Primer probe for nucleic acid detection and application thereof

Abstract

The invention relates to a primer probe for nucleic acid detection and application thereof. The primer probe disclosed by the invention can be used for simultaneously analyzing two dimensions of the fluorescence channel and the melting temperature in a single-tube reaction, that is, different targets can be detected by using the same fluorescence channel through the melting temperature characteristic; or different fluorescent channels are used, so that the target object type detection of the product of the number of the fluorescent channels and the melting temperature characteristic can be realized. The primer probe is used for nucleic acid detection, and has the advantages of low fluorescence background, high fluorescence intensity, adjustable melting temperature, good inclusiveness, low cost, single-tube reaction, simplicity and convenience in operation, difficulty in causing pollution, high sensitivity, wide application range and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer probe for nucleic acid detection and an application thereof. Background Art

[0002] Nucleic acid detection technology is a frequently used technique for analyzing mutations and polymorphisms in nucleic acid samples. Detection and typing of bacteria, viruses, and fungi (including infectious pathogens) is also achieved by detecting and analyzing their DNA or RNA.

[0003] In order to detect multiple target nucleic acids, that is, to achieve multiple or even super multiple nucleic acid detection, the prior art usually combines PCR detection technology with other methods, such as PCR combined with hybridization chips, and multiple target detection is performed with different amplification product lengths. As disclosed in the document CN107090519A, a method for a multiple RT-PCR combined gene chip detection kit for common respiratory pathogens utilizes the principle of nucleic acid molecule hybridization, and the single-stranded probes for each target are arranged and fixed on the gene chip in a specific order to form a probe array, and then the multiple PCR products to be tested are hybridized with them, so that the target amplification products are hybridized with the probes on the chip and emit fluorescent signals. Although this method can detect 20 respiratory pathogens at the same time, the gene chip preparation process is complicated and expensive, which is not conducive to clinical promotion and development, and the process is cumbersome, and multiple steps of opening and cleaning are required, which is easy to cause contamination and result in false positives.

[0004] Another example is a kit and a detection method for simultaneously detecting thirty diarrhea pathogens disclosed in document CN103074450B, which utilizes a combination of PCR amplification and capillary electrophoresis to extract the PCR amplification product and perform capillary electrophoresis analysis, and compare the obtained spectrum with the standard spectrum to determine the type of diarrhea pathogens. In addition to using PCR amplification detection equipment, this method also requires the use of capillary electrophoresis equipment for product analysis, which greatly increases the detection cost and is not conducive to clinical promotion and use.

[0005] In order to realize multiple nucleic acid detection, it is necessary to add multiple specific primers to the same reaction system, but this often results in weak positive signals, resulting in insufficient detection sensitivity.

[0006] Therefore, there is an urgent need for a method that can perform multiple molecular tests, which can quickly, easily and cost-effectively detect a variety of common clinical targets such as bacteria, viruses, gene mutations, etc., and provide stronger specific signals to achieve more sensitive nucleic acid detection. Summary of the invention

[0007] In view of the deficiencies of nucleic acid detection in the prior art, the present invention provides a primer probe for nucleic acid detection, which can significantly increase the intensity of positive signals, thereby improving the sensitivity of nucleic acid detection.

[0008] To this end, the first aspect of the present invention provides a primer probe for nucleic acid detection, comprising a first primer, a probe (P) and a second primer, wherein:

[0009] The first primer comprises a target sequence binding region 1; the target sequence binding region 1 is a sequence that can specifically pair and bind with the target sequence;

[0010] The probe (P) is a sequence that does not pair with any target sequence, and includes a probe signal detection region (H);

[0011] The second primer comprises an anti-primer signal detection region (h'), an amplification blocking region, a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 is a sequence that can specifically pair and bind to the target sequence, and the primer signal detection region (h) is a sequence that does not pair and bind to any target sequence; the primer signal detection region (h) is complementary to the anti-primer signal detection region (h'), and the complementary sequence of the primer signal detection region (h) specifically pairs and binds to the probe signal detection region (H) of the probe (P); and the amplification blocking region can prevent polymerase extension.

[0012] In the above-mentioned primer probe, after the first primer and the second primer specifically bind to the target (target nucleic acid) respectively, a single-stranded pre-amplification product having a complementary sequence to the primer signal detection region (h) is generated (due to the presence of the amplification blocking region in the second primer, the single-stranded pre-amplification product has a complementary sequence to the primer signal detection region (h), but does not have a complementary sequence to the anti-primer information detection region (h')), the single-stranded pre-amplification product specifically binds to the probe (P) and is extended by ≥0 bases to form a double-stranded product, and the formation of the double-stranded product causes the probe to produce a detectable signal change.

[0013] In the present invention, the "specific pairing binding" or "specific binding" refers to the base complementary pairing binding of two single-stranded nucleic acid molecules with complementary sequences under certain conditions (suitable temperature and ionic strength, etc.). It is well known to those skilled in the art that two single-stranded nucleic acid molecules do not need to be completely complementary to each other in order to specifically bind, and can also specifically bind in the presence of a small number of base mismatches.

[0014] In the present invention, the complementary sequence of the primer signal detection region (h) in the single-stranded pre-amplification product specifically binds to the probe (P) at the 5' end of the probe (P), or other means of blocking extension are set at the specific binding position, then specifically binds and extends 0 bases to form a double-stranded product; that is, after specific binding, hybridization forms a double-stranded product, and the double-stranded product is a hybridized double-stranded product.

[0015] In the present invention, the complementary sequence of the primer signal detection region (h) in the single-stranded pre-amplification product specifically binds to the probe (P) at the non-5' end of the probe (P) and there is no other special means to block extension, then specifically binds and extends > 0 bases to form a double-stranded product, that is, after specific binding, extension forms a secondary amplification double-stranded product.

[0016] In the above-mentioned cases of extension>0 bases and extension of 0 bases, the formation of the secondary amplification double-stranded product and the hybridization double-stranded product both cause a change in the probe signal, thereby generating a detectable signal change.

[0017] In the present invention, the single-stranded pre-amplification product and the probe (P) are specifically bound mainly by the specific pairing of the complementary sequence of the primer signal detection region (h) and the probe signal detection region (H), and are extended in the presence of a polymerase.

[0018] The term "from 5' to 3' end" of the present invention does not mean that there is no base gap between regions. In certain embodiments, there is a 0-20 base gap between adjacent regions, for example, there is a 0-20 base gap between the anti-primer signal detection region (h') and the amplification blocking region.

[0019] In other embodiments of the present invention, the first primer further comprises a probe anchor region (A); wherein the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is located at the 5' end of the target sequence binding region 1; the probe (P) further comprises a primer anchor region (A'); the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer.

[0020] That is, in some embodiments of the present invention, a primer probe for nucleic acid detection comprises a first primer, a probe (P) and a second primer, wherein:

[0021] The first primer comprises a probe anchor region (A) and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the target sequence binding region 1 is a sequence that can specifically pair with the target sequence;

[0022] The probe (P) is a sequence that does not pair with any target sequence, and includes a probe signal detection region (H) and a primer anchor region (A'), wherein the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer;

[0023] The second primer comprises an anti-primer signal detection region (h'), an amplification blocking region, a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 is a sequence that can specifically pair and bind to the target sequence, and the primer signal detection region (h) is a sequence that does not pair and bind to any target sequence; the primer signal detection region (h) is complementary to the anti-primer signal detection region (h'), and the complementary sequence of the primer signal detection region (h) specifically pairs and binds to the probe signal detection region (H) of the probe (P); and the amplification blocking region can prevent polymerase extension.

[0024] In some embodiments of the present invention, the “primer anchor region (A′) is complementary to the probe anchor region (A)” means that the two can be forward complementary or reverse complementary, as long as they can be complementary to each other.

[0025] In the above-mentioned primer probe, after the first primer and the second primer specifically bind to the target (target nucleic acid) respectively, a single-stranded pre-amplification product having complementary sequences of the probe anchor region (A) and the primer signal detection region (h) is generated (due to the presence of the amplification blocking region, the single-stranded pre-amplification product has a complementary sequence of the primer signal detection region (h), but does not have a complementary sequence of the anti-primer information detection region (h')), the single-stranded pre-amplification product specifically binds to the probe (P) and is extended by ≥0 bases to form a double-stranded product, and the formation of the double-stranded product causes the probe to produce a detectable signal change.

[0026] In other embodiments of the present invention, the first primer further comprises an amplification retardation region, wherein the amplification retardation region is located between the probe anchor region (A) and the target sequence binding region 1, and the amplification retardation region can prevent polymerase extension.

[0027] That is, in some embodiments of the present invention, a primer probe for nucleic acid detection comprises a first primer, a probe (P) and a second primer, wherein:

[0028] The first primer comprises a probe anchor region (A), an amplification blocking region and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the amplification blocking region can prevent polymerase extension; the target sequence binding region 1 is a sequence that can specifically pair with the target sequence;

[0029] The probe (P) is a sequence that does not pair with any target sequence, and includes a probe signal detection region (H) and a primer anchor region (A'), wherein the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer;

[0030] The second primer comprises an anti-primer signal detection region (h'), an amplification blocking region, a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 is a sequence that can specifically pair and bind to the target sequence, and the primer signal detection region (h) is a sequence that does not pair and bind to any target sequence; the primer signal detection region (h) is complementary to the anti-primer signal detection region (h'), and the complementary sequence of the primer signal detection region (h) specifically pairs and binds to the probe signal detection region (H) of the probe (P); and the amplification blocking region can prevent polymerase extension.

[0031] In the above-mentioned primer probe, after the first primer and the second primer specifically bind to the target (target nucleic acid), two single-stranded pre-amplification products are generated: one is a single-stranded pre-amplification product (referred to as: competitive pre-amplification product) containing an anti-primer signal detection region (h') and a primer signal detection region (h). In the competitive pre-amplification product, due to the presence of the amplification retardation region in the first primer, it does not have a complementary sequence to the probe anchor region, and the competition relationship with the probe is weakened; the other is a single-stranded pre-amplification product (referred to as: detection pre-amplification product) having a complementary sequence to the probe anchor region (A) and the primer signal detection region (h). Due to the presence of the amplification retardation region in the second primer, the detection pre-amplification product has a complementary sequence to the primer signal detection region (h), but does not have a complementary sequence to the anti-primer information detection region (h'). After the detection pre-amplification product specifically binds to the probe (P) and extends ≥0 bases to form a double-stranded product, the formation of the double-stranded product causes the probe to produce a detectable signal change.

[0032] In other embodiments of the present invention, the first primer further comprises an anti-probe anchor region (a') and an amplification retardation region; the amplification retardation region is located between the anti-probe anchor region (a') and the probe anchor region (A); the anti-probe anchor region (a') is complementary to the probe anchor region (A), and the amplification retardation region can prevent polymerase extension.

[0033] In some embodiments of the present invention, the first primer comprises, from the 5' end to the 3' end, an anti-probe anchor region (a'), an amplification retardation region, a probe anchor region (A), and a target sequence binding region 1. In other embodiments, the first primer comprises, from the 5' end to the 3' end, a probe anchor region (A), an amplification retardation region, an anti-probe anchor region (a'), and a target sequence binding region 1.

[0034] That is, in some embodiments of the present invention, a primer probe for nucleic acid detection comprises a first primer, a probe (P) and a second primer, wherein:

[0035] The first primer comprises, from the 5' end to the 3' end, an anti-probe anchor region (a'), an amplification retardation region, a probe anchor region (A) and a target sequence binding region 1; or, the first primer comprises, from the 5' end to the 3' end, a probe anchor region (A), an amplification retardation region, an anti-probe anchor region (a') and a target sequence binding region 1; the amplification retardation region is located between the anti-probe anchor region (a') and the probe anchor region (A); the anti-probe anchor region (a') is complementary to the probe anchor region (A), and the amplification retardation region can prevent polymerase extension;

[0036] The probe anchor region (A) is a sequence that does not pair with any target sequence, and the target sequence binding region 1 is a sequence that can specifically pair with the target sequence; the anti-probe anchor region (a') is complementary to the probe anchor region (A), and the amplification blocking region can prevent the polymerase from extending;

[0037] The probe (P) is a sequence that does not pair with any target sequence, and includes a probe signal detection region (H) and a primer anchor region (A'), wherein the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer;

[0038] The second primer comprises an anti-primer signal detection region (h'), an amplification blocking region, a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 is a sequence that can specifically pair and bind to the target sequence, and the primer signal detection region (h) is a sequence that does not pair and bind to any target sequence; the primer signal detection region (h) is complementary to the anti-primer signal detection region (h'), and the complementary sequence of the primer signal detection region (h) specifically pairs and binds to the probe signal detection region (H) of the probe (P); and the amplification blocking region can prevent polymerase extension.

[0039] In other embodiments of the present invention, a primer probe for nucleic acid detection comprises a first primer, a probe (P) and a second primer, wherein:

[0040] The first primer comprises an anti-probe anchor region (a'), an amplification retardation region, a probe anchor region (A) and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is complementary to the anti-probe anchor region (a'); the target sequence binding region 1 is a sequence that can specifically pair with the target sequence; the amplification retardation region can prevent polymerase extension; or,

[0041] The first primer comprises a probe anchor region (A), an amplification retardation region, an anti-probe anchor region (a') and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is complementary to the anti-probe anchor region (a'); the target sequence binding region 1 is a sequence that can specifically pair with the target sequence; the amplification retardation region can prevent the polymerase from extending;

[0042] The probe (P) is a sequence that does not pair with any target sequence, and includes a primer anchor region (A') and a probe signal detection region (H); the probe (P) is modified with a detection label; the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer;

[0043] The second primer comprises a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 is a sequence that can specifically pair and bind to the target sequence, the primer signal detection region (h) is a sequence that does not pair and bind to any target sequence, and the complementary sequence of the primer signal detection region (h) specifically pairs and binds to the probe signal detection region (H) of the probe (P).

[0044] In some embodiments of the present invention, a primer probe for nucleic acid detection comprises a first primer, a probe (P) and a second primer, wherein:

[0045] The first primer comprises a probe anchor region (A), an amplification blocking region and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the amplification blocking region can prevent polymerase extension; the target sequence binding region 1 is a sequence that can specifically pair with the target sequence;

[0046] The probe (P) is a sequence that does not pair with any target sequence, and includes a probe signal detection region (H) and a primer anchor region (A'), wherein the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer;

[0047] The second primer comprises a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 is a sequence that can specifically pair and bind with the target sequence, and the primer signal detection region (h) is a sequence that does not pair and bind with any target sequence; the complementary sequence of the primer signal detection region (h) specifically pairs and binds with the probe signal detection region (H) of the probe (P).

[0048] The primer probe of the present invention is arranged in such a way that when the first primer and the second primer specifically bind to the target (target nucleic acid) respectively, two single-stranded pre-amplification products are generated: one is a single-stranded pre-amplification product without a complementary sequence to the primer signal detection region (h) (referred to as: competitive pre-amplification product); the other is a single-stranded pre-amplification product with a complementary sequence to the primer signal detection region (h) (referred to as: detection pre-amplification product), the detection pre-amplification product utilizes the complementary sequence of the primer signal detection region (h), specifically binds to the probe (P) and extends ≥0 bases to form a double-stranded product, and the formation of the double-stranded product causes the probe to generate a detectable signal change.

[0049] In the above-mentioned competitive pre-amplification product, since the primer signal detection region (h) is blocked by the anti-primer signal detection region (h'), and / or there is no probe anchor region (A), the competition between the competitive pre-amplification product and the probe is reduced, so that the probe can more advantageously bind to the detection pre-amplification product containing the complementary sequence of the primer signal detection region (h), thereby increasing the detectable signal generated by the probe, making the positive signal higher and improving the detection sensitivity.

[0050] In addition to improving the structure of the competitive pre-amplification product, the structure of the detection pre-amplification product can also be improved. For example, by providing complementary anti-probe anchor regions (a') and probe anchor regions (A) in the detection pre-amplification product, competition can be reduced, the specific binding advantage between the probe and the detection pre-amplification product can be improved, the detection signal brightness can be increased, and the detection sensitivity can be improved.

[0051] The term "from the 5' end to the 3' end" in the present invention does not mean that there is no base gap between the regions. In certain embodiments, there is a gap of 0 to 20 bases between adjacent regions. For example, there is a gap of 0 to 20 bases between the anti-probe anchor region (a') and the target sequence binding region 1, there is a gap of 0 to 20 bases between the probe anchor region (A) and the target sequence binding region 1, and there is a gap of 0 to 20 bases between the primer signal detection region (h) and the target sequence binding region 2.

[0052] In some embodiments of the present invention, there is a 0-20 base interval between the amplification retardation zone and the adjacent region. For example, there is a 0-20 base interval between the amplification retardation zone and the anti-primer signal detection zone (h'), and / or, there is a 0-20 base interval between the amplification retardation zone and the primer signal detection zone (h), and / or, there is a 0-20 base interval between the amplification retardation zone and the anti-probe anchoring zone (a'), and / or, there is a 0-20 base interval between the amplification retardation zone and the probe anchoring zone (A). In some embodiments of the present invention, the above-mentioned 0-20 bases used for spacing are also called "connection sequences", and in some embodiments, the bases used for spacing are A bases or T bases.

[0053] In some embodiments of the present invention, the detection label is a detection group.

[0054] In some embodiments of the present invention, the detection label includes a first detection group and a second detection group, and the first detection group and the second detection group generate a signal change by changing the distance; preferably, the first detection group and the second detection group are spaced 3-250 angstroms apart; preferably, the distance is 3-201 angstroms apart; more preferably, the distance is 3-140 angstroms apart; further preferably, the first detection group is a fluorescent reporter group, and the second detection group is a quenching group or other modification group that can generate a signal change with the first detection group through fluorescence resonance energy transfer.

[0055] In the present invention, the positions of the first detection group and the second detection group on the probe enable the single-stranded pre-amplification product to specifically bind to the probe (P) and extend ≥0 bases to form a double-stranded product. As long as the formation of the double-stranded product can cause the positions of the first detection group and the second detection group to change, the probe can be caused to produce a detectable signal change. The positions of the first detection group and the second detection group can be interchanged. Different restrictions can be made on the positions of the first detection group and the second detection group on the probe for different situations to achieve the effect of reducing the fluorescence background. For example: the probe (P) includes a primer anchor region (A') and a probe signal detection region (H) from the 5' end to the 3' end, and the positions of the first detection group and the second detection group on the probe (P) on the probe can be modified between the primer anchor region (A') and the probe signal detection region (H), respectively.

[0056] In some embodiments of the present invention, the positions of the first detection group and the second detection group on the probe enable the single-stranded pre-amplification product to specifically bind to the probe (P) and extend to form a secondary amplification double-stranded product, and the formation of the secondary amplification double-stranded product causes the probe to produce a detectable signal change. For example, at least one detection group is modified at the 5' end of the probe signal detection region (H) of the probe, or at least one detection group is modified within the probe signal detection region (H) of the probe.

[0057] In some embodiments of the present invention, the probe (P) comprises 1-2 primer anchor regions (A') and 1-2 probe signal detection regions (H);

[0058] In some embodiments of the present invention, the probe (P) is any one of the structures 1) to 4);

[0059] 1) The probe (P) comprises a primer anchor region (A') and a probe signal detection region (H);

[0060] 2) The probe (P) comprises a probe signal detection region (H) and two primer anchor regions (A'), the two primer anchor regions (A') are respectively located on both sides of the probe signal detection region (H), and the sequences of the two primer anchor regions (A') are different;

[0061] 3) The probe (P) comprises a primer anchor region (A') and two probe signal detection regions (H), the two probe signal detection regions (H) are respectively located on both sides of the primer anchor region (A'), and the sequences of the two probe signal detection regions (H) are different;

[0062] 4) The probe (P) comprises two primer anchor regions (A') and two probe signal detection regions (H), wherein the primer anchor regions (A') and the probe signal detection regions (H) are arranged at intervals, the sequences of the two primer anchor regions (A') are different, and the sequences of the two probe signal detection regions (H) are different.

[0063] In the above embodiment of the present invention, the relationship between the primer anchor region (A') and the probe signal detection region (H) on the probe (P) includes the following:

[0064] ① It only contains the probe signal detection region (H) but does not contain the primer anchor region (A'); correspondingly, the upstream primer (F) does not contain the probe anchor region (A);

[0065] ② Contains one probe signal detection region (H) and one primer anchor region (A'): the positional relationship between the two can be interchangeable (A'-H or H-A'), and there may or may not be a base gap between the two;

[0066] ③ Two probe signal detection regions (H) and one primer anchor region (A'): the probe signal detection region (H) is distributed on both sides of the primer anchor region (A') (A'-H-A'); there may be a base gap between the probe signal detection region (H) and one primer anchor region (A'), or there may not be one base gap;

[0067] ④ Two primer anchor regions (A') and two probe signal detection regions (H), wherein the primer anchor regions (A') and the probe signal detection regions (H) are arranged at intervals (H-A'-H-A' or A'-H-A'-H); there may or may not be a base interval between the probe signal detection region (H) and the primer anchor region (A').

[0068] In some embodiments of the present invention, the primer anchor region (A') of the probe (P) is located at the 5' end of the probe signal detection region (H); preferably, the detection group on the probe (P) is modified between the primer anchor region (A') and the probe signal detection region (H).

[0069] The above “the probe (P) comprises a primer anchor region (A') and a probe signal detection region (H) in sequence from the 5' end to the 3' end” does not mean that there is no base gap between the primer anchor region (A') and the probe signal detection region (H).

[0070] In other embodiments of the present invention, the primer anchor region (A') of the probe (P) is located at the 5' end of the probe signal detection region (H); preferably, the detection group on the probe (P) is modified at the 5' end of the probe signal detection region (H).

[0071] In some specific embodiments of the present invention, the probe (P) in the primer probe is a freely designed sequence that does not pair with any target sequence, and the probe (P) comprises a primer anchor region (A') and a probe signal detection region (H) from the 5' end to the 3' end; the sequence of the primer anchor region (A') is complementary to the sequence of the probe anchor region (A) of the first primer; the second detection group is modified at a base position 3-140 angstroms away from the first detection group, wherein the first detection group can be a fluorescent reporter group, and the second detection group can be a quenching group or other modified groups that can produce a signal change with the first detection group through fluorescence resonance energy transfer (FRET).

[0072] In some specific embodiments of the present invention, the probe (P) in the primer probe is a freely designed sequence that does not pair with any target sequence, and the probe (P) comprises a probe signal detection region (H) and a primer anchor region (A') from the 5' end to the 3' end; the sequence of the primer anchor region (A') is complementary to the sequence of the probe anchor region (A) of the first primer; the second detection group is modified at a base position 3-140 angstroms away from the first detection group, wherein the first detection group can be a fluorescent reporter group, and the second detection group can be a quenching group or other modified groups that can produce a signal change with the first detection group through fluorescence resonance energy transfer (FRET).

[0073] In some specific embodiments of the present invention, the probe (P) in the primer probe is a freely designed sequence that does not pair with any target sequence, and the probe (P) includes a primer anchor region (A') and two probe signal detection regions (H); the probe (P) includes a probe signal detection region (H1), a primer anchor region (A') and a probe signal detection region (H2) from the 5' end to the 3' end; the sequence of the primer anchor region (A') is complementary to the sequence of the probe anchor region (A) of the first primer; the second detection group is modified at a base position 3-140 angstroms away from the first detection group, wherein the first detection group can be a fluorescent reporter group, and the second detection group can be a quencher group or other modified groups that can produce signal changes with the first detection group through fluorescence resonance energy transfer (FRET).

[0074] In some embodiments of the present invention, the 3' end of the probe (P) contains a blocking region.

[0075] In the present invention, the "blocking region", also known as the "amplification blocking region", is a part used to prevent the extension of the nucleic acid chain by the DNA polymerase, thereby preventing the extension of the chain in, for example, the PCR process. The blocking region can be a 3'OH modified with 3'-Spacer C3, 3'-Phosphat, 3'-ddC, 3'-Inverted End, etc., so that its 3'OH is blocked, thereby preventing its extension reaction. The blocking region can also be a polymerase enzyme blocking group, a group with a functional property of blocking the further extension of the polymer. The blocking group can be any chemical group that can be connected to a nucleotide, which will allow the 5' end of the modified nucleotide to be connected to the 3' end of another nucleotide in the DNA chain but will not allow the nucleotide to be connected to the 3' hydroxyl group of the modified nucleotide. Suitably, the absence of the OH group at the 3' position will prevent further extension by the polymerase activity. In some embodiments, the blocking group is selected from acetyl, CH3, glycyl, leucyl and alanyl groups. In other embodiments, the blocking group can be in the form of a di- or tripeptide.

[0076] In some embodiments of the present invention, the Tm value of the primer signal detection region (h) is simultaneously smaller than the Tm value of the target sequence binding region 1 in the first primer and the Tm value of the target sequence binding region 2 in the second primer.

[0077] In other embodiments of the present invention, the Tm value of the primer anchor region (A') is greater than the Tm value of the primer signal detection region (h).

[0078] In the present invention, by limiting the Tm value of the primer signal detection region (h) and the Tm value of the primer anchor region (A') within the above range, the generation of primer dimers during amplification can be effectively avoided. It is worth noting that even if dimers are generated during amplification, the signals of dimers can be distinguished by the melting curve. If the signal is collected at a higher melting temperature, the signals of dimers will not be collected. The specific method adopted is: the signal change generated by the double-stranded product formed by the specific binding of the single-stranded pre-amplification product containing the complementary sequence of the primer signal detection region and the probe (P) is different from the signal change generated by the secondary amplification double-stranded product formed by the specific binding of the single-stranded pre-amplification product containing the complementary sequence of the primer signal detection region and the probe (P) and extension. The different signal changes refer to different signal strengths and / or different melting temperatures.

[0079] In some embodiments of the present invention, there is a 0-20 base interval between the probe anchor region (A) and the target sequence binding region 1 in the first primer.

[0080] In some embodiments of the present invention, the lengths of the first primer and the second primer are each independently 15 to 80 bases; preferably, the Tm values ​​of the target sequence binding region 1 and the target sequence binding region 2 are each independently 40°C to 80°C, and the GC content is each independently 20% to 80%; the length of the primer signal detection region (h) is 5 to 65 bases, the Tm value is 30°C to 80°C, and the GC content is 20% to 80%.

[0081] In other embodiments of the present invention, the length of the probe (P) is 20 to 100 bases; preferably, the length of the probe signal detection region (H) is 5 to 65 bases, the Tm value is 30°C to 85°C, and the GC content is 20% to 80%.

[0082] In some embodiments of the present invention, the length of the probe anchor region (A) is 6 to 35 bases, the Tm value is 40° C. to 80° C., and the GC content is 30% to 80%.

[0083] In other embodiments of the present invention, the length of the primer anchor region (A') is 6 to 35 bases, the Tm value is 40°C to 85°C, and the GC content is 20% to 80%; preferably, the Tm value of the primer anchor region (A') is greater than the Tm value of the primer signal detection region (h).

[0084] In the present invention, the Tm value of the primer anchor region (A') is made greater than the Tm value of the primer signal detection region (h), so that the complementary sequence of the primer signal detection region needs to be bound to the probe with the help of the probe anchor region (A).

[0085] In a second aspect of the present invention, there is provided a primer probe set for nucleic acid detection, comprising 1 probe (P), at least 1 first primer and at least 2 second primers;

[0086] The first primer comprises a target sequence binding region 1, and different target sequence binding regions 1 are sequences that can specifically pair and bind with different target sequences;

[0087] The probe (P) is a sequence that does not pair with any target sequence, and includes a probe signal detection region (H); the probe (P) is modified with a detection label;

[0088] The at least two second primers are different from each other, and each independently comprises an anti-primer signal detection region (h'), an amplification blocking region, a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 in different second primers is a sequence that specifically pairs and binds to different target sequences; the primer signal detection region (h) in different second primers is a sequence that does not pair and bind to any target sequence, and the complementary sequence of the primer signal detection region (h) specifically pairs and binds to the probe signal detection region (H) of the probe (P); the sequences of the primer signal detection region (h) in different second primers are different from each other; the primer signal detection region (h) in the second primer is complementary to the anti-primer signal detection region (h') on the primer; and the amplification blocking region can prevent polymerase extension.

[0089] In the present invention, the first primer and the second primer respectively specifically bind to the target to generate a pre-amplification product, wherein the pre-amplification product contains a single-stranded pre-amplification product having a complementary sequence to the primer signal detection region (h), and the complementary sequence in the single-stranded pre-amplification product specifically binds to the probe (P) and extends ≥0 bases to form a double-stranded product, and the formation of the double-stranded product causes a change in the probe signal.

[0090] In the present invention, different second primers form different single-stranded pre-amplification products, different single-stranded pre-amplification products form different double-stranded products after combining with the probe (P), and different double-stranded products have different Tm values.

[0091] The “different Tm values” refer to different melting temperatures, that is, different double-stranded products can be separated from each other by melting curves.

[0092] In some embodiments of the present invention, the double-stranded product is a secondary amplified double-stranded product;

[0093] In the present invention, "different detection labels" means that detection is performed under different detection channels, and the detection labels include a first detection group and a second detection group, and the first detection group and the second detection group generate a signal change by a change in distance; one of the first detection group and the second detection group can be a fluorescent group, and the other can be a quenching group or other groups that can generate a signal change with the fluorescent group through fluorescence resonance energy transfer.

[0094] Different probes have different fluorescent groups, but the quenching groups may be the same.

[0095] In the present invention, "the sequences of the primer signal detection regions (h) in different second primers are different from each other" means that the sequences of the primer signal detection regions (h) of different second primers have different bases and / or different lengths, so that different single-stranded pre-amplification products containing reverse complementary sequences of different primer signal detection regions (h) combine with the probe and extend ≥0 bases to form double-stranded products, and the melting temperature curves of different double-stranded products can be separated from each other.

[0096] Since the melting temperature of the double-stranded product formed with the probe (P) is detected during the melting curve analysis, the bases and length of the sequence of the primer signal detection region (h) of the second primer can be adjusted to obtain a complementary sequence of the primer signal detection region of the single-stranded pre-amplification product (S1) that is partially or completely reverse complementary to the probe signal detection region (H) of the probe (P), thereby adjusting the melting temperature of the double-stranded product formed with the probe (P), for example, adjusting the length or bases of the sequence of the primer signal detection region (h), or adjusting the position where the complementary sequence of the primer signal detection region (h) is complementary to the probe signal detection region (H) of the probe (P), thereby increasing or decreasing the melting temperature of the double-stranded product formed with the probe (P).

[0097] In the present invention, the primer probe set includes multiple sets of first primers and second primers for different target sequences. When the primer probe set is used for multiplex PCR detection, different target sequences can be identified through a fluorescent channel or through a melting temperature. When different target sequences are identified through a fluorescent channel, different target sequences can use different probes (P); when different target sequences are identified through a melting temperature, different target sequences can share the same probe (P).

[0098] In some embodiments of the present invention, in the primer probe set for nucleic acid detection, at least one of the first primers further comprises a probe anchor region (A); wherein the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is located at the 5' end of the target sequence binding region 1;

[0099] The probe (P) further comprises a primer anchor region (A'); the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer.

[0100] In some embodiments of the present invention, at least one first primer comprises a probe anchor region (A) and a target sequence binding region 1 in sequence from the 5' end to the 3' end; wherein the probe anchor region (A) is a sequence that does not pair with any target sequence; and the sequences of the target sequence binding regions 1 in different first primers are different.

[0101] In some embodiments of the present invention, the primer probe set for nucleic acid detection is characterized in that at least one of the first primers further comprises a probe anchor region (A) and an amplification retardation region; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is located at the 5' end of the target sequence binding region 1; the amplification retardation region can prevent polymerase extension; the amplification retardation region is located between the probe anchor region (A) and the target sequence binding region 1;

[0102] The probe (P) further comprises a primer anchor region (A'); the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer.

[0103] In some embodiments of the present invention, in the primer probe set for nucleic acid detection, at least one of the first primers further comprises an anti-probe anchor region (a') and an amplification retardation region; the amplification retardation region is located between the anti-probe anchor region (a') and the probe anchor region (A); the anti-probe anchor region (a') is complementary to the probe anchor region (A), and the amplification retardation region can prevent polymerase extension.

[0104] In some embodiments of the present invention, in a primer probe set for nucleic acid detection, at least one of the first primers comprises an anti-probe anchor region (a'), an amplification retardation region, a probe anchor region (A) and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is complementary to the anti-probe anchor region (a'); the target sequence binding region 1 is a sequence that can specifically pair with the target sequence; the amplification retardation region can prevent polymerase extension; or,

[0105] The first primer comprises a probe anchor region (A), an amplification blocking region, an anti-probe anchor region (a') and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is complementary to the anti-probe anchor region (a'); the target sequence binding region 1 is a sequence that can specifically pair with the target sequence; the amplification blocking region can prevent polymerase extension.

[0106] In some embodiments of the present invention, a primer probe set for nucleic acid detection includes one probe (P), at least one first primer and at least two second primers; wherein,

[0107] The first primer comprises an anti-probe anchor region (a'), an amplification retardation region, a probe anchor region (A) and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is complementary to the anti-probe anchor region (a'); the target sequence binding region 1 is a sequence that can specifically pair with the target sequence; the amplification retardation region can prevent polymerase extension; or,

[0108] The first primer comprises a probe anchor region (A), an amplification retardation region, an anti-probe anchor region (a') and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is complementary to the anti-probe anchor region (a'); the target sequence binding region 1 is a sequence that can specifically pair with the target sequence; the amplification retardation region can prevent the polymerase from extending;

[0109] The probe (P) is a sequence that does not pair with any target sequence, and includes a primer anchor region (A') and a probe signal detection region (H); the probe (P) is modified with a detection label; the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer;

[0110] The at least two second primers are different from each other, and each independently comprises a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 in different second primers is a sequence that specifically pairs and binds to different target sequences, and the primer signal detection region (h) in different second primers is a sequence that does not pair and bind to any target sequence, and the complementary sequence of the primer signal detection region (h) specifically pairs and binds to the probe signal detection region (H) of the probe (P); the sequences of the primer signal detection regions (h) of different second primers are different from each other.

[0111] In some embodiments of the present invention, a primer probe set for nucleic acid detection includes one probe (P), at least one first primer and at least two second primers; wherein,

[0112] The first primer comprises a probe anchor region (A), an amplification retardation region and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence; the target sequence binding region 1 is a sequence that can specifically pair with the target sequence; the amplification retardation region can prevent polymerase extension;

[0113] The probe (P) is a sequence that does not pair with any target sequence, and includes a primer anchor region (A') and a probe signal detection region (H); the probe (P) is modified with a detection label; the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer;

[0114] The at least two second primers are different from each other, and each independently comprises a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 in different second primers is a sequence that specifically pairs and binds to different target sequences, and the primer signal detection region (h) in different second primers is a sequence that does not pair and bind to any target sequence, and the complementary sequence of the primer signal detection region (h) specifically pairs and binds to the probe signal detection region (H) of the probe (P); the sequences of the primer signal detection regions (h) of different second primers are different from each other.

[0115] In some embodiments of the present invention, the primer signal detection region (h) of the second primer and the target sequence binding region 2 are each independently separated by 0 to 20 bases.

[0116] In some embodiments of the present invention, the detection label is a detection group.

[0117] In some embodiments of the present invention, the detection label includes a first detection group and a second detection group, and the first detection group and the second detection group generate a signal change by changing the distance; preferably, the first detection group and the second detection group are spaced 3-250 angstroms apart; preferably, the distance is 3-201 angstroms apart; more preferably, the distance is 3-140 angstroms apart; further preferably, the first detection group is a fluorescent reporter group, and the second detection group is a quenching group or other modification group that can generate a signal change with the first detection group through fluorescence resonance energy transfer.

[0118] In some embodiments of the present invention, the probe (P) includes 1-2 primer anchor regions (A') and 1-2 probe signal detection regions (H).

[0119] In some embodiments of the present invention, the probe (P) is any one of the structures 1) to 4);

[0120] 1) The probe (P) comprises a primer anchor region (A') and a probe signal detection region (H);

[0121] 2) The probe (P) comprises a probe signal detection region (H) and two primer anchor regions (A'), the two primer anchor regions (A') are respectively located on both sides of the probe signal detection region (H), and the sequences of the two primer anchor regions (A') are different;

[0122] 3) The probe (P) comprises a primer anchor region (A') and two probe signal detection regions (H), the two probe signal detection regions (H) are respectively located on both sides of the primer anchor region (A'), and the sequences of the two probe signal detection regions (H) are different;

[0123] 4) The probe (P) comprises two primer anchor regions (A') and two probe signal detection regions (H), wherein the primer anchor regions (A') and the probe signal detection regions (H) are arranged at intervals, the sequences of the two primer anchor regions (A') are different, and the sequences of the two probe signal detection regions (H) are different.

[0124] In some embodiments of the present invention, the 3' end of the probe (P) contains a blocking region.

[0125] In the present invention, the primer probe set for nucleic acid detection contains at least two second primers for different target nucleic acids, that is, multiple specific primers are added in a single tube multiplex detection. Due to the special primer probe structure in the present invention, the binding ability of the pre-amplification product to be detected generated in the pre-amplification is improved, so that the positive signal is strong, thereby improving the detection sensitivity.

[0126] The third aspect of the present invention provides a primer probe composition for nucleic acid detection, which includes at least one group of primer probes for nucleic acid detection as described in the first aspect, and / or, includes at least one group of primer probes for nucleic acid detection as described in the second aspect. In some specific embodiments, the sequences of the probes (P) of different groups are different. In other specific embodiments, the sequences and detection labels of the probes (P) of different groups are different.

[0127] In the present invention, the primer probe set includes multiple sets of first primers and second primers for different target sequences. When the primer probe set is used for multiplex PCR detection, different target sequences can be identified through a fluorescent channel or through a melting temperature. When different target sequences are identified through a fluorescent channel, different target sequences can use different probes (P); when different target sequences are identified through a melting temperature, different target sequences can share the same probe (P).

[0128] In some preferred embodiments of the present invention, different probes (P) have different sequences of primer anchor regions (A') and / or probe signal detection regions (H), and have different modified detection groups.

[0129] In the present invention, the probe anchor regions (A) of different first primers may be the same or different. When the probe anchor regions (A) of different first primers are different, the sequences and / or lengths of the probe anchor regions (A) of different first primers and the primer signal detection regions of different reverse primers make different single-stranded pre-amplification products to be tested contain complementary sequences of different probe anchor regions (A) and primer signal detection regions (h), different single-stranded pre-amplification products to be tested form different double-stranded products after binding to the probe and extending, and the melting temperature curves of different double-stranded products can be separated from each other.

[0130] Since the melting temperature of the double-stranded product formed with the probe (P) is detected during the melting curve analysis, the melting temperature of the double-stranded product formed with the probe (P) can be adjusted by adjusting the bases and length of the sequence of the probe anchor region (A) of the first primer and / or the primer signal detection region (h) of the second primer. For example, the length of the probe anchor region (A) or the bases of the sequence, or the length of the primer signal detection region (h) or the bases of the sequence, or the position where the probe anchor region is complementary to the primer anchor region (A') of the probe (P), or the position where the complementary sequence of the primer signal detection region (h) is reversely complementary to the probe signal detection region (H) of the probe (P), thereby increasing or decreasing the melting temperature of the double-stranded product formed with the probe (P).

[0131] In certain embodiments, in the primer probe set, the sequences and detection labels of different groups of the probes (P) are different and can be identified by a fluorescent channel. Then, it is possible that the double-stranded products formed by different pre-amplification products to be tested and different probes have similar / same melting temperatures.

[0132] The fourth aspect of the present invention provides a kit for nucleic acid detection, comprising the primer probe for nucleic acid detection as described in the first aspect, and / or, comprising the primer probe group for nucleic acid detection as described in the second aspect, and / or, comprising the primer probe composition for nucleic acid detection as described in the third aspect; preferably, also comprising an amplification reagent; further preferably, the amplification reagent comprises DNA polymerase and dNTPs; further preferably, when the nucleic acid template is RNA, the amplification reagent also comprises reverse transcriptase.

[0133] In the present invention, the amplification reagent may also include some reagents that promote PCR reaction, such as KCl, MgCl2, Tris-HCl, dithiothreitol (DTT), (NH4)2SO4, etc.

[0134] The fifth aspect of the present invention provides a method for nucleic acid detection using the primer-probe combination of the third aspect or the kit of the fourth aspect, comprising the following contents:

[0135] The first primer and the second primer are specifically bound to the target nucleic acid respectively, and after amplification, a pre-amplification product having a complementary sequence to the primer signal detection region (h) but not having a complementary sequence to the anti-primer signal detection region (h') is obtained;

[0136] The complementary sequence of the primer signal detection region (h) in the pre-amplification product is specifically combined with the probe (P) and extended by ≥0 bases to form a secondary amplification double-stranded product;

[0137] The formation of the double-stranded product causes the probe to produce a detectable signal change, and the presence of the target nucleic acid is determined based on the signal change; and / or

[0138] The double-stranded product is heated, and the melting of the double-stranded product causes the probe to produce a detectable signal change, and the presence of the target nucleic acid is determined based on the signal change.

[0139] In the present invention, "raising the temperature of the double-stranded product" means raising the temperature of the double-stranded product to a value greater than the Tm value of the double-stranded product.

[0140] In some embodiments of the present invention, the heating rate is 0.05°C / s, the fluorescence signal is collected at 35-95°C, and it is determined whether the sample to be tested contains the target substance based on the signal change;

[0141] In some specific embodiments of the present invention, the heating rate is 0.05°C / s, the fluorescence signal is collected at 40-90°C, and whether the sample to be tested contains the target is determined based on the signal change; preferably, the heating rate is 0.05°C / s, the temperature is gradually increased from 40°C to 90°C, the fluorescence signal is collected in real time during the heating process, a melting curve is obtained after the heating is completed, and whether the sample to be tested contains the target is determined based on the signal change;

[0142] In some other specific embodiments of the present invention, the heating rate is 0.05°C / s, the fluorescence signal is collected under the condition of 56-90°C, and it is judged whether the sample to be tested contains the target substance based on the signal change; preferably, the heating rate is 0.05°C / s, the temperature is gradually increased from 56°C to 90°C, the fluorescence signal is collected in real time during the heating process, a melting curve is obtained after the heating is completed, and it is judged whether the sample to be tested contains the target substance based on the signal change.

[0143] Preferably, the signal change refers to a change in the fluorescence signal. For example, when the temperature at which the signal change is the largest is within the range of the Tm value of the double-stranded product formed by the probe (P) of the specific detection channel of the target in the sample to be tested, it indicates that the sample to be tested contains the target.

[0144] The above-mentioned specific detection channel is the fluorescence channel corresponding to the detection group on the probe (P).

[0145] The temperature at which the above signal changes the most is the Tm value of the peak position.

[0146] In some embodiments of the present invention, when the Tm value of the peak position of the melting curve obtained is within the range of the Tm value of the double-stranded product formed by the target in the sample to be tested and the probe (P) of a specific detection channel, it indicates that the sample to be tested contains the target; preferably, the specific detection channel is the fluorescent channel corresponding to the first detection group on the probe (P).

[0147] The sixth aspect of the present invention provides a method for nucleic acid detection using the primer-probe combination of the third aspect or the kit of the fourth aspect, comprising the following contents:

[0148] S1, mixing the first primer, the second primer and the probe (P), an amplification reagent and a sample to be tested to obtain a reaction system;

[0149] S2, performing PCR amplification on the reaction system;

[0150] S3, analyzing whether there is a target.

[0151] The PCR detection method of the present invention can be used for diagnostic and therapeutic purposes, or can be used for non-diagnostic and therapeutic purposes.

[0152] In some embodiments of the present invention, step S3 comprises the following steps:

[0153] S3-1, obtaining the signal change generated by the PCR amplification in step S2, and judging whether there is a target in the sample to be tested according to the signal change; and / or

[0154] S3-2, heating the product after PCR amplification in step S2, and judging whether the sample to be tested contains the target substance according to the signal change.

[0155] The above PCR detection method is a fluorescence PCR detection method.

[0156] In some embodiments of the present invention, in the reaction system obtained in step S2, the concentration of the first primer is 30nM-1000nM, the concentration of the second primer is 30nM-500nM, and the concentration of the probe (P) is 150nM-1200nM.

[0157] In some embodiments of the present invention, the sample to be tested is from a serum sample, a plasma sample, a whole blood sample, a sputum sample, a swab sample, a lavage fluid sample, a fresh tissue sample, or a formalin-fixed paraffin-embedded tissue (FFPE).

[0158] In some embodiments of the present invention, step S2 comprises the following steps:

[0159] S2-1, randomly distributing the reaction system into more than 500 reaction units, each reaction unit containing one target of the sample to be tested or not containing the target of the sample to be tested; and / or,

[0160] S2-2, PCR amplification of all reaction units;

[0161] And / or, step S3 comprises the following steps:

[0162] S3, obtaining the signal of each reaction unit after PCR amplification in step S2, and judging whether there is a target according to the presence or absence of the signal.

[0163] The above-mentioned PCR detection method is a digital PCR detection method.

[0164] In some embodiments of the present invention, S2-1, the reaction system is randomly distributed into 500 to 20,000 reaction units, each reaction unit contains one target of the sample to be tested or does not contain a target of the sample to be tested.

[0165] In other embodiments of the present invention, in the reaction system obtained in step S1, the concentration of the forward primer (F) is 30nM-1000nM, the concentration of the reverse primer (R) is 30nM-1000nM, and the concentration of the probe (P) is 100nM-1200nM.

[0166] In some embodiments of the present invention, in step S2, when the nucleic acid template is DNA, the procedure of the PCR amplification is as follows:

[0167] Pre-denaturation at 90℃~96℃ for 3~15 minutes; denaturation at 90℃~95℃ for 10~60 seconds, annealing and extension at 50℃~75℃ for 30~90 seconds, for 35~50 cycles; denaturation at 90℃~95℃ for 10~600 seconds, annealing and extension at 30℃~55℃ for 10~600 seconds, for 1~20 cycles.

[0168] In some embodiments of the present invention, in step S2, when the nucleic acid template is DNA, the procedure of the PCR amplification is as follows:

[0169] Pre-denaturation at about 85°C to about 105°C for about 0 to about 15 minutes; denaturation at about 85°C to about 105°C for about 1 to about 60 seconds, annealing and extension at about 40°C to about 75°C for about 3 to about 90 seconds, and about 20 to about 60 cycles; denaturation at about 85°C to about 105°C for about 1 to about 60 seconds, annealing and extension at about 20°C to about 75°C for about 3 to about 90 seconds, and 1 to 20 cycles.

[0170] In some specific embodiments of the present invention, in step S2, when the nucleic acid template is DNA, the PCR amplification procedure is as follows: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, for a total of 45 cycles; denaturation at 94°C for 150 seconds, annealing and extension at 40°C for 300 seconds, for 1 cycle.

[0171] In some embodiments of the present invention, in step S2, when the nucleic acid template is RNA, the procedure of the PCR amplification is as follows:

[0172] Reverse transcription at 50-60°C for 5-15 minutes; pre-denaturation at 90-96°C for 3-15 minutes; denaturation at 90-95°C for 10-60 seconds, annealing and extension at 50-75°C for 30-90 seconds, for 35-50 cycles; denaturation at 90-95°C for 10-600 seconds, annealing and extension at 30-55°C for 10-600 seconds, for 1-20 cycles.

[0173] In some embodiments of the present invention, in step S2, when the nucleic acid template is RNA, the procedure of the PCR amplification is as follows:

[0174] Reverse transcription at about 30-about 65°C for about 2-about 30 minutes; pre-denaturation at about 85°C-about 105°C for about 0-about 15 minutes; denaturation at about 85°C-about 105°C for about 1-about 60 seconds, annealing and extension at about 40°C-about 75°C for about 3-about 90 seconds, and 20-60 cycles; denaturation at about 85°C-about 105°C for about 1-about 60 seconds, annealing and extension at about 20°C-about 75°C for about 3-about 90 seconds, and 1-20 cycles.

[0175] In some specific embodiments of the present invention, in step S2, when the nucleic acid template is RNA, the PCR amplification procedure is as follows: reverse transcription at 55°C for 15 minutes; pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, for a total of 45 cycles; denaturation at 94°C for 150 seconds, annealing and extension at 40°C for 300 seconds, for 1 cycle.

[0176] The beneficial effects of the present invention are:

[0177] (1) Multiplex detection: The primer probe and primer probe set of the present invention can simultaneously perform analysis in two dimensions, namely, fluorescence channel and melting temperature, in a single tube reaction. That is, different targets can be detected by using the same fluorescence channel through melting temperature characteristics; or target type detection can be achieved by using different fluorescence channels multiplied by the number of fluorescence channels and melting temperature characteristics;

[0178] (2) Low fluorescence background: Only one probe is used in each fluorescence channel, and the amplified products can be distinguished by their different melting temperatures, which greatly reduces the fluorescence background in the PCR reaction and improves the reaction sensitivity;

[0179] (3) Strong fluorescence signal: The design of the primer probe of the present invention reduces the competitive ability of the pre-amplification product without the complementary sequence of the primer signal detection region (h), thereby increasing the binding ability of the probe with the pre-amplification product containing the complementary sequence of the primer signal detection region (h), thereby increasing the fluorescence intensity;

[0180] (4) Melting temperature can be adjusted: The detection method using the primer probe of the present invention utilizes the different melting temperatures of the secondary amplification products for differentiation. Therefore, by adjusting the length or sequence of the primer signal detection region (h), or adjusting the position of the reverse complement of the primer signal detection region (h) and the probe signal detection region (H) of the probe (P), the melting temperature of the secondary amplification double-stranded product formed with the probe (P) is increased or decreased;

[0181] (5) Good tolerance: The primer probe of the present invention has two parts of reverse complementary pairing with the target sequence, namely the forward primer (F) and the reverse primer (R). Compared with the Taqman hydrolysis probe method, which requires three parts of reverse complementary pairing with the template, when there are many highly variable regions in the target sequence, such as viral or bacterial genomes, the primer probe of the present invention has better tolerance and lower design difficulty;

[0182] (6) Low cost: The method of the present invention only needs to complete the detection in a single tube reaction through fluorescent quantitative PCR, without the need for additional downstream instrument assistance, and without the need to split the reaction through chips or multiple reaction tubes, which greatly reduces the cost of instruments and consumables. At the same time, the number of fluorescent probes in the reagents is reduced, which greatly reduces the reagent cost, making large-scale promotion possible;

[0183] (7) Single-tube reaction: It consumes less sample and is particularly suitable for the detection of rare samples. It can greatly increase the sample concentration added to the detection reaction and improve the detection sensitivity. At the same time, the single-tube reaction provides the possibility for subsequent extraction-free one-tube detection;

[0184] (8) Easy operation: Only fluorescence quantitative PCR instrument is needed for detection, and there is no subsequent step such as capillary electrophoresis or nucleic acid hybridization. At the same time, the preparation is simple and no operation is required after the instrument is installed;

[0185] (9) Not easy to cause pollution: The method of the present invention is completely closed after the sample is added, and there is no need to open the cover for subsequent testing, which greatly reduces the possibility of pollution to the experimental environment;

[0186] (10) High sensitivity: The method of the present invention has very low requirements on the length of the target sequence. When the target type is a short nucleic acid fragment, such as free nucleic acid, a shorter target sequence length has a higher sensitivity in the detection;

[0187] (11) Wide scope of application: The method of the present invention can be applied to nucleic acid detection of various sample types, including serum samples, plasma samples, whole blood samples, sputum samples, swab samples, lavage fluid samples, fresh tissue samples, and formalin-fixed paraffin-embedded tissue (FFPE). BRIEF DESCRIPTION OF THE DRAWINGS

[0188] The present invention will be further described below in conjunction with the accompanying drawings.

[0189] Figure 1 This is the melting curve diagram of human papillomavirus HPV type 16;

[0190] Figure 2 This is the melting curve diagram of human papillomavirus HPV type 59;

[0191] Figure 3 This is the melting curve diagram of human papillomavirus HPV type 33;

[0192] Figure 4 This is the melting curve diagram of human papillomavirus HPV type 31;

[0193] Figure 5 This is the melting curve diagram of human papillomavirus HPV type 39;

[0194] Figure 6-1 and Figure 6-2 These are all melting curves of human papillomavirus HPV 58, among which, Figure 6-1 Comparison of melting curves using F7 and F9; Figure 6-2 Comparison of melting curves using F8 and F9;

[0195] Figure 7 This is the melting curve of human papillomavirus HPV type 52. DETAILED DESCRIPTION

[0196] In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, which are merely illustrative and are not intended to limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained by commercial routes or conventional methods unless otherwise specified.

[0197] Example 1: Nucleic acid detection of human papillomavirus HPV type 16

[0198] (1) The base sequences of the primer probes for detecting human papillomavirus HPV type 16 are shown in Table 1.

[0199] Table 1: Base sequences of primers and probes

[0200]

[0201] Among the above primer probes, the forward primer F1 (SEQ ID NO: 3) is a specific primer designed for the human papillomavirus HPV type 16 target sequence. The total length of F1 is 38 bp, and the 1st to 20th bases at its 3' end are the target sequence binding region, and the 1st to 15th bases at its 5' end are the probe anchor region (A), which are reverse complementary to the 1st to 15th base sequence at the 5' end of probe P1 (SEQ ID NO: 1), i.e., the primer anchor region (A').

[0202] The reverse primer R1 (SEQ ID NO: 2) is a specific primer designed for the human papillomavirus HPV type 16 target sequence. The total length of R1 is 42 bp. The 1st to 17th bases at the 3' end are the target sequence binding region, the 1st to 9th bases at the 5' end are the reverse complementary sequence of the primer signal detection region (h) (i.e., the anti-primer signal detection region h'), and the 13th to 22nd bases at the 5' end are the primer signal detection region (h), which is the same as the 45th to 54th base sequence at the 5' end of the probe P1 (SEQ ID NO: 1);

[0203] When the pathogen target is specifically amplified, the forward primer (F1) and the reverse primer (R1) amplify the target nucleic acid to obtain a double-stranded preamplifier product, wherein the 5' end to the 3' end of one single-stranded preamplification product (S1) are the probe anchor region (A), the target sequence, and the reverse complementary region (h') of the primer signal detection region; the probe signal detection region (H) of the probe (P1) is complementary to the reverse complementary sequence (h') of the primer signal detection region of one of the single-stranded preamplification products (S1), and the primer anchor region (A') of the probe is reversely complementary to the probe anchor region (A) at the 5' end of the single-stranded preamplification product (S1). After the amplification is completed, a melting curve analysis is performed. During the temperature change process, the double-stranded product formed by the probe (P1) and the single-stranded product will melt at a certain temperature, and at this time, the probe (P1) with the first detection group and the second detection group will become a single-stranded state, so that the fluorescent signal changes and can be detected by the instrument.

[0204] (2) Using the above primers and probes, single-plex PCR was performed to detect human papillomavirus HPV type 16.

[0205] The reaction system used in the detection is shown in Table 2.

[0206] Table 2: Human papillomavirus HPV 16 PCR detection reaction system

[0207]

[0208] Note: 2× PCR reaction buffer includes 3 mM MgCl 2 , 30 mM Tris-HCl buffer, pH 8.3, 0.5 mM dNTPs, and 70 mM (NH 4 ) 2 SO 4 .

[0209] The nucleic acid template is a human papillomavirus HPV16 plasmid, synthesized by General Biotechnology (Anhui) Co., Ltd., quantified and diluted, and mixed with the reaction system at 50 copies / reaction. At the same time, a no-template control (NTC) is set up, that is, ultrapure water or 1XTE Buffer is used instead of the nucleic acid template. Then PCR amplification and melting curve analysis are performed. The presence of HPV16 in the sample can be determined based on the characteristic Tm value of the target in a specific detection channel.

[0210] Reaction preparation:

[0211] After the nucleic acid template is prepared, the reaction system is configured according to the ratio described in Table 2.

[0212] PCR amplification and melting curve analysis:

[0213] After sealing the PCR tube cap, gently mix the sample, then centrifuge briefly and let it stand at room temperature for 5 minutes. Place the PCR tube in a handheld centrifuge again, centrifuge briefly and transfer it to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. The program used is: 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 10 seconds, 56℃ annealing and extension for 30 seconds, for a total of 45 cycles; 45-85℃ melting curve analysis, heating rate of 0.05℃ per second, light collection.

[0214] Data Analysis:

[0215] The SLAN real-time fluorescence quantitative PCR analysis software produced by Shanghai Hongshi Medical Technology Co., Ltd. was used to interpret the results based on the Tm value of the melting curve. Figure 1 Melting curves of positive and negative samples (no template control) for human papillomavirus HPV type 16.

[0216] from Figure 1 It can be seen that the control well without template has no melting peak, while the positive reaction well with HPV16 plasmid added has a melting peak with a Tm value of 79° in the FAM channel. It can be seen that the primer probe of the present invention has a strong fluorescent signal and can well identify the target nucleic acid.

[0217] Example 2: Nucleic acid detection of human papillomavirus HPV type 59

[0218] (1) The base sequences of the primer probes for detecting human papillomavirus HPV type 59 are shown in Table 3.

[0219] Table 3: Base sequences of primer probes

[0220]

[0221]

[0222] Among the above primer probes, the forward primer F2 (SEQ ID NO: 5) is a specific primer designed for the human papillomavirus HPV type 59 target sequence, and the total length of F2 is 37 bp, the 1st to 19th bases at the 3' end thereof are the target sequence binding region, and the 1st to 15th bases at the 5' end thereof are the probe anchor region (A), which are reverse complementary to the 1st to 15th base sequence at the 5' end of the probe P1 (SEQ ID NO: 1), i.e., the primer anchor region (A');

[0223] Reverse primer R2 (SEQ ID NO: 4) is a specific primer designed for the human papillomavirus HPV59 target sequence. R2 is 49 bp in length. The 1st to 25th bases at its 3' end are the target sequence binding region, the 1st to 9th bases at its 5' end are the reverse complementary sequence of the primer signal detection region (h) (i.e., the anti-primer signal detection region h'), and the 13th to 22nd bases at its 5' end are the primer signal detection region (h), which is the same as the 45th to 54th base sequence at the 5' end of probe P1 (SEQ ID NO: 1).

[0224] (2) Using the above primers and probes, single-plex PCR was performed to detect human papillomavirus HPV type 59.

[0225] The reaction system used in the detection is shown in Table 4.

[0226] Table 4: Human papillomavirus HPV 59 PCR detection reaction system

[0227]

[0228] Note: 2× PCR reaction buffer includes 3 mM MgCl 2 , 30 mM Tris-HCl buffer, pH 8.3, 0.5 mM dNTPs, and 70 mM (NH 4 ) 2 SO 4 .

[0229] The nucleic acid template is a human papillomavirus HPV59 plasmid, synthesized by General Biotechnology (Anhui) Co., Ltd., quantified and diluted, and mixed with the reaction system at 50 copies / reaction. At the same time, a no-template control (NTC) is set up, that is, ultrapure water or 1×TE Buffer is used instead of the nucleic acid template, and then PCR amplification and melting curve analysis are performed. The presence of HPV 59 in the sample can be determined based on the characteristic Tm value of the target in a specific detection channel.

[0230] Reaction preparation:

[0231] After the nucleic acid template is prepared, the reaction system is configured according to the ratio described in Table 4.

[0232] PCR amplification and melting curve analysis:

[0233] After sealing the PCR tube cap, gently mix the sample, then centrifuge briefly and let it stand at room temperature for 5 minutes. Place the PCR tube in a handheld centrifuge again, centrifuge briefly and transfer it to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. The program used is: pre-denaturation at 95℃ for 2 minutes; denaturation at 94℃ for 10 seconds, annealing and extension at 56℃ for 30 seconds, for a total of 45 cycles; melting curve analysis at 40-95℃, heating rate of 0.05℃ per second, and fluorescence signal collection.

[0234] Data Analysis:

[0235] The SLAN real-time fluorescence quantitative PCR analysis software produced by Shanghai Hongshi Medical Technology Co., Ltd. was used to interpret the results based on the Tm value of the melting curve. Figure 2 Melting curves of positive and negative samples (no template control) for human papillomavirus HPV type 59.

[0236] from Figure 2 It can be seen that the control well without template has no melting peak, while the positive reaction well with HPV59 plasmid added has a melting peak with a Tm value of 81° in the FAM channel. It can be seen that the primer probe of the present invention has a strong fluorescent signal and can well identify the target nucleic acid.

[0237] Example 3: Nucleic acid detection of human papillomavirus HPV type 33

[0238] (1) The base sequences of the primer probes for detecting human papillomavirus HPV type 33 are shown in Table 5.

[0239] Table 5: Base sequences of primer probes

[0240]

[0241] Among the above primer probes, the forward primer F3 (SEQ ID NO: 8) is a specific primer designed for the human papillomavirus HPV33 target sequence. The total length of F3 is 41 bp, and the 1st to 25th bases at its 3' end are the target sequence binding region, and the 1st to 15th bases at its 5' end are the probe anchor region (A), which are reverse complementary to the 1st to 15th base sequence at the 5' end of probe P1 (SEQ ID NO: 1), i.e., the primer anchor region (A').

[0242] The reverse primer R3 (SEQ ID NO: 6) is a specific primer designed for the human papillomavirus HPV33 target sequence. The total length of R3 is 48 bp. The 1st to 23rd bases at its 3' end are the target sequence binding region, the 1st to 9th bases at its 5' end are the reverse complementary sequence of the primer signal detection region (h) (i.e., the anti-primer signal detection region h'), and the 13th to 22nd bases at its 5' end are the primer signal detection region (h), which is the same as the 45th to 54th base sequence at the 5' end of the probe P1 (SEQ ID NO: 1);

[0243] Reverse primer R4 (SEQ ID NO: 7) is a specific primer designed for the human papillomavirus HPV33 target sequence. The total length of R4 is 35 bp. The 1st to 23rd bases at its 3' end are the target sequence binding region, and the 1st to 9th bases at its 5' end are the primer signal detection region (h), which is the same as the 45th to 54th base sequence at the 5' end of probe P1 (SEQ ID NO: 1), and serves as a control primer with primer R3;

[0244] When the pathogen target is specifically amplified, the forward primer (F3) and the reverse primer (R3 or R4) amplify the target nucleic acid to obtain a double-stranded preamplifier product, wherein the 5' end to the 3' end of one single-stranded preamplification product (S1) are the probe anchor region (A), the target sequence, and the reverse complementary region of the primer signal detection region; the probe signal detection region (H) of the probe (P1) is complementary paired with the reverse complementary sequence of the primer signal detection region of one of the single-stranded preamplification products (S1), and the primer anchor region (A') of the probe is reversely complementary paired with the probe anchor region (A) at the 5' end of the single-stranded preamplification product (S1). After the amplification is completed, a melting curve analysis is performed. During the temperature change process, the double-stranded product formed by the probe (P1) and the single-stranded product will be melted at a certain temperature, and at this time, the probe (P1) with the first detection group and the second detection group will become a single-stranded state, so that the fluorescent signal changes and can be detected by the instrument.

[0245] (2) Using the above primers and probes, single-plex PCR was performed to detect human papillomavirus HPV type 33.

[0246] The reaction system used in the detection is shown in Table 6.

[0247] Table 6: Human papillomavirus HPV 33 PCR detection reaction system

[0248]

[0249]

[0250] Note: 2× PCR reaction buffer includes 3 mM MgCl 2 , 30 mM Tris-HCl buffer, pH 8.3, 0.5 mM dNTPs, and 70 mM (NH 4 ) 2 SO 4 .

[0251] The nucleic acid template is human papillomavirus type 33 plasmid, synthesized by General Biotechnology (Anhui) Co., Ltd., quantified and diluted, and mixed with the reaction system at 50 copies / reaction. At the same time, a control primer (CT) is set, that is, the reverse primer (R4) is used instead of the reverse primer (R3) for the experiment, and then PCR amplification and melting curve analysis are performed. According to the characteristic Tm value of the target in a specific detection channel, it can be judged whether the human papillomavirus HPV33 plasmid exists in the sample.

[0252] Reaction preparation:

[0253] After the nucleic acid template is prepared, the reaction system is configured according to the ratio described in Table 6.

[0254] PCR amplification and melting curve analysis:

[0255] After sealing the PCR tube cap, gently mix the sample, then centrifuge briefly and let it stand at room temperature for 5 minutes. Place the PCR tube in a handheld centrifuge again, centrifuge briefly and transfer it to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. The program used is: 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 10 seconds, 56℃ annealing and extension for 30 seconds, for a total of 45 cycles; 45-85℃ melting curve analysis, heating rate of 0.05℃ per second, light collection.

[0256] Data Analysis:

[0257] The SLAN real-time fluorescence quantitative PCR analysis software produced by Shanghai Hongshi Medical Technology Co., Ltd. was used to interpret the results based on the Tm value of the melting curve. Figure 3 This is the melting curve of human papillomavirus HPV type 33.

[0258] from Figure 3It can be seen that: using two kinds of reverse primers (R3 and R4), both can detect the target nucleic acid. However, using the primer design method of the present invention (containing spacer C3 and anti-primer signal detection zone in the reverse primer), the peak height of the melting peak is significantly improved. It can be seen that using the primer probe of the present invention, the fluorescence signal intensity can be significantly improved and the target nucleic acid can be better identified.

[0259] Example 4: Nucleic acid detection of human papillomavirus HPV type 31

[0260] (1) The base sequences of the primer probes for detecting human papillomavirus HPV type 31 are shown in Table 7.

[0261] Table 7: Base sequences of primer probes

[0262]

[0263] In the above primer probe, the forward primer F4 (SEQ ID NO: 11) is a specific primer designed for the human papillomavirus HPV31 type target sequence, F4 is 38 bp in length, the 1st to 20th bases at its 3' end are the target sequence binding region, and the 1st to 15th bases at its 5' end are the probe anchor region (A), which are reverse complementary to the 1st to 15th base sequence at the 5' end of the probe P1 (SEQ ID NO: 1) (i.e., the primer anchor region (A')); and spacer C3 is modified between the 18th base and the 19th base of the forward primer F4.

[0264] The forward primer F5 (SEQ ID NO: 12) is a specific primer designed for the human papillomavirus HPV31 target sequence. The total length of F5 is 38 bp. The 1st to 20th bases at the 3' end are the target sequence binding region, and the 1st to 15th bases at the 5' end are the probe anchor region (A), which are reverse complementary to the 1st to 15th base sequence at the 5' end of the probe P1 (SEQ ID NO: 1) (i.e., the primer anchor region (A')); and there is no amplification retardation modification in the forward primer F5. It is used as a control primer with primer F4.

[0265] The reverse primer R5 (SEQ ID NO: 9) is a specific primer designed for the human papillomavirus HPV31 target sequence. The total length of R5 is 44 bp. The 1st to 20th bases at the 3' end are the target sequence binding region, the 1st to 9th bases at the 5' end are the reverse complementary sequence of the primer signal detection region (h) (i.e., the anti-primer signal detection region h'), and the 13th to 22nd bases at the 5' end are the primer signal detection region (h), which is the same as the 45th to 54th base sequence at the 5' end of the probe P1 (SEQ ID NO: 1);

[0266] The reverse primer R6 (SEQ ID NO: 10) is a specific primer designed for the human papillomavirus HPV31 target sequence. The total length of R6 is 32 bp. The 1st to 20th bases at the 3' end are the target sequence binding region, and the 1st to 9th bases at the 5' end are the primer signal detection region (h), which is the same as the 45th to 54th base sequence at the 5' end of the probe P1 (SEQ ID NO: 1), and serves as a control primer with primer R5;

[0267] When the pathogen target is specifically amplified, the forward primer (F4 or F5) and the reverse primer (R5 or R5) amplify the target nucleic acid to obtain a double-stranded preamplifier product, wherein the 5' end to the 3' end of one single-stranded preamplification product (S1) are respectively the probe anchor region (A), the target sequence, and the reverse complementary region of the primer signal detection region; the probe signal detection region (H) of the probe (P1) is complementary paired with the reverse complementary sequence of the primer signal detection region of one of the single-stranded preamplification products (S1), and the primer anchor region (A') of the probe is reversely complementary paired with the probe anchor region (A) at the 5' end of the single-stranded preamplification product (S1). After the amplification is completed, a melting curve analysis is performed. During the temperature change process, the double-stranded product formed by the probe (P1) and the single-stranded product will be melted at a certain temperature, and at this time, the probe (P1) with the first detection group and the second detection group will become a single-stranded state, so that the fluorescent signal changes and can be detected by the instrument.

[0268] (2) Using the above primers and probes, single-plex PCR was performed to detect human papillomavirus HPV type 31.

[0269] The reaction system used in the detection is shown in Table 8.

[0270] Table 8: Human papillomavirus HPV 31 PCR detection reaction system

[0271]

[0272] Note: 2× PCR reaction buffer includes 3 mM MgCl 2 , 30 mM Tris-HCl buffer, pH 8.3, 0.5 mM dNTPs, and 70 mM (NH 4 ) 2 SO 4 .

[0273] The nucleic acid template is human papillomavirus type 31 plasmid, synthesized by General Biotechnology (Anhui) Co., Ltd., quantified and diluted, and mixed with the reaction system at 50 copies / reaction. At the same time, a control primer (CT) is set, that is, the reverse primer (R6) is used instead of the reverse primer (R5), and the forward primer (F5) is used instead of the forward primer (F4) for the experiment, and then PCR amplification and melting curve analysis are performed. According to the characteristic Tm value of the target in a specific detection channel, it can be judged whether the human papillomavirus HPV31 plasmid exists in the sample.

[0274] Reaction preparation:

[0275] After the nucleic acid template is prepared, the reaction system is configured according to the ratio described in Table 8.

[0276] PCR amplification and melting curve analysis:

[0277] After sealing the PCR tube cap, gently mix the sample, then centrifuge briefly and let it stand at room temperature for 5 minutes. Place the PCR tube in a handheld centrifuge again, centrifuge briefly and transfer it to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. The program used is: 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 10 seconds, 56℃ annealing and extension for 30 seconds, for a total of 45 cycles; 45-85℃ melting curve analysis, heating rate of 0.05℃ per second, light collection.

[0278] Data Analysis:

[0279] The SLAN real-time fluorescence quantitative PCR analysis software produced by Shanghai Hongshi Medical Technology Co., Ltd. was used to interpret the results based on the Tm value of the melting curve. Figure 4 This is the melting curve of human papillomavirus HPV type 31.

[0280] from Figure 4 It can be seen that: using two sets of forward primers and reverse primers (F4 and R5, F5 and R6), the target nucleic acid can be detected. However, using the primer design method of the present invention (there is spacer C3 in the forward primer, and spacer C3 and anti-primer signal detection zone in the reverse primer), the peak height of the melting peak is significantly improved. It can be seen that using the primer probe of the present invention, the fluorescence signal intensity can be significantly improved and the target nucleic acid can be better identified.

[0281] By adopting the primer design method of the present invention, the fluorescence signal is significantly improved. The reason may be that when the primers (F4 and R5) of the present invention amplify the target nucleic acid to obtain double-stranded amplicon products, one of the single-stranded pre-amplification products contains the reverse complementary region of the primer signal detection region. Since F4 contains spacer C3, the 5' end to the 3' end of the other single-stranded pre-amplification product are the target sequence, the primer signal detection region (h), spacer C3 and the anti-primer signal detection region (h') in sequence. Since the single-stranded pre-amplification product does not contain the complementary sequence of the probe anchor region, and the primer signal detection region (h) and the anti-primer signal detection region (h') contained in it can complement each other, the competition between the single-stranded pre-amplification product and the probe is reduced, making it easier for the probe to perform a second amplification and / or detection with the single-stranded pre-amplification product containing the reverse complementary region of the primer signal detection region. After F5 and R6 amplify the target nucleic acid, the obtained single-stranded pre-amplification product that does not contain the reverse complementary region of the primer signal detection region has a strong competitive relationship with the probe, affecting the complementary pairing of the probe and the single-stranded pre-amplification product containing the reverse complementary region of the primer signal detection region, thereby affecting the detected fluorescence intensity.

[0282] Example 5: Nucleic acid detection of human papillomavirus HPV type 39

[0283] (1) The base sequences of the primer probes for detecting human papillomavirus HPV type 39 are shown in Table 9.

[0284] Table 9: Base sequences of primer probes

[0285]

[0286]

[0287] In the above primer probe, the forward primer F6 (SEQ ID NO: 14) is a specific primer designed for the human papillomavirus HPV39 target sequence, and the full length of F6 is 25 bp.

[0288] The reverse primer R7 (SEQ ID NO: 15) is a specific primer designed for the human papillomavirus HPV39 target sequence. The total length of R7 is 45 bp. The 1st to 21st bases at the 3' end are the target sequence binding region, the 1st to 9th bases at the 5' end are the reverse complementary sequence (h') of the primer signal detection region (h), and the 13th to 21st bases at the 5' end are the primer signal detection region (h), which is the same as the 46th to 54th base sequence at the 5' end of the probe P2 (SEQ ID NO: 13);

[0289] Reverse primer R8 (SEQ ID NO: 16) is a specific primer designed for the human papillomavirus HPV39 target sequence. R8 is 33 bp in length. The 1st to 21st bases at its 3' end are the target sequence binding region, and the 1st to 9th bases at its 5' end are the primer signal detection region (h), which is the same as the 46th to 54th bases at the 5' end of probe P2 (SEQ ID NO: 13), and serves as a control primer with primer R7;

[0290] When the pathogen target is specifically amplified, the forward primer (F6) and the reverse primer (R7 or R8) amplify the target nucleic acid to obtain a double-stranded preamplifier product, wherein the 5' end to the 3' end of a single-stranded preamplification product (S1) are the target sequence and the reverse complementary region of the primer signal detection region; the probe signal detection region (H) of the probe (P2) is complementary to the reverse complementary sequence of the primer signal detection region of the single-stranded preamplification product (S1), and a melting curve analysis is performed after the amplification is completed. During the temperature change process, the double-stranded product formed by the probe (P2) and the single-stranded product will melt at a certain temperature, and at this time, the probe (P2) with the first detection group and the second detection group will become a single-stranded state, so that the fluorescent signal changes and can be detected by the instrument.

[0291] (2) Using the above primers and probes, single-plex PCR was performed to detect human papillomavirus HPV type 39.

[0292] The reaction system used in the detection is shown in Table 10.

[0293] Table 10: Human papillomavirus HPV 39 PCR detection reaction system

[0294]

[0295] Note: 2× PCR reaction buffer includes 3 mM MgCl 2 , 30 mM Tris-HCl buffer, pH 8.3, 0.5 mM dNTPs, and 70 mM (NH 4 ) 2 SO 4 .

[0296] The nucleic acid template is human papillomavirus type 39 plasmid, synthesized by General Biotechnology (Anhui) Co., Ltd., quantified and diluted, and mixed with the reaction system at 50 copies / reaction. At the same time, a control primer (CT) is set, that is, the reverse primer (R8) is used instead of the reverse primer (R7) for the experiment, and then PCR amplification and melting curve analysis are performed. According to the characteristic Tm value of the target in a specific detection channel, it can be judged whether the human papillomavirus HPV39 plasmid exists in the sample.

[0297] Reaction preparation:

[0298] After the nucleic acid template is prepared, the reaction system is configured according to the ratio described in Table 10.

[0299] PCR amplification and melting curve analysis:

[0300] After sealing the PCR tube cap, gently mix the sample, then centrifuge briefly and let it stand at room temperature for 5 minutes. Place the PCR tube in a handheld centrifuge again, centrifuge briefly and transfer it to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. The program used is: 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 10 seconds, 56℃ annealing and extension for 30 seconds, for a total of 45 cycles; 45-85℃ melting curve analysis, heating rate of 0.05℃ per second, light collection.

[0301] Data Analysis:

[0302] The SLAN real-time fluorescence quantitative PCR analysis software produced by Shanghai Hongshi Medical Technology Co., Ltd. was used to interpret the results based on the Tm value of the melting curve. Figure 5 This is the melting curve of human papillomavirus HPV type 39.

[0303] from Figure 5 It can be seen that: using two kinds of reverse primers (R7 or R8), both can detect the target nucleic acid. However, using the primer design method of the present invention (containing spacer C3 and anti-primer signal detection zone in the reverse primer), the peak height of the melting peak is significantly improved. It can be seen that using the primer probe of the present invention, the fluorescence signal intensity can be significantly improved and the target nucleic acid can be better identified.

[0304] Example 6: Nucleic acid detection of human papillomavirus HPV type 58

[0305] (1) The base sequences of the primer probes for detecting human papillomavirus HPV type 58 are shown in Table 11.

[0306] Table 11: Base sequences of primer probes

[0307]

[0308] Among the above primer probes, the forward primer F7 (SEQ ID NO: 18) is a specific primer designed for the human papillomavirus HPV type 58 target sequence. F7 is 59 bp in length, and the 1st to 23rd bases at its 3' end are the target sequence binding region, and the 1st to 15th bases at its 5' end are the probe anchor region (A'), that is, the 1st to 15th base sequence at the 5' end of the probe P3 (SEQ ID NO: 17), and the 19th to 33rd bases at its 5' end are reverse complementary to the 1st to 15th base sequence at the 5' end of the probe P3 (SEQ ID NO: 17), that is, the primer anchor region (A').

[0309] The forward primer F8 (SEQ ID NO: 19) is a specific primer designed for the human papillomavirus HPV type 58 target sequence. The total length of F8 is 59 bp. The 1st to 23rd bases at its 3' end are the target sequence binding region, and the 1st to 15th bases at its 5' end are the probe anchor region (A), which are reverse complementary to the 1st to 15th base sequence at the 5' end of the probe P3 (SEQ ID NO: 17). The 19th to 33rd bases at its 5' end are the 1st to 15th base sequence at the 5' end of the probe P3 (SEQ ID NO: 17), i.e., the primer anchor region (A').

[0310] The forward primer F9 (SEQ ID NO: 20) is a specific primer designed for the human papillomavirus HPV type 58 target sequence. The total length of F9 is 41 bp. The 1st to 23rd bases at its 3' end are the target sequence binding region, and the 1st to 15th bases at its 5' end are the probe anchor region (A), which are reverse complementary to the 1st to 15th base sequence at the 5' end of the probe P3 (SEQ ID NO: 17) and serve as a control primer for the forward primer (F7 or F8).

[0311] Reverse primer R10 (SEQ ID NO: 20) is a specific primer designed for the human papillomavirus HPV58 target sequence. R10 is 44 bp in length. The 1st to 20th bases at its 3' end are the target sequence binding region, the 1st to 9th bases at its 5' end are the reverse complementary sequence (h') of the primer signal detection region (h), and the 13th to 21st bases at its 5' end are the primer signal detection region (h), which is the same as the 46th to 54th base sequence at the 5' end of probe P3 (SEQ ID NO: 17);

[0312] When the pathogen target is specifically amplified, the forward primer (F7 or F8 or F9) and the reverse primer (R10) amplify the target nucleic acid to obtain a double-stranded preamplifier product, wherein the 3' end of a single-stranded preamplification product (S1) has a reverse complementary region of the primer signal detection region; the probe signal detection region (H) of the probe (P3) is complementary to the reverse complementary sequence of the primer signal detection region of the single-stranded preamplification product (S1), and a melting curve analysis is performed after the amplification is completed. During the temperature change process, the double-stranded product formed by the probe (P3) and the single-stranded product will melt at a certain temperature, and at this time, the probe (P3) with the first detection group and the second detection group will become a single-stranded state, so that the fluorescent signal changes and can be detected by the instrument.

[0313] (2) Using the above primers and probes, single-plex PCR was performed to detect human papillomavirus HPV type 58.

[0314] The reaction system used in the detection is shown in Table 12.

[0315] Table 12: Human papillomavirus HPV 58 PCR detection reaction system

[0316]

[0317] Note: 2× PCR reaction buffer includes 3 mM MgCl 2 , 30 mM Tris-HCl buffer, pH 8.3, 0.5 mM dNTPs, and 70 mM (NH 4 ) 2 SO 4 .

[0318] The nucleic acid template is human papillomavirus type 58 plasmid, synthesized by General Biotechnology (Anhui) Co., Ltd., quantified and diluted, and mixed with the reaction system at 50 copies / reaction. At the same time, a control primer (CT) is set, that is, the forward primer (F9) is used to replace two different forward primers (F7 or F8) for the experiment, and then PCR amplification and melting curve analysis are performed. According to the characteristic Tm value of the target in a specific detection channel, it can be judged whether the human papillomavirus HPV58 plasmid exists in the sample.

[0319] Reaction preparation:

[0320] After the nucleic acid template is prepared, the reaction system is configured according to the ratio described in Table 12.

[0321] PCR amplification and melting curve analysis:

[0322] After sealing the PCR tube cap, gently mix the sample, then centrifuge briefly and let it stand at room temperature for 5 minutes. Place the PCR tube in a handheld centrifuge again, centrifuge briefly and transfer it to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. The program used is: 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 10 seconds, 56℃ annealing and extension for 30 seconds, for a total of 45 cycles; 45-85℃ melting curve analysis, heating rate of 0.05℃ per second, light collection.

[0323] Data Analysis:

[0324] The SLAN real-time fluorescence quantitative PCR analysis software produced by Shanghai Hongshi Medical Technology Co., Ltd. was used to interpret the results based on the Tm value of the melting curve. Figure 6-1 and Figure 6-2 All of them are melting curves of human papillomavirus HPV 58. When other reaction reagents and reaction procedures are the same, Figure 6-1 Comparison of melting curves using F7 and F9; Figure 6-2 The following are comparison diagrams of the melting curves using F8 and F9 respectively.

[0325] from Figure 6-1 and Figure 6-2 It can be seen that: using three kinds of forward primers (F7 or F8 or F9), the target nucleic acid can be detected. However, using the primer design mode of the present invention (containing spacer C3 and anti-probe anchoring region in the forward primer, and simultaneously, containing spacer C3 and anti-primer signal detection region in the reverse primer), the peak height of the melting peak is significantly improved. It can be seen that using the primer probe of the present invention, the fluorescence signal intensity can be significantly improved and the target nucleic acid can be better identified.

[0326] Example 7: Nucleic acid detection of human papillomavirus HPV type 52

[0327] (1) The base sequences of the primer probes for detecting human papillomavirus HPV type 52 are shown in Table 13.

[0328] Table 13: Base sequences of primer probes

[0329]

[0330] Among the above primer probes, the forward primer F10 (SEQ ID NO: 23) is a specific primer designed for the human papillomavirus HPV type 52 target sequence. F10 is 58 bp in length, and the 1st to 22nd bases at its 3' end are the target sequence binding region, and the 1st to 15th bases at its 5' end are the probe anchor region (A'), that is, the 1st to 15th base sequence at the 5' end of the probe P4 (SEQ ID NO: 22), and the 19th to 33rd bases at its 5' end are reverse complementary to the 1st to 15th base sequence at the 5' end of the probe P4 (SEQ ID NO: 22), that is, the primer anchor region (A').

[0331] The forward primer F11 (SEQ ID NO: 24) is a specific primer designed for the human papillomavirus HPV type 52 target sequence. The total length of F11 is 40 bp. The 1st to 22nd bases at its 3' end are the target sequence binding region, and the 1st to 15th bases at its 5' end are the probe anchor region (A'), that is, the 1st to 15th base sequence at the 5' end of the probe P4 (SEQ ID NO: 22), which is reverse complementary to the forward primer F10.

[0332] The reverse primer R11 (SEQ ID NO: 25) is a specific primer designed for the human papillomavirus HPV52 target sequence. The total length of R11 is 37 bp. The 1st to 25th bases at its 3' end are the target sequence binding region, and the 1st to 9th bases at its 5' end are the primer signal detection region (h), which is the same as the 51st to 59th base sequence at the 5' end of the probe P4 (SEQ ID NO: 22);

[0333] When the pathogen target is specifically amplified, the forward primer (F10 or F11) and the reverse primer (R11) amplify the target nucleic acid to obtain a double-stranded preamplifier product, wherein a single-stranded preamplification product (S1) contains a probe anchor region (A) and a reverse complementary region (h') of the primer signal detection region; the probe signal detection region (H) of the probe (P4) is complementary to the reverse complementary sequence of the primer signal detection region of the single-stranded preamplification product (S1), and the primer anchor region (A') of the probe is reversely complementary to the probe anchor region (A) at the 5' end of the single-stranded preamplification product (S1). After the amplification is completed, a melting curve analysis is performed. During the temperature change process, the double-stranded product formed by the probe (P4) and the single-stranded product will be melted at a certain temperature, and at this time, the probe (P4) with the first detection group and the second detection group will become a single-stranded state, so that the fluorescent signal changes and can be detected by the instrument.

[0334] (2) Using the above primers and probes, single-plex PCR was performed to detect human papillomavirus HPV type 52.

[0335] The reaction system used in the detection is shown in Table 14.

[0336] Table 14: Human papillomavirus HPV 52 PCR detection reaction system

[0337]

[0338] Note: 2× PCR reaction buffer includes 3 mM MgCl 2 , 30 mM Tris-HCl buffer, pH 8.3, 0.5 mM dNTPs, and 70 mM (NH 4 ) 2 SO 4 .

[0339] The nucleic acid template is human papillomavirus type 52 plasmid, synthesized by General Biotechnology (Anhui) Co., Ltd., quantified and diluted, and mixed with the reaction system at 50 copies / reaction. At the same time, a control primer (CT) is set, that is, the forward primer (F11) is used instead of the forward primer (F10) for the experiment, and then PCR amplification and melting curve analysis are performed. According to the characteristic Tm value of the target in a specific detection channel, it can be judged whether the human papillomavirus HPV52 plasmid exists in the sample.

[0340] Reaction preparation:

[0341] After the nucleic acid template is prepared, the reaction system is configured according to the ratio described in Table 14.

[0342] PCR amplification and melting curve analysis:

[0343] After sealing the PCR tube cap, gently mix the sample, then centrifuge briefly and let it stand at room temperature for 5 minutes. Place the PCR tube in a handheld centrifuge again, centrifuge briefly and transfer it to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. The program used is: 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 10 seconds, 56℃ annealing and extension for 30 seconds, for a total of 45 cycles; 45-85℃ melting curve analysis, heating rate of 0.05℃ per second, light collection.

[0344] Data Analysis:

[0345] The SLAN real-time fluorescence quantitative PCR analysis software produced by Shanghai Hongshi Medical Technology Co., Ltd. was used to interpret the results based on the Tm value of the melting curve. Figure 7 This is the melting curve of human papillomavirus HPV type 52.

[0346] from Figure 7 It can be seen that: using two forward primers (F10 or F11), both can detect the target nucleic acid. However, using the primer design method of the present invention (containing spacer C3 and anti-probe anchor region in the forward primer), the peak height of the melting peak is significantly improved. It can be seen that using the primer probe of the present invention can significantly improve the fluorescence signal intensity and better identify the target nucleic acid.

Claims

1. A primer probe for nucleic acid detection, comprising a first primer, a probe (P) and a second primer, characterized in that: The first primer comprises a target sequence binding region 1; The target sequence binding region 1 is a sequence that can specifically pair and bind with the target sequence; The probe (P) is a sequence that does not pair with any target sequence, and includes a probe signal detection region (H); The second primer comprises an anti-primer signal detection region (h'), an amplification blocking region, a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 is a sequence that can specifically pair and bind to the target sequence, and the primer signal detection region (h) is a sequence that does not pair and bind to any target sequence; the primer signal detection region (h) is complementary to the anti-primer signal detection region (h'), and the complementary sequence of the primer signal detection region (h) specifically pairs and binds to the probe signal detection region (H) of the probe (P); and the amplification blocking region can prevent polymerase extension.

2. The primer probe according to claim 1, characterized in that The first primer further comprises a probe anchor region (A); wherein the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is located at the 5' end of the target sequence binding region 1; The probe (P) further comprises a primer anchor region (A'); the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer.

3. The primer probe according to claim 1, characterized in that The first primer further comprises an amplification blocking region, which is located between the probe anchor region (A) and the target sequence binding region 1, and can prevent polymerase extension.

4. The primer probe according to claim 1, characterized in that The first primer also comprises an anti-probe anchor region (a') and an amplification retardation region; the amplification retardation region is located between the anti-probe anchor region (a') and the probe anchor region (A); the anti-probe anchor region (a') is complementary to the probe anchor region (A), and the amplification retardation region can prevent polymerase extension.

5. A primer probe for nucleic acid detection, comprising a first primer, a probe (P) and a second primer, characterized in that: The first primer comprises an anti-probe anchor region (a'), an amplification retardation region, a probe anchor region (A) and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is complementary to the anti-probe anchor region (a'); the target sequence binding region 1 is a sequence that can specifically pair with the target sequence; the amplification retardation region can prevent polymerase extension; or, The first primer comprises a probe anchor region (A), an amplification retardation region, an anti-probe anchor region (a') and a target sequence binding region 1 from the 5' end to the 3' end; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is complementary to the anti-probe anchor region (a'); the target sequence binding region 1 is a sequence that can specifically pair with the target sequence; the amplification retardation region can prevent the polymerase from extending; The probe (P) is a sequence that does not pair with any target sequence, and includes a primer anchor region (A') and a probe signal detection region (H); the probe (P) is modified with a detection label; the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer; The second primer comprises a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 is a sequence that can specifically pair and bind to the target sequence, the primer signal detection region (h) is a sequence that does not pair and bind to any target sequence, and the complementary sequence of the primer signal detection region (h) specifically pairs and binds to the probe signal detection region (H) of the probe (P).

6. The primer probe according to any one of claims 1 to 5, characterized in that: There is a 0-20 base interval between the amplification blocking region and the adjacent region.

7. The primer probe according to any one of claims 1 to 5, characterized in that: The detection label includes a first detection group and a second detection group, and the first detection group and the second detection group generate a signal change by changing the distance; preferably, the first detection group and the second detection group are spaced 3-250 angstroms apart; preferably, the distance is 3-201 angstroms apart; more preferably, the distance is 3-140 angstroms apart; further preferably, the first detection group is a fluorescent reporter group, and the second detection group is a quenching group or other modification group that can generate a signal change with the first detection group through fluorescence resonance energy transfer.

8. The primer probe according to any one of claims 2 to 5, characterized in that: The probe (P) includes 1-2 primer anchor regions (A') and 1-2 probe signal detection regions (H); Preferably, the probe (P) is any one of the structures 1) to 4); 1) The probe (P) comprises a primer anchor region (A') and a probe signal detection region (H); 2) The probe (P) comprises a probe signal detection region (H) and two primer anchor regions (A'), the two primer anchor regions (A') are respectively located on both sides of the probe signal detection region (H), and the sequences of the two primer anchor regions (A') are different; 3) The probe (P) comprises a primer anchor region (A') and two probe signal detection regions (H), the two probe signal detection regions (H) are respectively located on both sides of the primer anchor region (A'), and the sequences of the two probe signal detection regions (H) are different; 4) The probe (P) comprises two primer anchor regions (A') and two probe signal detection regions (H), wherein the primer anchor regions (A') and the probe signal detection regions (H) are arranged at intervals, the sequences of the two primer anchor regions (A') are different, and the sequences of the two probe signal detection regions (H) are different.

9. The primer probe according to any one of claims 2 to 5, characterized in that: The primer anchor region (A') of the probe (P) is located at the 5' end of the probe signal detection region (H); preferably, the detection group on the probe (P) is modified between the primer anchor region (A') and the probe signal detection region (H).

10. The primer probe according to any one of claims 2 to 5, characterized in that: The primer anchor region (A') of the probe (P) is located at the 3' end of the probe signal detection region (H); preferably, the detection group on the probe (P) is modified at the 5' end of the probe signal detection region (H).

11. A primer probe set for nucleic acid detection, characterized in that: It includes 1 probe (P), at least 1 first primer and at least 2 second primers; The first primer comprises a target sequence binding region 1, and different target sequence binding regions 1 are sequences that can specifically pair and bind with different target sequences; The probe (P) is a sequence that does not pair with any target sequence, and includes a probe signal detection region (H); the probe (P) is modified with a detection label; The at least two second primers are different from each other, and each independently comprises an anti-primer signal detection region (h'), an amplification blocking region, a primer signal detection region (h) and a target sequence binding region 2 from the 5' end to the 3' end; wherein the target sequence binding region 2 in different second primers is a sequence that specifically pairs and binds to different target sequences; the primer signal detection region (h) in different second primers is a sequence that does not pair and bind to any target sequence, and the complementary sequence of the primer signal detection region (h) specifically pairs and binds to the probe signal detection region (H) of the probe (P); the sequences of the primer signal detection region (h) in different second primers are different from each other; the primer signal detection region (h) in the second primer is complementary to the anti-primer signal detection region (h') on the primer; and the amplification blocking region can prevent polymerase extension.

12. The primer probe set according to claim 11, characterized in that: At least one of the first primers further comprises a probe anchor region (A); wherein the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is located at the 5' end of the target sequence binding region 1; The probe (P) further comprises a primer anchor region (A'); the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer.

13. The primer probe set according to claim 11, characterized in that: At least one of the first primers further comprises a probe anchor region (A) and an amplification retardation region; the probe anchor region (A) is a sequence that does not pair with any target sequence, and the probe anchor region (A) is located at the 5' end of the target sequence binding region 1; the amplification retardation region can prevent polymerase extension; the amplification retardation region is located between the probe anchor region (A) and the target sequence binding region 1; The probe (P) further comprises a primer anchor region (A'); the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the first primer.

14. The primer probe set according to claim 12, characterized in that: At least one of the first primers further comprises an anti-probe anchor region (a') and an amplification retardation region; the amplification retardation region is located between the anti-probe anchor region (a') and the probe anchor region (A); the anti-probe anchor region (a') is complementary to the probe anchor region (A), and the amplification retardation region can prevent polymerase extension.

15. A primer-probe composition for nucleic acid detection, characterized in that: It comprises at least one group of primer probes for nucleic acid detection according to any one of claims 1 to 10, and / or comprises at least one group of primer probe sets for nucleic acid detection according to any one of claims 11 to 14; preferably, the sequences of the probes (P) in different groups are different; preferably, the sequences and detection labels of the probes (P) in different groups are different.

16. A kit for nucleic acid detection, characterized in that: It comprises the primer probe for nucleic acid detection as described in any one of claims 1 to 10, and / or, comprises the primer probe group for nucleic acid detection as described in any one of claims 11 to 14, and / or, comprises the primer probe composition as described in claim 15; preferably, it also comprises an amplification reagent; further preferably, the amplification reagent comprises DNA polymerase and dNTPs; further preferably, when the nucleic acid template is RNA, the amplification reagent also comprises reverse transcriptase.

17. A method for nucleic acid detection using the primer-probe combination of claim 15 or the kit of claim 16, characterized in that: It includes the following: The first primer and the second primer are specifically bound to the target nucleic acid respectively, and after amplification, a pre-amplification product having a complementary sequence to the primer signal detection region (h) but not having a complementary sequence to the anti-primer signal detection region (h') is obtained; The complementary sequence of the primer signal detection region (h) in the pre-amplification product is specifically combined with the probe (P) and extended by ≥0 bases to form a secondary amplification double-stranded product; The formation of the double-stranded product causes the probe to produce a detectable signal change, and the presence of the target nucleic acid is determined based on the signal change; and / or The double-stranded product is heated, and the melting of the double-stranded product causes the probe to produce a detectable signal change, and the presence of the target nucleic acid is determined based on the signal change.

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