Primer group for rapid and sensitive detection of monkeypox based on rpa-cr ispr technology and application thereof

Abstract

The present application relates to a primer composition for detecting monkeypox virus and its use, the primer composition comprises: a primer pair for amplifying N3R gene and a crRNA targeting N3R gene; and / or a primer pair for amplifying N4R gene and a crRNA targeting N4R gene. The monkeypox virus is detected by using the primer composition in the RPA-CRISPR technology, specific, sensitive, simple, rapid on-site detection can be carried out for the characteristics of wide spread and stable of monkeypox virus, which is helpful for clinical diagnosis and detection.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology based on CRISPR technology, and more particularly to a primer composition based on RPA-CRISPR technology for detecting monkeypox virus and its uses. Background Technology

[0002] Poxviruses are a class of enveloped, large linear double-stranded DNA viruses, including vaccinia virus, variola virus, cowpox virus, and monkeypox virus. Poxviruses possess large genomes, containing up to 328 open reading frames (ORFs) within a 130–375 kbp sequence across different species.

[0003] Poxviruses are a widespread class of pathogens, with most members capable of infecting humans and various animals, causing death. They can spread from person to person through close contact with contaminated objects such as skin lesions, bodily fluids, respiratory droplets, and bedding. Smallpox, one type of poxvirus, has caused tens of thousands of deaths throughout human history. In the 19th and 20th centuries, the use of vaccinia virus as a vaccine for mass vaccination played a crucial role in the eradication of smallpox.

[0004] After smallpox was eradicated, infections with other zoonotic poxviruses, such as monkeypox virus, were subsequently discovered. Monkeypox infection has a long incubation period, generally 10-14 days, and clinical manifestations mainly include rash, fever, and swollen lymph nodes. These characteristics indicate that this round of monkeypox virus transmission is insidious, carries a high risk of human-to-human transmission, and makes blocking monkeypox virus infection even more challenging.

[0005] Given the strong infectivity and transmissibility of monkeypox virus, achieving rapid and accurate diagnosis to effectively isolate and control infected patients and safeguard public health is of paramount importance and urgency. Therefore, the development of an effective, rapid, sensitive, and accurate detection method for monkeypox virus is urgently needed. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a primer composition for detecting monkeypox virus and its uses. Using this primer composition to detect monkeypox virus with RPA-CRISPR technology can provide specific, sensitive, simple and rapid on-site detection, taking advantage of the wide spread and relatively stable characteristics of monkeypox virus, which is helpful for clinical diagnosis and detection.

[0007] To achieve the above objectives, a first aspect of the present invention provides a primer composition for detecting monkeypox virus, comprising:

[0008] Primer pairs for amplifying the N3R gene and crRNA targeting the N3R gene; and / or

[0009] Primer pairs for amplifying the N4R gene and crRNA targeting the N4R gene;

[0010] The primer pairs used to amplify the N3R gene are selected from any of the following primer pairs:

[0011] (1) Primer pair 11 consisting of the sequences shown in SEQ ID NO.37 and SEQ ID NO.40;

[0012] (2) Primer pair 12 consisting of the sequences shown in SEQ ID NO.37 and SEQ ID NO.41;

[0013] (3) Primer pair 13 consisting of the sequences shown in SEQ ID NO.38 and SEQ ID NO.40;

[0014] (4) Primer pair 14 consisting of the sequences shown in SEQ ID NO.38 and SEQ ID NO.41; and

[0015] (5) Primer pair 15 consisting of the sequences shown in SEQ ID NO.38 and SEQ ID NO.42;

[0016] The crRNA targeting the N3R gene is selected from any of the sequences shown in SEQ ID NO.15-19;

[0017] The primer pairs used to amplify the N4R gene are selected from any of the following primer pairs:

[0018] ① Primer pair 16 consisting of the sequences shown in SEQ ID NO.44 and SEQ ID NO.47;

[0019] ② Primer pair 17 consisting of the sequences shown in SEQ ID NO.44 and SEQ ID NO.48;

[0020] ③ Primer pair 18 consisting of the sequences shown in SEQ ID NO.45 and SEQ ID NO.46;

[0021] ④ Primer pair 19 consisting of the sequences shown in SEQ ID NO.45 and SEQ ID NO.47; and

[0022] ⑤ Primer pair 20 consisting of the sequences shown in SEQ ID NO.45 and SEQ ID NO.48;

[0023] The crRNA targeting the N4R gene is selected from any of the sequences shown in SEQ ID NO.20-24.

[0024] In a preferred embodiment of the present invention, the primer composition further comprises:

[0025] Primer pairs and crRNA targeting the D6R gene for amplification; and / or

[0026] Primer pairs for amplifying the E9L gene and crRNA targeting the E9L gene;

[0027] The primer pairs used to amplify the D6R gene are selected from any of the following primer pairs:

[0028] a. Primer pair 1 consisting of the sequences shown in SEQ ID NO.25 and SEQ ID NO.28;

[0029] b. Primer pair 2 consisting of the sequences shown in SEQ ID NO.28 and SEQ ID NO.29;

[0030] c. Primer pair 3 consisting of the sequences shown in SEQ ID NO.25 and SEQ ID NO.30;

[0031] d. Primer pair 4 consisting of the sequences shown in SEQ ID NO. 26 and SEQ ID NO. 28; and

[0032] e. Primer pair 5 consisting of the sequences shown in SEQ ID NO.27 and SEQ ID NO.28;

[0033] The crRNA targeting the D6R gene is selected from any of the sequences shown in SEQ ID NO. 5-9;

[0034] The primer pairs used to amplify the E9L gene are selected from any of the following primer pairs:

[0035] I. Primer pair 6 of the sequences shown in SEQ ID NO. 31 and SEQ ID NO. 35;

[0036] II. Primer pair 7 of the sequences shown in SEQ ID NO.31 and SEQ ID NO.36;

[0037] III. Primer pair 8 of the sequences shown in SEQ ID NO.32 and SEQ ID NO.34;

[0038] IV. Primer pair 9 of the sequences shown in SEQ ID NO. 32 and SEQ ID NO. 35; and

[0039] Primer pair 10 of the sequences shown in SEQ ID NO. 32 and SEQ ID NO. 36;

[0040] The crRNA targeting the E9L gene is selected from any of the sequences shown in SEQ ID NO.10-14.

[0041] In a preferred embodiment of the present invention, the primer pair used for amplifying the N3R gene is:

[0042] (2) Primer pair 12 consisting of the sequences shown in SEQ ID NO.37 and SEQ ID NO.41;

[0043] The crRNA targeting the N3R gene is the sequence shown in SEQ ID NO.15;

[0044] The primer pair used to amplify the N4R gene is:

[0045] ③ Primer pair 18 consisting of the sequences shown in SEQ ID NO.45 and SEQ ID NO.46;

[0046] The crRNA targeting the N4R gene is the sequence shown in SEQ ID NO.20.

[0047] In a preferred embodiment of the present invention, the primer pair used for amplifying the D6R gene is:

[0048] a. Primer pair 1 consisting of the sequences shown in SEQ ID NO.25 and SEQ ID NO.28;

[0049] The crRNA targeting the D6R gene has the sequence shown in SEQ ID NO.5;

[0050] The primer pair used to amplify the E9L gene is:

[0051] V. Primer pair 10 consisting of the sequences shown in SEQ ID NO.32 and SEQ ID NO.36;

[0052] The crRNA targeting the E9L gene is the sequence shown in SEQ ID NO.13.

[0053] A second aspect of the present invention provides a kit comprising the above-described primer composition.

[0054] In a preferred embodiment of the present invention, the kit further comprises Cas12a protein, a signal reporter probe, and a detection system reaction buffer.

[0055] Preferably, the signal reporting probe is a single-stranded DNA with a fluorescent group labeled at one end and a quenching group labeled at the other end;

[0056] More preferably, the signal reporting probe is 5'-(FAM)TTATTATT(BHQ1)-3'.

[0057] In a preferred embodiment of the present invention, the kit further comprises RPA enzyme premix, magnesium acetate, and amplification reaction buffer.

[0058] A third aspect of the invention provides the use of the above-described primer compositions or kits in the development and / or preparation of products for detecting and / or diagnosing monkeypox virus.

[0059] A fourth aspect of the present invention provides a method for detecting monkeypox virus, comprising the following steps:

[0060] Sample extraction: Take the sample to be tested and extract the genome from it;

[0061] One-step detection: Add the above primer composition, RPA enzyme premix, magnesium acetate, amplification reaction buffer, Cas12a protein, signal reporter probe and detection system reaction buffer to the genome of the sample to be tested obtained above, and perform RPA amplification and CRISPR reaction detection simultaneously, and read the detection signal to interpret the detection result.

[0062] In a preferred embodiment of the present invention, the conditions for the step detection are: constant temperature reaction at 37±1℃ for 20-40 min;

[0063] The working concentrations of the primer pairs used for amplifying the N3R gene, the N4R gene, the D6R gene, and the E9L gene are all 1–20 μM.

[0064] The working concentrations of crRNAs targeting the N3R gene, N4R gene, D6R gene, and E9L gene were 300–600 nM.

[0065] The beneficial effects of this invention are as follows:

[0066] This invention provides a primer composition based on RPA-CRISPR technology. Using this primer composition to detect monkeypox virus with RPA-CRISPR technology shortens the detection time of monkeypox virus, which can be completed within 20-40 minutes, and has the advantages of high sensitivity and strong specificity.

[0067] In addition, the use of this primer composition for CRISPR detection of monkeypox virus does not require complex temperature changes, thus eliminating the dependence on complex temperature-dependent amplification instruments such as qPCR instruments. This makes CRISPR-Cas technology a promising candidate for point-of-care diagnosis of monkeypox, enabling rapid, sensitive, and accurate detection of monkeypox virus. Attached Figure Description

[0068] Figure 1 This is a target sequence screening diagram from Example 1.

[0069] Figure 2 The graph shows the results of crRNA efficiency screening in Example 2.

[0070] Figure 3 The results are the crRNA target sensitivity detection results in Example 3.

[0071] Figure 4 This is a screening diagram of RPA primer amplification in Example 4.

[0072] Figure 5 This is the specific detection result of crRNA target detection in Example 5. Detailed Implementation

[0073] It should be noted that, unless otherwise defined, the technical or scientific terms used in this application shall have the ordinary meaning as understood by one of ordinary skill in the art.

[0074] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all medicinal materials and reagents used in the following examples are commercially available products.

[0075] When used herein and in the appended claims, the singular forms “a,” “an,” “another,” and “the” include the plural referents unless the context clearly indicates otherwise.

[0076] In this application, the term "comprising / including" generally means including the explicitly specified features, but does not exclude other elements.

[0077] The term “and / or” as used herein includes any and all combinations of one or more of the related listed items.

[0078] Recombinase-polymerase amplification (RPA) is a rapidly emerging isothermal amplification technique. It utilizes recombinases that bind to single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA-binding proteins (SSBs), and DNA polymerases with strand displacement capabilities to obtain detectable amplified nucleic acids at room temperature (optimal reaction temperature 37℃~42℃). However, the fundamental nature of this single-stage amplification reaction remains unchanged, and compared to most qPCR detection methods currently on the market, there is no substantial improvement in sensitivity.

[0079] Clustered, spaced, regular short palindromic repeats (CRISPR) and their associated Cas proteins are collectively referred to as the CRISPR / Cas system. This system is primarily functioned by its effector proteins. There are various types of Cas proteins, differing in size, structure, composition, and targets. CRISPR / Cas systems are classified into two main categories based on their Cas proteins: Type I CRISPR / Cas systems contain multiple proteins that work synergistically, including types I, III, and IV; Type II CRISPR / Cas systems are characterized by a single protein performing multiple functions, including types II, V, and VI.

[0080] In CRISPR / Cas12a-based rapid detection technologies, Cas12a is a type V effector protein of the CRISPR system. Studies have shown that this protein only requires the participation of a single crRNA (guide RNA) to function. Guided by the crRNA, Cas12a can bind to a specific target sequence to form a trimer complex and then cleave it. In addition to this ability to cleave target genes, this trimer complex can also cleave non-specific single-stranded DNA. Therefore, this non-specific cleavage ability depends on the presence of the target sequence in the system; that is, Cas12a's non-specific cleavage ability is activated when the specific target sequence is present and deactivated when the target sequence is absent.

[0081] This invention utilizes the non-specific cleavage capability of Cas12a, which can be "switched" by the target sequence. Considering that the optimal temperature for Cas12a to perform nuclease function is similar to that of RPA (both are 37°C), the two can be used together to develop a method for detecting monkeypox virus based on RPA-CRISPR technology. Specifically, RPA is responsible for isothermal amplification of the target sequence to increase the copy number of the target sequence, while Cas12a is responsible for specifically identifying the enzyme-cleaved target sequence and relying on a fluorescent reporter probe to visualize the detection results. In the detection method of this invention, Cas12a in the system and crRNA targeting the designed DNA fragment form a dimer complex. If the sample to be tested contains the target DNA fragment, the Cas12a-crRNA dimer complex will form a Cas12a-crRNA-target DNA trimer complex. At the same time, the non-specific cleavage ability of Cas12a is activated, cleaving the ssDNA (single-stranded DNA) probe in the system, thereby enabling the system to emit fluorescence. It can degrade a large number of probes in a very short time, thereby emitting a strong fluorescence signal. The ssDNA probe is an ssDNA with fluorescent and quenching groups labeled at both ends. When the probe is intact, it does not emit fluorescence. After being cleaved, the fluorescent and quenching groups move away from each other, thereby emitting fluorescence.

[0082] The Cas12a in the system converts the presence or absence of target DNA in the system into the presence or absence of a fluorescent signal by cleaving the fluorescent reporter probe. Therefore, in practical applications, the presence or absence or magnitude of the fluorescent signal can be used to determine whether the sample contains the target gene.

[0083] This invention provides four primer pairs and crRNA for detecting monkeypox virus based on RPA-CRISPR technology, namely:

[0084] 1) Primer pairs for amplifying the D6R gene (whose nucleotide sequence is shown in SEQ ID NO.1) and crRNA targeting the D6R gene;

[0085] 2) Primer pairs for amplifying the E9L gene (whose nucleotide sequence is shown in SEQ ID NO.2) and crRNA targeting the E9L gene;

[0086] 3) Primer pairs for amplifying the N3R gene (whose nucleotide sequence is shown in SEQ ID NO.3) and crRNA targeting the N3R gene;

[0087] 4) Primer pairs for amplifying the N4R gene (whose nucleotide sequence is shown in SEQ ID NO.4) and crRNA targeting the N4R gene;

[0088] The N3R and N4R genes are specific to monkeypox virus. Primer pairs and crRNA designed for the N3R and N4R genes can specifically detect monkeypox virus. Therefore, the presence of monkeypox virus in a sample can be confirmed by the detection of the N3R and / or N4R genes. The D6R and E9L genes are conserved genes of the orthopoxvirus genus (including monkeypox virus, smallpox virus, vaccinia virus, and cowpox virus, etc.), meaning they are present in all orthopoxviruses. The detection of the D6R and / or E9L genes in a sample only confirms the presence of an orthopoxvirus (possibly including monkeypox virus), but does not definitively confirm the presence of monkeypox virus. Further testing for the N3R and N4R genes is required. Only the detection of at least one of the D6R and E9L genes, and at least one of the N3R and N4R genes, confirms the presence of monkeypox virus in the sample.

[0089] Therefore, the primer composition for detecting monkeypox virus of the present invention may contain only 3) primer pair for amplifying the N3R gene and crRNA targeting the N3R gene, or only 4) primer pair for amplifying the N4R gene and crRNA targeting the N4R gene; it may contain both 3) and 4); or it may contain at least one of 3) and 4) and at least one of 1) primer pair for amplifying the D6R gene and crRNA targeting the D6R gene and 2) primer pair for amplifying the E9L gene and crRNA targeting the E9L gene.

[0090] The primer composition of this invention can be applied to the CRISPR-Cas12a system. Its detection principle is as follows: Guided by guide RNA (crRNA), the Cas12 protein targets the target sequence and initiates its own "collateral cleavage" activity. If a fluorescent reporter probe (a commonly used reporter probe is an oligonucleotide sequence with a fluorescent group at one end and a quencher group at the other; normally, due to quenching, the complete reporter molecule will not be detected, but when the oligonucleotide molecule is hydrolyzed, the free fluorescent signal can be detected) is added to the system, utilizing the collateral cleavage activity of the Cas enzyme to convert the target sequence information into a fluorescent signal. Furthermore, through the coupling of RPA and CRISPR-Cas, a two-stage amplification process—"sequence amplification" (completed by RPA) plus "enzyme cascade" (completed by the Cas enzyme)—can be achieved, thus surpassing the sensitivity of single-stage amplification like qPCR.

[0091] Furthermore, because RPA amplification does not require complex temperature changes, it eliminates the reliance on complex temperature-dependent amplification instruments such as qPCR instruments. This makes CRISPR-Cas technology a promising candidate for point-of-care diagnosis of monkeypox, with results available in as little as 40 minutes. This significantly shortens the detection time, helps prevent wider spread of the disease, and offers substantial advantages.

[0092] In a preferred embodiment of the present invention, the primer pair used for amplifying the N3R gene is:

[0093] (2) Primer pair 12 consisting of the sequences shown in SEQ ID NO.37 and SEQ ID NO.41;

[0094] The crRNA targeting the N3R gene is the sequence shown in SEQ ID NO.15;

[0095] The primer pair used to amplify the N4R gene is:

[0096] ③ Primer pair 18 consisting of the sequences shown in SEQ ID NO.45 and SEQ ID NO.46;

[0097] The crRNA targeting the N4R gene is the sequence shown in SEQ ID NO.20.

[0098] In a preferred embodiment of the present invention, the primer pair used for amplifying the D6R gene is:

[0099] a. Primer pair 1 consisting of the sequences shown in SEQ ID NO.25 and SEQ ID NO.28;

[0100] The crRNA targeting the D6R gene has the sequence shown in SEQ ID NO.5;

[0101] The primer pair used to amplify the E9L gene is:

[0102] V. Primer pair 10 consisting of the sequences shown in SEQ ID NO.32 and SEQ ID NO.36;

[0103] The crRNA targeting the E9L gene is the sequence shown in SEQ ID NO.13.

[0104] In CRISPR detection technology, the performance of the combination of crRNA and amplification primers has a significant impact on the detection effect. When designing crRNA and amplification primers, it is necessary to consider not only the conventional primer design principles but also whether there are suitable sequences within the primer amplification fragment range that can be used for crRNA design. Currently, commonly used primer design guidelines are insufficient to design good crRNA and amplification primer combinations based on conventional parameter requirements; more often than not, manual adjustments to the design are needed based on experience and experimental trials. Based on previous research, the inventors, through multiple screenings and comparisons, discovered that the above-mentioned combination of crRNA and amplification primers exhibits better detection results.

[0105] The present invention also provides a kit comprising the above-described primer composition.

[0106] In a preferred embodiment of the present invention, the kit further comprises Cas12a protein and a signal reporter probe;

[0107] Preferably, the signal reporting probe is a single-stranded DNA with a fluorescent group labeled at the 5' end and a quenching group labeled at the 3' end.

[0108] More preferably, the signal reporter probe is 5'-(FAM)TTATTATT(BHQ1)-3'. When the signal reporter probe is added to the reaction system, and the cas12a protein, with the help of crRNA, recognizes the target DNA with the target sequence, the activated cas12a enzyme can degrade the signal reporter probe, thereby releasing a fluorescent signal and achieving detection.

[0109] In a preferred embodiment of the present invention, the kit further comprises an RPA enzyme premix (containing recombinant enzymes, single-stranded DNA binding proteins, and DNA polymerases with strand displacement function required for the RPA reaction) and magnesium acetate (an enzyme performance cofactor).

[0110] This invention also provides the use of the above-described primer compositions or kits in the development and / or preparation of products for detecting and / or diagnosing monkeypox virus. It is understood that the above-described products can be reagents or integrated detection devices, etc.

[0111] The present invention also provides a method for detecting monkeypox virus for non-diagnostic purposes, comprising the following steps:

[0112] Sample extraction: Take the sample to be tested and extract the genome from it;

[0113] One-step detection: Add the above primer combination, RPA enzyme premix, magnesium acetate, Cas12a protein and signal reporter probe to the genome of the sample to be tested obtained above, and perform RPA amplification and CRISPR reaction detection at the same time, and read the detection signal to interpret the detection result.

[0114] In a preferred embodiment of the present invention, the conditions for the step detection are: constant temperature reaction at 37±1℃ for 20-40 min;

[0115] The working concentrations of the primer pairs used for amplifying the N3R gene, the N4R gene, the D6R gene, and the E9L gene are all 1–20 μM.

[0116] The working concentrations of crRNA targeting the N3R gene, the N4R gene, the D6R gene, and the E9L gene are 300–600 nM. In practice, the inventors have found that the concentration used in the above primer compositions for monkeypox virus detection has a certain impact on the detection effect. If the concentration is unsuitable, reduced sensitivity or poor specificity may occur. The primer compositions using the above working concentrations exhibit good sensitivity and specificity.

[0117] It is understandable that the working concentration refers to the concentration of the primer sequence in the reaction system. The concentration of the primer composition during preparation, storage and transportation can be adjusted according to specific requirements. As long as the above concentration is maintained in the reaction system, a good detection effect can be achieved.

[0118] All of the above detection steps are completed under constant temperature conditions, without the need for complex temperature changes, thus eliminating the dependence on precision instruments such as Q-PCR instruments and having a broad application prospect.

[0119] Understandably, the above fluorescence signals can be read using a fluorescence detector with specific FAM fluorescence reading capabilities, such as a fluorescence PCR instrument or an enzyme-linked immunosorbent assay (ELISA) reader. When using a fluorescence PCR instrument, such as the ABI 7500 (Thermo Fisher) or the BIO-RADCFX96™ Real-Time System, the reaction conditions and time are as follows: react at 37°C for 20-40 minutes, and read the FAM fluorescence value every 1 minute.

[0120] Compared with traditional detection methods, the method for detecting monkeypox virus in this invention shortens the detection time and can complete the detection within 20-40 minutes.

[0121] This invention obtained specific sequence combinations as primer compositions for detection through screening, and also screened the detection conditions. The combination of the two to detect monkeypox virus has the advantages of high sensitivity and strong specificity. Its detection limit can reach 10 copies, and only monkeypox virus can be specifically detected in the specificity experiment, which meets the requirements of clinical testing.

[0122] The technical solution provided by the present invention will be further described below with reference to specific embodiments. The following embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention.

[0123] The materials and instruments involved in the following embodiments are as follows:

[0124] 1. Experimental materials:

[0125] The plasmids, primers, and signal reporter probes containing the target sequence were synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd., and the guide RNA (crRNA) was synthesized by GenScript Biotech Co., Ltd. The amino acid-containing Cas12a protein (AsCas12a protein) was expressed and purified by GenScript Biotech Co., Ltd. The RPA kit was purchased from Beijing Dongfang Nobel Technology Development Co., Ltd. Other biochemical reagents were either imported and repackaged or domestically produced analytical grade.

[0126] 2. Experimental apparatus

[0127] Centrifuges, vortex mixers, BIO-RAD CFX96TM Real-Time System fluorescence detection instruments, etc.

[0128] Example 1: Design of crRNA primer sequences for monkeypox virus detection

[0129] 1. Target sequence selection

[0130] Monkeypoxvirus belongs to the genus Orthopoxvirus in the family Poxviridae. It has two distinct genetic branches: the Central African (Congo Basin) branch and the West African branch. The inventors analyzed the sequences of the monkeypoxvirus circulating in 2022 and concluded that D6R and E9L are conserved genes in orthopoxviruses, and detection methods can be designed based on these conserved sequences to detect orthopoxviruses (including monkeypoxviruses). N3R and N4R are monkeypoxvirus-specific genes, and detection methods can be designed based on these specific sequences to specifically detect monkeypoxviruses (see [link to relevant documentation]). Figure 1 Based on the D6R, E9L, N3R, and N4R gene sequence regions, crRNA and RPA amplification primers were designed, and the sensitivity of the detection method was tested.

[0131] The test samples in this embodiment were plasmids pUC57-D6R and pUC57-E9L, which respectively contained conserved orthopoxvirus sequences D6R and E9L, and plasmids pUC57-N3R and pUC57-N4R, which contained monkeypoxvirus-specific sequences N3R and N4R (synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd.). The D6R, E9L, N3R, and N4R gene sequences were inserted between the BamHI and EcoRI sites of the pUC57 plasmid, respectively, to construct plasmids pUC57-D6R, pUC57-E9L, pUC57-N3R, and pUC57-N4R. The inserted D6R gene sequence is shown in SEQ ID NO.1. The inserted E9L gene sequence is shown in SEQ ID NO.2. The inserted N3R gene sequence is shown in SEQ ID NO.3. The inserted N4R gene sequence is shown in SEQ ID NO.4.

[0132] 2. Design of crRNA

[0133] crRNA primers were designed based on the target sequences described above. The crRNA primer sequences are shown in Table 1 below.

[0134] crRNA of D6R, E9L, N3R and N4R genes

[0135]

[0136]

[0137] Example 2: Screening for crRNA sequence detection efficiency.

[0138] To screen for the most effective crRNA sequences, plasmids containing the gene sequences of the two conserved target sites D6R and E9L of orthopoxvirus and the two specific target sites N3R and N4R of monkeypoxvirus, as described in Example 1, were used as standard test samples. Genes from these standard test samples were extracted using conventional methods, and five crRNAs each for D6R, E9L, N3R, and N4R were detected and screened. The template concentration was 10... 10 copies / μL.

[0139] 1.1 Method

[0140] Reaction system: 1 μL of genomic sample to be tested, 1.5 μL of AsCas12a protein (667 nM) (GenScript), 2 μL of 10x detection system reaction buffer, 1 μL of signal reporter probe (10 μM), 2.5 μL of crRNA (500 nM), and bring the total volume to 20 μL with DEPC water (Thermofisher).

[0141] Reaction conditions: Reaction at 37℃ for 100 min, with FAM fluorescence values ​​read every 1 min.

[0142] In this embodiment, the positive signal is a fluorescent signal. The signal reporter probe is characterized by RNA with fluorescent and quenching groups. When the Cas12a protein recognizes the target RNA with the help of crRNA, the activated Cas12a enzyme can degrade the signal reporter probe with the signal, thereby releasing the fluorescent signal and realizing detection.

[0143] Result Interpretation: The obtained cumulative fluorescence value is taken as the signal intensity and analyzed and judged according to the following criteria:

[0144] Negative judgment criteria: The fluorescence value is less than or equal to twice the fluorescence value of the negative control. The negative control here refers to any one of the following four negative control groups, but the fluorescence value of the negative control mainly refers to the fluorescence value of the group using DEPC water (Thermofisher) as a template (△Template) as the negative control group.

[0145] Positive judgment criteria: The fluorescence value is greater than twice the fluorescence value of the negative control. The negative control here refers to any one of the following four negative control groups, but the fluorescence value of the negative control mainly refers to the fluorescence value of the group using DEPC water (Thermofisher) as a template (△Template) as the negative control group.

[0146] The negative control group consisted of four groups corresponding to each experimental group: the Cas12a deletion group (△Cas), the negative control probe group (△gRNA), the DEPC water template group (△Template), and the probe deletion group (△probe).

[0147] 1.2 Results

[0148] In this embodiment, the BIO-RAD CFX96TM Real-Time System instrument was used to detect and screen targets D6R, E9L, N3R, and N4R respectively. Finally, one crRNA with good detection efficiency was selected for each gene target.

[0149] The filtering results are as follows Figure 2 As shown in the figure, the horizontal axis represents time, and the vertical axis represents the detected and collected FAM fluorescence signal values.

[0150] The results showed that the above-mentioned crRNAs all had good guiding effects and targeted activation of AsCas12a.

[0151] In this embodiment, plasmid DNA containing conserved viral genes of orthopoxvirus and monkeypox-specific genes was used as a template to screen different crRNAs. Ultimately, a set of highly specific and sensitive crRNAs targeting different gene sites were selected for monkeypox virus detection. The crRNA sequences for these four genes are as follows:

[0152] D6R gene: D6R-1crRNA shown in SEQ ID NO: 5;

[0153] E9L gene: E9L-4crRNA shown in SEQ ID NO: 13;

[0154] N3R gene: N3R-1crRNA shown in SEQ ID NO: 15;

[0155] N4R gene: N4R-1crRNA shown in SEQ ID NO: 20.

[0156] Example 3: Sensitivity Detection Based on Cas12a

[0157] Using plasmids containing the conserved D6R and E9L genes of orthopoxvirus and the N3R and N4R genes specific to monkeypoxvirus, respectively, as templates, and with dilutions of 10-1, the results were calculated. 9 copies / μL, 10 8 copies / μL, 10 7 copies / μL, 10 6 Four gradients of copies / μL were used as templates for sensitivity detection, with 1 μL of template added for each reaction.

[0158] 1. Method

[0159] Following the method described in Example 2 above, sensitivity analyses were performed on D6R, E9L, N3R, and N4R, with a negative control included. The crRNAs used were D6R-1 (SEQ ID NO. 5), E9L-4 (SEQ ID NO. 13), N3R-1 (SEQ ID NO. 15), and N4R-1 (SEQ ID NO. 20).

[0160] Reaction conditions: Reaction at 37℃ for 100 min, with FAM fluorescence values ​​read every 1 min.

[0161] The results are interpreted according to the result interpretation method in Example 2 above.

[0162] 2. Results

[0163] Fluorescence detection of templates at different concentration gradients was performed using a BIO-RAD CFX96TM Real-Time System instrument. Results are as follows: Figure 3 As shown, the horizontal axis represents time, and the vertical axis represents the collected FAM fluorescence signal values. The limits of detection (LODs) for amplification-free FAM RNA of D6R-1, E9L-4, N3R-1, and N4R-1crRNA were 10 Ω·cm. 6 copies / μL, 10 8 copies / μL, 10 8 copies / μL and 10 6 copies / μL.

[0164] Example 4: Design and screening of RPA amplification primer pairs

[0165] 1. Design of RPA amplification primer pairs

[0166] The experiment required designing and optimizing multiple pairs of RPA primers to amplify target sequences for different genes and crRNA targets. The designed primers produced amplification products ranging from 110 to 260 bp in length. Specifically, three upstream and three downstream primers were designed for the crRNA D6R-1 target sequence; three upstream and three downstream primers were designed for the crRNA E9L-4 target sequence; three upstream and three downstream primers were designed for the crRNA N3R-1 target sequence; and three upstream and three downstream primers were designed for the crRNA N4R-1 target sequence.

[0167] The primer sequences are shown in Table 2 below.

[0168]

[0169]

[0170] Note: In the table above, F represents the upstream primer and R represents the downstream primer.

[0171] 2. Screening of RPA primer pairs

[0172] To screen primers for Cas12a RPA amplification, plasmids containing the gene sequences of two conserved target sites D6R and E9L of orthopoxvirus and two specific target sites N3R and N4R of monkeypoxvirus, as described in Example 1, were used as standard test samples. Genes of the standard test samples were extracted using conventional methods. Primers targeting the D6R-1 target sequence (primers shown in Table 2 above) were combined in pairs with D6R-1crRNA, primers targeting the E9L-4 target sequence (primers shown in Table 2 above) were combined in pairs with E9L-4crRNA, primers targeting the N3R-1 target sequence (primers shown in Table 2 above) were combined in pairs with N3R-1crRNA, and primers targeting the N4R-1 target sequence (primers shown in Table 2 above) were combined in pairs with N4R-1crRNA. The template concentration was 100 copies / μL.

[0173] 2.1 Method

[0174] Reaction system: 0.5 μL upstream primer (10 μM), 0.5 μL downstream primer (10 μM), 6 μL amplification reaction buffer (for RPA amplification), 0.5 μL magnesium acetate (280 mM), 1 μL genomic sample to be tested, 1.5 μL AsCas12a protein (667 nM) (GenScript), 2 μL 10x detection system reaction buffer (which is the CRISPR detection system buffer), 1 μL signal reporter probe (10 μM), 2.5 μL crRNA (500 nM), and brought to 20 μL with DEPC water (Thermofisher).

[0175] Reaction conditions: React at 37℃ for 40 min, with FAM fluorescence values ​​read every 1 min.

[0176] The results are interpreted according to the result interpretation method in Example 2 above.

[0177] 2.2 Results

[0178] In this embodiment, the BIO-RAD CFX96TM Real-Time System instrument was used to detect and screen primers (primers shown in Table 2 above) for targets D6R-1, E9L-4, N3R-1 and N4R-1 in pairs. Finally, a pair of primers with good amplification efficiency was selected for each gene target.

[0179] The filtering results are as follows Figure 4 As shown in the figure, the horizontal axis represents different primer combinations, and the vertical axis represents the Fold-change value at 40 min (i.e., Fold-change value = fluorescence signal value of the test sample / fluorescence signal value of the negative control).

[0180] The results showed that the D6R-1 target gene had the best amplification efficiency using primer pair 1, consisting of the sequences shown in SEQ ID NO. 25 and SEQ ID NO. 28; the E9L target gene had the best amplification efficiency using primer pair 10, consisting of the sequences shown in SEQ ID NO. 32 and SEQ ID NO. 36; the N3R target gene had the best amplification efficiency using primer pair 12, consisting of the sequences shown in SEQ ID NO. 37 and SEQ ID NO. 41; and the N4R target gene had the best amplification efficiency using primer pair 18, consisting of the sequences shown in SEQ ID NO. 45 and SEQ ID NO. 46.

[0181] Example 5: Specificity Validation of Monkeypox Virus Detection Based on RPA Amplification Combined with Cas12a

[0182] Using monkeypox virus, vaccinia virus (VACV), Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Candida albicans, varicella-zoster virus, rubella virus, herpes simplex virus-1 / -2, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, measles virus, enterovirus, Treponema pallidum, and dengue virus as detection templates, each genomic sample was diluted to 10⁻⁶. 1 The specificity of the D6R, E9L, N3R, and N4R gene target systems was tested using human genomic DNA as a negative control at copies / μL.

[0183] 1. Method

[0184] Reaction system: 0.5 μL upstream primer (10 μM), 0.5 μL downstream primer (10 μM), 6 μL reaction buffer, 0.5 μL magnesium acetate (280 mM), 1 μL genomic sample to be tested, 2 μL AsCas12a protein (25 nM) (GenScript), 2 μL 10x reaction buffer, 2 μL signal reporter probe (250 nM), 1 μL crRNA (62.5 nM), and bring the total volume to 20 μL with DEPC water (Thermofisher).

[0185] Reaction conditions: React at 37℃ for 40 min, with FAM fluorescence values ​​read every 1 min.

[0186] The results are interpreted according to the result interpretation method in Example 2 above.

[0187] 2. Results Analysis

[0188] The amplification products were detected for fluorescence using a BIO-RAD CFX96™ Real-Time System instrument. The experimental results are as follows: Figure 5 As shown, the vertical axis represents the Fold-change value over 40 minutes (i.e., Fold-change value = fluorescence signal value of the test sample / fluorescence signal value of the negative control). NC represents negative samples, and PC represents positive samples (VACV and monkeypox virus). The results show that vaccinia virus (VACV) and monkeypox virus can be specifically detected targeting the conserved regions D6R-1 and E9L-4 of orthopoxvirus; targeting the monkeypox virus-specific N3R-1 and N4R-1, only monkeypox virus can be specifically detected, with no obvious signal in other samples.

[0189] This embodiment demonstrates good specificity for detecting monkeypox virus based on RPA combined with Cas12a.

[0190] Example 6: Method for detecting monkeypox virus using a kit

[0191] I. A kit for rapid detection of monkeypox virus, comprising:

[0192] (1) Primers and crRNA targeting the D6R gene, E9L gene, N3R gene, and N4R gene, as detailed below:

[0193] D6R-1crRNA (SEQ ID NO.5):

[0194] 5'-UAAUUUCUACUCUUGUAGAUAUGUCCGAAGAGACGAUA GAUUU-3', with a concentration of 500 nM;

[0195] D6R-1 forward primer (SEQ ID NO.25):

[0196] 5'-CTACTATTGTCTGGATCTCCCATTACTAAC-3', concentration 10 μM; D6R-1 reverse primer (SEQ ID NO. 28):

[0197] The concentration of 5'-CGTGATATCTTATCGTTGGTAGATCTTTATCAG-3' is 10 μM;

[0198] E9L-4crRNA (SEQ ID NO.13):

[0199] 5'-UAAUUUCUACUCUUGUAGAUUAUGGACGAUAGAGAUAUU AAUC-3', concentration 500 nM;

[0200] E9L-4 forward primer (SEQ ID NO.32):

[0201] 5'-CACGGAGTCGGTATCTCCATACACGCTAC-3', concentration 10 μM; E9L-4 reverse primer (SEQ ID NO. 36):

[0202] 5'-CTCATACGCTTCGGCTAAGAGTTGCACATC-3', concentration 10 μM; N3R-1crRNA (SEQ ID NO. 15):

[0203] 5'-UAAUUUCUACUCUUGUAGAUUUACCGACCAUGAACGCGA UAGA-3', concentration 500 nM;

[0204] N3R-1 forward primer (SEQ ID NO.37):

[0205] 5'-TATTGCTGGACAATTATCCAATTATGACC-3', concentration 10 μM; N3R-1 reverse primer (SEQ ID NO. 41):

[0206] 5'-TTCCTCGTATTGACTACTTACAGATACTCG-3', concentration 10 μM;

[0207] N3R-1crRNA (SEQ ID NO.15):

[0208] 5'-UAAUUUCUACUCUUGUAGAUUUACCGACCAUGAACGCGA UAGA-3', concentration 500 nM;

[0209] N3R-1 forward primer (SEQ ID NO.37):

[0210] 5'-TATTGCTGGACAATTATCCAATTATGACC-3', concentration 10 μM;

[0211] N3R-1 reverse primer (SEQ ID NO.41):

[0212] 5'-TTCCTCGTATTGACTACTTACAGATACTCG-3', concentration 10 μM;

[0213] N4R-1crRNA (SEQ ID NO.20):

[0214] 5'-UAAUUUCUACUCUUGUAGAUAGAUGUGCUUCCCUACGG GCGAU-3', with a concentration of 500 nM;

[0215] N3R-1 forward primer (SEQ ID NO.45):

[0216] 5'-GGATATACATGTCTTGACGTGGCCGTGGATAG-3', concentration 10 μM;

[0217] N3R-1 reverse primer (SEQ ID NO.46):

[0218] 5'-CAGTCAATAGACAATGGTTCTCTAAGCA-3', concentration 10 μM;

[0219] (2) RPA enzyme premix: commercially available, including creatine phosphate (concentration 20-80mM), creatine kinase (concentration 50-150mM), dNTPs (concentration 100-300μM), ATP (concentration 20-80mM), DTT (concentration 1-10mM), potassium acetate (concentration 50-200mM), recombinant enzyme uxsX (50-300ng / μL), uxsY (10-100ng / μL), single-chain binding protein (200-1000ng / μL), Bsu polymerase (10-100ng / μL);

[0220] (3) Magnesium acetate: concentration 280mM;

[0221] (4) AsCas12a protein: concentration of 667 nM;

[0222] (5) Signal reporting probe: 5'-(FAM)TTATTATT(BHQ1)-3', concentration 10 μM;

[0223] (6) Amplification reaction buffer;

[0224] (7) 10x detection system reaction buffer;

[0225] Understandably, the above reagents and concentrations can be adjusted according to transportation, storage, and other needs, and should be adjusted according to the testing requirements during use.

[0226] II. Method for monkeypox detection using the above-mentioned reagent kit

[0227] (1) Genomic extraction of the test sample.

[0228] The test samples can be cultured virus strains, clinical samples (mainly including blood samples, upper respiratory tract samples, lower respiratory tract samples, bronchoalveolar lavage fluid, sputum, pharyngeal swabs, nasopharyngeal swabs, etc.), or other scientific research samples.

[0229] (2) RPA amplification and CRISPR reaction detection.

[0230] Refer to the method in Example 5 above.

[0231] (3) Results analysis.

[0232] The fluorescence detector is used for detection, and the obtained cumulative fluorescence value is taken as the signal intensity. The analysis and judgment are based on the following standards:

[0233] Negative judgment criteria: The fluorescence intensity is less than or equal to twice the fluorescence intensity of the negative control. The negative control here refers to any one of the following four negative control groups, but the fluorescence value of the negative control mainly refers to the fluorescence value of the group using DEPC water (Thermofisher) as a template (△Template) as the negative control group.

[0234] Positive judgment criteria: The fluorescence intensity is greater than twice that of the negative control. The negative control here refers to any one of the following four negative control groups, but the fluorescence value of the negative control mainly refers to the fluorescence value of the group using DEPC water (Thermofisher) as a template (△Template) as the negative control group.

[0235] The negative control group consisted of four groups corresponding to each experimental group: the Cas12a deletion group (△Cas), the negative control probe group (△gRNA), the DEPC water template group (△Template), and the deletion probe group (△probe).

[0236] If both N3R and N4R genes of a sample are positive, the sample is determined to be positive for monkeypox virus.

Claims

1. A primer composition for detecting monkeypox virus, comprising: Primer pairs for amplifying the N3R gene and crRNA targeting the N3R gene; and Primer pairs for amplifying the N4R gene and crRNA targeting the N4R gene; in, The primer pair used to amplify the N3R gene is: Primer pair 12 consisting of the sequences shown in SEQ ID NO. 37 and SEQ ID NO. 41; The crRNA targeting the N3R gene has the sequence shown in SEQ ID NO. 15; The primer pair used to amplify the N4R gene is: Primer pair 18 consisting of the sequences shown in SEQ ID NO. 45 and SEQ ID NO. 46; The crRNA targeting the N4R gene is the sequence shown in SEQ ID NO.

20.

2. The primer composition according to claim 1, wherein, Also includes: Primer pairs and crRNA targeting the D6R gene for amplification; and / or Primer pairs for amplifying the E9L gene and crRNA targeting the E9L gene; The primer pairs used to amplify the D6R gene are selected from any of the following primer pairs: a. Primer pair 1 consisting of the sequences shown in SEQ ID NO. 25 and SEQ ID NO. 28; b. Primer pair 2 consisting of the sequences shown in SEQ ID NO. 25 and SEQ ID NO. 29; c. Primer pair 3 consisting of the sequences shown in SEQ ID NO. 25 and SEQ ID NO. 30; d. Primer pair 4 consisting of the sequences shown in SEQ ID NO. 26 and SEQ ID NO. 28; and e. Primer pair 5 consisting of the sequences shown in SEQ ID NO. 27 and SEQ ID NO. 28; The crRNA targeting the D6R gene has the sequence shown in SEQ ID NO. 5; The primer pairs used to amplify the E9L gene are selected from any of the following primer pairs: I. Primer pair 6 of the sequences shown in SEQ ID NO. 31 and SEQ ID NO. 35; II. Primer pair 7 of the sequences shown in SEQ ID NO. 31 and SEQ ID NO. 36; III. Primer pair 8 of the sequences shown in SEQ ID NO. 32 and SEQ ID NO. 34; IV. Primer pair 9 of the sequences shown in SEQ ID NO. 32 and SEQ ID NO. 35; and V. Primer pair 10 of the sequences shown in SEQ ID NO. 32 and SEQ ID NO. 36; The crRNA targeting the E9L gene is shown in SEQ ID NO.

13.

3. The primer composition according to claim 2, wherein: The primer pair used to amplify the D6R gene is: a. Primer pair 1 consisting of the sequences shown in SEQ ID NO. 25 and SEQ ID NO. 28; The crRNA targeting the D6R gene has the sequence shown in SEQ ID NO. 5; The primer pair used to amplify the E9L gene is: V. Primer pair 10 consisting of the sequences shown in SEQ ID NO. 32 and SEQ ID NO. 36; The crRNA targeting the E9L gene is shown in SEQ ID NO.

13.

4. A kit comprising the primer composition according to any one of claims 1-3.

5. The kit according to claim 4, wherein, It also contains the Cas12a protein, a signal reporter probe, and a detection system reaction buffer.

6. The kit according to claim 5, wherein, The signal reporting probe is a single-stranded DNA with a fluorescent group labeled at one end and a quenching group labeled at the other end.

7. The kit according to claim 6, wherein, The signal reporting probe is 5'-(FAM)TTATTATT(BHQ1)-3'.

8. The kit according to claim 5, wherein, It also contains RPA enzyme premix, magnesium acetate, and amplification reaction buffer.

9. Use of the primer composition according to any one of claims 1-3 or the kit according to any one of claims 4-8 in the development and / or preparation of products for detecting and / or diagnosing monkeypox virus.

10. A non-diagnostic method for detecting monkeypox virus, comprising the following steps: Sample extraction: Take the sample to be tested and extract the genome from it; One-step detection: Add the primer composition, RPA enzyme premix, magnesium acetate, amplification reaction buffer, Cas12a protein, signal reporter probe and detection system reaction buffer of the extracted sample genome as described in claim 1, and perform RPA amplification and CRISPR reaction detection simultaneously, and read the detection signal to interpret the detection result; The conditions for the first step of detection are: constant temperature 37±1℃ reaction for 20-40 min; The working concentrations of primer pairs used for amplifying the N3R gene and primer pairs used for amplifying the N4R gene are both 1–20 μM. The working concentrations of crRNA targeting the N3R gene and crRNA targeting the N4R gene were both 300–600 nM.

11. A non-diagnostic method for detecting monkeypox virus, comprising the following steps: Sample extraction: Take the sample to be tested and extract the genome from it; One-step detection: Add the primer composition, RPA enzyme premix, magnesium acetate, amplification reaction buffer, Cas12a protein, signal reporter probe and detection system reaction buffer as described in claim 2 or 3 to the genome of the sample to be tested obtained above, and perform RPA amplification and CRISPR reaction detection at the same time, and read the detection signal to interpret the detection result; The conditions for the first step of detection are: constant temperature 37±1℃ reaction for 20-40 min; The working concentrations of the primer pairs used for amplifying the N3R gene, the N4R gene, the D6R gene, and the E9L gene are all 1–20 μM. The working concentrations of crRNA targeting the N3R gene, crRNA targeting the N4R gene, crRNA targeting the D6R gene, and crRNA targeting the E9L gene were all 300–600 nM.

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