneal on the target, has not lost the ability to prime nucleic acid synthesis on the target, and possesses one or more modified bases, or modified sugar moiety(s), or one or more base analogs.

[0061] A further aspect of the present invention provides a method, wherein a positive control template and a labeled primer pair or a labeled primer and probe pair according to any one of claims 6 to 17, 28, 29, 34, 35, 46 specific for the positive control template are further provided during the amplification reaction.

[0062] Yet another aspect of the present invention provides a method, wherein multiple pairs of donor fluorophore-labelled primers and acceptor fluorophore / quencher-labelled primers or multiple pairs of donor fluorophore-labelled probes and acceptor fluorophore / quencher-labelled primers according to any one of claims 6 to 17, 28 to 29, 34 to 35 and 46 of the present invention are used in a multiplex reaction for simultaneous detection and / or quantification of multiple target sequences.

[0063] A further aspect of the invention provides a method, wherein one or more or a multiplicity of donor fluorophore labeled primers or donor fluorophore labeled probes are attached or covalently linked or tethered via a multi-carbon atom organic linker or polyethylene glycol or hybrid linker or polythymidine oligonucleotide with or without additional organic linkers of sufficient length to a glass or glass wafer, or plastics such as polystyrene, polyethylene, polypropylene, or transparent or semi-transparent solid surface such as dextran, cellulose, nylon, etc., and a microfluidic channel is used for detection of single or multiple or multiple nucleic acid targets in a single amplification reaction.

[0064] Another aspect of the invention provides a method, wherein the nucleic acid amplification reaction comprises any known nucleic acid amplification reaction, preferably the polymerase chain reaction, and comprises at least the steps of adding to the sample an effective amount of amplification primers or an effective amount of a labeled primer and a labeled probe or a labeled primer, as well as at least one polymerase enzyme, a reaction buffer, and deoxynucleoside triphosphates; cycling the sample during at least a denaturation step, an annealing step, an extension step or a single combined annealing and extension step, or in an isothermal reaction step; exciting the reaction mixture with donor fluorophore excitation light or radiation; and measuring the emission of the donor fluorophore or the acceptor fluorophore.

[0065] Further aspects of the invention include double-stranded DNA intercalating dyes, including but not limited to the intercalating dyes ethidium bromide, SYBR Green 1™, Picogreen™, YOPRO 1™, SYTO 9™, acridine orange, asymmetric cyanine dyes, in which a donor fluorophore, an acceptor fluorophore and a quencher enhance fluorescence upon intercalation into double-stranded DNA, and the dyes fluorescein, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 6-FAM(azido), 2'7'-dimethoxy-4'5-6-carboxyfluorescein (JOE), 5-(4,6-dichlorotriazin-2yl)aminofluorescein (DTAF), fluorescein isothiocyanate, HEX (hexachlorofluorescein), TET (tetrachlorofluorescein), VIC (Victoria Blue), MAX, which has a spectral profile nearly identical to VIC. VIC, SUN™, which is the equivalent of A VIC™ (ThermoFisher Scientific), TYE™ 563, TYE 665, TYE 705, NED, fluorescamine, pyrene, pyrene butyrate, succimidyl 1 pyrene butyrate, rhodamine (Rhod), rhodamine 123, rhodamine B, sulforhodamine, 6-carboxyrhodamine (R6G), 6-carboxy-X rhodamine (ROX), sulforhodamine b, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), Texas Red™-X, SIMA dye™, Texas Red™-X, TEX615 N',N',N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), TAMRA™ (azide), Rhodamine Green(TM)-X, RhodamineRed™, tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC), terbium chelate, europium chelate, quantum dots, graphene quantum dots, 5-(2'-aminoethyl)aminonaphthylamide-3,5 disulfonate (Lucifer Yellow vs), 7-amino-4-methylcoumarin (amc, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151), Cyanine 2, Cyanine 3, Cyanine 3.5, Cyanine 5, Cyanine 5. Cyanine dyes (sulfonated or non-sulfonated), including but not limited to cyanine 5, cyanine 7, [5-dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), DABSYL, 4-dimethylaminophenylazophenyl)-4'-isothiocyanate (DABITC), IAEDANS (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalenesulfonic acid), EDANS, QST 7, QSY9, QSY 21, QSY 35 (QSY dyes are diaryl-rhodamine derivatives), BIODIPY dyes, including but not limited to BIODIPY FL, Alexa fluor dyes, including but not limited to Alexa fluor 350, 488, 546, 555, 568, 594, 647, 660, 750 dyes, ATTO dyes, including but not limited to ATTO™ 488 (NHS ester), ATTO™ 532, ATTO™ 550, ATTO™ 565, ATTO™ 590, ATTO™ 633, ATTO™ 647N, ATTO™ Rho101, ATTO™ 647N (NHS ester), Yakima Yellow, MGB dyes (major or minor groove binding dyes), LI-COR's IRDyes®, IRDye® 700, IRDye® 800, IRDye® 800CW, Lightcycler® 640, Dy750, non-radioactive quenchers (Nanogold Particle, Blackhole Quencher 0, Blackhole Quencher 1, Blackhole Quencher 2, Blackhole Quencher 3, EclipseIn one embodiment, the quencher is selected from the group including, but not limited to, IDT quenchers Iowa Black™ RQ, Iowa Black FQ, ZEN™, TAO and / or ZEN®, nanoparticle quenchers, single stranded binding proteins.

[0066] Yet another aspect of the invention provides a method, wherein the polymerase enzyme(s) used in the nucleic acid amplification reaction is an enzyme that is a natural or modified or chimeric DNA polymerase with or without strand displacement activity or template independent primer activity or base extension activity, exonuclease activity, or a reverse transcriptase, or a polymerase with both reverse transcriptase activity and DNA polymerase activity, or an RNA polymerase, or an RNA polymerase and a DNA polymerase, wherein the polymerase can be a thermostable, enzyme active at ambient or below ambient temperature, a hot start polymerase (which becomes active after the polymerase is heated to an elevated temperature, preferably the primer annealing temperature).

[0067] A further aspect of the invention is a method used to detect a nucleic acid (methylated or unmethylated) or non-nucleic acid target, where a first binding moiety having a very high affinity for the nucleic acid or non-nucleic acid target is used to capture the nucleic acid or non-nucleic acid target, and a second binding moiety, which may be the same first binding moiety or may be a different binding moiety having a very high affinity for the nucleic acid or non-nucleic acid target, is used to bind to the captured nucleic acid or non-nucleic acid target, or a third binding moiety is used which binds to the second binding moiety with very high affinity, where the second binding moiety or third binding moiety is provided with a synthetic or natural nucleic acid target molecule appended thereto, and the bound second binding moiety or third binding moiety is provided with a nucleic acid appended unbound second binding moiety or third binding moiety. and after washing off the nucleic acid moiety, detected and quantified by nucleic acid amplification using a primer pair or a probe and primer pair labeled with a donor fluorophore and an acceptor fluorophore / quencher, optionally comprising any one of claims 6-17, 28-29, 34-35, 46, 54; The method provides a method in which the binding moieties are preferably selected from the binding pairs antigen-antibody, protein-antiprotein antibody, antibody-antibody, antibody-anti-IgG antibody, primary antibody-secondary antibody, protein A-antibody, protein G-antibody, biotin-avidin, biotin-streptavidin, lectin-sugar, nucleic acid-nucleic acid, protein-nucleic acid, peptide nucleic acid, aptamer-aptamer, aptamer-nucleic acid, aptamer-protein, hapten-antihapten antibody, and the hapten is a small molecule, including but not limited to fluorescent dyes, bromo-d-UTP, aflatoxins and other mycotoxins, peptides, sugars.

[0068] Another aspect of the present invention provides a method for detection and / or quantification of multiple m-RNAs or c-DNAs, comprising providing a first amplification primer specific to each m-RNA or c-DNA (DNA complementary to an mRNA), and providing as a second amplification primer a common primer (common to all m-RNAs or c-DNAs in a sample) selected from a sequence added to the m-RNA or c-DNA, and further providing a probe specific to each m-RNA or c-DNA, wherein the first amplification primer, the second common amplification primer and the probe are primers and probes selectively including 6-17, 28-29, 34-35 and 46.

[0069] A further aspect of the invention provides a method in which a donor fluorophore-labeled probe is provided attached or linked to one of the primers via a non-nucleotidic organic linker, hexamethylene, hexapolyethylene glycol, or chimeras or longer lengths thereof, and the probe hybridizes to a nascent nucleic strand generated through extension of the linked primer.

[0070] In another aspect, the present invention provides a kit for performing a nucleic acid amplification reaction, comprising: (a) an oligonucleotide primer labeled with at least a donor fluorophore and an oligonucleotide primer labeled with an acceptor fluorophore or a quencher according to any one of claims 8 to 17, 28, 34, 41, 43, 48 to 50, and 54 to 56; (b) at least a donor fluorophore-labeled oligonucleotide primer(s) and an acceptor fluorophore or quencher-labeled oligonucleotide primer(s), optionally including any one of claims 28, 43, 48 and 50, and SEQ ID NOs: 21-22; (c) at least an oligonucleotide probe(s) labeled with a donor fluorophore and an oligonucleotide primer(s) labeled with an acceptor fluorophore or a quencher, optionally comprising any one of claims 19, 23, 24-25, 29, 35, 48-50; (d) a first primer carrying a promoter sequence and a target-specific second primer, labeled with at least a donor fluorophore and an acceptor fluorophore / quencher, optionally comprising one of claims 41; (e) at least a positive control template and a primer pair or a primer and a probe labeled with a donor fluorophore and an acceptor fluorophore / quencher specific for the positive control template, optionally comprising any one of claims 6-17, 24-35, 41-43, 48-50, 54-56; (f) A labeled primer pair, wherein a donor fluorophore-labeled probe is provided attached or linked to an acceptor fluorophore or quencher-labeled primer via a non-nucleotidic organic linker, hexamethylene, hexapolyethylene glycol, or chimeras or longer lengths thereof, and the probe hybridizes to a nascent nucleic acid strand generated through extension of the linked primer. The present invention provides a kit comprising, in one or more containers, at least an oligonucleotide probe labeled with a donor fluorophore and an oligonucleotide primer(s) labeled with an acceptor fluorophore or a quencher, optionally comprising any one of claims 6, 10-17, 26-33, 35, 42, 49-50, and 54-56.

[0071] In another aspect, the invention provides a kit comprising a reaction buffer, a plurality of deoxynucleoside triphosphates, a polymerase enzyme or enzymes, a positive control template, and labeled primer pairs for each of the positive templates.

[0072] Yet another aspect of the invention provides that the target nucleic acid is a purified or partially purified or unpurified nucleic acid selected from natural, synthetic or semi-synthetic single-stranded or double-stranded DNA or RNA, single-stranded or double-stranded c-DNA, genomic DNA, methylated DNA, mitochondrial DNA, exosomal DNA, plasmid DNA, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (m-RNA), small RNA (including but not limited to microRNA, sRNA, stRNA, snoRNA, ncRNA), stem cell-derived DNA including very small embryonic-like stem cells, viral DNA or RNA, or cancer cell DNA (from any source including but not limited to body fluids, biopsy samples, tumors, pus, saliva, feces, cancer stem cells), and single-stranded or double-stranded synthetic or semi-synthetic DNA or RNA generated by adding one or two non-targeting synthetic sequences to the ends of the target nucleic acid. Furthermore, the target nucleic acid need not constitute an entire nucleic acid molecule, and the presence or absence or mutations (single base changes or deletions or insertions of a few bases or longer sequences) of genomic sequences of infectious agents, or genomic sequences of human bacteria, yeast, fungi, plants, animals, humans, parasites and their viruses and any other organisms, living or dead, are correlated with the presence of a disease or disorder, or susceptibility to infection or disease or disorder, or suitability for disease treatment, prenatal diagnosis, genetic traits, genotypes, allele types, SNP detection, cell types, tissue types, species or strain types, cancer types or cancer subtypes, cancer detection, disease typing or subtyping, expressed genes.

[0073] A thorough understanding of the present invention's implementation can be obtained by reference to the following figures. [Brief description of the drawings]

[0074] [Figure 1]Figure 1 shows a schematic diagram of PCR amplification using primers labeled with an internal donor fluorophore and an internal acceptor fluorophore / quencher. The donor fluorophore is excited with donor-specific excitation light, and the emission of the donor fluorophore is measured. Fluorescence of the donor fluorophore is enhanced upon target amplification, and there is simultaneous enhancement and quenching of fluorescence of the donor fluorophore upon formation of nonspecific primer dimers. If the enhancement and quenching of fluorescence of the donor fluorophore are balanced, there is no net fluorescence or net quenching of the donor fluorophore, resulting in no signal from the donor fluorophore and therefore no signal from the primer dimers. Target DNA (1) is separated by denaturation (2); primer and polymerase (4); primer extension (5); target amplification product (6); primer dimer formation and primer dimers (7, 8). [Diagram 2] Figure 2 shows a schematic diagram of PCR amplification using primers labeled with an internal acceptor fluorophore / quencher and a probe labeled with a donor fluorophore. The donor fluorophore is excited with the donor fluorophore-specific excitation light, and the emission of the donor fluorophore is measured. Fluorescence of the donor fluorophore is enhanced upon target amplification and hybridization of the probe, and fluorescence enhancement and quenching of the donor fluorophore occur simultaneously upon formation of nonspecific primer-dimer-like products and hybridization of the donor fluorophore-labeled probe. When the fluorescence enhancement and quenching of the donor fluorophore are balanced, there is no net fluorescence or net quenching of the donor fluorophore, resulting in no signal from the donor fluorophore and therefore no signal from the primer-dimer-like nonspecific products. Additionally, the initially formed primer dimer-like non-specific product is extended toward the strand complementary to the strand to which the probe hybridizes, generating an amplifiable primer dimer-like non-specific product. [Diagram 3]Figures 3-26 are amplification curves, which are graphical representations of fluorescent signal versus amplification cycle number. The Cq value is the amplification cycle number at which the fluorescent signal of the amplification reaction exceeds a threshold fluorescent signal set by the machine and the cycle number at which the slope of the curve crosses the threshold line. The Cq value is linearly correlated with the number of target copies in the sample and is an indication of the number of target copies present in the sample. Reactions are run in triplicate for each sample and control reaction, so there are three curves for each, and in some figures four or five curves. Figures 3-8 are amplification curves of fluorescein and Blackhole quencher 1 labeled primer pair based amplification of E. coli housekeeping gene threonine synthase, Figure 21 is an amplification curve of fluorescein and BHQ1 dual labeled Taqman probe based assay of E. coli housekeeping gene homoserine kinase using 1 ng DNA, and Figure 22 is a no template control reaction of Taqman assay performed simultaneously to compare the sensitivity and specificity of the labeled primer probe pair and labeled primer pair based detection of the method of the present invention with the sensitivity and specificity of the gold standard Taqman probe assay in the field. Figures 3, 5 and 7 are amplification curves of sample (1 ng DNA) and primer sequences SEQ ID NO: 8 and 10; SEQ ID NO: 9 and 10; SEQ ID NO: 7 and 10, respectively. Figures 3A, 4A, 5A, 6A, 7A, 8A are the respective melting curves. [Figure 4] Figures 4, 6 and 8 are amplification curves of no template control reactions using primer sequences SEQ ID NOs: 8 and 10; SEQ ID NOs: 9 and 10; SEQ ID NOs: 7 and 10, respectively. [Diagram 5] See above [Figure 6] See above [Figure 7] See above [Figure 8] See above [Figure 9] Figure 9 shows the amplification curves of template-free amplification using FAM-labeled primer SEQ ID NO: 9 at a concentration of 0.4 μM. Figure 9A is the melting curve. [Figure 10]FIG. 10 shows the amplification curves and FIG. 10A shows the melting curves for template-free amplification using FAM-labeled primers, SEQ ID NO:9 at a concentration of 0.1 μM, and SEQ ID NO:10 at a concentration of 0.3 μM. [Figure 11] FIG. 11 shows the amplification curves and FIG. 11A shows the melting curves for template-free amplification using FAM-labeled primers, SEQ ID NO:9 at a concentration of 0.2 μM, and SEQ ID NO:10 at a concentration of 0.3 μM. [Figure 12] FIG. 12 shows the amplification curves and FIG. 12A shows the melting curves for template-free amplification using FAM-labeled primers, SEQ ID NO:9 at a concentration of 0.2 μM, and SEQ ID NO:10 at a concentration of 0.2 μM. [Figure 13] FIG. 13A shows the amplification curves and FIG. 13B shows the melting curves for amplification with 1 ng of template DNA using FAM-labeled primers, SEQ ID NO:9 at a concentration of 0.1 μM, and SEQ ID NO:10 at a concentration of 0.3 μM. [Figure 14] FIG. 14A shows the amplification curves and FIG. 14B shows the melting curves for amplification of 1 ng of template DNA using FAM-labeled primers, SEQ ID NO:9 at a concentration of 0.2 μM, and SEQ ID NO:10 at a concentration of 0.3 μM. [Figure 15] 15-19 are amplification curves for fluorescein-labeled probe and Blackhole quencher 1-labeled primer-based amplification of the E. coli housekeeping gene threonine synthase. [Figure 16] Same as above [Figure 17] Same as above [Figure 18] Same as above [Figure 19] Same as above [Figure 20] See Figure 3 [Figure 21] See Figure 3 [Figure 22] See Figure 3 [Diagram 23] Figures 23 and 24 are amplification curves of sample and control reactions for amplification of the E. coli threonine synthase gene target using a common non-targeting primer pair. [Figure 24] Same as above [Diagram 25]Figures 25 and 26 show amplification curves of sample and control reactions for amplification of the E. coli threonine synthase gene target to generate FRET signals (donor fluorophore is excited and acceptor fluorophore emission is measured) with well-separated melting curves for higher specificity. [Figure 26] Same as above

[0075] The present invention will now be described in detail with reference to the accompanying drawings and the following description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] Thus, according to a basic aspect of the present invention, a nucleic acid amplification method for target detection is provided that has higher sensitivity and higher specificity compared to other existing methods. The present invention does not attempt to reduce the formation of primer dimers or primer dimer-like non-specific amplification products, but rather reduces or eliminates the signal from primer dimers and primer dimer-like non-specific amplification products. As a result, the amplification reaction does not contain any non-specific signals, and thus is more specific and sensitive (specificity and sensitivity are interdependent, and an attempt to increase specificity reduces sensitivity and vice versa). The use of currently available hot start techniques to reduce the formation of non-specific amplification products is not necessary, but can be used additionally with the solution of the present invention. The present invention discloses a detection method that exhibits a specificity of 95% or more, and a sensitivity higher than the sensitivity of Taqman Chemistry, which is the gold standard in the field (70-80%), i.e. more than 80%, preferably more than 90%. The method increases sensitivity by increasing the signal of the target amplification products in addition to increasing specificity.

[0077] The primers and probes of existing / current methods and other methods in the art generate signals from non-specific primer dimers and primer dimer-like amplification products in addition to the target amplification products. The increased signal is an increase in both the target amplification products and the non-specific amplification products, but the signal-to-noise ratio is not affected in any way. On the other hand, the primers and probes of the method of the present invention are specifically designed to generate signals only from the target amplification products and avoid signal generation from non-specific primer dimers and primer dimer-like amplification products. Furthermore, the primers and probes of the method of the present invention are further designed to generate higher signals from the target amplification products depending on the position and adjacent base sequence of the base labeled with the donor fluorophore of the labeled primer or labeled probe. Thus, the increased signal from the target amplification products in addition to zero or close to zero signals from primer dimers and primer dimer-like non-specific products results in a higher signal-to-noise ratio, and thus higher detection sensitivity and specificity.

[0078] The primers and probes of the methods of the invention are labeled with a donor fluorophore and / or an acceptor fluorophore (radioactive quencher) / quencher and are selected, designed and labeled such that the donor fluorophore and the acceptor fluorophore (radioactive quencher) / quencher are sufficiently separated in the target amplification product and maintained beyond their FRET (energy transfer) distance, such that there is no energy transfer interaction between them and a fluorescent signal is generated upon target amplification by enhanced fluorescence of the donor fluorophore.

[0079] On the other hand, in primer dimer or primer dimer-like non-specific amplification products, the donor fluorophore and the acceptor fluorophore (radioactive quencher) / quencher are within their FRET (energy transfer) distance, resulting in energy transfer from the donor fluorophore to the acceptor fluorophore (radioactive quencher) / quencher and quenching of the fluorescence of the donor fluorophore. In such products, the fluorescence of the donor fluorophore is enhanced and quenched simultaneously. The degree of quenching of the fluorescence of the donor fluorophore depends on the spectral properties of the donor fluorophore and the acceptor fluorophore (radioactive quencher) / quencher and their separation in the primer dimer or primer dimer-like product, as well as the length of the linker used to attach the donor fluorophore and the acceptor fluorophore (radioactive quencher) / quencher.

[0080] If the degree of quenching is greater than the enhancement, no signal will be generated from these non-specific products. As a result, the specificity is high, but the sensitivity is reduced because the excessive quenching in primer dimer products or primer dimer-like products reduces the overall fluorescent signal, and thus the signal generated by the target amplification products. In this case, the specificity is highest, but the sensitivity is slightly reduced.

[0081] If the fluorescence enhancement and quenching of the donor fluorophore in primer dimer or primer dimer-like non-specific products are equal or balanced, no signal will be generated by these non-specific products and the fluorescence signal of the target amplification products will not be lost, resulting in the highest possible specificity and sensitivity.

[0082] On the other hand, if quenching is less than enhancement, there will be signal from these non-specific products, resulting in reduced specificity but not reduced sensitivity. In this case, specificity will be slightly reduced, but sensitivity will be the highest. This choice should be the last of the three options available in the method of the present invention, but it can be used in certain situations. Due to this balance between enhancement and quenching of the fluorescence of the donor fluorophore, the labeled primers and probes of the present invention are named Libra primer pairs and Libra primer-probe pairs. This mechanism does not work with existing primers and probes, which is unique to the design and method of the present invention.

[0083] Primers and probes are labeled with donor and acceptor fluorophores and are selected, designed and labeled such that in the target amplification product the donor and acceptor fluorophores are positioned within FRET distance of each other, such that energy transfer occurs from the donor fluorophore to the acceptor fluorophore and a FRET signal from the acceptor fluorophore (the donor fluorophore is excited with radiation of a wavelength specific to the excitation of the donor fluorophore and the fluorescence of the acceptor fluorophore is measured) is generated upon target amplification. Also, in primer-dimer or primer-dimer-like non-specific amplification products, the donor and acceptor fluorophores are within their short-range energy transfer distance, i.e., static / contact quenching distance (the bases labeled with the donor and acceptor fluorophores are positioned opposite each other or separated by 0-3 bases, preferably 0-1 bases), resulting in energy transfer from the donor fluorophore to the acceptor fluorophore and quenching of the fluorescence of the donor fluorophore and the fluorescence of the acceptor fluorophore, resulting in zero or near-zero signal from the primer-dimer or primer-dimer-like product.

[0084] The target nucleic acid sequence may be any single or double stranded nucleic acid sequence (natural or artificial), a nucleic acid amplification product, methylated DNA, mitochondrial DNA, C-DNA (complementary DNA), exosomal DNA, or a sequence of an infectious agent, genomic DNA (gDNA), a mutation in a genomic sequence (a single base change or deletion or insertion of a few bases or a long sequence), or a genomic sequence of bacteria, yeast, human, animal, plant and their pathogens (including viruses) or any other organism, living or dead, RNA, messenger RNA (m-RNA), ribosomal RNA (r-RNA), or a sequence of a nucleic acid of a living or dead organism, such as bacteria, yeast, human, animal, plant and their pathogens (including viruses), or any other organism. -RNA), small RNA, transfer RNA (t-RNA), microRNA (mi-RNA), microRNA precursor (pre- / pri-mi-RNA), the presence or absence of which is associated with, but is not limited to, the presence of an infectious agent, disorder or disease, or susceptibility to an infectious disease or disease or disorder, or suitability for disease treatment, genetic traits, prenatal diagnosis, genotype, cell type, tissue type, allele type, SNP detection species or strain type, cancer type, cancer subtype, cancer detection, disease type or subtype, expressed genes, and the like.

[0085] Another objective is to detect polynucleotides (nucleic acids) or non-nucleic acid analytes present in biological or non-biological samples, including but not limited to clinical samples (including but not limited to blood, urine, lymph, saliva, cerebrospinal (CSF) fluid, bronchial washings, sweat, peritoneal fluid, amniotic fluid, sputum, feces, pus, semen, bodily fluids such as vaginal swabs, throat and nasal swabs, nodes, tissue samples, tumor samples, biopsy samples, liquid cytology samples, circulating tumor cells, circulating stem cells, very small embryonic-like stem cells, stem cells, cancer cells, cancer stem cells, culture media, fermentation broths, soil, water, food, oil well samples, forensic samples, etc.). The polynucleotides / nucleic acids, non-nucleic acid analytes may be unpurified or may be purified or partially purified by any of the known nucleic acid purification or extraction methods or other methods.

[0086] The following definitions are provided for specific terms used in the following description and appended claims to more clearly and concisely explain and clarify the subject matter of this disclosure.

[0087] Unless otherwise specified, the words "a" or "an" herein mean at least one, and the singular includes the plural. By way of example and not limitation, "a target nucleic acid" means more than one or more copies of a particular species of target nucleic acid species, and two or more different target nucleic acid species. "And / or" means that the terms before and after the slash may be taken together or separately. By way of example and not by way of limitation, A and / or B can mean A and B, or A or B.

[0088] As used herein, the term "zero signal" means no detectable signal, and "near zero signal" means minimal or insubstantial signal.

[0089] It will be understood that the terms "about" and "approximately" are implicit before concentrations, distances, times, amounts, temperatures, and the like discussed herein, and that minor and insubstantial deviations are to be construed as being within the scope of the inventive teachings herein.

[0090] Furthermore, the use of the words "contain," "contains," "containing," "include," "includes," and "including," "comprise," "comprises," "comprising," "consist," "consists," and "consisting" are not intended to limit the scope of the present teachings. It is to be understood that the foregoing general description and detailed description are exemplary and explanatory only and are not intended to limit the present teachings.

[0091] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to single- and double-stranded polymers of nucleotide monomers, including but not limited to entirely 2'-deoxynucleotides (DNA) or entirely ribonucleotides (RNA), or chimeric mixtures thereof, and may contain nucleotide analogs, which are linked by internucleotide phosphodiester bond linkages or internucleotide analogs and associated counterions, including H+, NH4+, trialkylammonium, Mg2+, Na+, and the like.

[0092] The terms "strand" and "nucleic acid strand," used interchangeably, refer to a single polynucleotide chain of deoxynucleotides or ribonucleotides.

[0093] Synthetic or semi-synthetic nucleic acids are synthesized chemically or biochemically in a laboratory (by PCR or non-PCR biochemical reactions, or by adding a synthetic sequence to a natural sequence) using their nucleotide building blocks.

[0094] An oligonucleotide is a short-length ribose or deoxyribose nucleic acid polymer that is chemically synthesized in the laboratory using these building blocks or biochemically produced in the laboratory from natural nucleic acid molecules. It has two hydroxyl moieties at the two ends, namely the 5' and 3' ends. Polynucleotides typically range in size from 5 to 40 nucleotides, and are sometimes referred to in the art as oligonucleotides, and can be several thousand monomer units in size. Unless otherwise stated, whenever a polynucleotide is shown, it is understood that the nucleotides are in the 5'-3' direction from left to right, with "A" representing deoxy-adenosine, "G" representing deoxy-guanosine, "C" representing deoxycytosine, "U" representing deoxy-uridine, and "T" representing deoxy-thymidine. Additionally, ribose or deoxyribose nucleic acid is synonymous with ribonucleotide and 2'-deoxynucleotide.

[0095] The term "nucleotide" refers to a phosphate ester, e.g., triphosphate, of a nucleoside, with the most common site of esterification being the C5 position of the pentose sugar. The term "nucleoside" refers to a compound containing a purine or deazapurine base, such as adenine, guanine, deazadenine, deazaguanine, or a pyrimidine base, such as cytosine, uracil, pseudouracil, thymine, inosine, linked at its 1' position to a pentose sugar, including 2' deoxy and 2' hydroxyl forms in which the pentose base is attached to the 9 position of the purine base and the 1 position of the pyrimidine base.

[0096] Artificial nucleotides are synthetic modified nucleotides in which either the purine-pyrimidine base or the sugar moiety or the phosphate group is modified. Exemplary base analogs include pseudouridine, 2,6 diaminopurine, hypoxanthine, isoguanine, isocytosine, 2-thiopyrimidine, C-5 propylene, and exemplary sugar analogs include hydrogen, hydroxy, alkoxy, such as methoxy, ethoxy, allyloxy, butoxy, isobutoxy, isopropoxy, and phenoxy, azido, amino or alkylamino, fluoro, chloro, and bromo modified sugar 2' or 3' positions, including locked nucleotides (ribonucleotides in which the 2' and 4' hydroxyl groups of the ribose sugar moiety are linked together and thus locked (LNA)). Phosphate analogs have one or more oxygen atoms replaced with non-oxygen moieties, such as sulfur, selenium, and boron. Exemplary phosphate analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilinothioates, phosphoranilidates, phosphoramidites, boranophosphates, including associated counterions and peptide nucleotides (PNAs in which a neutral peptide, such as an N-(2-aminoethyl)glycine backbone, is attached to the phosphate group).

[0097] Hapten: A hapten is a small molecule that does not elicit an immune response by itself, but does elicit an immune response when attached to a carrier molecule, such as a protein or peptide, which may or may not itself be immunogenic.

[0098] Aptamer: Aptamers are short lengths of single-stranded DNA or RNA or synthetic nucleic acids that are modified at the base or sugar moiety(s).

[0099] The terms "amplified product," "amplification product," or "amplicon" refer to a piece of DNA that has been amplified by a polymerase using a pair of primers in an amplification method such as PCR.

[0100] The terms "target amplification product," "specific amplification product," "target specific amplification product," "specific amplification product," and "specific target amplification product" are used interchangeably and refer to a specific nucleic acid fragment amplified by a polymerase using a pair of primers in an amplification method such as PCR.

[0101] The terms "target," "target sequence," "target nucleic acid," "specific target," "target specific," "target nucleic acid sequence," "specific sequence," "target specific sequence," "specific nucleic acid," "specific nucleic acid sequence," "nucleic acid of interest," "template" are used interchangeably and refer to a desired region or regions of a particular nucleic acid that is to be amplified or detected, or both, or amplified and quantified.

[0102] The terms "identical" and "identical sequence" are used interchangeably and refer to two nucleic acid sequences having the same sequence or complementary sequences.

[0103] A "complementary sequence" is a complement of a nucleic acid sequence in which the nucleotide bases of the complementary sequence pair with each complementary base of the nucleotide sequence, e.g., guanine bases are complementary to cytosine bases, and adenine bases are complementary to thymidine or uracil bases. Base complementarity can be complete or partial, as long as the hybrid is stable. Base complementarity is the ability to form base pairs. Two sequences are complementary, meaning that one sequence complements the other.

[0104] "Polymerization" and "nucleic acid synthesis," used interchangeably, refer to the process of extending the sequence of a primer through the use of a nucleic acid template, a polymerase and nucleotides, or through the sequential addition of nucleotides.

[0105] A "primer" having a freely extendable 3' end with a 3' hydroxyl (OH) group refers to an oligonucleotide, chemically synthesized or generated from a larger nucleic acid molecule, capable of priming or initiating a synthesis or polymerization reaction that extends the primer and produces a primer extension product complementary to the nucleic acid strand in the presence of deoxyribonucleotides such as G, C, A and T, and a polymerase enzyme such as DNA polymerase or reverse transcriptase, and a suitable buffer containing cofactors or substituents that affect pH, ionic strength, etc., at a suitable temperature for a sufficient period of time. The selected primers are at least substantially complementary to hybridize to the respective strands of each specific nucleic acid sequence to be amplified. Primers are usually complementary, except that non-complementary nucleotides may be present at a given sequence position, such as the primer termini as described.

[0106] In some embodiments, the primer can be single-stranded. In some embodiments, a non-complementary nucleotide sequence or fragment can be attached to the 5' end of the primer, where the remainder of the primer sequence is complementary or sufficiently complementary to the target region of the target nucleic acid. In some embodiments, the non-complementary nucleotide sequence is referred to as a non-target sequence.

[0107] A "probe" is a non-extendable oligonucleotide attached to a fluorescent reporter dye, or to a fluorescent reporter dye and a quencher such as biotin, which oligonucleotide is complementary to a strand of a target nucleic acid that is amplified in a nucleic acid amplification reaction.

[0108] The terms "annealing" or "anneal" and "hybridizing" or "hybridize" or "hybridization" refer to the pairing of nucleotide bases between one nucleic acid and another, resulting in the formation of a duplex or higher order structure using, but not limited to, A:T, A:U, and G:C Watson-Crick base pairing.

[0109] The terms "amplification," "target amplification," and "nucleic acid amplification" are used interchangeably and refer to the use of any amplification process or procedure to increase the concentration of a specific nucleic acid sequence within a mixture of nucleic acid sequences.

[0110] The term "thermal cycling", "thermal cycles", "thermal cycle" as typically used in the nucleic acid amplification process polymerase chain reaction (PCR) refers to repeated cycles of temperature change from a full denaturation temperature to an annealing / hybridization temperature to an extension temperature and back to the full denaturation temperature. In some embodiments, the annealing / hybridization temperature and the extension temperature are combined into a single temperature. The term also refers to repeated cycles of the above temperature change cycles. In some embodiments, the term "single cycle" or "single round of cycling" may be used to refer to one round of the above series of temperature changes (single or multiple). In some embodiments, a single round of cycling may include a denaturation temperature, repeated cycles of a first annealing / hybridization temperature and a first extension temperature, and repeated cycles of a second annealing / hybridization temperature and a second extension temperature, where the first annealing temperature and extension temperature are different from the second annealing temperature and extension temperature, or the first annealing temperature is different from the second annealing temperature. The number of cycles can be one or any number up to 45. The complete denaturation temperature allows the double-stranded DNA fragments to unwind into two single strands, the annealing temperature allows the primers to anneal or hybridize to the complementary strands of the separated strands of the DNA fragments, and the extension temperature allows the synthesis of nascent DNA strands of the amplification product or amplicon.

[0111] In isothermal amplification processes, only a single temperature is used to amplify the target sequence, and separation of the two strands of double-stranded DNA is achieved by using primers with a loop structure that allows the primer to anneal to the loop (loop-mediated amplification (LAMP)), or by using DNA double-strand unwinding enzymes such as recombinases (recombinase-polymerase amplification (RPA)), helicases (helicase-polymerase amplification (HPA)), topoisomerases (topoisomerase-polymerase amplification (TPA)), and the polymerases used in LAMP that synthesize the nascent strand of nucleic acid also have strand-displacing enzyme activity.

[0112] Quantitative PCR (qPCR) or quantitative isothermal amplification refers to amplification to quantify nucleic acids in a reaction mixture or nucleic acid sample, where an amplification signal is obtained when the signal crosses a threshold value during the exponential phase of the amplification reaction.

[0113] Hot start PCR generally refers to limiting the availability of essential reaction components, such as a polymerase, at a first temperature (typically a relatively low temperature) until a second temperature (typically a relatively high temperature that is the annealing temperature of the primers) is reached at which the essential components are able to participate in the amplification reaction.

[0114] Reverse transcription polymerase chain reaction (RT-PCR) is a process in which a reverse transcriptase enzyme is used to synthesize a DNA strand from an RNA strand, followed by PCR on the synthesized DNA strand. The process uses either a combination of reverse transcriptase and DNA polymerase, or an enzyme that has both reverse transcriptase and polymerase activities.

[0115] In allelic polymerase chain reaction (allele PCR), one of the two primers in an amplification reaction carries a base mismatch (single or multiple) with the target sequence at one or two bases 1-4 bases away from its 3' end, and the alleles of the target sequence differ in sequence by one or two bases, preferably by one base that is the second base from the 3' end of one of the primers. Furthermore, one of the primers is an allelic primer labeled with an acceptor or quencher moiety, and a probe labeled with a donor fluorophore hybridizes to one of the two strands of the amplified target.

[0116] In methylation-specific nucleic acid amplification, including methylation status PCR or methylation-specific PCR, methylated DNA is treated with a bisulfite reagent, which converts methylcytosine bases in the methylated DNA to uracil bases, altering the sequence of the DNA, which is then amplified using primers specific for the altered sequence.

[0117] In emulsion PCR, PCR amplification of a target sequence is carried out in an oil-water emulsion, in which the aqueous PCR reaction mixture is diluted with oil to the extent that each of the water droplets formed contains a small number, preferably a single nucleic acid molecule.

[0118] In bead PCR, a bead or beads carry a primer that binds to and captures a target nucleic acid and, in combination with another target-specific primer, amplifies the target nucleic acid. Either the beads are used in emulsion PCR, or each bead with a captured target nucleic acid is captured in a pore and subjected to PCR amplification.

[0119] In droplet PCR or digital PCR (dPCR), a PCR reaction mixture complete with all necessary components, reaction buffer, nucleotides, enzyme(s), primers or primers and probes, and target nucleic acid is dispensed into thousands of individual reaction droplets, and an amplification signal is obtained at the end point of the amplification reaction. dPCR allows detection of extremely low copy number targets and absolute quantification of targets.

[0120] The terms "non-specific signal," "non-specific fluorescence," and "background fluorescence," used interchangeably, refer to detectable signals emitted from fluorophores or nucleic acid-binding dye molecules associated with double-stranded nucleic acids (non-specific amplification products or non-specific amplicons) other than the desired amplification products or amplicons, including amplification products of target nucleic acids, and in some embodiments, internal standard or positive control sequences that may be included in the reactions of some embodiments.

[0121] Non-specific amplification is an amplification event in which an amplification product or products other than the intended amplification product (the specific target amplification product) are generated or produced; the unintended amplification product is referred to as the non-specific amplification product.

[0122] Primer dimer: This is a non-specific amplification product that is generated or produced when two primer molecules overlap each other at their extendable 3' ends and the polymerase of the amplification reaction extends the 3' ends of the two primer molecules using one as a template for the other.

[0123] A primer dimer-like amplification product is a non-specific amplification product that is generated or produced when the 3' terminal sequence of one or both amplification primers overlaps with the non-extendable 3' terminal sequence of a probe used to monitor the amplification reaction, and the polymerase of the amplification reaction extends the 3' end of the primer(s) molecule using the probe as a template. The primer dimer-like product thus formed is further extended in the amplification process against the target strand complementary to the strand to which the probe hybridizes, producing an amplifiable primer dimer-like product.

[0124] The terms "denaturing" and "denaturation" as used herein refer to a process in which double-stranded nucleic acids / polynucleotides, including but not limited to genomic DNA (gDNA) fragments, at least one target nucleic acid, double-stranded amplification products or amplicons, double-stranded polynucleotide fragments, as appropriate, are converted into two single-stranded polynucleotides or single-stranded or substantially single-stranded polynucleotides. Melting temperature, or "Tm," is a measure of the stability of a nucleic acid duplex and is the temperature at which half of the base pairs of a particular nucleic acid duplex dissociate. A "melting curve" or "melt curve" is a curve or profile of the dissociation and association of base pairs of a particular nucleic acid duplex, usually a plot of -dF / dT versus T, where "F" is a measure of the fluorescence of the nucleic acid duplex and "T" is temperature.

[0125] The term "label" as used herein refers to any atom or molecule that can be used to provide or assist in providing a detectable or quantifiable signal and that can be attached to a nucleic acid or oligonucleotide, where the detectable signal is a fluorescent signal, a label that produces detectable fluorescence is referred to herein as a "fluorophore" or "reporter dye" or "fluorescent dye" or "dye," and a label that absorbs or reduces the fluorescent signal of a fluorophore or quenches the fluorescence of a fluorophore or fluorescent dye is a quencher. A quencher can be a fluorophore (acceptor fluorophore or radioactive quencher) or a non-radioactive quencher or dark quencher (which absorbs the fluorescent signal but does not emit any light or radiation). A fluorophore emits energy or electromagnetic radiation, including light, when irradiated with electromagnetic radiation, including light, and the emission of this energy or electromagnetic radiation or light is the emission of a fluorophore. The energy or electromagnetic radiation or light emitted by a fluorophore is different from the energy or electromagnetic radiation irradiated to the fluorophore, which is the fluorescence of the fluorophore. A fluorophore absorbs light or radiation of a specific wavelength(s) and emits a unique light or radiation of a different wavelength(s). The fluorescence maximum or emission maximum is the wavelength at which the fluorescence emission of a fluorophore is maximum. A fluorophore that transfers its energy or light or radiation to an acceptor fluorophore or quencher is a donor fluorophore. An acceptor fluorophore absorbs the energy of a donor fluorophore and emits a unique energy or electromagnetic radiation or light that is different from that of the donor, and is also called a radiative quencher. On the other hand, a dark quencher or a non-radiative quencher absorbs the energy or electromagnetic radiation or light of the donor and does not emit any electromagnetic radiation or light.

[0126] The terms "reporter dye", "fluorescent dye" and "dye" also include "nucleic acid binding dye" or "intercalating dye", which terms are used interchangeably. Nucleic acid binding dye refers to a fluorescent molecule specific for double-stranded polynucleotides or at least a fluorescent molecule that exhibits a substantially greater fluorescence enhancement when associated with a double-stranded polynucleotide than with a single-stranded polynucleotide. Non-limiting examples of nucleic acid binding dyes include ethidium bromide, Hoechst dye 33342, Hoechst dye 33258, DAPI, lanthanide chelates (NDI-(BH-HCT-Eu3+ chelate) and the asymmetric cyanine dyes SYBR® Green and Pico® Green, Avagreen®, YOPRO 1, SYTO 9, acridine orange.

[0127] The excitation spectrum of a fluorophore is the graphical representation of the energy of electromagnetic radiation absorbed by the fluorophore over the entire spectral wavelength range of electromagnetic radiation, and the emission spectrum of a fluorophore is the energy of electromagnetic radiation emitted by the fluorophore over the entire spectral wavelength range of electromagnetic radiation.

[0128] Spectral overlap is the overlap of the excitation spectrum of one fluorophore or quencher with the emission spectrum of another fluorophore.

[0129] Polymerase in this specification is an enzyme that catalyzes the polymerization reaction of nucleotides. Polymerase can be DNA polymerase or RNA polymerase depending on whether deoxyribonucleotides or ribonucleotides are polymerized. DNA polymerase can be DNA-dependent DNA polymerase or RNA-dependent DNA polymerase. For RNA polymerase, when RNA polymerase synthesizes RNA molecule by polymerizing ribonucleotides using single-stranded DNA as template (this process is called RNA transcription), or reverse transcriptase when it synthesizes DNA strand by polymerizing deoxynucleotides using RNA strand as template (this process is called reverse transcription). Some reverse transcriptase has both reverse transcriptase activity and DNA polymerase enzyme activity (Thermus thermophilus or Tth polymerase). Some DNA polymerase can have strand displacement activity, exonuclease enzyme activity, template-independent polymerase activity. A polymerase is a meso-thermostable polymerase if it exhibits enzymatic activity at temperatures above 370C and up to above 60°C, and a thermostable polymerase if it is active at temperatures above 600°C. A polymerase is an ambient temperature polymerase (37°C-250°C) if it exhibits enzymatic activity at temperatures below 370°C, or a low temperature polymerase if it exhibits enzymatic activity at temperatures below 250°C. A polymerase can be a hot start polymerase, which is a polymerase that exhibits polymerization activity at the annealing temperature of the primer. The nucleic polymerase can be selected from the group consisting of, but not limited to, Taq DNA polymerase, Pfu DNA polymerase, Vent™ DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, Tth DNA polymerase, Tli DNA polymerase, thermostable polymerases with helicase activity or DNA unwinding activity.

[0130] Further provided herein is a kit for practicing or performing the method taught in the present invention. The term "kit" herein refers to a packaged set of one or more related components or compositions, typically in one or more containers or vessels. The kit can typically include at least one pair of oligonucleotides as a primer pair for polymerizing and / or amplifying at least one target nucleic acid from a sample, where one member of the oligonucleotide pair is labeled with a detectable label, such as a fluorophore or an intercalating dye, and the other member of the pair is labeled with a quencher. The kit also typically includes at least one pair of oligonucleotides as a primer pair for polymerizing and / or amplifying at least one target nucleic acid from a sample (wherein at least one member of the oligonucleotide primer pair is labeled with a quencher), and another non-extendable oligonucleotide with a detectable label, a fluorophore or an intercalating dye, as a probe for monitoring the amplification reaction. The kit can also typically include at least one pair of oligonucleotides as a primer pair for polymerizing and / or amplifying at least one target nucleic acid from a sample, the oligonucleotides being labeled with a detectable label, such as a fluorophore or an intercalating dye, one component of the fluorophore being a donor fluorophore and the other component being an acceptor fluorophore. The kit can also typically include at least one pair of oligonucleotides as a primer pair for polymerizing and / or amplifying at least one target nucleic acid from a sample (at least one component of the oligonucleotide primer pair being labeled with a detectable label) and another non-extendable oligonucleotide labeled with another detectable label as a probe for monitoring the amplification reaction, the detectable label being a fluorophore or an intercalating dye, the two labels being a donor and acceptor pair. The kit can also contain a sample containing a predefined nucleic acid used for a control reaction.The kits can also optionally include a reaction mixture or PCR master mix that contains all of the components, other than the primers and probes, required to amplify at least one target nucleic acid from a nucleic acid template, or buffers, salts, divalent metal ions (Mg, Mn, Stock solutions of dNTPs (dA, dC, dG, dT, dU), enzymes, glycerol, BSA (bovine serum albumin), gelatin, one or more surfactants, PVP (polyvinylpyrrolidone), PEG (polyethylene glycol) may also be included, and the range of concentrations used for each component is well known in the art and can be further optimized by those skilled in the art. In some embodiments, the kit may include multiple primer sets or primer probe sets. In other embodiments, specific systems or kits that will be understood by those skilled in the art are also included or contemplated. The kit may also contain a reaction vessel(s) for carrying out the amplification reaction, including but not limited to microtubes or microcentrifuge tubes (0.2 / 0.5 ml) and the like commonly used in molecular biology laboratories, wells of multi-well plates, spots on glass slides or silicon wafers, channels or chambers of microfluidic devices.

[0131] The section headings used herein are merely for organizational purposes and should not be construed as limiting the desired subject matter in any manner. All patents and other references incorporated herein are expressly incorporated by reference in their entirety for any purpose. In the event that any of the incorporated documents conflict with any term defined herein, the present specification shall control. While the teachings of the present invention have been described in conjunction with various embodiments, it is not intended that the teachings of the present invention be limited to such embodiments. Rather, the teachings of the present invention encompass various alternatives, modifications, and equivalents that will be understood by those skilled in the art. Throughout this specification, illustrations of specific terms should be considered as non-limiting examples.

[0132] Thus, the invention provides a process for nucleic acid amplification for enhancement of donor fluorophore fluorescence to generate a target amplification signal, and for balanced fluorescence enhancement to offset quenching of donor fluorophore fluorescence or balanced quenching to offset donor fluorophore enhancement to generate zero or near zero signal from primer dimer or primer dimer-like non-specific nucleic acid amplification products.

[0133] Another objective is to balance the simultaneous enhancement and quenching of donor fluorophore fluorescence in nonspecific primer dimers (formed by overlap and extension of the 3' end of a donor fluorophore-labeled primer with the 3' end of an acceptor fluorophore / quencher-labeled primer by the polymerase used in the amplification reaction) and primer dimer-like amplification products (formed by overlap of the 3' end of a donor fluorophore-labeled probe with the 3' end of an acceptor fluorophore / quencher-labeled primer and extension of the labeled primer by the polymerase using the probe as a template), to prevent signal generation from these nonspecific products.

[0134] Yet another object of the present invention is to use linear primers labeled with a donor fluorophore moiety and linear primers labeled with an acceptor fluorophore / quencher moiety for target nucleic acid amplification, the primers being selected and labeled such that the donor fluorophore moiety and the acceptor fluorophore / quencher moiety are sufficiently separated from one another in the target amplification product such that there is no significant energy transfer between the two moieties, incorporation of the donor fluorophore-labeled primer into the target amplification product significantly enhances the fluorescence of the donor fluorophore, and a melting curve and melting temperature (Tm) are also generated by the target amplification product.

[0135] However, in nonspecific primer dimer amplification products, the donor fluorophore and the acceptor fluorophore / quencher are within their FRET distance, the acceptor fluorophore / quencher moiety quenches the fluorescence of the donor fluorophore moiety (Figure 1), and the positions of the donor fluorophore and the acceptor fluorophore / quencher on the two primers are selected such that the amount of quenching of the donor fluorophore's fluorescence by the acceptor fluorophore / quencher is equal or nearly equal to the amount of enhancement of the donor fluorophore's fluorescence by the incorporation of the donor fluorophore-labeled primer into the primer dimer product. As a result, no signal is generated upon the formation of nonspecific primer dimer products, the sensitivity of target detection is not affected by the quenching of the donor fluorophore's fluorescence in the primer dimer, and the melting curve is also generated by the primer dimer.

[0136] Another objective is to appropriately select the placement of the donor fluorophore and acceptor fluorophore / quencher moieties on the two primers at any base (except the 5'-terminal base of the donor fluorophore-labeled primer) at least two bases away from the 3' end, and preferably more bases away. A pair of donor fluorophore-labeled and acceptor fluorophore-labeled primers is a libra primer pair.

[0137] Another objective is to arrange the donor fluorophore and acceptor fluorophore / quencher on the primers such that the fluorescence of the donor fluorophore in the non-specific primer-dimer amplification product is quenched by the acceptor fluorophore / quencher by 1-50 percent, preferably 17-45 percent, more preferably 30-40 percent, depending on the degree of enhancement of the donor fluorophore's fluorescence upon corresponding probe hybridization, which is 1-100 percent, preferably 20-80 percent, more preferably 40-70 percent, respectively.

[0138] Another objective is that the number of bases separating the 3' end of the donor fluorophore labeled primer from the donor fluorophore labeled base plus the number of bases separating the 3' end of the acceptor fluorophore / quencher labeled primer from the acceptor fluorophore / quencher labeled base minus the number of bases at the 3' ends of the labeled primers that overlap with each other in the primer dimer equals the number of bases corresponding to a distance of energy transfer between the donor fluorophore moiety and the acceptor fluorophore / quencher moiety of 1 to 50 percent, preferably 17 to 45 percent, and more preferably 30 to 40 percent.

[0139] Another objective is that the distance between the donor fluorophore labeled base and the 3' end of the donor fluorophore labeled primer plus the distance between the acceptor fluorophore / quencher labeled base and the 3' end of the acceptor fluorophore / quencher labeled primer minus the overlap distance between the bases at the 3' end of the primer in the primer dimer equals 1 to 50 percent, preferably 17 to 45 percent, and more preferably 30 to 40 percent energy transfer distance between the donor fluorophore moiety and the acceptor fluorophore / quencher moiety.

[0140] In another embodiment, the donor fluorophore-labeled primer is labeled with two fluorophores, which can be the same or different entities, allowing FRET between the two fluorophores, further increasing the enhanced signal of the amplification product.

[0141] Another object of the invention is to use a linear probe and a pair of linear primers labeled with a donor fluorophore moiety for target nucleic acid amplification, where an acceptor fluorophore / quencher moiety is located on one or both primers. The primers and probes are selected and labeled such that the donor fluorophore moiety and the acceptor fluorophore / quencher moiety are sufficiently separated from each other that when the probe hybridizes to either the target amplicon strand incorporating the acceptor / quencher labeled primer or the target strand to which the acceptor fluorophore / quencher labeled primer hybridizes / anneals, there is no significant energy transfer between the two moieties, and there is a significant enhancement in the fluorescence of the donor fluorophore upon hybridization of the labeled probe. The labeled probe generates a melting curve and melting temperature specific to melting of the probe from the target amplicon.

[0142] However, in non-specific primer-dimer-like amplification products (formed by overlap of the 3' ends of the labeled probe and the labeled primer(s) and extension of the labeled primer by the polymerase used in the amplification reaction using the probe as a template), the acceptor fluorophore / quencher-labeled primer is incorporated into one strand of the primer-dimer-like product, and the donor fluorophore-labeled probe hybridizes to the strand of the primer-dimer-like product that incorporates the acceptor / quencher-labeled primer (Figure 2). Hybridization of the donor fluorophore-labeled probe enhances the fluorescence of the donor fluorophore, bringing the donor fluorophore and acceptor fluorophore / quencher within their FRET / energy transfer distance, and the acceptor fluorophore / quencher moiety quenches the fluorescence of the donor fluorophore moiety. The positions of the donor fluorophore and acceptor fluorophore / quencher on the probe and primer(s) are selected such that the amount of quenching of the donor fluorophore's fluorescence by the acceptor fluorophore / quencher is equal or nearly equal to the amount of enhancement of the donor fluorophore's fluorescence. As a result, no signal is generated upon formation of nonspecific primer-dimer-like products, and the sensitivity of target detection is not affected by quenching of the donor fluorophore's fluorescence in primer-dimer-like products.

[0143] Additionally, the probe labeled with the donor fluorophore can further have an MGB dye (minor groove binder), and the probe is an MGB probe. As used herein, the term "minor groove binder" refers to a small molecule that fits into the minor groove of double-stranded DNA, possibly in a sequence-specific manner. In general, minor groove binders are long, flat molecules that can adopt a crescent-like shape, so that they fit snugly into the minor groove of the double helix, often displacing water. Minor groove binders are typically composed of several aromatic rings (such as, but not limited to, furan, benzene, or pyrrole rings) connected by bonds with torsional freedom.

[0144] Another object is to appropriately select the placement of the donor fluorophore moiety on the probe at the 3' end or at any base away from the 3' end except the 5' end base position, and the placement of the acceptor fluorophore / quencher moiety on the acceptor fluorophore / quencher labeled primer at any base away at least 2 bases, and preferably more, from the 3' end of the acceptor fluorophore / quencher labeled primer. A donor fluorophore and acceptor fluorophore / quencher labeled probe primer pair is a libra probe-primer pair.

[0145] A further objective is to use both primers for probe-based detection labeled with acceptor-fluorophore / quencher moieties, where the acceptor-fluorophore / quencher moieties on the two primers can be the same or different.

[0146] In another embodiment, the donor fluorophore and acceptor fluorophore / quencher are positioned on the probe and primer, respectively, such that upon hybridization of the donor fluorophore-labeled probe to non-specific primer-dimer-like amplification products, the fluorescence of the donor fluorophore is quenched by the acceptor fluorophore / quencher by 1-50 percent, preferably 17-45 percent, more preferably 30-40 percent, depending on the degree of enhancement of the donor fluorophore's fluorescence upon corresponding probe hybridization, which is 1-100 percent, preferably 20-80 percent, more preferably 40-70 percent, respectively.

[0147] Another objective is that the number of bases separating the 3' end of the donor fluorophore-labeled probe from the donor fluorophore-labeled base plus the number of bases separating the 3' end of the acceptor fluorophore / quencher-labeled primer from the acceptor fluorophore / quencher-labeled base minus the number of bases at the 3' end of the acceptor fluorophore / quencher-labeled primer that overlap with the 3' terminal base of the donor fluorophore-labeled probe in the primer-dimer-like product equals the number of bases corresponding to a resonance energy transfer / energy transfer distance between the donor fluorophore moiety and the acceptor / quencher moiety of 1 to 50 percent, preferably 17 to 45 percent, and more preferably 30 to 40 percent.

[0148] Another objective is that the distance between the donor fluorophore labeled base and the 3' end of the donor fluorophore labeled probe plus the distance between the acceptor / quencher labeled base and the 3' end of the acceptor / quencher labeled primer minus the overlap distance between the 3' terminal base of the labeled primer and the 3' terminal base of the probe in the primer-dimer-like product equals 1 to 50 percent, preferably 17 to 45 percent, more preferably 30 to 40 percent resonance energy transfer distance between the donor fluorophore moiety and the acceptor / quencher moiety.

[0149] In a particular extension of the embodiment, a donor fluorophore-labeled probe is provided that is linked to one of the two amplification primers by a non-nucleotide organic linker such as hexamethylene or hexa-polyethylene glycol, preferably hexa-polyethylene glycol or chimeras thereof or longer lengths thereof (the non-nucleotide organic linker displaces the advancing polymerase from the template), and the probe is designed to hybridize to a nascent target amplification strand or target amplicon strand synthesized by the primer linked to the probe.

[0150] In another extension, the donor fluorophore-labeled probe is labeled with two fluorophores, which can be the same or different entities, resulting in FRET and further increasing the enhanced signal of the fluorophore-labeled probe.

[0151] The fluorescence of the donor fluorophore is typically enhanced by 1-100 percent, preferably 20-80 percent, and more preferably 40-70 percent upon incorporation of the donor fluorophore-labeled primer into the amplification product or upon hybridization of the donor fluorophore-labeled probe to the amplification product or target strand, depending on the nucleotide sequence of the primer or probe surrounding the donor fluorophore-labeled base, the presence of guanine bases surrounding the donor fluorophore-labeled base, the position of the donor fluorophore on the donor fluorophore-labeled primer or probe relative to the 3' end of the donor fluorophore-labeled primer or probe, the linker used to attach the donor fluorophore and acceptor fluorophore / quencher to the primer or probe, the secondary structure of the donor fluorophore-labeled primer or probe, the labeling efficiency of the primer or probe, and the reaction conditions applied.

[0152] For non-specific primer-dimer or primer-dimer-like amplification products, the fluorescence of the donor fluorophore of the primer or probe is quenched by the acceptor fluorophore / quencher of the primer by 1-50 percent, preferably 17-45 percent, more preferably 30-40 percent, to avoid any signal generation by the non-specific amplification products. The degree of quenching of the fluorescence of the donor fluorophore by the acceptor fluorophore / quencher not only depends on the spectral properties of the donor and acceptor fluorophores / quenchers and their distance or separation, but also on the linkers used to attach the donor and acceptor fluorophores / quenchers to the primers and probes.

[0153] Another objective is to place at the 5' end of the donor fluorophore-labeled primer or probe an additional quencher for the donor fluorophore or a 4-8 base non-target sequence (with or without a quencher) that is complementary to the bases surrounding the donor fluorophore-labeled base. In such a condition, when the donor fluorophore-labeled primer or probe is incorporated into or hybridized to an amplification product, the fluorescence of the donor fluorophore increases approximately 3-8 fold. The positions of the donor fluorophore and acceptor fluorophore / quencher on the primer, or on the primer and probe, are selected such that the fluorescence of the donor fluorophore is quenched by 65-90 percent in the primer-dimer or primer-dimer-like product. The number of bases separating the 3' end of a donor fluorophore-labeled primer or probe from the base bearing the donor fluorophore plus the number of bases separating the 3' end of an acceptor fluorophore / quencher-labeled primer from the base bearing the acceptor fluorophore / quencher, minus the number of bases overlapping between the 3' ends of the labeled primers in the primer-dimer or primer-dimer-like products or between the 3' ends of the labeled primers and the 3' ends of the labeled probes, equals the number of bases corresponding to a distance of 65 to 90 percent resonance energy transfer / energy transfer between the donor fluorophore and acceptor fluorophore / quencher moieties.

[0154] The distance between the base bearing a donor fluorophore and the 3' end of the donor fluorophore-labeled primer or probe, plus the distance between the base labeled with the acceptor fluorophore / quencher and the 3' end of the acceptor fluorophore / quencher-labeled primer, minus the distance of overlap between the bases at the 3' end of the labeled primer in the primer-dimer or primer-dimer-like product or between the bases at the 3' end of the labeled primer and the bases at the 3' end of the labeled probe, equals 65-90 percent resonance energy transfer / energy transfer distance between the donor fluorophore moiety and the acceptor fluorophore / quencher moiety.

[0155] Energy transfer is the process of energy transfer from a first fluorophore moiety to a second fluorophore moiety or quencher moiety upon excitation with appropriate electromagnetic radiation, when the excitation spectrum of the second fluorophore or quencher overlaps significantly with the emission spectrum of the first fluorophore and the two moieties are in close proximity. The first fluorophore that transfers energy is called the donor fluorophore or donor, the second fluorophore is called the acceptor fluorophore or acceptor of the donor fluorophore, and the quencher is called the acceptor or quencher of the donor fluorophore. Energy transfer that occurs over a short distance from the donor fluorophore to the acceptor fluorophore or quencher when they are in close proximity or contact with each other is a short-range energy transfer process, and the resulting quenching of the donor fluorophore is called static or contact quenching. Fluorescence resonance energy transfer (FRET) is a long-range energy transfer that occurs in the solution phase without any contact between a donor fluorophore and an acceptor fluorophore or quencher.

[0156] The value of the Förster radius gives an indication of the distance between the donor fluorophore and the acceptor fluorophore / quencher for a certain energy transfer efficiency. Significant energy transfer occurs up to a distance of 2R0. Furthermore, the distance "R" between the donor and acceptor for the energy transfer efficiency "E" is given by the formula R=(1 / E-1)1 / 6 R0, where R0 is the distance between the donor and acceptor for 50% energy transfer efficiency.

[0157] The Förster radius R0 is R0=[8.8×1023×k2×n4×QYD×J(λ)]1 / 6 Å where k2 = dipole direction factor, n = refractive index, εA = extinction coefficient of the acceptor, QYD = fluorescence quantum yield of the donor in the absence of acceptor, J(λ) = spectral overlap integral = integral [εA(λ).FD(λ).(λ)d(λ)] cm3 M, where FD = fluorescence emission intensity of the donor as a function of total integrated intensity, and Å = Angstroms.

[0158] The Förster radius R0 is longer when the spectral overlap or the integral of the spectral overlap is small, i.e., when the emission maximum of the donor fluorophore and the excitation maximum of the acceptor fluorophore / quencher are not very close, and shorter when the spectral overlap or the integral of the spectral overlap is large, i.e., when the emission maximum of the donor fluorophore and the excitation maximum of the acceptor fluorophore / quencher are very close. The Förster radius typically falls within the range of 22-75 Å. Förster radii for many donor and acceptor fluorophore pairs are available in the published literature (Penguang Wu et al, Analytical Biochemistry vol - 218, pages 1 - 13, 1994; Robert H Fairclough et al Methods in Enzymology, vol - 48, pages - 347 - 379, 1978) or from the catalogs / handbooks or websites of suppliers of donor and acceptor fluorophore / quencher dyes, especially Molecular Probes. In addition, newer donor and acceptor fluorophores / quenchers are constantly being developed.

[0159] Donor fluorophore and acceptor fluorophore / quencher molecules are primarily hydrophobic and tend to collapse upon each other when in close proximity, resulting in static or contact quenching interactions in addition to FRET interactions. Furthermore, single-stranded oligonucleotides form secondary structures and fold back. As a result, at close distances, two energy transfer mechanisms (FRET and static / contact quenching) operate, and the measured R0 value for oligonucleotides labeled with donor fluorophore and acceptor fluorophore / quencher will depend on the length and type of linker used to connect the donor fluorophore and acceptor fluorophore / quencher to the oligonucleotide. For this reason, an accurate or better estimation of the R0 value requires the measurement of the energy transfer efficiency "E" over the 2R0 distance to eliminate any contribution of static / contact quenching energy transfer. It may be advantageous to measure quenching of any donor fluorophore and acceptor fluorophore / quencher pair by providing a specific separation between the two moieties in a double-stranded structure that has no secondary structure or folds.

[0160] The number of bases separating the 3' end of the donor fluorophore-labeled primer / probe from the base carrying the donor fluorophore plus the number of bases separating the 3' end of the acceptor / quencher-labeled primer(s) from the base carrying the acceptor fluorophore / quencher will usually be within the range of 6-35 bases, preferably 8-30 bases, more preferably 10-25 bases, to avoid signal generation from non-specific primer-dimers or primer-dimer-like amplification products, depending on the donor fluorophore-acceptor fluorophore / quencher pair and the degree of overlap of the 3' ends of the donor fluorophore and acceptor fluorophore / quencher-labeled primer or primer-probe pairs, taking into account a maximum of 6 bases of overlap between the 3' ends of the labeled primers and between the 3' ends of the labeled primers and the 3' ends of the labeled probes. If the overlap between the 3' ends of the labeled primers and between the 3' end of the labeled primer and the 3' end of the labeled probe exceeds 6 bases, the longer end in the range of 6 to 35 bases is further extended.

[0161] If, in addition to the donor fluorophore of the donor fluorophore-labeled primer, a quencher or 4-8 bases of non-target sequence is also placed at the 5'-end of the donor fluorophore-labeled primer / probe, to avoid signal generation from non-specific primer-dimers or primer-dimer-like amplification products, the number of bases separating the 3'-end of the donor fluorophore-labeled primer / probe from the base carrying the donor fluorophore plus the number of bases separating the 3'-end of the acceptor / quencher-labeled primer from the base carrying the acceptor fluorophore / quencher is about 5-25 bases, preferably 10-20 bases, depending on the donor fluorophore-acceptor fluorophore / quencher pair and the overlap of the 3'-ends of the donor fluorophore and acceptor fluorophore / quencher-labeled primer pair or primer-probe pair.

[0162] Another object is to detect a target nucleic acid by applying nested nucleic acid amplification, where a first primer pair amplifies a first segment of the target nucleic acid and a second primer pair amplifies a second segment of the first segment. Either the second primer pair is a libra primer pair labeled with a donor fluorophore and an acceptor fluorophore of the invention, or one or both components of the second primer pair are labeled with an acceptor fluorophore or a quencher, and a donor fluorophore labeled probe is additionally used that hybridizes to the second segment, and the donor fluorophore labeled probe and the acceptor fluorophore or quencher labeled second primer pair is a donor fluorophore moiety labeled probe and acceptor fluorophore / quencher labeled primer(s) of the invention (libra primer-probe pair) as described above. Additionally, the first primer pair can also be provided labeled with an acceptor fluorophore / quencher. Additionally, a third primer pair can be used to amplify a larger segment of the target nucleic acid from which the first segment can be amplified. Furthermore, the amplification reaction can be performed in one or two steps, with the amplicon generated in the first step being amplified in the second step.

[0163] Another object is to detect the target nucleic acid by applying semi-nested nucleic acid amplification, where the first and second primers amplify a first segment of the target nucleic acid and the third primer together with the first primer amplifies a second segment of the first segment. Either the first and third primers are a libra primer pair labeled with a donor fluorophore and an acceptor fluorophore or quencher of the invention, or one or both of the first and third primers are labeled with an acceptor fluorophore or quencher, and a donor fluorophore-labeled probe that hybridizes to the second segment is additionally used, where the probe and the first and third primers are a donor fluorophore moiety-labeled probe and acceptor fluorophore / quencher-labeled primer(s) of the invention (libra primer-probe pair) as described above. Furthermore, the amplification reaction can be performed in one or two steps, where the amplicon generated in the first step is amplified in the second step.

[0164] Another objective is to detect nucleic acid or non-nucleic acid targets including but not limited to proteins, antigens, antibodies, lipids, glycosylated biomolecules, live or dead cells, cancer cells, stem cells, miniature embryonic-like stem cells, cancer stem cells, cancer proteins and small molecules such as DNA markers, methylated DNA, transcription factors, cytokines, carbohydrates / sugars, haptens, etc., where a first binding moiety with extremely high affinity for the nucleic acid or non-nucleic acid target is used to capture the nucleic acid or non-nucleic acid target, and a second binding moiety, which may be the same first binding moiety or may be a different binding moiety with extremely high affinity for the nucleic acid or non-nucleic acid target, is used to bind to the captured nucleic acid or non-nucleic acid target, or a third binding moiety that binds to the second binding moiety with extremely high affinity is used. The second or third binding moiety is provided with a synthetic or natural nucleic acid target molecule attached thereto that can be detected by nucleic acid amplification using a libra primer pair or a libra probe and primer pair labeled with a donor fluorophore and an acceptor fluorophore / quencher of the present invention after washing away any unbound second or third binding moiety to which a nucleic acid has been attached.

[0165] The binding moieties may be selected from, but are not limited to, the group of binding pairs antigen-antibody, protein-antiprotein antibody, antibody-antibody, antibody-anti-IgG antibody, primary antibody-secondary antibody, protein A-antibody, protein G-antibody, biotin-avidin, biotin-streptavidin, lectin-sugar, nucleic acid-nucleic acid, protein-nucleic acid, protein-protein, aptamer-aptamer, aptamer-nucleic acid, aptamer-protein, hapten-antihapten antibody, where the hapten is a small molecule, including but not limited to, fluorescent dyes, bromo-d-UTP, aflatoxins and other mycotoxins, peptides, sugars.

[0166] Another object is to capture cells or target nucleic acids (methylated or unmethylated) using a binding moiety, which can be an antibody, protein, biotin, avidin, streptavidin, aptamer or nucleic acid, and the captured cells or captured target nucleic acids are detected, with or without purification, by nucleic acid amplification using a libra primer pair or a libra probe and primer pair labeled with a donor fluorophore and an acceptor fluorophore / quencher of the present invention.

[0167] Another object is to use a first primer labeled with a first donor fluorophore, a second primer labeled with an acceptor fluorophore / quencher and a probe labeled with a second donor fluorophore, where the positions of the first donor fluorophore and the acceptor fluorophore / quencher on the two primers are selected such that the first donor fluorophore generates a first fluorescent signal upon target amplification. The primer labeled with the first donor fluorophore and the primer labeled with the acceptor fluorophore / quencher are the libra primer pair of the present invention.

[0168] Furthermore, the first and second donor fluorophores and the acceptor fluorophore / quencher are selected and their positions on the primer and the probe are selected such that the second donor fluorophore of the probe generates a second fluorescent signal when the probe labeled with the second donor fluorophore hybridizes to the target amplification product. The probe labeled with the second donor fluorophore, the primer labeled with the first donor fluorophore and the primer labeled with the acceptor fluorophore / quencher are a libra primer-probe pair of the invention, where either the first donor fluorophore is the donor of the second donor fluorophore or, preferably, the second donor fluorophore is the donor of the first donor fluorophore.

[0169] Another object is to amplify a target nucleic acid sequence using a primer labeled with a donor fluorophore and a primer labeled with an acceptor fluorophore. The donor fluorophore and the acceptor fluorophore labeled primers are selected and labeled such that the energy transfer between the donor and the acceptor in the target amplification product is 20-70 percent, preferably 30-40 percent. The separation between the donor and the acceptor fluorophore in the target amplification is preferably 8-25 bases, more preferably 12-20 bases, depending on the spectral properties of the donor and acceptor fluorophore pair, such that the target amplification product produces a melting curve or melting temperature that is sufficiently separated from the melting curve or melting temperature of the primer dimer. The donor fluorophore is excited and the emission of the acceptor fluorophore (FRET signal) is measured to provide an estimate of the target amplification.

[0170] The number of bases separating the donor fluorophore-labeled base and the 3' end of the donor fluorophore-labeled primer plus the number of bases separating the acceptor fluorophore-labeled base and the 3' end of the acceptor fluorophore-labeled primer minus the number of bases that may overlap between the 3' ends of the two primers is plus or minus three, preferably plus or minus one, or zero, or more preferably, the two labeled bases are positioned opposite each other in the primer dimer such that there is static or contact quenching between the donor and acceptor fluorophores and there is energy transfer from the donor fluorophore to the acceptor fluorophore resulting in quenching of the donor while there is quenching of the acceptor fluorophore emission by static or contact quenching, resulting in zero or near zero signal from the primer dimer.

[0171] In another extension of this embodiment, instead of a pair of donor fluorophore-labeled and acceptor-labeled primers, a donor fluorophore-labeled probe and an acceptor fluorophore-labeled primer are used in the same format.

[0172] Another object is to use the donor fluorophore-labeled primer and the acceptor fluorophore / quencher-labeled primer of the present invention in allele-specific amplification, preferably allele PCR, where one of the primers is allele-specific, the second base from its 3' end is the allele base (targeted mutant base), the donor fluorophore- or acceptor fluorophore / quencher-labeled base of the allele primer is a thymine base 2-5 bases, preferably 3-4 bases away from the 3' end of the allele primer, and further, the donor fluorophore- or acceptor fluorophore / quencher-labeled T base may have a base mismatch of G to T or C to T or A to T, preferably G to T or C to T, with the corresponding base of the target sequence for better discrimination between the two alleles. Furthermore, the above two labeled primers are libra primers labeled with the donor fluorophore and acceptor fluorophore of the present invention.

[0173] In another extension, a donor fluorophore-labeled probe is used in conjunction with an acceptor fluorophore / quencher-labeled primer of the invention, the acceptor fluorophore / quencher-labeled primer being preferably an allele-specific primer.Furthermore, with the same specifications as above, the labeled probe and primer are a libra probe-primer pair labeled with a donor fluorophore and an acceptor fluorophore / quencher-labeled primer of the invention.

[0174] Another object of the present invention is to provide at least a positive control template(s), as well as at least a donor fluorophore-labeled primer and an acceptor fluorophore / quencher-labeled primer [libra primer pair] or at least a donor fluorophore-labeled probe and an acceptor fluorophore / quencher-labeled primer(s) [libra primer-probe pair(s)] of the present invention for the positive control template(s).

[0175] Another object is to use multiplexing using multiple libra primer pairs labeled with donor fluorophores and acceptor fluorophore / quencher labels of the present invention, or multiple libra probes and primer pairs labeled with donor fluorophores and acceptor fluorophore / quencher labels, for detection and / or quantification of multiple target sequences.

[0176] Another object is to amplify at least one target sequence using one non-target primer in conjunction with a target specific primer or using two non-target primers, where the non-target primer sequence(s) are added or incorporated at one or two ends of the target sequence by any of the methods known in the art, and the combination of one target specific primer and one non-target primer and the combination of two non-target primers are libra primer pairs labeled with donor fluorophore and acceptor fluorophore of the present invention. Additionally, the non-target sequence(s) can be added to the target sequence(s) using any of the methods known in the art, including, but not limited to, adding the non-target sequence(s) to the 5' end and extending them in a polymerization or PCR reaction (including Tail PCR reaction, etc.), PCR primer(s), or hybridizing a first target-specific single-stranded oligonucleotide carrying the sequence of the first non-target sequence at its 5' end and a second target-specific single-stranded oligonucleotide carrying the sequence of the second non-target sequence at its 3' end to one strand of the target sequence and ligating the first target-specific oligonucleotide to the second target-specific oligonucleotide using a ligase enzyme (the two target-specific single-stranded oligonucleotides are two consecutive sequences, one of which has a phosphate group for ligation). Alternatively, the method includes using a polymerase to extend a first single-stranded sequence having a first non-target sequence at its 5' end and a few bases at its 3' end that overlap with the 3' end of the single-stranded RNA or microRNA or m-RNA or single-stranded or double-stranded DNA target, and similarly extending a second single-stranded sequence having a second non-target sequence at its 5' end and a few bases at its 3' end that overlap with the 3' end of the extension product (on the single-stranded RNA or DNA target).

[0177] Moreover, in tail PCR, a particular objective is to use two tail primers, each of which contains an amplification primer or priming sequence and a tail sequence at the 5' end of the primer or priming sequence, where the tail sequence is not a target specific sequence, and another pair of primers corresponding to the two non-target tail sequences are used to initiate and drive the target amplification. The primer pairs corresponding to the two tail sequences are the libra primer pairs labeled with the donor fluorophore and acceptor fluorophore / quencher of the present invention.

[0178] A further object is to provide a non-target primer as described above labeled with a donor fluorophore moiety, which is quenched by additionally providing an acceptor fluorophore / quencher or 4-8 non-target sequences at its 5' end.

[0179] Another object is to amplify the target using a first non-target primer carrying a promoter sequence at the 5' end and a poly-T sequence at the 3' end followed by one or two non-thymine bases or not (preferably) and a second target-specific primer, the first non-target primer and the second target-specific primer being a libra primer pair labeled with a donor fluorophore and an acceptor fluorophore of the present invention. The first non-target primer is extended on the target sequence (RNA / DNA, preferably messenger RNA, m-RNA), and the second target-specific primer is located on the extended strand and extended by a polymerase(s) including a reverse transcriptase and a DNA polymerase, generating a template for the RNA polymerase to transcribe the RNA sequence, and the RNA is transcribed. The primer extension and RNA transcription are repeated many times to linearly amplify the target sequence. In another extension, a double-stranded adapter carrying a promoter sequence and a 3'-end overhang of several bases is used instead of the first non-target primer sequence described above. The target DNA may be single-stranded or double-stranded. In a further extension, a probe labeled with a donor fluorophore is also used, the labeled probe and primer(s) being a libra primer-probe pair of the invention.

[0180] Another object is to covalently attach donor fluorophore or acceptor fluorophore / quencher labeled primers or donor fluorophore labeled probes (through their 5' or 3' ends or internal base / internal linkages) to a solid surface such as glass or glass wafer or plastic (transparent or semi-transparent) or well or spot, and provide other primer(s) in a reaction mixture in contact with the solid surface of a reaction chamber for nucleic acid amplification. A large number of labeled primers or probes for a single nucleic acid target sequence or multiple nucleic acid target sequences can be covalently attached to the solid surface of a reaction chamber(s) [well(s)] for detecting single or multiple nucleic acid targets.

[0181] Another object is to use the donor fluorophore and acceptor fluorophore / quencher labeled libra primer pairs or donor fluorophore and acceptor fluorophore / quencher labeled libra probe-primer pairs of the present invention for absolute quantification of target sequence(s). One aspect of absolute quantification of nucleic acid targets by amplification is to apply a Poisson distribution to the nucleic acid amplification reaction. Applying a Poisson distribution requires that the probability of a single target sequence being present during the amplification reaction is less than 1, and the same amplification reaction mixture is divided into thousands of droplets of sub-nanoliter volume, and the amplification reaction signal of each droplet is measured at the end point of the amplification reaction, in sharp contrast to the measurement of the signal in the logarithmic phase of amplification used in PCR or qPCR. This is known as digital PCR or droplet PCR. The problem with digital PCR is that the use of end point measurements and the use of low amounts of primers or primers and probes for this purpose causes many amplification reactions to fail. As a result, 20,000-40,000 droplets are generated for good quantification, which requires expensive dedicated equipment. The use of libra primer pairs or libra probe-primer pairs allows more primers and probes to be used, resulting in fewer failed reactions, and therefore fewer droplets or fewer very small volume reactions to achieve good absolute quantification of the target sequence(s).

[0182] In certain embodiments, a method for synthesizing a nucleic acid molecule is provided, the method comprising contacting a target nucleic acid sequence with a mixture of at least one primer labeled with a donor fluorophore and one or more primers labeled with an acceptor fluorophore or quencher, one or more nucleoside triphosphates and / or deoxynucleoside triphosphates, and a thermostable or non-thermostable polymerase, wherein signals from non-specific primer dimer-like products are eliminated or substantially eliminated. In certain embodiments, a method for synthesizing a nucleic acid molecule is provided, the method comprising contacting a target nucleic acid sequence with a mixture of at least one probe labeled with a donor fluorophore and one or more primers labeled with an acceptor fluorophore or quencher, one or more nucleoside triphosphates and / or deoxynucleoside triphosphates, and a thermostable or non-thermostable polymerase, wherein signals from non-specific primer dimer-like products are eliminated or substantially eliminated. In certain embodiments, the polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.

[0183] In certain embodiments, kit(s) for carrying out certain methods of the invention are provided. The kit(s) comprise at least one primer pair separately labeled with a donor fluorophore or an acceptor fluorophore or a non-radioactive quencher in one or more containers. In certain embodiments, kit(s) for carrying out certain methods of the invention are provided, the kit(s) comprise at least one primer pair separately labeled with a donor fluorophore or an acceptor fluorophore or a non-radioactive quencher in one or more containers, the primer pair being a common universal primer pair for amplifying any target sequence. In certain embodiments, the kit(s) comprise at least one probe labeled with a donor fluorophore and at least one or more primers labeled with an acceptor fluorophore or a non-radioactive quencher. In addition, the kit(s) may also contain a reaction mixture containing all necessary components (PCR mix or PCR master mix or amplification reaction mix), one or more nucleoside triphosphates and / or deoxynucleoside triphosphates, reaction buffer, thermostable or non-thermostable polymerase(s) or thermostable or non-thermostable ligase, enzymes topoisomerase, recombinase, thermostable or non-thermostable helicase, thermostable or non-thermostable single-stranded binding protein(s). In addition, the kit(s) may also contain at least one positive control template and a labeled primer pair or a labeled primer probe pair for amplifying the positive control template.

[0184] Primers and probes are oligonucleotides 10-50 bases in length, preferably 15-35 bases in length, and more preferably 20-30 bases in length, and may be perfectly or imperfectly complementary to the target sequence, and may contain one or more modified bases or modified sugar moieties (singular or plural), so long as the desired properties resulting from the complementarity are obtained, i.e., the ability to hybridize to or prime the target is not lost.

[0185] The target nucleic acid is selected from natural, synthetic or semi-synthetic single-stranded or double-stranded DNA or RNA, single-stranded or double-stranded c-DNA, genomic DNA, methylated DNA, mitochondrial DNA, exosomal DNA, plasmid DNA, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (m-RNA), small RNA (including but not limited to microRNA, sRNA, stRNA, snoRNA, ncRNA), stem cell-derived DNA including very small embryonic-like stem cells, viral DNA or RNA, or cancer cell DNA (from any source including but not limited to body fluids, biopsy samples, tumors, pus, saliva, feces, cancer stem cells), and single-stranded or double-stranded synthetic or semi-synthetic DNA or RNA generated by adding one or two non-targeting synthetic sequences to the ends of the target nucleic acid. Moreover, the target nucleic acid does not have to constitute the entire nucleic acid molecule.

[0186] The donor fluorophore and acceptor fluorophore / quencher labels on internal bases can be on any of the four bases of the deoxynucleotide, on the deoxyuridine or, preferably, on the thymine base.

[0187] Another object is to select nucleic acid amplification from polymerase chain reaction (PCR) including but not limited to quantitative PCR (qPCR), reverse transcriptase PCR (RT-PCR), allelic PCR, nested PCR, semi-nested PCR, methylation status PCR, emulsion PCR, tail PCR, droplet PCR or digital PCR (dPCR), in-situ PCR, isothermal nucleic acid amplification including but not limited to loop-mediated amplification (LAMP), recombinase polymerase amplification (RPA), helicase polymerase amplification (HPA), NASBA, and variations of isothermal amplification including but not limited to allelic primer or primer-probe pairs, nested or semi-nested primer pairs, nested or semi-nested primer-probe pairs variations of isothermal amplification.

[0188] The length of the target amplification product in PCR amplification is 40-200 base pairs, preferably 70-120 base pairs. The length of the target amplification product in isothermal loop-mediated nucleic acid amplification (LAMP) is 100-600 base pairs, preferably 150-280 base pairs, and the inner or loop primers are labeled as libra primer pairs. In recombinase polymerase amplification (RPA), the length of the target amplification product can be as long as 1000 base pairs, but is preferably 100-200 base pairs, and the primers are labeled as libra primer pairs. In helicase polymerase amplification (HPA), the length of the target amplification product can be as long as 1000 base pairs, but is preferably 100-200 base pairs.

[0189] The donor fluorophores, acceptor fluorophores and quenchers of the present invention may be any of a variety of dyes, including reporter dyes or dyes, fluorescein, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 6-FAM (azide), 2'7'-dimethoxy-4'5-6-carboxyfluorescein (JOE), 5-(4,6-dichlorotriazin-2yl)aminofluorescein (DTAF), fluorescein isothiocyanate, HEX (hexachlorofluorescein), TET (tetrachlorofluorescein), VIC (Victoria Blue), MAX™ VIC, A VIC® (ThermoFisher Blue), which has a nearly identical spectral profile to VIC. SUN™, TYE™ 563, NED, fluorescamine, pyrene, pyrene butyrate, succimidyl 1-pyrene butyrate, rhodamine (Rhod), rhodamine 123, rhodamine B, sulforhodamine, 6-carboxyrhodamine (R6G), 6-carboxy-X rhodamine (ROX), sulforhodamine b, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), Texas Red®-X, Texas Red®-X, TEX615 N',N',N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), TAMRA™ (azide), Rhodamine Green™-X, Rhodamine Red™, tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC), terbium chelate, europium chelate, quantum dots, graphene quantum dots, 5-(2'-aminoethyl)aminonaphthylamide-3,5 disulfonate (Lucifer Yellow vs), 7-amino-4-methylcoumarin (amc, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151), cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.Cyanine dyes (sulfonated or non-sulfonated), including but not limited to Cyanine 5, Cyanine 7, [5-dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), DABSYL, 4-dimethylaminophenylazophenyl)-4'-isothiocyanate (DABITC), IAEDANS (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalenesulfonic acid), EDANS, QST 7, QSY9, QSY 21, QSY 35 (QSY dyes are diaryl-rhodamine derivatives), BIODIPY dyes, including but not limited to BIODIPY FL, Alexafluor Alexafluor dyes, including but not limited to 350, 488, 546, 555, 568, 594, 647 dyes; ATTO dyes, including but not limited to ATTO™ 488, ATTO™ 532, ATTO™ 550, ATTO™ 565, ATTO™, ATTO™ 590, ATTO™ 633, ATTO™ 647N; Yakima Yellow, LI-COR IRDyes®, IRDye® 700, IRDye® 800, IRDye® 800CW, Lightcycler® 640, Dy750, non-radioactive quenchers (Nanogold, Blackhole Quencher 0, Blackhole Quencher 1, Blackhole Quencher 2, Blackhole Quencher 3, Eclipse Quencher, Dark Quencher, IDT Quencher Iowa) The dyes may be selected from the group consisting of, but not limited to, Iowa Black RQ, Iowa Black FQ, ZEN™, TAO and / or ZEN, nanoparticle quenchers, MGB dyes.

[0190] Further dyes can be selected from those listed in the references (Penguang Wu et al, Analytical Biochemistry vol - 218, pages 1 - 13, 1994; Robert H Fairclough et al Methods in Enzymology, vol - 48, pages - 347 - 379, 1978) and can be found at www.ncbi.nlm.nih.govsite for common FRET pairs and Molecular Probes' Hand Book of Fluorescent Probes and Research Products. New fluorophores and quenchers are constantly being developed and can be used without special requirements. In fact, there are a large number of fluorophores and quenchers, which would make the complete list quite large.

[0191] The general method is as follows.

[0192] 1) All oligonucleotides were purchased HPLC purified from Eurogentec and other commercial vendors or could be chemically synthesized by solid phase phosphotriester chemistry.

[0193] 2) Preparation of chromosomal DNA: E. coli cells were grown overnight in LB medium, centrifuged at 5000 rpm for 10 min, washed with wash buffer (20 mM Tris-HCl pH-7.5, 50 mM NaCl, 1 mM EDTA) and centrifuged at 5000 rpm for 5 min.

[0194] The cell pellet was used for chromosomal DNA isolation using a Qiagen chromosomal DNA purification kit according to the kit's protocol. Purified DNA was estimated spectrophotometrically by measuring the optical density at 260 nm.

[0195] 3) Synthesis of primers and probes labeled with fluorophores or quenchers: Primers and probes labeled with internal donor fluorophores or acceptor fluorophore / quenchers are chemically synthesized using four deoxynucleotide phosphoramidites and donor fluorophore or acceptor fluorophore / quencher labeled thymidine (dT) phosphoramidites (Proc. Natl. Acad. Sci. USA, 1995, vol - 92, pages 9347 - 9351) for internal labeling by solid phase phosphotriester chemistry on an oligo synthesizer.

[0196] Using a 3' end-labeled phosphoramidite labeled with a donor fluorophore, synthesize a probe labeled with a donor fluorophore at the 3' end.

[0197] 5'-terminated phosphoramidites labeled with donor or acceptor fluorophore / quencher or biotin are used to synthesize probes labeled with a donor or acceptor fluorophore / quencher or biotin at the 5' end.

[0198] Primers and probes labeled with donor fluorophores or acceptor fluorophores / quenchers can be obtained from commercial vendors.

[0199] 4) Purification of oligonucleotides: The primers and probes used are purified by HPLC or polyacrylamide gel electrophoresis (PAGE).

[0200] HPLC purification of oligonucleotides is typically performed on a C-18 reverse phase column using a linear gradient of 0.1 M triethylammonium acetate (pH-6.5) in 75% acetonitrile and 0.1 M triethylammonium acetate (pH-6.5).

[0201] PAGE purification is performed by applying the desalted oligonucleotide preparation from the oligo synthesizer to an 8% polyacrylamide gel and applying high voltage (500-1000 volts depending on the length of the gel).

[0202] The slowest migrating band is excised from the gel, crushed and soaked in elution buffer (ammonium acetate buffer) or in a gel elution apparatus, after which the purified oligonucleotide is eluted from the gel.

[0203] Many methods for HPLC and PAGE purification of oligonucleotides are available in the art. Commercial vendors supplying unlabeled and labeled oligonucleotides will provide the necessary purification of oligonucleotides if requested.

[0204] Various types of purification are available, including cartridge purification, HPLC purification or PAGE (polyacrylamide gel electrophoresis) purification.

[0205] All labeled oligonucleotides as well as unlabeled oligonucleotides and synthetic sequences were purified by the vendor by HPLC or PAGE.

[0206] 5. PCR amplification conditions (using primer pairs or primer-probe pairs): Amplification reactions were performed in 10 or 15 μl amplification reaction volumes using 2× Kapa PCR Master Mix supplied by commercial vendor Kapa Corporation, labeled and unlabeled primers at a concentration of 0.2 μM, and labeled probe.

[0207] Alternatively, a PCR reaction mix containing 20 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.01% gelatin, and 2.0 or 3.0 units of Taq polymerase can be used in place of the commercially available reaction master mix.

[0208] The thermal cycling parameters used for PCR were initial denaturation at 95°C for 2 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 45 s, and extension at 72°C for 15 s.

[0209] A final denaturation at 94°C for 20 min was also analyzed.

[0210] PCR amplification was performed in a Bio Rad series 1000 cycler equipped with a CFX 384 RT PCR block.

[0211] All primers and probes were designed and analyzed using primer premier on the IDT website, or Gene script software, or IDT Oligoanalyzer software.

[0212] This advance is further illustrated below by the following non-limiting illustrative examples. The following Examples 1-8 illustrate features of the present invention. EXAMPLES

[0213] Example 1: A distance or separation between a donor fluorophore and an acceptor fluorophore / quencher such that energy transfer between the two moieties is minimal The separation between the donor and acceptor fluorophore / quencher moieties, at which energy transfer from the donor fluorophore to the acceptor fluorophore / quencher is minimal, varies from donor fluorophore to acceptor fluorophore / quencher pair to donor fluorophore / acceptor fluorophore / quencher pair. Every donor fluorophore and acceptor fluorophore / quencher pair has its own Förster radius (R0) (the separation at which the efficiency of energy transfer between the donor fluorophore and the acceptor fluorophore / quencher is 0.5), which ranges from 22 Å to 75 Å, and energy transfer between donor fluorophores and acceptor fluorophores / quenchers separated by 2R0 is negligible. Thus, for separations greater than 2R0, energy transfer is minimal, with corresponding separations of 12 bases to 40 bases. However, because of the extremely high level of amplification that occurs in PCR, a detectable signal can be generated with a small amount of energy transfer. In addition, the length of the linkers used to attach the donor fluorophore and acceptor fluorophore / quencher to the oligonucleotide primers and probes is also important in determining the actual separation between the two moieties. Furthermore, the donor fluorophore and acceptor fluorophore / quencher molecules are mostly hydrophobic and tend to interact with each other when placed in close proximity. Therefore, if donor and acceptor fluorophore / quencher pairs are selected from the lower end of the R0 value range, the separation needs to be even greater than their 2R0 distance. Therefore, for separations where the energy transfer is small, it is better to add a small additional separation to the 2R0 separation of the donor and acceptor / quencher pairs.

[0214] A separation of 40 bases or more between the donor fluorophore and the acceptor fluorophore / quencher in a target amplification reaction can be safely used without any adverse effects when designing labeled primers and labeled probes for amplification of target sequences. In this example, the donor fluorophore FAM and the BHQ1 quencher were used with a separation of more than 40 bases / base pairs in the target amplicon.

[0215] Example 2: Measurement of fluorescence enhancement of donor fluorophore FAM-labeled primers and probes For this purpose, the fluorescence of oligonucleotide primers and probes (2.5 pmol each) labeled with the donor fluorophore FAM was measured in a BioRad series 1000 cycler equipped with a CFX 384 RT PCR block in a buffer of 20 mM Tris-HCl Ph-8.4, 50 mM KCl and 2.0 mM MgCl2 in a total volume of 15 μl, both without template (F1) and hybridized to the HPLC-purified synthetic template 3, SEQ ID NO: 18, complementary to these oligonucleotides (12.5 pmol each) by first heating at 95 °C for 2 min and then reducing the temperature to 25 °C at a rate of 0.1 °C / s (F2).

[0216] The difference in fluorescence values ​​between the two measurements gave the degree of fluorescence enhancement of the fluorophore FAM upon hybridization to the synthetic template. The oligonucleotides used were SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:12. The percentage enhancement of the fluorescence of the donor fluorophore upon hybridization of the FAM-labeled oligo to the synthetic template sequence was calculated from the two fluorescence signal measurements above (enhancement = [{(F2-F1) / F1} x 100 percent]). Different protocols and different synthetic templates were used. There was some variation between tubes, but the best results were taken into account. Measurements on a spectrofluorometer give a better estimate of the enhancement percentage than using a real-time PCR machine.

[0217] result: The % enhancement is as follows (using synthetic template-3, SEQ ID NO:18): 1) SEQ ID NO:5 - (-) 2.0% 2) SEQ ID NO:6 - 17% 3) SEQ ID NO:7 - 15% 4) SEQ ID NO:8 - 45% 5) SEQ ID NO:9 - 40% 6) SEQ ID NO:11 - (-) 4% 7) SEQ ID NO:12 - (-) 28%

[0218] Conclusion: The degree of enhancement of FAM fluorescence of FAM-labeled oligonucleotides varies depending on the location of the FAM-labeled base at the 3'-end or 5'-end or at an internal base of the FAM-labeled oligonucleotide. Furthermore, the value of the enhancement rate also depends on the purity of the HPLC-purified synthetic template.

[0219] It is known that the presence of G bases in the vicinity of the fluorophore-labeled base plays an important role in quenching the fluorescence of fluorophore-labeled oligonucleotides, which further depends on the base sequence, the primary and secondary structure of the oligonucleotide, the local sequence and the length of the linker, and changes when the labeled oligonucleotide hybridizes to a complementary sequence or is extended (Seidel et al, J Phys Chem, 1996, Vol-100: 5541 - 5553; Kelly et al, Science, 1999, Vol - 283: 375 - 381; Nazarenko et al, Nucleic Acid Research, 2002, Vol. 30, No. 9, 2089 - 2195).

[0220] Example 3: Measurement of the degree of quenching between the donor fluorophore FAM and the quencher BHQ1 For this purpose, the fluorescence emission of oligonucleotide primers and probes (2.5 pmol each) labeled with the donor fluorophore FAM was measured in a buffer of 20 mM Tris-HCl Ph-8.4, 50 mM KCl and 2.0 mM MgCl2 in a total volume of 15 μl, either unhybridized (F1) or hybridized separately to HPLC-purified synthetic templates (12.5 pmol) with complementarity to these oligonucleotides for 10 min at 55 °C (F2), as well as hybridized separately to HPLC-purified synthetic templates (12.5 pmol each) with complementarity to both oligonucleotides (F3) at 55 °C for 10 min with FAM-labeled oligonucleotides (2.5 pmol each) and BHQ1 quencher-labeled oligonucleotides (3.75 pmol each). The difference between F2 and F1 values ​​indicates the enhancement. F2-F1 values ​​indicate the enhanced fluorescence values, and F3-F2 indicates the net quenching. The quenching rate was calculated as [(F3-F2) / F2] x 100%. The experiment was performed using a Bio-Rad series 1000 cycles equipped with a CFX384 RT-PCR block.

[0221] There was some variation between tubes, but this was taken into account as the best possible outcome. A spectrofluorometer is a better instrument for this measurement than a real-time PCR machine. Also, hybridization of FAM-labeled oligos with unlabeled complementary oligos and BHQ1-labeled oligos gives better results.

[0222] The oligonucleotides and synthetic templates used and the quenching rates for the various separations are as follows: 1) Template 1 - SEQ ID NO:16, SEQ ID NO:5 and SEQ ID NO:4, Separation - 6 bases, and Quenching - 26%; 2) Template 1 - SEQ ID NO:16, SEQ ID NO:6 and SEQ ID NO:4, separation - 9 bases, and quenching - 21%; 3) Template 2 - SEQ ID NO:17, SEQ ID NO:5 and SEQ ID NO:4, separation - 8 bases, and quenching - 23%; 4) Template 2 - SEQ ID NO:17, SEQ ID NO:6 and SEQ ID NO:4, separation - 11 bases, and quenching - 16%.

[0223] The sequences of the synthetic templates used in this experiment are template-1 (SEQ ID NO: 16) and template-2 (SEQ ID NO: 17).

[0224] The relative orientation of the FAM fluorophore-labeled oligonucleotide and the BHQ1 quencher-labeled oligonucleotide on the synthetic template is shown in FIG.

[0225] Template-1, SEQ ID NO:16: 5' TTC TAC GGT TTA CCG AAT GTG A / AG AAT GGT CAC TGG CTT ATC ACC C 3' 3' AAG ATG CCA AAT GGC TTA CAC T / TC TTA CCA GAG ACC GAA TAG TGG G 5' Template-2, SEQ ID NO:17: 5' TTC TAC GGT TTA CCG AAT GTG A / AT / AG AAT GGT CAC TGG CTT ATC ACC C 3' 3' AAG ATG CCA AAT GGC TTA CAC T / TA / TC TTA CCA GAG ACC GAA TAG TGG G 5' Template-3, SEQ ID NO:18: 5' CAT CAC CAA TAA ACG CCG AGA / AGA ATG GTC ACT GGC TTA TCA CCC 3' 3' GTA GTG GTT ATT TGC GGC TCT / TCT TAC CAG TGA CCG AAT AGT GGG 5' Red - sequence number 4, Blue - sequence numbers 5 / 6, Magenta - sequence numbers 7 / 8 / 9

[0226] The individual sequences (5-9) are color-coded to show the relative alignment of SEQ ID NOs: 4, 5, 6, 7, 8, 9 to each other when hybridized to different templates. SEQ ID NOs: 5 and 6 have the same sequence with different label positions. SEQ ID NOs: 7, 8, and 9 have the same sequence with different label positions.

[0227] Example 4: Amplification of the threonine synthase gene of the bacterium Escherichia coli using FAM-labeled and BHQ1-labeled primers Target sequence: SEQ ID NO: 26 gatcctctcg gcgtttattg gtgatgaaat cccacaggaa atcctggaag agcgcgtgcg cgcggcgttt gccttcccgg ctccggtcgc caatgttgaa agcgatgtcg gttgtctgga

[0228] Amplification reactions were performed in triplicate in 15 μl or 10 μl reaction volumes using 0.2 μM each of the FAM-labeled forward primers (SEQ ID NO: 7, 8, and 9) and 0.2 μM of the BHQ1-labeled reverse primer (SEQ ID NO: 10) separately, with 2× Kapa PCR master mix, 1 ng of E. coli chromosomal DNA preparation or no DNA (as control), to amplify a 111 base pair segment of the E. coli threonine synthase gene. In any one set of amplification reactions (set of reactions with or without template), any one of the FAM-labeled primers and the BHQ1-labeled reverse primer were used.

[0229] For comparison, amplification reactions were performed in triplicate in 15 μl or 10 μl reaction volumes using the same 1 ng amount of E. coli chromosomal DNA or no DNA (control reaction) to amplify a 145 base pair segment of the E. coli homoserine kinase gene using 0.2 μM Taqman probe (SEQ ID NO: 15) carrying FAM at the 5' end and quencher BHQ1 at the 3' end, 0.6 μM each of forward primer (SEQ ID NO: 13) and reverse primer (SEQ ID NO: 14) and 2× Kapa PCR master mix.

[0230] SEQ ID NOs: 7, 8 and 9 have the same nucleotide sequence but different FAM label positions. Heterodimer analysis of the FAM-labeled forward primer and the BHQ1-labeled reverse primer showed that their 3' ends overlapped by 9 bases, of which 4 bases were identical, and a heterodimer was formed with ΔG = (-) 5.0 kcal / mol.

[0231] For SEQ ID NOs: 7, 8, and 9, the sum of the separation of the FAM-labeled base from the 3' end of the FAM-labeled primer and the separation of the BHQ1-labeled base from the 3' end of the BHQ1-labeled reverse primer is 21 bases, 11 bases, and 14 bases, respectively, and the estimated separation of the FAM-labeled base and the BHQ1-labeled base in the primer dimer is 12 bases, 2 bases, and 5 bases, respectively.

[0232] The amplification reactions using the forward primer of SEQ ID NO:8 and the reverse primer of SEQ ID NO:10 showed an average amplification Cq value of 19.8 in the amplification reactions using template DNA (Figure 3) and a Cq value of 0 in the no template control reactions (Figure 4). Figures 3A and 4A show the respective melting curves.

[0233] Amplification reactions using the forward primer of SEQ ID NO:9 and the reverse primer of SEQ ID NO:10 showed an average amplification Cq value of 19.2 in the amplification reactions using template DNA (Figure 5), and Cq values ​​of 37.4, 38.6, 40.4, and 0 in the no template control reactions (Figure 6). Figures 5A and 6A are the respective melting curves.

[0234] The amplification reactions using the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 10 showed an average amplification Cq value of 21.8 in the amplification reactions using template DNA (Figure 7) and a Cq value of 0 in the no template control reactions (Figure 8). Figures 7A and 8A are the respective melting curves.

[0235] Discussion: For the forward primer SEQ ID NO:9 and SEQ ID NO:10, the total separation of the FAM-labeled base from the 3' end of SEQ ID NO:9 and the BHQ1-labeled base from the 3' end of the SEQ ID NO:10 reverse primer is 14 bases. These two primers can form a heterodimer with 9 bases overlap and ΔG=(-)5.0 kcal / mol (in silico analysis). As a result, the donor fluorophore Fam of the forward primer and the quencher BHQ1 of the reverse primer are expected to be in close proximity in the non-specific primer dimer, separated by 5 intervening bases (6 base distance). As demonstrated in Example 2, approximately 40% enhancement is observed upon hybridization of the FAM-labeled primer SEQ ID NO:9 to the complementary sequence. The degree of quenching required to balance the 40% enhancement is approximately 28.6%. The degree of quenching between the FAM fluorophore and the BHQ1 quencher at a 5-base separation should be greater than 26% when measured at a 6-base separation (Example 3), and may be less than the 28.6% quenching required to precisely balance the enhancement of FAM fluorescence in the primer dimer. As a result, there is an effective net fluorescence emission from the primer dimer, resulting in a signal from the primer dimer. Thus, the no-template control reaction showed amplification Cq values ​​of 37.4, 38.6, 40.4, and 0 using the primers SEQ ID NO:9 and SEQ ID NO:10 of the present invention. The placement of the FAM fluorophore and the quencher BHQ1 on the primer is on the higher side of the optimal placement, which results in zero signal from the primer dimer. However, this signal is near zero signal, and can be used for target amplification, even if it is not the optimal primer pair to zero signal. The 40% enhancement measured for SEQ ID NO:9 may be slightly less than the actual enhancement.

[0236] Furthermore, regarding the issue of the forward primer of SEQ ID NO:9 forming a primer dimer instead of a forward primer and a reverse primer, amplification reactions were performed without a template, with a forward primer at a concentration of 0.4 μM (twice the concentration normally used) and without a reverse primer (Figure 9) [Figure 9A is a melting curve], with a forward primer at a concentration of 0.1 μM and a reverse primer at a concentration of 0.2 μM, with a forward primer at a concentration of 0.1 μM (half the concentration normally used) and a reverse primer at a concentration of 0.3 μM (1.5 times the concentration normally used) (Figure 10, Figure 10A is a melting curve), and with a forward primer at a concentration of 0.2 μM and a reverse primer at a concentration of 0.2 μM (normally used for labeled primers) (Figure 12, Figure 12A is a melting curve). There was no formation of primer dimers in these reactions. However, in amplification reactions using 0.2 μM (normal) concentration of forward primer and 0.3 μM (1.5 times the normal concentration of reverse primer) without any template DNA, primer dimers were formed (Figure 11, Figure 11A). Since no primer dimers were formed with FAM-labeled forward primer at twice the normally used concentration, the primer dimers formed in the no-template control reaction (Figure-6) were due to the formation of primer dimers between the FAM-labeled forward primer SEQ ID NO:9 and the BHQ1-labeled reverse primer SEQ ID NO:10, and not due to homodimer formation by the FAM-labeled forward primer. Furthermore, by using the FAM-labeled forward primer at half the normally used concentration and increasing the concentration of the BHQ1-labeled reverse primer, or by using the FAM-labeled forward primer at the normally used concentration and increasing the concentration of the BHQ1-labeled reverse primer, the formation of primer dimers between the FAM-labeled forward primer and the BHQ1-labeled reverse primer can be biased against self-dimers of the FAM-labeled forward primer. According to this policy, an amplification reaction was carried out using 0.1 μM of FAM-labeled forward primer (half the concentration normally used) and 0.3 μM of BHQ1-labeled reverse primer (1.5 times the normal concentration) (FIG. 13, FIG. 13A is the melting curve).The target sequence was amplified well without a significant decrease in sensitivity. On the other hand, the use of 0.2 μM (normally used concentration) of FAM-labeled forward primer and 0.3 μM (1.5 times the normal concentration) of BHQ1-labeled reverse primer (FIG. 14, FIG. 14A is the melting curve) showed slightly better amplification in comparison. Therefore, FAM-labeled forward primer (SEQ ID NO: 9) 0.2 μM (normally used concentration) and 0.3 μM of BHQ1-labeled reverse primer (1.5 times the normal concentration) can be used for this bias purpose without many adverse effects and homodimerization of FAM-labeled primer. It should be noted that the primers were designed to bias the formation of non-specific primer dimers with 4 base complementarity. These high base complementarities and ΔG=-5.0 KCal / mol are undesirable, and the ΔG value for heterodimer formation should be less than -3.0 KCal / mol.

[0237] For the primer pair SEQ ID NO:8 and SEQ ID NO:10, the total separation of the FAM-labeled base from the 3' end of SEQ ID NO:8 and the BHQ1-labeled base from the 3' end of SEQ ID NO:10 is 11 bases. There is a 9-base overlap, and the formation of the heterodimer can have a ΔG value of -5.0 KCal / mol (in silico analysis). As a result, the donor fluorophore FAM of the forward primer and the quencher BHQ1 of the reverse primer are expected to be in close proximity in the non-specific primer dimer, separated by two intervening bases. As demonstrated in Example (2), approximately 45% enhancement is observed upon hybridization of the FAM-labeled primer (sequence 8) to its complementary sequence. The degree of quenching required to balance the 45% enhancement is approximately 31%.

[0238] The degree of quenching between the FAM fluorophore and the BHQ1 quencher at a separation of 2 bases should be greater than 26% when measured at a separation of 6 bases (Example 3) and greater than 28.6% (for SEQ ID NO: 9 and SEQ ID NO: 10), which may be less than the 31% quenching required to precisely balance the enhancement of FAM fluorescence in the primer dimer. As a result, the primer dimer formed will either have roughly balanced enhancement and quenching or only slightly more quenching, resulting in no signal from the primer dimer or a slightly reduced signal due to excess quenching. It may be noted that at close separations of 2 and 5 bases, the degree of quenching may not be significantly different, considering the involvement of contact quenching at such short separations. The amplification curves (Figure 3) and melting curves (Figure 3A) of the amplification reactions of BHQ2 with SEQ ID NO: 8 and SEQ ID NO: 10 support this. As a result, the Cq value (19.8) is slightly increased compared to the Cq value (19.2) of the primer pair SEQ ID NO:9 and SEQ ID NO:10, indicating a slightly lower sensitivity compared to SEQ ID NO:9 and SEQ ID NO:10. This is because there is a slight net enhancement in the case of amplification with SEQ ID NO:9 and SEQ ID NO:10.

[0239] For the primer pair SEQ ID NO:7 and SEQ ID NO:10, the total separation of the FAM-labeled base from the 3' end of SEQ ID NO:7 and the BHQ1-labeled base from the 3' end of SEQ ID NO:10 is 21 bases. There is a 9-base overlap, and the formation of the heterodimer can have a ΔG value of -5.0 KCal / mol. As a result, the donor fluorophore FAM of the forward primer and the quencher BHQ1 of the reverse primer are expected to be in close proximity in the non-specific primer dimer, separated by 12 intervening bases. As demonstrated in Example (2), approximately 15% enhancement is observed upon hybridization of the FAM-labeled primer (SEQ ID NO:7) to the complementary sequence. The degree of quenching required to balance the 15% enhancement is approximately 13.05%.

[0240] The degree of quenching between the FAM fluorophore and the BHQ1 quencher at a separation of 12 bases should be slightly less than the 16% measured at a separation of 11 bases (Example 3), and would exceed the 13.05% required to precisely balance the enhancement of FAM fluorescence in the primer dimer. As a result, there is a net quenching of the primer dimers formed, resulting in no signal from the primer dimers and a reduction in signal due to excess quenching. The target amplification curves of the amplification using SEQ ID NO:7 and SEQ ID NO:10 (Figure 7) and the amplification curve of the no template control (Figure 8) confirm this. After 35 cycles of amplification of the no template control, a gradual downward reduction in the fluorescent signal or a negative curvature of the amplification curve can be observed. As a result, the Cq value (21.8) is increased compared to the Cq value of the primer pair SEQ ID NO:9 and SEQ ID NO:10 (19.2), indicating a lower sensitivity compared to the primer pair SEQ ID NO:9 and SEQ ID NO:10 and the Taqman probe amplification (Cq-21.2) (Figure 21). FIG. 22 is the no template control reaction of the Taqman assay.

[0241] Furthermore, the average Cq value of 19.2 for the primer pair of SEQ ID NO:9 and SEQ ID NO:10 is 2.0 units lower than the Cq value of 21.2 for the Taqman probe assay (SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15). The lower the Cq value, the higher the yield of the target amplification product and the higher the detection sensitivity. That is, the amplification of the primer pair of SEQ ID NO:9 and SEQ ID NO:10 demonstrates that the target amplification using SEQ ID NO:9 and SEQ ID NO:10 of the present disclosure is more sensitive than the Taqman assay. Typically, a difference in Cq value of 2.0 units corresponds to about a 5- to 7-fold difference in target quantity.

[0242] The Cq value of 19.8 for the primer pair SEQ ID NO:8 and SEQ ID NO:10 is 1.4 units lower than the Cq value of the Taqman probe assay (SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15) and 0.6 units higher than the Cq value of the primer pair SEQ ID NO:9 and SEQ ID NO:10. Amplification of the primer pair SEQ ID NO:8 and SEQ ID NO:10 demonstrates that the Taqman assay is also sensitive in target amplification, but less sensitive than the primer pair SEQ ID NO:9 and SEQ ID NO:10. The results for both primer pairs (SEQ ID NO:9 and SEQ ID NO:10 and SEQ ID NO:8 and SEQ ID NO:10) are consistent with the concept of offset / balance of fluorescence enhancement and quenching of the present invention.

[0243] [Table 1]

[0244] Furthermore, while the fluorescence enhancement for primer sequence SEQ ID NO:9 / SEQ ID NO:8 is only 40 / 45%, the degree of enhancement can be as high as 60%-80% as reported in the literature. In that case, the current Cq value difference of 2.0 units compared to the gold standard Taqman assay can be increased to 3.0-4.0 units, which is a huge leap in the target amplification signal and in the sensitivity of target detection or quantification by nucleic acid amplification using the disclosed method. The additional placement of a quencher at the 5' end of the FAM-labeled primer increases the fluorescence enhancement and thus the sensitivity is better.

[0245] It can be seen that the FAM fluorophore in SEQ ID NO:8 and SEQ ID NO:9 is approximately in the middle of the primer sequence, while in SEQ ID NO:7 it is near the 5' end of the sequence. In the reverse primer SEQ ID NO:10 the quencher BHQ1 is 5 bases away from the 3' end. Furthermore, it can be seen that to achieve zero or near zero signal the quencher BHQ1 needs to be closer to the FAM fluorophore to balance fluorescence enhancement and quenching in the non-specific primer dimer products, which requires a long base overlap (9 bases) between the 3' end of the forward primer sequence SEQ ID NO:8 or SEQ ID NO:9 and the 3' end of the reverse primer SEQ ID NO:10, and placement of BHQ1 near the 3' end in the reverse primer SEQ ID NO:10. Higher fluorescence enhancement is required for more sensitive target detection, which requires the donor fluorophore to be located near the middle of the donor fluorophore-labeled primer, and the acceptor or quencher moiety of the acceptor or quencher-labeled primer to be labeled near its 3' end. The fluorescence enhancement of the donor fluorophore is 1-20 percent less when it is placed near the 3' end, which can be seen in the case of the donor fluorophore FAM-labeled probes SEQ ID NO:5 and SEQ ID NO:6 in Example 5. If the donor fluorophore is placed between the middle and 5' end of the donor fluorophore-labeled primer (SEQ ID NO:7), far from the 3' end of the fluorophore-labeled primer, the acceptor or quencher must be placed near the 3' end of the acceptor- or quencher-labeled primer, and there is a longer overlap between the fluorophore-labeled forward primer (SEQ ID NO:7) and the BHQ1-labeled reverse primer (SEQ ID NO:10).

[0246] New lots of labeled primers and probes give better results. New lots always work better because these labeled oligonucleotides are not very stable unless stabilized, which some companies do with the right composition. The formation of non-specific primer-dimer products is a random / stochastic phenomenon, which may form at one time and not at another. Furthermore, the formation of non-specific products depends on the quality of the labeled primers and probes used and the presence of inhibitors. Labeled primers and probes were obtained in two different lots.

[0247] Example 5: Amplification of the threonine synthase gene of the bacterium Escherichia coli using a FAM-labeled probe and a BHQ1-labeled primer target DNA SEQ ID NO:3 tggcacaaat gctgacccat attgcgggtg ataagccagt gaccattctg accgcgacct ccggtgatac cggagcggca gtggctcatg ctttctacgg tttaccgaat gtgaaagtgg

[0248] Amplification reactions were performed in triplicate in 10 / 15 μl reaction volumes using 0.2 μM each of unlabeled forward primer (SEQ ID NO:1), separately using BHQ1-labeled reverse primers (SEQ ID NO:4, SEQ ID NO:2), separately using FAM-labeled probes (SEQ ID NO:5 and SEQ ID NO:6), and using 2× Kapa PCR Master Mix (Kapa Corporation) with 1 ng of E. coli chromosomal DNA preparation or without DNA (as control) to amplify a 113 base pair segment of the E. coli threonine synthase gene. In either set of amplification reactions (set of reactions with or without template), either one of the FAM-labeled probes and either one of the BHQ1-labeled reverse primers was used in combination with forward primer sequence 1.

[0249] For comparison, amplification reactions were performed in triplicate in 10 / 15 μl reaction volumes using the same 1 ng amount of E. coli chromosomal DNA or no DNA (control reaction) to amplify a 145 base pair segment of the E. coli homoserine kinase gene using 0.2 μM Taqman probe (SEQ ID NO: 15) carrying FAM at the 5' end and quencher BHQ1 at the 3' end, 0.6 μM each of forward primer (SEQ ID NO: 13) and reverse primer (SEQ ID NO: 14) and 2× Kapa PCR master mix.

[0250] The probe sequences SEQ ID NO:5 and SEQ ID NO:6 have the same nucleotide sequence but differ in the position of the FAM label. The reverse primers SEQ ID NO:4 and SEQ ID NO:2 have the same nucleotide sequence but differ in the position of the BHQ1 label. Heterodimer analysis of the FAM-labeled probe and BHQ1-labeled reverse primer shows that they overlap by 4 bases at their 3' ends and match by 4 bases at their 3' ends, forming a heterodimer with ΔG=(-)5.12 kcal / mol.

[0251] The sum of the separation of the FAM labeled base from the 3' end of the FAM labeled probe (SEQ ID NO:5) and the BHQ1 labeled base from the 3' end of the BHQ1 labeled reverse primer (SEQ ID NO:4) is 15 bases, with an estimated separation of the FAM labeled base from the BHQ1 labeled base in the primer dimer-like non-specific product being 11 bases. The sum of the separation of the FAM labeled base from the 3' end of the FAM labeled probe (SEQ ID NO:6) and the BHQ1 labeled base from the 3' end of the BHQ1 labeled reverse primer (SEQ ID NO:4) is 18 bases, with an estimated separation of the FAM labeled base from the BHQ1 labeled base in the primer dimer-like non-specific product being 14 bases. The sum of the separation of the FAM-labeled base from the 3' end of the FAM-labeled probe (SEQ ID NO: 6) and the separation of the BHQ1-labeled base from the 3' end of the BHQ1-labeled reverse primer (SEQ ID NO: 2) is 7 bases, and the estimated separation of the BHQ1-labeled base from the 3' end of the BHQ1-labeled reverse primer in the primer dimer-like non-specific product is 3.

[0252] Amplification reactions using the probe (SEQ ID NO:5) and reverse primer (SEQ ID NO:4) yielded an average amplification Cq value of 21.8 for the amplification reactions using template DNA (Figure 15), and Cq values ​​of 41.07, 40.25, and 0 for the no-template control reactions (Figure 16). Figure 16A shows the melting curves for the no-template control reactions.

[0253] Amplification reactions using the probe (SEQ ID NO:6) and reverse primer SEQ ID NO:4 had an average amplification Cq value of 20.4 in the amplification reactions using template DNA (Figure 17) and a Cq value of 0 in the no template control reactions (Figure 18). Two of the reactions were aberrant and were ignored.

[0254] Amplification reactions using the probe (SEQ ID NO: 6) and reverse primer SEQ ID NO: 2 showed an average amplification Cq value of 24.0 in the amplification reactions using template DNA (Figure 19) and a Cq value of 0 in the no template control reactions (Figure 20).

[0255] Discussion: For the probe (SEQ ID NO:5) and reverse primer (SEQ ID NO:4), the total separation of the FAM-labeled base from the 3' end of SEQ ID NO:5 and the BHQ1-labeled base from the 3' end of SEQ ID NO:4 is 15 bases. These two labeled probes and labeled primers form a heterodimer with 4-base overlap and ΔG=(-)5.0 kcal / mol (in silico analysis). As a result, the donor fluorophore FAM of the probe and the quencher BHQ1 of the reverse primer are expected to be in close proximity, separated by 11 intervening bases, in the non-specific primer dimer-like product. As demonstrated in Example 2, the enhancement measurement test did not observe any significant enhancement upon hybridization of the FAM-labeled probe to the complementary sequence. However, the Cq value of 21.8 for the target amplification indicates that the fluorescence of the FAM-labeled probe (SEQ ID NO:5) is enhanced. The measured degree of quenching between the FAM fluorophore and the BHQ1 quencher at a separation of 11 bases (Example 3) was approximately 16%, which is greater than the quenching required to balance the enhancement of FAM fluorescence upon hybridization of the probe (SEQ ID NO:5).

[0256] A net quenching of the fluorescence emission from the primer dimer-like products is expected. Only one amplification curve shows a plateau with a Cq value of 0 and no non-specific product formation, while three show Cq values ​​of 36, 40.25, and 41.07 (Figure 19) [Figure 19A, melting curves], so it can also be considered a slight net quenching. Only one melting curve shows a good negative peak, while the remaining three melting curves do not show a positive peak, but these two melting curves show a gradual melting in the negative direction without a sharp negative peak, which may be slightly different from the plateau. This may be due to the formation of non-specific products in the later cycles of the amplification reaction. The placement of the FAM fluorophore and quencher BHQ1 on the probe and primer is less than or borderline optimal for zeroing the signal from the primer dimer. The label configuration of the present invention can be used for target amplification, even if it is not the best primer pair for zeroing the signal. An increase of one or two bases in the separation between FAM and BHQ1 in the primer dimer-like product can result in zero or near zero signal.

[0257] For the probe (SEQ ID NO:6) and reverse primer SEQ ID NO:4, the total separation of the FAM-labeled base from the 3' end of SEQ ID NO:6 and the BHQ1-labeled base from the 3' end of SEQ ID NO:4 is 18 bases, and there may be a 4-base overlap between the 3' end of the FAM-labeled probe and the 3' end of the BHQ1-labeled reverse primer. As a result, the donor fluorophore FAM of the forward primer and the quencher BHQ1 of the reverse primer are expected to be in close proximity, separated by 14 intervening bases, in the non-specific primer-dimer-like product. As demonstrated in Example 2, approximately 17% enhancement is seen upon hybridization of the FAM-labeled probe (SEQ ID NO:6) to a complementary sequence. The degree of quenching required to balance the 17% enhancement is approximately 15%. The degree of quenching between the FAM fluorophore and the BHQ1 quencher at a separation of 11 bases (Example 3) is 16%, where quenching occurs with a separation of 14 intervening bases, which is approximately equal to the 15% quenching required to precisely balance the enhancement of FAM fluorescence in the primer dimer. The amplification curves of the amplifications using SEQ ID NO:6 and SEQ ID NO:4 (Figure 17) and the amplification curve of the no-template control (Figure 18) confirm this. Furthermore, the no-template control reaction showed a Cq value of 0, indicating either a near balance between enhancement and quenching or no formation of non-specific products.

[0258] For the probe (SEQ ID NO:6) and reverse primer (SEQ ID NO:2), the total separation of the FAM labeled base from the 3' end of SEQ ID NO:6 and the BHQ1 labeled base from the 3' end of SEQ ID NO:2 is 7 bases. Considering the possible overlap of 4 bases between the 3' end of the probe (SEQ ID NO:6) and the 3' end of the reverse primer (SEQ ID NO:2), the donor fluorophore FAM of the probe and the quencher BHQ1 of the reverse primer are expected to be in close proximity, separated by 3 intervening bases, in the non-specific primer dimer-like product. As measured and demonstrated in Example 2, hybridization to complementary sequences shows approximately 17% enhancement upon hybridization of the FAM labeled probe (SEQ ID NO:6). The degree of quenching required to balance the 17% enhancement is 15%. The degree of quenching between the FAM fluorophore and the BHQ1 quencher at a separation of three bases (Example 3) is expected to be greater than 26%, which is greater than the 15% quenching required to balance enhancement and quenching. There is net quenching of primer dimer-like non-specific products, which reduces the sensitivity of target detection. The target amplification curve (Figure 19) and the no-template control amplification curve (Figure 20) confirm this. It can be noted that the no-template control amplification curve shows a negative slope after 35 cycles.

[0259] New lots of labeled primers and probes give better results. New lots always work better. This is because these labeled oligonucleotides are not very stable unless stabilized, which some companies do with the right composition. The FAM in the FAM-labeled probe of this example is located at or near the 3' end of the probe. Placing the FAM in the FAM-labeled probe farther away from the 3' end of the probe results in higher fluorescence enhancement of the FAM, resulting in higher sensitivity of target detection. The formation of non-specific primer-dimer products is a random / stochastic phenomenon, which may form at some times and not at other times. Furthermore, the formation of non-specific products depends on the quality of the labeled primers and probes used and the presence of inhibitors.

[0260] It can be seen that the FAM fluorophore is at the 3' end of probe sequence 5 or 4 bases away from the 3' end of probe sequence 6, and the BHQ1 quencher is 15 bases away from the 3' end of quencher-labeled primer sequence 4 and 5 bases away from the 3' end of BHQ1-labeled reverse primer sequence 2. Thus, with the fluorophore located at or near the 3' end, the fluorescence enhancement of hybridization of probe sequence 6 to the target sequence is about 17 percent. To balance the 17 percent enhancement in the non-specific primer dimer-like product, 15 percent quenching of the FAM fluorophore by the quencher BHQ1 of the reverse primer is required. In the case of probe sequence 5, the enhancement is much less, and quenching of the FAM fluorophore of sequence 5 in the primer dimer-like non-specific product requires much less than the 15 percent quenching by the BHQ1 quencher of the BHQ1-labeled reverse primer. To reduce quenching of FAM in non-specific primer dimer-like products, the fluorophore FAM and the BHQ1 quencher must be very far apart. The 11 base separation between FAM and BHQ1 in the non-specific product using probe sequence 5 and reverse primer sequence 4 results in a net quenching and unbalanced fluorescence enhancement of FAM in this non-specific product. The BHQ1 label in sequence 4 is 15 bases away from the 3' end, which is between the middle and 5' end of sequence 4. Similarly, the 14 base separation between FAM and BHQ1 in the non-specific product using probe sequence 6 and reverse primer sequence 4 results in roughly balanced quenching of the fluorescence enhancement of FAM in the non-specific product, where FAM is 4 bases away from the 3' end of sequence 6 and BHQ1 is 15 bases away from the 3' end, which is between the middle and 5' end of sequence 4. Therefore, if the donor fluorophore FAM is placed at or near the 3' end of the FAM-labeled probe, the quencher BHQ1 should be placed between the middle and the 5' end of the BHQ1-labeled primer, far from the 3' end of the BHQ1-quencher-labeled primer.Furthermore, in probe SEQ ID NO:6, the FAM fluorophore is 4 bases away from the 3' end, and in BHQ1 quencher-labeled primer SEQ ID NO:2, BHQ1 is placed 5 bases from the 3' end. In both the labeled probe and the labeled primer, the label is near the 3' end. There is no non-specific signal, but there is a significant reduction in target amplification signal, thus reducing sensitivity. This is an issue when placing the donor fluorophore and quencher at or near the 3' end.

[0261] [Table 2]

[0262] Example 6: Comparison of specificity and sensitivity between Taqman and Libra assays Sensitivity and specificity were compared using 90 positive and 84 negative mock clinical samples. Human nasal swab extracts were enriched in Escherichia coli chromosomal DNA (copy number 2 × 10 5 , 2×10 4 , 2×10 3 , 2×10 2 , 2×10 1 , 2×10 0 Mock clinical samples were prepared by spiking 1 ng to 10 fg of DNA into the sham sample.

[0263] The nasal swab extract was prepared by suspending the nasal swabs in 20 mM Tri-HCl Ph-7.5, adding 200 μl of chromosomal DNA extraction buffer and 200 μl of absolute ethanol, each from a commercial chromosomal DNA extraction kit, to 200 μl of the nasal swab suspension, loading onto a silica DNA extraction column, washing with a wash buffer containing 60 percent ethanol and 80 percent ethanol, and finally eluting the column with 100 μl of Milli-Q water. The eluent was the nasal swab extract used in the experiment.

[0264] E. coli chromosomal DNA preparations were serially diluted 10-fold in water. 2X Kapa Master Mix from Kapa Corporation was used for the experiments. Amplification reactions were performed in a Bio Rad Thermal Cycler Series 1000 CFX 384 RT-PCR machine in a 12.5 μl reaction volume using a concentration of 0.2 μM of each labeled primer and probe and a concentration of 0.2 μM of unlabeled primers (with the exception of unlabeled primers at a concentration of 0.4 μM for the TaqMan assay).

[0265] The primers and probes used were SEQ ID NO:1, 6 and 4; SEQ ID NO:1, 6 and 2; SEQ ID NO:8 and 10 for amplifying the 113 bp and 111 bp segments of the E. coli threonine synthase gene, and SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 for amplifying the 145 bp segment of the E. coli homoserine kinase gene.

[0266] [Table 3]

[0267] Discussion: SEQ ID NO:1 is an unlabeled forward primer for amplifying a 113 bp segment of the E. coli threonine synthase gene, SEQ ID NO:2 and SEQ ID NO:4 are BHQ1-labeled reverse primers for amplifying the segment, and SEQ ID NO:6 is a FAM-labeled common probe for these two amplification reactions. SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 are unlabeled forward and reverse primers and FAM and BHQ1 double-labeled TaqMan probes for amplifying a 145 bp segment of the E. coli homoserine kinase gene, respectively. SEQ ID NO:8 is a FAM-labeled forward primer for amplifying a 111 bp segment of the E. coli threonine synthase gene, and SEQ ID NO:10 is a BHQ1-labeled reverse primer for amplifying the segment.

[0268] Amplification reactions using SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:6 showed approximately 5.5% higher sensitivity and 3.6% higher specificity compared to the TaqMan assay, whereas amplification reactions using SEQ ID NO:8 and SEQ ID NO:10 showed approximately 10.3% higher sensitivity and 5.9% higher specificity compared to the TaqMan assay.

[0269] The fluorescence enhancement of the FAM fluorophore of probe SEQ ID NO:6 is smaller than that of the FAM fluorophore of primer SEQ ID NO:8, and this difference therefore results in higher sensitivity in the amplification reaction using SEQ ID NO:8 and SEQ ID NO:10 compared to the amplification reaction using probe SEQ ID NO:6 and reverse primer 4.

[0270] Furthermore, in the case of the primer pair SEQ ID NO:8 and SEQ ID NO:10, there is relatively more quenching of FAM fluorescence by BHQ1 in non-specific amplification products compared to the case of the FAM-labeled probe SEQ ID NO:6 and the BHQ1-labeled reverse primer SEQ ID NO:4. This is the reason why the amplification reaction of the primer pair SEQ ID NO:8 and SEQ ID NO:10 (descent of the curve of the no-template control after 35 cycles) is more specific compared to the amplification reaction of the probe SEQ ID NO:6 and the reverse primer SEQ ID NO:4 (linear amplification curve of the no-template control).

[0271] The fluorescence enhancement of the FAM label of probe SEQ ID NO:6 is the same and common between the amplification reaction containing the FAM-labeled probe of SEQ ID NO:6 and the BHQ1-labeled reverse primer 2 and the amplification reaction containing the FAM-labeled probe SEQ ID NO:6 and the BHQ1-labeled reverse primer 4. The only difference is that BHQ1 is in close proximity to the 3' end of the reverse primer SEQ ID NO:2 (5 bases away from the 3' end) compared to the reverse primer SEQ ID NO:4 (16 bases away from the 3' end). In the former case (SEQ ID NO:2), there is much less separation between FAM and BHQ1 in the non-specific amplification products compared to the latter case (SEQ ID NO:4), and therefore the quenching of the FAM fluorophore by the quencher BHQ1 in the non-specific amplification products is much increased. This improves the specificity of the amplification reaction containing probe SEQ ID NO:6 and the reverse primer SEQ ID NO:2 (specificity -96.6%) compared to the specificity of the amplification reaction containing probe SEQ ID NO:6 and the reverse primer SEQ ID NO:4 (specificity -92.8%). Due to this excess quenching of FAM fluorescence by BHQ1 in non-specific amplification products, there is a net quenching that exceeds the balanced quenching required to generate non-substantial signal from non-specific amplification.This excess quenching in non-specific amplification products in this reaction reduces the overall fluorescence level of this amplification reaction, which reduces the signal from target amplification products, and therefore reduces sensitivity.As a result, this reaction containing labeled probe SEQ ID NO:6 and labeled reverse primer SEQ ID NO:2 has a significant reduction in target detection sensitivity (sensitivity-65.4%), which is lower than the latter case containing labeled probe SEQ ID NO:6 and labeled reverse primer SEQ ID NO:4 (sensitivity-81.3%), and all other reactions containing TaqMan assay (sensitivity-75.8%).

[0272] SEQ ID NOs: 2 and 6 are poor combinations of BHQ1-labeled reverse primer and FAM-labeled probe for amplification of this target.

[0273] Example 7: Amplification of the threonine synthase gene of the bacterium Escherichia coli using labeled non-targeting primers Three femtomoles each of a first oligonucleotide sequence (SEQ ID NO: 19) having a first non-target primer sequence at its 5' end and an E. coli threonine synthase gene sequence at its 3' end and a second oligonucleotide sequence (SEQ ID NO: 20) having an E. coli threonine synthase gene sequence at its 5' end and a second non-target primer sequence at its 3' end, wherein the 3' end of SEQ ID NO: 19 has a phosphate group, the threonine synthase gene sequences on the first and second oligonucleotides are two consecutive sequences, and the first and second oligonucleotide sequences are designed to hybridize to one strand, were added to 1 ng of E. coli chromosomal DNA in a ligation buffer containing a ligase enzyme and incubated at 8°C for 4 hours.

[0274] Aliquots of this ligation mixture were used for PCR amplification in triplicate in a 15 μl reaction volume using 3 pmoles of a first non-target primer (SEQ ID NO: 21) labeled with Blackhole quencher 1 and 3 pmoles of a second non-target primer (SEQ ID NO: 22) labeled with FAM using 2× Kapa PCR master mix, along with a control reaction containing no target DNA. The PCR amplification used annealing at 63° C. for 45 seconds and extension for 15 seconds. The E. coli threonine synthase gene sequence was successfully amplified with a Cq value of 20.4 ( FIG. 23 ), while the control reaction showed a Cq value of 0 ( FIG. 24 ).

[0275] The example of Example 7 is different from the examples of Examples 4 and 5. Examples 4 and 5 are about target detection using a labeled Libra primer pair and a labeled Libra primer-probe pair. The libra primer-probe pair and the libra primer pair in these two examples are different for each target, and are designed and labeled separately for each target. On the other hand, the common non-target primer pair in Example 7 is also a libra primer pair, but it is not specially designed for any specific target, but is specially designed so that the same primer pair can be used for amplifying any target with higher specificity and sensitivity, and at the same time, the primer pair does not amplify any non-specific target sequence, and the common non-target primer pair does not have a significant number of bases matching with any organism sequence. The primer pair SEQ ID NO: 21 and 22 are a pair of two universal primers, so it replaces the DNA double strand intercalating dye-based detection, which is a universal target detection method for any target, in which only two unlabeled target-specific amplification primers and a DNA intercalating dye are provided. This double-stranded DNA intercalating dye-based detection method lacks specificity and sensitivity, and is therefore not used for applications such as diagnostic applications where higher specificity and sensitivity are required. The solution of Example 6 is to achieve the usefulness of DNA intercalating dye-based detection, but with higher specificity and sensitivity. In fact, the labeled primer pair of Example 7 is a substitute for DNA intercalating dye. This detection strategy is new, simpler, cheaper, and designed for use as a ready-made amplification reagent, such as a DNA intercalating dye-based amplification reagent, and is specifically designed for superior specificity and sensitivity compared to DNA intercalating dye-based amplification reagents. This is a solution to the problem of the relatively low specificity and sensitivity of DNA intercalating dye-based detection. The design requirements of the primers of this Example 7 are the same as those of the libra primer pair and the libra primer-probe pair.That is, the number of bases separating the donor fluorophore-bearing base from the 3' end of the donor fluorophore-labeled primer plus the number of bases separating the acceptor fluorophore / quencher-bearing base from the 3' end of the acceptor fluorophore / quencher-labeled primer is in the range of 6-35 or 6-40 bases. Furthermore, these primers are designed not to have significant base matches with any organism's nucleotide sequence or with any sequence to avoid the formation of non-specific amplification products from non-target sequences.

[0276] The two primers in Example 6 are libra primers used in different strategies for different purposes (universal reagents), thus resulting in specificity and sensitivity similar to that of libra primer pair and libra primer-probe pair. The Cq value of target amplification of 20.4 and the Cq value of no target control reaction of 0 indicate high sensitivity and specificity, respectively, and the Cq values ​​of Taqman probe assay are 21.2 and 0, respectively. The primer pair in Example 7 is not the best libra primer pair, and a better labeled primer pair can be designed to achieve even better specificity and sensitivity.

[0277] In an alternative strategy, sequences corresponding to two labeled non-target primer sequences (SEQ ID NO:21 and SEQ ID NO:22) are separately added to the 5' ends of two PCR primers for the target sequence, and the target sequence is amplified using two target amplification primers with the non-target sequences added at 1 / 100th the normal PCR primer concentration and the two labeled non-target primers (SEQ ID NO:21 and SEQ ID NO:22) at the normal concentration.

[0278] Example 8: Amplification of the homoserine kinase gene of the bacterium Escherichia coli using labeled primers to generate a FRET signal Amplification reactions were performed in triplicate in 15 μl reaction volumes using 3 pmoles each of an unlabeled forward primer (SEQ ID NO:23, 5'-GATAAGCTGCCGTCAGAACC-3'), an internal fluorescein-labeled reverse primer (SEQ ID NO:24, 5'-AACAGGCACTGGAGCCTAAG-3') and an internal fluorescein-labeled probe (SEQ ID NO:25, 5'-CCA GTG GCG ATG ACC CTG GAA AAG AAT ATG-3') in a PCR master mix with 1 ng of E. coli chromosomal DNA preparation or without DNA (as a control) to amplify a 145 base pair segment of the E. coli homoserine kinase gene. The amplification reactions were monitored by exciting fluorescein at 465 nm and measuring fluorescein emission at 510 nm. The target amplification reaction showed a Cq value of 21.3 (Figure 25), whereas the control reaction containing no target DNA showed a Cq value of 0 (Figure 26).

[0279] The method or strategy of target detection in Example 8 is different from that in Examples 4-7 based on libra primer-probe pair and libra primer pair. Examples 4-7 use non-FRET signal generation for target detection, where donor fluorophore is excited and donor fluorophore emission is measured as signal. Conversely, in Example 8, FRET signal is generated for target detection, where donor fluorophore is excited and acceptor fluorophore emission is measured as signal. The advantage of this strategy is that it generates melting curves of target amplification products with low fluorescence background and distinguishable from primer dimers, which is an additional specificity that is not possible with Taqman probe-based detection. The sensitivity and specificity of the strategy or method in Example 8 is similar to or slightly better than that of Taqman probe-based target detection method, but is lower than that of the above three strategies (Examples 4-6 and 7) of the present invention including libra primer-probe pair and libra primer pair.

[0280] In the FRET-based signal generation method, the FRET primer pair and the FRET primer-probe pair are designed to maximize the energy transfer between the donor and the acceptor (70-80 percent) to obtain a higher signal from the acceptor fluorophore. It was observed that the higher signal from the acceptor does not make a significant difference in the target detection sensitivity and specificity, and the melting curves of the target amplicon of the primer pair labeled with the donor fluorophore and the acceptor fluorophore are indistinguishable from the melting curves of the non-specific primer dimers. On the other hand, in the method of Example 8, the primers and probes are labeled and configured such that there is only 30-50 percent energy transfer between the donor fluorophore and the acceptor fluorophore in the target amplicon, resulting in a melting curve of the target amplicon that is distinguishable from the melting curve of the non-specific primer dimers by further separating the labeled primer pair or the labeled primer-probe pair. Thus, further specificity is achieved. Additionally, the primers are labeled such that the donor and acceptor fluorophores are separated by 3 nucleotides or less in the primer dimer or primer dimer-like nonspecific amplification products, resulting in zero or near-zero signal from the primer dimer or primer dimer-like nonspecific amplification products.

[0281] In this Example 8, unlabeled primers and fluorescein-labeled primers and fluorescein-labeled probes (where one fluorescein acts as the donor and the other as the acceptor) are used, but primer pairs labeled with donor and acceptor fluorophores (different from the donor fluorophore) can be used for target detection as well. The acceptor fluorophore is preferably different from the donor fluorophore. The labeled primers and probes are selected and labeled such that the donor and acceptor fluorophores are separated by 15-25 bases in the target amplification product. [Sequence List Free Text]

[0282] SEQ ID NO: 1: synthetic primer SEQ ID NO: 2: synthetic primer having BHQ1 DYE at base 18 SEQ ID NO: 4: synthetic primer - black hole quencher 1 at base 7 SEQ ID NO: 5: Synthetic oligonucleotide probe - Fluorescein dye at base 24 SEQ ID NO: 6: Synthetic oligonucleotide probe - Fluorescein dye at base 21 SEQ ID NO: 7: Synthetic primer - Fluorescein dye at base 3 SEQ ID NO: 8: Synthetic primer - Fluorescein dye at base 13 SEQ ID NO: 9: Synthetic primer - Fluorescein dye at base 10 SEQ ID NO: 10: synthetic primer - black hole quencher 1 at base 16 SEQ ID NO: 11: Synthetic oligonucleotide probe - Fluorescein dye at base 1 SEQ ID NO: 12: Synthetic primer - Fluorescein dye onBase1 at base 1 SEQ ID NO: 13: synthetic primer SEQ ID NO: 14: synthetic primer SEQ ID NO: 15: Synthetic oligonucleotide probe - fluorescein dye at base 15 (5' end) and black hole quencher 1 at base 21 (3' end) SEQ ID NO:16: Synthetic target sequence for hybridizing with SEQ ID NO:5 (probe), SEQ ID NO:6 (probe), and SEQ ID NO:4 (primer) to generate different separations between the fluorescein dye and the black hole quencher 1. SEQ ID NO:17: Synthetic target sequence for hybridizing with SEQ ID NO:5 (probe), SEQ ID NO:6 (probe), and SEQ ID NO:4 (primer) to generate different separations between the fluorescein dye and the black hole quencher 1. SEQ ID NO: 18: Synthetic target sequence for hybridizing with SEQ ID NO: 7 (primer), SEQ ID NO: 8 (primer) and SEQ ID NO: 9 (primer) to measure enhancement of fluorescein dye SEQ ID NO: 19: Non-target primer sequence at the 5' end and E. coli threonine synthase gene sequence at the 3' end SEQ ID NO: 20: Escherichia coli threonine synthase gene sequence at the 5' end and non-target primer sequence at the 3' end SEQ ID NO: 21: Synthetic primer - Black hole quencher 1 at base 23 SEQ ID NO: 22: Synthetic primer - Fluorescein dye at base 15 SEQ ID NO: 23: synthetic primer SEQ ID NO: 24: Synthetic primer - Fluorescein dye at base 17 SEQ ID NO: 25: Synthetic oligonucleotide probe - Fluorescein dye at base 27

Claims

1. A method for detecting and / or quantifying a nucleic acid target by nucleic acid amplification, comprising selectively controlling the signal enhancement and corresponding attenuation of any detectable signal from non-specific amplification, and further the loss of the target amplification signal under controlled attenuation, and comprising the following steps. Providing at least one target nucleic acid and at least one oligonucleotide, wherein the oligonucleotide is a non-extendable probe or an extendable primer and is labeled with at least one luminescent or signaling group / portion adapted to emit extra light when hybridizing to a nucleic acid molecule or when incorporated into a nucleic acid molecule.

2. In addition to the labeled non-extendable probe or extendable primer, comprising providing at least one additional extendable oligonucleotide primer, wherein the at least one additional extendable oligonucleotide primer is labeled with at least one converter or acceptor group / attenuator portion adapted to convert the color of the luminescent group / portion to a different color, or to thermalise it, or to attenuate its luminescence, and is incorporated into the amplification product(s) and is a linear oligonucleotide. The method according to claim 1, wherein the nucleic acid amplification is well controlled by a process comprising the following. Selectively controlling the signal enhancement and corresponding attenuation of any detectable signal from non-specific amplification, and further the loss of the target amplification signal under controlled attenuation, by selectively placing the luminescent / signaling portion at any of the bases at the 3'-end of the oligonucleotide probe or primer labeled with the luminescent / signaling portion, or at a position up to 30 bases away from the 3'-end, except for the 5'-end, and in the oligonucleotide primer labeled with the attenuator or acceptor portion, the attenuator or acceptor portion is placed at any base at a position at least 2 nucleotides and up to 30 bases away from its 3'-end, thereby the following products: (a) A target amplification product that avoids undesirable interactions and associated undesirable energy transfer between the luminescent or signaling moiety and the converter / attenuator moiety in a target amplification reaction, including separation of the distance between the luminescent or signaling moiety and the converter / attenuator moiety, such that energy transfer between the two moieties is significantly reduced or eliminated, and the two moieties in the target amplification product remain separated by a distance of at least 1.5 times the Förster radius (R0 value), and (b) Ensuring a desired target amplification product having a desired target-detectable signal specificity, wherein the degree of signal enhancement and corresponding attenuation of the detectable signal is under desired control, such that any unwanted net signal enhancement and net signal from non-specific products, net signal attenuation, and loss of the target amplification signal do not occur, and only by hybridizing or incorporating the luminescent group / luminescence moiety of the oligonucleotide labeled with the luminescent group to the target nucleic acid amplification product. The non-specific product used, wherein the luminescent or signaling moiety used is a donor fluorophore or fluorescent dye or luminescent moiety, and the attenuator or acceptor moiety used is an acceptor fluorophore or a non-radioactive acceptor or quencher moiety that receives energy but does not emit any energy or electromagnetic radiation, the signal is a fluorescence signal, and the quenching of the fluorescence signal of the donor fluorophore or the acceptor fluorophore is the key of the signal, and the donor fluorophore or fluorophore / fluorescent dye and the acceptor moiety form an energy transfer pair, and the step of ensuring the controlled generation of non-specific products.

3. The controlled generation of the non-specific product, where the degree of signal enhancement of the detectable signal and the corresponding attenuation are under the desired control, involves separation in the non-specific amplification product between the base labeled with the luminescence / signaling moiety and the base labeled with the attenuator or acceptor moiety in the oligonucleotide labeled with the luminescence / signaling moiety and the attenuator or acceptor moiety, based on the distance between them. This separation by distance results in no change in the signal of the signaling moiety, no net signal enhancement and no net signal in the non-specific amplification product, no attenuation of the net signal, and no loss of the target amplification signal, achieving the desired ideal and most balanced state. Control is provided to make the degree of signal attenuation in the non-specific amplification product equal to or approximately equal to the degree of signal enhancement of the luminescence / signaling moiety in the non-specific amplification product. In a less than ideal and unbalanced state, although undesirable but usable, where the enhancement exceeds the attenuation, the signal of the signaling moiety in the non-specific amplification product is large or increases to maintain specificity to the extent that usability is not impaired. In another less than ideal and unbalanced state, although undesirable but usable, where the enhancement is less than the attenuation, the signal of the signaling moiety in the non-specific amplification product is small or decreases to maintain the target amplification signal and / or sensitivity to the extent that usability is not impaired. The method according to claim 2.

4. The oligonucleotide labeled with the luminescence group / signaling moiety to be used is a non-extendable probe for monitoring nucleic acid amplification, which hybridizes to one strand of the target amplification product or the target nucleic acid. The luminescence / signaling moiety is located at the base at the 3'-end of the oligonucleotide probe labeled with the luminescence / signaling moiety, or at any base located up to 30 bases away from the 3'-end, excluding the 5'-end. The luminescence / signaling moiety used is a donor fluorophore or a fluorophore or a fluorescent dye or a luminescent moiety, the signal is a fluorescent signal, and the oligonucleotide is linear. The method according to claim 1, wherein the probe comprises or does not comprise at least a minor groove binding dye or a combination of a minor groove binding dye and a non-fluorescent quencher. **Claim 5** The oligonucleotide labeled with the luminescent group / signaling moiety used is a nucleic acid amplification primer that anneals to one of the two strands of the target amplification product or the target nucleic acid and is extended by a polymerase(s), wherein the luminescent or signaling moiety is disposed at any base, except the 5' end, at a position at least 2 nucleotides and up to 30 bases away from the 3' end of the oligonucleotide labeled with the luminescent / signaling moiety, and the luminescent / signaling moiety used is a donor fluorophore or a fluorophore or a fluorescent dye or a luminescent moiety, the signal is a fluorescent signal, and the oligonucleotide is linear. The method according to claim 1. **Claim 6** The method according to claim 1, wherein the oligonucleotide labeled with the luminescent or signaling moiety used is linear. **Claim 7** The non-extendable oligonucleotide labeled with the luminescent / signaling moiety or the donor fluorophore is a probe, and further comprises an acceptor fluorophore or a non-radioactive acceptor or quencher at least at or near its 5' end, or The extendable oligonucleotide labeled with the luminescent / signaling moiety or the donor fluorophore moiety is a primer, and further comprises an acceptor fluorophore / non-radioactive quencher at its 5' end, and the oligonucleotide is a linear oligonucleotide. The method according to claim 1. **Claim 8**: The oligonucleotide probe or primer labeled with the luminescent or signaling portion has a nucleotide sequence sufficiently complementary to the sequence of the oligonucleotide probe or primer labeled with the luminescent / signaling portion or donor fluorophore / fluorescent dye in the vicinity of the internal base that is a base labeled with the luminescent / signaling portion or donor fluorophore / fluorescent dye. The complementary nucleotide sequence forms a stem structure and may or may not have a quencher. There may or may not be a spacer / linker intervening between the primer or probe labeled with the signaling portion or donor fluorophore / fluorescent dye and the nucleotide sequence. The additional nucleotide sequence may hold a 5' quencher portion, and the probe may hold a minor groove binding dye. The method according to claim 1. **Claim 9** The luminescent / signaling portion or donor fluorophore or acceptor is arranged in the oligonucleotide labeled with the luminescent portion or donor fluorophore that is a primer or probe, and the oligonucleotide labeled with the acceptor or quencher portion that is a primer. The value obtained by adding the number of bases separating the base labeled with the signaling portion or donor fluorophore portion and the 3' end of the primer or probe labeled with the donor fluorophore to the number of bases separating the base labeled with the acceptor or quencher portion and the 3' end of the primer labeled with the acceptor or quencher portion is 6 to 40 bases (or 6 to 44 bases) when the Förster radius of the energy transfer pair between the donor fluorophore portion and the acceptor or quencher portion is in the range of 21 angstroms to 75 angstroms. The arrangement is carried out as described in claim 2. **Claim 10** An oligonucleotide labeled with the luminescent moiety or the donor fluorophore / fluorescent dye, which is a primer or a probe, and an oligonucleotide labeled with the acceptor fluorophore or non-radioactive quencher moiety, which is a primer, are arranged with the luminescent moiety or donor fluorophore / fluorescent dye and the acceptor fluorophore or non-radioactive quencher, and the number of bases separating the base labeled with the luminescent or signaling moiety or donor fluorophore moiety from the 3'-end of the primer or probe labeled with the luminescent moiety or donor fluorophore is added to the number of bases separating the base labeled with the acceptor or quencher moiety from the 3'-end of the primer labeled with the acceptor or quencher moiety, and the value is 2 bases or less when the Förster radius of the energy transfer pair between the donor fluorophore and the acceptor or quencher moiety is in the range of 10 angstroms to 21 angstroms, or static or contact quenching also plays a role in addition to Förster energy transfer in the attenuation of the luminescent moiety or donor fluorophore by the acceptor or quencher moiety of the energy transfer pair in non-specific primer dimers or similar products. The method according to claim 2.

11. When the primer labeled with the signaling or donor fluorophore / fluorescent dye moiety is incorporated into the target amplification product, or when the probe labeled with the signaling or donor fluorophore / fluorescent dye moiety hybridizes to the target amplification product, the degree of signal enhancement or fluorescence enhancement of the donor fluorophore / fluorescent dye exceeds 2-fold. The value obtained by adding the number of bases separating the base labeled with the signaling or donor fluorophore moiety and the 3'-end of the primer or probe labeled with the signaling or donor fluorophore to the number of bases separating the base labeled with the acceptor or quencher moiety and the 3'-end of the primer labeled with the acceptor or quencher moiety is 4 or more bases for a pair of donor and acceptor in the Förster radius range of 21 angstroms to 75 angstroms of the Förster radius (R 0 ) of a pair of a donor fluorophore and an acceptor that is commonly used, and the base labeled with the signaling or donor fluorophore moiety and the base labeled with the acceptor or quencher moiety are separated within the target amplification product by a distance that is 1.5 times the R 0 distance. The method according to claim 2.

12. The method according to claim 1, wherein two or more signaling moieties or donor fluorophores are disposed on the probe or primer labeled with the signaling moiety, and two or more acceptor moieties or quenchers are disposed on the primer(s) labeled with the acceptor moiety.

13. The oligonucleotide labeled with the luminescent or signaling group / moiety is either the donor fluorophore / fluorophore moiety-labeled nucleic acid amplification probe or primer of the method, and is used in nested and semi-nested nucleic acid amplification, transcription-mediated nucleic acid amplification, and absolute quantification of nucleic acid targets, either in combination with or without a converter or acceptor moiety comprising an oligonucleotide primer(s) labeled with an acceptor fluorophore or non-radioactive quencher moiety. The method according to claim 1.

14. To amplify at least one target nucleic acid sequence, a semi-synthetic target nucleic acid sequence is generated before or during an amplification reaction by incorporating or adding a first non-target sequence, or a first non-target sequence and a second non-target sequence, or a combination thereof, into the target nucleic acid sequence, and the amplification of the target nucleic acid is performed by at least one target-specific primer, and / or a primer derived from the first non-target sequence, or a primer derived from the first non-target sequence and a primer derived from the second non-target sequence, and / or a target-specific primer with an added non-target sequence(s), wherein the primer(s) for performing the amplification comprises any of the primer pairs described in the method, which appropriately comprises SEQ ID NO: 21 and SEQ ID NO: 22, and a probe selected from any of the signaling moieties or probes labeled with a donor fluorophore / fluorescent dye described in the method is provided together with a plurality of primers including the primer(s) for performing the amplification, and the probe hybridizes to the non-target sequence(s) incorporated or added to the synthetic target sequence generated before or during the amplification reaction, and the probe may hold a minor groove binding dye. The method according to claim 1.

15. A composition or reagent for use in a method for detecting and / or quantifying (at least one) nucleic acid target by nucleic acid amplification, at least a second extendable linear oligonucleotide primer internally labeled with an acceptor fluorophore or a non-radioactive quencher moiety, and / or an additional quencher at the 5'-end, or a 5'-end additional nucleotide sequence forming a stem structure that is sufficiently complementary to the sequence of the primer labeled with the donor fluorophore in the vicinity of the donor fluorophore-labeled base, which may or may not be provided with a quencher, and may or may not be provided with a spacer intervening between the primer labeled with the donor fluorophore and the nucleotide sequence, and comprising at least a first extendable linear oligonucleotide primer internally labeled with a fluorophore or a donor fluorophore group or moiety. The first oligonucleotide and the second oligonucleotide are two universal primers for a nucleic acid amplification reaction, preferably a polymerase chain reaction (PCR), for amplifying any target nucleic acid sequence, and the first oligonucleotide and the second oligonucleotide do not have sufficient complementarity to any known sequence of any organism and their viruses or their fractions / parts, and the first extendable oligonucleotide and the second extendable oligonucleotide are primers specific to a first non-target sequence and a second non-target sequence that are incorporated into or added to the target sequence. In the labeled first extendable oligonucleotide primer, the fluorophore or the donor fluorophore moiety is disposed at a base at any position at least 2 nucleotides away from the 3'-end of the labeled first extendable oligonucleotide primer, or at a base at a position up to 30 bases away from the 3'-end, excluding the 5'-end. In the labeled second extendable oligonucleotide primer, the acceptor or non-radioactive quencher moiety is disposed at any base at a position at least 2 nucleotides and up to 30 bases away from the 3'-end of the labeled second extendable oligonucleotide primer, or The composition or reagent includes at least a non-extendable linear oligonucleotide probe having a fluorophore or a donor-fluorophore moiety disposed at the 3'-end of the oligonucleotide or at a position up to 30 bases away from the 3'-end, excluding the 5'-end, for the detection and / or quantification of at least one target sequence, the probe being sufficiently complementary to a non-target sequence incorporated or added to the target sequence and hybridizing to the non-target sequence(s) incorporated or added to the target sequence generated before or during the amplification reaction, the labeled probe further optionally having an additional nucleotide sequence at the 5'-end that forms a stem structure sufficiently complementary to at least a minor groove binding dye and / or at least a quencher at the 5'-end or in the vicinity thereof, or to the sequence of the probe labeled with the donor-fluorophore in the vicinity of the donor-fluorophore labeled base, and the probe may or may not be provided with a quencher and may or may not be provided with a spacer intervening between the probe labeled with the donor-fluorophore and the nucleotide sequence. Optionally, (a) an amplification reaction buffer, (b) one or more thermostable polymerases (DNA polymerase, or DNA polymerase and reverse transcriptase, or a polymerase having both DNA polymerase activity and reverse transcriptase activity), which may be hot start or non-hot start, (c) a non-thermostable or thermostable DNA ligase, and (d) one or more nucleoside triphosphates, The composition or reagent further contains.

16. The oligonucleotide probe or primer labeled with the luminescent group is a probe or primer labeled with a donor fluorophore / fluorophore, and the probe labeled with the donor fluorophore is provided together with a primer labeled with an acceptor fluorophore or a converter moiety, or correspondingly, the primer labeled with the donor fluorophore is provided in combination with a probe or primer labeled with an acceptor fluorophore. Either the probe labeled with the donor fluorophore / fluorophore or the acceptor fluorophore may comprise a minor groove binding dye. The base labeled with the donor fluorophore / fluorophore and the base labeled with the acceptor fluorophore in the target amplification product are separated by 5 to 30 bases, preferably 12 to 20 bases, and upon excitation of the donor fluorophore of the target amplification product, the color or wavelength of the luminescence (accompanied by the emission of the acceptor fluorophore) changes. The oligonucleotide probe or primer labeled with the luminescent group or the donor fluorophore / fluorophore and the oligonucleotide primer or probe labeled with the acceptor fluorophore or the converter moiety are selected and labeled. The change in the luminescence, which is the emission of the acceptor fluorophore, is a measure of target amplification. In the non-specific amplification product, the luminescent group or the donor fluorophore is attenuated or quenched by the acceptor fluorophore or the converter. The base labeled with the luminescent group or the donor fluorophore and the base labeled with the acceptor fluorophore or the converter are separated by 2 or fewer nucleotides or bases in the non-specific amplification product. Due to the proximity of the donor fluorophore and the acceptor fluorophore, static or contact quenching occurs between the two parts of the donor fluorophore and the acceptor fluorophore. Due to the static or contact quenching, despite the high energy transfer, the signal from the acceptor fluorophore in the non-specific amplification product becomes zero or almost zero (the acceptor fluorophore becomes non-fluorescent). Alternatively, the oligonucleotide probe or primer labeled with the donor fluorophore further comprises, at its 5'-end or in the vicinity thereof, a non-radioactive quencher, or an additional nucleotide sequence at the 5'-end that forms a stem structure which is sufficiently complementary to the sequence of the labeled primer or probe in the vicinity of the base labeled with the donor fluorophore, preferably an internal base, and may or may not comprise a quencher, and may or may not comprise a spacer intervening between the primer labeled with the donor fluorophore and the additional nucleotide sequence, the method according to claim 1. **Claim 17** The primer labeled with the luminescent group or donor fluorophore / fluorophore is provided together with at least one primer comprising a primer labeled with an acceptor fluorophore / non-radioactive quencher, the primer comprising the primer labeled with the donor fluorophore and the primer labeled with the acceptor fluorophore / non-radioactive quencher being any of the labeled primer pairs according to the method, and being used for allele-specific nucleic acid amplification, preferably allele PCR, one of the labeled primers being allele-specific, the second base from its 3'-end being the allele base (the mutated or changed base of the allele of interest), and one or more bases located 2 to 5 bases away from the 3'-end of the allele primer having a base mismatch with the target sequence. Alternatively, a probe labeled with a luminescent group or a donor fluorophore is provided together with a plurality of primers including a primer (s) labeled with an acceptor fluorophore / non-radioactive quencher, and a primer containing a primer labeled with an acceptor fluorophore / non-radioactive quencher or one of the probes labeled with the donor fluorophore, preferably the allele-specific primer, is used for allele discrimination (allelism), and the allele primer has the same standard as the allele primer described above, and the allele discrimination or the probe labeled with a luminescent group or a donor fluorophore of allelism hybridizes to the target sequence including the sequence surrounding the allele base, and is any one of the donor fluorophore or the probe labeled with a fluorophore according to the method, and may include a minor groove binding dye, The method according to claim 1.

18. The nucleic acid amplification reaction includes any known nucleic acid amplification reaction, preferably polymerase chain reaction, and the nucleic acid amplification reaction is carried out on a sample with an effective amount of amplification primer or an effective amount of labeled primer or labeled probe or labeled primer and probe together with at least one polymerase and / or reverse transcriptase, a reaction buffer, and at least one deoxynucleoside triphosphate, and the sample / reaction mixture is circulated between at least a denaturation step, an annealing step, an extension step, or a single step combining annealing and extension, or subjected to a single isothermal reaction step, The primer hybridizes / anneals to each of the two strands of the segment of the double-stranded nucleic acid molecule towards the end of the segment, and the two primers are extended or elongated by nucleic acid synthesis by a polymerase enzyme using deoxynucleoside triphosphate, so that each replication of the corresponding strand of the segment is synthesized, and two double-stranded replications of the segment containing two primers and the nucleic acid between them are generated from one nucleic acid molecule. Furthermore, when using a labeled probe, the probe hybridizes to either one of the two strands of the double-stranded nucleic acid or amplification product within the segment, and the strands of the newly generated double-stranded segment of the nucleic acid are subjected to denaturation / strand separation, primer annealing / hybridization, and nucleic acid synthesis, the steps of denaturation / strand separation, primer annealing / hybridization, and nucleic acid synthesis are repeated at least once or multiple times, exciting the reaction mixture with donor fluorophore excitation light or radiation, and measuring a detectable signal emitted from the donor fluorophore / fluorescent dye or acceptor fluorophore, including hybridization of the probe labeled with the donor fluorophore / fluorescent dye or acceptor fluorophore to one of the two strands of the target amplification product, and / or incorporation of the acceptor fluorophore or non-radioactive quencher, and / or the donor fluorophore-labeled primer into the target amplification product, the method according to claim 1.

19. The nucleic acid amplification reaction includes quantitative polymerase chain reaction (qPCR) and / or real-time polymerase chain reaction, and the polymerase chain reaction (PCR) is polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), allele or allele-specific polymerase chain reaction (allele PCR), reverse transcription allele PCR, Tail PCR, reverse transcription Tail PCR, droplet PCR, reverse transcription droplet PCR, emulsion PCR, reverse transcription emulsion PCR, digital PCR, reverse transcription digital PCR, asymmetric PCR, reverse transcription asymmetric PCR, nested PCR, reverse transcription nested PCR, semi-nested PCR, reverse transcription semi-nested PCR, methylation status PCR, in-situ PCR, reverse transcription in-situ PCR, and the size of the target amplification product is 35 to 400 base pairs, preferably 50 to 150 base pairs, the method according to claim 1.

20. The nucleic acid amplification reaction includes isothermal nucleic acid amplification reactions such as loop-mediated isothermal nucleic acid amplification reaction (LAMP), recombinase polymerase amplification reaction (RPA), helicase polymerase amplification reaction (HPA), transcription-mediated nucleic acid amplification, nucleic acid sequence-based amplification (NASBA), etc. The loop primer used in the LAMP promotes DNA strand separation. Strand separation enzymes such as recombinase, helicase, gyrase, and topoisomerase are used in RPA and HPA for denaturation or strand separation in combination with single-strand binding (SSB) protein. The size of the target amplification product is 75 to 1000 base pairs, preferably 100 to 250 base pairs. The variant of the isothermal nucleic acid amplification reaction includes allele primers or primer-probe pairs, nested or semi-nested primer pairs, nested or semi-nested primer-probe pairs. The method according to claim 1.

21. The oligonucleotide primer and probe according to the method are selectively 10 to 50 bases in length, preferably 15 to 35 bases in length, more preferably 20 to 30 bases in length, are sufficiently complementary to the target sequence, have the ability to hybridize or anneal on the target and the ability to prime nucleic acid synthesis on the target, and may possess one or more artificial or modified synthetic nucleotides having modified bases or modified sugar moieties (s) or modified phosphate groups (s) or one or more nucleotide analogs. The method according to claim 1.

22. A positive control template and a labeled primer pair or a pair of a labeled primer and a probe according to any one of claims 2 to 11, 13, 14, 16, 17 specific to the positive control template are further provided during the amplification reaction. The method according to claim 1.

23. A plurality of pairs of a primer labeled with a donor fluorophore and a primer labeled with an acceptor fluorophore / quencher or a plurality of pairs of a probe labeled with a donor fluorophore and a primer labeled with an acceptor fluorophore / quencher according to any one of claims 2 to 11, 13, 14, 16, 17 are used in a multiplex reaction for simultaneous detection and / or quantification of a plurality of nucleic acid target sequences. The method according to claim 1.

24. A single, multiple types, or large array of primers labeled with a donor fluorophore / acceptor fluorophore or probes labeled with a donor fluorophore / acceptor fluorophore, with or without a multi-carbon atom organic linker, polyethylene glycol, hybrid linker, or additional organic linker of sufficient length, contains a poly-thymidine oligonucleotide, through which it is attached, covalently bonded, or tethered to a glass or glass wafer, plastic such as polystyrene, polyethylene, polypropylene, a transparent or translucent solid surface such as dextran, cellulose, nylon, a microfluidic channel, beads, wells, microwells or nanowells, and is used for the detection of single, multiple, or numerous nucleic acid targets in a single amplification reaction, the method according to claim 1.

25. The polymerase enzyme(s) used in the nucleic acid amplification reaction is / are natural, modified, or chimeric enzymes, which may or may not have strand displacement activity, template-independent primer activity, base extension activity, or exonuclease activity in addition to polymerase activity, or is / are reverse transcriptase, or a polymerase having both reverse transcriptase activity and DNA polymerase activity, or is / are RNA polymerase, or is / are RNA polymerase and DNA polymerase, and the polymerase can be a thermostable enzyme, an enzyme active at ambient temperature or below ambient temperature, a hot start polymerase, and the polymerase is activated after being heated at a high temperature, preferably the primer annealing temperature, the method according to claim 1.

26. The method according to claim 1, used for detecting (methylated or non-methylated) nucleic acid or non-nucleic acid targets, A first binding moiety having extremely high affinity for a nucleic acid or non-nucleic acid target is used to capture the nucleic acid or non-nucleic acid target, and a second binding moiety, identical or different from the first binding moiety, having extremely high affinity for the nucleic acid or non-nucleic acid target is used to bind the captured nucleic acid or non-nucleic acid target, or a third binding moiety that binds to the second binding moiety with extremely high affinity is used, and the second binding moiety or the third binding moiety in a state where a synthetic or natural nucleic acid target molecule is added is provided, and after washing away the unbound second binding moiety or third binding moiety in a state where nucleic acid is added, the bound second binding moiety or the third binding moiety is detected and quantified by nucleic acid amplification using a primer labeled with an acceptor fluorophore or a non-radioactive quencher according to any one of claims 2 to 11, 13, 14, 16, 17 and / or a primer pair labeled with a donor fluorophore / fluorophore, or a plurality of primers including a probe labeled with a donor fluorophore / fluorophore, or a primer(s) labeled with an acceptor fluorophore or a non-radioactive quencher and a probe labeled with an acceptor fluorophore and a primer pair labeled with a donor fluorophore / fluorophore, The method wherein the binding moiety is preferably selected from the following binding pairs: antigen - antibody, protein - anti-protein antibody, antibody - antibody, antibody - anti-IgG antibody, primary antibody - secondary antibody, protein A - antibody, protein G - antibody, biotin - avidin, biotin - streptavidin, lectin - sugar, nucleic acid - nucleic acid, protein - nucleic acid, peptide nucleic acid, aptamer - aptamer, aptamer - nucleic acid, aptamer - protein, hapten - anti-hapten antibody, and the hapten is a small molecule including but not limited to a fluorescent dye, bromo-d-UTP, aflatoxin and other mycotoxins, peptide, sugar.

27. The method according to claim 1, which is a method for detecting and / or quantifying a large number of m-RNAs or c-DNAs, providing a first amplification primer specific to each m-RNA or c-DNA, and providing, as a second amplification primer, a common primer (common to all m-RNAs or c-DNAs in a sample) selected from sequences added to the m-RNA or the c-DNA, wherein the first amplification primer and the second common amplification primer are primers according to the method comprising primers labeled with an acceptor fluorophore / quencher, and / or primers labeled with a luminescent group or a donor fluorophore according to any one of claims 2 to 11, 13, 14, 16, 17, further comprising providing a probe specific to each m-RNA or c-DNA target, wherein the first amplification primer, the second common amplification primer, and the specific probe are any of the labeled primer-probe pairs according to the method. **Claim 28** Any of the probes according to the invention labeled with a donor fluorophore is provided attached or linked via a non-nucleotide organic linker comprising hexamethylene, hexapolyethylene glycol, or a chimera thereof, or a longer length thereof, to a primer labeled with an acceptor fluorophore or a non-radioactive quencher moiety, the probe hybridizing to a nascent nucleic acid strand generated through extension of the primer linked to the probe, the probe labeled with the donor fluorophore / fluorophore may further comprise at least an additional quencher at or near its 5'-end, or a 5'-end additional nucleic acid sequence sufficiently complementary to a sequence near the donor fluorophore / fluorescent dye / luminescent moiety-labeled base of the labeled oligonucleotide probe, and the probe may or may not comprise a minor groove binding dye, the method according to claim 1. **Claim 29** A kit (singular or plural) for performing a nucleic acid amplification reaction, which selectively contains any one of: in one or more containers, at least a non-extendable linear oligonucleotide probe labeled with a donor fluorophore / fluorescent dye / luminescent / signaling moiety, an extendable linear oligonucleotide primer labeled with a donor fluorophore / fluorescent dye / luminescent / signaling moiety, an extendable linear oligonucleotide primer (plural available) labeled with an acceptor fluorophore or an attenuator or a non-radioactive quencher, or a non-extendable linear oligonucleotide probe labeled with an acceptor fluorophore. The oligonucleotide probe labeled with the signaling moiety or the donor fluorophore / fluorescent dye / luminescent moiety or the acceptor fluorophore is a probe for monitoring nucleic acid amplification that hybridizes to either the target amplification product or one strand of the target nucleic acid. The oligonucleotide primer labeled with the acceptor fluorophore or the acceptor / attenuator or the non-radioactive quencher moiety used is one or both of the nucleic acid amplification primers used for amplifying the target sequence, and is labeled with an acceptor fluorophore moiety or an acceptor moiety or an attenuator / non-radioactive quencher moiety. In an oligonucleotide probe labeled with a signaling or donor fluorophore / fluorescent dye moiety, the signaling or donor fluorophore / fluorescent dye moiety is disposed at a base at the 3'-end, or at any base, excluding the 5'-end, located up to 30 bases away from the 3'-end. In an oligonucleotide primer labeled with a signaling or donor fluorophore / fluorescent dye moiety, the donor fluorophore / fluorescent dye moiety is disposed at a base located 2 nucleotides away from the 3'-end, or at any base, excluding the 5'-end, located up to 30 bases away from the 3'-end. In an oligonucleotide primer(s) or probe(s) labeled with an acceptor fluorophore or attenuator or acceptor or non-radioactive quencher moiety, the acceptor fluorophore or attenuator or acceptor or non-radioactive quencher moiety(ies) is disposed at any base located at least 2 bases and up to 30 bases away from the 3'-end. Further, the labeled probe may additionally carry a minor groove binding dye, or a minor groove binding dye having a non-radioactive quencher. The signaling moiety used is a donor fluorophore, and the attenuator or acceptor moiety used is an acceptor fluorophore or a non-radioactive acceptor or quencher moiety that receives energy but does not emit any energy or electromagnetic radiation. The signal is a fluorescence signal, and signal attenuation is the quenching of the fluorescence signal of the donor fluorophore / fluorescent dye / luminescent / signal moiety. The donor fluorophore / fluorescent dye / luminescent / signal moiety and the acceptor fluorophore or non-radioactive acceptor or quencher moiety are an energy transfer pair and are used in the implementation of the method. Further, the probe or primer labeled with the donor fluorophore / fluorescent dye / luminescence is provided with a label by at least an acceptor or quencher at or near the 5'-end of the probe or primer, or a 5'-end additional nucleic acid sequence that is sufficiently complementary to the sequence near the labeled base of the donor fluorophore / fluorescent dye partial probe or primer may be further provided, with or without a non-radioactive acceptor or a radiolabeled quencher, either ligated or unligated. More selectively, a) oligonucleotide probes and primers selectively containing at least a donor fluorophore / fluorescent dye moiety, selectively containing oligonucleotide primer(s) labeled with an acceptor fluorophore or a non-radioactive quencher, wherein the oligonucleotide probe labeled with the donor fluorophore moiety is a probe for monitoring nucleic acid amplification and hybridizes to one strand of the target amplification product or the target nucleic acid, and the oligonucleotide primer labeled with the acceptor fluorophore moiety or the non-radioactive quencher moiety used is one of the nucleic acid amplification primers used for amplifying the sequence of the target nucleic acid. In the oligonucleotide probe labeled with the donor fluorophore moiety, the donor fluorophore moiety is disposed at any base of the 3'-terminal base or a position up to 30 bases away from the 3'-terminal, excluding the 5'-terminal. In the primer labeled with the acceptor fluorophore or non-radioactive quencher moiety, the acceptor fluorophore or non-radioactive quencher moiety is disposed at any base at a position at least a base and up to 30 bases away from the 3'-terminal. The signal is a fluorescence signal, the donor fluorophore is the signaling moiety, and the donor fluorophore moiety and the acceptor fluorophore or non-radioactive quencher moiety are an energy transfer pair. The probe or primer labeled with the donor fluorophore may additionally be provided with an acceptor or non-radioactive quencher moiety that receives energy but does not emit any energy or electromagnetic radiation and at least labels the 5'-terminal or the vicinity thereof of the probe or primer, or has a 5'-terminal additional nucleotide sequence that is sufficiently complementary to the sequence in the vicinity of the labeled base of the oligonucleotide probe labeled with the donor fluorophore / fluorescent dye / light-emitting moiety, and may or may not be provided with a quencher, and the probe labeled with the donor fluorophore may hold a minor groove binding dye. Probes and primers. b) At least one or more primers including an oligonucleotide primer labeled with an acceptor fluorophore or an acceptor / attenuator or a quencher, and / or an oligonucleotide primer labeled with a donor fluorophore / fluorescent dye / light-emitting moiety, wherein the oligonucleotide labeled with the signaling or the donor fluorophore moiety used and the oligonucleotide labeled with the attenuator or acceptor moiety anneal separately to two strands of the target amplification product or the target nucleic acid and are extended by a polymerase(s). Two nucleic acid amplification primers. In the oligonucleotide primer labeled with the signaling or donor fluorophore moiety, the signaling or donor fluorophore moiety is disposed at any base at a position at least 2 nucleotides or bases and up to 30 bases away from the 3'-end thereof. In the oligonucleotide primer labeled with the attenuator or acceptor moiety, the attenuator or acceptor moiety is disposed at any base at a position at least 2 bases and up to 30 bases away from the 3'-end thereof. The signaling moiety used is a donor fluorophore, and the attenuator or acceptor moiety used is an acceptor fluorophore or a non-radioactive acceptor or quencher moiety that accepts energy but does not emit any energy or electromagnetic radiation. The signal is a fluorescence signal, the signal attenuation is quenching of the fluorescence signal of the donor fluorophore, and the donor fluorophore moiety and the acceptor / quencher moiety are an energy transfer pair. The primer labeled with the donor fluorophore may comprise an acceptor or quencher that labels its 5'-end, or may comprise a 5'-end additional nucleotide sequence that is sufficiently complementary to the sequence in the vicinity of the labeled base of the oligonucleotide probe labeled with the donor fluorophore / fluorescent dye / light-emitting moiety, and may or may not comprise a quencher. c) Two nucleic acid amplification primers, namely an oligonucleotide primer labeled with at least a donor fluorophore or a fluorescent dye and an oligonucleotide primer labeled with an acceptor fluorophore, wherein the oligonucleotide labeled with the donor fluorophore or the fluorescent dye moiety used and the oligonucleotide labeled with the acceptor fluorophore moiety used anneal separately to the two strands of the target amplification product or the target nucleic acid and are extended by a polymerase (s). The signal is a fluorescence signal, the signaling moiety is the acceptor fluorophore, and the donor fluorophore or fluorescent dye and the acceptor fluorophore moiety are an energy transfer pair. The donor fluorophore or the primer labeled with a fluorescent dye has an acceptor or quencher at its 5'-end, or in the primer labeled with the donor fluorophore or the fluorescent dye, it has a 5- to 8-base sequence that is sufficiently complementary to an internal base near the base labeled with the donor fluorophore or the fluorescent dye and forms a stem structure, and may or may not have a quencher, and may or may not have a spacer intervening between the primer labeled with the donor fluorophore or the fluorescent dye and the additional nucleotide sequence. A primer, d) At least one or more primers comprising an acceptor fluorophore or an oligonucleotide primer labeled with a non-radioactive quencher, and / or an oligonucleotide primer labeled with a donor fluorophore / fluorescent dye / light-emitting moiety, wherein the primer may additionally have an additional 5'-end quencher or a 5'-end additional nucleotide that is sufficiently complementary to a sequence near the labeled base of the oligonucleotide primer labeled with the donor fluorophore / fluorescent dye / light-emitting moiety, and may further include those with or without the above quencher, and the one or more primers have an added non-target sequence (s) that selectively contains the sequences of SEQ ID NOs: 21-22 and is appropriately labeled. A primer, e) At least one or more primers comprising an acceptor fluorophore or an oligonucleotide primer (s) labeled with a non-radioactive quencher, and / or an oligonucleotide probe labeled with a donor fluorophore / fluorescent dye / luminescent moiety, wherein the probe is a non-target sequence incorporated or added to a synthetic target sequence generated before or during the amplification reaction (s) hybridizes, and the oligonucleotide probe labeled with the donor fluorophore / fluorescent dye / luminescent moiety may further comprise a minor groove binding dye, at least an additional quencher at or near the 5 'end, or a 5' end additional nucleotide sufficiently complementary to the sequence near the labeled base of the oligonucleotide probe labeled with the donor fluorophore / fluorescent dye / luminescent moiety, and may further comprise an oligonucleotide probe with or without the above-described quencher added, and may further comprise a primer (s) to which a non-target sequence (s) is added, probe, f) An oligonucleotide probe and a corresponding acceptor fluorophore or oligonucleotide primer labeled with a fluorescent dye / donor fluorophore, at least labeled with a donor fluorophore / fluorescent dye or acceptor fluorophore, for nucleic acid target detection based on nucleic acid amplification, wherein either the donor fluorophore or acceptor fluorophore of the probe generates a signal, and the probe may further comprise a minor groove binding dye, and the oligonucleotide probe or primer labeled with the donor fluorophore may comprise at least an additional quencher at or near the 5 'end, or may further comprise a 5' end additional nucleotide sufficiently complementary to the sequence near the labeled base of the oligonucleotide probe or primer labeled with the donor fluorophore, and with or without an additional quencher, probe and primer, g) For transcription-mediated amplification (TMA) / nucleic acid sequence-based amplification (NASBA), provide at least acceptor fluorophore / non-radioactive quencher-labeled primers specific for the target and / or donor fluorophore-labeled promoter sequence-bearing primers, or vice versa, wherein the primer labeled with the donor fluorophore may additionally have at least an additional quencher at or near the 5'-end, or 5'-end additional nucleotides sufficiently complementary to the sequence near the labeled base of the oligonucleotide primer labeled with the donor fluorophore / fluorescent dye / light-emitting moiety, and may or may not further have the additional quencher described first, and may additionally provide appropriately labeled probes with or without a minor groove binding dye, h) The kit further contains in the one or more containers at least a positive control template and a pair of a primer labeled with a donor fluorophore specific for the positive control template and a primer labeled with an acceptor fluorophore / quencher, or a primer labeled with a donor fluorophore / acceptor fluorophore / quencher and a corresponding probe labeled with an acceptor fluorophore / donor fluorophore, In the oligonucleotide primer labeled with a donor fluorophore / acceptor fluorophore moiety specific for the positive control template, the donor fluorophore / acceptor fluorophore is located at any base, except the 5'-end, at a position at least 2 nucleotides and up to 30 bases away from the 3'-end. In the oligonucleotide primer labeled with an acceptor fluorophore moiety or a quencher / donor fluorophore moiety specific for the positive control template, the acceptor fluorophore moiety or the quencher moiety or the attenuator moiety is located at any base at a position at least 2 bases and up to 30 bases away from the 3'-end thereof, In the oligonucleotide probe labeled with a signaling moiety or a donor fluorophore moiety specific to the positive control template, the signaling or donor fluorophore moiety is disposed at any base at the 3'-end thereof, or at a position up to 30 bases away from the 3'-end, provided that it excludes the 5'-end. In the oligonucleotide primer(s) labeled with an acceptor fluorophore moiety or a quencher moiety, the acceptor fluorophore moiety or the quencher moiety (s) is disposed at any base at a position at least 2 bases and up to 30 bases away from the 3'-end thereof. Alternatively, in the oligonucleotide probe labeled with a donor fluorophore / acceptor fluorophore moiety specific to the positive control template, the donor fluorophore / acceptor fluorophore moiety is disposed at any base at the 3'-end thereof, or at a position up to 30 bases away from the 3'-end, provided that it excludes the 5'-end. In the oligonucleotide primer labeled with an acceptor fluorophore / donor fluorophore moiety specific to the positive control template, the acceptor fluorophore / donor fluorophore corresponding to any base at a position at least 2 bases and up to 30 bases away from the 3'-end thereof is disposed. i) An oligonucleotide primer or probe labeled with at least a signaling or donor fluorophore / fluorescent dye / light-emitting moiety, which may further be provided with at least an additional quencher at or near the 5'-end, or with additional 5'-end nucleotides sufficiently complementary to the sequence near the labeled base of the oligonucleotide primer or probe labeled with the donor fluorophore / fluorescent dye / light-emitting moiety, and which may or may not further be provided with the additional quencher described first. The probe may or may not be provided with a minor groove binding dye, primer or probe. j) Primers (single or plural) labeled with an acceptor fluorophore / non-radioactive quencher and / or primers or probes labeled with a donor fluorophore / fluorophore for nested or semi-nested nucleic acid amplification and absolute quantification of a target nucleic acid, at least a plurality of primers including the primers or probes labeled with the donor fluorophore / fluorophore, wherein the primers or probes labeled with the donor fluorophore / fluorophore may additionally have at least an additional quencher at or near the 5'-end, or 5'-end additional nucleotides sufficiently complementary to the sequence near the labeled base of the oligonucleotide primer or probe labeled with the donor fluorophore / fluorescent dye / luminescent moiety, and may or may not further have the additional quencher described first, and the probe may have a minor groove binding dye, primer, k) At least a plurality of primer pairs including primers (single or plural) labeled with an acceptor fluorophore / non-radioactive quencher and / or oligonucleotide primers or probes labeled with any donor fluorophore / fluorescent dye / luminescent moiety for detection and / or quantification of a plurality of nucleic acid targets, or probes labeled with the acceptor fluorophore described above and a plurality of pairs of primers with a donor fluorophore / fluorescent dye, l) The kit contains at least a primer pair, and a probe labeled with at least a donor fluorophore / fluorescent dye / light-emitting moiety is attached or linked to a primer labeled with an acceptor fluorophore or a non-radioactive quencher moiety via a non-nucleotide organic linker containing hexamethylene, hexapolyethylene glycol, or a chimeric linker, or a longer length thereof. The probe hybridizes to a nascent nucleic acid strand generated through the extension of the primer linked to the probe. The probe labeled with the donor fluorophore / fluorophore may contain an additional quencher at or near the 5'-end, or 5'-end additional nucleotides that are sufficiently complementary to the sequence near the labeled base of the oligonucleotide probe labeled with the donor fluorophore / fluorescent dye / light-emitting moiety, and the probe may be provided with or without a minor groove-binding dye. The claimed kit(s) may further comprise a reaction buffer, a plurality of deoxynucleoside triphosphates, a thermostable or non-thermostable polymerase enzyme(s), a thermostable or non-thermostable ligase enzyme, a positive control template, and a labeled primer pair or labeled primer-probe pair for each of the positive templates. Claim 30 The target nucleic acid is a purified or partially purified nucleic acid or an unpurified nucleic acid, and is a natural, synthetic or semi-synthetic single-stranded or double-stranded DNA or RNA, single-stranded or double-stranded c-DNA, genomic DNA, methylated DNA, mitochondrial DNA, exosomal DNA, plasmid DNA, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (m-RNA), small molecule RNA (including but not limited to microRNA, sRNA, stRNA, snoRNA, ncRNA), stem cell-derived DNA including very small embryonic-like stem cells, viral DNA or RNA, or cancer cell DNA (derived from any source including but not limited to body fluids, biopsy samples, tumors, pus, saliva, feces, cancer stem cells), and is selected from single-stranded or double-stranded synthetic or semi-synthetic DNA or RNA generated by adding one or two non-target synthetic sequences to the ends of the target nucleic acid, and the target nucleic acid does not have to constitute the entire nucleic acid molecule, and also, the genomic sequence of an infectious agent, mutations in the genomic sequence (single-base changes, deletions or insertions of several bases or long sequences), or the presence or absence of the genomic sequences of human bacteria, yeast, fungi, plants, animals, humans, parasites and their viruses and any other organisms, or mutations in them (single-base changes, deletions or insertions of several bases or long sequences), are associated with the presence of a disease or disorder, or susceptibility to an infection or disease or disorder, or suitability for disease treatment, prenatal diagnosis, genetic traits, genotypes, allelic types, SNP detection, cell types, tissue types, species or strain types, cancer types or cancer subtypes, cancer detection, disease typing or subtyping, expression genes, the method according to claim 1.

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