Method for improving synthesis of nucleic acid under non-diagnostic constant-temperature condition, kit and application

Abstract

The invention provides a method for improving synthesis of nucleic acid under a non-diagnostic constant-temperature condition, a kit and application, and relates to the technical field of bioengineering. Provided are: a nucleic acid having an F2 region capable of annealing with F2c in an F region on the same chain at the 3'end, and an R2c region capable of annealing with R2 in an R region on the same chain at the 5 'end; taking the nucleic acid as a template, taking the 3'end of the F1c region annealed with the F1 region as a synthesis starting point, and synthesizing a complementary nucleic acid chain of the nucleic acid; and performing complementary chain synthesis through a polymerase catalytic chain displacement type synthesis reaction so as to replace the complementary nucleic acid chain synthesized in the previous step. The method has the main advantage that a new combination of short-chain primers can be obtained through simple transformation to trigger rapid amplification of gene fragments by applying a single-enzyme constant-temperature system to short-chain nucleic acid fragments.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to a method and a kit and application for improving nucleic acid synthesis under constant temperature conditions for non-diagnostic purposes. Background Art

[0002] As a method for amplifying target genes, the core of LAMP technology (Nucleic Acids Res, 2000, 28 (12): e63.) is to use four specific primers designed for six regions on the target gene and rely on a highly active strand displacement DNA polymerase to make the strand displacement DNA synthesis in a continuous self-cycle. This technology can achieve 109-1010 times amplification within 15-60 minutes. The reaction can produce a large amount of amplification products, namely white precipitates of magnesium pyrophosphate. The presence of the target gene can be judged by observing the presence of white precipitates with the naked eye. Japan Rongyan Company has also developed a turbidity meter to achieve real-time monitoring of amplification reactions. In addition to high specificity and high sensitivity, the advantages of the LAMP method are that the operation is very simple. The requirements for instruments in the application stage are low. A simple constant temperature device can achieve the reaction. The result detection is also very simple. The white precipitate or green fluorescence can be directly observed with the naked eye. It is a method suitable for on-site and grassroots rapid detection. One of the limitations is that the high specificity and sensitivity of this method depend on the properties of the four primers. Obtaining the best primers usually requires sequence alignment, online primer design, primer screening and specificity testing, which is a very cumbersome process (J ChromatogrA, 2015, 1377: 13-26.). Summary of the invention

[0003] The purpose of the present invention is to provide a method for optimizing the primer design for nucleic acid synthesis by methods such as LAMP, CAMP and CDA. One of the advantages of the present invention is that only a pair of new primers after recombining a pair of conventional PCR primers can be used to achieve nucleic acid amplification. The second advantage of the present invention is that the length of the core primer used is about 30 bp, which is 10-15 bp shorter than the original core primer of about 40 bp used in LAMP, which can save about a quarter of the primer cost. At the same time. The present invention uses polymerase to catalyze the chain displacement type complementary chain synthesis without complex temperature control, which is beneficial to the synthesis of nucleic acids. The DNA polymerase is an enzyme used in methods such as SDA, RCA, LAMP, CAMP, CDA, etc.

[0004] The present inventors improved the known method for providing 3'-OH and found that by using an oligonucleotide having a specific structure, a 3'-OH structure can be provided without any additional enzyme reaction, thereby arriving at the present invention. That is, the present invention relates to a method for synthesizing nucleic acid, a method for amplifying nucleic acid by using the nucleic acid synthesis method, and a kit for synthesizing nucleic acid using the method.

[0005] In order to solve the above technical problems, the technical solution adopted in this application is:

[0006] The nucleic acid chain obtained by the present invention can autonomously pair and extend infinitely, and the F1 region at the 3' end of the nucleic acid chain anneals with the adjacent F1c region, triggering a continuous extension reaction of the nucleic acid chain.

[0007] As a preferred embodiment, the specific steps of synthesizing nucleic acid are shown in the following figure: Figure 1 As shown, the following steps are included:

[0008] 1) providing a nucleic acid, wherein the 3' end has an F2 region that can anneal to F2c of the F region on the same chain, and the 5' end of the nucleic acid has an R2c region that can anneal to R2 of the R region on the same chain, and the nucleic acid can spontaneously form a short-chain complementary neck loop structure;

[0009] 2) annealing the first oligonucleotide I with the F1c region of the nucleic acid provided in step 1), and then performing a synthesis step using the F1 region of the first oligonucleotide I as a synthesis starting point; wherein the first oligonucleotide I includes the F2c region and the F1 region;

[0010] 3) using the nucleic acid provided in step 1) as a template to synthesize its own complementary chain, and the synthesized nucleic acid sequence is referred to as the first nucleic acid;

[0011] 4) annealing the second oligonucleotide II with the R1c region of the first nucleic acid provided in step 3), and then performing a synthesis step using the R1 region of the second oligonucleotide II as a synthesis starting point; wherein the second oligonucleotide II includes the R2c region and the R1 region;

[0012] 5) Using the first nucleic acid provided in step 3) as a template, synthesizing its own complementary chain, wherein the nucleic acid chain has complementary nucleotide sequence regions that are alternately connected.

[0013] Wherein, the method for preparing nucleic acid in step 1) comprises the following steps:

[0014] 1-a) annealing step, annealing the F1 region of the first nucleotide with the F1c region of the template, wherein the template is composed of the Fc region and the R region in the 3' to 5' direction; the Fc region includes the F1c and F2c regions, the R region includes the R1 region and the R2 region, and the F region includes the F1 region and the F2 region; the first nucleotide is the F2c region and the F1 region from the 5' end to the 3' end;

[0015] 1-b) synthesizing a first nucleic acid using the F2 region of the first oligonucleotide as a synthesis starting point, wherein the second oligonucleotide comprises an R2c region and an R1 region from the 5' end to the 3' end; the first nucleic acid has a nucleotide sequence complementary to the template, and the 5' end of the first nucleic acid has an F2c region that can anneal to the F2 region on the same chain;

[0016] 1-c) annealing the R1 region of the second oligonucleotide to the R1c region of the first nucleic acid under constant temperature conditions, wherein the second oligonucleotide comprises the R2c region and the R1 region from the 5' end to the 3' end; the R region comprises the R1 region and the R2 region, and the Rc region comprises the R1c region and the R2c region;

[0017] 1-d) synthesizing a second nucleic acid using the R1 region of the second oligonucleotide as a synthesis starting point, i.e. obtaining the nucleic acid provided in step 1);

[0018] Among them, F region: a region having a nucleotide sequence complementary to the Fc region;

[0019] F1 region: a region having a nucleotide sequence complementary to the F1c region;

[0020] F2 region: a region having a nucleotide sequence complementary to the F2c region;

[0021] R region: a region having a nucleotide sequence complementary to the Rc region;

[0022] R1 region: a region having a nucleotide sequence complementary to the R1c region;

[0023] R2 region: a region having a nucleotide sequence complementary to the R2c region.

[0024] See also Figure 2 , is a diagram of the synthesis steps corresponding to a method for synthesizing a nucleic acid in the above step 1) of the present invention, namely Figure 2 The second nucleic acid in the sequence from 5' to 3' is composed of R2c, R1, R2, F2c, F1c, and F2 regions.

[0025] As a preferred embodiment, the template in step 1-a) is RNA, and the first nucleic acid in step 1-b) is synthesized by an enzyme having reverse transcriptase activity.

[0026] Preferably, the template in step 1-1) is RNA, and the first nucleic acid in step 1-2) is synthesized by an enzyme having reverse transcriptase activity. The present invention is applicable to various DNAs and RNAs, such as DNAs and RNAs of various animal and plant cells, bacteria and viruses, etc. For example, it is used for cDNA and RNA detection of H1 gene and N1 gene of H1N1 virus, etc.

[0027] Preferably, the nucleic acid fragments of the F1 region and the R1 region are both 10-30 bp. Further preferably, the nucleic acid fragments of the F1 region and the R1 region are both 15-25 bp.

[0028] Preferably, the nucleic acid fragments of the F region and the R region are both 20-50 bp. Further preferably, the nucleic acid fragments of the F region and the R region are both 20-40 bp.

[0029] According to the method for synthesizing nucleic acid of the present invention, the synthesized nucleic acid refers to a nucleic acid having a nucleotide sequence in which the first and last segments are complementary to different parts of the middle segment on one chain, and the nucleic acid is synthesized based on the nucleic acid under the action of the first oligonucleotide and the second oligonucleotide, see Figure 3 and Figure 4 .

[0030] According to the method for synthesizing nucleic acid of the present invention, the constant temperature means that the whole reaction process is synthesized within a temperature range of 60-65°C.

[0031] The polymerase used in the polymerase-catalyzed strand displacement reaction of the present invention is one or more of Bst DNA polymerase, Bca (exo-) DNA polymerase, DNA polymerase I Klenow fragment, Vent DNA polymerase, Vent (Exo-) DNA polymerase (Vent DNA polymerase lacking exonuclease activity), Deep Vent DNA polymerase, Deep Vent (Exo-) DNA polymerase (Deep Vent DNA polymerase lacking exonuclease activity), Φ29phage DNA polymerase, and MS-2phage DNA polymerase, etc. Among them, Bst DNA polymerase or Bst 2.0 DNA polymerase is preferably used.

[0032] According to the method for synthesizing nucleic acid of the present invention, a melting temperature regulator may be added to the polymerase-catalyzed strand displacement reaction, wherein the melting temperature regulator is preferably betaine, and further preferably, the concentration of betaine in the reaction solution is 0.2-3.0M.

[0033] The present invention also provides a kit for synthesizing nucleic acid, the kit comprising the following components:

[0034] A first oligonucleotide, wherein the first oligonucleotide comprises an F2c region and an F1 region from the 5' end to the 3' end;

[0035] A second oligonucleotide, wherein the second oligonucleotide comprises an R2c region and an R1 region from the 5' end to the 3' end;

[0036] Nucleic acid synthesis catalytic enzyme;

[0037] Nucleotides, which serve as substrates for the DNA polymerase;

[0038] Wherein, F1 region: a region having a nucleotide sequence complementary to F1c region;

[0039] F2 region: a region having a nucleotide sequence complementary to the F2c region;

[0040] R1 region: a region having a nucleotide sequence complementary to the R1c region;

[0041] R2 region: a region having a nucleotide sequence complementary to the R2c region.

[0042] As a preferred embodiment, the kit further comprises a detection reagent for detecting the nucleic acid synthesis reaction product, wherein the detection reagent is a dye with a green excitation wavelength that binds to the minor groove region of the dsDNA double helix, preferably Sybrgreen I and Eva green.

[0043] As a preferred embodiment, the kit further comprises a buffer capable of keeping the enzyme reaction at an appropriate pH value, necessary salts for annealing or maintaining the catalytic activity of the enzyme, and a medium for protecting the enzyme.

[0044] The present invention also provides the use of the kit for non-diagnostic purposes in synthesizing nucleic acids or detecting target nucleotide sequences in samples. The present invention is applicable to the detection of various DNA and RNA, such as DNA and RNA of various animal and plant cells, bacteria and viruses. For example, it is used for the detection of M13 gene DNA; for the detection of cDNA and RNA of influenza A virus H1N1, etc.

[0045] Based on the method for synthesizing nucleic acid under constant temperature conditions of the present invention, a method for detecting a target nucleotide sequence in a sample is provided, comprising using the target nucleotide as a template, amplifying by the method for synthesizing nucleic acid of the present invention, and observing whether an amplification product is generated.

[0046] A probe containing a nucleotide sequence complementary to the formed asymmetric loop structure is added to the above-mentioned amplification product, and the hybridization between the two can be observed. The probe can also be marked on the particle, and the aggregation reaction occurring through hybridization can be observed. The amplification method can be implemented in the presence of a nucleic acid detection reagent, and whether the amplification product is generated can be observed based on the change in the signal.

[0047] According to the kit of the present invention, the DNA polymerase is one or more of Bst DNA polymerase, Bca (exo-) DNA polymerase, DNA polymerase I Klenow fragment, Vent DNA polymerase, Vent (Exo-) DNA polymerase, Deep Vent DNA polymerase, Deep Vent (Exo-) DNA polymerase, Φ29phage DNA polymerase and MS-2phage DNA polymerase, etc. Preferably, Bst DNA polymerase or Bca (exo-) DNA polymerase is used.

[0048] The present invention provides the use of the kit in synthesizing nucleic acid or detecting target nucleotide sequences in samples.

[0049] Based on the method for synthesizing nucleic acid under constant temperature conditions of the present invention, a method for detecting a target nucleotide sequence in a sample is provided, comprising using the target nucleotide as a template, amplifying by the method for synthesizing nucleic acid of the present invention, and observing whether an amplification product is generated.

[0050] A probe containing a nucleotide sequence complementary to the formed shorter stem-loop structure is added to the above-mentioned amplification product, and the hybridization between the two can be observed. The probe can also be marked on the particle, and the aggregation reaction occurring through hybridization can be observed. The amplification method can be implemented in the presence of a nucleic acid detection reagent, and whether the amplification product is generated can be observed based on the change in the signal.

[0051] Similar to PCR amplification technology, based on the method for synthesizing nucleic acid of the present invention, a method for detecting mutations in a target nucleotide sequence in a sample can also be provided, comprising amplifying the target nucleotide as a template by the method for synthesizing nucleic acid under constant temperature conditions of the present invention. Among them, the mutation in the nucleotide sequence as the amplification object hinders the synthesis of any complementary chain constituting the amplification method, thereby inhibiting the related generation of signals, thereby detecting the mutation.

[0052] The single-stranded nucleic acid of a nucleotide sequence that can form a shorter stem-loop structure is the purpose of the present invention. The nucleic acid refers to a nucleic acid with a target nucleic acid sequence as a mutually complementary nucleotide sequence alternately connected in a certain order in a single strand. The nucleic acid synthesized by the present invention is basically composed of mutually complementary chains connected by a stem-loop structure. Figure 5 , which is a schematic diagram of an ideal amplified nucleic acid product formed by the synthesis method of the present invention.

[0053] Generally speaking, regardless of whether or not it partially involves complementary base pairing, a chain that cannot be separated into two or more molecules when the complementary paired bases are separated is called a single strand. Complementary nucleotide sequences in the same strand can form base pairing, and the present invention allows nucleic acids having nucleotide sequences linked end to end in a single strand to base pair within the same strand, thereby obtaining a product of intramolecular base pairing, which includes a region constituting an obvious double strand and a loop that does not involve base pairing.

[0054] That is, the nucleic acid of the nucleotide sequence with a shorter stem-loop structure of the present invention can be defined as a single-stranded nucleic acid, which contains a complementary nucleotide sequence that can anneal in the same chain. The nucleotides with complementary nucleotide sequences can anneal into a loop that does not involve base pairing. The looping sequence can be any nucleotide sequence. The looping sequence can base pair to start the synthesis of the complementary chain for replacement. And preferentially provided with a sequence different from the nucleotide sequence located in other regions to obtain specific annealing.

[0055] In the present invention, substantially identical nucleotide sequences are defined as follows: when a complementary chain synthesized using a certain sequence as a template is annealed to a target nucleotide sequence as a starting point for synthesizing a complementary chain, the sequence is substantially identical to the target nucleotide sequence. For example, a sequence identical to F1 not only completely includes a sequence identical to F1, but also includes a nucleotide sequence that can serve as a template, wherein the template can give a nucleotide sequence that anneals to F1 and can serve as a starting point for synthesizing a complementary chain. The term "annealing" in the present invention refers to nucleic acids that form a complementary structure through base pairing according to the Watson-Crick law. Therefore, even if the nucleic acid chain constituting the base pairing is a single strand, annealing will occur if the complementary nucleotide sequences in the molecule are base paired. The double-stranded structure is formed by base pairing nucleic acids, and the meanings expressed by annealing and hybridization in the present invention have overlapping parts.

[0056] The number of nucleotide sequence pairs constituting the nucleic acid of the present invention is at least 1. In the model desired by the present invention, the number of nucleotide sequence pairs can be an integral multiple of 1. In this case, there is no theoretical upper limit to the number of complementary nucleotide sequence pairs constituting the nucleotides of the present invention, and when the nucleic acid synthesized by the present invention is composed of multiple sets of complementary nucleotide sequences, the nucleic acid is composed of repeated identical nucleotide sequences.

[0057] The single-stranded nucleotide of the nucleotide sequence with a short-chain stem-loop structure synthesized by the present invention has a different structure from the naturally occurring nucleic acid, and it is generally known that when a nucleic acid is synthesized by a nucleic acid polymerase, if a nucleotide derivative is used as a substrate, a nucleic acid derivative can be synthesized. The nucleotide derivative used includes a radioactive isotope-labeled nucleotide or a ligand-labeled nucleotide derivative such as biotin or digoxin. These nucleotide derivatives can be used to label the product nucleic acid. Alternatively, if the substrate is a fluorescent nucleotide, the product nucleic acid may be a fluorescent derivative.

[0058] There are many reports on complementary chain synthesis reactions, in which a helical loop is formed, with the helical loop sequence itself as a template, and a hairpin loop is formed, with the hairpin loop sequence itself as a template, but the present invention provides a region in which a portion of the hairpin loop is capable of base pairing, and has a new feature of utilizing this region when synthesizing a complementary chain. By using this region as the starting point of synthesis, the complementary chain previously synthesized with the hairpin loop sequence itself as a template is replaced.

[0059] The nucleic acid referred to in the present invention generally includes both DNA and RNA, and nucleic acids or modified nucleotides whose nucleotides are replaced by artificial derivatives from natural DNA or RNA are also included in the scope of the nucleic acid of the present invention. Usually the nucleic acid of the present invention is contained in a biological sample, and the biological sample includes tissues, cells, cultures and secretions of animals, plants or microorganisms, and their extracts. The biological sample of the present invention includes intracellular parasite genomic DNA or RNA, such as viruses or mycoplasmas. The nucleic acid of the present invention is generally derived from the nucleic acid contained in the biological sample. For example, cDNA synthesized from mRNA, microRNA, etc., and nucleic acids amplified based on nucleic acids derived from biological samples are typical examples of nucleic acids of the present invention.

[0060] In the present invention, an oligonucleotide is a nucleotide that meets two requirements, that is, it must be able to form complementary base pairing, and provide an -OH group at the 3'-terminus as the starting point for the synthesis of a complementary chain. Therefore, its main chain is not necessarily limited to a phosphodiester bond. For example, it can be composed of a main chain of a thiophosphate derivative or a peptide nucleic acid based on a peptide connection, wherein the thiophosphate derivative is S instead of O. Bases refer to those bases that can be complementary. There are five bases in nature, namely A, C, T, G and U, and the base can also be an analog such as bromodeoxyuridine. Preferably, the oligonucleotide of the present invention can be used not only as a starting point for synthesis but also as a template for the synthesis of a complementary chain. The term polynucleotide of the present invention includes oligonucleotides. The chain length of the term "polynucleotide" used in the present invention is unlimited, and the term "oligonucleotide" used refers to a nucleotide polymer with a relatively short chain length.

[0061] In the various nucleic acid synthesis reactions described below, under given conditions, the oligonucleotide chain of the present invention has a length that can base pair with the complementary chain and maintain a certain specificity. Specifically, it consists of 5-200 bases, more preferably 10-50 base pairs. The chain length of the known polymerase is recognized to be at least 5 bases. The polymerase catalyzes a nucleic acid synthesis reaction that depends on the sequence. Therefore, the chain length of the annealed portion should be longer than this length. In addition, a length of 10 bases or longer is statistically expected to obtain target nucleotide specificity. On the other hand, since it is difficult to prepare too long nucleotide sequences by chemical synthesis, the above chain length is an example of the expected range. The exemplified chain length refers to the chain length of the part annealed with the complementary chain. As described below, the oligonucleotide of the present invention can eventually anneal with at least two districts respectively. Therefore, the chain length exemplified here should be understood as the chain length of each district that constitutes the oligonucleotide.

[0062] In addition, the oligonucleotides of the present invention can be labeled with known markers. Markers include binding ligands such as digoxigenin and biotin, enzymes, fluorescent materials, luminescent materials, and radioisotopes. It is well known that the technology of replacing the bases that make up the oligonucleotides by fluorescent analogs (WO95 / 05391, Proc. Natl. Acad. Sci. USA, 91, 6644-6648, 1994).

[0063] Other oligonucleotides of the present invention can also be attached to solid phase.Perhaps, any part of oligonucleotide can be labeled with binding ligand, such as biotin, indirectly fixed by binding ligand such as fixed avidin.When fixed oligonucleotide is the starting point of synthesis, the synthetic reaction product nucleic acid is captured by solid phase, which will facilitate its separation.Separated part can be detected by nucleic acid specific indicator or with hybridization of labeled probe.The nucleic acid product obtained by this method can be recovered by restriction enzyme digestion product for target nucleic acid fragment.

[0064] The term "template" used in the present invention refers to a nucleic acid used as a template when synthesizing a complementary chain. A complementary chain having a nucleotide sequence complementary to the template means a chain corresponding to the template. However, the relationship between the two is only relative. That is, the synthesized complementary chain can function as a template again. In other words, the complementary chain can also serve as a template.

[0065] In the present invention, if the target is RNA, it can be constructed by simply adding reverse transcriptase. That is, using RNA as a template, it is possible to synthesize a complementary chain by annealing F1 and F1c in the template by reverse transcriptase. When the reverse transcriptase uses DNA as a template to synthesize a complementary chain, all reactions to synthesize a complementary chain by reverse transcriptase include the synthesis of a complementary chain using R1 annealed to Rlc as the synthesis starting point, and the complementary chain serves as a template in the strand displacement reaction. The mode of obtaining the first single-stranded nucleic acid using RNA as a template as described above is a preferred mode of the present invention. On the other hand, if a DNA polymerase having both strand displacement activity and reverse transcriptase activity is used, such as Bca DNA polymerase and RTx reverse transcriptase, etc., not only the synthesis of the first single-stranded nucleic acid from RNA, but also the subsequent reaction using DNA as a template can be carried out similarly by the same enzyme.

[0066] The reaction is carried out in the presence of the following components: a buffer that can make the enzyme reaction at an appropriate pH value, a necessary salt for annealing or maintaining the catalytic activity of the enzyme, a medium for protecting the enzyme, and a regulator necessary for regulating the melting temperature (Tm). For the buffer, for example, Tris-HCl is used, which has a buffering effect in the neutral or weakly alkaline range. The pH value is adjusted according to the DNA polymerase used. For salt, KCl, NaCl, (NH4)2SO4, etc. are added in appropriate amounts to maintain the activity of the enzyme and regulate the melting temperature (Tm) of the nucleic acid. Bovine serum albumin or sugars are used as the medium for protecting the enzyme. In addition, dimethyl sulfoxide (DMSO) or formamide is generally used as a regulator of the melting temperature (Tm). By using the regulator of the melting temperature (Tm), the annealing of the oligonucleotide is regulated under limited temperature conditions. In addition, betaine (N, N, N-trimethylglycine) or tetraalkylammonium salts (tetraalkyl) are also effective for improving the efficiency of chain displacement through their isostabilization. The desired effect of the present invention on promoting nucleic acid amplification can be obtained by adding 0.2-3.0 M betaine to the reaction solution, preferably 0.5-1.5 M. Because these melting temperature regulators have the effect of lowering the melting temperature, the appropriate stringency and reactivity conditions should be determined empirically in combination with salt concentration, reaction temperature, etc.

[0067] The important feature of the present invention is that a series of reactions cannot proceed unless the positional relationship of many regions is maintained. Due to this feature, non-specific synthesis reactions accompanying non-specific synthesis of complementary chains are effectively prevented. That is, even if a non-specific reaction occurs, the possibility of the product being used as a starting material in the subsequent amplification step is also reduced. Moreover, by regulating the progress of the reaction by many regions, a detection system that can accurately identify the desired product in similar nucleotide sequences can be arbitrarily composed.

[0068] The nucleic acid synthesized by the present invention is a single strand, and as far as the single strand is concerned, it is composed of complementary nucleotide sequences, most of which are base paired. By utilizing this feature, the synthesized product can be detected. By implementing the method for synthesizing nucleic acid of the present invention, in the presence of a fluorescent pigment as a double-stranded specific intercalator (double-specific intercalater) such as ethidium bromide, SYBR Green I, Pico Green or Eva Green, the intensity of fluorescence can be observed to increase as the product increases. By monitoring the fluorescence intensity, it is possible to track the progress of the real-time synthesis reaction in a closed system. It is also possible to consider applying this type of detection system in the PCR method, but there are many problems because the product signal and the signal of the primer dimer cannot be distinguished. However, when the present invention applies this system, the ability to increase non-specific base pairing is very low, and therefore, it is expected that high sensitivity and low interference may be obtained at the same time, and similar to the application of a double-stranded specific intercalator (double-specific intercalater), the transfer of fluorescence energy can be used in the same system to realize the method of the detection system.

[0069] The method for synthesizing nucleic acid of the present invention is supported by a DNA polymerase catalyzing a chain displacement type complementary chain reaction. During the above reaction, a reaction step that does not require a chain displacement type polymerase is also included. However, for the simplification of the composition reagents and from the economical point of view, it is advantageous to use a DNA polymerase, and the following enzymes are known. In addition, various mutants of these enzymes can be utilized within the scope of the present invention, all of which have sequence-dependent activity and chain displacement activity for complementary chain synthesis. Among them, mutants refer to those that only have the structure that causes the catalytic activity required by the enzyme or those mutants that are modified by, for example, mutations in amino acids for catalytic activity, stability or thermal stability.

[0070] Bst DNA polymerase

[0071] Bst DNA Polymerase (Large Fragment)

[0072] Bst 2.0 DNA polymerase

[0073] BstWarmstart 2.0 DNA Polymerase

[0074] Bst 3.0 DNA polymerase

[0075] Bca(exo-)DNA polymerase

[0076] DNA polymerase I Klenow fragment

[0077] Vent DNA polymerase

[0078] Vent (Exo-) DNA polymerase (Vent DNA polymerase lacking exonuclease activity)

[0079] Deep Vent DNA Polymerase

[0080] Deep Vent (Exo-) DNA Polymerase (Deep Vent DNA Polymerase lacking exonuclease activity)

[0081] Φ29phage DNA polymerase

[0082] MS-2phage DNA polymerase

[0083] OmniAmp DNA Polymerase

[0084] Among these enzymes, Bst DNA polymerase, Bca (exo-) DNA polymerase, OmniAmp DNA polymerase are particularly desired enzymes because they have a certain degree of thermostability and high catalytic activity. In a preferred embodiment, the reaction of the present invention can be realized at a constant temperature, but due to the regulation of the melting temperature (Tm) etc., it is impossible to always utilize the required temperature conditions to maintain the stability of the enzyme. Therefore, it is one of the conditions required for the thermostability of the enzyme. Although isothermal reaction is feasible, thermal denaturation can provide nucleic acid as the initial template, and in this respect, the use of thermostable enzymes has broadened the selection of test protocols.

[0085] The various reagents necessary for the synthesis or amplification of nucleic acid of the present invention can be prepackaged and provided in the form of a test kit. Specifically, the test kit provided by the present invention comprises various oligonucleotides necessary for the primers for synthesizing complementary chains and the outer primers for displacement reaction, the substrate dNTP for the synthesis of complementary chains, the DNA polymerase for realizing the synthesis of chain displacement complementary chains, the buffer for providing suitable conditions for the enzyme reaction, and the necessary medium for detecting the synthesis reaction product. Specifically, in the preferred mode of the present invention, the reagents need not be added during the reaction, and thus the reagents necessary for the reaction after moving into the reaction vessel can be started by adding a sample. The system of the reaction product can be detected in the container by utilizing visible light signals or fluorescent signals. The container need not be opened and closed after the reaction. This is very advantageous for preventing pollution.

[0086] The present invention synthesizes a single-stranded nucleic acid having a nucleotide sequence with a short-chain stem-loop structure. The nucleic acid has the following uses, for example: the first feature is to take advantage of the specific structure of a molecule having a complementary sequence, which may be advantageous for detection, i.e., a system for detecting nucleic acids, in which the signal of its change depends on base pairing with a complementary nucleotide sequence. For example, by combining the method of using a double-stranded specific intercalator as a detection reagent as described above, a detection system that fully utilizes the characteristics of the synthetic product of the present invention can be realized. If the product of the synthesis reaction of the present invention undergoes a thermal denaturation in the detection system once and returns to the original temperature, intramolecular annealing occurs preferentially, thereby allowing rapid base pairing between complementary sequences. If non-specific products exist, they do not have complementary sequences in the molecules so that after being separated into two or more molecules by thermal denaturation, they cannot immediately return to the original double strand. By providing a thermal denaturation step before detection, interference associated with non-specific reactions is reduced. If the DNA polymerase used is not heat-resistant, the thermal denaturation step means that the reaction is terminated, and thus it is beneficial to control the reaction temperature.

[0087] The second characteristic is that it often forms a shorter hairpin loop capable of base pairing.

[0088] According to a preferred mode of the present invention, a large number of base-pairing rings are generated in a single-stranded nucleic acid. This means that a large number of probes can hybridize with a nucleic acid molecule to achieve highly sensitive detection. Therefore, it is possible not only to achieve improved sensitivity but also to achieve a method for detecting nucleic acids based on special reaction principles such as aggregation. For example, probes fixed on fine particles such as polystyrene latex or gold, silver, and platinum nanoparticles or two-dimensional nanomaterials are added to the reaction product of the present invention, and the aggregation of the latex, etc. is observed as the product hybridizes with the probe. The intensity of the aggregation can be measured optically for highly sensitive and quantitative observation. Alternatively, the aggregation can also be observed with the naked eye, so a reaction system that does not require an optical measuring device can also be established.

[0089] In addition, the reaction product of the present invention allows for some incorporable labels, wherein each nucleic acid molecule can be subjected to chromatographic detection. In the field of immunoassays, the actual application is an analytical method using a chromatographic medium with a visible detection label (immunochromatography). The method is based on the principle that the analyte is sandwiched between an antibody fixed on a chromatographic medium and a labeled antibody, and the unreacted labeled component is eluted. The reaction product of the present invention allows this principle to be applied to nucleic acid analysis. That is, a labeled probe for the loop portion is prepared and fixed on a chromatographic medium to prepare a capture probe for capture to allow analysis in the chromatographic medium. Capture probes whose sequences are complementary to the loop portion are utilized, and since the reaction product of the present invention has a large number of hairpin loops, the product is combined with a large number of labeled probes to achieve detection of signals that can be recognized by the naked eye.

[0090] The reaction product of the present invention can often provide a loop region for base pairing, which can broaden other various detection systems. For example, a system for detecting the loop portion using a fixed probe using the surface cytoplasmic genome is feasible. In addition, if the probe of the loop portion is labeled with a double-stranded specific inserter, a more sensitive fluorescence analysis can be performed. Or the ability of the present invention to synthesize nucleic acids can be actively utilized to form a hairpin capable of base pairing at the 3'- and 5'-sides. For example, a loop is designed so that it has a common nucleotide sequence between the normal type and the abnormal type, and other loops are designed so that differences are produced therein. When the presence of a gene in the common part is confirmed by the probe, and when the presence of an abnormality is confirmed in other regions, it is possible to form a characteristic analysis system. Because the reaction of synthesizing nucleic acids of the present invention can also be carried out isothermally, it is worth mentioning that real-time analysis can be performed by a general fluorescence photometer. Until this time, the structure of the nucleic acid to be annealed in the same chain is known. However, the nucleic acid in the single-stranded sequence having the head-to-tail annealing loop obtained by the present invention is new, and it contains a large number of loops capable of base pairing with other bases.

[0091] On the other hand, a large number of loops given by the reaction product of the present invention can be used as probes themselves. For example, in a DNA chip, probes are densely packed in a limited area, and the number of oligonucleotides that can be fixed in a certain area is limited in this technology. Therefore, by using the product of the present invention, a large number of probes that can be annealed can be fixed at a high density. That is, the reaction product of the present invention can be used as a fixed probe on a DNA chip. After amplification, the reaction product can be fixed by any technique known in the art, or the fixed oligonucleotide is used as the oligonucleotide of the amplification reaction of the present invention, resulting in the generation of a fixed reaction product. Therefore, by using the fixed probe, a large amount of sample DNA can be hybridized in a limited area, and as a result, a high signal value is expected to be obtained.

[0092] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0093] 1. The method of the present invention is a further improvement on LAMP and the previously invented CDA technology. Compared with the LAMP amplification technology, the minimum target fragment is 120 bp, while the target fragment of the method of the present invention can be only about 40 bp, which can easily realize the constant temperature amplification of nucleic acids.

[0094] 2. The primer design of the method of the present invention is very simple and only requires simple improvement of conventional PCR primers.

[0095] 3. The method of the present invention requires two oligonucleotides to initiate the reaction in order to simultaneously achieve the effects of the present invention, while the amplification method previously achieved by the inventors requires three oligonucleotides to initiate the reaction (CN 114317689B). Compared with this, the present invention is an improved method with a greater advantage.

[0096] 4. The present invention utilizes polymerase to catalyze strand displacement-type complementary strand synthesis without complicated temperature control, which is beneficial to nucleic acid synthesis and can achieve target nucleic acid synthesis more specifically than other constant temperature amplification methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0098] Figure 1 It is a diagrammatic illustration of the steps of the nucleic acid synthesis method of the present invention.

[0099] Figure 2 It is a diagrammatic illustration of the steps of the second nucleic acid synthesis process in the present invention.

[0100] Figure 3 It is a schematic diagram of the short-chain stem-loop structure formed by the single-stranded nucleic acid of the present invention and the subsequent amplification reaction.

[0101] Figure 4 It is a schematic diagram of the subsequent amplification reaction of nucleic acid in the present invention.

[0102] Figure 5 It is a schematic diagram of an ideal amplification product formed by the synthesis method of the present invention.

[0103] Figure 6 It is the positional relationship of each corresponding nucleotide sequence region in the M13 target nucleotide sequence in Example 1 of the present invention.

[0104] Figure 7 This is a photograph of the agarose electrophoresis result of the product obtained by the method for synthesizing single-stranded nucleic acid of the present invention using M13 as a template in Example 1 of the present invention; wherein, lane 1: DNA Marker; lane 2: 1 fmol M13 dsDNA.

[0105] Figure 8 This is a photograph of the agarose electrophoresis result of the product obtained by the method for synthesizing single-stranded nucleic acid of the present invention using H1N1 DNA as a template in Example 2 of the present invention; wherein, lane 1: DNA Marker; lane 2: 1 fmol H1N1 dsDNA.

[0106] Fig. 9 It is a real-time fluorescence curve diagram of the H1N1 target nucleotide sequence DNA amplification process under the action of primers in Example 3 of the present invention.

[0107] Fig.10This is a diagram of the endpoint monitoring based on color change of the reaction using HNB to amplify the H1N1 target nucleotide sequence DNA under the action of primers in Example 4 of the present invention.

[0108] Fig.11 It is a real-time fluorescence curve diagram during the RNA amplification process of in vitro transcription of the H1N1 target nucleotide sequence under the action of primers in Example 5 of the present invention. DETAILED DESCRIPTION

[0109] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer, and can be specifically referred to the specific methods listed in the book "PCR Technology Experiment Guide" (2nd Edition), or according to the kit and product instructions. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0110] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0111] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0112] Example 1

[0113] This example discloses the amplification of a fragment in M13.

[0114] The M13 gene is a DNA fragment used for concept verification in an article on the LAMP technology, and is also used in this application.

[0115] Two primers used in the experiment are M13-SF (nucleotide sequence is shown in SEQ ID NO.1) and M13-SR (nucleotide sequence is shown in SEQ ID NO.2). These are designed to anneal into loop regions by utilizing the adjacent stacking phenomenon. In addition, these primers are set to high concentrations so that annealing of M13-SF (or M13-SR) occurs preferentially.

[0116] By using the primers M13-SF and M13-SR, a shorter chain neck loop structure is synthesized at both ends of the target nucleotide M13. The composition of the reaction solution for the method of synthesizing the nucleic acid of the present invention by using these primers is shown below.

[0117] The reaction solution was composed as follows, and the rest was prepared with ddH 2 O to 25 μL

[0118] 20 mM Tris-HCl pH 8.8

[0119] 10mM KCl

[0120] 10mM (NH 4 ) 2 SO 4

[0121] 14 mM MgSO 4

[0122] 0.1% Triton X-100

[0123] 1M Betaine

[0124] 1.25mM dNTP

[0125] 8U Bst DNA polymerase (NEW ENGLAND Biolabs)

[0126] Primers:

[0127] 1600nM M13-SF

[0128] 1600nM M13-SR

[0129] Target nucleic acid: M13 dsDNA (nucleotide sequence as shown in SEQ ID NO.3). Figure 6 , is the positional relationship of each corresponding nucleotide sequence region in the M13 target nucleotide sequence.

[0130] The mixture was reacted at 61°C for 1 hour. After the reaction, the reaction was terminated by heating at 80°C for 10 minutes and then transferred back into ice-cooled water.

[0131] Confirmation of the reaction: 1 μL of conventional nucleic acid electrophoresis loading buffer (provided with Takara DNA ladder) was added to 5 μL of the above reaction solution, and the sample was electrophoresed at 90 mV on a 1% agarose gel (TAE dissolved) prestained with GelRed (Biotum) for 1 hour. Takara DL2,000 DNA Marker was used as a molecular weight marker. The gel after electrophoresis was used to verify the nucleic acid. The results are shown in Figure 7 It is shown in the figure that a nucleic acid product with a wide molecular weight distribution is obtained, which verifies that the nucleic acid obtained by the inventive method can be infinitely self-assembled, annealed and extended to obtain ultra-large nucleic acid molecules.

[0132] Example 2

[0133] This example discloses the amplification of DNA fragments in H1N1.

[0134] Influenza virus A (H1N1) belongs to the family Orthomyxoviridae and the genus Influenza virus A. The symptoms of influenza A (H1N1) are similar to those of the common cold. The first symptom of most infected people is only fever, which is very rapid and can reach 38°C in a few hours, accompanied by sore throat, runny nose, cough, headache, and general fatigue. Since the nucleic acid detection of H1N1 generally uses reverse transcription and then PCR technology to detect cDNA. Therefore, the new primers designed by the method of the present invention can also be applied to the detection of H1N1 virus. The nucleic acid of the present invention is tried using artificially synthesized H1N1 (from GenBank: GQ290690.1) as a template. Two primers used in the experiment are N1-SF (nucleotide sequence as shown in SEQ ID NO.4) and N1-SR (nucleotide sequence as shown in SEQ ID NO.5). These are designed into regions that anneal into loops by utilizing the adjacent stacking phenomenon. Furthermore, setting these primers at high concentrations allows annealing of N1-SF (or N1-SR) to occur preferentially.

[0135] The composition of the reaction solution for the method of synthesizing the nucleic acid of the present invention using these primers is shown below.

[0136] The reaction solution was composed as follows, and the rest was prepared with ddH 2 O to 25 μL

[0137] 20 mM Tris-HCl pH 8.8

[0138] 10mM KCl

[0139] 10mM (NH 4 ) 2 SO 4

[0140] 14 mM MgSO 4

[0141] 0.1% Triton X-100

[0142] 1M Betaine

[0143] 1.25mM dNTP

[0144] 8U Bst DNA polymerase (NEW ENGLAND Biolabs)

[0145] Primers:

[0146] 1600nMN1-SF

[0147] 1600nMN1-SR

[0148] Target nucleic acid: H1N1 dsDNA (nucleotide sequence as shown in SEQ ID NO.6). The mixture was reacted at 61°C for 1 hour, and then terminated at 80°C for 10 minutes, and then transferred back to ice-cold water.

[0149] Confirmation of the reaction: 1 μL of conventional nucleic acid electrophoresis loading buffer (provided with Takara DNA ladder) was added to 5 μL of the above reaction solution, and the sample was electrophoresed at 90 mV on a 1% agarose gel (TAE dissolved) prestained with GelRed (Biotum) for 1 hour. DNAMarker was used as a molecular weight marker. The gel after electrophoresis was used to verify the nucleic acid. The results were Figure 8 It is shown in the figure that a nucleic acid product with a wide molecular weight distribution is obtained, which verifies that the nucleic acid obtained by the inventive method can be infinitely self-assembled, annealed and extended to obtain ultra-large nucleic acid molecules.

[0150] Example 3

[0151] This example uses EvaGreen to verify the H1N1 gene amplification reaction product.

[0152] EvaGreen is similar to SYBR Green I. It is a dye with a green excitation wavelength that binds to the minor groove region of all dsDNA double helices. Its inhibition on nucleic acid amplification reactions such as PCR is much less than that of the latter. In the free state, EvaGreen emits weak fluorescence, but once it binds to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of EvaGreen is related to the amount of double-stranded DNA, and the amount of double-stranded DNA in the nucleic acid amplification system can be detected based on the fluorescence signal.

[0153] The combination of the reaction solution of the method for synthesizing the nucleic acid of the present invention by using primers N1-SF (nucleotide sequence shown in SEQ ID NO.4) and N1-SR (nucleotide sequence shown in SEQ ID NO.5) is as shown below.

[0154] The reaction solution was composed as follows, and the rest was prepared with ddH 2 O to 25 μL

[0155] 20 mM Tris-HCl pH 8.8

[0156] 10mM KCl

[0157] 10mM (NH 4 ) 2 SO 4

[0158] 14 mM MgSO 4

[0159] 0.1% Triton X-100

[0160] 1M Betaine

[0161] 1.25mM dNTP

[0162] 8U Bst DNA polymerase (NEW ENGLAND Biolabs)

[0163] 1X EvaGreen(Biotum)

[0164] Primers:

[0165] 1600nMN1-MF

[0166] 1600nMN1-MR

[0167] Target nucleic acid: H1N1 dsDNA (nucleotide sequence as shown in SEQ ID NO.6). 8 positive controls and 8 negative controls were set for the amplification reaction. The reaction temperature of Applied Biosystems Q3 real time PCR was set to 61°C and the reaction time was 60 min. The curve of fluorescence intensity changing with reaction time is shown in Fig. 9 The application of fluorescence detection can achieve the purpose of real-time monitoring, and the results can be judged in advance through the real-time amplification curve.

[0168] Example 4

[0169] In this example, hydroxynaphthol blue (HNB) was used to monitor the end point of the amplification reaction.

[0170] Hydroxynaphthol blue (HNB) is a metal ion indicator that displays different indicator colors to judge the results according to the changes in the amount of magnesium ions or manganese ions combined with the by-product pyrophosphate in the reaction.

[0171] The combination of the reaction solution of the method for synthesizing the nucleic acid of the present invention by using primers N1-SF (nucleotide sequence shown in SEQ ID NO.4) and N1-SR (nucleotide sequence shown in SEQ ID NO.5) is as shown below.

[0172] The reaction solution was composed as follows, and the rest was prepared with ddH 2 O to 25 μL

[0173] 20 mM Tris-HCl pH 8.8

[0174] 10mM KCl

[0175] 10mM (NH 4 ) 2 SO4

[0176] 14 mM MgSO 4

[0177] 0.1% Triton X-100

[0178] 1M Betaine

[0179] 1.25mM dNTP

[0180] 8U Bst DNA polymerase (NEW ENGLAND Biolabs)

[0181] 120 μM HNB

[0182] Primers:

[0183] 1600nMN1-SF

[0184] 1600nMN1-SR

[0185] Target nucleic acid: H1N1 dsDNA (nucleotide sequence as shown in SEQ ID NO.6). 8 positive controls and 8 negative controls were set for the amplification reaction. The constant temperature water bath was set to a constant temperature of 61°C and the reaction time was 60 min. The endpoint results of the positive and negative reactions were as follows: Fig.10 HNB is applied to enable the interpretation of reaction results without instrument assistance.

[0186] Example 5

[0187] This example uses real-time fluorescence based on EvaGreen to achieve RNA target gene amplification.

[0188] AMV reverse transcriptase can synthesize cDNA using RNA as a template, and can detect RNA in combination with Bst DNA polymerase.

[0189] cDNA was synthesized using RNA as a template using primers N1-SF (nucleotide sequence as shown in SEQ ID NO.4) and N1-SR (nucleotide sequence as shown in SEQ ID NO.5). The reaction solution was composed as follows, and the rest was purified by ddH 2 O to 25 μL

[0190] 20 mM Tris-HCl pH 8.8

[0191] 10mM KCl

[0192] 10mM (NH 4 ) 2 SO 4

[0193] 14 mM MgSO 4

[0194] 0.1% Triton X-100

[0195] 1M Betaine

[0196] 1.25mM dNTP

[0197] 8U Bst DNA polymerase (NEW ENGLAND Biolabs)

[0198] 5UAMV reverse transcriptase

[0199] 1X EvaGreen(Biotum)

[0200] Primers:

[0201] 1600nMN1-SF

[0202] 1600nMN1-SR

[0203] Target nucleic acid: H1N1 RNA (RNA nucleic acid sequence is shown in SEQ ID NO.7). The H1N1 RNA is transcribed in vitro from H1N1 dsDNA (sequence is shown in SEQ ID NO.6).

[0204] The reaction temperature of Applied Biosystems Q3 real-time PCR was set to 61°C and the reaction time was 60 min. The curve of fluorescence intensity versus reaction time is shown in Fig.11 This result shows that this method is also feasible for RNA detection.

[0205] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present application.

Claims

1. A method for improving the synthesis of nucleic acids under constant temperature conditions for non-diagnostic purposes, characterized in that: The following steps are involved: S1. Provide a nucleic acid, wherein the 3' end of the nucleic acid has an F2 region that can anneal to F2c of the F region on the same chain, and the 5' end of the nucleic acid has an R2c region that can anneal to R2 of the R region on the same chain; S2, using the nucleic acid described in step S1 as a template, taking the 3' end of the F1c region that has annealed to the F1 region as the synthesis starting point, and synthesizing its own complementary nucleic acid chain; S3. Synthesizing a complementary chain by a polymerase-catalyzed strand displacement synthesis reaction to displace the complementary nucleic acid chain synthesized in step S2.

2. A method for improving nucleic acid synthesis under constant temperature conditions for non-diagnostic purposes according to claim 1, characterized in that: The specific steps include: 1) providing a nucleic acid, wherein the 3' end has an F2 region that can anneal to F2c of the F region on the same chain, and the 5' end of the nucleic acid has an R2c region that can anneal to R2 of the R region on the same chain, and the nucleic acid can spontaneously form a short-chain complementary neck loop structure; 2) annealing the first oligonucleotide I with the F1c region of the nucleic acid provided in step 1), and then performing a synthesis step using the F1 region of the first oligonucleotide I as a synthesis starting point; wherein the first oligonucleotide I includes the F2c region and the F1 region; 3) using the nucleic acid provided in step 1) as a template to synthesize its own complementary chain, and the synthesized nucleic acid sequence is referred to as the first nucleic acid; 4) annealing the second oligonucleotide II with the R1c region of the first nucleic acid provided in step 3), and then performing a synthesis step using the R1 region of the second oligonucleotide II as a synthesis starting point; wherein the second oligonucleotide II includes the R2c region and the R1 region; 5) Using the first nucleic acid provided in step 3) as a template, synthesizing its own complementary chain, wherein the nucleic acid chain has complementary nucleotide sequence regions that are alternately connected.

3. A method for improving nucleic acid synthesis under constant temperature conditions for non-diagnostic purposes according to claim 2, characterized in that: The method for preparing nucleic acid in step 1) comprises the following steps: 1-a) annealing step, annealing the F1 region of the first nucleotide with the F1c region of the template, wherein the template is composed of the Fc region and the R region in the 3' to 5' direction; the Fc region includes the F1c and F2c regions, the R region includes the R1 region and the R2 region, and the F region includes the F1 region and the F2 region; the first nucleotide is the F2c region and the F1 region from the 5' end to the 3' end; Among them, F region: a region having a nucleotide sequence complementary to the Fc region; F1 region: a region having a nucleotide sequence complementary to the F1c region; F2 region: a region having a nucleotide sequence complementary to the F2c region; 1-b) synthesizing a first nucleic acid using the F2 region of the first oligonucleotide as a synthesis starting point, wherein the second oligonucleotide comprises an R2c region and an R1 region from the 5' end to the 3' end; the first nucleic acid has a nucleotide sequence complementary to the template, and the 5' end of the first nucleic acid has an F2c region that can anneal to the F2 region on the same chain; 1-c) annealing the R1 region of the second oligonucleotide to the R1c region of the first nucleic acid under constant temperature conditions, wherein the second oligonucleotide comprises the R2c region and the R1 region from the 5' end to the 3' end; the R region comprises the R1 region and the R2 region, and the Rc region comprises the R1c region and the R2c region; R region: a region having a nucleotide sequence complementary to the Rc region; R1 region: a region having a nucleotide sequence complementary to the R1c region; R2 region: a region having a nucleotide sequence complementary to the R2c region; 1-d) synthesizing a second nucleic acid using the R1 region of the second oligonucleotide as a synthesis starting point; R1 region: a region having a nucleotide sequence complementary to the R1c region; R2 region: a region having a nucleotide sequence complementary to the R2c region.

4. A method for improving nucleic acid synthesis under constant temperature conditions for non-diagnostic purposes according to claim 3, characterized in that: The template in step 1-a) is RNA, and the first nucleic acid in step 1-b) is synthesized by an enzyme having reverse transcriptase activity.

5. A method for improving nucleic acid synthesis under constant temperature conditions for non-diagnostic purposes according to any one of claims 2 to 4, characterized in that: The nucleic acid fragments of the F1 region, F2 region, R1 region and R2 region are all 10-30 bp.

6. A method for improving nucleic acid synthesis under constant temperature conditions for non-diagnostic purposes according to claim 1 or 2, characterized in that: The obtained nucleic acid chain can pair autonomously and extend indefinitely. The synthetic nucleic acid refers to a nucleic acid having nucleotide sequences on one chain in which the first and last segments are complementary to different parts of the middle segment; under appropriate conditions, the nucleic acid chain can form a shorter neck loop chain through base complementary pairing; the F2 region at the 3' end of the nucleic acid chain will pair with the complementary segment F2c region on the chain as the starting point of synthesis, using itself as a template to continuously extend the nucleic acid chain.

7. A kit for synthesizing nucleic acid, characterized in that: The nucleic acid has head-to-tail complementary nucleotide sequences on one strand, and the nucleic acid strand has alternately connected complementary nucleotide sequences; the kit comprises the following components: The first oligonucleotide I comprises an F2c region and an F1 region, wherein the F2c region is connected to the 5' side of the F1 region, wherein F1 region: a region having a nucleotide sequence complementary to the F1c region, and F2c region: a region of a nucleotide sequence complementary to the F2 region in the F region; The second oligonucleotide II comprises an R2c region and an R1 region, wherein the R2c region is connected to the 5' side of the R1 region, wherein F1 region: a region having a nucleotide sequence complementary to the F1c region, and F2c region: a region of a nucleotide sequence complementary to the F2 region in the F region; DNA polymerase that catalyzes strand displacement-type complementary strand synthesis reaction; Nucleotides, which serve as substrates for the DNA polymerase.

8. Use of the kit as claimed in claim 7 for non-diagnostic purposes in synthesizing nucleic acids or detecting target nucleotide sequences in samples.

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