Chain intervention type isothermal nucleic acid amplification system

Abstract

The invention relates to the technical field of molecular biological detection, in particular to a chain intervention type isothermal nucleic acid amplification system which comprises recombinase UvsX, a recombinase loading factor UvsY, single-chain binding protein gp32, a DNA (deoxyribonucleic acid) chain displacement polymerase Bsu large fragment, exonuclease III, an ATP (adenosine triphosphate) energy circulation system and a buffer system, and the dosage range of the single-chain binding protein gp32 configured according to enzyme activity is 0.4-4.5 U. According to the chain intervention type isothermal nucleic acid amplification system disclosed by the invention, the chain intervention type isothermal nucleic acid amplification system is established according to partial enzyme activity expression, so that the amplification effects of systems established by different batches of materials are consistent.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology detection, and particularly relates to a strand-displacement isothermal nucleic acid amplification system. Background Art

[0002] Due to the characteristics of instrument independence, rapidity, and simple operation of isothermal amplification reagents, they have obtained an increasingly large market share. However, this technology started late, and the system is complex, and there are various proteins required for the system reaction. The existing system adds each material according to the concentration, but there will be differences in purity, protein performance, etc. between different batches of each material. When different batches of materials are used to construct the system according to the same concentration, it will lead to differences in amplification between systems, thus affecting the reagent performance.

[0003] The protein species sources that satisfy the system reaction are diverse, and the proteins from different species sources play different roles in this system, resulting in differences in amplification effects when using different proteins to establish the system. In addition, the existing system adds each material according to the concentration, but there will be different differences between different batches of each material. When using different batches of materials to construct the system according to the same concentration, it will lead to differences in amplification between systems, that is, the amplification effects established by different batches of materials are inconsistent.

[0004] With the development of molecular diagnostic technology and the progress of enzymology, more and more enzymes have established corresponding enzyme activity evaluation standards, including the functional evaluation of enzymes and the quality evaluation of enzymes (including purity, content of specific interfering proteins, residual amount of host DNA, etc.) to ensure that there will be no interference in the use scenario. However, for raw materials such as recombinase, single-stranded binding protein, strand-displacement DNA polymerase, recombinase coenzyme, exonuclease III, etc. used in the strand-displacement isothermal nucleic acid amplification system, most of them have a short research and development history and there is no mature enzyme activity evaluation scheme. Summary of the Invention

[0005] The purpose of the present invention is to provide a strand-displacement isothermal nucleic acid amplification system to solve the technical problem that for raw materials such as recombinase, single-stranded binding protein, strand-displacement DNA polymerase, recombinase coenzyme, exonuclease III, etc. used in the strand-displacement isothermal nucleic acid amplification system in the prior art, most of them have a short research and development history and there is no mature enzyme activity evaluation scheme.

[0006] The present invention discloses a strand-displacement isothermal nucleic acid amplification system, including recombinase UvsX, recombinase loading factor UvsY, single-stranded binding protein gp32, large fragment of DNA strand-displacement polymerase Bsu, exonuclease III, ATP energy recycling system, and buffer system. The addition amount range of the single-stranded binding protein gp32 configured according to enzyme activity is 0.4 - 4.5U.

[0007] Furthermore, the single-stranded binding protein gp32, the large fragment of DNA strand displacement polymerase Bsu, and exonuclease III are used to establish a system according to the enzyme activities of each enzyme.

[0008] Furthermore, the addition amount range of the large fragment of DNA strand displacement polymerase Bsu configured according to the enzyme activity is 7.5 - 60 U.

[0009] Furthermore, the addition amount range of exonuclease III configured according to the enzyme activity is 8 - 60 U.

[0010] Furthermore, the concentration of recombinase UvsX in the system is 40 - 320 ng / μL.

[0011] Furthermore, the concentration of recombinase loading factor UvsY in the system is 10 - 135 ng / μL.

[0012] Furthermore, the ATP energy recycling system includes ATP, sodium hexametaphosphate, ppk2, and pap, and the buffer system includes Tris-Ac, KOAc, MgOAc, TCEP (tris(2-carboxyethyl)phosphine hydrochloride), and polyethylene glycol 35000.

[0013] Furthermore, recombinase UvsX and recombinase coenzyme UvsY are derived from phage or Escherichia coli.

[0014] Furthermore, the single-stranded binding protein gp32 is derived from T4 phage or Thermus thermophilus or Escherichia coli.

[0015] Furthermore, the large fragment of DNA strand displacement polymerase Bsu is derived from Bacillus subtilis.

[0016] Furthermore, exonuclease III is derived from Escherichia coli.

[0017] An application of a strand-invading isothermal nucleic acid amplification system, which is applied to the preparation of an isothermal amplification kit.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. For the strand-invading isothermal nucleic acid amplification system of the present invention, the strand-invading isothermal nucleic acid amplification system is established according to the partial enzyme activity performance, ensuring that the systems established by the materials between different batches have consistent amplification effects.

[0020] 2. For the strand-invading isothermal nucleic acid amplification system of the present invention, the addition amount range and sources of each enzyme in the system are explored. The optimal amplification system of the present invention is obtained, and the system can achieve 100% detection (20 times of repetition) for samples with an original copy number of 3 copies / T, with high sensitivity. Description of the Drawings

[0021] Figure 1 This is the reaction schematic diagram of the chain-mediated isothermal nucleic acid amplification system of the present invention;

[0022] Figure 2 This is the amplification performance of different concentrations of UvsY in the system of the present invention;

[0023] Figure 3 This is the amplification performance of different amplification multiples of UvsX, single-strand binding protein gp32, and UvsY in the system of the present invention;

[0024] Figure 4 This is the amplification performance of different enzyme activity addition amounts of the large fragment of DNA strand displacement polymerase Bsu in the system of the present invention;

[0025] Figure 5 This is the amplification performance of different enzyme activity addition amounts of exonuclease III in the system of the present invention;

[0026] Figure 6 This is a comparison chart of the best amplification effects of strand displacement polymerases from three different species sources;

[0027] Figure 7 This is the amplification performance of different sources of endonucleases with AP site cleavage activity in the system;

[0028] Figure 8 This is the amplification performance chart of the system configured according to the concentration of three batches of UvsX;

[0029] Figure 9 This is the amplification performance chart of the system configured according to the enzyme activity of three batches of UvsX of the present invention;

[0030] Figure 10 This is the amplification performance chart of the system configured according to the concentration of three batches of single-strand binding protein gp32;

[0031] Figure 11 This is the amplification performance chart of the system configured according to the enzyme activity of three batches of single-strand binding protein gp32 of the present invention;

[0032] Figure 12 This is the amplification performance chart of the system configured according to the enzyme activity of the batch with too low specific activity of single-strand binding protein gp32. Detailed implementation manners

[0033] To make the objectives, technical solutions, and advantages of the implementation manners of the present invention clearer, the technical solutions in the implementation manners of the present invention will be clearly and completely described below. Obviously, the described implementation manners are some, rather than all, of the implementation manners of the present invention.

[0034] The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably a polymeric molecule, containing ribonucleic acid, deoxyribonucleic acid or their analogue units. The nucleic acid can be single-stranded or double-stranded. The single-stranded nucleic acid can be a nucleic acid of one strand of denatured double-stranded DNA. Alternatively, the single-stranded nucleic acid can be a single-stranded nucleic acid not derived from any double-stranded DNA.

[0035] A strand-invading isothermal nucleic acid amplification system provided by the present invention includes recombinase UvsX, recombinase loading factor UvsY, single-stranded binding protein gp32, large fragment of DNA strand displacement polymerase Bsu, exonuclease III, ATP energy recycling system and buffer system. The buffer system includes Tris-Ac, KOAc, MgOAc, TCEP (tris(2-carboxyethyl)phosphine hydrochloride), polyethylene glycol 35000, and also includes primer-probes required for the reaction. In this system, recombinase UvsX binds to the primer in the system and invades the homologous region of the double-stranded template, and the primer and the template strand achieve base complementary pairing. At this time, there will be a triple-strand complex state. The large fragment of DNA polymerase Bsu will bind to the 3' end of the primer to achieve strand displacement extension. To prevent the displaced strand on the double-stranded template from renaturing, the single-stranded binding protein gp32 is needed to fix the displaced template strand. Since other parts of the template except the triple-strand complex are in the natural double-helix state, the strand displacement polymerase used cannot have 5-3' exonuclease activity. Therefore, the probe in this system generally needs to be specially designed. For example, the probe contains an abasic site, and a fluorophore and a quencher are respectively modified at both ends of the abasic site, and the number of bases between the two groups does not exceed 5. The length of the 5' end sequence of the abasic site is recommended to be 30-35 bases, and the length of the 3' end sequence is recommended to be about 15 bases, and the 3' end is end-blocked to avoid cleavage by exonuclease or a nucleic acid exonuclease with this function. This probe needs to be cleaved by exonuclease III (source or different mutants). The reaction principle of this system is as Figure 1 shown. The ATP energy recycling system functions continuously for the whole system during the amplification reaction process, and the buffer system is used to provide a suitable environment for the amplification reaction. Among them, recombinase UvsX, recombinase loading factor UvsY, single-stranded binding protein gp32, large fragment of DNA strand displacement polymerase Bsu, exonuclease (Exonuclese III) are from self-production or commercialization.

[0036] The composition addition amount in the strand-invading isothermal nucleic acid amplification system of the present invention is added according to the enzyme activity of each enzyme, so as to establish the isothermal amplification system of the present invention. Among them, the recombinant enzyme UvsX is added at a concentration of 40-320 ng / μL, the recombinant enzyme loading factor UvsY is added at a concentration of 10-135 ng / μL, the single-stranded binding protein gp32 is added at an enzyme activity of 0.4-4.5 U, the DNA strand displacement polymerase large fragment Bsu is added at an enzyme activity of 7.5-60 U, and the exonuclease III is added at an enzyme activity of 8-60 U.

[0037] Example 1 Study on the enzyme activity addition range

[0038] 1) Proportion study of UvsX, UvsY, and single-stranded binding protein gp32

[0039] The recombinant enzyme UvsX, as the main functional protein in the strand-invading nucleic acid amplification system, has multiple functions: single-stranded binding function, ATP binding function, single-stranded DNA-dependent ATPase function, etc. The functions it plays in vivo include DNA damage, DNA recombination, DNA repair, DNA replication, etc. In the present invention, the DNA recombination activity of this enzyme is mainly utilized.

[0040] The recombinant enzyme coenzyme UvsY, as the coenzyme of UvsX, mainly functions in the strand-invading nucleic acid amplification system to assist UvsX in competing with the single-stranded binding protein gp32 for primer-probe at the start of the reaction; both UvsX and the single-stranded binding protein gp32 can bind the single-stranded primer and probe in the system; since gp32 in the system not only binds the primer-probe but also needs to bind and fix the displaced template single-strand to prevent the displaced template from renaturing and blocking the extension; therefore, in terms of the amount of substance, the amount of the single-stranded binding protein gp32 in the system is much higher than that of UvsX. Although the content of gp32 is high, when gp32 binds the primer-probe, the reaction cannot be initiated. Therefore, the role of UvsY is that when gp32 binds to the single-strand, it binds to it and loosens the structure, so that gp32 is separated from the single-stranded nucleic acid, providing a suitable structure and position for UvsX to bind. Because UvsY affects two components, it is more complex to establish the system according to the enzyme activity and is affected by more interference factors. Therefore, in the present invention, the concentration of UvsY in the system is adjusted (such as Figure 2 ), when the addition amount in the system is too much, the fluorescence height of the amplification curve is too low. If there are inhibitors in the template or the template concentration is further reduced, the situation of undetectable may occur, thus affecting the specificity (increasing false negatives) and sensitivity of the system. Among them, the amplification effect is better at 40-100 g / μL, and there is no obvious influence on the detection rate of low-concentration templates, indicating that the system has a wide tolerance range for UvsY.

[0041] The present invention studies the addition range of other main enzyme materials in a system configuration manner according to the enzyme activity. Since UvsX, UvsY, and single-stranded binding protein gp32 are responsible for the homologous recombination process between the primer probe and the template, the three components are uniformly adjusted. The test ratio includes but is not limited to the ratio in Table 1 below. When screening the recombination-related ratio, 15U / T is used for the polymerase and 8U / T is used for the nuclease III; the gray area marked in the table is the better amplification ratio in the amplification ratio. It can be seen from the table that the ratio range of UvsX: single-stranded binding protein gp32 is 1:0.4 to 1:1, including equal magnifications related to the ratio, such as 2:0.8 and 4:1.6. In addition to the ratio of UvsX: single-stranded binding protein gp32, the concentration of UvsY will also affect the amplification effect, which is basically manifested as high concentration of UvsY inhibiting amplification, but the concentration of UvsY is related to the concentration of UvsX and gp32. When the concentrations of UvsX and gp32 in the system increase, the concentration that can be tolerated for UvsY is higher.

[0042] Table 1 UvsX, UvsY, single-chain binding protein gp32 ratio adjustment table

[0043]

[0044]

[0045]

[0046] In the above table, the ratio of UvsX (ng / μ): gp32 (U / T): UvsY (ng / μL) with better performance is selected as 240:2:60 as the acceptable amplification multiple in the system, and the addition amount of other components remains the same. The multiple setting relationship is shown in Table 2 below, and the corresponding amplification relationship is as follows Figure 3 The results showed that when the amplification multiple was 1.25, the onset time was the earliest, and the fluorescence height was the highest at 15-20 cycles. All amplification multiples were ranked according to amplification capacity, and the results showed that 1.25>1>1.5>1.75>2>2.25>0.75>0.5. Although the fluorescence height was relatively highest after 30 minutes of reaction at 0.75 and 0.5 multiples, the onset time was late, and there was insufficient detection for low-concentration templates. When the enzyme concentration was too low, there would be occasional non-specific onset of NTC (no template control) (results not shown). Therefore, it was not an option if the protein concentration was too low. Therefore, the addition range of the single-stranded binding protein gp32 (U / T) was 0.4-4.5U.

[0047] Table 2 UvsX, UvsY, single-chain binding protein gp32 ratio expansion table

[0048]

[0049]

[0050] 2) Study on the increased amount of large fragment of DNA strand displacement polymerase Bsu

[0051] In the present invention, at 37 °C, the amount of enzyme required to open 1 pmol of a specially designed probe in 30 min is defined as 1 U, that is, 1 enzyme activity unit. According to the aforementioned optimal ratio of recombinant-related enzymes, after using the selected optimal ratio in the system, the optimal concentration of strand displacement polymerase is screened. The concentration gradient and results are as follows Figure 4 , when the amount of strand displacement polymerase (large fragment of Bsu) in the system is 45 U / T, the effect is the best. The order of each addition amount according to the effect is 45 U > 60 U > 30 U > 15 U > 7.5 U, which conforms to the trend that within a certain addition amount range, the amplification ability increases with the increase in concentration. To ensure the amplification effect of the system, 45 U is recommended; when the concentration is too low, such as further lower than 7.5 U, weak signals may be generated due to insufficient effective collision probability caused by too low enzyme concentration, which cannot be recognized, or even no amplification signal can be generated, resulting in false negatives or decreased detection sensitivity; when the concentration is further increased above 60 U, an inhibitory effect will appear, and there is a phenomenon that non-specific starting values randomly appear in NTC, resulting in false positives.

[0052] 3) Study on the increased amount of exonuclease III

[0053] Exonuclease III mainly plays a role in cleaving the AP site of the probe in the reaction system. The addition amount of exonuclease III is screened under the optimal concentrations of the above-mentioned components. The results are as follows Figure 5 shown. When the concentration range is within 0 - 50 U / T, as the concentration of exonuclease III increases, the amplification effect is further enhanced. When the concentration exceeds 50 U / T, as the concentration further increases, the system begins to be inhibited and the amplification effect decreases. To ensure the best effect of the system, a concentration of 50 U / T is recommended. When the addition amount of exonuclease III further decreases, it may lead to false negatives. When further increased, it may lead to false positives or a decrease in the sensitivity of the reagent.

[0054] Screening of the sources of each enzyme in Example 2

[0055] 1) Screening of the species sources of recombinant enzyme UvsX and recombinant enzyme cofactor UvsY from different sources

[0056] In the present invention, UvsY serves as an auxiliary protein for UvsX, and close cooperation between the two is required. Therefore, it is recommended to use recombinases and recombinase auxiliary proteins from the same species source, such as the UvsX and UvsY combinations of T4 and T6 phages, the RecA and RecORF combinations in Escherichia coli, as well as recombinant proteins and auxiliary proteins with the same function in fungi. However, as the complexity of the species increases, more functional proteins are required to complete the same life activities, and the cooperation becomes more precise. Therefore, it is more recommended to use proteins related to gene recombination and replication in simple organisms for establishing a nucleic acid amplification system in vitro, preferably phages or Escherichia coli.

[0057] 2) Screening of single-stranded DNA-binding proteins gp32 from different sources

[0058] In terms of the principle of strand-invasion nucleic acid amplification technology, when single-stranded DNA-binding proteins perform functions in the system, they need to cooperate with UvsX and UvsY. Therefore, single-stranded DNA-binding proteins from the same species source can be selected. However, for phages, most of the proteins required for life activities are derived from the host. Although T4 phage can express gp32 protein with single-stranded DNA-binding protein function, some phages do not have the expression of this functional protein. Therefore, we believe that it is feasible to cooperate with single-stranded DNA-binding proteins from different sources to complete the in vitro amplification system. In the present invention, several single-stranded DNA-binding proteins from different sources were selected for enzyme activity testing. The specific test data are shown in Table 3 below. Among them, except for the gp32 protein from T4 phage, Tth SSB from Thermus thermophilus and SSB from Escherichia coli can all function in the existing system, but the recommended enzyme activities are inconsistent. Compared with gp32, the performance of each recommended enzyme activity is basically the same. For proteins from other sources, adjusting the concentration cannot function in the system.

[0059] Although this method can analyze the activity of single-stranded DNA-binding proteins, this enzyme activity detection method does not have the ability to specifically screen raw materials for strand-invasion nucleic acid amplification technology, that is, the method specificity is not strong, and it can detect all proteins with single-stranded binding function. Therefore, when constructing the system, it is necessary to first screen whether it is feasible in the system. For single-stranded DNA-binding proteins from different biological sources, the inconsistent optimal concentrations required in the system may be due to the cooperation problem between enzymes, which is shown as the optimal enzyme amount here, not the only feasible enzyme amount. (Note: All data were tested at 37 °C)

[0060] Table 3 Screening of single-stranded DNA-binding proteins gp32 from different sources

[0061]

[0062] 3) Screening of large fragment of DNA strand displacement polymerase Bsu from different sources

[0063] Strand displacement polymerases are applied in most isothermal amplification reaction systems. In the present invention, four strand displacement polymerases from different sources were explored. The specific information is shown in Table 4. Except that the optimal reaction temperature of the large fragment of Bst is 60 - 65 °C, the optimal reaction temperature of other strand displacement polymerases is 37 °C. However, the enzyme activity was uniformly tested at 37 °C, which may be one of the reasons for the relatively low specific activity of the large fragment of Bst. Several strand displacement polymerases were respectively added to the above-mentioned experimental group with the optimal screening, and the enzyme activity addition screening was carried out. Among them, several enzymes that can react at 37 °C can all function in the system, but there is no obvious correlation between the optimal addition amount and the specific activity performance, and the addition amounts in the system are inconsistent. However, even at the optimal addition amount, the performances of each enzyme are not completely the same. The results are as Figure 6 shown. The performances of the large fragment of Bsu and the large fragment of Sau are similar, but the effect of the large fragment of Klenow is not good. The reason may be that the specific activity of this enzyme measured at 37 °C is lower than that measured at 65 °C. When preparing the system, the enzyme activity is low and the addition amount is high, and the excessive number of proteins interferes with the normal operation of other proteins in the system.

[0064] Table 4 Screening of the large fragment of Bsu, a DNA strand displacement polymerase from different sources

[0065]

[0066] 4) Screening of the source of the tool enzyme with AP site cleavage activity

[0067] When screening the AP site cleavage enzyme, relevant enzymes that can be purchased on the market were selected and tested using the AP site cleavage activity detection method invented by the company. The specific specific activities are shown in Table 5 below. The following several enzymes with AP site cleavage activity can all exert activity in the strand intervention-based nucleic acid amplification technology of the present invention, but the efficiency of the three endonucleases is relatively low, and it is difficult to observe the "S"-shaped amplification curve (as Figure 7 ) in the curve, but it rises in a straight line form with a slower rate. Among the three endonucleases, Endonuclease IV performs relatively best.

[0068] Table 5 Screening of the source of the tool enzyme with AP site cleavage activity

[0069]

[0070] Example 3 Verification of the differences between batches of each enzyme in the amplification system of the present invention

[0071] 1) Differences in the system configuration according to the concentration of the recombinase UvsX

[0072] The recombinase UvsX, as an important functional enzyme for strand-invaded nucleic acid amplification, can bind single-stranded nucleic acids (primers, probes, etc.) in the amplification system, recognize the homologous double-stranded region of the template double-strand and intervene, realizing the re-pairing of primers and probes with the template strand, and the original paired strand is displaced. The initial intervention efficiency of primers and probes affects the start time and amplification efficiency of the entire amplification process. Therefore, the addition amount of recombinase is crucial for the amplification system. In this invention, the system was prepared according to the amplification system in Table 6 below according to the concentration and the system configuration effect was compared by adjusting the concentration. The primer and probe sequences are shown in Table 7 (source: Rapid detection method for African swine fever laboratory SN / T 5479-2022), and the protein information of three batches of UvsX is shown in Table 8 below.

[0073] Table 6 Sampling scheme for strand-invaded nucleic acid amplification system by concentration

[0074]

[0075]

[0076] Table 7 Primer and probe sequences used in the system of this invention

[0077]

[0078] Table 8 Details of information on three batches of UvsX

[0079]

[0080]

[0081] The above three batches of UvsX proteins were uniformly added to the system at 300 ng / μL in the system for testing, and the results are as Figure 8 shown, showing Batch 2 > Batch 3 > Batch 1. The reason for this difference is the difference in protein purity of the batches, or the difference in activity caused by slightly different purification conditions and environments. However, the concentration measurement parameters cannot reflect this phenomenon; therefore, when preparing the system with UvsX according to the concentration, different batches of UvsX cannot be prepared at a single fixed concentration. Before a new batch of UvsX is put into use, it is necessary to compare its amplification performance with the batches that have been optimized for concentration in the strand-invaded nucleic acid amplification system, as Figure 8, where Batch 1 is used as a control (which has undergone comprehensive performance verification and meets the requirements). Batch 2 and Batch 3 perform slightly worse. The concentrations of Batch 2 and Batch 3 are adjusted separately, with an upward and downward adjustment of 20 ng / μL as a unit, involving 340 ng / μL, 320 ng / μL, 300 ng / μL, 280 ng / μL, and 260 ng / μL respectively. After testing, Batch 2 has the best effect at 280 ng / μL, and Batch 3 has the best effect at 340 ng / μL. Then the concentration is continuously increased to 360 ng / μL and 380 ng / μL, and the result shows that 340 ng / μL has the best effect. The three batches are uniformly tested in the amplification system according to the optimal concentrations: Batch 1 (300 ng / μL), Batch 2 (280 ng / μL), and Batch 3 (340 ng / μL). The results show that there is no significant difference in UvsX among the three batches, indicating that even if the enzyme activity of UvsX is not measured, the optimal concentration test within the system should still be carried out. This method is also applicable to other proteins without enzyme activity data.

[0082] 2) Differences in the system configuration of single-stranded binding protein gp32 according to enzyme activity or concentration

[0083] In the strand-invasion nucleic acid amplification system, the whole reaction temperature is 40 - 45 °C. At this temperature, the template double-strand cannot be completely denatured, and template denaturation only occurs in the primer-probe strand-invasion region. Except for this region, the other parts of the template still maintain a stable double-stranded state. Such a structural feature leads to the easy renaturation of the template denaturation region, and the primer-probes that have achieved strand invasion are re-dissociated, which is not conducive to amplification. Therefore, a single-stranded binding protein is needed to bind the displaced strand of the template to prevent its renaturation. However, the single-stranded binding protein has no sequence specificity and can bind any single-strand in the reaction system, such as primer-probes. After binding, the single-stranded binding protein-primer-probe complex cannot achieve the strand-invasion function, and the reaction cannot be initiated. Therefore, the amount of single-stranded protein is crucial for the amplification system.

[0084] The enzyme activities of 3 batches of single-stranded binding protein gp32 are tested, and tests are carried out within the system according to concentration and enzyme activity respectively. The concentration and specific activity data of the three batches are shown in Table 9 below:

[0085] Table 9 Details of the information of the protein encoded by gene 32 in three batches

[0086]

[0087] The above 3 batches of proteins were respectively tested in the system according to the concentration and enzyme activity. The sample addition scheme is shown in Table 6. Among them, for UvsX, Batch 1 was used at a concentration of 300 ng / μL, and the gene 32 protein was tested at 2.5 U / T. According to Batch 1 for enzyme activity and concentration conversion, the addition amount was 1851 ng / μL. The specific addition amount is shown in Table 9. The template was diluted 1E-03 times with the extraction product of African swine fever clinical positive samples. The results are as Figures 10 - 11 shown. Batch 1 and Batch 3 showed similar performance, while Batch 2 was slightly worse. Its performance was related to the conversion of concentration into enzyme activity equivalent; when formulating the system according to enzyme activity ( Figure 10 ), the performance of the three batches was basically the same. The reason for the incomplete overlap of the amplification curves may be related to some differences in the binding efficiency of each component at the start of the isothermal system reaction. This difference objectively exists and can be ignored. This result shows that when formulating the system according to enzyme activity, it is more capable of reducing the differences between batches of the system. However, it should be noted that even when calculated according to concentration, based on the relative performance of the enzyme in each batch, the amount of enzyme used can be further increased or decreased to adjust an optimal concentration for use in the system. However, it is difficult to achieve the expected effect with just one adjustment in this way. It is necessary to repeatedly adjust until the addition amount is the same as or very close to the addition amount according to enzyme activity in the system to show the effect of formulating the system according to enzyme activity; this method is effective for most purified batches of gene 32 protein. However, we found that when the specific activity is lower than 60 U / mg, even when adding up to 1.3 U / T according to enzyme activity in the system, the amplification effect is still difficult to reach the level of high-specific-activity batches (such as Figure 12 ). Therefore, when formulating the system according to enzyme activity, in addition to referring to the enzyme activity data, a range of specific activity data also needs to be established. If the batch of protein with a specific activity of 58.02 U / mg is used according to the concentration addition method, it is difficult to achieve the best effect no matter how the addition amount is adjusted. Therefore, formulating the system according to enzyme activity will be more accurate.

[0088] 3) Differences in formulating the system of strand displacement polymerase Bsu large fragment according to enzyme activity or concentration

[0089] In the strand-displacement nucleic acid amplification system, due to the low reaction temperature, it is difficult for the template to be completely denatured under this reaction temperature condition. Therefore, a strand-displacement polymerase is required. This enzyme can displace the template strand first during the polymerization process and then perform polymerization amplification along the template strand. In other amplification systems, such as PCR, LAMP and other technologies, the tool enzymes are formulated according to the enzyme activity. For commercial strand-displacement polymerases, the polymerization ability enzyme activity is evaluated, but this evaluation method cannot cover the activity of "strand displacement". Therefore, our company has established a unique evaluation method for the activity of strand-displacement polymerase, which is defined as the amount of enzyme required to open 1 pmol of a specially designed probe at 37°C for 30 minutes is defined as 1 U. According to the scheme in Table 6, compare the amplification performance of three batches of Bsu large fragments according to the concentration (the enzyme activity conversion of batch two) and the enzyme activity in the system. The results are shown in Table 10 below:

[0090] Table 10 Details of the protein information of three batches of Bsu large fragments

[0091]

[0092]

[0093] The test results show that when the specific activity is too high or too low, and the system is formulated according to the concentration, the amplification effect is not ideal. When the system is formulated according to the enzyme activity, except for batch four with a specific activity of 6480 U / mg, the rest can pass the enzyme activity test. It shows that for Bsu large fragments, the specific activity lower than 6480 U / mg is unqualified. In the Bsu system, it also shows that there is no obvious batch-to-batch difference when the system is formulated according to the enzyme activity, and there is an obvious batch-to-batch difference when the system is formulated according to the concentration.

[0094] 4) Difference in the system formulation of exonuclease III according to enzyme activity or concentration

[0095] Exonuclease III is not essential for strand displacement nucleic acid amplification systems, but it is one of the optional solutions for fluorescence-based strand displacement nucleic acid amplification systems. In addition, tools such as NFo and fpg can also be used, but exonuclease III has the best effect. Like strand displacement polymerases, although commercial exonuclease III has an enzyme activity label, the main domain evaluated for exonuclease is the ability of the enzyme to cleave AP (apurinic / apyrimidinic) sites in strand displacement nucleic acid amplification systems. Therefore, according to the method for evaluating the AP site cleavage ability of exonuclease III in the company's invention patent, enzyme activity analysis was carried out, and the performance of the system formulated according to enzyme activity and concentration was analyzed. According to the scheme in Table 6, exonuclease was added to the system according to enzyme activity or concentration for testing, and the amplification performance of three batches of exonuclease III in the system according to concentration and enzyme activity was compared. The specific information of the three batches of exonuclease III is shown in Table 11. When the specific activity of exonuclease III is not higher than 3074.23 U / mg, formulating the system according to enzyme activity also results in poor performance. However, it should be noted that although the specific activity of Batch 1 is high, when tested in the system according to concentration, it shows qualified performance, which may be because the strand displacement nucleic acid amplification system has strong tolerance to exonuclease III, and the corresponding range for the best effect is relatively wide.

[0096] Table 11 Details of protein information and result performance of three batches of exonuclease III

[0097]

[0098] Example 4 Performance verification of the amplification system of the present invention

[0099] The minimum detection limit performance of the strand displacement nucleic acid amplification system was evaluated, and the results are shown in Table 12. For samples with 3 copies / T, 100% detection (20 repetitions) can be achieved, which is much lower than the nominal 10 - 100 copies per reaction of other isothermal amplification systems on the market, indicating that the amplification system is sensitive enough.

[0100] Table 12 Details of the minimum detection limit of the strand displacement nucleic acid amplification system

[0101]

[0102] The above are the implementation methods listed in this example. However, this example is not limited to the above optional implementation methods. Those skilled in the art can obtain many other implementation methods by arbitrarily combining the above methods. Anyone can obtain various other forms of implementation methods under the inspiration of this example. The above specific implementation methods should not be construed as limiting the protection scope of this example. The protection scope of this example should be defined by the claims, and the description can be used to interpret the claims.

Claims

1. A chain-mediated isothermal nucleic acid amplification system, characterized in that: It includes recombinase UvsX, recombinase loading factor UvsY, single-stranded DNA binding protein gp32, large fragment of DNA strand displacement polymerase Bsu, exonuclease III, ATP energy recycling system and buffer system. The addition amount range of the single-stranded DNA binding protein gp32 configured according to enzyme activity is 0.4 - 4.5 U.

2. The isothermal nucleic acid amplification system of the chain intervention type according to claim 1, wherein: The single-stranded DNA binding protein gp32, large fragment of DNA strand displacement polymerase Bsu, and exonuclease III are used to establish a system according to the enzyme activity configuration of each enzyme.

3. The isothermal nucleic acid amplification system with chain intervention according to claim 1, characterized in that: The addition amount range of the large fragment of DNA strand displacement polymerase Bsu configured according to enzyme activity is 7.5 - 60 U.

4. The isothermal nucleic acid amplification system of the chain intervention type according to claim 3, characterized in that: The addition amount range of the exonuclease III configured according to enzyme activity is 8 - 60 U.

5. The isothermal nucleic acid amplification system of the chain intervention type according to claim 1, characterized in that: The concentration of the recombinase UvsX in the system is 40 - 540 ng / μL.

6. The isothermal nucleic acid amplification system of the chain intervention type according to claim 1, wherein: The concentration of the recombinase loading factor UvsY in the system is 10 - 135 ng / μL.

7. The isothermal nucleic acid amplification system with chain intervention according to claim 1, wherein: The ATP energy recycling system includes ATP, sodium hexametaphosphate, ppk2 and pap, and the buffer system includes Tris-Ac, KOAc, MgOAc, TCEP and polyethylene glycol 35000.

8. A strand-invading isothermal nucleic acid amplification system according to claim 7, characterized in that: The recombinase UvsX and the recombinase coenzyme UvsY are derived from phage or Escherichia coli.

9. The isothermal nucleic acid amplification system with chain intervention according to claim 1, wherein: The single-stranded DNA binding protein gp32 is derived from T4 phage or Thermus thermophilus or Escherichia coli.

10. Use of an isothermal nucleic acid amplification system of the chain intervention type according to any one of claims 1-9, characterized in that: It is applied to the preparation of an isothermal amplification kit.

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