CN107446919B

CN107446919B - Method and kit for synthesizing nucleic acid under constant temperature condition

Info

Publication number: CN107446919B
Application number: CN201710828028.8A
Authority: CN
Inventors: 杜昱光; 毛瑞
Original assignee: Zhongke Xinrui Suzhou Biotechnology Co ltd
Current assignee: Hangzhou Tinker Biotechnology Co ltd
Priority date: 2017-09-14
Filing date: 2017-09-14
Publication date: 2020-04-28
Anticipated expiration: 2037-09-14
Also published as: CN107446919A

Abstract

The invention discloses a method and a kit for synthesizing nucleic acid under a constant temperature condition, wherein the method comprises the following steps: 1) providing a nucleic acid having at its 5 '-end an Nc region annealable to an N region on the same strand and at its 3' -end an Nc region annealable to an N region on the same strand, the Nc regions at its 5 '-end and 3' -end being in a competitive relationship with annealing to the N region on the same strand; 2) synthesizing a self complementary strand using the nucleic acid of step 1) as a template and the 3' end of the Nc region annealed with the N region as a synthesis origin; 3) complementary strand synthesis is performed by polymerase-catalyzed strand displacement-type complementary strand synthesis reaction to displace the complementary strand synthesized in step 2). The main advantage of the invention is that the rapid amplification of the gene can be realized by applying a single-enzyme constant temperature system aiming at short chain nucleic acid fragments (the ideal fragments can be only 60bp and are shorter than the minimum ideal fragments of LAMP by 120 bp).

Description

Method and kit for synthesizing nucleic acid under constant temperature condition

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a method for synthesizing nucleic acid under a constant temperature condition, in particular to a method for synthesizing nucleic acid which is composed of specific nucleotide sequences and can form a special structure, and a useful method for amplifying nucleic acid based on the specific nucleotide sequences.

Background

The most fundamental difference of organisms carrying genetic information of organisms, the analysis method based on nucleotide sequence complementarity can directly analyze the genetic characteristics carried by genes. This analysis is a very powerful method for identifying genetic diseases, canceration, microorganisms, etc. Since the target gene is not easily detected when the amount of the target gene in the sample is very small, the target gene must be amplified or its detection signal must be amplified. As a method for amplifying a target gene, the PCR method is considered to be the most classical method (Saiki, Gelfandet al 1988), and is also the most common technique for in vitro amplification of nucleic acid sequences. The exponential amplification result of the method ensures that the method has high sensitivity, establishes the position of the method in the field of molecular biological method detection, and develops the existing series of mature products after decades of development. In addition, the amplification product can be recycled and thus is widely used as an important tool for supporting genetic engineering techniques such as gene cloning and structure determination. However, the PCR method has obvious problems as follows: in actual operation, a special program temperature control system is required; the exponential rise of the amplification reaction makes it difficult to quantify; the sample and the reaction solution are susceptible to external contamination, and the problem of false positive is prominent.

For example: if the complementary strand is accidentally missynthesized in PCR, the product will be run as a template in the subsequent reaction, resulting in erroneous results. In practice, it is difficult to strictly control PCR if only one base at the end of the primer is different, so that it is necessary to improve the specificity so that PCR can be more preferably used for detection of SNPs. On the other hand, compared with the synthesis of nucleic acid by a complicated programmed temperature-controlled process, scientists have developed a technique for synthesizing nucleic acid under constant temperature conditions (Zhao, Chen et al.2015), which mainly includes the following: nucleic acid sequence dependent amplification (NASBA), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), loop mediated isothermal amplification (LAMP), Helicase Dependent Amplification (HDA), Recombinant Polymerase Amplification (RPA).

NASBA, also known as TMA (transcription-mediated amplification method), does not require complex temperature control. The method is synthesized by adding a probe linked to a T7 promoter to a target RNA as a template by a DNA polymerase, allowing a second probe to enter into double strands to produce a product, and then transcribing the double-stranded DNA produced by the DNA polymerase to amplify the double-stranded DNA to produce a large amount of RNA product. NASBA requires a heat denaturation step until double-stranded DNA is formed, but the subsequent transcription reaction is performed by T7RNA polymerase under isothermal conditions. It is necessary to use a combination of various enzymes such as reverse transcriptase, RNase H, DNA polymerase and T7RNA polymerase, however, the combination of various enzymes is disadvantageous in terms of cost. Meanwhile, due to the complicated setting of reaction conditions of various enzymes, the method is difficult to popularize as a general analysis method.

RCA (Rolling Circle Amplification) is intended to mimic the process of Rolling Circle replication of circular DNA in microorganisms, and for circular single-stranded DNA templates, Amplification of circular nucleic acids can only be achieved in the in situ method using primers that bind to the template. To make this method universally applicable to amplification of linear DNA, single-stranded DNA complementary to a padlock probe (padlock probe) or a loop probe was shown to be continuously synthesized in the presence of a target nucleotide, with a series of nucleotide sequences complementary to the padlock probe (padlock probe) or the loop probe (Lizardi, Huang et al 1998). This method also has a problem that a plurality of enzymes are required. Moreover, the initiation of complementary strand synthesis depends on the reaction of joining two adjacent regions, and the specificity thereof is substantially the same as that in LCR.

The Strand Displacement Amplification (SDA) method is also known as a method for amplifying a template DNA having a sequence complementary to a target sequence (Zhang, Cui et al 1992). The SDA method employs a specific DNA polymerase to synthesize a complementary strand from a primer complementary to the 3 '-side of a target nucleotide sequence to replace the sequence on the 5' -side of the double strand. This technique is called SDA method because the newly synthesized complementary strand replaces the double strand on the 5' -side. The restriction enzyme recognition sequence as a primer inserted into the annealing sequence in the SDA method can remove the temperature change step necessary for the PCR method. That is, the 3' -OH group is supplied as the origin of complementary strand synthesis by the restriction enzyme-generated nick, and the complementary strand synthesized first is released as a single strand by strand displacement synthesis and then used again as a template for the next complementary strand synthesis. However, SDA amplification products differ in structure from the native nucleic acids and have limitations for use of restriction enzymes to break or apply the amplification products to gene clones. This aspect is also the main reason for the higher cost. In addition, when the SDA method is applied to an unknown sequence, a nucleotide sequence identical to the recognition sequence of the restriction enzyme for introducing a nick may be present in the region to be synthesized, and thus synthesis of a completely complementary strand may be prevented.

Helicase-dependent Isothermal DNA amplification (HDA) is a novel Isothermal nucleic acid amplification technique invented by researchers of the American NEB company in 2004 (Vincent, Xu et al 2004). The technology simulates the natural process of DNA replication in a natural organism, unwinding a DNA double strand by helicase under the condition of constant temperature, simultaneously, stably unwinding a single strand by single-stranded DNA-binding protein (SSB), providing a binding template for a primer, and then catalytically synthesizing a complementary strand by DNA polymerase. The newly synthesized double strand is decomposed into single strand under the action of helicase, and the single strand is used as a template for the next round of synthesis to enter the cyclic amplification reaction, and finally the exponential growth of the target sequence is realized.

Recombinase Polymerase Amplification (RPA), whose main point is: the recombinase, in combination with the primer, forms a protein-DNA complex that is able to search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequences, the strand-displacing DNA polymerase then mediates the formation of strand-exchange reactions and initiates DNA synthesis, which exponentially amplifies the target region on the template. The replaced DNA strand binds to a single strand binding protein (SSB) to prevent further replacement. In this system, a single synthesis event is initiated by two opposing primers. The entire process is carried out very quickly and detectable levels of amplification product are typically obtained within ten minutes. However, in the whole process, a primer which can be combined with a recombinase and has good specificity needs to be screened, and meanwhile, the cost is greatly increased by using three enzymes, and the difficulty in designing the primer is also high.

At the heart of LAMP technology (Notomi, Okayama et al 2000), four specific primers are designed for six regions on the target gene, relying on a highly active strand displacement DNA polymerase, so that strand displacement DNA synthesis is constantly self-cycling. The technology can realize amplification of 109-1010 times within 15-60 minutes, the reaction can generate a large amount of amplification products, namely magnesium pyrophosphate white precipitate, whether the target gene exists can be judged by observing the existence of the white precipitate by naked eyes, and Japan Rongand research company also develops a turbidimeter in a targeted manner to realize real-time monitoring of the amplification reaction. The LAMP method has the advantages of high specificity and high sensitivity, is very simple to operate, has low requirements on instruments in the application stage, can realize reaction by using a simple constant temperature device, is very simple in result detection, can be used for directly observing white precipitates or green fluorescence by naked eyes, and is a method suitable for rapid detection on site and in basic level. One limitation is that the method relies on the properties of 4 primers for its high specificity and sensitivity, and the acquisition of the optimal primers usually requires sequence alignment, on-line primer design, primer screening and specificity tests, which are cumbersome. Meanwhile, the target gene segment required by LAMP technology is also large, and the LAMP technology is difficult to apply to short-chain nucleic acid. For example, 5S rRNA is composed of about 120 nucleotide units, and is an important structural and functional ribosomal subunit in all living bodies. 5S rRNA is highly conserved and is often selected as a marker for molecular detection. Because the nucleic acid sequence of 5S rRNA is short, PCR is generally adopted for detecting the marker at present. However, isothermal amplification of nucleic acids is simple and fast, but is difficult to be applied to detection of 5S rRNA. Taking loop-mediated isothermal amplification (LAMP) as an example, LAMP primers are composed of F3, B3, FIP and BIP, nucleic acid sequences required for designing the primers need about 120 bases, not to mention that the interval between F2 and B2 is designed, so that the harsh conditions for designing LAMP primers are difficult to meet, and the LAMP primers cannot be applied to detection of 5S rRNA.

Disclosure of Invention

The invention aims to provide a method for synthesizing nucleic acid, which is inspired by common double helix structure of DNA, molecular beacon probe and LAMP, redesigns and plans a primer working mode on the basis of PCR primer, forms a competitive stem-loop initial amplification structure, designs a forming process of the structural loop, and has the characteristic of optional use of outer primer. More particularly, to provide a novel, low-cost method for efficiently synthesizing nucleic acids by means of sequences. That is, the object of the present invention is to provide a method for synthesizing and amplifying nucleic acids by a single enzyme under isothermal conditions. Another object of the present invention is to provide a method for synthesizing nucleic acid, which can synthesize nucleic acid rapidly with high specificity, which is difficult to achieve by modifying the existing principles of nucleic acid synthesis reaction, and a method for amplifying nucleic acid using the synthesis method. One advantage of the invention is that the rapid amplification of the gene can be realized by applying a single-enzyme constant temperature system aiming at short chain nucleic acid fragments (the ideal fragments can be only 60bp and are shorter than the ideal fragments with the minimum LAMP by 120 bp). The present invention utilizes polymerase to catalyze strand displacement-type complementary strand synthesis without complicated temperature control, and is useful for nucleic acid synthesis. The DNA polymerase is an enzyme used in methods such as SDA, RCA and LAMP.

The present inventors improved the supply of 3 '-OH in the known method, and as a result, found that by using an oligonucleotide having a specific structure, the 3' -OH structure can be provided without any additional enzymatic reaction, thereby leading to the present invention. Namely, the present invention relates to a method for synthesizing nucleic acid, a method for amplifying nucleic acid by using the method for synthesizing nucleic acid, and a kit for synthesizing nucleic acid using the method.

The specific technical scheme of the invention is as follows:

the invention provides a method for synthesizing nucleic acid under a constant temperature condition, which comprises the following steps:

1) providing a nucleic acid having at its 5 '-end an Nc region annealable to an N region on the same strand and at its 3' -end an Nc region annealable to an N region on the same strand, the Nc regions at its 5 '-end and 3' -end being in a competitive relationship with annealing to the N region on the same strand;

2) synthesizing a self complementary strand using the nucleic acid of step 1) as a template and the 3' end of the Nc region annealed with the N region as a synthesis origin;

3) complementary strand synthesis is performed by polymerase-catalyzed strand displacement-type complementary strand synthesis reaction to displace the complementary strand synthesized in step 2).

The method for synthesizing the nucleic acid under the constant temperature condition comprises the following specific synthesis steps:

1) a step of providing a nucleic acid having an Nc region capable of annealing to an N region on the same strand at both the 5 '-end and the 3' -end; the Nc region at the 3' -end is capable of forming a loop when annealed to the N region, said loop comprising an F1c region capable of base pairing; the Nc region at the 5' -end is capable of forming a loop when annealed to the N region, said loop comprising an R1 region capable of base pairing; the Nc regions at the 5 '-end and 3' -end of the nucleic acid compete with the annealing of the N region on the same strand;

2) annealing a first oligonucleotide I to the F1c region of said nucleic acid provided in step 1), and then performing a synthesis step with the F1 region of said first oligonucleotide I as a synthesis origin; wherein the first oligonucleotide I comprises an N region and a Fl region;

3) synthesizing a complementary strand of itself using the nucleic acid provided in step 1) as a template, and using the 3' end of the Nc region annealed with the N region as a synthesis origin; the nucleic acid sequence after synthesis is called nucleic acid A;

4) annealing a second oligonucleotide II to the region R1c of said nucleic acid A provided in step 3), and then carrying out a synthesis step with the region R1 of said second oligonucleotide II as the origin of synthesis; wherein the second oligonucleotide II comprises a R1 region and an Nc region;

5) synthesizing its own complementary strand using said nucleic acid A provided in step 3) as a template, and using the N region to which the Nc region has annealed at the 3' -end as a synthesis origin to obtain a nucleic acid having a head-to-tail complementary nucleotide sequence on one strand thereof and having complementary nucleotide sequence regions alternately linked on the nucleic acid strand.

Referring to FIG. 1, there is shown a schematic diagram of the steps corresponding to the above-described synthesis of nucleic acids according to the present invention.

Preferably, the method for preparing nucleic acid according to the step 1) above, comprises the steps of:

1-1) an annealing step of annealing a first oligonucleotide I to a region F1c of a template, wherein the 3 'end of the template comprises a region F1c and a region N located 5' to the region F1c and the 5 'end of the template comprises a region R1, wherein said first oligonucleotide I comprises a region N to a region Fl, said region N being linked to the 5' side of the region F1, wherein,

region F1: a region having a nucleotide sequence complementary to the F1c region,

and an N region: a region of nucleotide sequence complementary to the Nc region;

1-2) synthesizing a first nucleic acid with the F1 region of the first oligonucleotide I as a synthesis origin; the first nucleic acid has a nucleotide sequence complementary to the template, the 5' -end of the first nucleic acid has an N region that can anneal to an Nc region on the same strand, and a stem loop can be formed by annealing of the Nc region to the N region;

1-3) utilizing polymerase to catalyze strand displacement reaction for displacement to obtain a first nucleic acid synthesized in the step 1-2);

1-4) an annealing step of annealing a second oligonucleotide II to the R1c region of the first nucleic acid obtained in the step 1-3), wherein the second oligonucleotide II includes an R1 region and an Nc region, and the Nc region is linked to the 5' -side of the R1 region; wherein the content of the first and second substances,

region R1: a region having a nucleotide sequence complementary to the R1c region,

nc region: a region of nucleotide sequence complementary to the N region;

1-5) synthesizing a second nucleic acid with the region R1 of the second oligonucleotide II as the starting point for the synthesis;

1-6) displacing said second nucleic acid by means of a polymerase catalyzed strand displacement reaction to obtain the nucleic acid of step 1).

Referring to FIG. 2, there is shown a schematic diagram of the steps corresponding to the second nucleic acid (i.e., the nucleic acid described in step 1) of the present invention) described above.

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. For example, the kit is used for detecting cDNA and RNA of H1 gene and N1 gene of H1N1 virus; for cDNA and RNA detection of MERS-CoV virus, such as: orf1a, orf1b segment of the RNA; the kit is used for DNA detection of the carp herpes virus III type and the like.

Preferably, the nucleic acid fragments of the F1c region, the N region and the R1 region are all 15-60 bp. Further preferably, the nucleic acid fragments of the F1c region, the N region and the R1 region are all 20 bp.

The method for synthesizing nucleic acid under isothermal conditions further comprises: the nucleic acid chain obtained in the step 5) can be infinitely extended by self-pairing, and the 3' end Nc region on the nucleic acid chain is paired with the complementary segment N region on the chain to be used as a synthesis starting point to continuously extend the nucleic acid chain by taking the nucleic acid chain as a template.

The method for synthesizing a nucleic acid according to the present invention, wherein the synthesized nucleic acid is a nucleic acid having a nucleotide sequence complementary end to end on one strand thereof.

The method for synthesizing nucleic acid according to the present invention, wherein the constant temperature means that the synthesis is performed at a temperature ranging from 60 to 65 ℃ throughout the reaction process.

According to the method for synthesizing a nucleic acid of the present invention, preferably, nucleic acid amplification is accelerated by introducing the acceleration primers X2 and/or Xin; wherein X2 is a segment located on the 5' side of the F1 region and R1 region of the complementary strand of the original nucleic acid, and Xin is a segment located in the middle of the F1c region to N region and the Nc region to R1 c. The polymerase used in the polymerase catalytic strand displacement reaction 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), phi 29 phase DNA polymerase, MS-2 phase DNA polymerase and the like. Among them, BstDNA polymerase or Bca (exo-) DNA polymerase is preferably used.

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

The kit for synthesizing nucleic acid under isothermal conditions according to the present invention is characterized by comprising:

a first oligonucleotide I comprising a region F1 and an N region linked to the 5' side of the region F1, wherein,

region F1: a region having a nucleotide sequence complementary to the F1c region, and

a second oligonucleotide II comprising a R1 region and an Nc region linked to the 5' side of the R1 region, wherein,

region R1: a region having a nucleotide sequence complementary to the R1c region, and

nc region: a region of nucleotide sequence complementary to the N region;

a DNA polymerase catalyzing a strand displacement-type complementary strand synthesis reaction, and,

a nucleotide that serves as a substrate for the DNA polymerase.

The kit according to the present invention, wherein the kit further comprises a detection reagent for detecting a product of the nucleic acid synthesis reaction.

The kit of the present invention further comprises accelerating primers X2 and/or Xin, wherein X2 is a segment located 5' to the F1 region and the R1 region of the complementary strand of the original nucleic acid, and Xin is a middle segment located from the F1c region to the N region and from the Nc region to the R1 c.

According to the kit, 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 (Exo-) DNA polymerase, phi 29phage DNA polymerase, MS-2phage DNA polymerase and the like. Among them, Bst DNA polymerase or Bca (exo-) DNA polymerase is preferably used.

The invention provides application of the kit in synthesizing nucleic acid or detecting a target nucleotide sequence in a sample.

Based on the method for synthesizing a nucleic acid of the present invention, there is provided a method for detecting a target nucleotide sequence in a sample, comprising amplifying by the method for synthesizing a nucleic acid of the present invention using a target nucleotide as a template, and observing whether or not an amplification product is produced.

A probe comprising a nucleotide sequence complementary to the stem-loop structure formed is added to the amplification product, and hybridization between the two is observed. The probe may also be labeled on a particle and the aggregation reaction by hybridization observed. The amplification method may be carried out in the presence of a nucleic acid detection reagent, and whether or not an amplification product is produced is observed based on a change in a signal of the detection reagent.

Based on the method for synthesizing nucleic acid of the present invention, there can also be provided a method for detecting a mutation in a target nucleotide sequence in a sample, comprising amplifying by the method for synthesizing nucleic acid of the present invention using the target nucleotide as a template. Wherein a mutation to be amplified in the nucleotide sequence inhibits synthesis of any of complementary strands constituting the amplification method, and the mutation is detected.

A single-stranded nucleic acid of a nucleotide sequence that can form a competitive stem-loop structure, which refers to a nucleic acid in which a target nucleic acid sequence is alternately linked in a single strand in a certain order with mutually complementary nucleotide sequences, is an object of the synthesis of the present invention. The nucleic acids synthesized by the present invention consist essentially of mutually complementary strands linked by a stem-loop forming structure. Referring to FIG. 5, a schematic representation of an ideal amplified nucleic acid product formed by the synthetic method of the present invention is shown.

In general, a strand that cannot be separated into two or more molecules upon base pairing separation is referred to as a single strand, regardless of whether a portion is involved in base pairing. Complementary nucleotide sequences in the same strand can form base pairing, and the present invention can obtain an intramolecular base-paired product comprising a region constituting a significant double strand and a loop not involved in base pairing by allowing nucleic acids having nucleotide sequences joined end to end in a single strand to base pair within the same strand.

That is, the nucleic acid having a nucleotide sequence of a competitive stem loop structure of the present invention can be defined as a single-stranded nucleic acid comprising a complementary nucleotide sequence capable of annealing in the same strand. Nucleotides having a complementary nucleotide sequence can anneal into loops not involved in base pairing. The loop forming sequence may be any nucleotide sequence. The loop-forming sequences are capable of base pairing to initiate synthesis of the complementary strand for displacement. And is preferably provided with a sequence different from the nucleotide sequence located in the other region to obtain specific annealing.

The nucleotide sequences that are substantially identical in the present invention are defined as follows: when a complementary strand synthesized with a certain sequence as a template anneals to a target nucleotide sequence to supply the origin of synthesizing the complementary strand, the certain sequence is substantially identical to the target nucleotide sequence. For example, a sequence substantially identical to F1 includes not only the sequence identical to F1 entirely but also a nucleotide sequence capable of serving as a template that gives a nucleotide sequence to which F1 anneals and can serve as a starting point for synthesizing a complementary strand. The term "annealing" according to the present invention refers to nucleic acids that form complementary structures by base pairing according to Watson-Crick's law. Therefore, even if a nucleic acid strand constituting base pairing is single-stranded, annealing occurs if the intramolecular complementary nucleotide sequence base pairs. Since the double-stranded structure is formed by base-pairing nucleic acids, the meaning expressed by annealing and hybridization according to the present invention is a coincidence.

The number of nucleotide sequence pairs constituting a nucleic acid of the present invention is at least 1. In the model contemplated by the present invention, the nucleotide sequence number of pairs may be an integer multiple of 1. In this case, there is no upper limit in the theoretical logarithm of the complementary nucleotide sequence of the constituent nucleotides of the present invention, and in the case of the synthetic product nucleic acid of the present invention composed of a plurality of sets of complementary nucleotide sequences, the nucleic acid is composed of nucleotide sequences that are identical in repetition.

The single-stranded nucleotides of the nucleotide sequence having a competitive stem loop structure synthesized in the present invention have a different structure from naturally occurring nucleic acids, and it is generally known that a nucleic acid derivative can be synthesized if a nucleotide derivative is used as a substrate when synthesizing a nucleic acid by the action of a nucleic acid polymerase. The nucleotide derivatives used include radioisotope-labeled nucleotides or nucleotide derivatives labeled with a binding ligand such as biotin or digoxigenin. These nucleotide derivatives are useful for labeling product nucleic acids. Alternatively, if the substrate is a fluorescent nucleotide, the product nucleic acid may be a fluorescent derivative.

The synthesis of a nucleic acid having the above structure is initiated by a DNA polymerase having a strand displacement activity and an Nc region annealed to a part of the N region on the same strand at the 3' -end to synthesize a complementary strand. There are many reports on complementary strand synthesis reactions in which a helical loop is formed using the sequence of the helical loop itself as a template and a hairpin loop is also formed using the sequence of the hairpin loop itself as a template, and the present invention provides a region in which a part of a competitive hairpin loop can base pair, and has a novel feature of utilizing the region in synthesizing a complementary strand. By using this region as the start of synthesis, the complementary strand previously synthesized with the hairpin loop sequence itself as a template is replaced.

The present invention uses the term "nucleic acid", which typically includes both DNA and RNA. However, the template for synthesizing nucleic acid of the present invention, nucleic acid in which a nucleotide derived from natural DNA or RNA is replaced with an artificial derivative or modified nucleotide is also included in the scope of the nucleic acid of the present invention. Typically, the nucleic acids of the invention are contained in biological samples, including tissues, cells, cultures and secretions of animals, plants or microorganisms, as well as extracts thereof. The biological sample of the invention comprises intracellular parasite genomic DNA or RNA, such as a virus or mycoplasma. The nucleic acids of the invention are generally derived from the nucleic acids contained in said biological sample. For example, cDNA synthesis from mRNA, nucleic acids amplified based on nucleic acids derived from biological samples, are typical examples of nucleic acids of the present invention.

The nucleic acid of the present invention is characterized in that Nc regions are provided at the 3 '-and 5' -ends, and can anneal to a part of N regions on the same strand, and a hairpin loop can be formed by annealing the Nc region to the N region on the same strand, and the nucleic acid can be obtained in various methods, and the Nc regions at the 5 '-end and 3' -end of the nucleic acid compete with the annealing of the N regions on the same strand. Furthermore, extension by DNA polymerase is possible only when the Nc region at the 3' -end anneals to the N region. FIG. 3 is a schematic diagram of the competitive hairpin structure formed by the single-stranded nucleic acid of the invention and the subsequent amplification. FIG. 4 is a schematic representation of a subsequent amplification reaction of nucleic acid. Wherein NF in FIGS. 3 and 4 is the first oligonucleotide I and NR in FIG. 4 is the second oligonucleotide II.

The terms "identical" and "complementary" used to make up the nucleotide sequence features based on the oligonucleotides of the invention do not imply absolute identity and absolute complementarity. That is, a sequence identical to a certain sequence includes a sequence complementary to a nucleotide sequence to which the certain sequence anneals. On the other hand, the complementary sequence is a sequence that can anneal under stringent conditions and is provided as the 3' -end of the origin of complementary strand synthesis.

In the present invention, an oligonucleotide is a nucleotide that satisfies two requirements, i.e., must be capable of forming complementary base pairing and supply an-OH group at the 3' -end as the origin of complementary strand synthesis. Therefore, the main chain thereof is not necessarily limited to phosphodiester bond-type linkage. For example, it may consist of a backbone of phosphorothioate derivatives which are S substituted for O or be peptide nucleic acids based on peptide linkages. Bases are those bases that can be complementarily paired. Five bases, namely a, C, T, G and U, occur naturally, and bases may also be analogs such as bromodeoxyuridine. Preferably, the oligonucleotide of the present invention can be used not only as an origin of synthesis but also as a template for complementary strand synthesis. The term polynucleotide of the present invention includes oligonucleotides. The term "polynucleotide" as used herein is not limited in its chain length, while the term "oligonucleotide" as used herein refers to a polymer of nucleotides having a relatively short chain length.

The oligonucleotide chain of the present invention has such a length that it can base-pair with a complementary chain and maintain a certain specificity under given conditions in various nucleic acid synthesis reactions described below. Specifically, it consists of 5 to 200 bases, more preferably 10 to 50 base pairs. The chain length recognizing the known polymerase is at least 5 bases. The polymerase catalyzes a sequence-dependent nucleic acid synthesis reaction. The chain length of the annealed portion should be longer than this length. In addition, a length of 10 bases or more is statistically expected to obtain the target nucleotide specificity. On the other hand, it is difficult to prepare a too long nucleotide sequence by chemical synthesis. Thus the above chain lengths are examples of desirable ranges. Exemplary chain lengths refer to those lengths that partially anneal to a complementary strand. As described below, the oligonucleotides of the invention can eventually anneal to at least two regions, respectively. Thus, chain lengths exemplified herein are to be understood as the chain length of each region that makes up the oligonucleotide.

Furthermore, the oligonucleotide of the present invention may be labeled with a known label. Labels include binding ligands such as digoxigenin and biotin, enzymes, fluorescers, luminophores, radioisotopes. The technique of replacing the bases constituting an oligonucleotide by fluorescent analogs is well known (W095/05391, Proc. Natl. Acad. Sci. USA, 91, 6644-.

Other oligonucleotides of the invention may also be bound to a solid phase. Alternatively, any part of the oligonucleotide may be labelled with a binding ligand, such as biotin, which is indirectly immobilised by the binding ligand, such as immobilised avidin. When the immobilized oligonucleotide is the starting point of synthesis, the nucleic acid of the product of the synthesis reaction is captured by a solid phase, which facilitates its isolation. The separated portions may be detected by nucleic acid specific indicators or hybridization to labeled probes. The nucleic acid product obtained by the present method is directed to a method wherein the target nucleic acid fragment is recovered by digestion of the product with a restriction enzyme.

The term "template" as used herein refers to a nucleic acid that serves as a template for the synthesis of a complementary strand. The complementary strand having a nucleotide sequence complementary to the template means a strand corresponding to the template. But the relationship is relative. I.e., the synthesized complementary strand can function as a template again. That is, the complementary strand may also serve as a template.

In the present invention, if the target is RNA, it may be constituted only by adding reverse transcriptase. That is, using RNA as a template, it is possible to synthesize a complementary strand by annealing F1 to F1c in the template by reverse transcriptase. When the reverse transcriptase carries out the reaction of synthesizing a complementary strand using DNA as a template, all the reactions of synthesizing a complementary strand by the reverse transcriptase include the synthesis of a complementary strand using R1 annealed to Rlc as a synthesis origin, which 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, such as Bca DNA polymerase, is used, the synthesis from not only the first single-stranded nucleic acid of RNA but also the subsequent reaction using DNA as a template can be similarly performed by the same enzyme.

The reaction is carried out in the presence of a buffer that allows the enzyme reaction to be at a suitable pH, salts necessary to anneal or maintain the enzymatic activity, mediators to protect the enzyme, and modulators necessary to control the melting temperature (Tm). As buffers, for example Tris-HCl is used which has a buffering action in the neutral or weakly alkaline range. Adjusting the pH value according to the DNA polymerase used, for salt, KCl, NaCl, (NH)₄)₂SO₄And adding proper amount to maintain the activity of the enzyme and regulate the melting temperature (Tm) of nucleic acid, wherein bovine serum albumin or saccharide is used as a medium for protecting the enzyme. In addition, dimethyl sulfoxide (DMSO) or formamide are typically used as regulators of the melting temperature (Tm). By using modulation of melting temperature (Tm)The annealing of the oligonucleotide under defined temperature conditions is controlled by the control. Further, betaine (N, N-trimethylglycine) or tetraalkylammonium salt (tetraalkyi) is also effective for improving the efficiency of strand displacement by its isostabilization (isostabilization). The desired promoting effect of the present invention on nucleic acid amplification can be obtained by adding 0.2 to 3.0M betaine, preferably 0.5 to 1.5M betaine, to the reaction solution. Since these melting temperature regulators have the function of lowering the melting temperature, those suitable stringent and reactive conditions are empirically determined in terms of the concentration of the binding salt, the reaction temperature, etc.

An 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, the non-specific synthesis reaction accompanying the non-specific synthesis of the complementary strand is effectively prevented. That is, even if a certain nonspecific reaction occurs, the possibility that the product will serve as a starting material in the subsequent amplification step of the synthesis is reduced, and, by regulating the progress of the reaction through many regions, it is possible to allow a detection system that can accurately identify the desired product in a similar nucleotide sequence to be composed arbitrarily.

The nucleic acid synthesized in the present invention is a single strand, and in terms of single strand, is composed of complementary nucleotide sequences, most of which are base-paired. By utilizing this feature, the synthesized product can be detected. By carrying out the method for synthesizing a nucleic acid of the present invention, an increase in the intensity of fluorescence is observed with an increase in the product in the presence of a fluorescent dye as a double-strand specific intercalator (e.g., ethidium bromide, SYBR Green I, Pico Green or Eva Green). By monitoring the fluorescence intensity, it is possible to track the progress of real-time (real-time) synthesis reactions in a closed system. It is also conceivable to apply this type of detection system in the PCR method, but there are many problems because it is impossible to distinguish between a product signal and a signal of primer-dimer, etc. However, when the present invention employs 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 simultaneously obtained, and a method for realizing a detection system using the transfer of fluorescence energy in the same system, similarly to the use of a double-strand-specific intercalator (double-strand-specific intercalator).

The method of synthesizing a nucleic acid of the present invention is supported by a complementary strand reaction of strand displacement type catalyzed by DNA polymerase. The reaction period also includes a reaction step in which a strand displacement-type polymerase is not required. However, in order to make up the reagent simple and from the economical viewpoint, it is advantageous to use a DNA polymerase, the following enzymes are known. Furthermore, various mutants of these enzymes, all having sequence-dependent activity and strand displacement activity for complementary strand synthesis, can be utilized within the scope of the present invention. Where mutants are meant to include those having only the structure that results in the desired catalytic activity of the enzyme or those modified for catalytic activity, stability or thermostability by, for example, mutation in an amino acid.

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)

Phi 29 phase DNA polymerase

MS-2 phase DNA polymerase

Omniamp DNA polymerase

Among these enzymes, Bst DNA polymerase, Bca (exo-) DNA polymerase, Omniamp DNA polymerase are particularly desirable 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 carried out isothermally, but it is not always possible to maintain the stability of the enzyme using a desired temperature condition due to adjustment of melting temperature (Tm) and the like. Therefore, it is one of the conditions required for the thermal stabilization of the enzyme. Although isothermal reactions are feasible, thermal denaturation can provide the nucleic acid as the initial template, in this regard, the use of thermostable enzymes broadens the choice of assay protocols.

The various reagents necessary for synthesizing or amplifying a nucleic acid of the present invention may be prepackaged and provided in the form of a kit, and specifically, the kit provided by the present invention comprises various oligonucleotides necessary as primers for synthesizing complementary strand synthesis and outer primers for displacement reaction, dntps as substrates for complementary strand synthesis, DNA polymerase for effecting strand displacement-type complementary strand synthesis, buffers for providing appropriate conditions for the enzymatic reaction, and media necessary for detecting the products of the synthesis reaction. In particular, in a preferred mode of the invention, no reagents need to be added during the reaction and thus the reagents necessary to be supplied for the subsequent reaction after transfer into the reaction vessel, wherein the reaction can be initiated by the addition of the sample alone. A system for detecting a reaction product in a container by using a visible light signal or a fluorescent signal. The vessel does not have to be opened and closed after the reaction. This is very advantageous for preventing contamination.

The present invention synthesizes a single-stranded nucleic acid having a nucleotide sequence of a competitive stem loop structure. The nucleic acid has, for example, the following uses: the first feature is the advantage of using a specific structure having a complementary sequence in a molecule, which may facilitate detection, i.e., there are known systems for detecting nucleic acids in which the signal to change depends on base pairing with a complementary nucleotide sequence. For example, by combining the methods using a double-strand specific intercalator as a detection reagent as described above, a detection system that takes full advantage of the characteristics of the synthesized product of the present invention can be realized. If the product of the synthetic reaction of the invention is heat denatured once in the detection system and returned to the original temperature, intramolecular annealing occurs preferentially and thus allows rapid base pairing between complementary sequences. If non-specific products are present, they have no complementary sequence in the molecule so that they cannot return to the original double strand immediately after separation into 2 or more molecules by heat denaturation. Interference accompanying non-specific reactions is reduced by the heat denaturation step provided prior to detection. If the DNA polymerase used is not resistant to heat, the heat denaturation step has the meaning of reaction termination and thus it is advantageous to control the reaction temperature.

The second feature is that competitive hairpin loops (i.e., stem-loop structures) are often formed that can base pair. The structure of competitive hairpin loops capable of base pairing is shown in FIG. 3. This loop, as seen in FIG. 3, is composed of the nucleotide sequence (from 3 'to 5' end) F1c, N, R1, Nc, and can undergo intramolecular annealing to form a hairpin loop.

According to a preferred mode of the present invention, a large number of loops capable of base pairing are provided in a single-stranded nucleic acid. This means that a large number of probes can hybridize to a molecular nucleic acid to allow for highly sensitive detection. It is thus possible to realize not only improved sensitivity but also a method for detecting nucleic acids based on a special reaction principle such as aggregation. For example, a probe immobilized on a fine particle such as polystyrene latex is added to the reaction product of the present invention, and aggregation of the latex particle is observed as hybridization of the product with the probe. The intensity of the aggregation can be observed with high sensitivity and quantitatively by optical measurement. Alternatively, the aggregation can also be observed by the naked eye, so that a reaction system without an optical measuring device can also be established.

Furthermore, the reaction products of the invention allow for some bindable labels, wherein each nucleic acid molecule can be detected chromatographically. In the field of immunoassays, analytical methods using chromatographic media with visible detection markers (immunochromatography) are in practical use. This method is based on the principle that an analyte is sandwiched between an antibody immobilized on a chromatography medium and a labeled antibody, and an unreacted labeled component is eluted. The reaction products of the present invention apply this principle to nucleic acid analysis. That is, a labeled probe for the loop portion is prepared and immobilized on a chromatographic medium to prepare a capture probe for capture to allow analysis in the chromatographic medium. Capture probes whose sequence is complementary to a portion of the loop are utilized, and since the reaction product of the present invention has a plurality of hairpin loops, the product binds to a plurality of labeled probes to give a visually recognizable signal.

The reaction products of the present invention are often capable of providing base-paired loop regions, allowing a wide variety of other detection systems. For example, a system for detecting the loop portion using the surface plasmon using an immobilized probe is available. Furthermore, if the probe of the loop portion is labeled with a double-strand specific intercalator, a more sensitive fluorescence analysis can be performed. Or positively utilize the ability of the present invention to synthesize nucleic acids on the 3 '-and 5' -sides to form a hairpin capable of base pairing. For example, one loop is designed to have a common nucleotide sequence between the normal and abnormal types, while the other loop is designed to make a difference therebetween. The presence of the gene in the common part is confirmed by the probe, and when the abnormal presence is confirmed in the other region, it is possible to constitute a characteristic analysis system. Since the reaction for synthesizing a nucleic acid of the present invention can be carried out isothermally, it is worth mentioning an advantage that real-time analysis can be carried out by a general fluorescence photometer. Until this time, the structure of the nucleic acid to be annealed in the same strand was known. However, the nucleic acid in the single-stranded sequence having a loop formed by head-to-tail annealing obtained by the present invention is novel and contains a large number of loops capable of base-pairing with other bases.

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 packed in a limited region in a high density, and the number of oligonucleotides that can be immobilized in a certain region is limited in this technique, so that a large number of probes that can be annealed can be immobilized in a high density by using the product of the present invention, that is, the reaction product of the present invention can be used as immobilized probes on a DNA chip, and the reaction product after amplification can be immobilized by any technique known in the art, or immobilized oligonucleotides can be used as oligonucleotides for the amplification reaction of the present invention, resulting in the formation of immobilized reaction products. Thus, by using the immobilized probe, a large amount of sample DNA is hybridized in a limited region, and as a result, a high signal value is expected.

Drawings

FIG. 1 is a schematic diagram of the steps of the nucleic acid synthesis method of the present invention.

FIG. 2 is a schematic diagram showing the steps of a second nucleic acid synthesis process in the present invention.

FIG. 3 is a schematic diagram of a competitive hairpin structure formed by single-stranded nucleic acids of the invention and subsequent amplification reactions.

FIG. 4 is a schematic diagram of a subsequent amplification reaction of nucleic acid according to the present invention.

FIG. 5 is a schematic representation of the ideal amplification product formed by the synthetic method of the present invention.

FIG. 6 is a positional relationship of each nucleotide sequence region corresponding to the target nucleotide sequence of PH5SR in example 1 of the present invention.

FIG. 7 is a graph showing the real-time fluorescence curve of the primer of example 1 of the present invention during the amplification of DNA having a target nucleotide sequence of pH5 SR.

FIG. 8 is a positional relationship of each of the corresponding nucleotide sequence regions in the MERS-orf1a target nucleotide sequence in example 2 of the present invention.

FIG. 9 is a photograph showing the result of agarose electrophoresis of a product obtained by the method for synthesizing a single-stranded nucleic acid of the present invention using MERS-orf1a as a template in example 2 of the present invention; wherein, lane 1: biyuntian O0107DNA Ladder; lane 2: 1fmolMERS-orf1a dsDNA.

FIG. 10 is a photograph showing the result of agarose gel electrophoresis of a restriction enzyme digestion product obtained in example 2 by the nucleic acid synthesis reaction of the present invention in example 3. Wherein the content of the first and second substances,

lane 1: XhoI digestion of purified product

Lane 2: HindIII digestion of the purified product

Lane 3: XhoI combined with HindIII digestion of the purified product

Lane 4: purification of the product

Lane 5: molecular weight marker DNA ladder.

FIG. 11 is a graph showing real-time fluorescence during DNA amplification of the target nucleotide sequence of MERS-orf1a under the action of primers in example 4 of the present invention.

FIG. 12 is a graph showing real-time fluorescence during RNA amplification of in vitro transcription of the target nucleotide sequence MERS-orf1a by the primers in example 5 of the present invention.

FIG. 13 is a schematic diagram showing the principle site of action for amplification of a target nucleotide by addition of an acceleration probe in example 6 of the present invention.

FIG. 14 is a graph showing the fluorescence intensity of amplification with respect to MERS-orf1a system as a function of reaction time under the effect of different combinations of the accelerated probe-primer combinations in example 6 of the present invention.

FIG. 15 is a real-time fluorescence curve diagram of the influenza A virus H1 gene-containing target nucleotide sequence DNA during amplification under the action of the designed influenza A virus H1 target nucleotide primer in example 7 of the present invention.

FIG. 16 is a real-time fluorescence curve diagram of Cyprinus herpesvirus type III target nucleotide sequence-containing DNA amplification process under the action of Cyprinus herpesvirus type III target nucleotide primer in example 8 of the present invention.

Detailed Description

The experimental procedures used in the following examples are conventional unless otherwise specified, and may be specifically carried out by the methods specified in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to kits and product instructions; materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 amplification of the rRNA fragment of Pangasianodon hyphthalmus 5S

In the method for synthesizing nucleic acid under isothermal condition provided by the invention, the minimum target fragment can be a nucleic acid sequence of only 60 bases. In the examples, 5S rRNA gene (Genbank: HQ681111) of a crystal Papanicodan hypothalamus in the literature (Food chem.2011; 129: 1860-4) is selected as a target gene, and the constant temperature amplification of the target gene is realized by applying the method disclosed by the invention.

The nucleic acid having a complementary strand ligated to a single strand in the form of a helical loop of the present invention was attempted using artificial Pangasonian hyphthalmus 5S rRNA (abbreviated as PH5SR) (from GenBank: HQ681111) into which an enzyme cleavage site was inserted as a template. Two primers, PH5SRNF (nucleotide sequence shown in SEQ ID NO. l) and PH5SRNR (nucleotide sequence shown in SEQ ID NO. 2), were used in the experiment. These are designed to anneal into the ring-like regions by exploiting the proximity stacking phenomenon. In addition, setting these primers to high concentrations allows annealing of pH5SRNF (or pH5SRNR) to occur preferentially.

The positional relationship of each region of the target nucleotide sequence is shown in FIG. 2. By the primers, PH5SRNF and PH5SRNR, two Nc segments are synthesized at both ends of the target nucleotide PH5SR to compete with the N segment on the target nucleotide sequence to form a hairpin loop, and the synthesis process is shown in FIG. 2. The combination of reaction solutions for the method of synthesizing the nucleic acid of the present invention by these primers is shown below.

The reaction solutions were combined as follows, the remainder being ddH₂O to 25. mu.L

20mM Tris-HCl pH8.8

10mM KCl

10mM(NH₄)₂SO₄

14mM MgSO₄

0.1％Triton X-100

1M betaine

1.25mM dNTP

8U Bst DNA polymerase (NEW ENGLAND Biolabs)

1X EvaGreen(Biotum)

Primer:

1600nM PH5SRNF

1600nM PH5SRNR

target nucleic acid PH5SR dsDNA (nucleotide sequence shown in SEQ ID NO. 3). Referring to FIG. 6, the positional relationship of each nucleotide sequence region corresponding to the target nucleotide sequence of PH5SR is shown.

The ABI StepOne real time PCR reaction temperature is set to be 63 ℃ constantly, and the reaction time is set to be 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 7. The fluorescence detection is applied to the target that real-time monitoring can be realized, and the result can be judged in advance through a real-time amplification curve.

Example 2 amplification of the fragment in MERS-orf1a

The nucleic acid having a complementary strand ligated to a single strand in the form of a helical loop of the present invention was attempted using artificial MERS-orf1a (available from GenBank: KX108946.1) designed with an enzyme cleavage site inserted therein as a template. In the experiment, two primers are Mo1aNF (the nucleotide sequence is shown as SEQ ID NO. 4) and Mo1aNR (the nucleotide sequence is shown as SEQ ID NO. 5). These are designed to anneal into the ring-like regions by exploiting the proximity stacking phenomenon. In addition, setting these primers to high concentrations allows annealing of Mo1 ainf (or Mo1 arnr) to occur preferentially.

By the primers Mo1aNF and Mo1aNR, two Nc segments synthesized at both ends of the target nucleotide MERS-orf1a compete with the N segment on the target nucleotide to form a hairpin loop. Combinations of reaction solutions for the method of synthesizing the nucleic acid of the present invention by these primers are shown below.

20mM Tris-HCl pH8.8

10mM KCl

10mM(NH₄)₂SO₄

14mM MgSO₄

0.1％Triton X-100

1M betaine

1.25mM dNTP

8U Bst DNA polymerase (NEW ENGLAND Biolabs)

Primer:

1600nM Mo1aNF

1600nM Mo1aNR

target nucleic acid MERS-orf1a dsDNA (nucleotide sequence shown in SEQ ID NO. 6). Referring to FIG. 8, the positional relationship of each of the corresponding nucleotide sequence regions in the MERS-orf1a target nucleotide sequence is shown.

The mixture was reacted at 63 ℃ for 1 hour, after which the reaction was terminated at 80 ℃ for 10 minutes and then transferred again to ice-precooled water.

Confirmation of the reaction: mu.L of a conventional nucleic acid electrophoresis loading buffer (Biyuntian DNA ladder gift) was added to 5. mu.L of the above reaction solution, and the sample was electrophoresed on 90mV 1% agarose gel (TAE lysis) prestained in GelRed (Biotum) for 1 hour. The molecular weight marker was O0107 DNAlader from Biyuntian. Gel after electrophoresis to verify nucleic acids. The results are shown in FIG. 9, where nucleic acid products with broad molecular weight distribution were obtained, i.e., it was verified that the nucleic acids obtained by the inventive method could be annealed and extended indefinitely self-assembling to give very large nucleic acid molecules.

Example 3 confirmation of the reaction product by digestion with restriction enzymes

In order to clarify the nucleic acid structure obtained in example 2 of the present invention having complementary nucleotide sequences linked in a circular structure within a single strand, the product was digested with restriction enzymes. If a theoretical fragment can be generated by digestion, without the presence (dispear) of high molecular weight as observed in example 2, producing an unclear striped pattern and electrophoresed bands, any of these products would be expected to be nucleic acids of the invention with complementary sequences alternately ligated in single strands.

The reaction solution in example 2 was precipitated and purified by treatment with phenol and precipitation with ethanol, the resulting precipitate was recovered and redissolved in ultrapure water, digested with the restriction enzymes HindIII, XhoI and the combination of these two enzymes at 37 ℃ for 2 hours, and the sample was electrophoresed on a 90mV 1% agarose gel (TAE dissolution) prestained in GelRed (Biotum) for 1 hour. The molecular weight marker was O0107 DNAlader from Biyuntian. Gel after electrophoresis to verify nucleic acids. The results are shown in FIG. 10, the obtained nucleic acid product can be cut into small fragments by enzyme from large fragments, and the products are obtained by target nucleic acid amplification, and non-specific amplification does not occur, thereby confirming the specificity of the method of the invention.

Example 4 validation of reaction products using EvaGreen

Like SYBR Green I, EvaGreen is a dye with Green excitation wavelength and combined with all the dsDNA double helix minor groove regions, and the inhibition of the dye on nucleic acid amplification reactions such as PCR is far smaller than that of the dye. In the free state, EvaGreen emits weak fluorescence, but once bound to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of EvaGreen is correlated with the amount of double-stranded DNA, and the amount of double-stranded DNA present in the nucleic acid amplification system can be detected from the fluorescence signal.

The combination of the reaction solutions of the method for synthesizing the nucleic acid of the present invention by the primers Mo1aNF (nucleotide sequence shown by SEQ ID NO. 4) and Mo1aNR (nucleotide sequence shown by SEQ ID NO. 5) is shown below.

20mM Tris-HCl pH8.8

10mM KCl

10mM(NH₄)₂SO₄

14mM MgSO₄

0.1％Triton X-100

1M betaine

1.25mM dNTP

8U Bst DNA polymerase (NEW ENGLAND Biolabs)

1X EvaGreen(Biotum)

Primer:

1600nM Mo1aNF1600nM Mo1aNR

target nucleic acid MERS-orf1a dsDNA (nucleotide sequence shown in SEQ ID NO. 6). The ABI StepOnereal time PCR reaction temperature is set to be constant at 63 ℃ and the reaction time is set to be 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 11. The fluorescence detection is applied to the target that real-time monitoring can be realized, and the result can be judged in advance through a real-time amplification curve.

Example 5 RNA target Gene amplification Using real-time EvaGreen-based fluorescence

The AMV reverse transcriptase can synthesize cDNA by taking RNA as a template, and the detection of the RNA can be realized by matching with Bst DNA polymerase.

Synthesizing cDNA by using primers Mo1aNF (nucleotide sequence is shown as SEQ ID NO. 4) and Mo1aNR (nucleotide sequence is shown as SEQ ID NO. 5) and taking RNA as a template, combining reaction solutions, and using ddH for the rest₂O to 25. mu.L

20mM Tris-HCl pH8.8

10mM KCl

10mM(NH₄)₂SO₄

14mM MgSO₄

0.1％Triton X-100

1M betaine

1.25mM dNTP

8U Bst DNA polymerase (NEW ENGLAND Biolabs)

5U AMV reverse transcriptase

1X EvaGreen(Biotum)

Primer:

1600nM Mo1aNF

1600nM Mo1aNR

target nucleic acid MERS-orf1a RNA (RNA nucleic acid sequence shown in SEQ ID NO. 7). The MERS-orf1a RNA is obtained by in vitro transcription of MERS-orf1a (the sequence is shown as SEQ ID NO. 6).

The ABI StepOne real time PCR reaction temperature is set to be 63 ℃ constantly, and the reaction time is set to be 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 12. This result demonstrates that the method is equally applicable to RNA detection.

Example 6 amplification of MERS-orf1a dsDNA target Gene Using an accelerated Probe

Combinations of reaction solutions for the method of synthesizing the nucleic acid of the present invention by these primers are shown below.

The accelerating probe combination is divided into four groups, and only the primer combination is different (wherein the accelerating probe 1 comprises primers F2 and R2, and the accelerating probe 2 comprises primers Fin and Rin):

a, no acceleration probe 1, no acceleration probe 2

b, with the acceleration probe 1 and without the acceleration probe 2

c, no acceleration probe 1 and acceleration probe 2

d, with acceleration probe 1, with acceleration probe 2

Referring to FIG. 13, there is shown a schematic view of the principle site of amplification of a target nucleotide with the addition of an acceleration probe.

20mM Tris-HCl pH8.8

10mM KCl

10mM(NH₄)₂SO₄

14mM MgSO₄

0.1％Triton X-100

1M betaine

1.25mM dNTP

8U Bst DNA polymerase (NEW ENGLAND Biolabs)

1X EvaGreen(Biotum)

a primer:

1600nM Mo1aNF (shown in SEQ ID NO. 4)

1600nM Mo1aNR (SEQ ID NO. 5)

b, primer:

1600nM Mo1aNF (shown in SEQ ID NO. 4)

1600nM Mo1aNR (SEQ ID NO. 5)

200nM Mo1aF2 (SEQ ID NO. 8)

200nM Mo1aR2 (SEQ ID NO. 9)

c, primer:

1600nM Mo1aNF (shown in SEQ ID NO. 4)

1600nM Mo1aNR (SEQ ID NO. 5)

800nM Mo1aFin (SEQ ID NO. 10)

800nM Mo1aRin (SEQ ID NO. 11)

d, primer:

1600nM Mo1aNF (shown in SEQ ID NO. 4)

1600nM Mo1aNR (SEQ ID NO. 5)

200nM Mo1aF2 (SEQ ID NO. 8)

200nM Mo1aR2 (SEQ ID NO. 9)

800nM Mo1aFin (SEQ ID NO. 10)

800nM Mo1aRin (SEQ ID NO. 11)

The target nucleic acid corresponding to each primer group a, b, c and d is MERS-orf1a dsDNA (the sequence is shown as SEQ ID NO. 6). The ABI StepOne real time PCR reaction temperature is set to be 63 ℃ constantly, and the reaction time is set to be 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 14. Ct values of the group a and the group b are compared, and the acceleration probe 1 plays an acceleration effect; comparing the Ct values of the group a and the group c, which shows that the acceleration probe 2 has an acceleration effect; meanwhile, Ct values of the group a, the group b and the group c are compared, and the acceleration effect of the acceleration probe 2 is better than that of the acceleration probe 1; and simultaneously comparing the Ct values of the group a, the group b, the group c and the group c, which shows that the acceleration probe 1 and the acceleration probe 2 are matched with each other to play a synergistic effect.

Example 7 amplification of influenza A Virus H1dsDNA target Gene

The type A H1N1 virus belongs to orthomyxoviridae (0 rtmoylovidae), influenza A virus (influenza A), and the symptoms of type A H1N1 influenza are similar to those of cold, and patients can have fever, cough, fatigue, inappetence and the like. H1N1 was widely prevalent in 2009, causing a degree of panic. For H1N1 nucleic acid detection, reverse transcription is generally used, and then PCR is used for detecting cDNA. Therefore, the new primer designed by the method can also be applied to the detection of the H1N1 virus.

20mM Tris-HCl pH8.8

10mM KCl

10mM(NH₄)₂SO₄

14mM MgSO₄

0.1％Triton X-100

1M betaine

1.25mM dNTP

8U Bst DNA polymerase (NEW ENGLAND Biolabs)

1X EvaGreen(Biotum)

Primer:

1600nM H1-NF (SEQ ID NO. 12)

1600nM H1-NR (SEQ ID NO. 13)

The target nucleic acid is H1dsDNA (the sequence is shown as SEQ ID NO. 14)

The ABI StepOne real time PCR reaction temperature is set to be 63 ℃ constantly, and the reaction time is set to be 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 15. The amplification curve shows that the method can be applied to the detection application field of the influenza A virus.

Example 8 amplification of Cyprinidae herpesvirus III target Gene

Cyprinid herpesvirus disease, herpesvirus hematopoietic necrosis disease, cyprinid pox disease caused by cyprinid herpesvirus infection seriously threaten cyprinid fish breeding. The virus has the characteristics of extremely high pathogenicity and extremely high infectivity, so that the virus is popular in the world, and the death rate of infected fishes can reach 80-100%. The disease has attracted high attention of the international animal health Organization (OIE), is listed as a key epidemic disease catalogue, is also listed as a second class animal epidemic disease in China, and has been carried out daily monitoring work. It is very important to develop corresponding detection technology to realize the rapid detection of relevant epidemic diseases and to deal with the epidemic situation. Therefore, the cyprinid herpesvirus III is selected and used as a potential application object by the method.

20mM Tris-HCl pH8.8

10mM KCl

10mM(NH₄)₂SO₄

14mM MgSO₄

0.1％Triton X-100

1M betaine

1.25mM dNTP

8U Bst DNA polymerase (NEW ENGLAND Biolabs)

1X EvaGreen(Biotum)

Primer:

1600nM CyHVIII-NF (shown in SEQ ID NO. 15)

1600nM CyHVIII-NR (SEQ ID NO. 16)

The target nucleic acid is CyHVIII dsDNA (shown as SEQ ID NO. 17)

The ABI StepOne real time PCR reaction temperature is set to be 63 ℃ constantly, and the reaction time is set to be 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 16. The amplification curve shows that the method can be applied to the detection application field of aquatic product prevention and control such as cyprinid herpesvirus.

The above description is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the spirit of the invention, and any changes and modifications made are within the scope of the invention.

Sequence listing

<110> Zhongkekuei (Suzhou) Biotechnology Co., Ltd

<120> method and kit for synthesizing nucleic acid under constant temperature

<130>2017

<160>17

<170>SIPOSequenceListing 1.0

<210>1

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

gatcggacga gatcgggcgt agcttacggc cataccagcc tg 42

<210>2

<211>41

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

tacgcccgat ctcgtccgat ccccgaccct gcttagcttc c 41

<210>3

<211>122

<212>DNA

<213>Pangasianodon hypophthalmus 5S rRNA

<400>3

gcttacggcc ataccagcct gaatacgccc gatctcgtcc gatctcggaa gctaagcagg 60

gtcgggcctg gttagtactt ggatgggaga ccgcctggga ataccaggtg ctgtaagctt 120

tt 122

<210>4

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

cacaggcaac aagaaaagtg tcatttgtga ctatggcctt cg 42

<210>5

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

gacacttttc ttgttgcctg tgggagtagt gggctcgtag ac 42

<210>6

<211>301

<212>DNA

<213>MERS-orf1a

<400>6

tctagacacg tgtaatacga ctcactatag gtatgtgata atcttacaag ccactaaatt 60

tactttgtgg aactacttgt ttgagactat tccatatgcc acacagttgt tcccactctt 120

atttgtgact atggccttcg ttatgttgtt ggttctcgag aaacacaaac acaccttttt 180

gacacttttc ttgttgcctg tggctatttg tttgacttat gcaaagctta acatagtcta 240

cgagcccact actcccattt cgtcagcgct gattgcagtt gcaaattggg ttaacgaatt 300

c 301

<210>7

<211>301

<212>RNA

<213>MERS-orf1a

<400>7

ucuagacacg uguaauacga cucacuauag guaugugaua aucuuacaag ccacuaaauu 60

uacuuugugg aacuacuugu uugagacuau uccauaugcc acacaguugu ucccacucuu 120

auuugugacu auggccuucg uuauguuguu gguucucgag aaacacaaac acaccuuuuu 180

gacacuuuuc uuguugccug uggcuauuug uuugacuuau gcaaagcuua acauagucua 240

cgagcccacu acucccauuu cgucagcgcu gauugcaguu gcaaauuggg uuaacgaauu 300

c 301

<210>8

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

attcccacac agttgttc 18

<210>9

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

tgcaatcagc gctgacgaa 19

<210>10

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ggttaaacac aaacacacc 19

<210>11

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

gcataagtca aacaaatagc 20

<210>12

<211>41

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

cgaaagcata ccatggtggg agacactata acatttgaag c 41

<210>13

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

caccatggta tgctttcggt gaactggagc atctgatg 38

<210>14

<211>356

<212>DNA

<213>Influenza virus H1N1

<400>14

tttcagttgg atcgtcaaaa tacaaccgaa gattcgctcc ggaaatagca gctagaccta 60

aagttagagg acaggcaggc agaatgaact attattggac actattagac caaggagaca 120

ctataacatt tgaagccact gggaatttga tagcaccatg gtatgctttc gcattgaata 180

aggggtctga ctctggaatt ataacatcag atgctccagt tcacaattgt gacacaaggt 240

gccaaacccc tcatggggct ttgaacagca gccttccttt tcagaatgta catcctatca 300

ctattggaga atgtcccaaa tacgtcaaga gcaccaaact aagaatggca acagga 356

<210>15

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

aggtcgggga agaactgtca cctgtacgag gtgatgcagc 40

<210>16

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

gacagttctt ccccgacctc cgccacgatc acgtacttg 39

<210>17

<211>322

<212>DNA

<213>Cyprinid herpes virus-3

<400>17

cgggcgacgc tgcatcgccg tcaagcacgc catagaccag cgctacaccg aagagtccaa 60

ggtggccatg cacagcggcg cgacctaccc ggccatctcc gcgggttacc tgtacgaggt 120

gatgcagcgt ctggaggaat acgacgccgt ggccgtcgac gagggacagt tcttccccga 180

cctctacgag ggagtcgtgc agctgctgac cgcgggcaag tacgtgatcg tggcggcgct 240

ggacggggac tttatgcagc agcccttcaa gcaggtgacg gcgttggtgc ccatggcgga 300

caagctggac aagctgacgg cg 322

Claims

1. A method for synthesizing a nucleic acid under isothermal conditions of non-diagnostic interest, comprising the steps of:

2) synthesizing a complementary nucleic acid strand of itself using the nucleic acid of step 1) as a template and the 3' end of the Nc region annealed with the N region as a synthesis origin;

3) complementary strand synthesis is performed by polymerase-catalyzed strand displacement-type complementary strand synthesis reaction to displace the complementary nucleic acid strand synthesized in step 2).

2. The method for synthesizing nucleic acid according to claim 1, comprising the following steps:

5) synthesizing its own complementary strand using said nucleic acid A provided in step 3) as a template, and using the N region whose 3' -end has been annealed to the Nc region as a synthesis origin to obtain a nucleic acid strand having a head-to-tail complementary nucleotide sequence on one strand thereof and having complementary nucleotide sequence regions alternately connected thereto.

3. The method for synthesizing nucleic acid under isothermal conditions according to claim 1, wherein the method for preparing nucleic acid according to step 1) comprises the following steps:

1-1) an annealing step of annealing a first oligonucleotide I to a F1c region of a template, wherein the 3 'end of the template comprises a F1c region and an N region located 5' to the F1c region and the 5 'end of the template comprises a R1 region, wherein the first oligonucleotide I comprises an N region linked to a Fl region, said N region being linked 5' to the F1 region, wherein,

nc region: a region of nucleotide sequence complementary to the N region;

4. The method for synthesizing a nucleic acid according to claim 3, wherein: 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.

5. The method for synthesizing a nucleic acid according to any one of claims 2 to 4, wherein: the nucleic acid fragments of the F1c region, the N region and the R1 region are all 15-60 bp.

6. The method for synthesizing a nucleic acid under isothermal conditions according to claim 1 or 2, characterized in that: the obtained nucleic acid chain can be infinitely extended by self-pairing, and the Nc region at the 3' end of the nucleic acid chain is paired with the N region of the complementary segment on the chain to be used as a synthesis starting point to continuously extend the nucleic acid chain by taking the nucleic acid chain as a template.

7. The method for synthesizing a nucleic acid according to claim 2, wherein: in the method for synthesizing nucleic acid, the nucleic acid amplification is accelerated by introducing an accelerating primer X2 and/or Xin; wherein X2 is a segment located on the 5' side of the F1 region and R1 region of the complementary strand of the original nucleic acid, and Xin is a segment located in the middle of the F1c region to N region and the Nc region to R1 c.

8. A kit for synthesizing a nucleic acid having a head-to-tail complementary nucleotide sequence on one strand thereof and complementary nucleotide sequences alternately linked on the nucleic acid strand; the kit comprises the following components:

nc region: a region of nucleotide sequence complementary to the N region;

a DNA polymerase catalyzing a strand displacement-type complementary strand synthesis reaction;

a nucleotide that serves as a substrate for the DNA polymerase.

9. The kit according to claim 8, wherein the kit further comprises accelerating primers X2 and/or Xin, wherein X2 is a segment located on the 5' side of the F1 region and the R1 region of the complementary strand of the original nucleic acid, and Xin is a middle segment located from the F1c region to the N region and from the Nc region to the R1 c.

10. Use of a kit according to claim 8 or 9 for non-diagnostic purposes in the synthesis of nucleic acids or in the detection of target nucleotide sequences in a sample.