Tubular self-amplification system, kit and method for detecting monkey pox virus

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a one-tube self-amplification system, kit and method for detecting monkey pox virus. The one-tube self-amplification system comprises a DF probe for detecting DNA of a monkey pox virus B6R gene and crRNA (Complementary Ribonucleic Acid); the nucleotide sequence of the DF probe is as shown in SEQ ID NO: 3, and the nucleotide sequence of the crRNA is as shown in SEQ ID NO: 4. The trans-cleavage activity of Cas12a and affinity difference between phi29 DNA polymerase and single-chain and double-chain states of a probe are utilized, so that the problem of substrate competition of two enzymes in a co-reaction system is effectively solved, and efficient synergy of a double-signal amplification system in the space-time dimension is realized. The monkey pox virus DNA ultrafast signal amplification can be achieved within 8 min, the lowest detection limit is 87.68 aM (52.8 copies / microliter), and by combining a virus nucleic acid thermal cracking method, sample entering and result obtaining can be achieved within 10 min.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and specifically relates to a one-tube self-amplification system, kit and method for detecting monkeypox virus. Background Art

[0002] Monkeypox virus (MPXV) is an enveloped double-stranded deoxyribonucleic acid virus of the genus Orthopoxvirus in the family Poxviridae, which is transmitted through body fluids such as blood and respiratory droplets or contaminated personal items of patients. In recent years, it has spread in 120 countries, causing more than 100,000 people to be infected. The mortality rate of monkeypox virus combined with severe underlying diseases even exceeds 10%. Moreover, the monkeypox virus is continuously mutating during human-to-human transmission, which may make the virus more lethal. Therefore, the World Health Organization (WHO) has elevated the prevention and control of the MPXV epidemic to the same level as the COVID-19 pandemic and recommends that infected individuals be quarantined at home or in the hospital to contain the spread of the virus. Similar to the novel coronavirus, the quarantine of suspected infected individuals will occupy a large amount of social and medical resources. A rapid, accurate and sensitive detection method helps to identify infected patients and quickly release the occupied resources.

[0003] The clustered regularly interspaced short palindromic repeats (CRISPR) / Cas diagnostic system has the advantages of high detection efficiency, excellent sensitivity, strong specificity and simple reaction conditions, etc., and is expected to achieve ultra-rapid detection of infectious pathogens. Unfortunately, the detection limit of the single CRISPR / Cas system is only at the pM level, far from meeting the requirements of clinical detection. To improve the sensitivity of the reaction and avoid the risk of aerosol contamination of amplification products, the one-tube detection technology combining isothermal amplification and CRISPR is a current research hotspot. However, the target is competitively bound by CRISPR and isothermal amplification enzymes as the reaction substrate, resulting in a detection sensitivity lower than expected. Although this effect can be overcome by physically isolating the nucleic acid amplification reaction first and then mixing it with the CRISPR cleavage reaction system through methods such as sucrose density gradient, the detection time is long. In addition, some researchers have designed crRNAs with suboptimal PAM sites to reduce the affinity of the Cas12a-crRNA complex (RNP) for the target, making it easier for the amplification enzyme to bind to the target and accumulate more amplicons, thereby improving the detection sensitivity. However, the screening of crRNAs with suboptimal PAM sites is relatively complex. Therefore, there is still a need to develop a more rapid and universal detection technology. Summary of the Invention

[0004] The purpose of the present invention is to provide a one-tube self-amplification system, kit and method for detecting monkeypox virus, so as to achieve high-specificity and high-sensitivity detection of monkeypox virus.

[0005] To this end, the present invention provides the following technical solutions.

[0006] One aspect of the present invention provides a one-tube self-amplification system for detecting monkeypox virus, and the one-tube self-amplification system includes a DF probe for detecting the DNA of the B6R gene of monkeypox virus and crRNA; the nucleotide sequence of the DF probe is as shown in SEQ ID NO: 3, and the nucleotide sequence of the crRNA is as shown in SEQ ID NO: 4.

[0007] As a preferred embodiment, the 5'-end of the DF probe is modified with a fluorophore, and the 3'-end is modified with a quencher.

[0008] As a preferred embodiment, the fluorophore is selected from CY5, HEX, FAM or ROX; the quencher is selected from TAMRA, BHQ2 or BHQ1.

[0009] As a preferred embodiment, the one-tube self-amplification system further includes CRISPR / Cas12a protein, DNA polymerase, dNTP, RNase inhibitor and reaction buffer.

[0010] As a preferred embodiment, the Cas12a is LbCas12a.

[0011] As a preferred embodiment, the DNA polymerase is phi29 DNA polymerase.

[0012] Another aspect of the present invention provides a kit for detecting monkeypox virus, and the kit contains the one-tube self-amplification system as described above.

[0013] Still another aspect of the present invention provides the use of the one-tube self-amplification system or kit as described above in the preparation of a reagent for detecting monkeypox virus.

[0014] Yet another aspect of the present invention provides a method for detecting monkeypox virus, and the method includes the following steps: (1) Extract the DNA of the monkeypox virus sample to be detected; (2) Use the one-tube self-amplification system or kit as described above to amplify the DNA sample including the DNA extracted in step (1), and qualitatively judge and / or quantitatively detect the DNA situation in the sample to be detected according to the amplification result.

[0015] As a preferred embodiment, in step (1), the DNA is extracted from the monkeypox virus sample to be detected by thermal lysis.

[0016] As a preferred embodiment, in step (2), the amplification reaction system comprises the following components with a total volume of 20 μL: 1 μL of 1 μM circular DNA template, 0.5 μL of 1 U / μL phi29 DNA polymerase, 2 μL of 10× reaction buffer, 1 μL of 1 μM Cas12a, 0.5 μL of 10 mM dNTP, 1 μL of 1 μM crRNA, 2 μL of 1 μM DF probe, 1 μL of 40 U / μL RNase inhibitor, 5 μL of the sample to be tested, and 6 μL of DNPC-treated water.

[0017] As a preferred embodiment, in step (2), the amplification reaction program is: incubation at 37 °C for 8 minutes.

[0018] By the above technical solution, the present invention has at least the following advantages: The present invention constructs a one-tube self-amplification system (AURORA) driven by cleavage-gated terminal exposure based on CRISPR and rolling circle amplification (RCA), which can achieve ultra-fast and sensitive detection of MPXV. The present invention designs a bifunctional single-stranded reporter probe (DF probe). When the target is present, the single-stranded probe will be highly affinity-bound and cleaved by Cas12a, which initiates the cleavage-gated exposure of the 3'-end on the probe. The cleaved DF probe can hybridize with circular template DNA and be highly affinity-bound by phi29 DNA polymerase as a primer to initiate RCA. The RCA product has a Cas12a recognition site, which can activate Cas12a to cleave the DF probe again, thus forming a positive feedback loop self-amplification signal amplification system. This method utilizes the trans-cleavage activity of Cas12a and the affinity difference of phi29 DNA polymerase for the single-stranded and double-stranded states of the probe, effectively solving the substrate competition problem of the two enzymes in the co-reaction system, and realizing the efficient coordination of the double-signal amplification system in the spatio-temporal dimension. The detection system of the present invention can achieve ultra-fast signal amplification of monkeypox virus DNA within 8 minutes, and the lowest detection limit is 87.68 aM (52.8 copies / μL). Combining with the viral nucleic acid thermal lysis method, it can achieve results from sample input to result output within 10 min. The AURORA detection system of the present invention, as a double-enzyme collaborative self-amplification system, greatly improves the detection efficiency of MPXV DNA, can meet the requirements of ultra-fast detection speed and high sensitivity during the epidemic to prevent and control the epidemic, and is especially suitable for areas such as customs, railway stations, and hospitals.

[0019] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it according to the content of the specification, the following provides a detailed description of the preferred embodiments of the present invention as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shows the schematic diagram of AURORA for detecting MPXV.

[0021] Figure 2 Shows the feasibility verification of AURORA for MPXV detection; among them, (A) constructs the fluorescence intensity of the AURORA system under different experimental conditions, (B) the end fluorescence intensity of AURORA under different experimental conditions, (C) the schematic diagram of the AURORA workflow.

[0022] Figure 3 Shows the gel electrophoresis diagram of circular DNA templates.

[0023] Figure 4 Shows the optimization of AURORA reaction conditions; among them, (A) optimization of DF probe concentration, (B) optimization of Cas12a RNP concentration, (C) optimization of phi29 DNA polymerase concentration, (C) optimization of dNTP concentration, (E) optimization of circular DNA template concentration, (F) optimization of reaction time, (n = 3 technical replicates, error bars represent mean ± standard deviation).

[0024] Figure 5 Shows the AURORA analytical performance evaluation; among them, (A) sensitivity detection of MPXV by the AURORA detection system, (B) linear relationship between fluorescence intensity and target DNA concentration, (C) stability study of MPXV target DNA by the AURORA detection system, (D) specificity detection of target DNA by the AURORA detection system, (n = 3 technical replicates, error bars represent mean ± standard deviation).

[0025] Figure 6 Shows the analysis of verifying the AURORA detection system using clinical MPXV specimens; among them, (A) schematic diagram of using the AURORA detection system and qPCR to detect MPXV in clinical samples, (B) identification of MPXV in 36 clinical samples by qPCR, ND means not detected, (C) situation of identifying MPXV in 36 clinical samples by the AURORA detection system, (D) heatmap showing the comparison of MPXV detection results of 36 samples with qPCR and Aurora, "+" and "-" represent qPCR positive and negative samples respectively, and the heatmap of AURORA represents the normalized average fluorescence value, (E) correlation between qPCR and AURORA measurements of MPXV, performing linear fitting on the data points to generate a linear correlation curve, (n = 3 technical replicates, error bars represent mean ± standard deviation).

[0026] Figure 7Shows a comparison of the AURORA detection system with other detection methods; among them, (A) shows a schematic diagram of the step-by-step reaction workflow: Step 1, in vitro CRISPR / Cas12a cleavage; Step 2, rolling circle amplification; Step 3, in vitro CRISPR / Cas12a cleavage amplification generation, (B) sensitivity of only Cas12a assay (only step 1), (C) sensitivity of the two-step method (including step 1 and step 2), (D) sensitivity of the three-step method (including step 1, step 2 and step 3), (n = 3 technical replicates, error bars represent mean ± standard deviation). Detailed implementation mode

[0027] In order to make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0028] The one-tube self-amplification system (abbreviated as AURORA) is a one-tube self-amplification system driven by cleavage-gated terminal exposure based on CRISPR and rolling circle amplification (RCA). The working principle of this system is as Figure 1As shown, the ingenuity of this system lies in the design of a dual-functional reporter probe to construct a self-amplifying signal amplification system. This probe consists of a fluorescent and a quenching group, a primer functional region (RNA), and a cleavage functional region (DNA). Based on the above principles, the present invention designed the DF probe shown in Table 1. Due to the high affinity of the trans-cleavage activity of Cas12a for single-stranded DNA, after the target activates Cas12a, it can quickly cleave the DNA region (TTATT) of the DF probe, exposing the primer functional region on the probe. The cleaved DF probe hybridizes with the circular DNA template through base complementary pairing and is then highly affinity-bound by phi29 DNA polymerase to initiate RCA. The circular DNA template includes a primer binding region and a Cas12a detection region. The single-stranded RCA product can also be recognized by Cas12a, thus activating the trans-cleavage activity of Cas12a to cleave more DF probes. Therefore, the cleaved DF probe serves as an RCA primer, forming a positive feedback loop of self-amplification between RCA and CRISPR-Cas, achieving ultra-fast signal amplification. When no target is present, Cas12a lacks trans-cleavage activity and cannot trigger the cleavage gating. The intact DF probe cannot activate phi29 DNA polymerase to initiate RCA because it is not completely complementary to the circular DNA template, thus unable to initiate self-amplification. The present invention utilizes the collateral cleavage activity of Cas12a and the affinity difference of phi29 DNA polymerase for the single-stranded and double-stranded states of the probe, successfully avoiding the problem of competition for substrates between the two enzymes and truly achieving the efficient coordination of the dual-signal amplification system in the spatio-temporal dimension.

[0029] AURORA is a one-tube self-amplifying system created by combining RCA based on the recognition and cleavage activities of CRISPR-Cas12a. As Figure 2 shown in A, when the target DNA is added to the one-pot reaction, Cas12a is activated to cleave the DF probe. This initiates the self-amplification reaction, resulting in a rapid increase in the fluorescence signal and reaching a plateau within 15 minutes. In contrast, the absence of target DNA only leads to a low fluorescence value. When Cas12a, crRNA, and phi29 DNA polymerase are absent, the detected fluorescence value is negligible ( Figure 2 B). These results indicate that AURORA can be used to detect MPXV, and its high detection performance depends on the synergistic effect between Cas12a and phi29 DNA polymerase. The workflow and reaction procedure of AURORA are shown in Figure 2 C. It should be noted that compared with the stepwise reaction, AURORA does not require repeatedly opening the lid and multiple sample transfers.

[0030] The following are mentioned and used in the following examples: 1. Positive control and / or standard Target DNA was selected from the B6R gene of monkeypox virus (MPXV), and a positive standard was constructed by inserting the sequence located at 165115 - 166068 in the MPXV genome (GenBank: ON563414.3) into the plasmid pUC57.

[0031] 2. The relevant component sequence information is shown in Table 1. Table 1

[0032] Example 1: Preparation, verification and purification of circular DNA template First, phosphorylated linear padlock (1.2 μM) and adapter (1.2 μM) were mixed and incubated at 85 °C for 5 minutes, and then gradually cooled to room temperature. Subsequently, the above product was mixed with 1 μL of T4 DNA ligase (40 U / μL), 5 μL of 10× T4 DNA ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT) and DEPC-treated water in a total volume of 50 μL, incubated at 25 °C for 2 hours, and then inactivated at 65 °C for 15 minutes. Then it was inactivated at 65 °C for 15 minutes. Exonuclease I and Exonuclease III were used to digest the linear single-stranded DNA that did not form circular DNA, and the enzymes were heated at 80 °C for 20 minutes to inactivate the enzymes.

[0033] Subsequently, a 15% urea-polyacrylamide gel was used to verify whether the circular DNA template was successfully prepared. To prepare 15% urea-PAGE, 20 μL of the above product was mixed with DNA / RNA loading buffer (2X, for denaturing PAGE), then the above mixture was added to the 15% PAGE wells and run at 120 V for 1 hour in 0.5× TBE buffer. The gel was taken out and immersed in 1× SYBR Gold nucleic acid gel stain for 15 minutes. The gel results were exposed using a Bio-rad imager as shown in Figure 3. As Figure 3 shown, the circular DNA template was successfully prepared. To purify the circular DNA template, the PAGE gel containing the circular DNA template was cut with a disposable scalpel blade, and then the prepared circular DNA template was purified according to the classical gel DNA elution and extraction procedure. Finally, the concentration of the purified circular DNA template was analyzed by NanoDrop spectrophotometer and stored at -20 °C for later use.

[0034] Example 2: Construction and optimization of the reaction system for detecting monkeypox virus To achieve the best performance of AURORA detection, it is crucial to optimize key factors, including Cas12a, crRNA, phi29 DNA polymerase, dNTP, circular DNA template, and DF probe concentration. The DF probe is a key factor for the tandem use of Cas12a and phi29 DNA polymerase. It plays two important roles in the reaction: (i) reporting fluorescence; (ii) serving as a primer to initiate self-amplification and generating a large number of amplicons after cleavage.

[0035] Based on the above, in this example, each condition in the AURORA detection system was optimized to obtain the best composition of the AURORA detection system. The specific operations include: Use the composition in Table 1 to perform a one-tube self-amplification reaction on the positive plasmid standard constructed in Example 1 to determine the best reaction system and conditions. Among them, the temperature used during the optimization process was 37 °C, and the time was 2 - 10 min. The reaction system composition was: circular DNA template (10 - 100 nM), 2 U phi29 DNA polymerase (0.00625 - 0.1 U / μL), 2 μL 10× reaction buffer, Cas12a (5 - 75 nM), dNTP (50 - 1000 μM), 1 μM crRNA, DF probe (50 - 750 nM), and RNase inhibitor. During the reaction, fluorescence emission measurements were recorded every minute at 635 nm using ABI-7500. The results are shown in Figure 4 。

[0036] As Figure 4 shown, as shown in Figure 4A, the detection performance of the DF probe is best at a concentration of 100 nM. As the probe concentration increases, although the positive signal increases significantly, the signal-to-noise ratio (S / N) decreases due to non-specific reactions caused by excessive probes. This may be related to the 3'-5' exonuclease activity of phi29 DNA polymerase. Excessive probes increase the probability of collision with the circular DNA template, which may trigger the correction of phi29 DNA polymerase and result in non-specific amplification. Therefore, the present invention selected a DF probe concentration of 100 nM for subsequent experiments. The non-specific cleavage of Cas12a on the circular DNA template greatly reduces the effect of self-amplification. Then, the present invention optimized the concentration of Cas12a / crRNA. As Figure 4As shown in B, the S / N increased significantly with the increase in RNP concentration. When the RNP concentration was higher than 50 nM, the S / N did not continue to increase. This is because excessive Cas12a / crRNA would not only rapidly cleave the probe and amplification products but also extensively cleave the circular DNA template, thereby reducing the amplification efficiency. Therefore, a Cas12a RNP concentration of 50 nM was selected for subsequent experiments. AURORA relies on the synergistic effect of Cas12a and phi29 DNA polymerase. As shown in Figure 4C, with the increase in phi29 DNA polymerase, the RCA amplification efficiency gradually increased, and the best detection performance was achieved at a phi29 DNA polymerase concentration of 0.025 U / μL. However, excessive phi29 DNA polymerase significantly increased the non-specificity of the reaction. This may be because excessive phi29 DNA polymerase has excessive proofreading activity, which, through self-amplification cycles, causes the rapid occurrence of non-specific amplification. To reduce the impact of non-specific amplification, a phi29 DNA polymerase concentration of 0.025 U / μL was selected for subsequent experiments. In addition, dNTP is an important component of RCA. According to Figure 4 D, when the dNTP concentration was 250 μM, the AURORA effect was the best. If there was too much dNTP, the reaction would be inhibited. In addition, the circular DNA template, as the amplification template for self-amplification, plays an important role in the amplification efficiency. As Figure 4 shown in E. When the concentration of the circular DNA template was 50 nM, it exhibited high detection performance. Excessive circular DNA templates led to a significant increase in RCA efficiency, generating a large amount of RCA products that competed with the DF probe for non-specific cleavage by Cas12a. This may have led to a decline in detection performance. Finally, the reaction time of AURORA was optimized, and the best detection performance was achieved at 8 min ( Figure 4 F).

[0037] Therefore, the optimal reaction system for the AURORA system to detect MPXV was finally determined as follows: 50 nM Cas12a, 0.025 U / μL phi29 DNA polymerase, 250 μM dNTP, 50 nM circular DNA template, and 100 nM DF probe. The reaction conditions were: 37 °C, 8 min.

[0038] The complete 20 μL reaction system for AURORA detection is shown in Table 2: Table 2

[0039] Note: RNase inhibitor (NEB Catalog: M0314L).

[0040] Example 3: Sensitivity and Specificity of the AURORA Detection System The analytical performance of AURORA was evaluated by detecting the optimal conditions for serial dilution of the MPXV positive standard plasmid (as the test sample) using the optimal AURORA detection reaction system and reaction conditions determined in Table 2 of Example 2. The results are shown in Figure 5 . As Figure 5 shown in A, as the concentration of the MPXV positive standard plasmid decreased, the fluorescence intensity also decreased simultaneously. The fluorescence intensity was logarithmically and linearly correlated with the DNA concentration from 1 fM to 100 pM (R 2 = 0.9452). The linear regression equation was Y = 3.458*X - 4.912 ( Figure 5 B). Using the amplitude signal of the control plus three times the standard deviation, the lowest detection limit LOD was calculated to be 87.68 aM (52.8 copies / μL). In addition, after 20 repeated experiments, the standard deviation of the AURORA detection reaction system was as low as 3%, indicating its excellent stability and reliability ( Figure 5 C).

[0041] To evaluate the specificity of the optimal AURORA detection reaction system determined in Table 2 of Example 2, this example also extended the detection of some non-target DNA, including VAVR (smallpox virus), VACV (vaccinia virus), and CPXV (cowpox virus) as test samples for AURORA detection. The results are shown in Figure 5 . As Figure 5 shown in D, significant fluorescence signals could only be generated when the test sample was MPXV, while the signals of the three non-target DNA samples (VAVR, VACV, and CPXV) were consistent with the signals of the negative control. The above results indicate that AURORA exhibits high detection specificity for DNA targets with different sequences.

[0042] Example 5: Detection of MPXV in Samples by AURORA To investigate the practicality and reliability of AURORA for analyzing real clinical samples, MPXV was tested in 36 clinical skin swabs in this example. The collection and processing of clinical samples were carried out strictly in accordance with the MPXV standard procedures recommended by the World Health Organization. First, MPXV was detected in the samples using qPCR, and samples with Ct values greater than 35 and less than 35 were defined as negative and positive, respectively. At the same time, the optimal AURORA detection reaction system and reaction conditions determined in Table 2 of Example 2 were used to detect MPXV in clinical samples. The results are shown in Figure 6 . As Figure 6As shown in A, in terms of detection time, the qPCR assay based on virus lysis takes nearly 1.5 h, while the AURORA detection system of the present invention only takes 10 min, which is significantly superior to qPCR in terms of detection time. In addition, for all clinical MPXV samples, the detection results of AURORA are 100% consistent with those of qPCR ( Figure 6 B-D). This means that the AURORA detection system and reaction conditions determined by the present invention accurately identify MPXV in the sample with 100% sensitivity and 100% specificity. As Figure 6 shown in E, there is a strong correlation between the AURORA detection system of the present invention and the gold standard qPCR detection of MPXV. The above research shows that the viral load in the skin swabs of monkeypox patients is greater than 172 copies / μL, which is higher than the detection limit of AURORA. Therefore, the AURORA detection of the present invention has more excellent performance such as sensitivity and specificity in the detection of clinical samples.

[0043] Example 6: Comparison with stepwise detection In this example, the effects of stepwise methods (only Cas12a detection, two-step method, three-step method) and the AURORA detection system determined in the above example for detecting MPXV were further compared. The operation procedures of the stepwise methods are shown in Figure 7 A, and the specific operation process is as follows: (1) The 20 μL reaction system for only Cas12a determination includes: 2 μL of 10× reaction buffer, 1 μL of Cas12a (1 μM), 1 μL of crRNA (1 μM), 1 μL of DF probe (10 μM), 1 μL of RNase inhibitor (40 U / μL), 5 μL of positive standard, and 6.8 μL of DNPC-treated water. React the above system at 37°C for 1 hour, and inactivate it at 65°C for 10 minutes after the reaction. Use ABI-7500 (Thermo, USA) to record the fluorescence emission measurement values once per minute at 635 nm. The results are shown in Figure 7 B.

[0044] (2) In the two-step method, mix the above reaction product with 1 μL of circular DNA template (1 μM), 0.2 μL of phi29 DNA polymerase (10 U / μL), 1 μL of dNTP (10 mM), 0.55 μL of 10× reaction buffer, and 1.25 μL of 20× SYBRGreen II, and prepare a 25 μL reaction solution with DEPC-treated water. React at 37°C for 1 hour. Inactivate it at 65°C for 10 minutes after the reaction. And use ABI-7500 to record the fluorescence emission measurement values once per minute at 518 nm. The results are shown in Figure 7 C.

[0045] (3) In the three-step method, 1 μL of Cas12a (1 μM), 1 μL of crRNA (1 μM), 1 μL of DF probe (10 μM), 1 μL of RNase inhibitor (40 U / μL), 0.5 μL of 10× reaction buffer, and 0.5 μL of DNPC-treated water were added to the reaction product of the above two-step method to form a 30 μL reaction system. The reaction was carried out at 37 °C for 1 hour. After the reaction, it was inactivated at 65 °C for 10 minutes. Fluorescence emission measurements were recorded every minute at 635 nm using ABI-7500. The results are shown in Figure 7 D.

[0046] As Figure 7 shown in B-D, only Cas12a detection and the two-step method showed consistent performance with a limit of detection (LOD) of 10 pM. However, due to the tandem combination of CRISPR and RCA, the three-step method improved the detection sensitivity to reach an LOD of 1 pM. It is worth noting that the lowest detection limit of the AURORA detection system of the present invention is 87.68 aM (52.8 copies / μL), which is significantly lower than the above-mentioned Cas12a detection, two-step method, or three-step method detection limits. The above results indicate that the AURORA detection system of the present invention not only reduces the operation steps and contamination risks, but also shortens the turnaround time and improves the reaction sensitivity.

[0047] As mentioned above, it is only a preferred embodiment of the present invention and does not impose any formal limitations on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or equivalent variations within the scope of the technical solution of the present invention by using the disclosed methods and technical contents. However, any simple modifications, equivalent variations, and decorations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A one-tube self-amplification system for detecting monkeypox virus, characterized in that, The one-tube self-amplification system described above includes a DF probe and a crRNA for detecting the DNA of the monkeypox virus B6R gene; the nucleotide sequence of the DF probe is as shown in SEQ ID NO: 3, and the nucleotide sequence of the crRNA is as shown in SEQ ID NO:

4.

2. The one-tube self-amplification system for detecting monkeypox virus according to claim 1, wherein The 5' end of the DF probe is modified with a fluorophore, and the 3' end is modified with a quencher.

3. The one-tube self-amplification system for detecting monkeypox virus according to claim 1, wherein, The fluorophore is selected from CY5, HEX, FAM or ROX; the quencher is selected from TAMRA, BHQ2 or BHQ1.

4. The one-tube self-amplification system for detecting monkeypox virus according to claim 1, characterized in that, The one-tube self-amplification system described above further includes a CRISPR / Cas12a protein, a DNA polymerase, dNTPs, an RNase inhibitor and a reaction buffer.

5. The one-tube self-amplification system for detecting monkeypox virus according to claim 1, wherein The Cas12a protein is LbCas12a; The DNA polymerase is phi29 DNA polymerase.

6. A kit for detecting monkeypox virus, characterized in that, The kit contains the one-tube self-amplification system according to any one of claims 1-5.

7. A detection method for monkeypox virus, characterized in that, The method includes the following steps: (1) Extract the DNA of the monkeypox virus sample to be detected; (2) Use the one-tube self-amplification system according to claims 1-5 or the kit according to claim 6 to amplify the DNA sample including the DNA extracted in step (1), and qualitatively judge and / or quantitatively detect the DNA situation in the sample to be detected according to the amplification result.

8. The detection method according to claim 7, characterized in that, In step (1), DNA is extracted from the monkeypox virus sample to be detected by a thermal lysis method.

9. The detection method according to claim 7, wherein In step (2), the reaction system for amplification includes the following components with a total volume of 20 μL: 1 μL of 1 μM circular DNA template, 0.5 μL of 1 U / μL phi29 DNA polymerase, 2 μL of 10× reaction buffer, 1 μL of 1 μM Cas12a, 0.5 μL of 10 mM dNTPs, 1 μL of 1 μM crRNA, 2 μL of 1 μM DF probe, 1 μL of 40 U / μL RNase inhibitor, 5 μL of the test sample and 6 μL of DNPC-treated water.

10. The detection method according to claim 7, characterized in that, In step (2), the reaction program for amplification is: incubate at 37°C for 8 minutes.

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