Detection kit for monkey pox virus branch I based on RPA and CRISPR / Cas12a and application of detection kit
By combining RPA and CRISPR/Cas12a systems, specific primers and crRNA were designed to achieve rapid, sensitive and specific detection of monkeypox virus branch I, solving the problems of complex detection and cross-contamination in existing technologies. This method is suitable for disease control sites or primary laboratories.
Patent Information
- Application Number
- CN202511261946.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-05
- Publication Date
- 2025-11-14
AI Technical Summary
Existing technologies are insufficient for rapid, sensitive, and specific on-site detection of monkeypox virus branch I. Traditional methods are complex and pose a risk of cross-contamination.
By combining recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system, specific primers and crRNA were designed to achieve isothermal amplification and visual detection of monkeypox virus branch I, avoiding cross-contamination.
This method enables rapid, sensitive, and specific detection of monkeypox virus branch I without the need for sophisticated equipment, avoids aerosol contamination, and is suitable for disease control sites or primary laboratories.
Smart Images
Figure CN120945131A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a detection kit for monkeypox virus branch I based on RPA and CRISPR / Cas12a and its application. Background Technology
[0002] Monkeypox virus (MPXV) belongs to the family Poxviridae, genus Orthopoxvirus, and is a double-stranded DNA virus. It includes branch I and branch II. MPXV branch I has stronger virulence and transmissibility. Laboratory detection methods for MPXV include: etiological detection, serological detection, immunofluorescence staining, and nucleic acid detection. Virus isolation and culture is the gold standard for virological detection; this method has high specificity but is cumbersome and time-consuming. Serological detection methods include enzyme-linked immunosorbent assay (ELISA). While immunofluorescence staining is feasible, it poses a high risk of infection to operators.
[0003] Currently, the most widely used laboratory diagnostic method is nucleic acid testing. However, traditional nucleic acid testing methods, such as PCR, are complex to operate, require professional personnel and specialized equipment, and have a long testing time, making them unsuitable for rapid on-site testing. Isothermal amplification methods, such as recombinase polymerase amplification (RPA), are used in conjunction with the CRISPR / Cas system for diagnosis. However, opening the amplification reaction tube may cause cross-contamination, and placing the amplification reagents and the CRISPR / Cas system in the same system will reduce amplification efficiency and affect detection sensitivity.
[0004] Therefore, those skilled in the art are eager to develop an efficient, specific, and rapid on-site detection method for MPXV branch I, in order to achieve rapid detection of MPXV branch I at the disease site or in grassroots laboratories. Summary of the Invention
[0005] The purpose of this invention is to achieve rapid detection of MPXV branch I.
[0006] This invention provides a detection kit for monkeypox virus branch I based on RPA and CRISPR / Cas12a. The detection kit includes a CRISPR / Cas12a reaction system and an RPA reaction system. The CRISPR / Cas12a reaction system includes crRNA, LbCas12a, and ssRNA probes. The RPA reaction system includes DNA of the sample to be tested, the forward primer shown in SEQ ID NO.5, and the reverse primer shown in SEQ ID NO.6.
[0007] Further specifying, the sequences amplified by the forward and reverse primers are shown in SEQ ID NO.9.
[0008] Further specifying, the sequence of crRNA is shown in SEQ ID NO.8.
[0009] Further specifying, the sequence of the ssRNA probe is shown as FAM-TTATT-BHQ1.
[0010] Furthermore, the volume ratio of the CRISPR / Cas12a reaction system to the RPA reaction system is 1:1.
[0011] Further defining the RPA reaction system, the following is provided: 5 μL of DNA sample to be tested, 2.4 μL each of 10 μM forward and reverse primers, 29.5 μL of rehydration buffer, 8.2 μL of RNase-free ddH2O, mixed well, and then 2.5 μL of 280 mM magnesium acetate solution is added.
[0012] Further specifying the reaction system, the CRISPR / Cas12a reaction system is as follows: 6.25 μL of 1.5 μM LbCas12a, 0.625 μL of 2 μM crRNA, 5 μL of 10 μM ssRNA probe, 5 μL of 10× Buffer, and ddH2O to a final volume of 20 μL.
[0013] The present invention provides a detection system for monkeypox virus branch I, the system comprising the above-described detection kit and visualization detection system.
[0014] This invention provides a method using the above-described detection system, characterized by the following specific steps: Step 1: Configure the RPA reaction system and the CRISPR / Cas12a reaction system; Step 2: The volume ratio of the RPA reaction system to the CRISPR / Cas12a reaction system is 1:1; Step 3: Perform RPA amplification reaction at 39℃ for 20 min, centrifuge, and then react with CRISPR / Cas12a at 37℃ for 20 min to obtain the reactants. Use a blue light spectrometer to visualize and detect the reactants. If green fluorescence is observed under the blue light spectrometer, it indicates that the sample contains monkeypox virus branch I; if no fluorescence is observed under the blue light spectrometer, it indicates that the sample does not contain monkeypox virus branch I.
[0015] The present invention provides the application of the above-described reagent kit or detection system in the diagnostic detection of monkeypox virus branch I.
[0016] Beneficial Effects: This invention provides a system, kit, and application for monkeypox virus branch I, including an RPA forward primer and an RPA reverse primer. The nucleotide sequence of the RPA forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the RPA reverse primer is shown in SEQ ID NO.2. It also includes crRNA designed based on monkeypox virus branch I nucleic acid; the crRNA nucleotide sequence is shown in SEQ ID NO.3. This invention further provides a detection kit containing the above primer pairs and crRNA.
[0017] 1) This invention involved gene sequence alignment of nine MPXV branch I strains, screening to identify nucleotide sequences from positions 48456 to 48701 in GenBank: NC_003310.1 as target sequences to ensure the specificity of primers and crRNA. Primers suitable for RPA isothermal amplification and crRNA suitable for CRISPR / Cas12a reactions were designed targeting the target sequences of MPXV branch I.
[0018] 2) The kit for detecting monkeypox virus branch I provided by this invention: First, the kit provided by this invention combines RPA technology with the CRISPR / Cas12a system, eliminating the need for sophisticated temperature-controlled equipment. Effective amplification of the MPXV branch I target gene can be achieved under constant temperature conditions. The detection of MPXV branch I is achieved by specifically recognizing the crRNA of MPXV branch I within the detection system. The entire process is carried out in fully enclosed centrifuge tubes, which not only visualizes the results but also avoids false positive results caused by aerosols, enabling rapid and visual detection at the disease site.
[0019] 3) The kit provided by this invention has a detection sensitivity of 4.37 × 10⁻⁶ samples. 1 Copies / μL of MPXV branch I recombinant plasmid. It is evident that the detection kit provided by this invention has high sensitivity for the detection of monkeypox virus branch I.
[0020] In summary, the detection method provided by this invention does not require specialized instruments and equipment or laboratory personnel, and can achieve rapid detection of MPXV branch I at the epidemic site or in grassroots laboratories. Attached Figure Description
[0021] Figure 1 This is a graph showing the screening results of RPA reaction primer pairs; Figure 2 The image shows the results of crRNA screening in the CRISPR / Cas12a reaction; Figure 3 Figure showing the results of optimizing Cas12a protein concentration in the CRISPR / Cas12a reaction; Figure 4Figure showing the results of crRNA concentration optimization in the CRISPR / Cas12a reaction; Figure 5 The figure shows the results of probe concentration optimization in the CRISPR / Cas12a reaction; Figure 6 Figure showing the results of RPA reaction temperature optimization; Figure 7 The graph shows the sensitivity test results of the reagent kit. Figure 8 This is a graph showing the specific detection results of the kit; Figure 9 This is a graph showing the simulated clinical sample test results of the reagent kit. Detailed Implementation
[0022] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.
[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.
[0024] The monkeypox virus (MPXV) branch I recombinant plasmid used in the following examples was synthesized by General Biotech (Anhui) Co., Ltd.; the RPA kit was purchased from TwistDx (UK); and the nucleic acid extraction kit was purchased from Takara Bio Engineering (Dalian) Co., Ltd.
[0025] Example 1: Preparation of monkeypox virus branch I primer pairs and crRNA 1. Preparation of primers for detecting monkeypox virus branch I In this embodiment, the SnapGene software was used to align the gene sequences of nine MPXV branch I strains isolated from different countries, regions, and times, including existing variants, published in the GeneBank database. The strain information is shown in Table 1. Based on the sequence alignment results, the nucleotide sequence at positions 48456-48701 of GenBank: NC_003310.1 was selected as the final detection target, as shown in Table 2.
[0026] RPA primers were designed for the target sequences of MPXV branch I, as shown in Table 3.
[0027] Table 1
[0028] Table 2
[0029] Table 3
[0030] 2. Screening of RPA reaction primer pairs The RPA primers prepared in this embodiment (nucleotide sequences shown in SEQ ID NO. 1-6) were screened, and an RPA amplification system was prepared on ice (as shown in Table 4) with a concentration of 4.37 × 10⁻⁶. 2 copies / μl up to 4.37×10 4 A recombinant plasmid of MPXV branch I, at a ratio of copies / μl, was used as the target for RPA reaction. A negative control group was set up, using ddH2O instead of the target. Amplification was performed for 20 min in each case. The RPA products were analyzed by agarose gel electrophoresis to determine the amplification efficiency of different primer pairs.
[0031] The results are as follows Figure 1 As shown in section A, the amplification efficiency of primer pairs F3 and R3 is superior to that of other primer pairs. Therefore, primers F3 and R3 were selected for the amplification of monkeypox virus branch I.
[0032] Table 4
[0033] 3. Design of crRNA in the CRISPR / Cas12a reaction system: (1) Based on the screening of target sequences containing PAM sites in the conserved sequences of MPXV branch I, two CRISPR / Cas12a crRNAs (nucleotide sequences shown in SEQ ID NO. 7-8) specifically recognizing MPXV branch I were designed according to the design principle of Cas12acrRNA. The specific sequence information is shown in Table 5. The crRNAs were obtained by in vitro transcription.
[0034] Table 5
[0035] (2) Screening of crRNA in the CRISPR / Cas12a reaction system: In this embodiment, the optimal crRNA was screened in a 20 µL CRISPR / Cas12a reaction system. The CRISPR / Cas12a amplification system was prepared on ice (as shown in Table 6). The concentration was 4.37 × 10⁻⁶. 9 Using MPXV branch I recombinant plasmid (copies / μl) as the target, the two crRNAs mentioned above were added to the system, with each crRNA having 3 replicates. ddH2O was used as a negative control. The reaction system was placed in a real-time PCR instrument and incubated at 37°C for 40 min. Fluorescence signals were collected every 1 min. By comparing the fluorescence reaction time and fluorescence signal intensity, the optimal crRNA in the CRISPR / Cas12a system was selected.
[0036] Table 6
[0037] The results are as follows Figure 2 As shown, crRNA-2 can generate a strong fluorescent signal in the CRISPR / Cas12a reaction system. Therefore, crRNA2 was selected for the CRISPR / Cas12a reaction system for detecting monkeypox virus branch I.
[0038] Example 2: Preparation of a kit for detecting monkeypox virus branch I The kit described in this embodiment includes an RPA primer pair and crRNA; the primer pair includes an RPA forward primer and an RPA reverse primer, the nucleotide sequence of the RPA forward primer is shown in SEQ ID NO.5, the nucleotide sequence of the RPA reverse primer is shown in SEQ ID NO.6, and the nucleotide sequence of the crRNA is shown in SEQ ID NO.8.
[0039] The RPA amplification system in the kit is as follows: 5 μL of the nucleic acid sample to be tested, 2.4 μL each of 10 μM forward and reverse primers, 29.5 μL of rehydration buffer, 8.2 μL of RNase-free ddH2O, mixed well, and then 2.5 μL of 280 mM magnesium acetate solution is added. The CRISPR / Cas12a reaction system in the kit is as follows: 6.25 μL of 1.5 μM LbCas12a, 0.625 μL of 2 μM crRNA, 5 μL of 10 μM ssRNA reporter molecule, 5 μL of 10× Buffer, and ddH2O to bring the total to 20 μL. The sequence of the ssRNA reporter molecule is FAM-TTATT-BHQ1.
[0040] Example 3: Application of a kit for detecting monkeypox virus branch I in the visual detection of monkeypox virus branch I 1. The detection of monkeypox virus branch I using the kit prepared in Example 2 includes the following steps: Take 45 µl of the premixed RPA amplification system solution and transfer it to the bottom of a new centrifuge tube. Add 5 µL of the target substance to be tested for the RPA amplification reaction. Prepare the CRISPR / Cas12a reaction system and place it at the bottom of the tube. Then, invert the tube into the centrifuge tube containing the RPA reaction system, seal the centrifuge tube, and place it in a thermal cycler or water bath at 39°C for the RPA amplification reaction. After centrifugation, perform the CRISPR / Cas12a reaction to obtain the reactants. A closed reaction tube can avoid aerosol contamination. Use a blue light spectrometer to visualize the above reactants and judge the results. All detection reactions are repeated 3 times. The RPA reaction temperature is 39°C and the time is 20 min. The CRISPR / Cas12a reaction temperature is 37°C and the time is 20 min.
[0041] The criteria for judging the results of the visualization test are as follows: if green fluorescence is observed under a blue light meter using a mobile phone or the naked eye, it indicates that the sample contains monkeypox virus branch I; if no fluorescence is observed under a blue light meter, it indicates that the sample does not contain monkeypox virus branch I.
[0042] 2. Optimization of the CRISPR / Cas12a reaction system (1) Optimization of Cas12a protein concentration in CRISPR / Cas12a reaction system: In this embodiment, to determine the optimal Cas12a protein concentration, a concentration of 4.37 × 10⁻⁶ was used. 9 Using MPXV branch I recombinant plasmid (copies / μL) as the target, Cas12a protein concentrations of 75 nM, 112.5 nM, 150 nM, 187.5 nM, and 225 nM were set. A negative control group (NTC) was also set up for each Cas12a protein concentration, using ddH2O instead of the target. A total of 10 groups were validated, with 3 replicates for each condition. Then, 0.25 μL of 2 μM crRNA, 2 μL of 10 μM ssRNA reporter, and 2 μL of 10× Buffer were added to the system, and ddH2O was added to bring the total to 20 μL. The reaction system was placed in a real-time PCR instrument and incubated at 37°C for 40 min, with fluorescence signals collected every 1 min. The optimal Cas12a protein concentration was determined by comparing the fluorescence signal intensity.
[0043] The results are as follows Figure 3As shown, the fluorescence signal intensity was highest when the Cas12a protein concentration in the CRISPR / Cas12a system was 187.5 nM. Therefore, a Cas12a protein concentration of 187.5 nM is optimal for the CRISPR / Cas12a reaction system.
[0044] (2) Optimization of crRNA concentration in the CRISPR / Cas12a reaction system: In this embodiment, to determine the optimal crRNA concentration, 4.37 × 10⁻⁶ was used. 9 Using MPXV branch I recombinant plasmid (copies / μL) as the target, crRNA concentrations of 25 nM, 50 nM, 100 nM, and 200 nM were set. A negative control group (NTC) was also set up for each crRNA concentration, using ddH2O instead of the target. A total of 8 groups were validated, with 3 replicates for each condition. Then, 2.5 μL of 1.5 μM LbCas12a, 2 μL of 10 μM ssRNA reporter, 2 μL of 10× Buffer, and ddH2O were added to the system, bringing the total to 20 μL. The reaction system was placed in a real-time PCR instrument and incubated at 37°C for 40 min, with fluorescence signals collected every 1 min. The optimal crRNA concentration was determined by comparing the fluorescence signal intensity.
[0045] The results are as follows Figure 4 As shown, the highest fluorescence signal intensity was observed in the CRISPR / Cas12a system when the crRNA concentration was 25 nM. Therefore, a crRNA concentration of 25 nM is optimal for the CRISPR / Cas12a reaction system.
[0046] (3) Optimization of probe concentration in CRISPR / Cas12a reaction system In this embodiment, to determine the optimal probe concentration, 4.37 × 10⁻⁶ was used. 9 Using MPXV branch I recombinant plasmid (copies / μL) as the target, probe concentrations of 0.5 μM, 0.75 μM, 1 μM, 1.25 μM, and 1.5 μM were set up. A negative control group was also set up for each probe concentration, using ddH2O instead of the target. A total of 10 groups were validated, with 3 replicates for each condition. Then, 2.5 μL of 1.5 μM LbCas12a, 0.25 μL of 2 μM crRNA, 2 μL of 10× Buffer, and ddH2O were added to the system, bringing the total to 20 μL. The reaction system was placed in a real-time PCR instrument and incubated at 37°C for 40 min, with fluorescence signals collected every 1 min. The optimal probe concentration was screened by comparing the fluorescence signal intensity.
[0047] The results are as follows Figure 5 As shown, the fluorescence signal is better with increasing probe concentration, and the negative background value is within an acceptable range. Considering both effectiveness and cost, a 1 μM probe concentration is the optimal choice for the CRISPR / Cas12a reaction system.
[0048] 3. Optimization of RPA amplification reaction Optimization of RPA amplification reaction procedure: Based on the above 45 μl RPA reaction system, take 20 μl of the solution and transfer it to another test tube, then add 5 μl of 4.37 × 10⁻⁶ solution. 0 -4.37×10 4 MPXV branched I recombinant plasmids (copies / μl) were amplified for 20 min at different temperatures (42℃, 39℃, 37℃). The amplified template was then mixed with the prepared CRISPR / Cas12a system and reacted with CRISPR / Cas12a at 37℃ for 20 min.
[0049] The results are as follows Figure 6 As shown, the amplification efficiency at 39°C was significantly better than that at 37°C and 42°C, indicating that 39°C is the optimal temperature for RPA amplification.
[0050] Effect Experiment: In this embodiment, to evaluate the detection efficacy of the kit prepared in Example 2 for monkeypox virus branch I, sensitivity testing, specificity testing, and simulated clinical sample evaluation were performed.
[0051] 1. Sensitivity detection To evaluate the sensitivity of the above detection method, the MPXV branched I recombinant plasmid was serially diluted tenfold (4.37 × 10⁻⁶). 0 -4.37×10 4 The sensitivity of the detection method was evaluated using MPXV branched I recombinant plasmids at different concentrations as targets. The RPA reaction was carried out at 39℃ for 20 min, followed by brief centrifugation and then a CRISPR / Cas12a reaction at 37℃ for 20 min. After the reaction, the reaction tubes were placed under a blue light analyzer, and the results were observed visually or via mobile phone. Images were also captured using a smartphone to collect and analyze the pixel intensity of the green and red light channels.
[0052] The results are as follows Figure 7 As shown, the sensitivity can reach 4.37 × 10⁻⁶. 1 The copies / μL indicates that the kit provided by this invention has high sensitivity for the detection of monkeypox virus branch I.
[0053] 2. Specific detection To evaluate the specificity of the above detection method, nucleic acids of pathogens associated with monkeypox virus branch I were detected, including MPXV branch II, BV, CPXV, VVT, and VZV. These pathogen nucleic acids were placed in the kit reaction system, with a negative control group using ddH2O instead of the target. The RPA reaction was performed at 39°C for 20 min, followed by brief centrifugation and a CRISPR / Cas12a reaction at 37°C for 20 min. After the reaction, the reaction tubes were placed under a blue light analyzer, and the results were observed visually or via mobile phone. Images were also captured using a smartphone to collect and analyze the pixel intensity of the green and red light channels.
[0054] The results are as follows Figure 8 As shown, MPXV branch I group produced green fluorescence, while MPXV branch II, BV, CPXV, VVT, and VZV experimental groups and the negative control did not produce fluorescence signals. Therefore, the kit provided by this invention has a certain specificity for the detection of MPXV branch I.
[0055] 3. Simulated clinical sample evaluation Total DNA was extracted from African green monkey kidney cells (Vero cells) and oropharyngeal swabs from healthy humans, respectively. Subsequently, different concentrations of the pcDNA3.1-MPXV-I recombinant plasmid were mixed into the total DNA. Of the 32 simulated samples prepared, 14 were Vero cell DNA samples with the added recombinant plasmid, 14 were healthy human oropharyngeal swab DNA samples with the added recombinant plasmid, 2 were normal Vero cell DNA samples, and 2 were healthy human oropharyngeal swab DNA samples. All samples were evaluated using the kit provided in this invention and the qPCR detection method.
[0056] The results are as follows Figure 9 As shown, the positive detection rate of the kit provided by this invention for both types of samples was 71.43% (10 / 14), while the detection rate of the qPCR method for Vero cell samples was 57.14% (8 / 14), and the detection rate for human oropharyngeal swab samples was 71.43% (10 / 14). This indicates that the kit for detecting monkeypox virus branch I provided by this invention is feasible in simulated clinical screening and identification of suspected MPXV branch I positive samples, and can meet the needs of simulated clinical testing.
[0057] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A detection kit for monkeypox virus branch I based on RPA and CRISPR / Cas12a, characterized in that, The detection kit includes a CRISPR / Cas12a reaction system and an RPA reaction system; the CRISPR / Cas12a reaction system includes crRNA, LbCas12a and ssRNA probes; the RPA reaction system includes the DNA of the sample to be tested, the forward primer shown in SEQ ID NO.5 and the reverse primer shown in SEQ ID NO.
6.
2. The detection kit according to claim 1, characterized in that, The sequences amplified by the forward and reverse primers are shown in SEQ ID NO.
9.
3. The detection kit according to claim 1, characterized in that, The sequence of crRNA is shown in SEQ ID NO.
8.
4. The detection kit according to claim 1, characterized in that, The sequence of the ssRNA probe is shown in FAM-TTATT-BHQ1.
5. The detection kit according to claim 1, characterized in that, The volume ratio of the CRISPR / Cas12a reaction system to the RPA reaction system is 1:
1.
6. The detection kit according to claim 1, characterized in that, The RPA reaction system consisted of 5 μL of the DNA sample to be tested, 2.4 μL each of 10 μM forward and reverse primers, 29.5 μL of rehydration buffer, and 8.2 μL of RNase-free ddH2O. After mixing, 2.5 μL of 280 mM magnesium acetate solution was added.
7. The detection kit according to claim 1, characterized in that, The CRISPR / Cas12a reaction system consisted of: 1.5 μL MbCas12a (6.25 μL), 2 μM crRNA (0.625 μL), 10 μM ssRNA probe (5 μL), 10× Buffer (5 μL), and ddH2O added to a final volume of 20 μL.
8. A detection system for monkeypox virus branch I, characterized in that, The system includes the detection kit and visualization detection system as described in any one of claims 1-7.
9. A method using the detection system of claim 8, characterized in that, The specific steps are as follows: Step 1: Configure the RPA reaction system and the CRISPR / Cas12a reaction system; Step 2: The volume ratio of the RPA reaction system to the CRISPR / Cas12a reaction system is 1:1; Step 3: RPA amplification reaction was performed at 39℃ for 20 min. After centrifugation, CRISPR / Cas12a reaction was performed at 37℃ for 20 min to obtain the reactants. The reactants were visualized and detected using a blue light spectrometer. If green fluorescence was observed under the blue light spectrometer, it indicated that the sample contained monkeypox virus branch I. If no fluorescence is observed under a blue light spectrometer, it indicates that the sample does not contain monkeypox virus branch I.
10. The use of the kit according to any one of claims 1-7 or the detection system according to claim 8 in the diagnostic detection of monkeypox virus branch I.