Thermostable recombinant enzyme polymerase amplification reaction system and application thereof

Abstract

The present application relates to the technical field of biology, in particular to a heat-resistant stable recombinant enzyme polymerase amplification reaction system and application, the present application provides a heat-resistant RPA reaction system for the first time, compared with ordinary RPA technology, the most important feature of the method of the present application is that the method in the present application can realize isothermal amplification in a wider temperature range (37-75 DEG C). The non-specific amplification and primer dimer caused by low temperature of ordinary RPA technology can be well made up. When non-specific amplification and primer dimer appear in the reaction, only need to place the reaction in a higher isothermal environment, and the non-specific amplification and primer dimer can be eliminated, so that the target DNA can be better specifically amplified.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a thermostable recombinase polymerase amplification reaction system and its application, achieving high sensitivity and high specificity amplification of target DNA under in vitro isothermal conditions. Background Technology

[0002] Recombinase polymerase amplification (RPA), developed by ASM in the UK in 2006 (US7666598B2), is a relatively widely used isothermal nucleic acid amplification technique. Its reaction system includes a pair of primers and three key enzymes: recombinases (uvsX, uvsY) that bind to oligonucleotide primers, a single-stranded DNA binding protein (GP32), and a strand displacement DNA polymerase (Bsu or Bst). At the start of the reaction, the primers bind to the recombinase to form a primer-recombinase complex, which binds complementary to the corresponding sites on the template strand, causing a conformational change in the double-stranded DNA. The homologous strand in the double-stranded template DNA is displaced and binds to the single-stranded binding protein. Under the catalysis of the DNA polymerase with strand displacement properties, a new, complete double strand is formed. After completing the strand displacement process, the recombinase detaches from the bound primer. With the help of ATP, the recombinase can then re-displace and synthesize new double-stranded DNA. The reaction temperature is typically 37–42°C. Although the lower reaction temperature makes it easier to promote in clinical practice, the lower reaction temperature also makes it easier for primers and templates, and primers to form higher-order structures, which can lead to non-specific amplification or primer dimers, ultimately affecting the amplification results [1].

[0003] PCR technology is currently the most widely used and mature nucleic acid amplification technology. When eliminating higher-order structures between primers and templates, and between primers, it is often necessary to add some chemical reagents, such as DMSO and betaine, which can reduce the formation of higher-order structures to a certain extent. However, these reagents have compatibility issues with RPA reactions. For example, DMSO has been reported to inhibit RPA reactions [2]. In recent years, SAMRS technology has been reported to significantly reduce the occurrence of various non-specific amplifications in RPA reactions [3,4]. However, it requires the use of a large number of modified nucleotide molecules, making it too costly in practical applications. Finding a simple and feasible method to eliminate non-specific amplifications in RPA reactions is an important direction for improving RPA reactions. However, since RPA reactions are completed by a series of enzyme-catalyzed reactions, there are still many uncertainties in its reaction mechanism, making its improvement difficult.

[0004] Theoretically, increasing the reaction temperature can reduce non-specific amplification to some extent. However, the activity of various enzymes in the RPA reaction decreases sharply above 45°C. Using thermostable enzymes might increase the specificity and efficiency of amplification simply by raising the reaction temperature. However, using new enzymes requires addressing the synergistic effects between them, particularly identifying recombinant enzymes and single-stranded binding proteins that react synergistically. A completely new reaction system also needs to be established for these enzymes. This system must achieve two goals: 1. Compatibility with all thermostable enzymes used, ensuring they reach the required activity. 2. A reversible reaction process involving the dissociation and recombination of the recombinant enzyme-primer complex, directing the reaction in a direction more favorable to RPA synthesis. Achieving RPA amplification under high-temperature conditions requires a complete re-screening of enzymes and the establishment of a new, suitable reaction system. Therefore, significant unknowns and technical challenges exist.

[0005] This invention screened out an enzyme system that maintains its activity and works synergistically under high-temperature conditions, and established its reaction conditions. The new reaction technique is named Thermostable Recombinase Polymerase Amplification (TsRPA). This invention also verified that TsRPA technology can effectively achieve highly efficient and specific amplification of target genes at higher reaction temperatures.

[0006] References

[0007] 1.Li J,Macdonald J,von Stetten F:Review:a comprehensive summary of adecade development of the recombinase polymerase amplification.The Analyst2018,144(1):31-67.

[0008] 2.Kojima K,Juma KM,Akagi S,Hayashi K,Takita T,O'Sullivan CK,FujiwaraS,Nakura Y,Yanagihara I,Yasukawa K: Solvent engineering studies on recombinasepolymerase amplification.Journal of bioscience and bioengineering 2021,131(2):219-224.

[0009] 3.Sharma N, Hoshika S, Hutter D, Bradley KM, Benner SA: Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS). Chembiochem: a European journal of chemical biology 2014, 15(15): 2268-2274.

[0010] 4.Hoshika S, Chen F, Leal NA, Benner SA: Self-Avoiding Molecular Recognition Systems (SAMRS). Nucleic acids symposium series 2008(52):129-130. Summary of the Invention

[0011] To address the above problems, this invention provides a thermostable recombinase polymerase amplification reaction system. This system can reduce or eliminate non-specific amplification and primer dimers during isothermal nucleic acid amplification, thereby obtaining highly specific amplification products.

[0012] Another objective of this invention is to provide the application of a thermostable recombinase polymerase amplification reaction system in the preparation of an RPA detection kit.

[0013] To achieve the above objectives, the present invention adopts the following technical measures:

[0014] The thermostable recombinase polymerase amplification reaction system is as follows:

[0015]

[0016] The sequence of the TthRecA protein is shown in SEQ ID NO.1;

[0017] The sequence of the TthSSB protein is shown in SEQ ID NO.2.

[0018] When using the system described above, preferably, the reaction temperature of the system is ≤75℃ and ≥50℃;

[0019] The pH of the Tris buffer is preferably 7.5-9.0, and in some preferred embodiments, the pH is 8.3. The molecular weight of the polyethylene glycol is preferably 15,000-35,000.

[0020] The primers are 30–35 bp in length;

[0021] The scope of protection of this invention also includes the application of the above system in the preparation of RPA detection kits.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] This invention provides a thermally stable RPA reaction system for the first time. Compared to conventional RPA technology, the most significant feature of this method is that it can achieve isothermal amplification over a wider temperature range (37–75°C). This effectively compensates for the non-specific amplification and primer dimers caused by low temperatures in conventional RPA technology. When non-specific amplification and primer dimers occur, simply placing the reaction in a higher isothermal environment can eliminate these phenomena, resulting in better specific amplification of the target DNA.

[0024] In this reaction system, Tris buffer provides a stable pH environment suitable for the reaction. Potassium acetate and magnesium acetate provide the necessary basic ions for the reaction. β-mercaptoethanol provides a reducing environment for the reaction. Polyethylene glycol provides a crowded environment for the reaction. ATP, polyphosphate, and polyphosphokinase provide sustained energy for the reaction. With sufficient ATP, the TthRecA protein can displace the primer with the DNA template, allowing the primer to recognize a pairing site on the template strand. The displaced single strand is further recognized by the single-strand binding protein TthSSB. The strand displacement DNA polymerase Bst3.0 can specifically extend the displaced primer according to the complementary pairing principle with the template DNA, and this process can be repeated continuously, thereby achieving efficient amplification of the target DNA. Attached Figure Description

[0025] Figure 1 The effect of thermostable recombinases from different sources on amplification efficiency.

[0026] TthSSB was used as the single-chain binding protein in the TsRPA reaction to test the effect of thermostable recombinases from different sources on amplification efficiency. M was the DL2000 marker. Lane 1 contained TthRecA protein from *Thermophilic bacterium*; lane 2 contained RadA protein from *Thermophilic sulfide-leaved leaf bacterium*; lane 3 contained RadA protein from *Methanococcus wari*; lane 4 contained RadA protein from *Extremely thermophilic archaea*; and lane 5 contained Rad51 protein from *Hyperthermophilic primitive bacteria*.

[0027] Figure 2 Electrophoresis image of the amplified product of the chicken infectious anemia virus (CIAV) antigen gene VP1;

[0028] Where M is the DL2000 marker, and lanes 1-3 are the amplification products of the VP1 sequence when the amount of TthRecA protein used in this experiment is 50 ng / μl, 100 ng / μl, and 200 ng / μl, respectively.

[0029] Figure 3 Electrophoresis image of the amplified product of the chicken infectious anemia virus (CIAV) antigen gene VP1;

[0030] Where M is the DL2000 marker, lane 1 is the negative control amplified using the commercial TwistDx kit; lane 2 is the specific product amplified using the commercial TwistDx kit; and lane 3 is the specific product amplified using the method described in this invention.

[0031] Figure 4 Gel electrophoresis images of H9 subtype influenza virus nucleic acid amplified using the TwistDx commercial kit and the isothermal nucleic acid amplification method described in this patent, followed by product recovery. M represents the DL2000 marker; lanes 1-5 represent bands obtained at 40℃, 45℃, 50℃, 55℃, and 60℃, respectively, after reacting for 30 min using the method of this invention. Lane 6 represents the product amplified using the TwistDx commercial kit at 39℃.

[0032] Figure 5 The effect of different reaction temperatures on the amplification of the African swine fever (ASFV) pp220 gene;

[0033] Where M is the DL2000 marker; lanes 1-6 are the bands obtained by reacting for 30 min using the method of this invention under isothermal conditions of 75℃, 70℃, 65℃, 55℃, 45℃ and 35℃, respectively. Detailed Implementation

[0034] The present invention will be described in detail below with reference to the embodiments. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial sources. In the embodiments of this invention, the molecular weight of polyethylene glycol is 15,000 to 35,000.

[0035] Example 1:

[0036] Screening for thermostable recombinases that match the single-chain binding protein TthSSB:

[0037] Considering the need for heat resistance in this invention, the applicant directly replaced the single-chain binding protein from GP32 with TthSSB (Accession No. BAW02114). Based on this, the applicant conducted a large-scale screening of heat-resistant proteins reported or suspected of possessing recombinase properties from NCBI; these enzymes were then incubated at 75 degrees Celsius to examine their tolerance, ultimately identifying five heat-resistant recombinases:

[0038] The TthRecA protein of *Thermus thermophiles* (Accession No. BAW01838), the SsoRadA protein of *Sulfolobus sofataricus* (Accession No. WP_009990512), the MvoRadA protein of *Methanococcus voltae* (Accession No. AF008421), the PfuRadA protein of *Pyrococcus furiosus* (Accession No. AF052597), and the KodRad51 protein of *Thermococcus kodakaraensis* (Accession No. BAD86088.1).

[0039] The gene sequences encoding these proteins were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the synthesized recombinase genes were cloned into the expression vector pET-28a(+). The constructed recombinant expression vectors were then transformed into the *E. coli* expression host strain BL21(DE3) to obtain recombinase expression strains. Single clones were picked and cultured in LB broth at 37°C for 4 hours. Subsequently, IPTG was added to a final concentration of 1 mM and the culture was continued at 28°C for 5 hours to induce the expression of the target proteins.

[0040] After inducing expression, the strain was pressure-disrupted, and the target protein was purified using a nickel affinity purification column, followed by elution with 400 mM imidazole solution. The eluted target protein was then dialyzed using dialysis buffer (20 mM Tris buffer (pH 7.4), 50 mM NaCl, 1 mM DTT, 10% glycerol) and stored at -80°C for later use.

[0041] The VP1 sequence of the porcine circovirus 2 (PCV2) capsid gene (Accession No. MW561985) was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the synthesized VP1 gene was ligated into the pMD19-T vector. The synthesized vector containing the PCV2 antigen gene VP1 gene sequence was heat-shocked into Escherichia coli strain DH5α, and the transformed strain was plated on ampicillin-resistant plates and incubated at 37°C for 12 h. Single colonies were picked and cultured in LB liquid medium containing ampicillin resistance at 37°C for 12 h. After extracting the E. coli plasmid using the alkaline lysis method, the extracted plasmid was diluted to 1 ng / μL and used as a template for isothermal amplification and PCR reactions.

[0042] Using the reaction system described in this invention, the thermostable recombinase in the system was modified. The specific amplification efficiency of different recombinases for the PCV2 capsid protein at 65°C was then compared.

[0043] Primers were designed and synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. The primer sequences are as follows:

[0044]

[0045]

[0046] The reaction system and conditions for this isothermal amplification method are as follows:

[0047]

[0048] Add 1 μl of diluted template to the prepared reaction system, mix well, and react in a 65°C water bath for 30 min. The amplified product can be recovered by a recovery kit before electrophoresis identification.

[0049] Depend on Figure 1 It is known that the RecA protein from *Thermophilus thermophilus* can effectively synergize with the single-stranded binding protein TthSSB, thereby efficiently amplifying the target fragment. However, TthSSB does not synergize well with other thermostable recombinases, resulting in relatively weak or no amplification. Besides TthRecA, only KodRad51 can achieve a small amount of target fragment amplification in the system described in this invention. The sequence of the TthRecA protein is shown in SEQ ID NO.1; the sequence of the TthSSB protein is shown in SEQ ID NO.2.

[0050] Example 2: Screening of working concentrations of thermostable recombinase in RPA reaction systems

[0051] Using the chicken infectious anemia virus (CIAV) antigen gene VP1 sequence as a template (Accession No. MH186140), the gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and ligated into the pMD19-T vector. The synthesized vector containing the CIAV antigen gene VP1 gene sequence was heat-shocked into *Escherichia coli* strain DH5α, and the transformed strains were plated on ampicillin-resistant plates and incubated at 37°C for 12 h. Single clones were picked and cultured in LB broth containing ampicillin resistance at 37°C for 12 h using a shaker. Plasmids were extracted from the *E. coli* using the alkaline lysis method and diluted to 1 ng / μL as templates for isothermal amplification and PCR reactions.

[0052] Primers were designed and synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. The primer sequences are as follows:

[0053]

[0054] In this embodiment, the amounts of most components were kept constant, and different concentrations of TthRecA protein were tested to screen for the optimal working concentration of TthRecA protein. The isothermal amplification system and conditions are as follows:

[0055]

[0056] The TthRecA protein dosage Xng / μl represents a concentration gradient of 50ng / μl, 100ng / μl, and 200ng / μl. Add 1μl of diluted template to the prepared reaction system, mix well, and react in a 65℃ constant temperature water bath for 30 min. The amplified product can be recovered by a recovery kit before electrophoresis identification.

[0057] The electrophoresis results of the three groups of experiments amplifying the VP1 gene sequence are as follows: Figure 2 The amplified band size was 189 bp. The results show that under the RPA reaction conditions in this experiment, when the TthRecA protein concentration was 50 ng / μl, the electrophoretic band after amplification product recovery was not obvious, indicating that only a small amount of amplified product could be obtained. However, when the TthRecA protein concentration was increased to 100 ng / μl and 200 ng / μl, the amplification efficiency of the RPA reaction increased significantly in a stepwise manner. Among the three groups of experiments, the RPA reaction amplification efficiency was highest when the TthRecA protein concentration was 200 ng / μl.

[0058] In further experiments, effective amplification of the VP1 gene was achieved at TthRecA protein concentrations up to 500 ng / µl, but the highest efficiency was achieved at 200 ng / µl (data not shown).

[0059] Example 3:

[0060] Detection of chicken infectious anemia virus (CIAV) antigen gene VP1

[0061] The chicken infectious anemia virus (CIAV) antigen gene VP1 sequence (Accession No. MH186140) was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the synthesized VP1 gene was ligated into the pMD19-T vector. The synthesized vector containing the CIAV antigen gene VP1 gene sequence was heat-shocked into Escherichia coli strain DH5α, and the transformed strain was plated on ampicillin-resistant plates and incubated at 37°C for 12 h. Single colonies were picked and cultured in LB liquid medium containing ampicillin resistance at 37°C for 12 h. After extracting the E. coli plasmid using the alkaline lysis method, the extracted plasmid was diluted to 1 ng / μL and used as a template for isothermal amplification and PCR reactions.

[0062] Two methods were used to detect the VP1 gene. One method used a commercially available isothermal amplification kit from TwistDx. The procedure was performed according to the manufacturer's instructions, with a reaction temperature of 39°C. Water was used as a negative control instead of the DNA template to assess non-specific amplification in the commercial kit. The other method used was the method described in this invention, performed at 65°C.

[0063] Primers were designed and synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. The primer sequences are as follows:

[0064]

[0065] The reaction system and conditions for this isothermal amplification method are as follows:

[0066]

[0067]

[0068] Add 1 μl of diluted template to the prepared reaction system, mix well, and react in a 65°C water bath for 30 min. The amplified product can be recovered by a recovery kit before electrophoresis identification.

[0069] Electrophoresis results of the two methods for amplifying the VP1 gene sequence are as follows: Figure 3The amplified band size was 189 bp. The results show that using a commercial RPA kit, the target band could not be amplified under these conditions. The negative control showed that a large number of primer dimers formed at 39°C. It is speculated that the formation of these primer dimers interfered with the normal amplification of the RPA reaction. However, using the method described in this invention, the target band could be amplified at 65°C with almost no primer dimer formation. Therefore, the amplification method of this invention has certain advantages over commercial kits.

[0070] Example 4:

[0071] Detection of the M gene of H9 subtype influenza virus

[0072] Infected chicken liver tissue samples were taken, cut into small pieces, ground, and repeatedly frozen and thawed to extract H9 influenza virus RNA contained in the chicken liver tissue. The RNA was reverse transcribed into cDNA using a TaKaRa reverse transcription kit. The cDNA template was diluted and used for amplification reactions.

[0073] Primers were designed and synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. The primer sequences are as follows:

[0074]

[0075] The TwistDx isothermal amplification kit can be used to amplify and detect the M gene according to its instructions. The reaction temperature is 39℃ and the reaction time is 30 min.

[0076] The amplification system and reaction conditions for this method are as follows:

[0077]

[0078]

[0079] Add 1 μl of diluted template to the prepared reaction system, mix well, and react at 40℃, 45℃, 50℃, 55℃, and 60℃ for 30 min respectively. The amplified products are recovered by a recovery kit and identified by electrophoresis.

[0080] like Figure 4 As shown, using the method of this invention, amplification of the target band can be completed at temperatures ranging from 40 to 60°C, but the amplification efficiency gradually increases with increasing temperature. When the temperature is increased to 60°C, the amplification efficiency is close to that of commercial kits. However, the products amplified by commercial kits show a small number of non-specific amplification bands. The amplification specificity of this method is higher than that of commercial kits.

[0081] Example 5

[0082] Amplification of the African swine fever virus (ASFV) pp220 gene

[0083] The African swine fever antigen gene pp220 sequence (Accession No. MN393476.1 (118460~125890bp)) was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the synthesized VP1 gene was ligated into the pMD19-T vector. The synthesized vector containing the African swine fever antigen gene pp220 sequence was heat-shocked into Escherichia coli strain DH5α, and the transformed strains were plated on ampicillin-resistant plates and cultured at 37°C for 12 h. Single colonies were picked and cultured in LB liquid medium containing ampicillin resistance at 37°C for 12 h on a shaker. After extracting the E. coli plasmid using the alkaline lysis method, the extracted plasmid was diluted to 1 ng / μL and used as a template for isothermal amplification and PCR reactions.

[0084] Primers were designed and synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. The primer sequences are as follows:

[0085]

[0086] The reaction system and conditions for this isothermal amplification method are as follows:

[0087]

[0088] Add 1 μl of diluted template to the prepared reaction system, mix well, and then place in a constant temperature water bath at 35℃, 45℃, 55℃, 65℃, 70℃ and 75℃ for 30 min respectively. The amplified product can be identified by electrophoresis after being recovered by the recovery kit.

[0089] Depend on Figure 5 It is evident that placing the reaction system at 35℃ will not yield amplification products. As the reaction temperature increases, the amount of amplification products gradually increases. The amount of reaction products is highest when the reaction temperature reaches 70–75℃, indicating the maximum amplification efficiency. Therefore, for primers and templates with particularly high GC content (i.e., those that readily produce higher-order structures under low-temperature conditions), efficient amplification can be achieved simply by increasing the reaction temperature.

Claims

1. Thermostable recombinase polymerase amplification reaction system: ， The sequence of the TthRecA protein is shown in SEQ ID NO.1, and the sequence of the TthSSB protein is shown in SEQ ID NO.

2.

2. The reaction system as described in claim 1, characterized in that, When using the reaction system, the reaction temperature of the system is ≤75℃ and ≥50℃.

3. The application of the reaction system according to claim 1 in the preparation of an RPA detection kit.

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