RPA-CRISPR technology-based multi-channel detection system for monkey pox virus and application

Abstract

The invention discloses a reagent for specifically detecting a monkey pox virus. The reagent comprises an RPA (recombinase polymerase amplification) primer pair aiming at an N3R gene of the monkey pox virus, N3R-crRNA (ribonucleic acid) and N3R-ssRNA reporter molecules, the primer pair comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.4; the N3R-crRNA comprises an anchoring sequence and a guide sequence, wherein the anchoring sequence is used for being combined with Cas13a protein; the guide sequence is used for targeting a monkey pox virus N3R gene target sequence; the N3R-crRNA comprises a nucleotide sequence as shown in SEQ ID NO.7; the N3R-ssRNA reporter molecule comprises a nucleotide sequence as shown in SEQ ID NO. 11. The N3R-ssRNA reporter molecule has a nucleotide sequence as shown in SEQ

Description

Technical Field

[0001] The present invention belongs to the technical field of biological reagents, and specifically relates to a monkeypox virus multi-channel detection system and application based on RPA-CRISPR technology. Background Art

[0002] Monkeypox is a viral zoonosis caused by monkeypox virus (MPXV), with clinical manifestations mainly including fever, rash, and swollen lymph nodes. According to the latest classification of the International Committee on Taxonomy of Viruses (ICTV), monkeypox virus (MPXV) belongs to the genus Orthopoxvirus in the family Pox viridae, subfamily Chordopoxvirinae, order Chitovirales. There are 12 species of viruses in the genus Orthopoxvirus, including Vaccinia virus (VACV), Variola virus (VARV), Cowpox virus (CPXV), Monkey poxvirus (MPXV) and camelpox virus, which can cause infectious diseases in humans, as well as Ectromeliavirus (ECTV) which is only transmitted among animals, as well as raccoonpox virus, skunkpox virus, taterapoxvirus, Volepox virus, Abatinomacacapox virus and Akhmeta virus.

[0003] Since the eradication of smallpox virus in the 1980s, monkeypox virus has become one of the members of the Poxviridae family with the greatest impact on public health, mainly prevalent in central and western Africa. Since May 2022, monkeypox outbreaks have occurred in more than 100 countries and regions around the world. Epidemics in many countries have shown that monkeypox has spread from person to person and has spread widely to countries and regions outside Africa, with a case fatality rate of about 0.1%. In September 2022, my country reported its first imported case of monkeypox, and local monkeypox outbreaks began in June 2023. Currently, more than 20 provinces in the country have reported monkeypox cases, triggering new local secondary outbreaks and hidden transmission. In order to prevent and control the monkeypox virus, many diagnostic methods have emerged, including nucleic acid detection, sequencing, antibody and antigen detection, virus isolation and electron microscopy observation. Among them, the most widely used is nucleic acid detection, and the mainstream method is real-time quantitative PCR (RT-qPCR). However, although this technology is sensitive, it still requires expensive thermal cyclers, special experimental conditions and skilled technicians, which is not conducive to rapid diagnosis. In comparison, the advantages of the new isothermal amplification technology are highlighted, and it has become a promising on-site detection method.

[0004] The main isothermal amplification technologies for MPXV detection include loop-mediated isothermal amplification (LAMP), recombinase-mediated isothermal amplification (RAA), recombinase polymerase amplification (RPA), and RPA-CRISPR technology. However, the existing isothermal detection methods have various defects in the actual application and implementation process. For example, RPA / RAA technology can theoretically achieve rapid (5-30 minutes), low temperature (37-42°C), and multiple (2-4 channels) detection capabilities, but RPA / RAA itself also has the problems of low specificity and easy cross-contamination; especially because the conserved genes F3L, J1L, J2L, B2R, B6R, B7R, etc. in the 12 members of the Orthopoxvirus genus are highly conserved among species, it is very difficult to achieve specific detection of monkeypox virus or even typing detection through this segment target. The loop-mediated isothermal amplification (LAMP) technology can solve the problem of poor interspecies detection specificity (cross-contamination) of RPA / RAA to a certain extent due to its high reaction temperature (65°C) and multiple specific reaction primers (4 to 6). However, this technical method has high requirements for primer design, is prone to nonspecific amplification and contamination, and the multiple reaction system is unstable and susceptible to inhibitors and impurities. There are two different combinations of RPA-CRISPR / Cas technology, including RPA-CRISPR / Cas12a and CRISPR / Cas13a systems. Cas12 and Cas13 proteins have trans-cleavage activity. Under the guidance of crRNA, the protein specifically targets double-stranded DNA / single-stranded RNA (dsDNA / RNA) and activates its trans-cleavage activity, indiscriminately cutting nonspecific single-stranded DNA / RNA (ssDNA / ssRNA). Each targeted shearing will lead to more subsequent nonspecific targeted shearing activities, amplifying the signal, and the specifically enhanced fluorescence signal can be detected in a short time. Among them, the Cas12 system recognizes double-stranded DNA and trans-cuts to recognize single-stranded DNA; the Cas13 system recognizes RNA, and trans-cuts to recognize RNA. Therefore, the Cas12 and Cas13 systems have developed rapidly in the field of detection. Although this technical method has high sensitivity and specificity, it cannot be used in multi-channel detection due to the non-specific cutting function of the system itself.

[0005] Chinese invention patent CN116064950A discloses a crRNA target and CRISPR-Cas13a system for detecting monkeypox virus. The patent combines the RPA reaction system and the CRISPR-Cas13a reaction system. By adding the RPA amplification product to the CRISPR-Cas13a system for cutting, the highly sensitive and highly specific detection of monkeypox virus is achieved. However, the system provided by the patent has two defects. The first defect is that the entire system needs to be carried out in two steps, that is, the RPA reaction is carried out first, and then the product is subjected to the CRISPR-Cas13a cutting reaction. This method makes it impossible to complete it in a single reaction tube, resulting in cumbersome operation, long time, and practical application difficulties; the second defect is that the signal is based on the non-specific shearing activity of CRISPR-Cas13a, resulting in the entire system can only collect single channel fluorescence, limiting the number of fluorescence channels. Chinese invention patent CN115820939A discloses a crRNA, nucleic acid molecule composition, detection system and application for monkeypox virus detection, which combines the RPA reaction system with CRISPR-Cas12a and CRISPR-Cas13 systems to achieve dual-channel detection. The improvement of the technical solution of this patent over the Chinese invention patent CN116064950A is that it adds a CRISPR-Cas12a system and introduces a fluorescence detection under this system, which can achieve dual-channel detection, but the implementation scheme of this patent is also carried out in two steps, which cannot be completed in a single reaction tube, resulting in cumbersome operation, long time and practical application difficulties; in addition, the scheme contains a lot of enzyme proteins involved in the two reaction systems, resulting in high cost of the finished kit; in addition, the primer set sequences of the two reaction systems are long, which is difficult in the design process; more importantly, this combination can only achieve dual-channel detection at most, and cannot meet the detection needs of more channels.

[0006] Therefore, there is an urgent need to develop an economical, effective, and simple diagnostic method that can detect monkeypox virus in multiple channels to quickly screen for monkeypox virus, intervene in the early stages of transmission, and block transmission. Summary of the invention

[0007] In view of the shortcomings of the prior art and actual needs, the present invention proposes a reagent and a detection method for detecting monkeypox virus, which comprises two reaction systems, a basic RPA-CRISPR / Cas13a and a fluorescent RPA. The basic RPA-CRISPR / Cas13a reaction system is used as a separate reaction channel to detect the conserved genes of monkeypox virus, and the dual specificity of the RPA primer set and the crRNA sequence of CRISPR / Cas13a and the CRISPR / Cas13a signal amplification mechanism are utilized to achieve highly sensitive and ultra-specific detection of monkeypox virus, thereby avoiding interspecies interference. The RPA fluorescent system can be designed with two or more fluorescent channels for different targets according to actual needs, thereby achieving the purpose of low-cost multiple detection.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a reagent for specific detection of monkeypox virus, the reagent comprising an RPA amplification primer pair for the N3R gene of the monkeypox virus, N3R-crRNA, and N3R-ssRNA reporter molecules;

[0010] The primer pair includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.4; the N3R-crRNA includes an anchor sequence for binding to the Cas13a protein and a guide sequence targeting the monkeypox virus N3R gene target sequence, which includes a nucleotide sequence as shown in SEQ ID NO.7; the N3R-ssRNA reporter molecule includes a nucleotide sequence as shown in SEQ ID NO.11.

[0011] In one or more embodiments, the reagent also includes an RPA amplification primer pair and a probe for the orthopoxvirus E9L gene; the primer pair includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.13 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.14; the probe includes a nucleotide sequence as shown in SEQ ID NO.15.

[0012] In one or more embodiments, the reagent also includes an RPA amplification primer pair and a probe for the human HBB gene; the primer pair includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.17 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.18; the probe includes a nucleotide sequence as shown in SEQ ID NO.19.

[0013] In a second aspect, the present invention provides a multi-channel detection system for monkeypox virus based on RPA-CRISPR technology, which includes the reagent described in any one of the present invention, Cas13a protein and detection system reaction buffer, and the multi-channel detection system comprises three fluorescence channel combinations.

[0014] In one or more embodiments, the three fluorescence channels in the detection system are combined into CY5-ssRNA-BHQ2 (monkeypox virus N3R target), FAM-DNA-BHQ2 (orthopoxvirus E9L target), and HEX-DNA-BHQ1 (human gene HBB).

[0015] In a third aspect, the present invention provides use of any of the above reagents or detection systems in the preparation of a detection agent for detecting monkeypox virus present in a sample and / or for distinguishing monkeypox virus from other orthopoxviruses present in a sample.

[0016] In a fourth aspect, the present invention provides a method for detecting monkeypox virus or distinguishing monkeypox virus from other orthopoxviruses in a sample for non-disease diagnosis and treatment purposes, the method comprising the following steps:

[0017] (1) Extracting the genome from the sample to be tested;

[0018] (2) configuring the extracted genome and the components in the multi-channel detection system according to claim 4 or 5 into a reaction system, performing a constant temperature amplification reaction, and collecting fluorescence multiple times during the reaction;

[0019] (3) Determine the detection result based on the fluorescence signal.

[0020] In one or more embodiments, the isothermal amplification reaction in step (2) is specifically as follows: the prepared reaction system is centrifuged and mixed, placed in a multi-color fluorescence quantitative PCR instrument, incubated at 42° C. for 20 minutes, and fluorescence is collected every minute.

[0021] In one or more embodiments, the reaction system in step (2) is prepared by configuring the extracted genome with the components in the multi-channel detection system of claim 5.

[0022] In one or more embodiments, the method for determining the test result in step (3) is:

[0023] If there is no amplification signal in the HEX channel, FAM channel and CY5 channel of the detection well at the same time, it means that the experimental results are invalid and need to be re-measured;

[0024] If there is no signal in the HEX channel, but any of the other two channels has obvious amplification, it means that the result is reliable: if only the CY5 channel has obvious amplification and the FAM channel has no signal, it means that the monkeypox test result is positive; if only the FAM channel has obvious amplification and the CY5 channel has no signal, it means that the monkeypox test result is negative, and other orthopoxviruses are positive;

[0025] If there is no amplification line in the CY5 channel of the detection well and there is an amplification line in the HEX channel, it means that the monkeypox test result is negative; if there is no amplification line in the CY5 channel of the detection well, there is an amplification curve in the FAM channel, and there is an amplification line in the HEX channel, it means that the test result of orthopoxvirus other than monkeypox is positive.

[0026] The present invention realizes the detection requirements of three channels and above based on the single-tube RPA-CRISPR / Cas13a reaction system. Among them, the basic RPA-CRISPR / Cas13a reaction system is used as a separate detection channel to detect the conserved gene of monkeypox virus N3R, and the balance between the low signal produced by the CY5-ssRNA-BHQ2 probe labeling and the CRISPR / Cas13a signal amplification mechanism produces a comprehensive effect, which is convenient for the selection of more channels; at the same time, the dual specificity of the RPA primer set and the crRNA sequence of CRISPR / Cas13a is used to realize the highly sensitive and ultra-specific detection of monkeypox virus, avoiding interspecies interference. The nucleic acid molecule composition of the E9L gene is used as another separate detection channel to detect the conserved gene of the orthopoxvirus genus E9L, preferably the FAM-DNA-BHQ2 probe combination labeling, and the signal intensity generated by the fluorescent probe is relatively high and can be equal to the signal of the CY5-ssRNA-BHQ2 probe. The nucleic acid molecule composition of the human gene HBB is used as the third separate detection channel and can be used as the quality control of the detection system to determine whether the detection results are valid. The preferred probe combination is labeled HEX-DNA-BHQ1. The signal intensity generated by this fluorescent probe is relatively high and can be on par with the fluorescent signals of the plateau phase of the other two channels mentioned above.

[0027] Compared with the existing monkeypox isothermal detection system, the present invention combines the advantages of high specificity, high sensitivity, low cost, multi-channel and low cost, and can meet the clinical demand for rapid detection of monkeypox virus. In addition, the present invention can detect the presence of monkeypox virus or other members of the genus Orthopoxvirus in a short time, which is also conducive to the portable detection and promotion of monkeypox virus in customs, airports, railway stations or remote areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram of the screening results of the monkeypox N3R gene Cas13a-crRNA in Example 2.

[0029] Figure 2This is a graph showing the screening results of primer pairs for the monkeypox N3R gene in Example 3.

[0030] Figure 3 This is a graph showing the sensitivity test results of the E9L gene primer-probe combination in the RPA-CRISPR / Cas13a system in Example 5.

[0031] Figure 4 This is a graph showing the sensitivity test results of the HBB gene primer-probe combination in the RPA-CRISPR / Cas13a system in Example 7.

[0032] Figure 5 This is a graph showing the sensitivity test results of the three targets in the RPA-CRISPR / Cas13a system in Example 8. DETAILED DESCRIPTION

[0033] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0034] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0035] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0036] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0037] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0038] The invention discloses a monkeypox virus multi-channel detection system based on RPA-CRISPR technology, which comprises an RPA amplification primer pair for the monkeypox virus N3R gene, N3R-crRNA, an N3R-ssRNA reporter molecule, an RPA amplification primer pair and a probe for the orthopoxvirus E9L gene, an RPA amplification primer pair and a probe for the human HBB gene, a Cas13a protein and a detection system reaction buffer, wherein the multi-channel detection system comprises three fluorescence channel combinations.

[0039] Among them, the primer pair for the monkeypox virus N3R gene includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.4; the N3R-crRNA includes an anchor sequence for binding to the Cas13a protein and a guide sequence targeting the monkeypox virus N3R gene target sequence, which includes a nucleotide sequence as shown in SEQ ID NO.7; the N3R-ssRNA reporter molecule includes a nucleotide sequence as shown in SEQ ID NO.11.

[0040] Among them, the primer pair for the orthopoxvirus E9L gene includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.13 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.14; and the probe includes a nucleotide sequence as shown in SEQ ID NO.15.

[0041] Among them, the primer pair for the human HBB gene includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.17 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.18; and the probe includes a nucleotide sequence as shown in SEQ ID NO.19.

[0042] Among them, the three fluorescence channel combinations in the detection system are CY5-ssRNA-BHQ2 (monkeypox virus N3R target), FAM-DNA-BHQ2 (orthopoxvirus E9L target), and HEX-DNA-BHQ1 (human gene HBB).

[0043] The multi-channel detection system of the present invention can be used to detect monkeypox virus present in a sample and / or to distinguish monkeypox virus from other orthopoxviruses present in a sample.

[0044] The present invention also discloses a detection method for detecting monkeypox virus based on RPA-CRISPR technology, and the specific steps are as follows:

[0045] Step 1: Sample extraction

[0046] Take the sample to be tested and extract the genome therein; in a specific embodiment of the present invention, a plasmid synthesized by a third party is used to simulate the sample for testing.

[0047] Step 2: Amplification reaction

[0048] 1) Primer probe combination dissolution and Mix preparation: Use H 2 O (PCR Grade) dilutes the primer and probe compositions of all the nucleotide sequences above. Specifically, a 10× primer and probe composition Mix is ​​prepared for subsequent experiments, wherein the concentrations of the components in the 10× composition Mix are as follows:

[0049] A. N3R-RPA-F: 1 μM

[0050] B. N3R-RPA-R: 2 μM

[0051] C.N3R-crRNA: 250nM

[0052] D.N3R-ssRNA reporter: 20 μM

[0053] E.E9L-RPA-F: 2 μM

[0054] F.E9L-RPA-R: 2 μM

[0055] G.E9L-RPA-P: 1 μM

[0056] H.HBB-RPA-F: 2 μM

[0057] I. HBB-RPA-R: 2 μM

[0058] J.HBB-RPA-P: 1 μM

[0059] 2) Prepare the reaction system:

[0060] Components Final concentration Components Final concentration Step 2: 10×Mix 1× NTPMix (20 mM each) 2mM Cas13a protein 25nM dNTPs 200μM T7 RNA polymerase 0.2U / μL ATP 3mM T4UvsXRecombinase 240 ng / μL Potassium acetate 100mM T4UvsYProtein 60ng / μL RNase inhibitors 0.5U / μL T4gene32protein 500ng / μL Creatine Phosphate 50mM BsuDNAPolymerase 0.25 U / μL Magnesium acetate 20mM ExonucleaseⅢ 2U / μL Polyethylene glycol 20000 5%(V / V) Creatine kinase 100ng / μL Tris((pH7.9)) 50mM Step 1 Extracted nucleic acid /

[0061] 3) Perform a constant temperature amplification reaction: centrifuge the prepared reaction system to mix evenly, place it in a multi-color fluorescence quantitative PCR instrument, incubate it at 42° C. for 20 minutes, and collect fluorescence every minute.

[0062] Result analysis: If there is no amplification signal in the three channels of the HEX channel, FAM channel and CY5 channel of the detection well at the same time, it means that the experimental results are invalid and need to be re-measured. If there is no signal in the HEX channel, but any of the other two channels has obvious amplification, it means that the result is credible; if only the CY5 channel has obvious amplification and the FAM channel has no signal, it means that the monkeypox test result is positive; if only the FAM channel has obvious amplification and the CY5 channel has no signal, it means that the monkeypox test result is negative and other orthopoxviruses are positive. If there is no amplification line in the CY5 channel of the detection well and there is an amplification line in the HEX channel, it means that the monkeypox test result is negative; if there is no amplification line in the CY5 channel of the detection well, there is an amplification curve in the FAM channel, and there is an amplification line in the HEX channel, it means that the orthopoxvirus test result other than monkeypox is positive.

[0063] The present invention will be further described below in conjunction with embodiments:

[0064] Example 1: Design of primer-probe combination based on RPA-CRISPR / Cas13a monkeypox virus detection method

[0065] The reference genome information of various viruses in the genus Orthopoxvirus was retrieved in the NCBI database. After comparison and analysis, it was found that the N3R gene can effectively distinguish monkeypox virus from other orthopoxviruses. According to the conservation of the N3 gene sequences of different monkeypox viruses compared, primers and probes specifically targeting monkeypox virus were designed and searched in the NCBI blast database to further ensure its specificity in targeting monkeypox virus. The primers, RPA probes and plasmids of the kit of the present invention were synthesized by Sangon Biotech Co., Ltd., and the crRNA and ssRNA used in this experiment were synthesized by GenScript Biotech Co., Ltd.

[0066] All nucleotide sequences involved in the experiment are as follows:

[0067]

[0068]

[0069] Example 2: Screening and verification of the best Cas13a-crRNA for the N3R gene

[0070] According to the final concentration in the table below, the reaction volume was set to 18 μL, and then 2 μL of N3R plasmid templates with concentrations of 5×10^3 copies / μL, 5×10^2 copies / μL, 5×10^1 copies / μL, and 5×10^0 copies / μL were added for testing. The reaction conditions were 42°C, 40 min, and CY5 fluorescence was collected every 1 min. Figure 1 From the screening results of crRNA shown, it can be seen that the best Cas13a-crRNA for the N3R gene is N3R-crRNA1.

[0071]

[0072]

[0073] Example 3: Screening and verification of the best primer pair for N3R gene

[0074] The reaction volume was set to 15 μL according to the final concentration in the table below, and then 5 μL of N3R plasmid templates with concentrations of 2×10^2 copies / μL, 2×10^1 copies / μL, and 2×10^0 copies / μL were added for testing. The reaction conditions were 42°C, 40 min, and CY5 fluorescence was collected every 1 min. Figure 2 The screening results shown show that the best primer pair for N3R gene is N3R-RPA-F1 and N3R-RPA-R1.

[0075] Components Final concentration Components Final concentration N3R-RPA-F 200nM NTPMix (20 mM each) 2mM N3R-RPA-R 200nM dNTPs 200μM N3R-crRNA1 250nM ATP 3mM N3R-ssRNA reporter 2μM Potassium acetate 100mM Cas13a protein 25nM RNase inhibitors 0.5U / μL T7 RNA polymerase 0.2U / μL Creatine Phosphate 50mM T4UvsXRecombinase 240 ng / μL Magnesium acetate 20mM T4UvsYProtein 60ng / μL Polyethylene glycol 20000 5%(V / V) T4gene32protein 500ng / μL Tris((pH7.9)) 50mM BsuDNAPolymerase 0.25 U / μL Creatine kinase 100ng / μL ExonucleaseⅢ 2U / μL

[0076] Example 4: Design of orthopoxvirus primer probe based on RPA fluorescence system

[0077] The reference genome information of various viruses in the genus Orthopoxvirus was retrieved in the NCBI database. After comparison and analysis, it was found that the E9L gene was highly conserved among species. RPA primers and probes specifically targeting the E9L gene were designed and searched in the NCBI blast database to further ensure their specificity.

[0078] All nucleotide sequences involved in the experiment are as follows:

[0079]

[0080]

[0081] Example 5: Testing the sensitivity of the E9L gene primer probe set in the RPA-CRISPR / Cas13a system

[0082] According to the final concentration in the table below, the reaction volume was set to 15 μL, and then 5 μL of E9L plasmid template at concentrations of 2×10^2 copies / μL, 2×10^1 copies / μL, and 2×10^0 copies / μL were added for testing. The reaction conditions were 42°C, 40 min, and FAM fluorescence was collected every 1 min. Figure 3 The screening results shown show that the primer-probe combination for the E9L gene has high amplification sensitivity.

[0083] Components Final concentration Components Final concentration E9L-RPA-F 200nM NTPMix (20 mM each) 2mM E9L-RPA-R 200nM dNTPs 200μM E9L-RPA-P 250nM ATP 3mM N3R-RPA-F 200nM Potassium acetate 100mM N3R-RPA-R 200nM RNase inhibitors 0.5U / μL N3R-crRNA1 250nM Creatine Phosphate 50mM N3R-ssRNA reporter 2μM Magnesium acetate 20mM Cas13a protein 25nM Polyethylene glycol 20000 5%(V / V) T7 RNA polymerase 0.2U / μL Tris((pH7.9)) 50mM T4UvsXRecombinase 240 ng / μL Creatine kinase 100ng / μL T4UvsYProtein 60ng / μL BsuDNAPolymerase 0.25 U / μL T4gene32protein 500ng / μL ExonucleaseⅢ 2U / μL

[0084] Example 6: Design of primer probes for human genes based on RPA fluorescence system

[0085] The reference genome sequence of human HBB gene was downloaded from NCBI database and specific RPA primers and probes were designed and searched in NCBI blast database to ensure their specificity.

[0086] All nucleotide sequences involved in the experiment are as follows:

[0087]

[0088]

[0089] Example 7: Testing the sensitivity of the HBB gene primer probe set in the RPA-CRISPR / Cas13a system

[0090] The reaction volume was set to 15 μL according to the final concentration in the table below, and then 5 μL of HBB plasmid templates with concentrations of 2×10^3 copies / μL, 2×10^2 copies / μL, and 2×10^1 copies / μL were added for testing. The reaction conditions were 42°C, 40 min, and HEX fluorescence was collected every 1 min. Figure 4 The screening results shown show that the primer-probe combination for the HBB gene has a higher amplification sensitivity.

[0091] Components Final concentration Components Final concentration HBB-RPA-F 200nM NTPMix (20 mM each) 2mM HBB-RPA-R 200nM dNTPs 200μM HBB-RPA-P 250nM ATP 3mM N3R-RPA-F 200nM Potassium acetate 100mM N3R-RPA-R 200nM RNase inhibitors 0.5U / μL N3R-crRNA1 250nM Creatine Phosphate 50mM N3R-ssRNA reporter 2μM Magnesium acetate 20mM Cas13a protein 25nM Polyethylene glycol 20000 5%(V / V) T7 RNA polymerase 0.2U / μL Tris((pH7.9)) 50mM T4UvsXRecombinase 240 ng / μL Creatine kinase 100ng / μL T4UvsYProtein 60ng / μL BsuDNAPolymerase 0.25 U / μL T4gene32protein 500ng / μL ExonucleaseⅢ 2U / μL

[0092] Example 8: Testing the sensitivity of the RPA-CRISPR / Cas13a system in the three-target channel

[0093] Prepare a 15μL system with a final concentration according to the table below, and then add 3 gradients of 5μL of mixed plasmid templates containing genes N3R, E9L and HBB. The concentrations of the mixed plasmid templates of N3R, E9L and HBB in the first gradient template are 2×10^2 copies / μL, 2×10^2 copies / μL and 2×10^3 copies / μL, respectively, the concentrations of the mixed plasmid templates of N3R, E9L and HBB in the second gradient template are 2×10^1 copies / μL, 2×10^1 copies / μL and 2×10^2 copies / μL, respectively, the concentrations of the mixed plasmid templates of N3R, E9L and HBB in the first gradient template are 2×10^0 copies / μL, 2×10^0 copies / μL and 2×10^1 copies / μ, respectively. The reaction conditions are 42°C, 30min, and fluorescence is collected every 1min. Figure 5 As shown in the screening results, the N3R gene of the CY5 channel, the E9L gene of the FAM channel, and the HBB gene of the HEX channel can all be amplified with high reaction sensitivity.

[0094]

[0095]

[0096] The above is only an implementation method of the present invention. It should be pointed out that, for ordinary technicians in this field, changes can be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A reagent for specific detection of monkeypox virus, characterized in that: The reagents include an RPA amplification primer pair for the N3R gene of monkeypox virus, N3R-crRNA, and N3R-ssRNA reporter molecules; The primer pair includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.4; The N3R-crRNA includes an anchor sequence for binding to the Cas13a protein and a guide sequence targeting the monkeypox virus N3R gene target sequence, which includes a nucleotide sequence as shown in SEQ ID NO.7; The N3R-ssRNA reporter molecule includes the nucleotide sequence shown in SEQ ID NO.

11.

2. The reagent according to claim 1, characterized in that The reagents also include an RPA amplification primer pair and a probe for the orthopoxvirus E9L gene; The primer pair includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.13 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.14; The probe includes the nucleotide sequence shown in SEQ ID NO.

15.

3. The reagent according to claim 2, characterized in that The reagents also include an RPA amplification primer pair and a probe for the human HBB gene; The primer pair includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.17 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.18; The probe includes the nucleotide sequence shown in SEQ ID NO.

19.

4. A multi-channel detection system for monkeypox virus based on RPA-CRISPR technology, characterized in that: It includes the reagent according to claim 3, Cas13a protein and detection system reaction buffer, and the multi-channel detection system includes three fluorescence channel combinations.

5. The multi-channel detection system according to claim 4, characterized in that: The three fluorescence channel combinations in the detection system are CY5-ssRNA-BHQ2 (monkeypox virus N3R target), FAM-DNA-BHQ2 (orthopoxvirus E9L target), and HEX-DNA-BHQ1 (human gene HBB).

6. Use of the reagent according to any one of claims 1 to 3 or the detection system according to any one of claims 4 to 5 in the preparation of a detection agent for detecting monkeypox virus present in a sample and / or for distinguishing monkeypox virus from other orthopoxviruses present in a sample.

7. A method for detecting monkeypox virus or distinguishing monkeypox virus from other orthopoxviruses present in a sample for non-disease diagnosis and treatment purposes, characterized in that: The method comprises the following steps: (1) Extracting the genome from the sample to be tested; (2) configuring the extracted genome and the components in the multi-channel detection system according to claim 4 or 5 into a reaction system, performing a constant temperature amplification reaction, and collecting fluorescence multiple times during the reaction; (3) Determine the detection result based on the fluorescence signal.

8. The method according to claim 7, characterized in that The isothermal amplification reaction in step (2) is specifically as follows: the prepared reaction system is centrifuged and mixed, placed in a multi-color fluorescence quantitative PCR instrument, incubated at 42° C. for 20 minutes, and fluorescence is collected every minute.

9. The method according to claim 7, characterized in that The reaction system in step (2) is formed by configuring the extracted genome and the components in the multi-channel detection system described in claim 5.

10. The method according to claim 9, characterized in that The method for determining the test result in step (3) is: If there is no amplification signal in the HEX channel, FAM channel and CY5 channel of the detection well at the same time, it means that the experimental results are invalid and need to be re-measured; If there is no signal in the HEX channel, but any of the other two channels has obvious amplification, it means that the result is reliable: if only the CY5 channel has obvious amplification and the FAM channel has no signal, it means that the monkeypox test result is positive; if only the FAM channel has obvious amplification and the CY5 channel has no signal, it means that the monkeypox test result is negative, and other orthopoxviruses are positive; If there is no amplification line in the CY5 channel of the detection well and there is an amplification line in the HEX channel, it means that the monkeypox test result is negative; if there is no amplification line in the CY5 channel of the detection well, there is an amplification curve in the FAM channel, and there is an amplification line in the HEX channel, it means that the test result of orthopoxvirus other than monkeypox is positive.

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