Chain intervention type isothermal nucleic acid amplification product specificity analysis method

Abstract

The invention relates to the technical field of biological detection, in particular to a chain intervention type isothermal nucleic acid amplification product specificity analysis method which comprises the following steps: S1, establishing an isothermal amplification PCR (Polymerase Chain Reaction) system, and carrying out fluorescent PCR amplification; s2, melting curve reaction: recording the melting curve reaction carried out on the amplification product in the previous step by a melting curve analysis program; and S3, after the reaction is finished, analyzing a melting curve of an amplification product, and judging a positive / negative sample according to whether a corresponding melting peak exists or not. According to the chain intervention type isothermal nucleic acid amplification product specificity analysis method disclosed by the invention, isothermal amplification and a melting curve method are combined, and specific products can be distinguished and the amplification sensitivity can be improved without adding dyes before a melting reaction.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a chain-intervention type isothermal nucleic acid amplification product specific analysis method. Background Art

[0002] The detection methods of chain-involved nucleic acid amplification product results usually include fluorescence method, lateral test strip method, electrophoresis method, etc. The fluorescence method has a short detection time and needs to be matched with a professional fluorescence detection instrument (such as a fluorescence quantitative PCR instrument). Its sensitivity is the highest among the common methods. Gel electrophoresis is a common method for PCR product analysis, but it is not suitable for chain-involved nucleic acid amplification systems, because this system is generally difficult to amplify fragments larger than 500bp, and the shorter the amplicon, the higher the amplification efficiency, so the generally recommended amplification product length is 100-200bp; and the primer length required for chain-involved nucleic acid amplification technology itself is generally recommended to be 28-38p. Longer primers will increase the probability of primer dimer formation. If ordinary agarose gel electrophoresis is used for distinction, it is limited by resolution, resulting in the inability to distinguish primer probe dimers from amplification products.

[0003] When a specially modified probe is introduced into the amplification system (such as Tables 6 and 7 in the specification of patent CN202311851314.8), PC and no template control (NTC) are clearly distinguished, indicating that the probe can improve the specificity of the chain-intervening nucleic acid amplification system. Compared with gel electrophoresis, the addition of the probe prevents the amplification system from reacting to the accumulation of primer dimers, thereby effectively shielding the interference of primer dimers.

[0004] Although the probe can effectively shield the interference of primer dimers, the probe method is also limited by the inherent defects of the chain-intervening nucleic acid amplification system itself, that is, when the temperature is low, the frequency of Brownian motion of molecules decreases and effective molecular collisions decrease. Although there are crowding agents such as high molecular weight polyethylene glycol in the amplification system, compared with high-temperature temperature cycling PCR technology, the sensitivity is generally more than 90% of PCR, and the fluorescence of high-concentration templates is high. As the template further decreases, the fluorescence height gradually decreases. Sometimes, even if low-concentration templates are amplified, they cannot be detected by the machine due to insufficient signal accumulation. Therefore, for critical positive samples, they are sometimes not effectively detected. Summary of the invention

[0005] The purpose of the present invention is to provide a strand-intervention isothermal nucleic acid amplification product-specific analysis method to solve the technical problem in the prior art that critical positive samples sometimes cannot be effectively detected.

[0006] The present invention discloses a strand-intervention isothermal nucleic acid amplification product specific analysis method, comprising the following steps: S1: Establish an isothermal amplification PCR system and perform fluorescent PCR isothermal amplification; S2: melting curve reaction, the melting curve analysis program records the melting curve reaction of the amplified product in the above steps; S3: After the reaction is completed, the melting curve of the amplified product is analyzed, and the positive / negative samples are judged by the presence or absence of corresponding melting peaks.

[0007] Furthermore, the reaction temperature of the fluorescent PCR isothermal amplification is 37-48°C, fluorescence is collected per minute per cycle, and the reaction is repeated for 15 cycles. Preferably, the isothermal amplification temperature is 42-44°C, and preferably, the isothermal amplification temperature is 44°C.

[0008] Furthermore, the melting curve reaction is carried out at a heating and cooling rate of 2-6°C / s, from room temperature to 95-98°C, maintained for 30s-1min, then cooled to 50°C, maintained for 10-15s, and then gradually increased in temperature from 50°C to 85-95°C, collecting a fluorescent signal every time the temperature increases by 0.5-1°C.

[0009] Preferably, the melting curve reaction is performed at a heating and cooling rate of 6°C / s, from room temperature to 98°C, maintained for 1 min, then cooled to 50°C, maintained for 10 s, and then gradually increased in temperature from 50°C to 85°C, collecting a fluorescent signal every time the temperature increases by 1°C.

[0010] Furthermore, the primer probes used in the isothermal amplification PCR system are ASFV-F, ASFV-R and ASFV-P.

[0011] Furthermore, the isothermal amplification PCR system includes recombinase UvsX, recombinase loading factor UvsY, single-strand binding protein gp32, DNA strand displacement polymerase Bsu large fragment, nuclease III, ATP energy circulation system and buffer system.

[0012] Furthermore, the recombinase UvsX, the single-strand binding protein gp32, the DNA strand displacement polymerase Bsu large fragment, and the exonuclease III are configured to establish a system according to the enzymatic activity of each enzyme; Furthermore, the dosage range of the recombinant enzyme UvsX according to the enzyme activity configuration is: 0.2~6.75U; Furthermore, the ratio of the enzyme activity addition of the recombinant enzyme UvsX to gp32 is 0.2:0.4~6.75:4.5; Furthermore, the single-chain binding protein gp32 is added in an amount ranging from 0.4 to 4.5 U according to the enzyme activity configuration; Furthermore, the amount of the DNA strand displacement polymerase Bsu large fragment prepared according to the enzyme activity ranges from 7.5 to 60 U; Furthermore, the amount of exonuclease III added according to the enzyme activity is in the range of 8-60U; Furthermore, the concentration of the recombinase loading factor UvsY in the system is 10-135 ng / μL; Furthermore, the ATP energy cycle system includes ATP, sodium hexametaphosphate, ppk2 and pap, and the buffer system includes Tris-Ac, KOAc, MgOAc, TCEP and polyethylene glycol 35000.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. A strand-intervention isothermal nucleic acid amplification product specific analysis method of the present invention combines isothermal amplification with a melting curve method, and can distinguish specific products without adding dyes before the melting reaction, thereby increasing amplification sensitivity and effectively detecting critical positive samples; 2. In the chain-intervention isothermal nucleic acid amplification system of the present invention, all enzyme components in the isothermal amplification system are prepared in an amount according to the enzyme activity, which can ensure that the amplification effects of different batches are consistent. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only represent some embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 It is the amplification curve diagram of the isothermal amplification system PC and NTC of the present invention; Figure 2 The melting reaction curve of the isothermal amplification system of the present invention with dyes PC and NTC added; Figure 3 The melting peak curve diagram of the isothermal amplification system of the present invention with dyes PC and NTC added; Figure 4 The melting reaction curves of the isothermal amplification system of the present invention when the dyes PC and NTC are added without the probe; Figure 5 It is a melting peak curve diagram of the isothermal amplification system of the present invention when the dyes PC and NTC are added without a probe; Figure 6 The amplification curve diagram of the isothermal amplification system of the present invention with the addition of dyes PC and NTC; Figure 7 This is a melting reaction curve diagram of the isothermal amplification system of the present invention without adding dyes PC and NTC; Figure 8This is the melting peak curve diagram of the isothermal amplification system of the present invention without adding dyes PC and NTC; Fig. 9 It is a melting reaction curve diagram of the isothermal amplification system of the present invention without forward and reverse primers and template; Fig.10 It is a melting peak curve diagram of the isothermal amplification system of the present invention without forward and reverse primers and template; Fig.11 It is a melting reaction curve diagram of the isothermal amplification system of the present invention in which the melting reaction is directly performed without amplification; Fig.12 It is a melting peak curve diagram of the isothermal amplification system of the present invention in which a melting reaction is directly performed without amplification; Fig.13 The amplified fluorescence height distribution diagram of 314 NTCs and 336 low-concentration templates of the present invention; Fig.14 This is the optimum temperature trend diagram of the isothermal amplification program of the present invention. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0017] The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably a polymeric molecule, comprising ribonucleic acid, deoxyribonucleic acid or analog units thereof. The nucleic acid may be single-stranded or double-stranded. A single-stranded nucleic acid may be a nucleic acid of one strand of a denatured double-stranded DNA. Alternatively, a single-stranded nucleic acid may be a single-stranded nucleic acid that is not derived from any double-stranded DNA.

[0018] Example 1 Exploration of the combination of chain-intervention isothermal amplification and melting curve method During the experimental research, the inventors explored a method combining chain-intervention isothermal amplification with the melting curve method, and no dye needs to be added before the melting reaction, and the amplification products of low-concentration templates and positive templates can be specifically distinguished. The template mentioned in the present invention refers to the nucleic acid extracted from the sample, and the sample can be but not limited to pharyngeal / nasal swabs, blood, feces, cerebrospinal fluid, pleural effusion, etc. The template in this embodiment refers to the nucleic acid product extracted from the nasal swab sample with African swine fever virus.

[0019] The exploratory tests used by the applicant are as follows: 1. Study on the chain-intervention isothermal amplification system and melting curve method The fluorescence height of the amplification curve of the chain-intervention nucleic acid detection system decreases as the template concentration decreases. When the final concentration is close to the Linyang template, it is often difficult to distinguish it from the NTC due to the low fluorescence increase of the amplification curve, and it is impossible to determine whether the product has been effectively amplified. To solve this problem, we need to further detect the amplification product of the Linyang template. However, due to the low accumulation of the amplification product of the Linyang template, there are not many methods for secondary detection of its product. The applicant tried to use saturated dyes to detect the product. In order to simultaneously evaluate the amplification product of the Linyang template and NTC, the detection method is as follows: Experiment 1. Adding dye The amplification reaction was performed according to the system in Table 1. The amplification group was divided into two groups. Group A added 5ul of the corresponding template, and group B added 5ul of water as NTC. The amplification program used 44°C, and fluorescence was collected every minute per cycle. A total of 15 cycles were set. After the reaction was completed, the amplified product was transferred to the electrophoresis laboratory, and 2.5ul of picogreen dye (Yisheng Biotechnology, 12641ES01) diluted to 20x according to the operating instructions was added to the product. The melting curve program was set as shown in Table 2, where the primer probe sequences are shown in Table 3 (Source: African swine fever laboratory rapid detection method SN / T 5479-2022). The amplification results are shown in Figure 1 As shown, the melting curve and dissolution peak curve results are as follows Figure 2 , 3 As shown, there is a distinction between adding template and not adding template, but through melting peak curve analysis, there is no peak in group A and group B; it can be found that adding dye to the chain-intervening nucleic acid amplification system cannot distinguish between the amplified group and the non-amplified group.

[0020] Table 1 Isothermal amplification reaction system

[0021] Table 2 Melting curve reaction program

[0022] Table 3 Primer probe sequences used in the system of the present invention

[0023] Experiment 2. Add dye and remove probe Considering that there are two luminescent systems, probes, and dyes in the system, and the probes need to be cut under the action of Exonuclease III, the applicant supplemented the following verification experiments to verify whether the above results are due to the mutual interference between the two luminescent systems. In the amplification system of Table 1, Exoclease III and probes were removed, and the test was repeated under the same procedure and dye concentration. The experiment with template added was taken as Group A, and the experiment without template added was taken as Group B. The melting curve is shown in Figure 4Its melting peak curve is shown in Figure 5 As shown in the figure, it can be seen that group A has a clear melting curve peak, while group B has a peak, but the peak shape is poor and continuous, indicating the presence of complex types of primer dimers. In addition, in the amplification results of the experimental group with added dye but no probe and Exoclease III (such as Figure 6 ), there is no difference in the amplification curves between group A and group B, which may also indicate that double-stranded nucleic acid was produced in group B during the amplification process, otherwise no amplification curve should be produced.

[0024] comprehensive Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 The results show that the use of dyes in the fluorescent chain-mediated nucleic acid amplification reaction is inconsistent with PCR, and the fluorescence generated by the fluorescent chain-mediated nucleic acid amplification system will affect the interpretation of the melting curve after the addition of the dye.

[0025] Experiment 3: No dye added To solve this problem, the applicant directly performed a melting curve analysis on the product of fluorescent chain-intervention nucleic acid amplification without adding dye, and also used the system in Table 1, with the template group as group A and the template-free group as group B. The melting curve results are as follows: Figure 7 As shown, the melting peak curve is Figure 8 As shown, it can be found that, unexpectedly, in group A of the fluorescent chain-intervention nucleic acid amplification system, there is an obvious melting peak, which is a single peak, and there is no peak in group B. That is, this method can specifically distinguish the amplification products of the positive template.

[0026] Although the fluorescent signal of the melting curve and the signal generated by amplification should theoretically have the same intensity, because the product generated in the amplification step is consistent with the amount of product in the melting curve analysis, the fluorescent signal generated by the same amount of probe cut will change with the change of temperature. For example, as the temperature rises, the fluorescent signal decreases. When it is superimposed with the signal generated by the system due to amplification, the fluorescent signal in the melting curve experiment will decrease more with the increase of melting temperature. This can explain to a certain extent why no fluorescent signal that can be judged as positive is generated during the amplification process, but the melting peak corresponding to the product can be analyzed in the melting curve step. However, the change of temperature on the fluorescent signal must be superimposed with the amplification product. When there is no amplification product, there is only a change in temperature, and no corresponding melting peak will appear, thereby ensuring specificity.

[0027] To verify that this phenomenon did not occur by chance, the applicant conducted two sets of experimental verifications.

[0028] Verification 1: The generation of the melting peak is not caused by the cleavage of the [THF] site of the probe.

[0029] Because the probe structure in the chain-involved nucleic acid amplification system is relatively special, the entire chain is 46-54bp long, and there is a [THF] site at 30-38p near the 5' end. A group of fluorescent groups and corresponding quenching groups are modified on the two T bases about 2-3 bases on both sides of the site, and the 3' end is blocked with C3 Spacer or other blocking groups. Because the [THF] site is a baseless site, the connection here is not as stable as a normal base, and it is possible that the free probe breaks during the test of the melting curve, resulting in fluorescence release. Therefore, based on Table 1, the forward and reverse primers are removed and the test is performed without adding a template. The results are as follows Fig. 9 and Fig.10 As shown, when there is only a probe in the system, there is no melting curve. This phenomenon indicates that the melting peak of the normal amplification system is not caused by the breakage of the [THF] site at high temperature. In addition, as the temperature rises, if the THF site is broken, then the fluorescence height of the melting curve should rise rapidly near the melting peak, rather than falling. Furthermore, if the melting curve changes due to the breakage of the [THF] site under high temperature conditions, both Group A and Group B should produce the same melting curve. The actual performance is as follows Figure 7 and Figure 8 As shown, there should be a difference between Group A and Group B. Therefore, the melting peak generated in the method of the present invention is not caused by the cleavage of the [THF] site of the probe.

[0030] Verification 2: The generation of melting peaks is indeed correlated with the amount of amplified products To verify the correlation between the melting peak and the amplified product, we compared the amplification results and melting curve results of high-concentration template, low-concentration template, and NTC. The results are shown in Table 4. It can be seen that when the template concentration is high, the melting peak is high. Even if NTC has a certain degree of amplified fluorescence height, there is no melting curve. This result shows that the existence and height of the melting peak are related to the amplification amount, and the melting curve can be used as a supplementary method to further determine whether there is an amplified product.

[0031] Table 4 Comparison of different template concentrations, NTC amplification results and melting curve results

[0032] To verify that the result is not related to the amount of template, but the accumulation of the real reaction amplification product, the applicant did not perform amplification reaction based on the system in Table 1, and directly entered the melting curve reaction program. The test results are as follows: Fig.11 and Fig.12As shown, there is no melting curve peak. This result shows that although the peak height of the melting curve is correlated to the amount of template, no melting curve is generated if it does not participate in amplification, and the melting peak reflects the accumulation of the real amplification product. So far, a chain-intervention isothermal amplification of the present invention is combined with the melting curve method, and a method for specifically distinguishing the amplification products of low-concentration templates and positive templates without adding dyes before the melting reaction has been confirmed.

[0033] Comprehensive verification test results analysis shows that the probe in the method of the present invention can immediately react to the accumulation of amplified products, which is also the essential difference between the present invention and the real-time fluorescent PCR probe / dye melting curve method. The probe and dye in the real-time fluorescent PCR probe / dye melting curve method are specially added to adapt to the release of fluorescent signals when the temperature rises during the melting curve reaction and the double-stranded DNA is unwound; and the dye melting curve method has very high requirements for the specificity of the system, because the dye binds to double-stranded DNA without sequence specificity. Therefore, if primer dimers exist in the amplified product, the dye method cannot accurately reflect the amplification effect. The present invention can not only specifically display the amplification effect of the sample, but also perform melting curve analysis without opening the lid and without adding any additional raw materials.

[0034] In addition, when comparing the melting peak with the amplification curve (fluorescence height values ​​in Table 4), the fluorescence height of the high-concentration template and the melting peak are consistent, but there is controversy when it comes to low-concentration templates, because the fluorescence height of the amplification curve of individual low-concentration templates intersects with the fluorescence height of NTC, making it difficult to judge. However, judging by the melting curve, no amplicon is produced. We counted 314 NTCs and 336 low-concentration template amplifications, and the distribution of their fluorescence heights is shown in the figure below. Fig.13 As shown, the NTC fluorescence height distribution range is -20~160, and the data is concentrated in 0-60, so the NTC judgment standard should be set to a minimum of 160, but this setting is too extreme, so for this system, it is temporarily set to a threshold of 200.

[0035] According to this standard, some starting values ​​of low-concentration templates cannot be judged as positive based on the fluorescence height. We tracked and observed the melting curves of 112 NTCs without melting peaks and 96 low-concentration templates, and compared the melting curve test results with those of competing reagents. The consistency comparison results are shown in Table 5 (high melting curve positivity rate). Among the 50 negative samples tested in the competing reagent (Anpu Future, DNA Constant Temperature Rapid Amplification Kit (Fluorescence Type)) (tested according to the competing test method), 5 were tested as positive by chain-intervention nucleic acid amplification reagent combined with melting curve, and these 5 samples were retested and were also positive. This result shows that the detection sensitivity of the melting curve is higher.

[0036] Table 5 Comparison of the actual test results of the method of the present invention and competing products

[0037] Example 2 Study on the dosage of each enzyme in the system 1. Study on the dosage of recombinase UvsX Because the specific activity results of UvsX in different batches are inconsistent, and the test results of the melting curve are more sensitive than the amplification curve results, the amount of UvsX in the chain-intervening nucleic acid amplification system is tested based on the results of the melting curve, and the other components are consistent with Table 1. Because the amounts of UvsX, gp32, and UvsY affect each other, it is necessary to re-screen the optimal amount of UvsX based on the enzyme activity test results.

[0038] According to the system in Table 1, when the gp32 addition amount was 2.5U / T and the UvsY addition amount was 56ng / μL, the UvsX enzyme amount screening was performed, as shown in Table 6 for details.

[0039] Table 6 Test of adding amount of recombinant enzyme UvsX in the system (high concentration)

[0040] It can be seen from Table 6 that when the concentration of UvsX in the system is too high, there is an amplification curve but no melting curve. When the template is replaced with water for testing, a corresponding amplification curve will also be generated. Therefore, when the concentration of UvsX in the system is too high, nonspecific amplification will occur.

[0041] The concentrations of gp32 and UvsY in the system were adjusted downward in equal proportions to 1.3U / T, 30ng / μL, and the concentration of UvsX was tested again. The results are shown in Table 7 below.

[0042] Table 7 Test of adding amount of recombinant enzyme UvsX in the system (low concentration)

[0043] When the amounts of gp32 and UvsY were simultaneously downregulated, the concentration of UvsX could be further reduced. When the concentration was as low as 0.2U / T, the system had neither an amplification curve nor a melting curve.

[0044] Example 3 Temperature exploration of isothermal amplification system The present invention explores the optimum temperature of the isothermal amplification system. Fig.14As shown, when the amplification temperature is 37-48°C, fluorescent signals are generated. When the amplification temperature is 50°C, the fluorescence intensity drops rapidly. It is possible that this temperature is close to the inactivation temperature of a single enzyme or multiple enzymes, resulting in insufficient effective enzyme concentration in the system, or complete inactivation of the enzyme, so the amplification performance is seriously reduced. When the temperature is 42-44°C, the fluorescence intensity is the highest, and at 44°C, the fluorescence intensity reaches the highest. It can be concluded that the isothermal amplification temperature of the present invention is 37-48°C, preferably 42-44°C, and the optimal temperature is 44°C, which can be adjusted within this range according to different projects.

[0045] Example 4 Exploration of melting reaction conditions In order to shorten the reaction time and improve the test efficiency, the present invention explores the conditions of the melting reaction. The melting curve reaction program involves heating rate, denaturation temperature, denaturation temperature maintenance time, annealing temperature, annealing temperature maintenance time, endpoint temperature, and signal acquisition frequency. The change of each condition will cause the total time of the melting curve reaction to be different. In the present invention, the melting reaction conditions are shown in Table 8 below, and the heating rate is 2-6°C / s, the denaturation temperature is between 95-98°C, the denaturation temperature maintenance time is 30s-1min, the annealing temperature is 50°C, the annealing temperature maintenance time is 10-15s, the endpoint temperature is between 85-95°C, and the signal acquisition frequency is 0.5-1°C for every increase. When the signal acquisition frequency is 2°C or more for every increase, the melting curve is unstable and there is no peak, which may be due to the small number of signals collected at the melting peak. It can be obtained from Table 8 that the shortest program of the present invention is the program of sequence number 3, which takes only 13 minutes.

[0046] Table 8 Melting curve reaction schedule

[0047] The above are the implementation methods listed in this embodiment, but this embodiment is not limited to the above optional implementation methods. Those skilled in the art can arbitrarily combine the above methods to obtain other various implementation methods. Anyone can derive other various forms of implementation methods under the inspiration of this embodiment. The above specific implementation methods should not be understood as limiting the scope of protection of this embodiment. The scope of protection of this embodiment shall be based on the definition in the claims, and the description can be used to interpret the claims.

Claims

1. A method for specific analysis of strand-intervention isothermal nucleic acid amplification products, characterized in that: The following steps are involved: S1: Establish an isothermal amplification PCR system and perform fluorescent PCR isothermal amplification. The primer probes used in the isothermal amplification PCR system are ASFV-F, ASFV-R and ASFV-P; S2: melting curve reaction, the melting curve analysis program records the melting curve reaction of the amplified product in the above steps; S3: After the reaction is completed, the melting curve of the amplified product is analyzed, and the positive / negative samples are judged by the presence or absence of corresponding melting peaks.

2. The method for specific analysis of strand-intervention isothermal nucleic acid amplification products according to claim 1, characterized in that: The reaction temperature of the fluorescent PCR isothermal amplification is 37-48° C., fluorescence is collected per minute per cycle, and the reaction is repeated for 15 cycles.

3. A strand-intervention isothermal nucleic acid amplification product specific analysis method according to claim 2, characterized in that: The fluorescence PCR isothermal amplification reaction is performed at 42-44°C.

4. The method for specific analysis of strand-intervention isothermal nucleic acid amplification products according to claim 1, characterized in that: The melting curve reaction is to increase the temperature from room temperature to 95-98°C at a heating and cooling rate of 2-6°C / s, maintain for 30s-1min, then cool to 50°C, maintain for 10-15s, and then gradually increase the temperature from 50°C to 85-95°C, and collect a fluorescent signal every time the temperature increases by 0.5-1°C.

5. The method for specific analysis of strand-intervention isothermal nucleic acid amplification products according to claim 1, characterized in that: The isothermal amplification PCR system comprises recombinase UvsX, recombinase loading factor UvsY, single-strand binding protein gp32, DNA strand displacement polymerase Bsu large fragment, nuclease exonuclease III, ATP energy circulation system and buffer system.

6. A strand-intervention isothermal nucleic acid amplification product specific analysis method according to claim 5, characterized in that: The recombinase UvsX, the single-strand binding protein gp32, the DNA strand displacement polymerase Bsu large fragment, and the nuclease III are configured to establish a system according to the enzymatic activity of each enzyme.

7. A strand-intervention isothermal nucleic acid amplification product specific analysis method according to claim 6, characterized in that: The dosage range of the recombinant enzyme UvsX according to the enzyme activity configuration is: 0.2~6.75U.

8. The method for specific analysis of strand-intervention isothermal nucleic acid amplification products according to claim 6, characterized in that: The enzyme activity addition ratio of the recombinant enzyme UvsX to gp32 is 0.2:0.4~6.75:4.

5.

9. A strand-intervention isothermal nucleic acid amplification product specific analysis method according to claim 6, characterized in that: The concentration of the recombinase loading factor UvsY in the system is 10-135 ng / μL.

10. The method for specific analysis of strand-intervention isothermal nucleic acid amplification products according to claim 5, characterized in that: The ATP energy cycle system comprises ATP, sodium hexametaphosphate, ppk2 and pap, and the buffer system comprises Tris-Ac, KOAc, MgOAc, TCEP and polyethylene glycol 35000.

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