A method for analyzing the presence and type of mutations in a target nucleic acid

Abstract

The present application provides a method for analyzing the presence and type of mutations in a target nucleic acid. In addition, the present application also provides a kit that can be used to implement the method of the present invention.

Description

Technical Field

[0001] The present application relates to the field of molecular biology, particularly the field of nucleic acid detection and analysis. In particular, the present application provides a method for analyzing the presence and type of mutations in a target nucleic acid. In addition, the present application also provides a kit that can be used to implement the method of the present invention. Background Art

[0002] In contemporary biology and medicine, nucleic acid amplification is an indispensable biotechnology. Currently, nucleic acid amplification technology has been widely used in clinical diagnosis, basic research, epidemiological research, transgenic research, archaeological research and other fields. Polymerase chain reaction (PCR) is the first established in vitro nucleic acid amplification technology, which is of epoch-making significance. This technology has been widely used in biological and medical fields. However, when using PCR technology and PCR-related technologies (such as real-time PCR and multiplex PCR) for nucleic acid amplification, it is limited by laboratory conditions and relies on complex and expensive thermal cycling instruments. In addition, the determination of PCR results (i.e., the detection of amplified products) is relatively complicated and requires a set of complex processes and equipment. These disadvantages limit the widespread application of this technology, especially in economically backward areas and in the field of rapid diagnosis.

[0003] To overcome the shortcomings of PCR-related amplification techniques, a number of isothermal amplification technologies have emerged. Compared to PCR-related techniques, isothermal amplification techniques do not rely on thermal cycling equipment, offer faster reaction speeds, and exhibit greater sensitivity. Therefore, isothermal amplification techniques facilitate rapid amplification, on-site diagnosis, and convenient testing. To date, over ten different isothermal amplification techniques have been developed, with the most widely used including loop-mediated isothermal amplification (LAMP), cross-primer amplification (CPA), rolling circle amplification (RCA), strand displacement amplification (SDA), and helicase-dependent isothermal amplification (HDA).

[0004] Cross-primer amplification (CPA) is an isothermal amplification technology with a simple design and sensitive detection. The primer amplification system mainly includes cross primers, stripping primers, detection primers, and DNA polymerases with chain displacement function. In this method, five specific primers are usually designed for the target gene, and they are isothermally amplified under the action of chain displacement DNA polymerase (such as Bst DNA polymerase). Isothermal amplification technology stands out among many detection technologies with its advantages such as fast response and low requirements for detection equipment. Compared with traditional polymerase chain reaction, isothermal amplification technology can perform rapid amplification at a constant temperature, the instruments and equipment used are simple, the amplification time is greatly shortened, and the results are easy to interpret. Therefore, cross-primer isothermal amplification technology is currently widely used in genetically modified crop detection, food safety inspection and quarantine, and medical disease safety inspection and quarantine.

[0005] However, cross-primer amplification also has its drawbacks. For one thing, traditional CPA methods have poor specificity and strong nonspecific signals, which may interfere with result interpretation. Furthermore, traditional CPA methods can only detect the presence of nucleic acid variants or mutations, but cannot distinguish the type of variant. For example, when a gene segment contains multiple variants or mutations, traditional CPA methods cannot distinguish between these different variants or mutations.

[0006] Therefore, there is still a need in this field to develop new methods for detecting nucleic acid variations to solve the problems existing in the traditional CPA method. Summary of the Invention

[0007] Based on in-depth research, the inventors of this application have developed a new method for detecting target nucleic acid molecules. The method of this application has the characteristics of simplicity, rapidity, and high efficiency.

[0008] Detection method

[0009] Therefore, in one aspect, the present invention provides a method for detecting the presence and type of a mutation in a target nucleic acid, comprising the steps of:

[0010] (1) providing a first primer, a second primer, and a third primer; wherein,

[0011] The first primer comprises an annealing sequence and a crossover sequence; wherein the annealing sequence is located downstream or at the 3' end of the crossover sequence, and the two are directly connected or connected via a nucleotide linker; the annealing sequence is capable of hybridizing or annealing with the first region of the first strand (a strand) of the target nucleic acid under conditions that allow nucleic acid annealing or hybridization, and is capable of initiating an extension reaction and generating a nucleic acid strand containing a complementary sequence (Ts sequence) to the nucleic acid sequence to be detected (Ta sequence) under conditions that allow nucleic acid synthesis or amplification; the Ta sequence or Ts sequence is suspected of containing a mutation;

[0012] The second primer and the third primer can hybridize or anneal with the complementary strand of the a strand (the s strand) under conditions that allow nucleic acid annealing or hybridization; and the second primer can anneal or hybridize with a second region of the s strand that is located downstream or 3' of the Ts sequence; the third primer can anneal or hybridize with a third region of the s strand that is located downstream or 3' of the Ts sequence; and the second region is located downstream or 3' of the third region; and,

[0013] The crossover sequence contains a sequence capable of annealing or hybridizing to the second region;

[0014] Optionally, a fourth primer and / or a fifth primer are further provided, wherein the fourth primer can anneal or hybridize to a fourth region of the a chain located downstream or 3' of the first region under conditions that allow nucleic acid annealing or hybridization; and the fifth primer can anneal or hybridize to a fifth region of the s chain located downstream or 3' of the second region under conditions that allow nucleic acid annealing or hybridization;

[0015] (2) contacting the first, second and third primers, and optionally the fourth and / or fifth primers, with a target nucleic acid and a nucleic acid polymerase (e.g., incubating) under conditions that allow nucleic acid synthesis or amplification, and generating a nucleic acid amplification product; the nucleic acid amplification product includes: (i) a first amplified chain containing the sequence of the first primer, the Ts sequence, the sequence of the third region, and the sequence of the second region; and (ii) a second amplified chain containing the complementary sequence of the second region, the complementary sequence of the third region, the Ta sequence, and the complementary sequence of the first primer;

[0016] (3) performing melting curve analysis on the first amplified chain and / or the second amplified chain using one or more detection probes; wherein,

[0017] The detection probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridized with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence;

[0018] Furthermore, the detection probe contains a Ts capture sequence capable of hybridizing or annealing to a Ts sequence, and optionally, contains a first region complementary-capture sequence capable of hybridizing or annealing to a complementary sequence of the first region and / or a third region capture sequence capable of hybridizing or annealing to a third region; or, the detection probe contains a Ta capture sequence capable of hybridizing or annealing to a Ta sequence, and optionally, contains a first region capture sequence capable of hybridizing or annealing to a first region and / or a third region complementary-capture sequence capable of hybridizing or annealing to a complementary sequence of the third region;

[0019] (4) Determine the presence and type of mutations in the target nucleic acid based on the results of the melting curve analysis.

[0020] In the method of the present invention, the target nucleic acid is not limited to its sequence composition or length. For example, the target nucleic acid can be DNA, RNA, DNA / RNA complex, or a mixture thereof. In addition, the target nucleic acid can exist in single-stranded or double-stranded form, for example, the target nucleic acid is selected from single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or DNA / RNA hybrid duplex.

[0021] In certain preferred embodiments, before step (2), the target nucleic acid may be pre-treated, for example, purified, enriched or reverse transcribed.

[0022] For example, when the target nucleic acid is RNA, in certain embodiments, a reverse transcription reaction is performed before performing the method of the present invention to obtain cDNA complementary to the RNA. A detailed description of the reverse transcription reaction can be found in, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001).

[0023] The target nucleic acid can be obtained from any source, including but not limited to prokaryotes, eukaryotes (e.g., protozoa, parasites, fungi, yeasts, plants, animals including mammals and humans) or viruses (e.g., Herpes virus, HIV, influenza virus, Epstein-Barr virus, hepatitis virus, polio virus, etc.), viroids or nucleic acid molecule libraries. The target nucleic acid can also be any form of nucleic acid sequence, such as a genomic sequence, an artificially isolated or fragmented sequence, a synthetic sequence, etc.

[0024] In the method of the present invention, the length of the Ts sequence or Ta sequence is not limited. In certain preferred embodiments, the length of the Ts sequence or Ta sequence is 1-100nt, such as 1-3nt, 3-5nt, 5-8nt, 8-10nt, 10-13nt, 13-15nt, 15-18nt, 18-20nt, 20-25nt, 25-30nt, 30-35nt, 35-40nt, 40-45nt, 45-50nt, 50-55nt, 55-60nt, 60-65nt, 65-70nt, 70-75nt, 75-80nt, 80-85nt, 85-90nt, 90-95nt, 95-100nt.

[0025] In the method of the present invention, the type of mutation suspected to be contained in the Ts sequence or Ta sequence is not limited. In certain preferred embodiments, the mutation is selected from addition, deletion, substitution, or any combination thereof.

[0026] First primer

[0027] In the method of the present invention, the first primer contains an annealing sequence and a crossover sequence.

[0028] The annealing sequence is not limited by its composition and length, as long as it can specifically hybridize with the first region of the first strand (a strand) of the target nucleic acid. For example, the length of the annealing sequence is 5-50 nt, such as 5-8 nt, 8-10 nt, 10-13 nt, 13-15 nt, 15-18 nt, 18-20 nt, 20-25 nt, 25-30 nt, 30-35 nt, 35-40 nt, 40-45 nt, 45-50 nt.

[0029] In certain preferred embodiments, the annealing sequence is capable of annealing or hybridizing with the first region of the a chain located downstream or at the 3' end of the Ta sequence.

[0030] In certain preferred embodiments, the annealing sequence contains a sequence that is complementary (eg, completely complementary) to the nucleotide sequence of the first region or a fragment thereof.

[0031] In certain preferred embodiments, the annealing sequence is located at the 3' end of the first primer.

[0032] In certain preferred embodiments, the annealing sequence comprises or consists of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.

[0033] In certain preferred embodiments, the annealing sequence comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the annealing sequence comprises a non-natural nucleotide, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0034] In the method of the present invention, the crossover sequence is not limited by its composition and length, as long as it can specifically hybridize with the second region. For example, the length of the crossover sequence is 5-100 nt, such as 5-10 nt, 10-14 nt, 14-20 nt, 20-25 nt, 25-30 nt, 30-35 nt, 35-40 nt, 40-45 nt, 45-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt.

[0035] The crossover sequence contains a sequence that is complementary (eg, completely complementary) to the nucleotide sequence of the second region or a fragment thereof.

[0036] In certain preferred embodiments, the crossover sequence contains the sequence of the second primer.

[0037] In certain preferred embodiments, the crossover sequence is identical to the sequence of the second primer.

[0038] In certain preferred embodiments, the crossover sequence comprises or consists of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.

[0039] In certain preferred embodiments, the crossover sequence comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the crossover sequence comprises a non-natural nucleotide, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0040] Nucleotide linker

[0041] In the methods of the present invention, in certain embodiments, the annealing sequence and the crossover sequence of the first primer are connected by a nucleotide linker.

[0042] In certain preferred embodiments, the length of the nucleotide linker is 5-20 nt, such as 5-10 nt, 10-15 nt, or 15-20 nt.

[0043] In certain preferred embodiments, the nucleotide linker comprises or consists of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.

[0044] In certain preferred embodiments, the nucleotide linker comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the nucleotide linker comprises a non-natural nucleotide, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0045] Second primer

[0046] In the method of the present invention, the second primer is not limited by its composition and length, as long as it can specifically hybridize to the second region. For example, the length of the second primer is 5-100 nt, such as 5-10 nt, 10-14 nt, 14-20 nt, 20-25 nt, 25-30 nt, 30-35 nt, 35-40 nt, 40-45 nt, 45-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt.

[0047] In certain preferred embodiments, the second primer contains a sequence that is complementary (eg, completely complementary) to the nucleotide sequence of the second region or a fragment thereof.

[0048] In certain preferred embodiments, the second primer comprises or consists of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.

[0049] In certain preferred embodiments, the second primer comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the second primer comprises a non-natural nucleotide, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0050] The third primer

[0051] In the method of the present invention, the third primer is not limited by its composition and length, as long as it can specifically hybridize to the third region. For example, the length of the third primer is 5-100nt, such as 5-10nt, 10-13nt, 13-17nt, 17-20nt, 20-25nt, 25-30nt, 30-35nt, 35-40nt, 40-45nt, 45-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt.

[0052] In certain preferred embodiments, the third primer contains a sequence that is complementary (eg, completely complementary) to the nucleotide sequence of the third region or a fragment thereof.

[0053] In certain preferred embodiments, the third primer comprises or consists of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.

[0054] In certain preferred embodiments, the third primer comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the third primer comprises a non-natural nucleotide, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0055] The fourth primer

[0056] In the method of the present invention, the fourth primer is not limited by its composition and length, as long as it can specifically hybridize to the fourth region. For example, the length of the fourth primer is 5-100nt, such as 5-10nt, 10-13nt, 13-17nt, 17-20nt, 20-23nt, 23-25nt, 25-30nt, 30-35nt, 35-40nt, 40-45nt, 45-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt.

[0057] In certain preferred embodiments, the fourth primer contains a sequence that is complementary (eg, completely complementary) to the nucleotide sequence of the fourth region or a fragment thereof.

[0058] In certain preferred embodiments, the fourth primer comprises or consists of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.

[0059] In certain preferred embodiments, the fourth primer comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the fourth primer comprises a non-natural nucleotide, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0060] Fifth primer

[0061] In the method of the present invention, the fifth primer is not limited by its composition and length, as long as it can specifically hybridize to the fifth region. For example, the length of the fifth primer is 5-100 nt, such as 5-10 nt, 10-13 nt, 13-17 nt, 17-20 nt, 20-22 nt, 22-25 nt, 25-30 nt, 30-35 nt, 35-40 nt, 40-45 nt, 45-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt.

[0062] In certain preferred embodiments, the fifth primer contains a sequence that is complementary (eg, completely complementary) to the nucleotide sequence of the fifth region or a fragment thereof.

[0063] In certain preferred embodiments, the fifth primer comprises or consists of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.

[0064] In certain preferred embodiments, the fifth primer comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the fifth primer comprises a non-natural nucleotide, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0065] Regarding steps (2), (3) and (4)

[0066] It is readily understood that the method of the present invention is not limited to the number of detection probes used. In step (3), at least one, at least two, at least three, at least four, at least five, at least six, at least eight, or at least ten detection probes may be used to perform melting curve analysis on the first amplified strand and / or the second amplified strand.

[0067] In certain preferred embodiments, the reporter groups contained in the detection probes are different from each other.

[0068] It is easy to understand that the detection probe can be added to the reaction system of step (2) before the nucleic acid amplification product is generated (for example, before nucleic acid synthesis or amplification); or, the detection probe can be contacted with the nucleic acid amplification product generated in step (2) after the nucleic acid amplification product is generated (for example, after step (2) is completed).

[0069] In certain preferred embodiments, in step (3), when performing melting curve analysis on the first amplified chain and / or the second amplified chain, the signal of the reporter group of each detection probe is monitored in real time, thereby obtaining one or more melting curves each corresponding to the signal of a reporter group; subsequently, in step (4), the presence and type of mutation are determined based on the signal type of the reporter group and the melting peak (melting point) in the melting curve.

[0070] As discussed above, melting curve analysis can be performed by using detection probes labeled with a reporter group and a quencher group.

[0071] In certain embodiments, the product of step (2) can be gradually heated and the signal emitted by the reporter group on the detection probe can be monitored in real time to obtain a curve showing how the signal intensity of the product of step (2) changes with temperature. For example, the product of step (2) can be gradually heated from 45°C or lower (e.g., no more than 45°C, no more than 40°C, no more than 35°C, no more than 30°C, no more than 25°C) to 75°C or higher (e.g., at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C), and the signal emitted by the reporter group on the detection probe can be monitored in real time to obtain a curve showing how the signal intensity of the reporter group changes with temperature. The rate of heating can be routinely determined by those skilled in the art. For example, the rate of temperature increase can be: 0.01-1°C (e.g., 0.01-0.05°C, 0.05-0.1°C, 0.1-0.5°C, 0.5-1°C, 0.04-0.4°C, such as 0.01°C, 0.02°C, 0.03°C, 0.04°C, 0.05°C, 0.06°C, 0.07°C, 0.08°C, 0.09°C, 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C or 1.0°C) per step, and each step is maintained for 0.5-15s (e.g., 0.5-1s , 1-2s, 2-3s, 3-4s, 4-5s, 5-10s, 10-15s); or the temperature is increased by 0.01-1°C per second (for example, 0.01-0.05°C, 0.05-0.1°C, 0.1-0.5°C, 0.5-1°C, 0.04-0.4°C, for example, 0.01°C, 0.02°C, 0.03°C, 0.04°C, 0.05°C, 0.06°C, 0.07°C, 0.08°C, 0.09°C, 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C or 1.0°C).

[0072] In certain embodiments, the product of step (2) can be gradually cooled and the signal emitted by the reporter group on the detection probe can be monitored in real time to obtain a curve showing that the signal intensity of the product of step (2) changes with temperature. For example, the product of step (2) can be gradually cooled from a temperature of 75°C or higher (e.g., at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C) to a temperature of 45°C or lower (e.g., no more than 45°C, no more than 40°C, no more than 35°C, no more than 30°C, no more than 25°C), and the signal emitted by the reporter group on the detection probe can be monitored in real time to obtain a curve showing that the signal intensity of the reporter group changes with temperature. The rate of cooling can be routinely determined by those skilled in the art. For example, the cooling rate can be: cooling by 0.01-1°C per step (e.g., 0.01-0.05°C, 0.05-0.1°C, 0.1-0.5°C, 0.5-1°C, 0.04-0.4°C, such as 0.01°C, 0.02°C, 0.03°C, 0.04°C, 0.05°C, 0.06°C, 0.07°C, 0.08°C, 0.09°C, 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C or 1.0°C), and maintaining for 0.5-15s per step (e.g., 0.5-1s , 1-2s, 2-3s, 3-4s, 4-5s, 5-10s, 10-15s); or cooling by 0.01-1°C per second (for example, 0.01-0.05°C, 0.05-0.1°C, 0.1-0.5°C, 0.5-1°C, 0.04-0.4°C, for example, 0.01°C, 0.02°C, 0.03°C, 0.04°C, 0.05°C, 0.06°C, 0.07°C, 0.08°C, 0.09°C, 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C or 1.0°C).

[0073] Subsequently, the obtained curve can be differentiated to obtain the melting curve of the product of step (2). Based on the melting peak (melting point) in the melting curve, the presence of the mutation type corresponding to the melting peak (melting point) can be determined.

[0074] Without being bound by theory, the resolution or accuracy of melting curve analysis can reach 0.1°C or higher. In other words, melting curve analysis can distinguish two melting peaks with a melting point difference of only 0.1°C or lower. Therefore, in certain embodiments of the method of the present invention, the melting point difference between any two duplexes (e.g., different duplexes formed by different probes and the same amplification product; or, different duplexes formed by the same probe and different amplification products) can be at least 0.1°C, so that the arbitrary two duplexes can be distinguished and discriminated by melting curve analysis. However, for the purpose of facilitating distinction and discrimination, a larger melting point difference between the two duplexes is preferred in some cases. Therefore, in certain embodiments of the method of the present invention, the melting point difference between the two duplexes can be any desired value, as long as the melting point difference can be distinguished and discriminated by melting curve analysis.

[0075] According to the method of the present invention, the conditions allowing nucleic acid synthesis or amplification in step (2) can be routinely determined by those skilled in the art.

[0076] In certain preferred embodiments, step (2) is performed under conditions that allow for isothermal amplification of the nucleic acid.

[0077] In certain preferred embodiments, in step (2), the first, second and third primers, and optionally the fourth and / or fifth primers, are contacted (e.g., incubated) with the target nucleic acid and nucleic acid polymerase at a temperature of 55°C to 65°C (e.g., 55°C to 58°C, 58°C to 60°C, 60°C to 63°C, 63°C to 65°C), and a nucleic acid amplification product is generated.

[0078] In certain preferred embodiments, in step (2), the first, second and third primers, and optionally the fourth and / or fifth primers, are contacted (e.g., incubated) with the target nucleic acid and nucleic acid polymerase for a period of at least 10 min, at least 20 min, at least 30 min, at least 40 min, at least 50 min, or at least 60 min.

[0079] In certain preferred embodiments, the concentration of the target nucleic acid is at least 0.01×10 3 copies / μL (e.g. at least 0.01×10 3 copies / μL, at least 0.05×10 3 copies / μL, at least 0.1×10 3 copies / μL, at least 0.5×10 3 copies / μL, at least 1.0×10 3 copies / μL, at least 2.0×10 3 copies / μL).

[0080] In certain preferred embodiments, the first amplified chain comprises, starting from the 5' end, the sequence of the first primer, the Ts sequence, the sequence of the third region, and the sequence of the second region.

[0081] In certain preferred embodiments, the second amplified chain contains, starting from the 5' end, a complementary sequence to the second region, a complementary sequence to the third region, a Ta sequence, and a complementary sequence to the first primer.

[0082] probe

[0083] In the method of the present invention, the detection probe is not limited by its length. For example, the length of the detection probe is 10-500nt, such as 10-15nt, 15-20nt, 20-25nt, 25-30nt, 30-35nt, 35-40nt, 40-45nt, 45-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-150nt, 150-200nt, 200-250nt, 250-300nt, 300-350nt, 350-400nt, 400-450nt, 450-500nt.

[0084] In certain preferred embodiments, the detection probe contains a Ts capture sequence capable of hybridizing or annealing to the Ts sequence, and optionally, contains a first region complementary-capture sequence capable of hybridizing or annealing to the complementary sequence of the first region and / or a third region capture sequence capable of hybridizing or annealing to the third region.

[0085] For example, the detection probe contains a Ts capture sequence capable of hybridizing or annealing with a Ts sequence; or, the detection probe contains a Ts capture sequence capable of hybridizing or annealing with a Ts sequence, and a first region complementary-capture sequence capable of hybridizing or annealing with a complementary sequence of the first region, and the first region complementary-capture sequence is located downstream or at the 3' end of the Ts capture sequence; or, the detection probe contains a Ts capture sequence capable of hybridizing or annealing with a Ts sequence, and a third region capture sequence capable of hybridizing or annealing with a third region, and the third region capture sequence is located upstream or at the 5' end of the Ts capture sequence; or, the detection probe contains a Ts capture sequence, a first region complementary-capture sequence, and a third region capture sequence; and the third region capture sequence is located upstream or at the 5' end of the Ts capture sequence, and the first region complementary-capture sequence is located downstream or at the 3' end of the Ts capture sequence.

[0086] When a melting curve analysis is performed on the first amplified chain and / or the second amplified chain using multiple detection probes, in certain preferred embodiments, different detection probes hybridize or anneal to different regions of the first amplified chain and / or the second amplified chain.

[0087] In certain preferred embodiments, the detection probe comprises or consists of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.

[0088] In certain preferred embodiments, the detection probe comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the detection probe comprises a non-natural nucleotide, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0089] In certain preferred embodiments, the detection probe has a 3'-OH end, or its 3'-end is blocked to inhibit its extension. The 3'-end of a nucleic acid (e.g., a detection probe) can be blocked by various methods. For example, the 3'-OH of the last nucleotide of the detection probe can be modified to block the 3'-end of the detection probe. In certain embodiments, the 3'-end of the detection probe can be blocked by adding a chemical moiety (e.g., biotin or an alkyl) to the 3'-OH of the last nucleotide of the detection probe. In certain embodiments, the 3'-end of the detection probe can be blocked by removing the 3'-OH of the last nucleotide of the detection probe, or replacing the last nucleotide with a dideoxynucleotide.

[0090] As described above, the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence.

[0091] In such embodiments, when the detection probe is not hybridized with other sequences, the quencher group is located at a position that can absorb or quench the signal of the reporter group (for example, the quencher group is located adjacent to the reporter group), thereby absorbing or quenching the signal emitted by the reporter group. In this case, the detection probe does not emit a signal. Further, when the detection probe is hybridized with its complementary sequence, the quencher group is located at a position that cannot absorb or quench the signal of the reporter group (for example, the quencher group is located at a position away from the reporter group), thereby being unable to absorb or quench the signal emitted by the reporter group. In this case, the detection probe emits a signal.

[0092] In the method of the present invention, the reporter group and the quencher group can be any suitable group or molecule known in the art, and specific examples thereof include but are not limited to Cy2 TM (506),YO-PRO TM -l(509),YOYO TM -l(509),Calcein(517),FITC(518),FluorX TM (519),Alexa TM (520),Rhodamine 110(520),Oregon Green TM 500(522),Oregon Green TM 488(524),RiboGreen TM (525),Rhodamine Green TM (527),Rhodamine123(529),Magnesium Green TM (531),Calcium Green TM (533),TO-PRO TM -l(533),TOTOl(533),JOE(548),BODIPY530 / 550(550),Dil(565),BODIPY TMR(568),BODIPY558 / 568(568),BODIPY564 / 570(570),Cy3 TM (570),Alexa TM 546(570),TRITC(572),MagnesiumOrange TM (575),Phycoerythrin R&B(575),Rhodamine Phalloidin(575),CalciumOrange TM(576),PyroninY(580),Rhodamine B(580),TAMRA(582),Rhodamine Red TM (590),Cy3.5 TM (596),ROX(608),Calcium Crimson TM (615),Alexa TM 594(615),Texas Red(615),Nile Red(628),YO-PRO TM -3(631),YOYO TM -3(631),R-phycocyanin(642),C-Phycocyanin(648),TO-PRO TM -3(660),T0T03(660),DiD DilC(5)(665),Cy5 TM (670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), Biosearch Blue (447), CALFluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CALFluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705), and Quasar 705 (610). The numbers in parentheses indicate the maximum emission wavelength in nm.

[0093] In addition, various suitable pairs of reporter and quencher groups are known in the art, see, for example, Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Color AND Constitution of Oiganic Molecules (Academic Press, New York, 1976); Bishop, editor, Indicators (Peigamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1993); New York, 1949); Haugland, RP, Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene, Oreg., 1996); U.S. Patents 3,996,345 and 4,351,760.

[0094] In certain preferred embodiments, the reporter group is a fluorescent group. In such embodiments, the signal emitted by the reporter group is fluorescence, and the quencher group is a molecule or group that can absorb / quench the fluorescence (e.g., another fluorescent molecule that can absorb the fluorescence, or a quencher that can quench the fluorescence). In certain preferred embodiments, the fluorescent group includes but is not limited to various fluorescent molecules, such as ALEX-350, FAM, VIC, TET, CAL Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705, etc. In certain preferred embodiments, the quencher group includes but is not limited to various quenchers, such as DABCYL, BHQ (such as BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA, etc.

[0095] The design of such self-quenching detection probes is within the capabilities of those skilled in the art. For example, the detection probe is labeled with a reporter group at its 5' end or upstream and a quencher group at its 3' end or downstream, or with a reporter group at its 3' end or downstream and a quencher group at its 5' end or upstream.

[0096] Therefore, when the detection probe exists alone, the reporter group and the quencher group are close to each other and interact with each other, so that the signal emitted by the reporter group is absorbed by the quencher group, thereby causing the detection probe to not emit a signal; and when the detection probe hybridizes with its complementary sequence, the reporter group and the quencher group are separated from each other, so that the signal emitted by the reporter group cannot be absorbed by the quencher group, thereby causing the detection probe to emit a signal.

[0097] However, it should be understood that the reporter group and the quencher group do not have to be marked at the end of the detection probe. The reporter group and / or the quencher group can also be marked inside the detection probe, as long as the signal emitted by the detection probe when hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence. For example, the reporter group can be marked upstream (or downstream) of the detection probe, and the quencher group can be marked downstream (or upstream) of the detection probe, and the two can be separated by a sufficient distance (e.g., 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, or a longer distance). Thus, when the detection probe exists alone, due to the free curling of the probe molecule or the formation of a secondary structure (such as a hairpin structure) of the probe, the reporter group and the quencher group approach each other and interact with each other, so that the signal emitted by the reporter group is absorbed by the quencher group, thereby causing the detection probe to not emit a signal; and, when the detection probe hybridizes with its complementary sequence, the reporter group and the quencher group are separated by a sufficient distance so that the signal emitted by the reporter group cannot be absorbed by the quencher group, thereby causing the detection probe to emit a signal. In certain preferred embodiments, the reporter group and the quencher group are 10-80nt or longer apart, for example, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt. In certain preferred embodiments, the reporter group and the quencher group are no more than 80nt, no more than 70nt, no more than 60nt, no more than 50nt, no more than 40nt, no more than 30nt, or no more than 20nt apart. In certain preferred embodiments, the reporter group and the quencher group are at least 5 nt, at least 10 nt, at least 15 nt, or at least 20 nt apart.

[0098] Therefore, the reporter group and the quencher group can be labeled at any suitable position of the detection probe, as long as the signal emitted by the detection probe when hybridized with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence. However, in certain preferred embodiments, at least one of the reporter group and the quencher group is located at the end (e.g., 5' or 3' end) of the detection probe. In certain preferred embodiments, one of the reporter group and the quencher group is located at the 5' end of the detection probe or at a position 1-10nt away from the 5' end, and the reporter group and the quencher group are at a suitable distance such that the quencher group can absorb or quench the signal of the reporter group before the detection probe hybridizes with its complementary sequence. In certain preferred embodiments, one of the reporter group and the quencher group is located at the 3' end of the detection probe or at a position 1-10nt away from the 3' end, and the reporter group and the quencher group are at a suitable distance such that the quencher group can absorb or quench the signal of the reporter group before the detection probe hybridizes with its complementary sequence. In certain preferred embodiments, the reporter group and the quencher group can be at a distance as defined above (e.g., 10-80nt or longer). In certain preferred embodiments, one of the reporter group and the quencher group is located at the 5' end of the detection probe and the other is located at the 3' end.

[0099] In the method of the present invention, the detection probe can also be modified, for example, so that it has resistance to nuclease activity (e.g., 5' nuclease activity, e.g., 5' to 3' exonuclease activity). For example, modifications that resist nuclease activity can be introduced into the backbone of the detection probe, such as phosphorothioate bonds, alkyl phosphotriester bonds, aryl phosphotriester bonds, alkyl phosphonate bonds, aryl phosphonate bonds, hydrogenated phosphate bonds, alkyl phosphoramidate bonds, aryl phosphoramidate bonds, 2'-O-aminopropyl modifications, 2'-O-alkyl modifications, 2'-O-allyl modifications, 2'-O-butyl modifications, and 1-(4'-thio-PD-ribofuranosyl) modifications.

[0100] In the method of the present invention, the detection probe can be linear or can have a hairpin structure. In certain preferred embodiments, the detection probe is linear. In certain preferred embodiments, the detection probe has a hairpin structure. The hairpin structure can be natural or artificially introduced. In addition, conventional methods in the art can be used to construct a detection probe with a hairpin structure. For example, two complementary oligonucleotide sequences can be added to the two ends (5' end and 3' end) of the detection probe so that the detection probe can form a hairpin structure. In such embodiments, the two complementary oligonucleotide sequences constitute the arms (stems) of the hairpin structure. The arms of the hairpin structure can have any desired length, for example, the length of the arms can be 2-15nt, for example 3-7nt, 4-9nt, 5-10nt, 6-12nt.

[0101] In certain preferred embodiments, the melting temperature of the duplex formed by the detection probe and the nucleic acid amplification product is higher than the melting temperature of the duplex formed by the first, second or third primer and the nucleic acid amplification product.

[0102] In certain preferred embodiments, the nucleic acid polymerase used in step (2) has strand displacement activity and / or high fidelity.

[0103] In certain preferred embodiments, the nucleic acid polymerase also has reverse transcription activity.

[0104] In certain preferred embodiments, the nucleic acid polymerase has no 5' to 3' exonuclease activity or has significantly reduced 5' to 3' exonuclease activity.

[0105] In certain preferred embodiments, the nucleic acid polymerase is selected from Bst polymerase, Bsm polymerase, Phi29 polymerase, exo-Klenow polymerase, BsobⅠ polymerase and exo-Bca polymerase or a combination thereof.

[0106] Reagent test kit

[0107] In another aspect, the present invention provides a kit comprising a first primer, a second primer, a third primer, and one or more detection probes as defined above.

[0108] In certain preferred embodiments, the kit further comprises a fourth primer, and / or a fifth primer as defined above.

[0109] In certain preferred embodiments, the kit further comprises a nucleic acid polymerase. In certain preferred embodiments, the nucleic acid polymerase is as defined above.

[0110] It will be readily understood that such kits can be used to implement the methods of the present invention described in detail above. Therefore, the various technical features described in detail above with respect to the first primer, second primer, third primer, fourth primer, fifth primer, and detection probe are also applicable to the first primer, second primer, third primer, fourth primer, fifth primer, and detection probe in the kit. Furthermore, such kits may further comprise other reagents required for implementing the methods of the present invention.

[0111] In certain preferred embodiments, the kit further comprises: a reagent for performing nucleic acid hybridization, a reagent for performing nucleic acid extension, a reagent for performing nucleic acid amplification, or any combination thereof. Such reagents can be routinely determined by those skilled in the art and include, but are not limited to, a working buffer for an enzyme (e.g., a nucleic acid polymerase), dNTPs, water, a buffer containing ions (e.g., Mg), 2+) solution, single-stranded DNA-binding protein (SSB), or any combination thereof.

[0112] Those skilled in the art may modify, replace, or combine the various technical features of the technical solutions of the present invention based on the principles described in detail in this application without departing from the spirit and scope of the present invention. All such technical solutions and their variations are intended to be within the scope of the claims of this application or their equivalents.

[0113] Terminology

[0114] In this application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the nucleic acid chemistry laboratory procedures used herein are conventional procedures widely used in the relevant field. At the same time, in order to better understand the present invention, definitions and explanations of relevant terms are provided below. Unless specifically limited or described differently elsewhere in this article, the following terms and descriptions related to the present invention should be understood according to the definitions given below.

[0115] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0116] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0117] As used herein, the term "target nucleic acid" refers to nucleic acid to be detected. The target nucleic acid can be DNA, RNA, DNA / RNA complex, or a mixture thereof, and can exist in any form. For example, the target nucleic acid can exist in a single-stranded, double-stranded, or hybrid double-stranded form. In certain preferred embodiments, the target nucleic acid is selected from single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or a DNA / RNA hybrid double-stranded.

[0118] The source of the target nucleic acid is not limited and can be derived from an organism (e.g., a prokaryote, a eukaryote, a virus, a viroid) or a non-organism (e.g., a nucleic acid molecule library). In some cases, the target nucleic acid can be obtained by a pre-treatment step (e.g., purification, amplification, enrichment, enzymatic hydrolysis, denaturation, etc.) as needed.

[0119] As used herein, the term "complementary" means that two nucleic acid sequences can form hydrogen bonds between each other according to the base pairing principle (Waston-Crick principle), and thus form a duplex. In the present application, the term "complementary" includes "substantially complementary" and "completely complementary". As used herein, the term "completely complementary" means that each base in a nucleic acid sequence can be paired with the base in another nucleic acid chain without mispairing or gaps. As used herein, the term "substantially complementary" means that most of the bases in a nucleic acid sequence can be paired with the base in another nucleic acid chain, which allows the presence of mispairing or gaps (e.g., mispairing or gaps of one or several nucleotides). Typically, under conditions allowing nucleic acid hybridization, annealing or amplification, two nucleic acid sequences that are "complementary" (e.g., substantially complementary or completely complementary) will selectively / specifically hybridize or anneal, and form a duplex.

[0120] As used herein, the terms "hybridization" and "annealing" refer to the process by which complementary single-stranded nucleic acid molecules form double-stranded nucleic acids. In this application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Generally, two nucleic acid sequences that are completely complementary or substantially complementary can hybridize or anneal. The complementarity required for hybridization or annealing of two nucleic acid sequences depends on the hybridization conditions used, particularly the temperature.

[0121] As used herein, "conditions that allow nucleic acid annealing or hybridization" have the meaning commonly understood by those skilled in the art and can be determined by conventional methods. For example, two nucleic acid molecules with complementary sequences can hybridize under appropriate hybridization conditions. Such hybridization conditions may involve factors such as temperature, the pH, composition, and ionic strength of the hybridization buffer, and may be determined based on the length and GC content of the two complementary nucleic acid molecules. For example, when the two complementary nucleic acid molecules are relatively short in length and / or have a relatively low GC content, low-stringency hybridization conditions may be employed. When the two complementary nucleic acid molecules are relatively long in length and / or have a relatively high GC content, high-stringency hybridization conditions may be employed. Such hybridization conditions are well known to those skilled in the art and can be found, for example, in Joseph Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc., NY (1999). In this application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Accordingly, the expressions "conditions that allow nucleic acid hybridization" and "conditions that allow nucleic acid annealing" also have the same meaning and are used interchangeably.

[0122] As used herein, the expression "conditions that allow nucleic acid synthesis or amplification" has the meaning commonly understood by those skilled in the art, and refers to conditions that allow a nucleic acid polymerase (e.g., DNA polymerase) to synthesize another nucleic acid chain using one nucleic acid chain as a template and form a duplex. Such conditions are well known to those skilled in the art and may involve factors such as temperature, pH, composition, concentration, and ionic strength of the hybridization buffer. Suitable nucleic acid amplification conditions can be determined by conventional methods (see, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)). In this application, "synthesis" and "amplification" have the same meaning and are used interchangeably. In the methods of the present invention, "conditions that allow nucleic acid synthesis or amplification" are preferably the working conditions of a nucleic acid polymerase (e.g., DNA polymerase). The working conditions of various enzymes can be determined by those skilled in the art by conventional methods and may generally involve factors such as temperature, pH, composition, concentration, and ionic strength of the buffer. Alternatively, the conditions recommended by the enzyme manufacturer may be used.

[0123] As used herein, the term "upstream" is used to describe the relative positional relationship of two nucleic acid sequences and has a meaning generally understood by those skilled in the art. For example, the expression "a nucleic acid sequence is located upstream of another nucleic acid sequence" means that, when arranged in a 5' to 3' direction, the former is located at a more forward position (i.e., closer to the 5' end) than the latter. As used herein, the term "downstream" has the opposite meaning to "upstream."

[0124] As used herein, the term "fluorescent probe" refers to an oligonucleotide that carries a fluorescent group and is capable of generating a fluorescent signal. In this application, the fluorescent probe is used as a detection probe.

[0125] As used herein, a nucleic acid polymerase with "strand displacement activity" refers to a nucleic acid polymerase that, in the process of extending a new nucleic acid chain, if encounters a downstream nucleic acid chain complementary to the template chain, can continue the extension reaction and strip off (rather than degrade) the nucleic acid chain complementary to the template chain.

[0126] As used herein, a nucleic acid polymerase with "high fidelity" refers to a nucleic acid polymerase that has a lower probability of introducing an erroneous nucleotide (i.e., an error rate) during nucleic acid amplification than a wild-type Taq enzyme (e.g., a Taq enzyme whose sequence is shown in UniProt Acession: P19821.1).

[0127] As used herein, a nucleic acid polymerase having "significantly reduced 5' to 3' exonuclease activity" refers to a nucleic acid polymerase having about or less than 10%, or preferably about or less than 5% or 1%, of the 5' to 3' exonuclease activity of a wild-type Taq enzyme (e.g., a Taq enzyme whose sequence is shown in UniProtAcession: P19821.1).

[0128] As used herein, the term "melting curve analysis" has a meaning generally understood by those skilled in the art, and refers to a method for analyzing the presence or identity of a double-stranded nucleic acid molecule by measuring the melting curve of the double-stranded nucleic acid molecule, which is generally used to assess the dissociation characteristics of the double-stranded nucleic acid molecule during heating. Methods for performing melting curve analysis are well known to those skilled in the art (see, for example, The Journal of Molecular Diagnostics 2009, 11 (2): 93-101). In this application, the terms "melting curve analysis" and "melting analysis" have the same meaning and are used interchangeably.

[0129] In certain preferred embodiments of the present application, melting curve analysis can be performed by using detection probes labeled with reporter groups and quencher groups. In short, at ambient temperature, the detection probe can form a duplex with its complementary sequence through base pairing. In this case, the reporter group (e.g., fluorescent group) and the quencher group on the detection probe are separated from each other, and the quencher group cannot absorb the signal (e.g., fluorescent signal) emitted by the reporter group. At this time, the strongest signal (e.g., fluorescent signal) can be detected. As the temperature rises, the two chains of the duplex begin to dissociate (i.e., the detection probe gradually dissociates from its complementary sequence), and the detection probe under dissociation is in a single-stranded free curl state. In this case, the reporter group (e.g., fluorescent group) and the quencher group on the dissociated detection probe are close to each other, and the signal (e.g., fluorescent signal) emitted by the reporter group (e.g., fluorescent group) is absorbed by the quencher group. Therefore, as the temperature rises, the detected signal (e.g., fluorescent signal) gradually weakens. When the two chains of the duplex are completely dissociated, all detection probes are in a single-stranded free curl state. In this case, the signals (e.g., fluorescent signals) emitted by the reporter groups (e.g., fluorescent groups) on all detection probes are absorbed by the quenching group. Therefore, it is basically impossible to detect the signals (e.g., fluorescent signals) emitted by the reporter groups (e.g., fluorescent groups). Therefore, by detecting the signals (e.g., fluorescent signals) emitted by the duplex containing the detection probe during the heating or cooling process, the hybridization and dissociation process of the detection probe and its complementary sequence can be observed, forming a curve in which the signal intensity changes with temperature. Further, the obtained curve is subjected to derivative analysis to obtain a curve with the signal intensity change rate as the ordinate and the temperature as the abscissa (i.e., the melting curve of the duplex). The peak in the melting curve is the melting peak, and the temperature corresponding thereto is the melting point (T m Generally speaking, the higher the degree of match between the detection probe and the complementary sequence (for example, the fewer mismatched bases and the more matched bases), the higher the T value of the duplex. m Therefore, by detecting the T m The value of the melting point can determine the presence and identity of the sequence complementary to the detection probe in the duplex. m Value" have the same meaning and can be used interchangeably.

[0130] Advantageous Effects of the Invention

[0131] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0132] (1) The method of the present invention uses isothermal amplification (cross-primer amplification method) to amplify the target nucleic acid molecule, which greatly shortens the time required for the amplification step, making it possible to quickly (for example, within 1 hour) complete the amplification and detection of the target nucleic acid molecule.

[0133] (2) The method of the present invention uses melting curve analysis to analyze and detect the isothermal amplification products after the amplification is completed. This not only greatly improves the sensitivity and specificity of the analysis and detection, but also enables efficient and accurate analysis of the presence and type of mutations in the target nucleic acid molecule.

[0134] In addition, the inventors of the present application have unexpectedly discovered that when performing melting curve analysis on the isothermal amplification products, the detection probe designed for the amplification product of the first primer (i.e., the first amplification chain) has a detection ability that is significantly better than the detection probe designed for the amplification product of the second primer (i.e., the second amplification chain).

[0135] Therefore, the present invention provides a simple, rapid and efficient method for detecting target nucleic acid molecules, which solves the problems existing in the traditional cross-primer amplification method.

[0136] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1 An exemplary scheme for detecting mutations in a target nucleic acid using the method of the present invention is shown. Briefly, the exemplary scheme comprises the following steps.

[0138] First, a first primer, a second primer, and a third primer are provided; wherein,

[0139] The first primer comprises an annealing sequence and a crossover sequence; wherein the annealing sequence is located downstream or at the 3' end of the crossover sequence, and the two are directly connected or connected via a nucleotide linker; the annealing sequence is capable of hybridizing or annealing with the first region of the first strand of the target nucleic acid (hereinafter referred to as the a strand) under conditions that allow nucleic acid annealing or hybridization, and is capable of initiating an extension reaction and generating a nucleic acid strand containing a complementary sequence (hereinafter referred to as the Ts sequence) to the nucleic acid sequence to be detected (hereinafter referred to as the Ta sequence) under conditions that allow nucleic acid synthesis or amplification;

[0140] The second primer and the third primer can hybridize or anneal with the complementary strand of the a strand (hereinafter referred to as the s strand) under conditions that allow nucleic acid annealing or hybridization; and the second primer can anneal or hybridize with a second region of the s strand that is located downstream or 3' of the Ts sequence; the third primer can anneal or hybridize with a third region of the s strand that is located downstream or 3' of the Ts sequence; and the second region is located downstream or 3' of the third region; and,

[0141] The crossover sequence contains a sequence capable of annealing or hybridizing to the second region.

[0142] In certain preferred embodiments, the annealing sequence is capable of annealing or hybridizing with the first region of the a chain located downstream or 3' of the Ta sequence. In certain preferred embodiments, the annealing sequence contains a sequence that is complementary (e.g., completely complementary) to the nucleotide sequence of the first region or a fragment thereof. In certain preferred embodiments, the annealing sequence is located at the 3' end of the first primer.

[0143] In certain preferred embodiments, the second primer contains a sequence that is complementary (e.g., completely complementary) to the nucleotide sequence of the second region or its fragment. In certain preferred embodiments, the third primer contains a sequence that is complementary (e.g., completely complementary) to the nucleotide sequence of the third region or its fragment. In certain preferred embodiments, the intersection sequence contains a sequence that is complementary (e.g., completely complementary) to the nucleotide sequence of the second region or its fragment. In certain preferred embodiments, the intersection sequence contains a sequence that is complementary (e.g., completely complementary) to the nucleotide sequence of the second region or its fragment. In certain preferred embodiments, the intersection sequence contains the sequence of the second primer. In certain preferred embodiments, the intersection sequence is identical to the sequence of the second primer.

[0144] In certain preferred embodiments, optionally, a fourth primer and a fifth primer are further provided, wherein the fourth primer can anneal or hybridize with the fourth region located downstream or at the 3' end of the first region in the a chain under conditions that allow nucleic acid annealing or hybridization; and the fifth primer can anneal or hybridize with the fifth region located downstream or at the 3' end of the second region in the s chain under conditions that allow nucleic acid annealing or hybridization.

[0145] In certain preferred embodiments, the fourth primer contains a sequence that is complementary (e.g., completely complementary) to the nucleotide sequence of the fourth region or a fragment thereof. In certain preferred embodiments, the fifth primer contains a sequence that is complementary (e.g., completely complementary) to the nucleotide sequence of the fifth region or a fragment thereof.

[0146] Next, the first, second and third primers (and, optionally, the fourth and fifth primers) are contacted (eg, incubated) with the target nucleic acid molecule and a nucleic acid polymerase under conditions that allow nucleic acid synthesis or amplification, and generate nucleic acid amplification products. Figure 1 A schematically shows the process of amplifying the target nucleic acid molecule using the above five primers.

[0147] During nucleic acid amplification, the nucleic acid polymerase can extend the first primer (e.g. Figure 1 A shown in (1)), and generates a first amplified chain, which starts from the 5' end and contains the sequence of the first primer (including the cross sequence and the annealing sequence), the Ts sequence, the sequence of the third region and the sequence of the second region (as shown in Figure 1 (as shown in (2) in A).

[0148] Without being limited by theory, the nucleic acid polymerase can also extend the fourth primer using the a chain of the target nucleic acid molecule as a template; therefore, when the nucleic acid polymerase used has chain displacement activity, the addition and use of the fourth primer will facilitate the peeling of the first amplified chain from the a chain of the target nucleic acid molecule to form a single-stranded nucleic acid molecule.

[0149] After the first amplified strand is generated, the nucleic acid polymerase can use the first amplified strand as a template to extend the second and third primers (e.g. Figure 1 A (2)), and generate the second and third amplified chains, wherein the second amplified chain starts from the 5' end and contains the complementary sequence of the second region, the complementary sequence of the third region, the Ta sequence, and the complementary sequence of the first primer (including the complementary sequence of the annealing sequence and the complementary sequence of the cross sequence) (as shown in Figure 1 A (3) shown); the third amplified chain starts from the 5' end and contains the complementary sequence of the third region, the Ta sequence, and the complementary sequence of the first primer (including the complementary sequence of the annealing sequence and the complementary sequence of the cross sequence) (as shown in FIG. Figure 1 (as shown in (4) in A).

[0150] Without being limited by theory, the nucleic acid polymerase can also extend the fifth primer using the first amplified chain as a template; therefore, when the nucleic acid polymerase used has chain displacement activity, the addition and use of the fifth primer will facilitate the peeling of the second and third amplified chains from the first amplified chain to form a single-stranded nucleic acid molecule.

[0151] After the second amplified chain is generated, the nucleic acid polymerase can use the second amplified chain as a template to extend the first and second primers and generate a double-stranded nucleic acid molecule (e.g., Figure 1 As shown in (5) in A). Since the first amplified chain contains a crossover sequence, an annealing sequence, a Ts sequence, a sequence of the third region and a sequence of the second region, and the crossover sequence can anneal or hybridize with the second region (e.g., be complementary), the first amplified chain can spontaneously form a stem-loop structure (e.g., Figure 1 Similarly, the second amplified chain can also spontaneously form a stem-loop structure (as shown in (8) in A). Figure 1(shown in (7) in A).

[0152] Subsequently, the second amplified strand in the double-stranded nucleic acid molecule can be used as a template to extend the first and second primers (e.g. Figure 1 A (7)), generating a new first amplified chain; and the first amplified chain can be used as a template for extending the second and third primers (as shown in Figure 1 A (8) shows), generating new second and third amplified chains. Thus, through multiple rounds of nucleic acid amplification, the first amplified chain containing the Ts sequence and the second amplified chain containing the Ta sequence, each of which can form a stem-loop structure, are synthesized and enriched in large quantities.

[0153] In addition, it should be noted that after generating the third amplified chain, the nucleic acid polymerase can also extend the second primer using the third amplified chain as a template and generate another double-stranded nucleic acid molecule (such as Figure 1 As shown in (6) in A). The double-stranded nucleic acid molecule is a by-product of the amplification reaction, and the two nucleic acid chains contained therein are unable to form a stem-loop structure.

[0154] Various nucleic acid polymerases can be used to implement the method of the present invention. In certain preferred embodiments, the nucleic acid polymerase is a DNA polymerase. In certain preferred embodiments, the DNA polymerase has strand displacement activity and / or does not have 5' to 3' exonuclease activity.

[0155] Method of the present invention can be used for detecting various target nucleic acid molecules. In some preferred embodiments, the target nucleic acid molecule can be a double-stranded nucleic acid (for example, double-stranded DNA). In some preferred embodiments, the target nucleic acid molecule can be a single-stranded nucleic acid (for example single-stranded DNA, single-stranded RNA).

[0156] After generating the first amplified chain and the second amplified chain each capable of forming a stem-loop structure, the Ts sequence contained in the first amplified chain and / or the Ta sequence contained in the second amplified chain can be analyzed or identified by melting curve analysis. Figure 1 B schematically shows an exemplary scheme for performing melting curve analysis on the Ts sequence contained in the first amplified chain using a detection probe.

[0157] In this detection scheme, a detection probe is provided, which contains a Ts capture sequence capable of hybridizing or annealing with a Ts sequence, and optionally, contains a first region complementary-capture sequence capable of hybridizing or annealing with a complementary sequence of the first region and / or a third region capture sequence capable of hybridizing or annealing with a third region; and, the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and, the signal emitted by the detection probe when hybridized with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence.

[0158] In certain preferred embodiments, the detection probe contains a Ts capture sequence capable of hybridizing or annealing to a Ts sequence. In certain preferred embodiments, the detection probe contains a Ts capture sequence capable of hybridizing or annealing to a Ts sequence, and a first region complementary-capture sequence capable of hybridizing or annealing to the complementary sequence of the first region, and the first region complementary-capture sequence is located downstream or at the 3' end of the Ts capture sequence. In certain preferred embodiments, the detection probe contains a Ts capture sequence capable of hybridizing or annealing to a Ts sequence, and a third region capture sequence capable of hybridizing or annealing to a third region, and the third region capture sequence is located upstream or at the 5' end of the Ts capture sequence. In certain preferred embodiments, the detection probe contains a Ts capture sequence, a first region complementary-capture sequence, and a third region capture sequence; and the third region capture sequence is located upstream or at the 5' end of the Ts capture sequence, and the first region complementary-capture sequence is located downstream or at the 3' end of the Ts capture sequence.

[0159] Melting curve analysis can be performed by using detection probes labeled with reporter groups and quencher groups. In short, at ambient temperature, the detection probe can form a duplex with its complementary sequence (e.g., the loop region of the first amplified chain) through base pairing. In this case, the reporter group (e.g., fluorescent group) and the quencher group on the detection probe are separated from each other, and the quencher group cannot absorb the signal (e.g., fluorescent signal) emitted by the reporter group. At this time, the strongest signal (e.g., fluorescent signal) can be detected. As the temperature rises, the two chains of the duplex begin to dissociate (i.e., the detection probe gradually dissociates from its complementary sequence), and the detection probe under dissociation is in a single-stranded free coil state. In this case, the reporter group (e.g., fluorescent group) and the quencher group on the dissociated detection probe are close to each other, and the signal (e.g., fluorescent signal) emitted by the reporter group (e.g., fluorescent group) is absorbed by the quencher group. Therefore, as the temperature rises, the detected signal (e.g., fluorescent signal) gradually weakens. When the two chains of the duplex are completely dissociated, all detection probes are in a single-stranded free coil state. In this case, the signals (e.g., fluorescent signals) emitted by the reporter groups (e.g., fluorescent groups) on all detection probes are absorbed by the quenching group. Therefore, it is basically impossible to detect the signals (e.g., fluorescent signals) emitted by the reporter groups (e.g., fluorescent groups). Therefore, by detecting the signals (e.g., fluorescent signals) emitted by the duplex containing the detection probe during the heating or cooling process, the hybridization and dissociation processes of the detection probe and its complementary sequence (e.g., the loop region of the first amplification chain) can be observed, forming a curve in which the signal intensity changes with temperature. Further, the obtained curve is subjected to derivative analysis to obtain a curve with the rate of change of signal intensity as the ordinate and the temperature as the abscissa (i.e., the melting curve of the duplex). The peak in the melting curve is the melting peak, and the temperature corresponding thereto is the melting point (T m Generally speaking, the higher the degree of match between the detection probe and the complementary sequence (for example, the fewer mismatched bases and the more matched bases), the higher the T value of the duplex. m Therefore, by detecting the T m The value of the melting point can determine the presence and identity of the sequence complementary to the detection probe in the duplex. m Value" have the same meaning and can be used interchangeably.

[0160] For example, the detection probe can be designed to contain a sequence that is completely complementary to the Ts sequence of the wild-type gene. Thus, when the target nucleic acid molecule to be detected does not contain a mutation (the target nucleic acid molecule contains the sequence of the wild-type gene), the detection probe will be completely complementary to the first amplified strand, and the duplex formed by the two will have the highest Ts. mWhen the target nucleic acid molecule to be detected contains one or more mutations, the detection probe will not be completely complementary to the first amplified chain (ie, the two are partially complementary), and the duplex formed by the two will have a reduced T value. m The more mutations the target nucleic acid molecule / first amplified chain contains, the greater the T value of the duplex formed. m Therefore, by measuring T m Melting curve analysis of the values ​​can determine whether the first amplified chain contains a mutation and determine the type of mutation. Conversely, the detection probe can also be designed to contain a sequence that is completely complementary to the Ts sequence of the mutant gene.

[0161] Figure 2 The amplification curves of isothermal amplification of a nucleic acid sample containing the wild-type A gene (black solid line) or the control gene (grey solid line) using five primers and detection probes 1 and 2 in Example 1 are shown.

[0162] Figure 3 The figure shows the results of isothermal amplification of a nucleic acid sample containing wild-type or mutant A gene using five primers and then melting curve analysis of the isothermal amplification products using two detection probes.

[0163] Figure 4 The graph shows the results of isothermal amplification of a nucleic acid sample containing the wild-type B gene using five primers, and then melting curve analysis of the isothermal amplification products using detection probe 3 or 4.

[0164] Sequence information

[0165] Information on the sequences involved in the present invention is provided in Table 1 below.

[0166] Table 1

[0167]

[0168]

[0169] DETAILED DESCRIPTION

[0170] The present invention will now be described with reference to the following examples which are intended to illustrate the present invention (but not to limit the present invention). Where specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are conventional products that can be obtained commercially. It will be appreciated by those skilled in the art that the examples describe the present invention by way of example and are not intended to limit the scope of protection claimed in this application.

[0171] Example 1: Amplification and detection of target nucleic acid

[0172] In this example, the ability of the present method to amplify and detect target nucleic acids was verified using the wild-type A gene as an example. The nucleotide sequence of the wild-type A gene is shown in SEQ ID NO: 1. In addition, the A gene has multiple mutants, including, for example, the following: 269C>T (SEQ ID NO: 2); 281A>C (SEQ ID NO: 6); 281A>G (SEQ ID NO: 7); 280G>C (SEQ ID NO: 4); 280G>A (SEQ ID NO: 3); and 280G>T (SEQ ID NO: 5).

[0173] Based on the nucleotide sequences of the wild type and mutant forms of gene A, with the high-frequency mutation region as the target region for detection (Ts sequence), the following five primers were designed: a first primer (SEQ ID NO: 8), a second primer (SEQ ID NO: 9), a third primer (SEQ ID NO: 10), a fourth primer (SEQ ID NO: 11), and a fifth primer (SEQ ID NO: 12), to amplify the nucleic acid molecules in the sample to be tested, generating a first amplified chain containing the Ts sequence (which is the amplification product of the first primer) and a second amplified chain containing the complementary sequence of the Ts sequence (Ta sequence) (which is the amplification product of the second primer). Furthermore, detection probe 1 (SEQ ID NO: 13) and detection probe 2 (SEQ ID NO: 14) were designed to perform amplification curve analysis and melting curve analysis on the first amplified chain.

[0174] Based on the nucleotide sequences of the detection probe, target gene, and primers, the expected T values ​​of the duplexes formed by the detection probe and the various first amplified strands can be calculated. m The values ​​are shown in Table 2.

[0175] Table 2: Expected T of the duplex formed by the detection probe and the first amplified strand m value

[0176]

[0177]

[0178] Prepare 25 μL of reaction system according to Table 3.

[0179] Table 3: Reaction system for amplification and detection of A gene

[0180] Components Final concentration 10×Bst buffer 1× MgSO4 (purchased from Sinopharm Chemical Reagent Co., Ltd., product number: 10013018) 2.0mM dNTPs (purchased from Thermo Fisher Scientific, catalog number: AM822G) 0.3mM BST DNA polymerase (purchased from Feipeng, product number: MD030) 8.0U Detection probe 1 (SEQ ID NO: 13) 0.14 μM Detection probe 2 (SEQ ID NO: 14) 0.14 μM First primer (SEQ ID NO: 8) 0.5μM Second primer (SEQ ID NO: 9) 0.3 μM Third primer (SEQ ID NO: 10) 0.3 μM Fourth primer (SEQ ID NO: 11) 0.15μM Fifth primer (SEQ ID NO: 12) 0.15μM Betaine (purchased from Sigma-Aldrich, product number: 14300-100G) 1.0 μM Nucleic acid sample to be tested (containing wild-type A gene or control gene) <![CDATA[2×10 3 copies / μL]]> water Add to 25 μL

[0181] Amplification was performed according to the following amplification reaction protocol: 63°C for 50 min (one cycle of 1 min, collecting fluorescence signals from the FAM and ROX channels); incubation at 95°C for 1 min (to inactivate the BST DNA polymerase). The instrument used in this example was a SLAN96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0182] Test results such as Figure 2 shown. Figure 2 Amplification curves are shown for isothermal amplification of a nucleic acid sample containing the wild-type A gene (black solid line) or a control gene (gray solid line) using five primers and detection probes 1 and 2. The results show that when the nucleic acid sample contains the A gene, an amplification curve can be detected; conversely, when the nucleic acid sample does not contain the A gene, an amplification curve cannot be detected. These results demonstrate that the primers and probes designed according to the present method can rapidly (within 1 hour), effectively, and specifically perform isothermal amplification of target nucleic acid molecules.

[0183] Example 2: Detection of wild-type genes and mutant genes

[0184] In this example, the five primers designed in Example 1 (first primer, second primer, third primer, fourth primer, fifth primer) were used to perform isothermal amplification on the nucleic acid molecules to be tested containing the wild-type A gene (WT) or the mutant A gene (269C>T, 281A>C, 281A>G, 280G>C, 280G>A and 280G>T), and the amplified products were subjected to melting curve analysis using detection probes 1 and 2 to verify the ability of the method of the present invention to detect and analyze mutations in target nucleic acid molecules.

[0185] Briefly, a 25 μL reaction system was prepared according to Table 4:

[0186] Table 4: Reaction system for amplification and detection of wild-type or mutant A genes

[0187]

[0188] Amplification was performed according to the following amplification reaction protocol: 63°C for 50 min (1 min per cycle, collecting fluorescence signals from the FAM and ROX channels); incubation at 95°C for 1 min (to inactivate BST DNA polymerase). Subsequently, melting curve analysis was performed from 40°C to 85°C at a heating rate of 0.4°C / s, and fluorescence signals from the FAM and ROX channels were collected. The instrument used in this example was a SLAN96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0189] Test results such as Figure 3 shown. Figure 3The results of melting curve analysis of the isothermal amplification products using two detection probes after isothermal amplification of the nucleic acid sample containing wild-type or mutant A gene using five primers are shown. The results show that the two detection probes form different T-terminal regions with the first amplified chain generated by the wild-type A gene (WT) or mutant A gene (269C>T, 281A>C, 281A>G, 280G>C, 280G>A and 280G>T). m Thus, by performing melting curve analysis on the isothermal amplification product, the T value of the duplex formed by the detection probe and the first amplified chain is determined. m The results show that the primers and probes designed according to the present invention can be used to detect and analyze the presence and type of mutations in target nucleic acid molecules.

[0190] Example 3: Design of detection probe

[0191] The isothermal amplification product obtained by the inventive method contains two kinds of nucleic acid single strands that can spontaneously form a stem-loop structure, that is, the first amplified chain produced by the extension of the first primer, and the second amplified chain produced by the extension of the second primer. Accordingly, in the melting curve analysis, a detection probe (s chain detection probe) can be designed for the first amplified chain, and a detection probe (a chain detection probe) can also be designed for the second amplified chain. In the present embodiment, taking the wild-type B gene as an example, the detection ability of two detection probes was evaluated.

[0192] In this example, detection probes 3 and 4 (SEQ ID NOs: 21 and 22) were designed to perform melting curve analysis on the amplified chains obtained from the wild-type B gene, respectively.

[0193] In brief, this example uses five primers (first primer, second primer, third primer, fourth primer, and fifth primer) designed for the wild-type B gene (SEQ ID NO: 15) to perform isothermal amplification on a nucleic acid sample containing the wild-type B gene; after amplification, the amplified product is subjected to melting curve analysis using detection probe 3 or detection probe 4, respectively.

[0194] Prepare 25 μL of reaction system according to Table 5:

[0195] Table 5: Reaction system for amplification and detection of B gene

[0196]

[0197] Amplification was performed according to the following protocol: 63°C for 50 min (1 min per cycle, with fluorescence signal from the ROX channel collected); incubation at 95°C for 1 min (to inactivate BST DNA polymerase). Subsequently, melting curve analysis was performed from 40°C to 85°C at a heating rate of 0.4°C / s, with fluorescence signal from the ROX channel collected. The instrument used in this example was a SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0198] Test results such as Figure 4 shown. Figure 4 The figure shows the results of isothermal amplification of a nucleic acid sample containing the wild-type B gene using five primers, and then melting curve analysis of the isothermal amplification products using detection probes 3 and 4. The results show that detection probes 3 and 4 respectively form a melting curve with the expected T value with the first amplified chain and the second amplified chain generated by the isothermal amplification of the wild-type B gene. m This indicates that the detection probes designed for the first amplified chain or the second amplified chain can be used in the method of the present invention.

[0199] also, Figure 4 The results also showed that the detection probe that binds to and detects the first amplified chain (s-chain detection probe, detection probe 3) can form a more specific melting peak and has a better detection effect than the detection probe that binds to and detects the second amplified chain (a-chain detection probe, detection probe 4). Therefore, without being limited by theory, in the method of the present invention, it is preferred to use a detection probe that binds to and detects the first amplified chain.

[0200] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Xiamen University <120> A method for analyzing the presence and type of mutations in a target nucleic acid <130> IDC200477 <160> twenty two <170> PatentIn version 3.5 <210> 1 <211> 425 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of wild-type A gene <400> 1 atgacagaca cgacgttgcc gcctgacgac tcgctcgacc ggatcgaacc ggttgacatc 60 gagcaggaga tgcagcgcag ctacatcgac tatgcgatga gcgtgatcgt cggccgcgcg 120 ctgccggagg tgcgcgacgg gctcaagccc gtgcatcgcc gggtgctcta tgcaatgttc 180 gattccggct tccgcccgga ccgcagccac gccaagtcgg cccggtcggt tgccgagacc 240 atgggcaact accacccgca cggcgacgcg tcgatctacg acagcctggt gcgcatggcc 300 cagccctggt cgctgcgcta cccgctggtg gacggccagg gcaacttcgg ctcgccaggc 360 aatgacccac cggcggcgat gaggtacacc gaagcccggc tgaccccgtt ggcgatggag 420 atgct 425 <210> 2 <211> 425 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of 269C>T mutant <400> 2 atgacagaca cgacgttgcc gcctgacgac tcgctcgacc ggatcgaacc ggttgacatc 60 gagcaggaga tgcagcgcag ctacatcgac tatgcgatga gcgtgatcgt cggccgcgcg 120 ctgccggagg tgcgcgacgg gctcaagccc gtgcatcgcc gggtgctcta tgcaatgttc 180 gattccggct tccgcccgga ccgcagccac gccaagtcgg cccggtcggt tgccgagacc 240 atgggcaact accacccgca cggcgacgtg tcgatctacg acagcctggt gcgcatggcc 300 cagccctggt cgctgcgcta cccgctggtg gacggccagg gcaacttcgg ctcgccaggc 360 aatgacccac cggcggcgat gaggtacacc gaagcccggc tgaccccgtt ggcgatggag 420 atgct 425 <210> 3 <211> 425 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the 280G>A mutant <400> 3 atgacagaca cgacgttgcc gcctgacgac tcgctcgacc ggatcgaacc ggttgacatc 60 gagcaggaga tgcagcgcag ctacatcgac tatgcgatga gcgtgatcgt cggccgcgcg 120 ctgccggagg tgcgcgacgg gctcaagccc gtgcatcgcc gggtgctcta tgcaatgttc 180 gattccggct tccgcccgga ccgcagccac gccaagtcgg cccggtcggt tgccgagacc 240 atgggcaact accacccgca cggcgacgcg tcgatctaca acagcctggt gcgcatggcc 300 cagccctggt cgctgcgcta cccgctggtg gacggccagg gcaacttcgg ctcgccaggc 360 aatgacccac cggcggcgat gaggtacacc gaagcccggc tgaccccgtt ggcgatggag 420 atgct 425 <210> 4 <211> 425 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of 280G>C mutant <400> 4 atgacagaca cgacgttgcc gcctgacgac tcgctcgacc ggatcgaacc ggttgacatc 60 gagcaggaga tgcagcgcag ctacatcgac tatgcgatga gcgtgatcgt cggccgcgcg 120 ctgccggagg tgcgcgacgg gctcaagccc gtgcatcgcc gggtgctcta tgcaatgttc 180 gattccggct tccgcccgga ccgcagccac gccaagtcgg cccggtcggt tgccgagacc 240 atgggcaact accacccgca cggcgacgcg tcgatctacc acagcctggt gcgcatggcc 300 cagccctggt cgctgcgcta cccgctggtg gacggccagg gcaacttcgg ctcgccaggc 360 aatgacccac cggcggcgat gaggtacacc gaagcccggc tgaccccgtt ggcgatggag 420 atgct 425 <210> 5 <211> 425 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of 280G>T mutant <400> 5 atgacagaca cgacgttgcc gcctgacgac tcgctcgacc ggatcgaacc ggttgacatc 60 gagcaggaga tgcagcgcag ctacatcgac tatgcgatga gcgtgatcgt cggccgcgcg 120 ctgccggagg tgcgcgacgg gctcaagccc gtgcatcgcc gggtgctcta tgcaatgttc 180 gattccggct tccgcccgga ccgcagccac gccaagtcgg cccggtcggt tgccgagacc 240 atgggcaact accacccgca cggcgacgcg tcgatctact acagcctggt gcgcatggcc 300 cagccctggt cgctgcgcta cccgctggtg gacggccagg gcaacttcgg ctcgccaggc 360 aatgacccac cggcggcgat gaggtacacc gaagcccggc tgaccccgtt ggcgatggag 420 atgct 425 <210> 6 <211> 425 <212> DNA <213> Artificial Sequence <220> Nucleotide sequence of the 281A>C mutant <400> 6 atgacagaca cgacgttgcc gcctgacgac tcgctcgacc ggatcgaacc ggttgacatc 60 gagcaggaga tgcagcgcag ctacatcgac tatgcgatga gcgtgatcgt cggccgcgcg 120 ctgccggagg tgcgcgacgg gctcaagccc gtgcatcgcc gggtgctcta tgcaatgttc 180 gattccggct tccgcccgga ccgcagccac gccaagtcgg cccggtcggt tgccgagacc 240 atgggcaact accacccgca cggcgacgcg tcgatctacg ccagcctggt gcgcatggcc 300 cagccctggt cgctgcgcta cccgctggtg gacggccagg gcaacttcgg ctcgccaggc 360 aatgacccac cggcggcgat gaggtacacc gaagcccggc tgaccccgtt ggcgatggag 420 atgct 425 <210> 7 <211> 425 <212> DNA <213> Artificial Sequence <220> Nucleotide sequence of the 281A>G mutant <400> 7 atgacagaca cgacgttgcc gcctgacgac tcgctcgacc ggatcgaacc ggttgacatc 60 gagcaggaga tgcagcgcag ctacatcgac tatgcgatga gcgtgatcgt cggccgcgcg 120 ctgccggagg tgcgcgacgg gctcaagccc gtgcatcgcc gggtgctcta tgcaatgttc 180 gattccggct tccgccccgga ccgcagccac gccaagtcgg cccggtcggt tgccgagacc 240 atgggcaact accacccgca cggcgacgcg tcgatctacg gcagcctggt gcgcatggcc 300 cagccctggt cgctgcgcta cccgctggtg gacggccagg gcaacttcgg ctcgccaggc 360 aatgacccac cggcggcgat gaggtacacc gaagcccggc tgaccccgtt ggcgatggag 420 atgct 425 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of upstream cross primer 1s <400> 8 gggtagcgca gcgaccaggg caactaccac ccgcac 36 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of downstream detection primer 2a <400> 9 gggtagcgca gcgacca 17 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of downstream detection primer 3a <400> 10 gctgggccat gcgcacc 17 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of upstream stripping primer 4s <400> 11 acgccaagtc ggcccggt 18 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of downstream stripping primer 5a <400> 12 gaagttgccc tggccgtcc 19 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Detection probe 1 nucleotide sequence <220> <221> misc_feature <222> (2)..(2) <223> n represents inosine deoxynucleotide residue <400> 13 gntgtcgcag attcaccacg tcgccgcgcg gc 32 <210> 14 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Detection probe 2 nucleotide sequence <400> 14 gcgcatcaac ctagccctag atcgacacgt cg 32 <210> 15 <211> 176 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of wild-type B gene <400> 15 tcgtcagctc ccactcgtag ccgtacagga tctcgaggaa actgttgtcc catttcgtcg 60 gggtgttcgt ccatacgacc tcgatgccgc tggtgatcgc gtccttaccg gttccggtgc 120 catacgagct cttccagccc aagcccatct gctccagcgg agcagcctcg ggttcg 176 <210> 16 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of upstream cross primer 1s of B gene <400> 16 ttgggctgga agagctcgta tgtcggggtg ttcgtccata cg 42 <210> 17 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of downstream detection primer 2a of gene B <400> 17 ttgggctgga agagctcgta t 21 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of downstream detection primer 3a of B gene <400> 18 gaaccggtaa ggacgcgat 19 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of upstream stripping primer 4s of gene B <400> 19 tcgtcagctc ccactcgtag ccgta 25 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of B gene downstream stripping primer 5a <400> 20 tgctccgctg gagcagatg 19 <210> twenty one <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Detection probe 3 nucleotide sequence <400> twenty one cgatcaccag cggcatcgag 20 <210> twenty two <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of detection probe 4 <400> twenty two ctcgatgccg ctggtgatcg 20

Claims

1. A method for detecting the presence and type of a mutation in a target nucleic acid for non-diagnostic and non-therapeutic purposes, comprising the following steps: (1) Providing a first primer, a second primer, and a third primer; wherein, The first primer comprises an annealing sequence and a crossover sequence; wherein the annealing sequence is located downstream or at the 3' end of the crossover sequence, and the two are directly connected or connected via a nucleotide linker; the annealing sequence can hybridize or anneal with the first region of the a chain of the target nucleic acid under conditions that allow nucleic acid annealing or hybridization, and can initiate an extension reaction and generate a nucleic acid chain containing a Ts sequence under conditions that allow nucleic acid synthesis or amplification, wherein the Ts sequence is a complementary sequence to the Ta sequence; the Ta sequence is the nucleic acid sequence to be tested, and the Ta sequence or the Ts sequence is suspected of containing a mutation; The second primer and the third primer can hybridize or anneal with the s strand under conditions that allow nucleic acid annealing or hybridization, and the s strand is the complementary strand of the a strand; and the second primer can anneal or hybridize with a second region of the s strand that is located downstream or 3' of the Ts sequence; the third primer can anneal or hybridize with a third region of the s strand that is located downstream or 3' of the Ts sequence; and the second region is located downstream or 3' of the third region; and, The crossover sequence contains a sequence capable of annealing or hybridizing to the second region; Furthermore, a fourth primer and / or a fifth primer are provided, wherein the fourth primer can anneal or hybridize to a fourth region of the a chain located downstream or 3' of the first region under conditions that allow nucleic acid annealing or hybridization; and the fifth primer can anneal or hybridize to a fifth region of the s chain located downstream or 3' of the second region under conditions that allow nucleic acid annealing or hybridization. (2) contacting the first, second and third primers, and the fourth and / or fifth primers with a target nucleic acid and a nucleic acid polymerase under conditions that allow nucleic acid synthesis or amplification, and generating a nucleic acid amplification product; the nucleic acid amplification product comprises: (i) a first amplified chain comprising a sequence of the first primer, a Ts sequence, a sequence of the third region and a sequence of the second region; and (ii) a second amplified chain comprising a complementary sequence of the second region, a complementary sequence of the third region, a Ta sequence, and a complementary sequence of the first primer; (3) performing melting curve analysis on the first amplified chain and / or the second amplified chain using one or more detection probes; wherein, The detection probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridized with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence; Furthermore, the detection probe contains a Ts capture sequence capable of hybridizing or annealing with the Ts sequence, and a first region complementary-capture sequence capable of hybridizing or annealing with the complementary sequence of the first region and / or a third region capture sequence capable of hybridizing or annealing with the third region; or, the detection probe contains a Ta capture sequence capable of hybridizing or annealing with the Ta sequence, and a first region capture sequence capable of hybridizing or annealing with the first region and / or a third region complementary-capture sequence capable of hybridizing or annealing with the complementary sequence of the third region; (4) Determine the presence and type of mutations in the target nucleic acid based on the results of the melting curve analysis.

2. The method according to claim 1, wherein The target nucleic acid is DNA, RNA, a DNA / RNA complex, or a mixture thereof.

3. The method according to claim 1, wherein The target nucleic acid exists in a single-stranded or double-stranded form.

4. The method according to claim 1, wherein The target nucleic acid is selected from single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or DNA / RNA hybrid duplex.

5. The method according to claim 1, wherein The target nucleic acid is obtained from a source selected from the group consisting of prokaryotes, eukaryotes, viruses, viroids, or a nucleic acid molecule library.

6. The method of claim 1, wherein Before performing step (2), the target nucleic acid is pretreated.

7. The method according to claim 6, wherein The pretreatment includes purification, enrichment or reverse transcription.

8. The method of claim 1, wherein The length of the Ts sequence or Ta sequence is 10-100 nt.

9. The method of claim 1, wherein The length of the Ts sequence or Ta sequence is 10-13 nt, 13-15 nt, 15-18 nt, 18-20 nt, 20-25 nt, 25-30 nt, 30-35 nt, 35-40 nt, 40-45 nt, 45-50 nt, 50-55nt, 55-60 nt, 60-65 nt, 65-70 nt, 70-75 nt, 75-80 nt, 80-85 nt, 85-90 nt, 90-95 nt, or 95-100 nt.

10. The method of claim 1, wherein The mutation is selected from addition, deletion, substitution, or any combination thereof.

11. The method of claim 1, wherein The method has one or more characteristics selected from the following: (a) the annealing sequence in the first primer is capable of annealing or hybridizing with the first region of the a chain located downstream or 3' of the Ta sequence; (b) the annealing sequence contains a sequence complementary to the nucleotide sequence of the first region or a fragment thereof; (c) the annealing sequence is located at the 3' end of the first primer; (d) the annealing sequence is 10-50 nt in length; (e) the annealing sequence comprises: naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (f) the crossover sequence in the first primer contains a sequence complementary to the nucleotide sequence of the second region or a fragment thereof; (g) the crossover sequence contains the sequence of the second primer; (h) the length of the crossover sequence is 10-100 nt; (i) the crossover sequence comprises: naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (j) the second primer contains a sequence complementary to the nucleotide sequence of the second region or a fragment thereof; (k) the length of the second primer is 10-100 nt; (1) the second primer comprises: naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (m) the third primer contains a sequence complementary to the nucleotide sequence of the third region or a fragment thereof; (n) the length of the third primer is 10-100 nt; (o) the third primer comprises: naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (p) the fourth primer contains a sequence complementary to the nucleotide sequence of the fourth region or a fragment thereof; (q) the fourth primer is 10-100 nt in length; (r) the fourth primer comprises: naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (s) the fifth primer comprises a sequence complementary to the nucleotide sequence of the fifth region or a fragment thereof; (t) the fifth primer is 10-100 nt in length; (u) the fifth primer comprises: naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (v) the nucleotide linker comprises: naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (w) The length of the nucleotide linker is 5-20 nt.

12. The method of claim 1, wherein The method has one or more characteristics selected from the following: (a) the length of the annealing sequence is 10-13 nt, 13-15 nt, 15-18 nt, 18-20 nt, 20-25 nt, 25-30 nt, 30-35 nt, 35-40 nt, 40-45 nt, or 45-50 nt; (b) the length of the crossover sequence is 10-14 nt, 14-20 nt, 20-25 nt, 25-30 nt, 30-35 nt, 35-40 nt, 40-45 nt, 45-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, or 90-100 nt; (c) the length of the second primer is 10-14 nt, 14-20 nt, 20-25 nt, 25-30 nt, 30-35 nt, 35-40 nt, 40-45 nt, 45-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, or 90-100 nt; (d) the length of the third primer is 10-13 nt, 13-17 nt, 17-20 nt, 20-25 nt, 25-30 nt, 30-35 nt, 35-40 nt, 40-45 nt, 45-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, or 90-100 nt; (e) the length of the fourth primer is 10-13 nt, 13-17 nt, 17-20 nt, 20-23 nt, 23-25 nt, 25-30 nt, 30-35 nt, 35-40 nt, 40-45 nt, 45-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, or 90-100 nt; (f) the length of the fifth primer is 10-13 nt, 13-17 nt, 17-20 nt, 20-22 nt, 22-25 nt, 25-30 nt, 30-35 nt, 35-40 nt, 40-45 nt, 45-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, or 90-100 nt; (g) the length of the nucleotide linker is 5-10 nt, 10-15 nt, or 15-20 nt; (h) The crossover sequence is identical to the sequence of the second primer.

13. The method of claim 1, wherein In step (3), at least one detection probe is used to perform melting curve analysis on the first amplified chain and / or the second amplified chain.

14. The method of claim 1, wherein The reporter groups contained in the detection probes are different from each other.

15. The method of claim 1, wherein The detection probe is added to the reaction system of step (2) before the nucleic acid amplification product is generated; or, the detection probe is contacted with the nucleic acid amplification product generated in step (2) after the nucleic acid amplification product is generated.

16. The method of claim 1, wherein In step (3), when performing melting curve analysis on the first amplified chain and / or the second amplified chain, the signal of the reporter group of each detection probe is monitored in real time, thereby obtaining one or more melting curves each corresponding to the signal of a reporter group; then, in step (4), the presence and type of mutation are determined based on the signal type of the reporter group and the melting peak in the melting curve.

17. The method of claim 1, wherein Step (2) is performed under conditions that allow isothermal amplification of the nucleic acid.

18. The method of claim 1, wherein In step (2), the first, second and third primers, and the fourth and / or fifth primers are contacted with the target nucleic acid and nucleic acid polymerase at a temperature of 55° C. to 65° C. to generate a nucleic acid amplification product.

19. The method of claim 18, wherein The duration of the contacting is at least 10 minutes.

20. The method of claim 18, wherein The concentration of the target nucleic acid is at least 0.1×10 3 copies / µL.

21. The method of claim 1, wherein The first amplified chain contains the sequence of the first primer, the Ts sequence, the sequence of the third region and the sequence of the second region starting from the 5' end.

22. The method of claim 1, wherein The second amplified chain contains, starting from the 5' end, a complementary sequence of the second region, a complementary sequence of the third region, a Ta sequence, and a complementary sequence of the first primer.

23. The method of claim 1, wherein The detection probe has one or more characteristics selected from the following: (1) The detection probe contains a Ts capture sequence capable of hybridizing or annealing to a Ts sequence, and a first region complementary-capture sequence capable of hybridizing or annealing to a complementary sequence of the first region and / or a third region capture sequence capable of hybridizing or annealing to a third region; (2) different detection probes hybridize or anneal to different regions of the first amplified strand and / or the second amplified strand; (3) The detection probe comprises: naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (4) The detection probe is 10-500 nt in length; (5) The detection probe has a 3'-OH end, or its 3'-end is blocked; (6) The detection probe is labeled with a reporter group at its 5' end or upstream and a quencher group at its 3' end or downstream, or a reporter group at its 3' end or downstream and a quencher group at its 5' end or upstream; (7) The distance between the reporter group and the quencher group contained in the detection probe is 10-80 nt or longer; (8) The reporter group in the detection probe is a fluorescent group; and the quencher group is a molecule or group that can absorb / quench the fluorescence; (9) The detection probe has resistance to nuclease activity; and / or the backbone of the detection probe contains a modification that resists nuclease activity; (10) The detection probe is linear or has a hairpin structure; (11) The melting temperature of the duplex formed by the detection probe and the nucleic acid amplification product is higher than the melting temperature of the duplex formed by the first, second or third primer and the nucleic acid amplification product.

24. The method of claim 1, wherein The detection probe contains a Ts capture sequence capable of hybridizing or annealing with a Ts sequence; or, the detection probe contains a Ts capture sequence capable of hybridizing or annealing with a Ts sequence, and a first region complementary-capture sequence capable of hybridizing or annealing with a complementary sequence of the first region, and the first region complementary-capture sequence is located downstream or at the 3' end of the Ts capture sequence; or, the detection probe contains a Ts capture sequence capable of hybridizing or annealing with a Ts sequence, and a third region capture sequence capable of hybridizing or annealing with a third region, and the third region capture sequence is located upstream or at the 5' end of the Ts capture sequence; or, the detection probe contains a Ts capture sequence, a first region complementary-capture sequence, and a third region capture sequence; and the third region capture sequence is located upstream or at the 5' end of the Ts capture sequence, and the first region complementary-capture sequence is located downstream or at the 3' end of the Ts capture sequence.

25. The method of claim 1, wherein The reporter group in the detection probe is a fluorescent group ALEX-350, FAM, VIC, TET, CAL Fluor® Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, or Quasar 705; and the quencher group in the detection probe is a molecule or group that can absorb / quench the fluorescence.

26. The method of claim 25, wherein The quenching group is DABCYL, BHQ, ECLIPSE, and / or TAMRA.

27. The method of claim 1, wherein The detection probe has resistance to 5' nuclease activity and / or 5' to 3' exonuclease activity.

28. The method of claim 1, wherein The nucleic acid polymerase has strand displacement activity and / or high fidelity.

29. The method of claim 1, wherein The nucleic acid polymerase also has reverse transcription activity.

30. The method of claim 1, wherein The nucleic acid polymerase has no 5' to 3' exonuclease activity or has significantly reduced 5' to 3' exonuclease activity.

31. The method of claim 1, wherein The nucleic acid polymerase is selected from Bst polymerase, Bsm polymerase, Phi29 polymerase, exo-Klenow polymerase, Bsob I polymerase and exo-Bca polymerase or a combination thereof.

32. The method of claim 1, wherein In step (3), the nucleic acid amplification product and the detection probe are gradually heated or cooled and the signal emitted by the reporter group on each detection probe is monitored in real time, thereby obtaining a curve showing how the signal intensity of each reporter group changes with temperature; subsequently, the obtained curve is derived to obtain a melting curve of the product of step (2).

33. A kit comprising a first primer, a second primer, a third primer, and one or more detection probes; wherein, The first primer, the second primer, and the third primer are defined in claim 1 or claim 11 or 12; and the detection probe is defined in any one of claims 1, 13-16, 23-27.

34. The kit according to claim 33, wherein The kit further comprises a fourth primer and / or a fifth primer, wherein the fourth primer and the fifth primer are as defined in claim 1 or claim 11 or 12.

35. The kit according to claim 33, wherein The kit also includes a nucleic acid polymerase.

36. The kit according to claim 35, wherein The nucleic acid polymerase is as defined in any one of claims 28-31.

37. The kit according to claim 33, wherein The kit further comprises: a reagent for nucleic acid hybridization, a reagent for nucleic acid extension, a reagent for nucleic acid amplification, or any combination thereof.

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