A primer probe set, method and kit for detecting nucleic acid

Abstract

The application provides a primer probe set, a method and a kit for detecting nucleic acid, the primer probe set comprises a first primer, a second primer and a probe; wherein the probe comprises a primer anchor region and a probe signal detection region, the primer anchor region is located at the 5' end of the probe signal detection region, and the probe is modified with a detection label; the first primer comprises a probe anchor region, a connection sequence and a target sequence binding region from 5' end to 3' end in sequence, and the second primer comprises a primer signal detection region, a connection sequence and a target sequence binding region from 5' end to 3' end in sequence. When the primer probe set is used for nucleic acid detection, the phenomenon of primer dimerization can be reduced, and false positive results caused by primer dimers or other non-specific amplification can be effectively avoided. The nucleic acid detection method can realize single-tube multiplex detection, has low requirements on the length of the target sequence and high detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically a primer and probe set, method, and kit for detecting nucleic acids. Background Technology

[0002] Polymerase chain reaction (PCR) is a molecular biology technique that replicates DNA enzymatically without the use of a living organism. PCR is commonly used in medical and biological research laboratories for a variety of tasks, such as the diagnosis of infectious diseases, gene cloning, phenotyping of laboratory animals, transcriptome research, detection of genetic diseases, identification of genetic fingerprints, and paternity testing. Due to its unparalleled replication and precision capabilities, PCR is considered by molecular biologists to be the preferred method for nucleic acid detection. In the late 1990s, the introduction of Real-Time Quantitative PCR (qPCR) technology and related products by ABI in the United States further developed PCR into a highly sensitive, highly specific, and precisely quantitative nucleic acid sequence analysis technique.

[0003] In nucleic acid testing, the presence of primer dimers can easily lead to false positives. This is especially true in single-tube multiplex testing, where the reaction system is more complex and multiple primer pairs need to be present in the same reaction system. It is difficult to completely avoid the homology between primers, and the amplification conditions may not be ideal for all primer pairs. Therefore, primer dimers are more likely to form, leading to more serious false positives.

[0004] Existing technologies have proposed some methods to reduce the generation of primer dimers, but the generation of primer dimers is difficult to completely avoid. How to further solve the false positive problem caused by the generation of primer dimers remains an urgent problem for those skilled in the art. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a primer-probe set, method, and kit for detecting nucleic acids. The primer-probe set of this invention reduces primer dimerization during nucleic acid detection and effectively avoids false positive results caused by primer dimers or other non-specific amplification products. The nucleic acid detection method of this invention enables multiplex detection in a single tube, has low requirements for target sequence length, and high detection sensitivity.

[0006] The objective of this invention is achieved through the following technical solutions.

[0007] In a first aspect, the present invention provides a primer-probe set for detecting nucleic acids, comprising a first primer, a second primer, and a probe; wherein,

[0008] The probe is a freely designed sequence that does not pair with any target sequence. It includes a primer anchoring region (A') and a probe signal detection region (H). The primer anchoring region (A') is located at the 5' end of the probe signal detection region (H). The probe is modified with a detection marker.

[0009] The first primer comprises, from its 5' end to its 3' end, a probe anchoring region (A), a linker sequence (L1), and a target sequence binding region (Ⅰ); wherein, the probe anchoring region (A) specifically binds to the primer anchoring region (A') of the probe; the linker sequence (L1) is a freely designed sequence that does not pair with any target sequence; and the target sequence binding region (Ⅰ) is a sequence that can specifically bind to the target.

[0010] The second primer comprises, from its 5' end to its 3' end, a primer signal detection region (h), a linker sequence (L2), and a target sequence binding region (II); wherein, the complementary sequence (h') of the primer signal detection region (h) specifically binds to the probe signal detection region (H) of the probe; part or all of the complementary sequence (L2') of the linker sequence (L2) specifically binds to part or all of the linker sequence (L1); and the target sequence binding region (II) is a sequence capable of specifically binding to a target.

[0011] The first primer and the second primer are primer pairs used to amplify the same target. That is, the first primer and the second primer are the forward primer and the reverse primer, respectively. They specifically bind to the same target through their respective target sequence binding regions (target sequence binding region (Ⅰ) and target sequence binding region (Ⅱ)), and amplify the amplified product containing the target sequence after extension.

[0012] In some embodiments of the present invention, there is a 0-20 base interval between the linker sequence (L1) of the first primer and the target sequence binding region (Ⅰ), preferably a 0-10 base interval.

[0013] In some embodiments of the present invention, the 0 to 20 base intervals between the linker sequence (L1) of the first primer and the target sequence binding region (I) indicate that the linker sequence (L1) of the first primer and the target sequence binding region (I) may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0014] In some embodiments of the present invention, the ligation sequence (L1) of the first primer is part of the probe anchoring region (A), or the ligation sequence (L1) is a sequence independent of the probe anchoring region (A).

[0015] In some embodiments of the present invention, there is a 0-20 base interval between the linker sequence (L2) of the second primer and the target sequence binding region (II), preferably a 0-10 base interval.

[0016] In some embodiments of the present invention, the 0 to 20 base intervals between the linker sequence (L2) of the second primer and the target sequence binding region (II) indicate that the linker sequence (L2) of the second primer and the target sequence binding region (II) may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0017] In some embodiments of the present invention, the linker sequence (L2) of the second primer is part of the primer signal detection region (h), or the linker sequence (L2) is a sequence independent of the primer signal detection region (h).

[0018] In some embodiments of the present invention, the complementary sequence (L2') of the linker sequence (L2) and the linker sequence (L1) have at least 3 consecutive pairs, preferably at least 4 pairs, of base sequences that can specifically bind; preferably, the complementary sequence (L2') of the linker sequence (L2) and the linker sequence (L1) have 3 to 20 consecutive pairs, preferably 4 to 12 pairs, of base sequences that can specifically bind; more preferably, the complementary pairing sequence (L2') of the linker sequence (L2) can completely and specifically bind to the linker sequence (L1).

[0019] In some embodiments of the present invention, the linker sequence (L2) and the linker sequence (L1) have at least 3, preferably at least 4 consecutive identical base sequences; preferably, the linker sequence (L2) and the linker sequence (L1) have 3 to 20, preferably 4 to 12 consecutive identical base sequences; more preferably, the linker sequence (L2) and the linker sequence (L1) are completely identical.

[0020] In some embodiments of the present invention, the length of the probe is 20 to 100 bases; preferably, the length of the primer anchoring region (A') is 6 to 35 bases, and the length of the probe signal detection region (H) is 5 to 65 bases.

[0021] In some embodiments of the present invention, the length of the probe is 20 to 100 bases, meaning the probe length can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 5 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 bases.

[0022] In some embodiments of the present invention, the length of the primer anchoring region (A') is 6 to 35 bases, meaning that the length of the primer anchoring region (A') can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases.

[0023] In some embodiments of the present invention, the length of the probe signal detection region (H) is 5 to 65 bases, meaning the length of the probe signal detection region (H) can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 bases.

[0024] In some embodiments of the present invention, the Tm value of the primer anchoring region (A') is 40°C to 80°C.

[0025] In some embodiments of the present invention, the GC content of the primer anchoring region (A') is 40% to 80%.

[0026] In some embodiments of the present invention, the Tm value of the probe signal detection region (H) is 40°C to 80°C.

[0027] In some embodiments of the present invention, the GC content of the probe signal detection region (H) is 40% to 80%.

[0028] In some embodiments of the present invention, the length of the first primer is 15 to 80 bases; preferably, the length of the linker sequence (L1) is 3 to 20 bases, and the length of the probe anchoring region (A) is 6 to 35 bases.

[0029] In some embodiments of the present invention, the length of the first primer being 15 to 80 bases means that the length of the first primer can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 bases.

[0030] In some embodiments of the present invention, the length of the linking sequence (L1) is 3 to 20 bases, meaning that the length of the linking sequence (L1) can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0031] In some embodiments of the present invention, the length of the probe anchoring region (A) is 6 to 35 bases, meaning that the length of the probe anchoring region (A) can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases.

[0032] In some embodiments of the present invention, the Tm value of the connection sequence (L1) is 40°C to 70°C.

[0033] In some embodiments of the present invention, the GC content of the connection sequence (L1) is 40% to 90%.

[0034] In some embodiments of the present invention, the Tm value of the probe anchoring area (A) is 40°C to 80°C.

[0035] In some embodiments of the present invention, the GC content of the probe anchoring region (A) is 40% to 80%.

[0036] In some embodiments of the present invention, the Tm value of the target sequence binding region (Ⅰ) is 50°C to 80°C.

[0037] In some embodiments of the present invention, the GC content of the target sequence binding region (Ⅰ) is 40% to 80%.

[0038] In some embodiments of the present invention, the second primer is 15 to 80 bases in length; preferably, the primer signal detection region (h) is 5 to 65 bases in length, and the linker sequence (L2) is 3 to 20 bases in length.

[0039] In some embodiments of the present invention, the length of the second primer being 15 to 80 bases means that the length of the second primer can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 bases.

[0040] In some embodiments of the present invention, the length of the primer signal detection region (h) is 5 to 65 bases, meaning that the length of the primer signal detection region (h) can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 bases.

[0041] In some embodiments of the present invention, the length of the linker sequence (L2) is 3 to 20 bases, meaning that the length of the linker sequence (L2) can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0042] In some embodiments of the present invention, the Tm value of the primer signal detection region (h) is 40℃~80℃.

[0043] In some embodiments of the present invention, the GC content of the primer signal detection region (h) is 40% to 80%.

[0044] In some embodiments of the present invention, the Tm value of the connection sequence (L2) is 40°C to 70°C.

[0045] In some embodiments of the present invention, the GC content of the connection sequence (L2) is 40% to 90%.

[0046] In some embodiments of the present invention, the Tm value of the target sequence binding region (II) is 50°C to 80°C.

[0047] In some embodiments of the present invention, the GC content of the target sequence binding region (II) is 40% to 80%.

[0048] In some embodiments of the present invention, the detection marker includes a first detection group and a second detection group, and the first detection group and the second detection group generate a signal change through a change in distance.

[0049] In some embodiments of the present invention, at least one detection group is modified at the 5' end of the probe signal detection region (H); preferably, both detection groups are modified at the 5' end of the probe signal detection region (H).

[0050] In some embodiments of the present invention, the first detection group and the second detection group are spaced 3 to 140 angstroms apart.

[0051] In some embodiments of the present invention, the first detection group is a fluorescent reporter group, and the second detection group is a quenching group or other modifying group that can generate a signal change with the first detection group through fluorescence resonance energy transfer.

[0052] In some embodiments of the present invention, the 3' end of the probe contains a blocking region, which is used to prevent further extension of DNA polymerase. When the 3' end of the probe contains a blocking region, it can achieve specific binding to the probe and extension only to the end of the probe, thereby helping to reduce non-specific amplification.

[0053] In some embodiments of the present invention, the blocking region may be modified with 3'OH by 3'-Spacer C3, 3'-Phosphat, 3'-ddC or 3'-Inverted End, etc., so that its 3'OH is blocked, thereby preventing its extension reaction.

[0054] In some embodiments of the invention, the blocking region is a blocking group of the polymerase (a group having the property of blocking further extension of the polymerase). The blocking group allows the 5' end of the modified nucleotide to connect to the 3' end of another nucleotide, but it prevents any further nucleotide from connecting to the 3' hydroxyl group of the modified nucleotide. That is, when DNA polymerase encounters this modified nucleotide, it can add this nucleotide to the synthesizing DNA chain, but cannot add more nucleotides to its 3' blocking group. In some specific embodiments, the blocking group is selected from groups such as acetyl, -CH3, glycyl, leucyl, and alanyl. In other specific embodiments, the blocking group may be in the form of a dipeptide or tripeptide.

[0055] Secondly, the present invention provides another primer-probe set for detecting nucleic acids, comprising one probe, at least one first primer, and at least two second primers; wherein

[0056] The probe is a freely designed sequence that does not pair with any target sequence. It includes a primer anchoring region (A') and a probe signal detection region (H). The primer anchoring region (A') is located at the 5' end of the probe signal detection region (H). The probe is modified with a detection marker.

[0057] The first primer comprises, from its 5' end to its 3' end, a probe anchoring region (A), a linker sequence (L1), and a target sequence binding region (Ⅰ); wherein, the probe anchoring region (A) specifically binds to the primer anchoring region (A') of the probe; the linker sequence (L1) is a freely designed sequence that does not pair with any target sequence; and the target sequence binding region (Ⅰ) is a sequence that can specifically bind to the target.

[0058] The at least two second primers are different from each other, each independently comprising, from the 5' end to the 3' end, a primer signal detection region (h), a linker sequence (L2), and a target sequence binding region (II); wherein, the complementary sequence (h') of the primer signal detection region (h) specifically binds to a portion of the probe signal detection region (H) of the probe, and the sequences of the primer signal detection regions (h) of different second primers are different from each other; a portion or all of the complementary sequence (L2') of the linker sequence (L2) specifically binds to a portion or all of the linker sequence (L1), and the sequences of the linker sequences (L2) of different second primers are the same; the target sequence binding region (II) is a sequence capable of specifically binding to a target, and the target sequence binding region (II) of different second primers specifically binds to different targets.

[0059] The first primer and the second primer are primer pairs used to amplify the same target. That is, the first primer and the second primer are the forward primer and the reverse primer, respectively. They specifically bind to the same target through their respective target sequence binding regions (target sequence binding region (Ⅰ) and target sequence binding region (Ⅱ)), and amplify the amplified product containing the target sequence after extension.

[0060] In some embodiments of the present invention, when the number of first primers in the primer-probe set is less than the number of second primers, some different second primers form different primer pairs with the same first primer to amplify different targets. For example, when the primer-probe set includes two first primers and three second primers, one first primer forms a primer pair with one of the second primers, and the other first primer forms different primer pairs with the other two second primers.

[0061] In some embodiments of the present invention, when there is only one type of first primer in the primer-probe set, the at least two types of second primers share the same first primer to form different primer pairs to amplify different targets. For example, when the primer-probe set is intended to be used to detect a sample containing two different targets, the first primers amplifying the two different targets are the same, but the second primers are different from each other.

[0062] In some embodiments of the present invention, there is a 0-20 base interval between the linker sequence (L1) of the first primer and the target sequence binding region (Ⅰ), preferably a 0-10 base interval.

[0063] In some embodiments of the present invention, the 0 to 20 base intervals between the linker sequence (L1) of the first primer and the target sequence binding region (I) indicate that the linker sequence (L1) of the first primer and the target sequence binding region (I) may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0064] In some embodiments of the present invention, the ligation sequence (L1) of the first primer is part of the probe anchoring region (A), or the ligation sequence (L1) is a sequence independent of the probe anchoring region (A).

[0065] In some embodiments of the present invention, there is a 0-20 base interval between the linker sequence (L2) of the second primer and the target sequence binding region (II), preferably a 0-10 base interval.

[0066] In some embodiments of the present invention, the 0 to 20 base intervals between the linker sequence (L2) of the second primer and the target sequence binding region (II) indicate that the linker sequence (L2) of the second primer and the target sequence binding region (II) may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0067] In some embodiments of the present invention, the linker sequence (L2) of the second primer is part of the primer signal detection region (h), or the linker sequence (L2) is a sequence independent of the primer signal detection region (h).

[0068] In some embodiments of the present invention, the complementary sequence (L2') of the linker sequence (L2) and the linker sequence (L1) have at least 3 consecutive pairs, preferably at least 4 pairs, of base sequences that can specifically bind; preferably, the complementary sequence (L2') of the linker sequence (L2) and the linker sequence (L1) have 3 to 20 consecutive pairs, preferably 4 to 12 pairs, of base sequences that can specifically bind; more preferably, the complementary pairing sequence (L2') of the linker sequence (L2) can completely and specifically bind to the linker sequence (L1).

[0069] In some embodiments of the present invention, the linker sequence (L2) and the linker sequence (L1) have at least 3, preferably at least 4 consecutive identical base sequences; preferably, the linker sequence (L2) and the linker sequence (L1) have 3 to 20, preferably 4 to 12 consecutive identical base sequences; more preferably, the linker sequence (L2) and the linker sequence (L1) are completely identical.

[0070] In some embodiments of the present invention, the length of the probe is 20 to 100 bases; preferably, the length of the primer anchoring region (A') is 6 to 35 bases, and the length of the probe signal detection region (H) is 5 to 65 bases.

[0071] In some embodiments of the present invention, the length of the probe is 20 to 100 bases, meaning the probe length can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 5 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 bases.

[0072] In some embodiments of the present invention, the length of the primer anchoring region (A') is 6 to 35 bases, meaning that the length of the primer anchoring region (A') can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases.

[0073] In some embodiments of the present invention, the length of the probe signal detection region (H) is 5 to 65 bases, meaning the length of the probe signal detection region (H) can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 bases.

[0074] In some embodiments of the present invention, the Tm value of the primer anchoring region (A') is 40°C to 80°C.

[0075] In some embodiments of the present invention, the GC content of the primer anchoring region (A') is 40% to 80%.

[0076] In some embodiments of the present invention, the Tm value of the probe signal detection region (H) is 40°C to 80°C.

[0077] In some embodiments of the present invention, the GC content of the probe signal detection region (H) is 40% to 80%.

[0078] In some embodiments of the present invention, the length of the first primer is 15 to 80 bases; preferably, the length of the linker sequence (L1) is 3 to 20 bases, and the length of the probe anchoring region (A) is 6 to 35 bases.

[0079] In some embodiments of the present invention, the length of the first primer being 15 to 80 bases means that the length of the first primer can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 bases.

[0080] In some embodiments of the present invention, the length of the linking sequence (L1) is 3 to 20 bases, meaning that the length of the linking sequence (L1) can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0081] In some embodiments of the present invention, the length of the probe anchoring region (A) is 6 to 35 bases, meaning that the length of the probe anchoring region (A) can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases.

[0082] In some embodiments of the present invention, the Tm value of the connection sequence (L1) is 40°C to 70°C.

[0083] In some embodiments of the present invention, the GC content of the connection sequence (L1) is 40% to 90%.

[0084] In some embodiments of the present invention, the Tm value of the probe anchoring area (A) is 40°C to 80°C.

[0085] In some embodiments of the present invention, the GC content of the probe anchoring region (A) is 40% to 80%.

[0086] In some embodiments of the present invention, the Tm value of the target sequence binding region (Ⅰ) is 50°C to 80°C.

[0087] In some embodiments of the present invention, the GC content of the target sequence binding region (Ⅰ) is 40% to 80%.

[0088] In some embodiments of the present invention, the second primer is 15 to 80 bases in length; preferably, the primer signal detection region (h) is 5 to 65 bases in length, and the linker sequence (L2) is 3 to 20 bases in length.

[0089] In some embodiments of the present invention, the length of the second primer being 15 to 80 bases means that the length of the second primer can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 bases.

[0090] In some embodiments of the present invention, the length of the primer signal detection region (h) is 5 to 65 bases, meaning that the length of the primer signal detection region (h) can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 bases.

[0091] In some embodiments of the present invention, the length of the linker sequence (L2) is 3 to 20 bases, meaning that the length of the linker sequence (L2) can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0092] In some embodiments of the present invention, the Tm value of the primer signal detection region (h) is 40℃~80℃.

[0093] In some embodiments of the present invention, the GC content of the primer signal detection region (h) is 40% to 80%.

[0094] In some embodiments of the present invention, the Tm value of the connection sequence (L2) is 40°C to 70°C.

[0095] In some embodiments of the present invention, the GC content of the connection sequence (L2) is 40% to 90%.

[0096] In some embodiments of the present invention, the Tm value of the target sequence binding region (II) is 50°C to 80°C.

[0097] In some embodiments of the present invention, the GC content of the target sequence binding region (II) is 40% to 80%.

[0098] In some embodiments of the present invention, the detection marker includes a first detection group and a second detection group, and the first detection group and the second detection group generate a signal change through a change in distance.

[0099] In some embodiments of the present invention, at least one detection group is modified at the 5' end of the probe signal detection region (H); preferably, both detection groups are modified at the 5' end of the probe signal detection region (H).

[0100] In some embodiments of the present invention, the first detection group and the second detection group are spaced 3 to 140 angstroms apart.

[0101] In some embodiments of the present invention, the first detection group is a fluorescent reporter group, and the second detection group is a quenching group or other modifying group that can generate a signal change with the first detection group through fluorescence resonance energy transfer.

[0102] In some embodiments of the present invention, the 3' end of the probe contains a blocking region, which is used to prevent further extension of DNA polymerase. When the 3' end of the probe contains a blocking region, it can achieve specific binding to the probe and extension only to the end of the probe, thereby helping to reduce non-specific amplification.

[0103] In some embodiments of the present invention, the blocking region may be modified with 3'OH by 3'-Spacer C3, 3'-Phosphat, 3'-ddC or 3'-Inverted End, etc., so that its 3'OH is blocked, thereby preventing its extension reaction.

[0104] In some embodiments of the invention, the blocking region is a blocking group of the polymerase (a group having the property of blocking further extension of the polymerase). The blocking group allows the 5' end of the modified nucleotide to connect to the 3' end of another nucleotide, but it prevents any further nucleotide from connecting to the 3' hydroxyl group of the modified nucleotide. That is, when DNA polymerase encounters this modified nucleotide, it can add this nucleotide to the synthesizing DNA chain, but cannot add more nucleotides to its 3' blocking group. In some specific embodiments, the blocking group is selected from groups such as acetyl, -CH3, glycyl, leucyl, and alanyl. In other specific embodiments, the blocking group may be in the form of a dipeptide or tripeptide.

[0105] Thirdly, the present invention provides another primer-probe set for detecting nucleic acids, comprising one probe, at least two first primers, and at least two second primers; wherein

[0106] The probe is a freely designed sequence that does not pair with any target sequence. It includes a primer anchoring region (A') and a probe signal detection region (H). The primer anchoring region (A') is located at the 5' end of the probe signal detection region (H). The probe is modified with a detection marker.

[0107] The at least two first primers are different from each other, each independently comprising, from its 5' end to its 3' end, a probe anchoring region (A), a linker sequence (L1), and a target sequence binding region (Ⅰ); wherein, the probe anchoring region (A) specifically binds to a portion of the primer anchoring region (A') of the probe; the linker sequence (L1) is a freely designed sequence that does not pair with any target sequence, and the linker sequences (L1) of different first primers are identical; the target sequence binding region (Ⅰ) is a sequence capable of specifically binding to a target, and the target sequence binding regions (Ⅰ) of different first primers specifically bind to different targets;

[0108] The at least two second primers are different from each other, each independently comprising, from the 5' end to the 3' end, a primer signal detection region (h), a linker sequence (L2), and a target sequence binding region (II); wherein, the complementary sequence (h') of the primer signal detection region (h) specifically binds to a portion of the probe signal detection region (H) of the probe, and the sequences of the primer signal detection regions (h) of different second primers are different from each other; a portion or all of the complementary sequence (L2') of the linker sequence (L2) specifically binds to a portion or all of the linker sequence (L1), and the sequences of the linker sequences (L2) of different second primers are the same; the target sequence binding region (II) is a sequence capable of specifically binding to a target, and the target sequence binding region (II) of different second primers specifically binds to different targets.

[0109] The first primer and the second primer are primer pairs used to amplify the same target. That is, the first primer and the second primer are the forward primer and the reverse primer, respectively. They specifically bind to the same target through their respective target sequence binding regions (target sequence binding region (Ⅰ) and target sequence binding region (Ⅱ)), and amplify the amplified product containing the target sequence after extension.

[0110] In some embodiments of the present invention, the probe anchoring regions (A) of different first primers have the same sequence and can specifically bind to the probe's primer anchoring region (A').

[0111] In some embodiments of the present invention, the sequences of the probe anchoring regions (A) of different first primers are different from each other, and can specifically bind to different positions of the primer anchoring regions (A') of the same probe.

[0112] In some embodiments of the present invention, when the number of first primers in the primer-probe set is less than the number of second primers, some different second primers form different primer pairs with the same first primer to amplify different targets. For example, when the primer-probe set includes two first primers and three second primers, one first primer forms a primer pair with one of the second primers, and the other first primer forms different primer pairs with the other two second primers.

[0113] In some embodiments of the present invention, when the number of first primers in the primer-probe set exceeds the number of second primers, some different first primers form different primer pairs with the same second primer to amplify different targets. For example, when the primer-probe set includes three first primers and two second primers, one first primer forms a primer pair with one of the second primers, and the other two first primers form different primer pairs with the other second primer. For example, in "classification without genotyping" nucleic acid detection, the two first primers target different targets (different genotypes), but share one second primer to produce the same detection signal (for the same category).

[0114] In some embodiments of the present invention, the primer probe set includes the same number of first primers and second primers, for example, including 2 types of first primers and 2 types of second primers or including 3 types of first primers and 3 types of second primers; so that the first primers and second primers are different for different targets.

[0115] In some embodiments of the present invention, there is a 0-20 base interval between the linker sequence (L1) of the first primer and the target sequence binding region (Ⅰ), preferably a 0-10 base interval.

[0116] In some embodiments of the present invention, the 0 to 20 base intervals between the linker sequence (L1) of the first primer and the target sequence binding region (I) indicate that the linker sequence (L1) of the first primer and the target sequence binding region (I) may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0117] In some embodiments of the present invention, the ligation sequence (L1) of the first primer is part of the probe anchoring region (A), or the ligation sequence (L1) is a sequence independent of the probe anchoring region (A).

[0118] In some embodiments of the present invention, there is a 0-20 base interval between the linker sequence (L2) of the second primer and the target sequence binding region (II), preferably a 0-10 base interval.

[0119] In some embodiments of the present invention, the 0 to 20 base intervals between the linker sequence (L2) of the second primer and the target sequence binding region (II) indicate that the linker sequence (L2) of the second primer and the target sequence binding region (II) may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0120] In some embodiments of the present invention, the linker sequence (L2) of the second primer is part of the primer signal detection region (h), or the linker sequence (L2) is a sequence independent of the primer signal detection region (h).

[0121] In some embodiments of the present invention, the complementary sequence (L2') of the linker sequence (L2) and the linker sequence (L1) have at least 3 consecutive pairs, preferably at least 4 pairs, of base sequences that can specifically bind; preferably, the complementary sequence (L2') of the linker sequence (L2) and the linker sequence (L1) have 3 to 20 consecutive pairs, preferably 4 to 12 pairs, of base sequences that can specifically bind; more preferably, the complementary pairing sequence (L2') of the linker sequence (L2) can completely and specifically bind to the linker sequence (L1).

[0122] In some embodiments of the present invention, the linker sequence (L2) and the linker sequence (L1) have at least 3, preferably at least 4 consecutive identical base sequences; preferably, the linker sequence (L2) and the linker sequence (L1) have 3 to 20, preferably 4 to 12 consecutive identical base sequences; more preferably, the linker sequence (L2) and the linker sequence (L1) are completely identical.

[0123] In some embodiments of the present invention, the length of the probe is 20 to 100 bases; preferably, the length of the primer anchoring region (A') is 6 to 35 bases, and the length of the probe signal detection region (H) is 5 to 65 bases.

[0124] In some embodiments of the present invention, the length of the probe is 20 to 100 bases, meaning the probe length can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 5 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 bases.

[0125] In some embodiments of the present invention, the length of the primer anchoring region (A') is 6 to 35 bases, meaning that the length of the primer anchoring region (A') can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases.

[0126] In some embodiments of the present invention, the length of the probe signal detection region (H) is 5 to 65 bases, meaning the length of the probe signal detection region (H) can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 bases.

[0127] In some embodiments of the present invention, the Tm value of the primer anchoring region (A') is 40°C to 80°C.

[0128] In some embodiments of the present invention, the GC content of the primer anchoring region (A') is 40% to 80%.

[0129] In some embodiments of the present invention, the Tm value of the probe signal detection region (H) is 40°C to 80°C.

[0130] In some embodiments of the present invention, the GC content of the probe signal detection region (H) is 40% to 80%.

[0131] In some embodiments of the present invention, the length of the first primer is 15 to 80 bases; preferably, the length of the linker sequence (L1) is 3 to 20 bases, and the length of the probe anchoring region (A) is 6 to 35 bases.

[0132] In some embodiments of the present invention, the length of the first primer being 15 to 80 bases means that the length of the first primer can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 bases.

[0133] In some embodiments of the present invention, the length of the linking sequence (L1) is 3 to 20 bases, meaning that the length of the linking sequence (L1) can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0134] In some embodiments of the present invention, the length of the probe anchoring region (A) is 6 to 35 bases, meaning that the length of the probe anchoring region (A) can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases.

[0135] In some embodiments of the present invention, the Tm value of the connection sequence (L1) is 40°C to 70°C.

[0136] In some embodiments of the present invention, the GC content of the connection sequence (L1) is 40% to 90%.

[0137] In some embodiments of the present invention, the Tm value of the probe anchoring area (A) is 40°C to 80°C.

[0138] In some embodiments of the present invention, the GC content of the probe anchoring region (A) is 40% to 80%.

[0139] In some embodiments of the present invention, the Tm value of the target sequence binding region (Ⅰ) is 50°C to 80°C.

[0140] In some embodiments of the present invention, the GC content of the target sequence binding region (Ⅰ) is 40% to 80%.

[0141] In some embodiments of the present invention, the second primer is 15 to 80 bases in length; preferably, the primer signal detection region (h) is 5 to 65 bases in length, and the linker sequence (L2) is 3 to 20 bases in length.

[0142] In some embodiments of the present invention, the length of the second primer being 15 to 80 bases means that the length of the second primer can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 bases.

[0143] In some embodiments of the present invention, the length of the primer signal detection region (h) is 5 to 65 bases, meaning that the length of the primer signal detection region (h) can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 bases.

[0144] In some embodiments of the present invention, the length of the linker sequence (L2) is 3 to 20 bases, meaning that the length of the linker sequence (L2) can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0145] In some embodiments of the present invention, the Tm value of the primer signal detection region (h) is 40℃~80℃.

[0146] In some embodiments of the present invention, the GC content of the primer signal detection region (h) is 40% to 80%.

[0147] In some embodiments of the present invention, the Tm value of the connection sequence (L2) is 40°C to 70°C.

[0148] In some embodiments of the present invention, the GC content of the connection sequence (L2) is 40% to 90%.

[0149] In some embodiments of the present invention, the Tm value of the target sequence binding region (II) is 50°C to 80°C.

[0150] In some embodiments of the present invention, the GC content of the target sequence binding region (II) is 40% to 80%.

[0151] In some embodiments of the present invention, the detection marker includes a first detection group and a second detection group, and the first detection group and the second detection group generate a signal change through a change in distance.

[0152] In some embodiments of the present invention, at least one detection group is modified at the 5' end of the probe signal detection region (H); preferably, both detection groups are modified at the 5' end of the probe signal detection region (H).

[0153] In some embodiments of the present invention, the first detection group and the second detection group are spaced 3 to 140 angstroms apart.

[0154] In some embodiments of the present invention, the first detection group is a fluorescent reporter group, and the second detection group is a quenching group or other modifying group that can generate a signal change with the first detection group through fluorescence resonance energy transfer.

[0155] In some embodiments of the present invention, the 3' end of the probe contains a blocking region, which is used to prevent further extension of DNA polymerase. When the 3' end of the probe contains a blocking region, it can achieve specific binding to the probe and extension only to the end of the probe, thereby helping to reduce non-specific amplification.

[0156] In some embodiments of the present invention, the blocking region may be modified with 3'OH by 3'-Spacer C3, 3'-Phosphat, 3'-ddC or 3'-Inverted End, etc., so that its 3'OH is blocked, thereby preventing its extension reaction.

[0157] In some embodiments of the invention, the blocking region is a blocking group of the polymerase (a group having the property of blocking further extension of the polymerase). The blocking group allows the 5' end of the modified nucleotide to connect to the 3' end of another nucleotide, but it prevents any further nucleotide from connecting to the 3' hydroxyl group of the modified nucleotide. That is, when DNA polymerase encounters this modified nucleotide, it can add this nucleotide to the synthesizing DNA chain, but cannot add more nucleotides to its 3' blocking group. In some specific embodiments, the blocking group is selected from groups such as acetyl, -CH3, glycyl, leucyl, and alanyl. In other specific embodiments, the blocking group may be in the form of a dipeptide or tripeptide.

[0158] The primer-probe set designs described in the first, second, and third aspects above all refer to the specific design of primers and probes within a single primer-probe set. Nucleic acid detection reagents or kits can contain multiple different primer-probe sets. For example, a nucleic acid detection reagent or kit may simultaneously contain one primer-probe set described in the first aspect, one primer-probe set described in the second aspect, and one primer-probe set described in the third aspect; or, a nucleic acid detection reagent or kit may simultaneously contain three primer-probe sets described in the first aspect, where the sequences of probes and primers in each primer-probe set are different from the sequences of probes and primers in the other two primer-probe sets.

[0159] Fourthly, the present invention provides a method for detecting nucleic acids, the method comprising the following steps:

[0160] (1) Mix the primer and probe set with the amplification reagent and the sample to be tested;

[0161] (2) Perform nucleic acid amplification on the mixture obtained in step (1), and determine whether there is a target in the sample to be tested based on the changes in the fluorescence signal generated by the amplification.

[0162] Optionally (3) performs melting curve analysis on the amplification product obtained in step (2) and determines whether there is a target in the sample to be tested based on the analysis results;

[0163] In step (1), the primer-probe set is selected from at least one set of primer-probe sets described in the first aspect, the second aspect, and the third aspect.

[0164] In some embodiments of the present invention, in the mixture obtained in step (1), in each set of primer probes, the concentration of the first primer is 30 nM to 1000 nM and the concentration of the second primer is 30 nM to 500 nM; preferably, the concentration of the first primer is greater than the concentration of the second primer; more preferably, the concentration of the first primer is 2 to 10 times the concentration of the second primer.

[0165] In some embodiments of the present invention, the concentration of the first primer being 30 nM to 1000 nM indicates that the concentration of the first primer can be selected from any of the following concentrations or any value between any two of them: 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 28 0, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000nM.

[0166] In some embodiments of the present invention, the concentration of the second primer being 30 nM to 500 nM means that the concentration of the second primer can be selected from any of the following concentrations or any value between any two of them: 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 nM.

[0167] In some embodiments of the present invention, in the mixture obtained in step (1), in each set of primers and probes, the concentration of the probe is 30 nM to 1200 nM, and the concentration of the first primer is 30 nM to 1000 nM; preferably, the concentration of the probe is equivalent to the concentration of the first primer; more preferably, the concentration of the probe is equal to the concentration of the first primer.

[0168] In some embodiments of the present invention, the concentration of the probe is 30 nM to 1200 nM, meaning that the concentration of the probe can be selected from any of the following concentrations or any value between any two of them: 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 31 0, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200nM.

[0169] In some embodiments of the present invention, the amplification reagent includes DNA polymerase and dNTPs.

[0170] In some embodiments of the present invention, when the nucleic acid is RNA, the amplification reagent further includes reverse transcriptase.

[0171] In some embodiments of the present invention, the amplification reagent further includes components that can promote PCR reactions, such as KCl, MgCl2, Tris-HCl, dithiothreitol (DTT), (NH4)2SO4, etc.

[0172] In some embodiments of the present invention, the conditions for nucleic acid amplification are: pre-denaturation at 85℃~105℃ for 0min~15min; denaturation at 85℃~105℃ for 1s~60s; annealing and extension at 40℃~75℃ for 3s~90s; 20~60 cycles.

[0173] In some embodiments of the present invention, the conditions for nucleic acid amplification are: pre-denaturation at 90℃~96℃ for 1min~15min; denaturation at 90℃~95℃ for 10s~60s; annealing and extension at 50℃~75℃ for 30s~90s; 35~50 cycles.

[0174] In some embodiments of the present invention, step (2) before nucleic acid amplification of the mixture further includes distributing the mixture into at least 2,000 reaction units, each of which contains one target of the sample to be tested or does not contain a target of the sample to be tested.

[0175] In some embodiments of the present invention, the melting curve analysis of the amplification product obtained in step (2) includes heating the amplification product between 35°C and 95°C to obtain a melting curve, with a heating rate of 0.05°C / s.

[0176] In some embodiments of the present invention, when a target is present in the sample to be tested, the amplification product obtained in step (2) is a secondary amplification double-stranded product formed by the specific binding and extension of the probe and the pre-amplification product, wherein the pre-amplification product is generated by the specific binding and amplification of the first primer and the second primer with the target, respectively; preferably, the pre-amplification product is a single-stranded nucleotide chain containing the reverse complementary sequence (L2') of the probe anchoring region (A), the linker sequence (L1), the linker sequence (L2), and the reverse complementary sequence (h') of the primer signal detection region (h).

[0177] In some embodiments of the present invention, heating between 35°C and 95°C to obtain a melting curve means heating within a certain temperature range between 35°C and 95°C to obtain a melting curve. For example, at temperatures such as 40°C to 95°C, 45°C to 90°C, 45°C to 85°C, 50°C to 95°C, 55°C to 90°C, and 50°C to 85°C, as long as the temperature range includes the melting temperature of the double-stranded product of the secondary amplification.

[0178] In the method of this invention, a fluorescence signal is only generated after the probe specifically binds to the pre-amplified product and extends to form a secondary amplified double-stranded product. This helps to minimize the fluorescence background of the fluorescence signal map.

[0179] In some embodiments of the present invention, step (3) of determining whether there is a target in the sample to be tested based on the analysis results includes when the obtained melting curve contains a characteristic peak and the temperature corresponding to the characteristic peak is the characteristic Tm of a certain target in a specific detection channel in the sample to be tested, indicating that the sample to be tested contains the target; preferably, the specific detection channel is the fluorescence channel corresponding to the detection group on the probe, and the characteristic Tm is the melting temperature of the secondary amplified double-stranded product.

[0180] In some embodiments of the present invention, when there are more than two targets in the sample to be tested, step (2) of determining whether there are targets in the sample to be tested also includes determining how many targets or which targets are present in the sample to be tested. Since different probes will show different fluorescence signals in the detection channel, different probes can be used to pair and bind with the pre-amplified products of different targets to generate different fluorescence signals, thereby determining how many targets or which targets are present in the sample to be tested.

[0181] In some embodiments of the present invention, when there are more than two targets in the sample to be tested, step (3) of determining whether there are targets in the sample to be tested also includes determining how many targets or which targets are present in the sample to be tested. Since the secondary amplified double-stranded products obtained by different targets will show different characteristic peaks when performing melting curve analysis, and the temperature corresponding to each characteristic peak is the melting temperature of the secondary amplified double-stranded product corresponding to that characteristic peak, when there are more than two targets in the sample to be tested, the number of targets or which targets are present in the sample to be tested can also be determined by the obtained melting curve.

[0182] In some specific embodiments of the present invention, the sample to be tested includes any one or more of the following: serum sample, plasma sample, whole blood sample, sputum sample, swab sample, irrigation fluid sample, fresh tissue sample, and formalin-fixed paraffin-embedded tissue (FFPE).

[0183] In some specific embodiments of the present invention, the sample to be tested may contain a target sequence of more than 40 bp.

[0184] Fifthly, the present invention provides a kit for detecting nucleic acids, comprising a primer-probe set selected from at least one set of primer-probe sets described in the first, second, and third aspects.

[0185] In some embodiments of the present invention, in the kit, in each set of primers and probes, the working concentration of the first primer is greater than the working concentration of the second primer; more preferably, the concentration of the first primer is 2 to 10 times the concentration of the second primer.

[0186] In some embodiments of the present invention, in the kit, in each set of primers and probes, the working concentration of the probe is equivalent to the working concentration of the first primer; more preferably, the working concentration of the probe is equal to the working concentration of the first primer.

[0187] In some embodiments of the present invention, the "working concentration" of each reagent component in the kit refers to the concentration of each reagent component in a single PCR reaction system for testing. For example, in a PCR reaction system with a total volume of 25 μL, the working concentration of the first primer is 30 nM to 1000 nM, the working concentration of the second primer is 30 nM to 500 nM, and the working concentration of the probe is 30 nM to 1200 nM. Due to practical needs such as convenient transportation and easy operation, the actual concentration of each reagent component in the kit may be higher than its working concentration, or even in lyophilized powder form; it is only necessary to prepare it to the corresponding working concentration during actual testing.

[0188] In some embodiments of the present invention, the working concentration of the first primer is 30 nM to 1000 nM, meaning that the working concentration of the first primer can be selected from any of the following concentrations or any value between any two of them: 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270. 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480 , 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000nM.

[0189] In some embodiments of the present invention, the working concentration of the second primer is 30 nM to 500 nM, meaning that the working concentration of the second primer can be selected from any of the following concentrations or any value between any two of them: 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 nM.

[0190] In some embodiments of the present invention, the working concentration of the probe is 30 nM to 1200 nM, meaning that the working concentration of the probe can be selected from any of the following concentrations or any value between any two of them: 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520 , 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200nM.

[0191] In some embodiments of the present invention, the kit further includes amplification reagents.

[0192] In some embodiments of the present invention, the amplification reagent includes DNA polymerase and dNTPs.

[0193] In some embodiments of the present invention, when the nucleic acid is RNA, the amplification reagent further includes reverse transcriptase.

[0194] In some embodiments of the present invention, the amplification reagent further includes components that can promote PCR reactions, such as KCl, MgCl2, Tris-HCl, dithiothreitol (DTT), (NH4)2SO4, etc.

[0195] In a sixth aspect, the present invention provides the application of the primer and probe set described in the first, second, or third aspect, the method described in the fourth aspect, and the kit described in the fifth aspect in detecting one or more nucleic acids for purposes other than disease diagnosis and treatment.

[0196] In the primer-probe combination of the present invention, a linker sequence is provided between the first primer and the second primer, and the linker sequence regions of the first primer and the second primer contain consecutive identical base sequences, thereby reducing primer dimerization. Furthermore, the pre-amplified product single-stranded nucleotide chain formed after the first primer, the second primer, and the target specifically bind and amplify contains complementary sequences [the inverse complementary sequence (L2') of the linker sequence (L1) and the linker sequence (L2)]. These complementary sequences undergo complementary pairing to form a stem-loop structure in the pre-amplified product single-stranded nucleotide chain. This stem-loop structure is as follows: Figure 1As shown (specific pre-amplification product); this stem-loop structure opens at the annealing temperature, allowing the single-stranded nucleotide chain to specifically bind to the probe, and further extends at the extension temperature to form a secondary amplified double-stranded product. The formation of the secondary amplified double-stranded product causes a detectable signal change in the probe, thus enabling the determination of the presence of a target in the sample by observing the signal change. When different probes are used to specifically bind to pre-amplified products targeting different targets, the number of different types of signals generated can be used to determine the number of targets present in the sample, or the different types of targets can be determined by the fluorescence color corresponding to the generated signals. When further analyzing the melting curve of the secondary amplified double-stranded product, the melting of the product during heating will also cause a detectable signal change in the probe. Moreover, when different targets are present in the sample (i.e., different secondary amplified double-stranded products are formed after amplification), the melting temperature of the secondary amplified double-stranded products obtained from different targets will be different during heating, which will be reflected in the melting curve with different characteristic peaks. Different characteristic peaks correspond to different temperatures, so the number of characteristic peaks generated by the melting curve can be used to determine how many targets are present in the sample, and the temperature corresponding to the characteristic peaks can be used to determine which types of targets are present in the sample.

[0197] Tm (melting temperature) refers to the temperature at which the secondary amplified double-stranded product undergoes half of its melting process during heating, i.e., the temperature at which 50% of the DNA molecules denature. The Tm value is related to the sequence, GC content, and fragment length of the secondary amplified double-stranded product. Theoretically, each nucleic acid amplification product has its own specific Tm value, which can be used to distinguish different amplification products and thus different targets, or to distinguish between target and non-specific amplification products.

[0198] In melting curve analysis, the actual melting temperature is also affected by the specific components in the amplification system (e.g., salt ion concentration, Mg). 2+ Due to the influence of concentration, etc., there may be some experimental error between the actual Tm value and the theoretical Tm value. However, it is generally believed that if the experimental conditions are properly controlled, the difference between the actual Tm value and the theoretical Tm value should be small enough to distinguish it from the theoretical Tm value of another target.

[0199] During nucleic acid amplification, if any two primers can produce primer dimers, for example:

[0200] (i) The first primer and the second primer produce a primer dimer. The resulting non-specific amplification product, from the 5' end to the 3' end, contains, sequentially, the probe anchoring region (A), the linker sequence (L1), the primer dimer region sequence (D), the inverse complementary sequence of the linker sequence (L2) (L2'), and the inverse complementary sequence of the primer signal detection region (h) (h'). Since this non-specific amplification product also contains mutually complementary sequences (the inverse complementary sequence of the linker sequence (L1) and the linker sequence (L2) (L2')), these complementary sequences will also specifically bind, causing the non-specific amplification product to form a stem-loop structure, such as... Figure 1 As shown (non-specific amplification product); however, since the primer dimer region sequence (D) in this non-specific amplification product only includes the target sequence binding region (Ⅰ) of the first primer and the reverse complementary target sequence binding region (Ⅱ') of the target sequence binding region (Ⅱ) of the second primer, and does not include other sequences further extended from these two region sequences, its length is relatively short; the stem-loop structure formed by this non-specific amplification product has a small loop, and the binding force of the reverse complementary sequence (L2') of the linking sequence (L1) and the linking sequence (L2) is very strong. At the annealing and extension temperatures, the absolute value of the Gibbs free energy of this stem-loop structure is large, the structure is relatively stable, and it cannot be opened. At this point, because the primer anchoring region (A') of the probe is located at the 5' end of the probe signal detection region (H), the primer dimer cannot bind to the probe and extend and stretch the probe. Therefore, the primer dimer cannot bind to the probe and extend to produce a detectable signal change. That is, even if primer dimers are generated during the amplification process, the primer dimer cannot bind to the probe and extend to produce a detectable signal change, so it will not affect the accuracy of determining whether there is a target in the sample by the detectable signal change.

[0201] (ii) Two identical first primers produce primer dimers, and the resulting non-specific amplification product contains, from the 5' end to the 3' end, a probe anchoring region (A), a linker sequence (L1), a primer dimer region sequence (D), an inverse complementary sequence of the linker sequence (L1) (L1'), and an inverse complementary sequence of the probe anchoring region (A) (A'). Similar to the primer dimer produced by the first and second primers, the stem-loop structure formed by this non-specific amplification product has a smaller loop. Compared to the primer dimer produced by the first and second primers, the stem-loop structure formed by the primer dimer produced by two identical first primers has a longer and more stable stem (not only does it exhibit specific binding between the linker sequence (L1) and its inverse complementary sequence (L1'), but it also exhibits specific binding between the probe anchoring region (A) and its inverse complementary sequence (A'). At annealing and extension temperatures, the absolute value of the Gibbs free energy of this stem-loop structure is larger, and the stem-loop structure is more difficult to open.

[0202] Two identical second primers produce primer dimers, and the resulting nonspecific amplification product, from the 5' end to the 3' end, contains the primer signal detection region (h), the linker sequence (L2), the primer dimer region sequence (D), the reverse complementary sequence of the linker sequence (L2) (L2'), and the reverse complementary sequence of the primer signal detection region (h) (h'). Similar to the primer dimer produced by the first and second primers, the stem-loop structure formed by this nonspecific amplification product has a smaller loop. Compared to the primer dimer produced by the first and second primers, the stem-loop structure formed by the primer dimer produced by two identical second primers has a longer and more stable stem (not only does it exhibit specific binding between the linker sequence (L2) and its reverse complementary sequence (L2'), but it also exhibits specific binding between the primer signal detection region (h) and its reverse complementary sequence (h'). At annealing and extension temperatures, the absolute value of the Gibbs free energy of this stem-loop structure is larger, making the stem-loop structure more difficult to open.

[0203] (III) Two different first primers produce primer dimers, and the non-specific amplification products formed therefrom the 5' end to the 3' end contain the first probe anchoring region (A1), the linker sequence (L1), the primer dimer region sequence (D), the reverse complementary sequence of the linker sequence (L1) (L1'), and the reverse complementary sequence of the second probe anchoring region (A2) (A2'). Similar to the primer dimers produced by the first and second primers, the stem-loop structure formed by this non-specific amplification product has a small loop, and the binding force between the linker sequence (L1) and the reverse complementary sequence of the linker sequence (L1) (L1') is very strong. At the annealing and extension temperatures, the absolute value of the Gibbs free energy of this stem-loop structure is large, the structure is relatively stable, and it cannot be opened.

[0204] Two different second primers produce primer dimers, and the resulting non-specific amplification product, from the 5' end to the 3' end, contains the primer signal detection region (h1), the linker sequence (L2), the primer dimer region sequence (D), the reverse complementary sequence of the linker sequence (L2) (L2'), and the reverse complementary sequence of the primer signal detection region (h2) (h2'). Similar to the primer dimer produced by the first and second primers, the stem-loop structure formed by this non-specific amplification product has a small loop, and the binding force between the linker sequence (L2) and the reverse complementary sequence of the linker sequence (L2) (L2') is very strong. At annealing and extension temperatures, the absolute value of the Gibbs free energy of this stem-loop structure is large, the structure is relatively stable, and it cannot be opened.

[0205] Melting curve analysis focuses on the melting temperature of the double-stranded product obtained from secondary amplification using the probe as a template. However, when any two primers dimerize, the stem-loop structure of the primer dimer is difficult to open, preventing secondary amplification using the probe as a template. Therefore, even if primer dimers are present during melting curve analysis, the unwinding of these dimers does not alter the distance between the detection groups on the probe, thus preventing the generation of characteristic peaks and not affecting the determination of the target's presence.

[0206] The beneficial effects of this invention are as follows:

[0207] All primers in the primer-probe set of this invention contain the same base sequence, which can reduce the phenomenon of primer dimerization. Even if primer dimerization occurs and primer dimers are generated, when determining whether there is a target in the sample by nucleic acid amplification or even by analyzing melting curves, the primer dimers will not specifically bind to the probe and generate false positive signals that can affect the judgment and quantification of target results. Therefore, it can effectively avoid false positive results caused by the presence of primer dimers.

[0208] The nucleic acid detection method of the present invention can be completed in a single tube reaction using a real-time fluorescence PCR instrument. It is simple to operate, has low cost of instruments and consumables, low requirements on target sequence length, and high detection sensitivity.

[0209] The primer and probe set of the present invention, the method for nucleic acid detection using the primer and probe set of the present invention, and the kit containing the primer and probe set of the present invention have promising applications in detecting one or more nucleic acids for purposes other than disease diagnosis and treatment. Attached Figure Description

[0210] Figure 1 This is a diagram of the stem-loop structure of the amplification product.

[0211] Figure 2 The melting curve is obtained according to the method in Example 1.

[0212] Figure 3 The melting curve is obtained according to the method in Example 2.

[0213] Figure 4 The melting curve was obtained according to the method in Example 3 (the nucleic acid template was EGFR exon 20 and NTC).

[0214] Figure 5 The melting curve was obtained according to the method in Example 3 (the nucleic acid template was EGFR exon 21 and NTC).

[0215] Figure 6 It is a melting curve obtained by the method of Comparative Example 1.

[0216] Figure 7 The melting curve was obtained using the method described in Comparative Example 2 (the nucleic acid template was EGFR exon 20 and NTC).

[0217] Figure 8 The melting curve was obtained using the method described in Comparative Example 2 (the nucleic acid template is EGFR exon 21 and NTC). Detailed Implementation

[0218] The following examples further illustrate the technology of the present invention. These examples are illustrative and exemplary of the present invention and do not limit the scope of the invention in any way.

[0219] In this paper, the term "sequentially containing" used in the definition of the first primer, the second primer, the probe, the preamplification product, etc., is only used to define the order between the two or more regions contained, and does not mean that there is no spacer sequence between two adjacent regions.

[0220] In this document, "pairing binding" or "specific binding" refers to the binding of two single-stranded nucleic acid molecules with complementary sequences under certain conditions (suitable temperature and ionic strength, etc.) through complementary base pairing. Those skilled in the art will know that two single-stranded nucleic acid molecules do not necessarily need to be perfectly complementary to bind specifically; specific binding can also occur even with a few base mismatches.

[0221] In this paper, the terms "first" and "second" are used only for distinction, and their sequence structures can be interchanged. For example, the first primer can be a forward primer, or the first primer can be a reverse primer.

[0222] In this article, "working concentration" refers to the concentration of each component in a single PCR reaction system for a test. For example, when preparing a PCR reaction system with a total volume of 25 μL, the concentration of the first primer (i.e., the working concentration) is 30–1000 nmol / L. Due to practical needs such as convenient transportation and operation, the actual concentration of each reagent component in the kit may be higher than its working concentration, or it may even be in lyophilized powder form. It is sufficient to prepare it to the corresponding working concentration for actual testing.

[0223] Example 1

[0224] A method for detecting the 16S gene in non-binding bacteria is as follows:

[0225] The probes and primers used in the detection method are shown in Table 1 below:

[0226] Table 1. Probes and primers used for detecting the 16S gene in non-binding bacteria.

[0227]

[0228] In Table 1 above, the first primer F1 (SEQ ID NO:2) is a specific primer designed for the target sequence of the non-binding bacillus 16S gene. F1 is 41 bp in length. The first to the 22nd bases at its 3' end are the target sequence binding region I; the 23rd to the 28th bases at its 3' end are the linker sequence L1; and the first to the 13th bases at its 5' end are the probe anchoring region A, which is inversely complementary to the sequence of the first to the 13th bases at the 5' end of the probe P1 (SEQ ID NO:1), i.e., the primer anchoring region A'.

[0229] In Table 1 above, the second primer R1 (SEQ ID NO:3) is a specific primer designed for the target sequence of the non-binding bacillus 16S gene. R1 is 40 bp in length. The first to the 23rd bases at its 3' end are the target sequence binding region II; the 24th to the 29th bases at its 3' end are the linker sequence L2; and the first to the 11th bases at its 5' end are the primer signal detection region h, which is the same as the first to the 11th bases at the 3' end of the probe P1 (SEQ ID NO:1), i.e., the probe signal detection region H.

[0230] The specific steps of the detection method are as follows:

[0231] (1) Prepare the test group reagents in the PCR tubes according to Table 2 below. Then, seal the PCR tubes and gently mix the reagents. Place the PCR tubes in a handheld centrifuge and centrifuge briefly. Then, let them stand at room temperature for 5 minutes. Place the PCR tubes in the handheld centrifuge again and centrifuge briefly. Then, transfer them to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. and amplify them according to the amplification program. Then, perform melting curve analysis on the amplified products according to the melting curve analysis program. The nucleic acid amplification program is: 95℃ pre-denaturation for 2 minutes; 94℃ denaturation for 10 seconds; 56℃ annealing extension for 30 seconds, for a total of 45 cycles. The melting curve analysis program is: heating from 40℃ to 95℃ to obtain the melting curve, with a heating rate of 0.05℃ / s.

[0232] (2) Prepare the control group reagents in the PCR tubes according to Table 2 below (without adding nucleic acid template, use 1X TE Buffer instead of nucleic acid template). Then seal the PCR tubes and gently mix the reagents. Place the PCR tubes in a handheld centrifuge and centrifuge briefly. Then let them stand at room temperature for 5 minutes. Place the PCR tubes in the handheld centrifuge again and centrifuge briefly. Then transfer them to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. and amplify them according to the amplification program. Then perform melting curve analysis on the amplified products according to the melting curve analysis program. The nucleic acid amplification program is: 95℃ pre-denaturation for 2 minutes; 94℃ denaturation for 10 seconds; 56℃ annealing extension for 30 seconds, for a total of 45 cycles. The melting curve analysis program is: heat from 55℃ to 90℃ to perform melting curve analysis, with a heating rate of 0.05℃ / s.

[0233] Table 2. Reagent components and concentrations used for detecting the 16S gene in non-binding bacteria.

[0234] reagent components concentration 2×PCR Reaction Buffer 1× DNA Polymerase 5U probe P1 400nM First primer F1 500nM Second primer R1 100nM Nucleic acid template 50ng Ultrapure water Add to 25 μL

[0235] In Table 2, the nucleic acid template is the non-binding bacillus 16S gene plasmid, synthesized by General Biotechnology (Anhui) Co., Ltd. 2×PCR Reaction Buffer is a buffer solution used for PCR reactions, including 3mM MgCl2, 30mM Tris-HCl at pH 8.3, 0.5mM dNTP, and 70mM (NH4)2SO4; "2×" indicates that the concentration of this buffer is twice the final concentration required for the reaction, meaning that only half the volume needs to be added when preparing the PCR reaction system.

[0236] The melting curve obtained by melting curve analysis is as follows: Figure 2 As shown, from Figure 2 It can be seen that the positive reaction wells containing the non-binding bacterial 16S gene plasmid formed a melting peak with a Tm value of 81.85℃ in the CY5 channel (Tm = 81.58℃, melting peak -1); while the control reaction wells without nucleic acid template did not show a melting peak (NTC-1) in the CY5 channel, indicating that there was no target in the control reaction wells. This demonstrates that the primer and probe design method used in this embodiment will not produce false positive signals that affect the judgment and quantification of target results during nucleic acid detection.

[0237] Example 2

[0238] A method for detecting the human papillomavirus (HPV) type 16 gene is as follows:

[0239] The probes and primers used in the detection method are shown in Table 3 below:

[0240] Table 3. Probes and primers used to detect human papillomavirus (HPV) type 16 gene.

[0241]

[0242]

[0243] In Table 3 above, the first primer F2 (SEQ ID NO:5) is a specific primer designed for the target sequence of human papillomavirus (HPV) type 16. F2 is 42 bp in length. The 1st to 19th bases at its 3' end are the target sequence binding region I. The 1st to 15th bases at its 5' end are the probe anchoring region A, which is inversely complementary to the 1st to 15th bases at the 5' end of the probe P2 (SEQ ID NO:4), i.e., the primer anchoring region A'. The 16th to 23rd bases at its 5' end are the linker sequence L1.

[0244] In Table 3 above, the second primer R2 (SEQ ID NO: 6) is a specific primer designed for the target sequence of human papillomavirus HPV16. R2 is 34 bp in length. The first to the 19th bases at its 3' end are the target sequence binding region II. The first to the 12th bases at its 5' end are the primer signal detection region h, which is the same as the first to the 12th bases at the 3' end of the probe P2 (SEQ ID NO: 4), i.e., the probe signal detection region H. The 5th to the 12th bases at its 5' end are the linker sequence L2.

[0245] The specific steps of the detection method are as follows:

[0246] Prepare reagents in the PCR tubes according to Table 4 below. After sealing the PCR tubes, gently mix the reagents and place them in a handheld centrifuge. After a brief centrifugation, allow them to stand at room temperature for 5 minutes. Place the PCR tubes in the handheld centrifuge again and centrifuge briefly. Transfer them to the tray of the SLAN real-time quantitative PCR instrument from Shanghai Hongshi Medical Technology Co., Ltd., and perform amplification according to the amplification program. Then, perform melting curve analysis on the amplified products according to the melting curve analysis program. The nucleic acid amplification program is as follows: 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 10 seconds; 56℃ annealing extension for 30 seconds, for a total of 45 cycles. The melting curve analysis program is as follows: heat from 55℃ to 90℃ to generate the melting curve, with a heating rate of 0.05℃ / s.

[0247] Table 4. Reagent components and concentrations used for detecting human papillomavirus (HPV) type 16 gene.

[0248] reagent components concentration 2×PCR Reaction Buffer 1× DNA Polymerase 3U probe P2 400nM First primer F2 500nM Second primer R2 100nM Nucleic acid template 50ng Ultrapure water Add to 25 μL

[0249] In Table 4, the nucleic acid template is the human papillomavirus (HPV) 16 plasmid, synthesized by General Biotechnology (Anhui) Co., Ltd. 2×PCR Reaction Buffer is a buffer solution used for PCR reactions, including 3mM MgCl2, 30mM Tris-HCl at pH 8.3, 0.5mM dNTP, and 70mM (NH4)2SO4; "2×" indicates that the concentration of this buffer is twice the final concentration required for the reaction, meaning that only half the volume needs to be added when preparing the PCR reaction system.

[0250] The melting curve obtained by melting curve analysis is as follows: Figure 3 As shown, from Figure 3 It can be seen that the positive reaction wells containing human papillomavirus (HPV) type 16 plasmid formed a melting peak with a Tm value of 75.05℃ in the FAM channel (Tm=75.05℃, melting peak-2).

[0251] Example 3

[0252] A method for detecting the H773_V774insH gene in exon 20 and / or the L861Q gene in exon 21 of EGFR in non-small cell lung cancer is as follows:

[0253] The probes and primers used in the detection method are shown in Table 5 below:

[0254] Table 5. Probes and primers used to detect the EGFR exon 20 H773_V774insH gene and / or exon 21 L861Q gene in non-small cell lung cancer.

[0255]

[0256] In Table 5 above, the forward primer F3 (SEQ ID NO:8) is a specific primer designed for the target sequence H773_V774insH of EGFR exon 20 in non-small cell lung cancer. F3 is 41 bp in length; the first to the 21st bases at its 3' end are the target sequence binding region I; the 22nd to the 27th bases at its 3' end are the spacer sequence L1; and the first to the 14th bases at its 5' end are the probe anchoring region A, which is inversely complementary to the first to the 14th bases at the 5' end of the probe P3 (SEQ ID NO:7), i.e., the primer anchoring region A'.

[0257] In Table 5 above, the reverse primer R3 (SEQ ID NO:9) is a specific primer designed for the target sequence H773_V774insH of EGFR exon 20 in non-small cell lung cancer. R3 is 33 bp in length; the 1st to 16th bases at its 3' end are the target sequence binding region II; the 17th to 22nd bases at its 3' end are the spacer sequence L2; and the 1st to 11th bases at its 5' end are the primer signal detection region h, which is the same as the 1st to 11th bases at the 3' end of the probe P3 (SEQ ID NO:7), i.e., part of the probe signal detection region H.

[0258] In Table 5 above, the forward primer F4 (SEQ ID NO:10) is a specific primer designed for the target sequence L861Q of EGFR exon 21 in non-small cell lung cancer. F3 is 42 bp in length; its 3' end, the first to the 22nd bases, is the target sequence binding region I; its 3' end, the 23rd to the 28th bases, is the spacer sequence L1; its 5' end, the first to the 14th bases, is the probe anchoring region A, which is inversely complementary to the 5' end, the first to the 14th bases, of probe P3 (SEQ ID NO:7), i.e., the primer anchoring region A'.

[0259] In Table 5 above, the reverse primer R4 (SEQ ID NO:11) is a specific primer designed for the target sequence L861Q of EGFR exon 21 in non-small cell lung cancer. R3 is 39 bp in length; the 1st to 19th bases at its 3' end are the target sequence binding region II; the 20th to 25th bases at its 3' end are the spacer sequence L2; and the 1st to 14th bases at its 5' end are the primer signal detection region h, which is the same as the 15th to 28th bases at the 3' end of probe P3 (SEQ ID NO:7), i.e., part of the probe signal detection region H.

[0260] The specific steps of the detection method are as follows:

[0261] (1) Prepare the test group reagents in the PCR tubes according to Table 6 below. Then, seal the PCR tubes and gently mix the reagents. Place the PCR tubes in a handheld centrifuge and centrifuge briefly. Then, let them stand at room temperature for 5 minutes. Place the PCR tubes in the handheld centrifuge again and centrifuge briefly. Then, transfer them to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. and amplify them according to the amplification program. Then, perform melting curve analysis on the amplified products according to the melting curve analysis program. The nucleic acid amplification program is as follows: 95℃ pre-denaturation for 2 minutes; 94℃ denaturation for 10 seconds; 56℃ annealing extension for 30 seconds, for a total of 45 cycles. The melting curve analysis program is as follows: heat from 55℃ to 90℃ to perform melting curve analysis, with a heating rate of 0.05℃ / s.

[0262] (2) Prepare the control group reagents in the PCR tubes according to Table 6 below without adding nucleic acid template (use 1X TE Buffer instead of nucleic acid template). Then seal the PCR tubes and gently mix the reagents. Place the PCR tubes in a handheld centrifuge and centrifuge briefly. Then let them stand at room temperature for 5 minutes. Place the PCR tubes in the handheld centrifuge again and centrifuge briefly. Then transfer them to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. and perform amplification according to the amplification program. Then perform melting curve analysis according to the melting curve analysis program. The nucleic acid amplification program is: 95℃ pre-denaturation for 2 minutes; 94℃ denaturation for 10 seconds; 56℃ annealing extension for 30 seconds, for a total of 45 cycles. The melting curve analysis program is: heat from 55℃ to 90℃ to perform melting curve analysis, with a heating rate of 0.05℃ / s.

[0263] Table 6. Reagent components and concentrations for detecting the H773_V774insH gene in exon 20 and / or the L861Q gene in exon 21 of non-small cell lung cancer EGFR.

[0264] reagent components concentration 2×PCR Reaction Buffer 1× DNA Polymerase 3U probe P3 400nM First primer F3 500nM Second primer R3 100nM First primer F4 500nM Second primer R4 100nM Nucleic acid template 50ng Ultrapure water Add to 25 μL

[0265] In Table 6, 2×PCR Reaction Buffer is a buffer solution used for PCR reactions, including 3mM MgCl2, 30mM Tris-HCl at pH 8.3, 0.5mM dNTP, 70mM (NH4)2SO4, etc.; "2×" indicates that the concentration of this buffer solution is twice the concentration required for the final reaction, that is, only half the volume needs to be added when preparing the PCR reaction system.

[0266] When the nucleic acid template in Table 6 is the EGFR exon 20 H773_V774insH plasmid for non-small cell lung cancer synthesized by General Biotech (Anhui) Co., Ltd., the melting curves obtained after amplification of the experimental group reagent containing the nucleic acid template and the control group reagent without the nucleic acid template are as follows: Figure 4 As shown. From Figure 4 It can be seen that the positive reaction wells containing the EGFR exon 20 H773_V774insH plasmid for non-small cell lung cancer formed a melting peak with a Tm value of 80.50℃ in the CY5 channel (Tm=80.50℃, melting peak-3); while the control reaction wells without nucleic acid template did not show a melting peak (NTC-3) in the CY5 channel, reflecting that there was no target in the control reaction wells.

[0267] When the nucleic acid template in Table 6 is the L861Q plasmid of EGFR exon 21 for non-small cell lung cancer synthesized by General Biotech (Anhui) Co., Ltd., the melting curves obtained after amplification of the experimental group reagent containing the nucleic acid template and the control group reagent without the nucleic acid template are as follows: Figure 5 As shown. From Figure 5 It can be seen that the positive reaction wells containing the EGFR exon 21 L861Q plasmid for non-small cell lung cancer formed a melting peak (melting peak-4) with a Tm value of 72.75℃ in the CY5 channel; while the control reaction wells without nucleic acid template did not show a melting peak (NTC-4) in the CY5 channel, indicating that there was no target in the control reaction wells.

[0268] Combination Figure 4 and Figure 5 As can be seen, when using the primers and probes of this invention for single-tube multiplex detection, no melting peak appears in the template-free control wells, and no false positive signals are generated that affect the judgment and quantification of target results. That is, the primer and probe set design method of this invention can reduce the interference of primer dimers and / or other non-specific products on the detection results.

[0269] Furthermore, the primer signal detection regions of the two primer pairs (F3 and R3 are one primer pair; F4 and R4 are one primer pair) are different (R3 is CAGACGAAGCG; R4 is GTAAGACCCACAAG), resulting in different specific binding sites of the single-stranded amplification products formed by the two pairs with the same probe P3, thus causing a significant difference in the Tm value of the melting peak. Figure 4 It is 80.50℃; Figure 5 (72.75℃), that is, by using the primer and probe design method of the present invention, different targets can be clearly distinguished by melting curve when performing various nucleic acid detections.

[0270] Comparative Example 1

[0271] A method for detecting the 16S gene in non-binding bacteria is as follows:

[0272] The probes and primers used in the detection method are shown in Table 7 below:

[0273] Table 7. Probes and primers used for detecting the 16S gene in non-binding bacteria.

[0274]

[0275] In Table 7 above, the first primer F5 (SEQ ID NO:7) is a specific primer designed for the target sequence of the non-binding bacillus 16S gene. F1 is 35 bp in length; its 3' end, from the 1st to the 22nd base, is the target sequence binding region I; its 5' end, from the 1st to the 13th base, is the probe anchoring region A, which is inversely complementary to the 5' end sequence of the probe P1 (SEQ ID NO:1), i.e., the primer anchoring region A'. The first primer F3 (SEQ ID NO:7) is equivalent to the first primer F1 (SEQ ID NO:2) without its linker sequence L1.

[0276] In Table 7 above, the second primer R5 (SEQ ID NO:8) is a specific primer designed for the target sequence of the non-binding bacillus 16S gene. R1 is 34 bp in length, with the first to 23rd bases at its 3' end forming the target sequence binding region II; the first to 11th bases at its 5' end form the primer signal detection region h, which is identical to the first to 11th bases at the 3' end of the probe P1 (SEQ ID NO:1), i.e., the probe signal detection region H. The second primer R3 (SEQ ID NO:8) is equivalent to the second primer R1 (SEQ ID NO:3) without its linker sequence L2.

[0277] The specific steps of the detection method are as follows:

[0278] (1) Prepare the test group reagents in the PCR tubes according to Table 8 below. Then, seal the PCR tubes and gently mix the reagents. Place the PCR tubes in a handheld centrifuge and centrifuge briefly. Then, let them stand at room temperature for 5 minutes. Place the PCR tubes in the handheld centrifuge again and centrifuge briefly. Then, transfer them to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. and amplify them according to the amplification program. Then, perform melting curve analysis on the amplified products according to the melting curve analysis program. The nucleic acid amplification program is: 95℃ pre-denaturation for 2 minutes; 94℃ denaturation for 10 seconds; 56℃ annealing extension for 30 seconds, for a total of 45 cycles. The melting curve analysis program is: heat from 55℃ to 90℃ to perform melting curve analysis at a rate of 0.05℃ / s.

[0279] (2) Prepare the control group reagents in the PCR tubes according to Table 8 below without adding nucleic acid template (use 1X TE Buffer instead of nucleic acid template). Then seal the PCR tubes and gently mix the reagents. Place the PCR tubes in a handheld centrifuge and centrifuge briefly. Then let them stand at room temperature for 5 minutes. Place the PCR tubes in the handheld centrifuge again and centrifuge briefly. Then transfer them to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. and perform amplification according to the amplification program. Then perform melting curve analysis according to the melting curve analysis program. The nucleic acid amplification program is: 95℃ pre-denaturation for 2 minutes; 94℃ denaturation for 10 seconds; 56℃ annealing extension for 30 seconds, for a total of 45 cycles. The melting curve analysis program is: heat from 55℃ to 90℃ to perform melting curve analysis at a heating rate of 0.05℃ / s.

[0280] The melting curve obtained by melting curve analysis is as follows: Figure 6 As shown, from Figure 6 It can be seen that the positive reaction wells containing the non-binding bacterial 16S gene plasmid formed a melting peak with a Tm value of 81.54℃ in the CY5 channel (Tm = 81.54℃, melting peak -5); while the control reaction wells without nucleic acid template also formed a melting peak with a Tm value of 81.79℃ in the CY5 channel (Tm = 81.79℃, NTC -5), and its Tm value (81.79℃) is close to the Tm value of the target (81.54℃). This indicates that when using the combination of the first primer F5 and the second primer R5 with the probe P1 for nucleic acid detection, non-specific amplification products (e.g., primer dimers) appeared in the detection reagent, and these non-specific amplification products also caused the probe to produce false positive signals that affected target identification and quantification.

[0281] Table 8. Reagent components and concentrations used for detecting the 16S gene in non-conjugating bacteria.

[0282] reagent components concentration 2×PCR Reaction Buffer 1× DNA Polymerase 5U probe P1 400nM First primer F5 500nM Second primer R5 100nM Nucleic acid template 50ng Ultrapure water Add to 25 μL

[0283] Comparative Example 2

[0284] A method for detecting EGFR exon 20 H773_V774insH and / or exon 21 L861Q in non-small cell lung cancer is as follows:

[0285] The probes and primers used in the detection method are shown in Table 9 below:

[0286] Table 9. Probes and primers for detecting EGFR exon 20 H773_V774insH and / or exon 21 L861Q in non-small cell lung cancer.

[0287]

[0288] In Table 9 above, the forward primer F6 (SEQ ID NO:12) is a specific primer designed for the target sequence of EGFR exon 20 H773_V774insH in non-small cell lung cancer. F3 is 39 bp in length; the first to the 21st bases at its 3' end are the target sequence binding region I; the first to the 14th bases at its 5' end are the probe anchoring region A, which is inversely complementary to the sequence of the first to the 14th bases at the 5' end of the probe P3 (SEQ ID NO:7), i.e., the primer anchoring region A'.

[0289] In Table 9 above, the reverse primer R6 (SEQ ID NO:13) is a specific primer designed for the target sequence H773_V774insH of EGFR exon 20 in non-small cell lung cancer. R3 is 31 bp in length; the first to the 16th bases at its 3' end are the target sequence binding region II; the first to the 11th bases at its 5' end are the primer signal detection region h, which is the same as the sequence of the first to the 11th bases at the 3' end of the probe P3 (SEQ ID NO:7), that is, part of the probe signal detection region H.

[0290] In Table 9 above, the forward primer F7 (SEQ ID NO:14) is a specific primer designed for the target sequence L861Q of EGFR exon 21 in non-small cell lung cancer. F4 is 39 bp in length; the first to the 22nd bases at its 3' end are the target sequence binding region I; the first to the 14th bases at its 5' end are the probe anchoring region A, which is inversely complementary to the sequence of the first to the 14th bases at the 5' end of the probe P3 (SEQ ID NO:7), i.e., the primer anchoring region A'.

[0291] In Table 9 above, the reverse primer R7 (SEQ ID NO:15) is a specific primer designed for the target sequence L861Q of EGFR exon 21 in non-small cell lung cancer. R4 is 37 bp in length; the 1st to 19th bases at its 3' end are the target sequence binding region II; the 1st to 14th bases at its 5' end are the primer signal detection region h, which is the same as the 15th to 28th bases at the 3' end of the probe P3 (SEQ ID NO:7), i.e., part of the probe signal detection region H.

[0292] The specific steps of the detection method are as follows:

[0293] (1) Prepare the test group reagents in the PCR tubes according to Table 10 below. Then, seal the PCR tubes and gently mix the reagents. Place the PCR tubes in a handheld centrifuge and centrifuge briefly. Then, let them stand at room temperature for 5 minutes. Place the PCR tubes in the handheld centrifuge again and centrifuge briefly. Then, transfer them to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. and perform amplification according to the amplification program. Then, perform melting curve analysis according to the melting curve analysis program. The nucleic acid amplification program is: 95℃ pre-denaturation for 2 minutes; 94℃ denaturation for 10 seconds; 56℃ annealing extension for 30 seconds, for a total of 45 cycles. The melting curve analysis program is: heat from 55℃ to 90℃ to perform melting curve analysis at a heating rate of 0.05℃ / s.

[0294] (2) Prepare the control group reagents in the PCR tubes according to Table 10 below without adding nucleic acid template (use 1X TE Buffer instead of nucleic acid template). Then seal the PCR tubes and gently mix the reagents. Place the PCR tubes in a handheld centrifuge and centrifuge briefly. Then let them stand at room temperature for 5 minutes. Place the PCR tubes in the handheld centrifuge again and centrifuge briefly. Then transfer them to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. and amplify them according to the amplification program. Then perform melting curve analysis on the amplified products according to the melting curve analysis program. The nucleic acid amplification program is: 95℃ pre-denaturation for 2 minutes; 94℃ denaturation for 10 seconds; 56℃ annealing extension for 30 seconds, for a total of 45 cycles. The melting curve analysis program is: heat from 55℃ to 90℃ to perform melting curve analysis, with a heating rate of 0.05℃ / s.

[0295] Table 10. Reagent components and concentrations for detecting exon 20 H773_V774insH and / or exon 21 L861Q in non-small cell lung cancer EGFR.

[0296]

[0297]

[0298] In Table 10, 2×PCR Reaction Buffer is a buffer solution used for PCR reactions, including 3mM MgCl2, 30mM Tris-HCl at pH 8.3, 0.5mM dNTP, 70mM (NH4)2SO4, etc. The "2×" indicates that the concentration of this buffer solution is twice the concentration required for the final reaction, that is, only half the volume needs to be added when preparing the PCR reaction system.

[0299] When the nucleic acid template in Table 10 is the EGFR exon 20 H773_V774insH plasmid for non-small cell lung cancer synthesized by General Biotech (Anhui) Co., Ltd., the melting curves obtained after amplification of the experimental group reagent containing the nucleic acid template and the control group reagent without the nucleic acid template are as follows: Figure 7 As shown. From Figure 7 It can be seen that the positive reaction wells containing the EGFR exon 20 H773_V774insH plasmid for non-small cell lung cancer formed a melting peak with a Tm value of 80.52℃ in the CY5 channel (Tm=80.52℃, melting peak-6); while the control reaction wells without nucleic acid template showed a melting peak in the CY5 channel (Tm=72.51℃, NTC-6), indicating that false positive signals appeared in the control reaction wells without the target.

[0300] When the nucleic acid template in Table 10 is the L861Q plasmid of EGFR exon 21 for non-small cell lung cancer synthesized by General Biotech (Anhui) Co., Ltd., the melting curves obtained after amplification of the experimental group reagent containing the nucleic acid template and the control group reagent without the nucleic acid template are as follows: Figure 8 As shown. From Figure 8 It can be seen that: in the positive reaction wells containing the EGFR exon 21 L861Q plasmid for non-small cell lung cancer, a melting peak with a Tm value of 72.76℃ (Tm=72.76℃, melting peak-7) was formed in the CY5 channel; however, in the control reaction wells without nucleic acid template, a melting peak also appeared in the CY5 channel (Tm=72.51℃, NTC-7), and its Tm value (72.51℃) was similar to that of the target (EGFR exon 21 L861Q) (72.76℃). The resulting false positive signal could easily interfere with the interpretation and quantification of the target.

[0301] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications having the same function.

Claims

1. A primer-probe set for detecting nucleic acids, characterized in that, It includes a first primer, a second primer, and a probe; among which, The probe is a freely designed sequence that does not pair with any target sequence. It includes a primer anchoring region (A') and a probe signal detection region (H). The primer anchoring region (A') is located at the 5' end of the probe signal detection region (H). The probe is modified with a detection marker. The first primer comprises, from its 5' end to its 3' end, a probe anchoring region (A), a linker sequence (L1), and a target sequence binding region (Ⅰ); wherein, the probe anchoring region (A) specifically binds to the primer anchoring region (A') of the probe; the linker sequence (L1) is a freely designed sequence that does not pair with any target sequence; and the target sequence binding region (Ⅰ) is a sequence that can specifically bind to the target. The second primer comprises, from its 5' end to its 3' end, a primer signal detection region (h), a linker sequence (L2), and a target sequence binding region (II); wherein, the complementary sequence (h') of the primer signal detection region (h) specifically binds to the probe signal detection region (H) of the probe; part or all of the complementary sequence (L2') of the linker sequence (L2) specifically binds to part or all of the linker sequence (L1); and the target sequence binding region (II) is a sequence capable of specifically binding to a target.

2. A primer-probe set for detecting nucleic acids, characterized in that, It includes one probe, at least one first primer, and at least two second primers; in The probe is a freely designed sequence that does not pair with any target sequence. It includes a primer anchoring region (A') and a probe signal detection region (H). The primer anchoring region (A') is located at the 5' end of the probe signal detection region (H). The probe is modified with a detection marker. The first primer comprises, from its 5' end to its 3' end, a probe anchoring region (A), a linker sequence (L1), and a target sequence binding region (Ⅰ); wherein, the probe anchoring region (A) specifically binds to the primer anchoring region (A') of the probe; the linker sequence (L1) is a freely designed sequence that does not pair with any target sequence; and the target sequence binding region (Ⅰ) is a sequence that can specifically bind to the target. The at least two second primers are different from each other, each independently comprising, from the 5' end to the 3' end, a primer signal detection region (h), a linker sequence (L2), and a target sequence binding region (II); wherein, the complementary sequence (h') of the primer signal detection region (h) specifically binds to a portion of the probe signal detection region (H) of the probe, and the sequences of the primer signal detection regions (h) of different second primers are different from each other; a portion or all of the complementary sequence (L2') of the linker sequence (L2) specifically binds to a portion or all of the linker sequence (L1), and the sequences of the linker sequences (L2) of different second primers are the same; the target sequence binding region (II) is a sequence capable of specifically binding to a target, and the target sequence binding region (II) of different second primers specifically binds to different targets.

3. A primer-probe set for detecting nucleic acids, characterized in that, It includes one probe, at least two first primers, and at least two second primers; in The probe is a freely designed sequence that does not pair with any target sequence. It includes a primer anchoring region (A') and a probe signal detection region (H). The primer anchoring region (A') is located at the 5' end of the probe signal detection region (H). The probe is modified with a detection marker. The at least two first primers are different from each other, each independently comprising, from its 5' end to its 3' end, a probe anchoring region (A), a linker sequence (L1), and a target sequence binding region (Ⅰ); wherein, the probe anchoring region (A) specifically binds to a portion of the primer anchoring region (A') of the probe; the linker sequence (L1) is a freely designed sequence that does not pair with any target sequence, and the linker sequences (L1) of different first primers are identical; the target sequence binding region (Ⅰ) is a sequence capable of specifically binding to a target, and the target sequence binding regions (Ⅰ) of different first primers specifically bind to different targets; The at least two second primers are different from each other, each independently comprising, from the 5' end to the 3' end, a primer signal detection region (h), a linker sequence (L2), and a target sequence binding region (II); wherein, the complementary sequence (h') of the primer signal detection region (h) specifically binds to a portion of the probe signal detection region (H) of the probe, and the sequences of the primer signal detection regions (h) of different second primers are different from each other; a portion or all of the complementary sequence (L2') of the linker sequence (L2) specifically binds to a portion or all of the linker sequence (L1), and the sequences of the linker sequences (L2) of different second primers are the same; the target sequence binding region (II) is a sequence capable of specifically binding to a target, and the target sequence binding region (II) of different second primers specifically binds to different targets.

4. The primer-probe set according to any one of claims 1 to 3, characterized in that, The first primer has a 0-20 base spacer between its linker sequence (L1) and the target sequence binding region (I), preferably a 0-10 base spacer; and / or The linker sequence (L2) of the second primer has a spacer of 0 to 20 bases between it and the target sequence binding region (II), preferably 0 to 10 bases; and / or The ligation sequence (L2) of the second primer is part of the primer signal detection region (h), or the ligation sequence (L2) of the second primer is a sequence independent of the primer signal detection region (h); and / or The complementary sequence (L2') of the linker sequence (L2) has at least three consecutive pairs of specifically binding base sequences with the linker sequence (L1); preferably, the complementary sequence (L2') of the linker sequence (L2) has four to twenty consecutive pairs of specifically binding base sequences with the linker sequence (L1); more preferably, the complementary pair (L2') of the linker sequence (L2) can bind completely specifically to the linker sequence (L1); and / or The linker sequence (L2) has at least three consecutive identical bases to the linker sequence (L1); preferably, the linker sequence (L2) has four to twenty consecutive identical bases to the linker sequence (L1); more preferably, the linker sequence (L2) is completely identical to the linker sequence (L1); and / or The probe is 20–100 bases long; preferably, the primer anchoring region (A') is 6–35 bases long, and the probe signal detection region (H) is 5–65 bases long; and / or The first primer is 15–80 bases in length; preferably, the linker sequence (L1) is 3–20 bases in length, and the probe anchoring region (A) is 6–35 bases in length; and / or The second primer is 15 to 80 bases in length; preferably, the primer signal detection region (h) is 5 to 65 bases in length, and the linker sequence (L2) is 3 to 20 bases in length.

5. The primer-probe set according to any one of claims 1 to 4, characterized in that, The detection marker includes a first detection group and a second detection group, and the first detection group and the second detection group generate signal changes through changes in distance; more preferably, At least one detection group is modified at the 5' end of the probe signal detection region (H); preferably, both detection groups are modified at the 5' end of the probe signal detection region (H); and / or The first detection group and the second detection group are spaced 3 to 140 angstroms apart; and / or The first detection group is a fluorescent reporter group, and the second detection group is a quencher group or other modifying group that can generate a signal change with the first detection group through fluorescence resonance energy transfer.

6. A method for detecting nucleic acids, characterized in that, The method includes the following steps: (1) The primer and probe set is mixed with the amplification reagent and the sample to be tested; the primer and probe set is selected from at least one set of primer and probe sets according to any one of claims 1 to 5; (2) Perform nucleic acid amplification on the mixture obtained in step (1), and determine whether there is a target in the sample to be tested based on the changes in the fluorescence signal generated by the amplification. Optionally (3) performs melting curve analysis on the amplification product obtained in step (2) and determines whether there is a target in the sample to be tested based on the analysis results; Preferably, in the mixture obtained in step (1), in each primer-probe group, the concentration of the first primer is 30 nM to 1000 nM, the concentration of the second primer is 30 nM to 500 nM, and the concentration of the probe is 30 nM to 1200 nM; more preferably, the concentration of the first primer is greater than the concentration of the second primer; even more preferably, the concentration of the first primer is 2 to 10 times the concentration of the second primer.

7. The method according to claim 6, characterized in that, The amplification reagents in step (1) include DNA polymerase and dNTPs; and / or Preferably, the nucleic acid amplification conditions are: pre-denaturation at 85℃~105℃ for 0min~15min; denaturation at 85℃~105℃ for 1s~60s, annealing and extension at 40℃~75℃ for 3s~90s, 20~60 cycles; and / or Preferably, the melting curve analysis of the amplification product obtained in step (2) includes heating the amplification product between 35°C and 95°C to obtain a melting curve, with a heating rate of 0.05°C / s; and / or In step (2), before nucleic acid amplification of the mixture, the mixture is further divided into at least 2000 reaction units, each of which contains one target of the sample to be tested or does not contain a target of the sample to be tested.

8. The method according to claim 6 or 7, characterized in that, When the target is present in the sample to be tested, the amplification product obtained in step (2) is a secondary amplification double-stranded product formed by the specific binding and extension of the probe and the pre-amplification product. The pre-amplification product is generated by the specific binding and amplification of the target by the first primer and the second primer, respectively. Preferably, the pre-amplification product is a single-stranded nucleotide chain containing the probe anchoring region (A), the linker sequence (L1), the reverse complementary sequence (L2') of the linker sequence (L2), and the reverse complementary sequence (h') of the primer signal detection region (h); and / or Step (3) determines whether a target exists in the sample to be tested based on the analysis results. This includes determining whether the sample to be tested contains a target if the obtained melting curve contains a characteristic peak and the temperature corresponding to the characteristic peak is the characteristic Tm of the target in a specific detection channel in the sample to be tested. Preferably, the specific detection channel is the fluorescence channel corresponding to the detection group on the probe, and the characteristic Tm is the melting temperature of the secondary amplified double-stranded product.

9. A reagent kit for detecting nucleic acids, characterized in that, It includes a primer-probe set, wherein the primer-probe set is selected from at least one set of primer-probe sets according to any one of claims 1 to 5; Preferably, in the kit, in each primer-probe set, the working concentration of the first primer is greater than the working concentration of the second primer; more preferably, the working concentration of the first primer is 2 to 10 times the working concentration of the second primer; and / or Preferably, the kit further includes amplification reagents; more preferably, the amplification reagents include DNA polymerase and dNTPs; even more preferably, the amplification reagents further include reverse transcriptase.

10. The use of the primer and probe set according to any one of claims 1 to 5, the method according to any one of claims 6 to 8, and the kit according to claim 9 in detecting one or more nucleic acids for non-disease diagnosis and treatment purposes.

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