Primers and their application in the detection of LAMP amplification products

Abstract

Primers and their application in the detection of LAMP amplification products are provided. The primers comprise: a single-stranded region including a first single-stranded sequence having a 3′ free end; and a double-stranded region, wherein the 3′ end of a complementary strand of the double-stranded region is connected to the 5′ end of the first single-stranded sequence.

Description

Primers and their application in detection of LAMP amplification products Technical Field

[0001] The present invention relates to the fields of biomedicine and nucleic acid detection. Specifically, the present application relates to primers and their application in LAMP amplification product detection. More specifically, the present application relates to a primer, an inner primer pair, a loop primer pair, a LAMP primer set, a kit, a nucleic acid amplification method, and a nucleic acid amplification product detection method. Background Art

[0002] In the fields of biological sample analysis and molecular diagnostics, amplification of target genes or nucleotide fragments is a commonly used technique to ensure sufficient sensitivity and specificity. Loop-mediated isothermal amplification (LAMP) is a rapid and efficient self-circulating strand displacement amplification technology developed by Eiken Chemical Co., Ltd. in Japan in 2000. This technology utilizes a specially designed primer combination to construct intermediate products with specific structures through multiple bidirectional strand displacements. These intermediates can form multiple primer annealing sites, thereby initiating superexponential amplification and DNA self-extension. LAMP utilizes a polymerase with strong displacement activity to achieve rapid extension and replication of DNA double strands under mild isothermal conditions (typically a constant temperature between 60 and 68°C). Due to its stable amplification performance and simple operation, LAMP technology has been widely used in infectious disease diagnosis, genetic screening, prenatal diagnosis, cancer detection, environmental monitoring, and other fields. The initial version of the LAMP reaction consists of four primers: F3, B3, FIP, and BIP. With the development of technology, the LAMP reaction rate was further improved by adding two primers, LF and LB, forming the current standard LAMP primer reaction system.

[0003] LAMP (Loop-Mediated Isothermal Amplification) is a nucleic acid amplification technique. Its unique molecular strand displacement amplification principle distinguishes it from qPCR reaction reagents, making it impossible to fluorescently label amplification products using polymerase exo-cleavage activity to cleave specific probes. Currently, detection and characterization methods for LAMP amplification products primarily include turbidimetric methods, color change methods, and nonspecific fluorescent dyes. In turbidimetric methods, pyrophosphate ions produced by the LAMP reaction combine with magnesium ions to form a white precipitate, and the yield of this precipitate is quantified in real time by the turbidity change of the reaction solution. Colorimetric methods use self-luminescent dyes such as hydroxynaphthol blue (HNB) and calcein. HNB binds to magnesium ions, resulting in an initial violet coloration of the reaction system. However, as the reaction proceeds, pyrophosphate ions combine with magnesium ions to form a precipitate, causing the HNB to change from violet to sky blue. This color change indicates the progress of the amplification reaction. Calcein operates on a similar principle, requiring the presence of manganese ions. As pyrophosphate ions produced by the LAMP reaction bind to manganese ions, free calcein, upon binding to magnesium ions, emits yellow-green fluorescence. The color change is then used to monitor the progress of the reaction. The nonspecific fluorescent dye method uses SYBR Green dye to bind to double-stranded DNA and emit light, but not single-stranded DNA, thereby marking the increase in amplification product during the LAMP reaction. Its fluorescence intensity is positively correlated with the increase in product. However, these detection methods share a common drawback: they cannot distinguish between specific product amplification and primer dimers or nonspecific amplification. This drawback can lead to false-positive results caused by nonspecific amplification, limiting the practical application of LAMP technology.

[0004] Therefore, the detection method of LAMP amplification products still needs to be improved.

[0005] Summary of the Invention

[0006] The present application aims to solve at least one of the problems in the prior art. To this end, the present application proposes a primer capable of specifically detecting LAMP amplification products.

[0007] In its first aspect, the present application provides a primer. According to embodiments of the present application, the primer comprises: a single-stranded region comprising a first single-stranded sequence having a free 3' end; and a double-stranded region, wherein the 3' end of a complementary strand of the double-stranded region is connected to the 5' end of the first single-stranded sequence. In some examples of the present application, the primer can be used for real-time detection of LAMP amplification products, exhibiting advantages such as short detection time, strong specificity, high sensitivity, the ability to stably quench short fragments, and strong versatility.

[0008] It should be noted that the phrase "the first single-stranded sequence has a 3' free end" in the present application means that there is a free nucleotide at the 3' end of the first single-stranded sequence. The free nucleotide cannot be modified with a fluorescent group or a quenching group, and cannot be linked to other nucleotide sequences.

[0009] In a second aspect of the present application, a set of inner primer pairs is provided. According to embodiments of the present application, at least one primer in the inner primer pair is as defined in the first aspect of the present application. In some examples of the present application, the inner primer pair can be used for real-time detection of LAMP amplification products, with advantages such as short detection time, strong specificity, high sensitivity, stable quenching of short fragments, and strong versatility.

[0010] In a third aspect of the present application, a set of loop primer pairs is provided. According to embodiments of the present application, at least one primer in the loop primer pair is as defined in the first aspect of the present application. In some examples of the present application, the loop primer pair can be used for real-time detection of LAMP amplification products, with advantages such as short detection time, strong specificity, high sensitivity, stable quenching of short fragments, and strong versatility.

[0011] In a fourth aspect of the present application, a LAMP primer set is provided. According to embodiments of the present application, the LAMP primer set includes an outer primer pair, an inner primer pair, and a loop primer pair, wherein at least one primer of at least one of the inner primer pair and the loop primer pair is as defined in the first aspect of the present application. In some examples of the present application, the LAMP primer set can be used in a LAMP amplification reaction. It can achieve real-time detection of LAMP amplification products and has advantages such as short detection time, strong specificity, high sensitivity, stable quenching of short fragments, and strong versatility.

[0012] In a fifth aspect, this application provides a kit. According to embodiments of this application, the kit includes: the primers described in the first aspect of this application; or the inner primer pair described in the second aspect; or the loop primer pair described in the third aspect; or the LAMP primer set described in the fourth aspect. In some examples of this application, the kit is used for real-time detection of LAMP amplification products, offering advantages such as simplicity and speed.

[0013] In its sixth aspect, the present application provides a nucleic acid amplification method. According to embodiments of the present application, the method comprises: using a nucleic acid sample to be amplified as a template, and performing an amplification reaction using the primers described in the first aspect, the inner primer pair described in the second aspect, the loop primer pair described in the third aspect, the LAMP primer set described in the fourth aspect, or the kit described in the fifth aspect. Using this method for LAMP amplification reactions enables real-time detection of LAMP amplification products with short detection times, strong specificity, and high sensitivity.

[0014] In its seventh aspect, the present application provides a method for detecting nucleic acids. According to embodiments of the present application, the method comprises: amplifying a test nucleic acid using the method described in the sixth aspect of the present application, detecting a fluorescent signal from the amplified product; and determining the test nucleic acid based on the fluorescent signal. In some examples of the present application, this method for detecting nucleic acid amplification products enables rapid, highly specific, and highly sensitive real-time detection of the amplified products.

[0015] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0017] FIG1 is a schematic diagram of the primer reaction and pairing principle of loop-mediated isothermal amplification (LAMP) according to one embodiment of the present application (hairpin primer);

[0018] FIG2 is a schematic diagram of the primer reaction and pairing principle of loop-mediated isothermal amplification (LAMP) according to one embodiment of the present application (double-labeled double-stranded structure primers);

[0019] FIG3 is a schematic diagram of the primer reaction and pairing principle of loop-mediated isothermal amplification (LAMP) according to one embodiment of the present application (double-labeled double-stranded + adapter structure primer);

[0020] FIG4 is a schematic diagram of the primer reaction and pairing principle of loop-mediated isothermal amplification (LAMP) according to one embodiment of the present application (dual-labeled adapter structure primers);

[0021] FIG5 is a schematic diagram of the detection results of the double-labeled hairpin loop primer method according to one embodiment of the present application;

[0022] FIG6 is a schematic diagram of the detection results of the double-labeled double-stranded structure primer method according to one embodiment of the present application;

[0023] FIG7 is a schematic diagram of the detection results of the double-labeled double-stranded structure primer method according to one embodiment of the present application;

[0024] FIG8 is a schematic diagram of the detection results of the double-labeled adapter structure primer method according to one embodiment of the present application.

[0025] The above figures are for illustrative purposes only and are not intended to limit the scope of the present invention. In the accompanying drawings, the dimensions of some components may be exaggerated and not drawn to scale for illustrative purposes. The dimensions and relative dimensions do not necessarily correspond to actual dimensions in the practice of the present invention. DETAILED DESCRIPTION

[0026] As used herein, unless otherwise indicated, the singular forms "a," "an," and the like include plural referents (more than one); "a set" or "a plurality" refers to two or more.

[0027] In this document, unless otherwise specified, the terms “comprise” or “include” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0028] In this document, unless otherwise specified, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated; features specified as "first", "second", etc. may explicitly or implicitly include one or more of the said features.

[0029] Unless otherwise indicated herein, the nucleotide sequences of primers, primer pairs or primer sets are written from left to right in the 5' to 3' direction.

[0030] Herein, unless otherwise specified, "upstream or downstream" refers to upstream or downstream at the 5' end or 3' end.

[0031] Currently, the detection methods of LAMP amplification products mainly include molecular beacon method, quenching of unincorporated amplification signal reporters (QUASR) method and assimilating probe method.

[0032] A molecular beacon is a target-specific dual-labeled probe modified with a fluorophore and a quencher at each end. In its free state, the 3' end is complementary to its 5' end, allowing the probe to spontaneously form a hairpin structure. In the absence of an amplicon, the molecular beacon exists as a closed ring due to intramolecular hybridization between the 5' and 3' ends. Here, the fluorophore and quencher are in close proximity, resulting in fluorescence quenching. In the presence of an amplicon, the probe hybridizes to the amplicon, causing the ring to open, separating the fluorophore and quencher and releasing fluorescence.

[0033] There are two approaches to detecting LAMP amplification products using molecular beacons: The first is that the molecular beacon does not participate in product amplification. This approach introduces a new molecular beacon probe, which increases the complexity of the primers and leads to decreased detection performance. Furthermore, the molecular beacon can be displaced and freed due to the chain displacement reaction principle of LAMP itself, thus affecting the final fluorescence signal. The second approach is that the molecular beacon participates in product amplification. The traditional molecular beacon design under this approach results in the 3' end of the hairpin structure being completely closed. After the temperature rises, some primers remain undissociated, resulting in a decrease in the number of primers involved in the reaction or insufficient reaction efficiency, which in turn increases the detection time and reduces the detection sensitivity.

[0034] The QUASR method is designed for LAMP endpoint detection schemes. The QUASR method uses a primer modified with a fluorescent group at its 5' end and a short quencher probe of about 7-13 nucleotides in length modified at the 3' end. The 3' end of the short quencher probe is complementary to the 5' end of the modified primer, and the half-melting temperature Tm of the double-stranded quencher reporter probe formed before the amplification is initiated must be significantly lower than the temperature of LAMP (such as below 50°C) to ensure that the short quencher probe is free ssDNA after LAMP is initiated to avoid affecting the amplification speed and efficiency. After LAMP is terminated, the reaction is cooled to room temperature, and the still free 5'-end fluorescent modified primer (if still present) and the short quencher probe will hybridize again. At this time, no fluorescent signal will be emitted due to the quenching effect. Once the target DNA is amplified, the 5'-end fluorescent modified primer will be incorporated into the amplicon, making it impossible for the short quencher probe to approach again, so a fluorescent signal will appear, thereby performing product characterization.

[0035] The disadvantage of the QUASR method is that it cannot maintain its own quenching state at 65°C, making it impossible to achieve real-time detection at a constant temperature of 65°C. The product can only be detected through the end-point fluorescence state.

[0036] The assimilation probe is a dimer composed of a pair of partially complementary primer probes. The 3' end of one of the long primer probes contains a specific target sequence and participates in the subsequent LAMP amplification reaction. Its 5' end contains a universal sequence modified with a fluorophore. This universal sequence complements the universal sequence of a short primer probe, forming a dimer. The 5' end of the short primer sequence is modified with a quencher. In the dimer state, the fluorophore and quencher groups are in close proximity, resulting in fluorescence quenching. During the LAMP reaction, the target-specific 3' primer sequence of the long primer probe participates in the amplification process. During the binding and extension of the reverse primer, the quencher-modified short primer strand is displaced, separating the quencher and fluorophore, leading to fluorescence release.

[0037] To ensure that assimilation probes remain in a dimer state at the LAMP reaction temperature of 65°C, they require a longer complementary pairing sequence, typically maintaining a sequence length of 20 to 30 base pairs. When using assimilation probes for LAMP nucleic acid amplification, the amplification rate is inhibited compared to traditional six-primer LAMP reactions, resulting in longer detection times and decreased sensitivity.

[0038] The various LAMP amplification product detection methods described above exhibit different characteristics due to their different principles. In addition to low sensitivity and specificity, these methods also face challenges such as long detection times, difficulty in probe design, support for only a single detection scheme (such as endpoint detection), and low versatility.

[0039] In order to solve at least one of the above problems. In one aspect of the present application, a primer is proposed. The primer comprises: a single-stranded region including a first single-stranded sequence, wherein the first single-stranded sequence has a 3' free end; and a double-stranded region, wherein the 3' end of a complementary strand of the double-stranded region is connected to the 5' end of the first single-stranded sequence. The inventors conducted in-depth research on existing LAMP amplification product detection methods and designed primers capable of real-time detection of LAMP amplification products. After extensive experimental verification, it was found that the primer has high sensitivity and specificity in the detection of LAMP amplification products.

[0040] In some examples of the present application, the double-stranded region of the primer includes a second single-stranded sequence, and the second single-stranded sequence is suitable for forming a stem-loop structure. The formation of the stem-loop structure can increase the Tm value of the primer and help improve the specificity of the reaction.

[0041] In some other examples of the present application, the double-stranded region of the primer includes a third single-stranded sequence and a fourth single-stranded sequence, and at least a portion of the third single-stranded sequence is suitable for complementary pairing with at least a portion of the fourth single-stranded sequence.

[0042] It should be noted that “at least partially complementary pairing” includes complementary pairing of a portion of the third single-stranded sequence with a portion of the fourth single-stranded sequence or complete complementary pairing of the third single-stranded sequence with the fourth single-stranded sequence.

[0043] In some examples of the present application, the primer is at least one of an inner primer pair and / or a loop primer pair of a LAMP reaction.

[0044] In some examples of the present application, the primer Tm value is selected from 64 to 81° C., preferably 68 to 75° C. Similarly, in other examples of the present application, the Tm value is optionally 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., or 81° C.

[0045] In some examples of the present application, the primer has a fluorescent group and a quencher group, and the quencher group is suitable for quenching the fluorescent group.

[0046] It should be noted that "the quencher is suitable for quenching the fluorescent group" means that the quencher in the primer is designed to interact with the fluorescent group to extinguish the fluorescent signal of the fluorescent group under certain conditions. The "certain conditions" mentioned above refer to the proximity of the fluorescent group and the quencher.

[0047] In some examples of the present application, the fluorescent group and the quencher group are independently disposed at positions no more than 5 bp away from the endpoints of the two complementary strands in the double-stranded region, preferably no more than 3 bp away. According to a specific embodiment of the present invention, the fluorescent group and the quencher group are independently disposed near the endpoints of the two complementary strands in the double-stranded region. For example, the fluorescent group is disposed at the 5' end of one complementary strand in the double-stranded region, and the quencher group is disposed at the 3' end of the other complementary strand in the double-stranded region. In this case, the fluorescent group and the quencher group disposed at the endpoints of the two complementary strands in the double-stranded region are close to each other, and the fluorescence is in a quenched state.

[0048] It should be noted that the bases labeled with the fluorescent group and quencher group can be located in either the double-stranded region or the single-stranded region. For example, the base labeled with the fluorescent group or quencher group can be located up to 5 bp to the left and right of the junction of the double-stranded region and the single-stranded region, such as 4 bp, 3 bp, 2 bp, or 1 bp. In this case, the fluorescent group or quencher group can be modified in either the single-stranded region or the double-stranded region.

[0049] In other examples of the present application, the fluorescent group and the quencher group are independently arranged at an end point of the double-stranded region and a position of no more than 5bp near the connection between the double-stranded region and the single-stranded region, preferably a position of no more than 3bp. In some specific examples of the present application, the fluorescent group is modified on a base at a position no more than 5bp away from the connection between the double-stranded region and the single-stranded region, and the quencher group is modified on the 5' end of the complementary chain in which the double-stranded region is not connected to the single-stranded region. In other specific examples of the present application, the quencher group is modified on a base at a position no more than 5bp away from the connection between the double-stranded region and the single-stranded region, and the fluorescent group is modified on the 5' end of the complementary chain in which the double-stranded region is not connected to the single-stranded region.

[0050] In other examples of the present application, the fluorescent group and the quencher group are independently arranged at one end point of the double-stranded region and the position of the T base closest to the connection between the double-stranded region and the single-stranded region.

[0051] By setting the positions of the fluorescent group and the quenching group as described above, it is ensured that in the absence of target DNA, the fluorescent group and the quenching group on the primer are close to each other in position and the fluorescence is in a quenched state. In the presence of target DNA, the complementary chain of the double-stranded region is opened, and the fluorescent signal is released or enhanced, thereby achieving accurate and sensitive real-time detection of the target DNA.

[0052] In the present application, the quencher is selected from at least one of BHQ1, BHQ2, BHQ3, MGB, BBQ 650, Dabcyl, DBQ1, TAMRA, and Eclipse; and the fluorescent group is selected from at least one of FAM, HEX, ROX, CY5Cy5, Atto 425, TET, JOE, VIC, R6G, Yakima Yellow, Quasar 570, Quasar 670, Cy3, NED, Cy5.5, Cy7, Texas Red, Atto 590, IR Dye 650, and IR Dye 750. In some examples of the present application, neither the fluorescent group nor the quencher affects the normal LAMP reaction. Furthermore, the fluorescent group and quencher must be used in pairs; the quencher attached to the primer can extinguish the fluorescence emitted by the fluorescent group.

[0053] In the present application, the Tm value of the primer can also be increased by modifying the nucleotides in the primer or replacing special nucleotides. In some examples of the present application, the primer includes at least one special nucleotide or modifying group. In some preferred examples of the present application, the double-stranded region of the primer includes at least one special nucleotide or modifying group. Wherein, the special nucleotide includes at least one selected from locked nucleic acid, peptide nucleic acid, ribonucleotide and deoxyinosinic acid; the modifying group includes at least one selected from phosphate group, methyl group and sugar group. By modifying the locked nucleic acid or adding a modifying group to increase the Tm value of the primer, real-time characterization of the LAMP amplification reaction by fluorescent signal can be achieved.

[0054] In some examples of the present application, the double-stranded region includes a second single-stranded sequence having a stem-loop structure, wherein at least a portion of the sequence upstream of the 3' end of the second single-stranded sequence is complementary to at least a portion of the sequence downstream of the 5' end of the second single-stranded sequence, and the 3' end of the second single-stranded sequence is connected to the 5' end of the first single-stranded sequence. One exemplary primer structure is shown in Figure 1.

[0055] In some examples of this application, the positions of the fluorescent group and the quenching group in the primer can be interchanged. This includes the following situations:

[0056] (1) The 5' end of the second single-stranded sequence has the fluorescent group, and the position of up to 5 bp near the junction of the double-stranded region and the single-stranded region has the quenching group;

[0057] (2) the 5′ end of the second single-stranded sequence has the quenching group, and the position of up to 5 bp near the junction of the double-stranded region and the single-stranded region has the fluorescent group;

[0058] (3) The 5′ end of the second single-stranded sequence has the fluorescent group, and the position of the T base closest to the junction of the double-stranded region and the single-stranded region has the quencher group;

[0059] (4) The 5' end of the second single-stranded sequence has the quencher group, and the position of the T base closest to the connection between the double-stranded region and the single-stranded region has the fluorescent group.

[0060] In some examples of the present application, in the primer of the double-stranded region having a stem-loop structure, the first single-stranded sequence has 1 to 30 nucleotides. Wherein, the first single-stranded sequence is optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In a preferred example of the present application, the first single-stranded sequence has 12 nucleotides. The first single-stranded sequence is of great significance for real-time characterization of the LAMP reaction.

[0061] In some examples of the present application, in a primer having a double-stranded region with a stem-loop structure, at least 6 to 30 nucleotides upstream of the 3' end of the second single-stranded sequence are complementary to at least 6 to 30 nucleotides downstream of the 5' end of the second single-stranded sequence. The nucleotide length of the complementary pairing region can optionally be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In a preferred example of the present application, the complementary pairing region is 10 nucleotides in length.

[0062] In a primer having a double-stranded region with a stem-loop structure, the second single-stranded sequence includes a stem and a loop structure. In some examples of the present application, under the premise that the stem and loop structure sequences are of the same length, increasing the length of the first single-stranded sequence can increase the LAMP reaction rate.

[0063] In some examples of the present application, in a primer having a double-stranded region with a stem-loop structure, the loop length of the stem-loop structure is selected from 2 to 10 nucleotides. The loop length of the stem-loop structure can optionally be 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In a preferred example of the present application, the stem-loop length is 4 nucleotides.

[0064] In some examples of the present application, in a primer having a double-stranded region with a stem-loop structure, under the premise that the stem length in the stem-loop structure is consistent with the length of the first single-stranded sequence, increasing the loop length in the stem-loop structure can increase the LAMP reaction rate.

[0065] In some examples of the present application, the primers have nucleotide sequences as shown in SEQ ID NOs: 1 to 9. The specific sequences are shown in Table 1.

[0066] In some examples of the present application, the double-stranded region includes a third single-stranded sequence and a fourth single-stranded sequence, at least a portion of the third single-stranded sequence is complementary to at least a portion of the fourth single-stranded sequence, and the 3' end of the third single-stranded sequence is connected to the 5' end of the first single-stranded sequence, or the 3' end of the fourth single-stranded sequence is connected to the 5' end of the first single-stranded sequence. One exemplary primer structure is shown in Figure 2 or Figure 4.

[0067] In some examples of this application, the positions of the fluorescent group and the quenching group in the primer can be interchanged. This includes the following situations:

[0068] (1) The 3' end of the third single-stranded sequence is connected to the 5' end of the first single-stranded sequence, the 5' end of the third single-stranded sequence has the fluorescent group, and the 3' end of the fourth single-stranded sequence has the quenching group;

[0069] (2) The 3' end of the third single-stranded sequence is connected to the 5' end of the first single-stranded sequence, the 5' end of the third single-stranded sequence has the quencher group, and the 3' end of the fourth single-stranded sequence has the fluorescent group;

[0070] (3) the 3' end of the fourth single-stranded sequence is connected to the 5' end of the first single-stranded sequence, the 5' end of the fourth single-stranded sequence has the fluorescent group, and the 3' end of the third single-stranded sequence has the quenching group;

[0071] (4) The 3' end of the fourth single-stranded sequence is connected to the 5' end of the first single-stranded sequence, the 5' end of the fourth single-stranded sequence has the quenching group, and the 3' end of the third single-stranded sequence has the fluorescent group.

[0072] In some examples of the present application, the third single-stranded sequence is composed of a first primer sequence, and the fourth single-stranded sequence is composed of a second primer sequence. One exemplary primer structure is shown in Figure 2. In this case, the first primer sequence and the second primer sequence are at least partially complementary.

[0073] In some examples of the present application, when the third single-stranded sequence consists of a first primer sequence and the fourth single-stranded sequence consists of a second primer sequence, the first single-stranded sequence has 1 to 30 nucleotides. Wherein, the first single-stranded sequence is optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In a preferred example of the present application, the first single-stranded sequence has 1 nucleotide. The first single-stranded sequence is important for real-time characterization of the LAMP reaction.

[0074] In some examples of the present application, when the third single-stranded sequence is composed of a first primer sequence and the fourth single-stranded sequence is composed of a second primer sequence, at least 6 to 30 nucleotides of the third single-stranded sequence are complementary to at least 6 to 30 nucleotides of the fourth single-stranded sequence. The nucleotide length of the complementary pairing region is optionally 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In a preferred example of the present application, the complementary pairing region is 8 to 20 nucleotides long. In a specific example of the present application, the complementary pairing region is 10 nucleotides long. By adjusting the length of the complementary region, the LAMP reaction rate is changed.

[0075] In some examples of the present application, at least a portion of the sequence downstream of the 5' end of the third single-stranded sequence or the fourth nucleic acid sequence includes at least one special nucleotide or modification group, wherein the special nucleotide or modification group remains the same as above and is not further described here.

[0076] In some examples of the present application, the primers have nucleotide sequences as shown in SEQ ID NOs: 10 to 21. The specific sequences are shown in Table 1.

[0077] In some examples of the present application, the third single-stranded sequence is composed of a first primer sequence and a first adapter, and the fourth single-stranded sequence is composed of a second primer sequence and a second adapter, wherein the first primer sequence is connected to the first adapter, and the second primer sequence is connected to the second adapter. One exemplary primer structure is shown in Figure 3. In this case, the first primer sequence and the second primer sequence are at least partially complementary, and the first adapter and the second adapter are at least partially complementary.

[0078] In some examples of the present application, when the third single-stranded sequence consists of a first primer sequence and a first adapter, and the fourth single-stranded sequence consists of a second primer sequence and a second adapter, the first single-stranded sequence has 1 to 30 nucleotides. Wherein, the first single-stranded sequence is optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In a preferred example of the present application, the first single-stranded sequence has 21 nucleotides. The first single-stranded sequence is of great significance for real-time characterization of the LAMP reaction.

[0079] In some examples of the present application, when the third single-stranded sequence is composed of a first primer sequence and a first adapter, and the fourth single-stranded sequence is composed of a second primer sequence and a second adapter, at least 6 to 30 nucleotides of the third single-stranded sequence are complementary to at least 6 to 30 nucleotides of the fourth single-stranded sequence. Wherein, the nucleotide length of the complementary pairing region is optionally 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In a preferred example of the present application, the complementary pairing region is 8 to 20 nucleotides in length. In a specific example of the present application, the complementary pairing region is 8 nucleotides in length. By adjusting the length of the complementary region, the LAMP reaction rate is changed.

[0080] In some examples of the present application, at least a portion of the third single-stranded sequence or the fourth nucleic acid sequence includes at least one special nucleotide or modification group, wherein the special nucleotide or modification group remains the same as above and is not further described here.

[0081] In some examples of the present application, the third single-stranded sequence is composed of a first adapter, and the fourth single-stranded sequence is composed of a second adapter. One exemplary primer structure is shown in Figure 4. In this case, the first adapter and the second adapter are at least partially complementary.

[0082] In some examples of the present application, the third single-stranded sequence is composed of a first linker, and the fourth single-stranded sequence is composed of a second linker. In some examples of the present application, the primer has a nucleotide sequence as shown in SEQ ID NOs: 22 to 37. The specific sequences are shown in Table 1.

[0083] The first single-stranded sequence has 1 to 30 nucleotides. The first single-stranded sequence can optionally be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In a preferred example of the present application, the first single-stranded sequence has 21 nucleotides. The first single-stranded sequence is important for real-time characterization of the LAMP reaction.

[0084] In some examples of the present application, at least 6 to 30 nucleotides of the third single-stranded sequence are complementary to at least 6 to 30 nucleotides of the fourth single-stranded sequence. The nucleotide length of the complementary pairing region is optionally 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In a preferred example of the present application, the complementary pairing region is 8 to 20 nucleotides in length. In a specific example of the present application, the complementary pairing region is 8 nucleotides in length. By adjusting the length of the complementary region, the LAMP reaction rate is changed.

[0085] In some examples of the present application, at least a portion of the third single-stranded sequence or the fourth single-stranded sequence includes at least one special nucleotide or modification group, wherein the special nucleotide or modification group remains the same as above and is not further described here.

[0086] Table 1

[0087] Note: Nucleotides marked with + indicate locked nucleic acid modification, and bases in italics indicate quencher moiety.

[0088] In another aspect of the present application, an inner primer pair is provided. At least one primer in the inner primer pair is as defined in the first aspect of the present application. In some examples of the present application, the inner primer pair can be used for real-time detection of LAMP amplification products, exhibiting advantages such as short detection time, strong specificity, high sensitivity, the ability to stably quench short fragments, and strong versatility.

[0089] In another aspect of the present application, a set of loop primer pairs is provided. At least one primer in the loop primer pair is as described above. In some examples of the present application, the loop primer pairs can be used for real-time detection of LAMP amplification products, exhibiting advantages such as short detection time, strong specificity, high sensitivity, the ability to stably quench short fragments, and strong versatility.

[0090] In another aspect of the present application, a LAMP primer set is provided. The LAMP primer set comprises an outer primer pair, an inner primer pair, and a loop primer pair, wherein at least one primer in at least one of the inner primer pair and the loop primer pair is as described above. In some examples of the present application, the LAMP primer set can be used in a LAMP amplification reaction. It can achieve real-time detection of LAMP amplification products and has advantages such as short detection time, strong specificity, high sensitivity, stable quenching of short fragments, and strong versatility.

[0091] In another aspect of the present application, a kit is provided. The kit includes: the primers described above; or the inner primer pair described above; or the loop primer pair described above; or the LAMP primer set described above. In some examples of the present application, the kit is used for real-time detection of LAMP amplification products, offering advantages such as simplicity and speed.

[0092] In some examples of the present application, the kit further comprises at least one selected from the group consisting of a strand-displacing DNA polymerase, a reverse transcriptase, a ribonuclease inhibitor, dNTPs, a pyrophosphatase, a protective agent, and a reducing agent.

[0093] In another aspect, the present application provides a nucleic acid amplification method. The method comprises: using a nucleic acid sample to be amplified as a template, and performing an amplification reaction using the aforementioned primers, the aforementioned inner primer pair, the aforementioned loop primer pair, the aforementioned LAMP primer set, or the aforementioned kit. Using this method for performing a LAMP amplification reaction enables real-time detection of LAMP amplification products with short detection times, strong specificity, and high sensitivity.

[0094] In some examples of the present application, the amplification reaction includes a step selected from loop-mediated amplification.

[0095] In some examples of the present application, the amplification reaction is carried out in an amplification reaction system, which includes a chain displacement DNA polymerase, dNTPs, bovine serum albumin, at least one of MgSO4, and an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer LF, and a loop primer LB.

[0096] In some examples of the present application, the concentration of the strand-displacing DNA polymerase in the amplification reaction system is 0.1 to 0.8 U / μL. In some examples of the present application, the concentration of the strand-displacing DNA polymerase can optionally be 0.1 U / μL, 0.2 U / μL, 0.3 U / μL, 0.4 U / μL, 0.5 U / μL, 0.6 U / μL, 0.7 U / μL, or 0.8 U / μL. The inventors have experimentally verified that when the concentration of the strand-displacing DNA polymerase is 0.27 U / μL, the LAMP reaction amplification efficiency is higher.

[0097] In some examples of the present application, the concentration of the dNTPs in the amplification reaction system is 1 to 3 mM. In some examples of the present application, the concentration of the dNTPs can optionally be 1 mM, 1.125 mM, 1.5 mM, 2 mM, or 3 mM. In a preferred example of the present application, the concentration of the dNTPs is 1.125 mM.

[0098] In some examples of the present application, the concentration of the bovine serum albumin in the amplification reaction system is 0.1 to 25 mg / mL. In some examples of the present application, the concentration of the bovine serum albumin can optionally be 0.1 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, or 25 mg / mL. In a preferred example of the present application, the concentration of the bovine serum albumin is 10 mg / mL.

[0099] In some examples of the present application, the concentration of the outer primer F3 in the amplification reaction system is 0.1 to 0.5 μM. In some examples of the present application, the concentration of the outer primer F3 is optionally 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, or 0.5 μM. In a preferred example of the present application, the concentration of the outer primer F3 is 0.2 μM.

[0100] In some examples of the present application, the concentration of the outer primer B3 in the amplification reaction system is 0.1 to 0.5 μM. In some examples of the present application, the concentration of the outer primer B3 is optionally 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, or 0.5 μM. In a preferred example of the present application, the concentration of the outer primer B3 is 0.2 μM.

[0101] In some examples of the present application, the concentration of the inner primer FIP in the amplification reaction system is 0.1 to 0.5 μM. In some examples of the present application, the concentration of the inner primer FIP is optionally 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, or 0.5 μM. In a preferred example of the present application, the concentration of the inner primer FIP is 0.16 μM.

[0102] In some examples of the present application, the concentration of the inner primer BIP in the amplification reaction system is 0.1 to 0.5 μM. In some examples of the present application, the concentration of the inner primer BIP is optionally 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, or 0.5 μM. In a preferred example of the present application, the concentration of the inner primer BIP is 0.16 μM.

[0103] In some examples of the present application, the concentration of the loop primer LF in the amplification reaction system is 0.1 to 1 μM. In some examples of the present application, the concentration of the loop primer LF is optionally 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1 μM. In a preferred example of the present application, the concentration of the loop primer LF is 0.8 μM.

[0104] In some examples of the present application, the concentration of the loop primer LB in the amplification reaction system is 0.1 to 1 μM. In some examples of the present application, the concentration of the loop primer LB is optionally 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM or 1 μM. In a preferred example of the present application, the concentration of the loop primer LB is 0.6 μM.

[0105] In some examples of this application, the amplification reaction is carried out at 60-68°C. At this reaction temperature, the complementary primer chains open to form two single-stranded DNAs (ssDNAs), generating a fluorescent signal. In some preferred examples of this application, the amplification reaction is carried out at 65°C. The inventors, taking into account the signal-to-noise ratio of the fluorescent signal and the time required for amplification, ultimately determined that the optimal amplification temperature is 65°C.

[0106] In another aspect, this application provides a method for detecting nucleic acids. The method comprises: amplifying a test nucleic acid using the method described above; detecting a fluorescence signal from the amplified product; and identifying the test nucleic acid based on the fluorescence signal. In some examples of this application, this method can be used to detect nucleic acid amplification products, enabling rapid, highly specific, and highly sensitive real-time detection of the amplified products.

[0107] Exemplarily, the nucleic acid amplification product detection method includes the following steps:

[0108] Template Preparation: Collect and preserve nucleic acid samples. Prepare the template solution for testing based on the sample type, such as RNA, cDNA, or dsDNA. Sample processing methods include RNA or DNA magnetic bead extraction, RNA or DNA silica gel spin column extraction, and reverse transcription of RNA samples into cDNA. Depending on the sample processing requirements, manual or automated platforms can be used for experimental operations.

[0109] Prepare the amplification reaction solution: Synthesize the aforementioned primers, dilute or reconstitute the primer solution to a predetermined volume and concentration, and mix with the template solution to form the amplification reaction solution. Alternatively, directly add the template solution and reconstitute to form an amplification reaction solution of the predetermined volume and concentration. The above amplification reaction solution preparation process can be performed manually or using an automated platform.

[0110] Real-time detection program setting: Different real-time detection programs are set according to the different types of samples to be detected. A separate reverse transcription program can be set for RNA samples, or the reaction temperature can be increased and combined with the loop-mediated amplification program into a single-step program. Normally, the temperature of the reverse transcription program is a constant temperature in the range of 40 to 65°C, which lasts for 2 to 20 minutes, and the temperature of the loop-mediated amplification program is a constant temperature in the range of 60 to 68°C, which lasts for 10 to 40 minutes. During this period, several fluorescence signal reading points can be set (the number is adjusted according to the time and performance requirements of the test kit, and considering the limitations of the real-time signal processing algorithm, it is usually 5 to 40 times), and the reading temperature is a constant temperature in the range of 20 to 68°C (this temperature can ensure that the fluorescent probe of the nucleotide length is paired with the modified primer to form a fluorescence-quenched double-stranded probe to mark the consumption of the modified primer in the loop-mediated amplification), and the reading time is greater than or equal to the inherent minimum time required by the equipment. The setting of the fluorescence signal detection channel is the same as that of the conventional multi-channel real-time fluorescence PCR program. The detection channel with the fluorescent probe wavelength supported by the hardware and software of the real-time detection equipment and corresponding to each sample to be detected in the amplification reaction solution is selected. For example, 1 to 4 detection channels such as FAM, HEX (VIC), ROX, CY5, etc. are selected for real-time signal acquisition.

[0111] On-device amplification: The amplification reaction solution is heated in situ and monitored in real time, or transferred to a device or apparatus equipped with constant temperature incubation and real-time detection capabilities to run the real-time detection process. This includes, but is not limited to, real-time fluorescence PCR instruments, various miniaturized nucleic acid detection devices, and custom-built fluorescence detection platforms, such as the ThermoFisher ABI 7500, Shanghai Hongshi SLAN-96S, Hangzhou Biori QuantGene 9600, and MGI GenCase. Amplification reaction solutions containing sufficient template content undergo full loop-mediated amplification under appropriate temperature conditions. Amplification reaction solutions containing template content below or equal to the detection limit have a certain probability of not undergoing loop-mediated amplification. The template content in a given reaction is positively correlated with the probability of amplification. The time required to complete amplification varies depending on the type and quantity of template, the performance of the primer set and probe, and the hardware and software capabilities of the equipment performing the real-time detection process, ranging from 5 to 90 minutes. Simultaneously with signal acquisition, the real-time detection signal data is recorded or saved.

[0112] Real-time signal processing: The recorded real-time detection signal data is processed using the equipment or device's accompanying software. This data is used to calculate and determine the amplification results, and to obtain parameters of interest related to the amplification reaction. This step can be performed after or concurrently with Step D. Beneficial effects

[0113] 1) The detection method of the present application realizes real-time fluorescence-specific detection of LAMP amplification products. During this process, the primers carrying fluorescent groups can participate in LAMP amplification without affecting the normal LAMP reaction, and the detection results can be displayed and judged in real time.

[0114] 2) By changing the structure of the primers, the rate and sensitivity of the LAMP reaction can be adjusted.

[0115] 3) The quenching group used in this application is relatively short. By introducing nucleotide modifications, the primer design is simplified, the versatility is improved, and the stability of the quenching is maintained.

[0116] The embodiments of the present invention will be described in more detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not specify the manufacturer are conventional products that can be obtained commercially.

[0117] Example 1: Real-time fluorescence LAMP detection of African swine fever virus - double-labeled hairpin structure loop primer method

[0118] 1. Primer and Probe Design: This example uses African swine fever virus (ASF) as the detection target and the B646L gene of ASF virus as the detection site for LAMP primer design. Based on the principles of LAMP primer design, three pairs of primers (outer primer, inner primer, and loop primer) were designed and selected for LAMP amplification. Their sequences are shown in Table 2. One of the loop primers, the LB primer, was further designed with a hairpin structure and modified with fluorescent and quenching groups.

[0119] Table 2

[0120] Note: LB is designed as a hairpin structure. The underlined portion of each primer in the table represents the complementary pairing sequence, which forms the stem of the hairpin structure. The 5' end of the nine primers, LB-2 to LB-11, is modified with the fluorescent group FAM. The T base near the 3' end (the tilted base) is labeled with the quencher BHQ1. LB-12 is modified with the fluorescent group FAM at the 5' end and the quencher BHQ1 at the 3' end.

[0121] 2. Experimental plan: Prepare reaction reagents based on the formula in Table 3, and prepare reaction reagents containing different LB primers in Table 2.

[0122] Table 3

[0123] A plasmid containing the African swine fever B646L gene, which had been accurately quantified by ddPCR, was used as a template. The plasmid concentration was diluted to 5000 cp / mL and used as the final detection template. 12 μL was added to each reaction.

[0124] The prepared reaction system was placed in a fluorescent real-time quantitative PCR instrument (Bioer FQD-96A model) for reaction. The amplification program is shown in Table 4. The test results were determined by the changes in the fluorescence signal and the CT value.

[0125] Table 4

[0126] The results are analyzed as follows (Figure 5):

[0127] 1) Comparison of LB-T2 and LB-T12, and LB-T3 and LB-T12, revealed that the LB-T12 probe is labeled with a BHQ1 group at its 3' end. After pairing with the template, the BHQ1 group at its 3' end hinders the polymerization reaction of the Bst enzyme, preventing LB-T12 from participating in the LAMP reaction and failing to characterize the reaction. Both LB-T2 and LB-T3 participated in the LAMP reaction and exhibited good real-time fluorescence signal changes. These results demonstrate that hairpin-structured loop primer probes can effectively characterize the LAMP reaction in real time, and that the presence of a free single-stranded nucleotide at the 3' end is crucial for this function.

[0128] 2) Comparison of the experimental data of LB-T5 and LB-T6 revealed that, while maintaining the same sequence of the stem and loop of the hairpin structure, increasing the length of the free single-stranded nucleotide at the 3' end can accelerate the LAMP reaction rate to a certain extent.

[0129] 3) Comparison of the experimental data of LB-T2 and LB-T7 revealed that while maintaining the consistency of the stem of the hairpin structure and the free single-stranded nucleotide at the 3' end, increasing the number of bases making up the loop can accelerate the LAMP reaction rate to a certain extent.

[0130] 4) Comparing the Tm values ​​of each data set reveals that within a certain range, Tm has little effect on the results. However, when Tm values ​​are too high, the reaction progress will be seriously affected, as in the case of LB-T10 and LB-T11.

[0131] In summary, when using this design for LAMP real-time labeling, the presence of free single-stranded nucleotides at the 3' end is extremely important for the reaction and labeling. At the same time, the length of the free single-stranded nucleic acid, the length of the loop structure, and the Tm value of the stem structure will all affect the reaction rate within a certain range. These three factors need to be comprehensively considered to achieve the optimal reaction rate.

[0132] Example 2: Real-time fluorescence LAMP detection of African swine fever virus - double-labeled double-stranded structure primer method

[0133] 1. Primer Design: This example uses African swine fever virus (ASF) as the detection target and the B646L gene of ASF virus as the detection site for LAMP primer design. Based on the principles of LAMP primer design, three pairs of primers (outer primer, inner primer, and loop primer) were designed and selected for LAMP amplification. Their sequences are shown in Table 5. One of the loop primers, the LB primer, was further designed with a locked nucleic acid-modified complementary double-stranded structure and modified with fluorescent and quenching groups.

[0134] Table 5

[0135] Note: When designing adapter primer sequences using LB, the underlined portions of the LB-TN and LB-TN-LNA primer sequences complement the LB-DNXX primer sequence. Nucleotides marked with + in the sequences are modified with locked nucleic acids to increase the double-strand Tm value after pairing. The LB-TN and LB-TN-LNA primer sequences are labeled with a quencher at their 5' end. The LB-DNXX primer sequence is labeled with a fluorescent group at its 3' end.

[0136] 2. Experimental plan: Prepare reaction reagents based on the formula in Table 6, and prepare reaction reagents containing different LBs in the above table.

[0137] Table 6

[0138] A plasmid containing the African swine fever B646L gene, which had been accurately quantified by ddPCR, was used as a template. The plasmid concentration was diluted to 1500 cp / mL and used as the final detection template. 12 μl was added to each reaction.

[0139] The prepared reaction system was placed in a fluorescent real-time quantitative PCR instrument (Bioer FQD-96A model) for reaction. The amplification program is shown in Table 7. The detection results were determined by the change in fluorescence signal and the peak cycle value.

[0140] Table 7

[0141] 3. Experimental results:

[0142] 1) Comparing the data sets of LB-DTN16 and LB-DTN16-LNA, and LB-DTN20 and LB-DTN20-LNA, it was found that the signals of LB-DTN16 and LB-DTN20 continued to increase during the heating process, and no amplification signal increased after reaching 65°C, making it impossible to achieve real-time characterization of the amplification reaction. However, the signals of LB-DTN16-LNA and LB-DTN20-LNA showed good curves and amplification signal growth. The results indicate that the Tm of the double-stranded primer structure after locked nucleic acid modification is increased, enabling real-time characterization of the amplification reaction by fluorescence signal.

[0143] 2) Comparing the data of LB-DTN12-LNA and LB-DTN16-LNA, and LB-DTN16-LNA and LB-DTN20-LNA, it can be found that the shortening of the LB-DNXX sequence will lead to an increase in background fluorescence but a shortened detection time.

[0144] In summary, when using this design for real-time LAMP labeling, the Tm value of the double-stranded structure can be adjusted through modifications such as locked nucleic acid, thereby achieving real-time changes in the fluorescence signal under constant temperature conditions. At the same time, shortening the length of the double-stranded structure can accelerate the reaction rate to a certain extent. LAM typically reacts between 55°C and 70°C, and the length of the double-stranded structure can be adjusted between 8bp and 20bp.

[0145] Example 3: Real-time fluorescence LAMP detection of African swine fever virus - double-labeled linker structure primer method

[0146] 1. Primer Design: This example uses African swine fever virus (ASF) as the detection target and the B646L gene of ASF virus as the detection site for LAMP primer design. Based on the principles of LAMP primer design, three pairs of primers (outer primer, inner primer, and loop primer) were designed and selected for LAMP amplification. Their sequences are shown in Table 8. One of the loop primers, the LB primer, was further designed with a linker structure and modified with fluorescence and quenching.

[0147] Table 8

[0148] Note: When designing adapter primer sequences using LB, the underlined sequence in the LB-TXX primer sequence is the introductory sequence and is complementary to the LB-DXX primer sequence. Nucleotides marked with + in the sequence are modified with locked nucleic acid to increase the double-stranded Tm value after pairing. The double-underlined sequence in LB-DTH11 represents the complementary pairing sequence between the LB primer and the detection target. The LB-TXX primer sequence is labeled with a quencher at its 5' end, and the LB-DXX primer sequence is labeled with a fluorescent group at its 3' end.

[0149] 2. Experimental plan: Prepare reaction reagents based on the formula in Table 9, and prepare reaction reagents containing different LBs in the above table.

[0150] Table 9

[0151] A plasmid containing the African swine fever B646L gene, which had been accurately quantified by ddPCR, was used as a template. The plasmid concentration was diluted to 1500 cp / mL and used as the final detection template. 12 μl was added to each reaction.

[0152] The prepared reaction system was placed in a fluorescent real-time quantitative PCR instrument (Bioer FQD-96A model) for reaction. The amplification program is shown in Table 10. The detection results were determined by the change in fluorescence signal and the peak cycle value.

[0153] Table 10

[0154] 3. Experimental results:

[0155] 1) All of the above reactions proceeded normally, and the progress of the LAMP reaction was characterized by fluorescence signals, demonstrating that the primer design scheme can participate in the reaction normally and achieve real-time signal changes.

[0156] 2) Comparing the experimental results of LB-DT20, LB-DT15, LB-DT13, LB-DT11-2, and LB-DT10 groups, it can be found that the increase in the length of the linker will lead to a slower LAMP reaction rate and a significant interference with the background fluorescence of the initial reaction.

[0157] 3) Comparing the experimental results of LB-DT11, LB-DT11-2 and LB-DT11-3, it can be found that under the same linker length, different sequences and modified linkers have an impact on the LAMP reaction rate and fluorescence intensity.

[0158] 4) Comparison of LB-DT10, LB-DT11, and LB-DTH11 revealed that using the target sequence as part of the double-stranded linker sequence can alleviate the problem of slower reaction rates due to linker length.

[0159] In summary, when using this design for LAMP real-time labeling, the effect of the linker length on the reaction rate and the initial silver light intensity should be fully considered. By adjusting the length of the linker fragment introduced by external aid, the modification of the base-locked nucleic acid in the linker, and other designs, the Tm value of the linker part can be adjusted to achieve the fastest LAMP reaction rate while having lower fluorescence and a larger fluorescence increment.

[0160] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A primer, characterized in that include: a single-stranded region comprising a first single-stranded sequence having a 3' free end; as well as The double-stranded region has a 3' end of one complementary strand connected to the 5' end of the first single-stranded sequence.

2. The primer according to claim 1, characterized in that The double-stranded region includes a second single-stranded sequence, and the second single-stranded sequence is suitable for forming a stem-loop structure; or The double-stranded region includes a third single-stranded sequence and a fourth single-stranded sequence, and at least a portion of the third single-stranded sequence is adapted to complementarily pair with at least a portion of the fourth single-stranded sequence.

3. The primer according to claim 1, characterized in that The primer is at least one of an inner primer pair and / or a loop primer pair of a LAMP reaction.

4. The primer according to claim 1, characterized in that The primer has a fluorescent group and a quencher group, and the quencher group is suitable for quenching the fluorescent group.

5. The primer according to claim 4, characterized in that The fluorescent group and the quenching group are independently disposed at a position no more than 5 bp away from the endpoints of the two complementary strands of the double-stranded region, preferably no more than 3 bp away from the endpoints; or The fluorescent group and the quenching group are independently disposed at one end point of the double-stranded region and a position at most 5 bp near the junction of the double-stranded region and the single-stranded region, preferably at most 3 bp; or The fluorescent group and the quenching group are independently arranged at one end point of the double-stranded region and the position of the T base closest to the connection between the double-stranded region and the single-stranded region.

6. The primer according to claim 4, characterized in that The quenching group is selected from at least one of BHQ1, BHQ2, BHQ3, MGB, BBQ 650, Dabcyl, DBQ1, TAMRA, and Eclipse; The fluorescent group is selected from at least one of FAM, HEX, ROX, CY5Cy5, Atto 425, TET, JOE, VIC, R6G, Yakima Yellow, Quasar570, Quasar670, Cy3, NED, Cy5.5, Cy7, Texas Red, Atto 590, IR Dye 650 and IR Dye 750.

7. The primer according to claim 4, characterized in that The primer includes at least one specific nucleotide or modification group.

8. The primer according to claim 4, characterized in that The double-stranded region of the primer includes at least one special nucleotide or modification group.

9. The primer according to claim 8, characterized in that The special nucleotides include at least one selected from locked nucleic acid, peptide nucleic acid, ribonucleotide and deoxyinosinic acid; The modification group includes at least one selected from a phosphate group, a methyl group and a glycosyl group.

10. The primer according to claim 2, characterized in that The double-stranded region includes a second single-stranded sequence, the second single-stranded sequence has a stem-loop structure, at least a portion of the sequence upstream of the 3' end of the second single-stranded sequence is complementary to at least a portion of the sequence downstream of the 5' end of the second single-stranded sequence, and the 3' end of the second single-stranded sequence is connected to the 5' end of the first single-stranded sequence.

11. The primer according to claim 6 or 10, characterized in that The 5' end of the second single-stranded sequence has the fluorescent group, and the position up to 5 bp near the junction of the double-stranded region and the single-stranded region has the quenching group; or The 5' end of the second single-stranded sequence has the quenching group, and the position up to 5 bp near the junction of the double-stranded region and the single-stranded region has the fluorescent group; or The 5' end of the second single-stranded sequence has the fluorescent group, and the position of the T base closest to the connection between the double-stranded region and the single-stranded region has the quencher group; The 5' end of the second single-stranded sequence has the quenching group, and the position of the T base closest to the connection between the double-stranded region and the single-stranded region has the fluorescent group.

12. The primer according to claim 10, characterized in that The first single-stranded sequence has 1 to 30 nucleotides, preferably 12 nucleotides.

13. The primer according to claim 10, characterized in that At least 6 to 30 nucleotides upstream of the 3' end of the second single-stranded sequence are complementary to at least 6 to 30 nucleotides downstream of the 5' end of the second single-stranded sequence, preferably 10 nucleotides.

14. The primer according to claim 10, characterized in that The loop length in the stem-loop structure is selected from 2 to 10 nucleotides, preferably 4 nucleotides.

15. The primer according to claim 10, characterized in that The primers have nucleotide sequences shown in SEQ ID NOs: 1 to 9.

16. The primer according to claim 2, characterized in that The double-stranded region includes a third single-stranded sequence and a fourth single-stranded sequence, at least a portion of the third single-stranded sequence is complementary to at least a portion of the fourth single-stranded sequence, the 3' end of the third single-stranded sequence is connected to the 5' end of the first single-stranded sequence, or the 3' end of the fourth single-stranded sequence is connected to the 5' end of the first single-stranded sequence.

17. The primer according to claim 6 or 16, characterized in that The 3' end of the third single-stranded sequence is connected to the 5' end of the first single-stranded sequence, the 5' end of the third single-stranded sequence has the fluorescent group, and the 3' end of the fourth single-stranded sequence has the quencher group; or The 3' end of the third single-stranded sequence is connected to the 5' end of the first single-stranded sequence, the 5' end of the third single-stranded sequence has the quencher group, and the 3' end of the fourth single-stranded sequence has the fluorescent group; or The 3' end of the fourth single-stranded sequence is connected to the 5' end of the first single-stranded sequence, the 5' end of the fourth single-stranded sequence has the fluorescent group, and the 3' end of the third single-stranded sequence has the quencher group; or The 3' end of the fourth single-stranded sequence is connected to the 5' end of the first single-stranded sequence. The 5' end of the fourth single-stranded sequence has the quenching group, and the 3' end of the third single-stranded sequence has the fluorescent group.

18. The primer according to claim 16, characterized in that The third single-stranded sequence consists of the first primer sequence, and the fourth single-stranded sequence consists of the second primer sequence.

19. The primer according to claim 16, characterized in that The third single-stranded sequence consists of a first primer sequence and a first linker, and the fourth single-stranded sequence consists of a second primer sequence and a second linker. The first primer sequence is connected to the first linker, and the second primer sequence is connected to the second linker.

20. The primer according to claim 16, characterized in that The third single-stranded sequence consists of a first linker, and the fourth single-stranded sequence consists of a second linker.

21. The primer according to claim 18, characterized in that The first single-stranded sequence has 1 to 30 nucleotides, preferably 1 nucleotide.

22. The primer according to claim 18, characterized in that At least 6 to 30 nucleotides of the third single-stranded sequence are complementary to at least 6 to 30 nucleotides of the fourth single-stranded sequence, preferably 8 to 20, more preferably 10.

23. The primer according to claim 18, characterized in that At least a portion of the sequence downstream of the 5' end of the third single-stranded sequence or the fourth nucleic acid sequence includes at least one special nucleotide or modification group.

24. The primer according to claim 18, characterized in that The primers have nucleotide sequences shown in SEQ ID NOs: 10 to 21.

25. The primer according to claim 19 or 20, characterized in that The first single-stranded sequence has 1 to 30 nucleotides, preferably 21 nucleotides.

26. The primer according to claim 19 or 20, characterized in that At least 6 to 30 nucleotides of the third single-stranded sequence are complementary to at least 6 to 30 nucleotides of the fourth single-stranded sequence, preferably 8 to 20 nucleotides, more preferably 8 nucleotides.

27. The primer according to claim 19 or 20, characterized in that At least a portion of the third single-stranded sequence or the fourth nucleic acid sequence includes at least one special nucleotide or modification group.

28. The primer according to claim 20, characterized in that The primers have nucleotide sequences shown in SEQ ID NOs: 22 to 37.

29. A LAMP primer set, characterized in that: The method comprises an outer primer pair, an inner primer pair and a loop primer pair, wherein at least one primer of at least one of the inner primer pair and the loop primer pair is as defined in any one of claims 1 to 28.

30. A kit, characterized in that include: The primer according to any one of claims 1 to 28; or The LAMP primer set according to claim 29.

31. The kit according to claim 30, characterized in that The method further comprises at least one selected from the group consisting of a strand displacement DNA polymerase, a reverse transcriptase, a ribonuclease inhibitor, dNTPs, a pyrophosphatase, a protecting agent, and a reducing agent.

32. A nucleic acid amplification method, characterized in that: include: The nucleic acid sample to be amplified is used as a template, and an amplification reaction is performed using the primers according to any one of claims 1 to 28, the LAMP primer set according to claim 29, or the kit according to any one of claims 30 to 31.

33. The method according to claim 32, characterized in that The amplification reaction comprises a step selected from loop-mediated amplification.

34. The method according to claim 32, wherein The amplification reaction is carried out in an amplification reaction system, which includes at least one of a strand displacement DNA polymerase, dNTPs, bovine serum albumin, and MgSO4, as well as outer primers F3 and B3, inner primers FIP and BIP, loop primers LF and LB.

35. The method according to claim 34, wherein The concentration of the strand displacement DNA polymerase in the amplification reaction system is 0.1 to 0.8 U / μL, preferably 0.27 U / μL; Optionally, the concentration of the dNTPs in the amplification reaction system is 1 to 3 mM, preferably 1.125 mM; Optionally, the concentration of bovine serum albumin in the amplification reaction system is 0.1 to 25 mg / mL, preferably 10 mg / mL; Optionally, the concentration of MgSO4 in the amplification reaction system is 1 to 10 mM, preferably 6.67 mM; Optionally, the concentration of the outer primer F3 in the amplification reaction system is 0.1 to 0.5 μM, preferably 0.2 μM; Optionally, the concentration of the outer primer B3 in the amplification reaction system is 0.1 to 0.5 μM, preferably 0.2 μM; Optionally, the concentration of the inner primer FIP in the amplification reaction system is 0.1 to 0.5 μM, preferably 0.16 μM; Optionally, the concentration of the inner primer BIP in the amplification reaction system is 0.1 to 0.5 μM, preferably 0.16 μM; Optionally, the concentration of the loop primer LF in the amplification reaction system is 0.1 to 1 μM, preferably 0.8 μM; Optionally, the concentration of the loop primer LB in the amplification reaction system is 0.1-1 μM, preferably 0.6 μM.

36. A method for detecting nucleic acid, characterized in that: include: Amplifying the nucleic acid to be tested by the method described in any one of items 32 to 35; Detecting the fluorescence signal of the amplified product; as well as Based on the fluorescent signal, the nucleic acid to be detected is determined.

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