Primer probe combination for detecting monkeypox virus and its branches and application thereof

Abstract

This invention discloses a primer-probe combination for detecting monkeypox virus and its branches, and its application, belonging to the field of virus detection technology. Based on the monkeypox virus genome, this invention successfully designed a set of specific primers and probes capable of detecting monkeypox virus and its branches. Based on these primers and probes, this invention establishes a method for detecting monkeypox virus and its branches using multiplex real-time fluorescent PCR. This method can accurately detect the pathogen in clinical monkeypox virus samples, and the detection results are completely consistent with the typing results determined by genome sequencing. The detection method provided by this invention is low-cost, effectively reduces labor and time costs, and improves detection efficiency, showing promising application prospects in laboratory diagnosis, on-site detection, and health assessment of viruses.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, specifically relating to a primer-probe combination for detecting monkeypox virus and its branches and its application. Background Technology

[0002] Monkeypox (mpox) is a zoonotic disease caused by the mpox virus (MPXV), and there is currently no specific treatment. The mpoxvirus belongs to the genus Orthopoxvirus in the family Poxviridae. It is a linear double-stranded DNA virus with a genome length of approximately 197 kb. Monkeypox virus is divided into two evolutionary branches: the Central African or Congolese branch (I) and the West African branch (II). The Central African or Congolese branch (I) is further divided into branches Ia and Ib; the West African branch (II) is further divided into branches IIa and IIb, etc.

[0003] Branch Ia primarily affects women and children, with a high case fatality rate of approximately 10%. Branch Ib, first discovered in Africa in April 2024, is mainly transmitted through sexual contact and close contact within households, with a case fatality rate of approximately 3.2%. Children under 15 years old account for 66% of infections and 82% of all deaths. Studies indicate that Ib may have spilled over from animals to humans as early as September 2023, acquiring the ability to spread through sexual contact, primarily via heterosexual contact, general household contact, and indirect contact. The large proportion of children and family members among monkeypox infections suggests that branch Ib monkeypox virus is also highly contagious within close-contact families and among other family members, raising global concerns about a potential resurgence of monkeypox. Due to the differences in epidemiological characteristics and pathogenicity between branch Ib and branches Ia and IIb, effective differentiation between these branches is crucial for disease prevention and treatment.

[0004] Currently, the commonly used method for distinguishing different branches of monkeypox virus is genome sequencing. While this method boasts high accuracy and specificity, it suffers from drawbacks such as high technical barriers, cumbersome procedures, long detection cycles, and expensive reagents, making it unsuitable for rapidly differentiating different branches of monkeypox virus. Therefore, establishing a rapid, effective, and sequencing-independent method for identifying different circulating branches of monkeypox virus is of significant value in providing technical support for monkeypox prevention and control.

[0005] Real-time fluorescent quantitative PCR (RT-PCR), which emerged at the end of the last century, is a technology that adds fluorescent groups to the PCR reaction system and uses the accumulation of fluorescence signals to monitor the entire PCR process in real time. This technology not only achieves qualitative and quantitative analysis of the template, but also has the characteristics of high sensitivity, good specificity, real-time performance, and accuracy. Moreover, it can perform multiple reactions in one tube, and has significant application advantages in establishing identification and detection methods for different monkeypox virus branches.

[0006] This invention provides a multiplex real-time fluorescent PCR method that can simultaneously detect the universal type, Ia, Ib and II branches of monkeypox virus. It can be used to detect whether a sample contains monkeypox virus while simultaneously and rapidly identifying different epidemic branches of monkeypox virus. This provides important technical support for the simultaneous identification of the virus and the identification of monkeypox serotypes, and effectively improves the efficiency of epidemic response. Summary of the Invention

[0007] This invention was supported by the Beijing Municipal Science and Technology Project for Health Development (2022-2G-30115). Firstly, based on the monkeypox virus genome, this invention successfully designed and synthesized a set of specific primers and probes for detecting monkeypox virus and its branches, as well as a kit including the primers and probes. Based on these primers and probes, this invention established a one-step multiplex real-time fluorescent PCR detection method. This method can accurately detect monkeypox virus in clinical samples and pathogens that can cause rash-like symptoms, with the detection results showing 100% consistency with the typing results determined by genome sequencing. Compared with currently commercially available single-multiplex real-time fluorescent PCR detection kits, the detection method provided by this invention can not only detect whether a sample contains monkeypox virus, but also simultaneously identify its typing. The two results corroborate each other, ensuring more accurate and efficient detection results.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a primer-probe combination for detecting monkeypox virus and its branches, characterized in that the primer-probe combination consists of the primer-probe groups shown in (1)-(4):

[0010] (1) A first primer and probe set for detecting a universal type of monkeypox virus, including an upstream primer as shown in SEQ ID NO:1, a downstream primer as shown in SEQ ID NO:2, and a probe as shown in SEQ ID NO:3;

[0011] (2) A second primer and probe set for detecting monkeypox virus Ia branch, including an upstream primer as shown in SEQ ID NO:4, a downstream primer as shown in SEQ ID NO:5, and a probe as shown in SEQ ID NO:6;

[0012] (3) A third primer and probe set for detecting monkeypox virus Ib branch, including an upstream primer as shown in SEQ ID NO:7, a downstream primer as shown in SEQ ID NO:8, and a probe as shown in SEQ ID NO:9;

[0013] (4) A fourth primer and probe set for detecting monkeypox virus branch II, including an upstream primer as shown in SEQ ID NO:10, a downstream primer as shown in SEQ ID NO:11, and a probe as shown in SEQ ID NO:12.

[0014] Furthermore, the 5' end of the probe is labeled with a fluorescent reporter group, and the fluorescent reporting signals of the probes do not interfere with each other. This invention does not specifically limit the fluorescent reporter group; those skilled in the art can select the fluorescent reporter group labeled at the 5' end of the probe according to actual conditions. The 3' end of the probe is labeled with a fluorescence quencher group. This invention does not specifically limit the fluorescence quencher group; those skilled in the art can select an appropriate fluorescence quencher group based on the fluorescent reporter group.

[0015] The "signal non-interference" mentioned in this invention means that the fluorescent groups used by the probes are different and will not affect each other's detection, that is, different channels can be used for detection. For example, ROX, CY5, FAM and VIC can be used. The absorbance values ​​of these groups are not close, and different channels can be selected. The signals of different channels will not interfere with each other. In addition, the signals of each fluorescent channel can be distinguished by adding different probe concentrations, thereby forming different cluster signals, which can be further distinguished by cluster analysis software without mutual interference.

[0016] In a specific implementation of the present invention, the fluorescent reporter group labeled at the 5' end of the probe is selected from one of ROX, CY5, FAM, and VIC.

[0017] In one specific embodiment of the present invention, the probe 5' end is marked ROX as shown in SEQ NO:3; CY5 as shown in SEQ NO:6; FAM as shown in SEQ NO:9; and VIC as shown in SEQ NO:12.

[0018] In a second aspect, the present invention provides a kit for detecting monkeypox virus and its branches, characterized in that the kit comprises the primer-probe combination described in the first aspect of the present invention.

[0019] The kit also includes a positive template control, a negative template control, and reagents required for real-time quantitative PCR detection. These reagents include at least one of high-fidelity Taq enzyme, UDG enzyme, magnesium ions, and dNTPs.

[0020] The positive template control consists of plasmids containing specific amplification sequences of monkeypox virus universal type, Ia branch, Ib branch, and II branch, respectively.

[0021] The negative template control was RNase-free water.

[0022] Thirdly, the present invention provides an application of the primer-probe combination described in the first aspect of the present invention and / or the kit described in the second aspect of the present invention in at least one of the following:

[0023] a) Application in the preparation of products for detecting monkeypox virus;

[0024] b) Application in the preparation of products for identifying monkeypox virus branches.

[0025] The monkeypox virus branches include monkeypox virus branch Ia, monkeypox virus branch Ib, and monkeypox virus branch II.

[0026] The products include, but are not limited to, reagents, test strips, membrane strips, chips, or testing platforms.

[0027] Fourthly, the present invention provides a method for detecting monkeypox virus and its branches based on multiplex real-time fluorescence PCR, wherein the method is not intended for disease diagnosis, and is characterized by comprising the following steps:

[0028] (1) Extract DNA or RNA from the sample to be tested;

[0029] (2) Configure a PCR amplification system containing the primer-probe combination described in the first aspect of the present invention;

[0030] (3) Set up four fluorescence channels for PCR amplification reaction;

[0031] (4) Determine whether the sample to be tested contains monkeypox virus and its branches based on the Ct value and / or amplification curve.

[0032] Preferably, in the PCR amplification system described in step (2), the final concentration of the primers is selected from 200-300 nM, and the final concentration of the probe is selected from 100-150 nM.

[0033] In a specific embodiment of the present invention, the result determination criteria are as follows: when the Ct value corresponding to the fluorescence channel is ≤38 and there is an obvious amplification curve, it is determined to be a positive result; when the Ct value is ≥38 or there is no obvious amplification curve, it is determined to be a negative result; specifically,

[0034] 1. When the result of the positive template control is negative or the result of the negative template control is positive, the test is deemed invalid;

[0035] 2. When the result of the positive template control is positive and the result of the negative template control is negative, the test is deemed valid, and the following determinations are made:

[0036] 2.1. All fluorescence channels showed negative results, indicating that the sample did not contain monkeypox virus;

[0037] 2.2. If the fluorescence channel corresponding to the general type is positive and other channels are negative, the sample is determined to contain monkeypox virus, but not from monkeypox virus Ia, Ib, or II branches;

[0038] 2.3. If the fluorescence channels corresponding to the general type and the Ia branch are positive, and the other channels are negative, the sample is determined to contain monkeypox virus, and it is monkeypox virus Ia branch;

[0039] 2.4. If the fluorescence channels corresponding to the general type and the Ib branch are positive, and the other channels are negative, the sample is determined to contain monkeypox virus, and it is monkeypox virus Ib branch;

[0040] 2.5. If the fluorescence channels corresponding to the general type and the II branch are positive, and the other channels are negative, the sample is determined to contain monkeypox virus, and it is monkeypox virus II branch.

[0041] The detection method described in this invention is not only applicable to the detection of samples from patients (humans or animals), but also includes the detection of various samples in the environment, such as water samples, food samples, air samples, microbial samples, and animal cell samples, for non-disease diagnostic purposes.

[0042] The technical solution provided by this invention has the following advantages based on existing technologies:

[0043] Monkeypox virus branch Ib was first discovered in Africa in April 2024. Investigations revealed its high transmissibility within close-contact families and among other members, indicating a potential for re-emergence. Furthermore, due to differences in epidemiological characteristics and pathogenicity between branch Ib and branches Ia and IIb, effective differentiation of these branches using non-sequencing methods is crucial for disease prevention and treatment. Against this backdrop, this invention provides a primer-probe combination that can effectively differentiate between monkeypox virus branches Ia, Ib, and II. Building upon this, the invention also incorporates universal primers and probes for detecting monkeypox virus into the detection system. This allows for simultaneous and rapid identification of different prevalent branches of monkeypox virus while simultaneously detecting the presence of the virus in a sample, providing crucial technical support for simultaneous virus identification and monkeypox serotype determination, and effectively improving the efficiency of epidemic response.

[0044] Furthermore, this invention establishes a method for detecting monkeypox virus and its branches based on multiplex real-time fluorescence PCR. This method can accurately detect the pathogen in clinical monkeypox virus samples, and the detection results are completely consistent with the typing results determined by genome sequencing. In addition to being highly specific, sensitive, efficient, and stable, the method for detecting monkeypox virus and its branches established in this invention is simple and rapid to operate, requiring no special instruments or procedures. The amount of viral DNA required for detection is less than that of singleton PCR with multiple reaction systems, resulting in lower detection costs, effectively reducing labor and time costs, and improving detection efficiency. It shows promising application prospects in laboratory diagnosis, on-site detection, and health assessment of viruses. Attached Figure Description

[0045] Figure 1 Design sites for universal detection primers and probes for monkeypox virus.

[0046] Figure 2 Primer and probe design sites for monkeypox virus Ia branch detection.

[0047] Figure 3 Primer and probe design sites for monkeypox virus Ib branch detection.

[0048] Figure 4 Design sites for primers and probes for monkeypox virus clade II detection.

[0049] Figure 5 Specific detection results of a one-step multiplex real-time fluorescence PCR detection method.

[0050] Figure 6 Sensitivity detection results of the one-step multiplex real-time fluorescence PCR detection method; A: Monkeypox virus universal type; B: Monkeypox virus Ia branch; C: Monkeypox virus Ib branch; D: Monkeypox virus II branch.

[0051] Figure 7 PCR amplification diagram of candidate primers and probes for universal detection of monkeypox virus: A: MPXV_OPG002; B: MPXV_OPG210; C: MPXV_OPG065; D: MPXV_OPG195.

[0052] Figure 8 PCR amplification diagram of candidate primers and probes for monkeypox virus clade II detection, A: II_OPG002; B: II_OPG031-034.

[0053] Figure 9 PCR amplification diagram of candidate primers and probes for monkeypox virus Ia branch detection: A: Ia-OPG037-038; B: Ia_OPG032; C: Ia_OPG032-1.

[0054] Figure 10 PCR amplification diagram of candidate primers and probes for monkeypox virus Ib branch detection: A: Ib_ΔOPG032; B: Ib_OPG045; C: Ib_OPG190-191. Detailed Implementation

[0055] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] Unless otherwise specified, the experimental methods described in the following examples are generally performed under conventional conditions and methods, such as those described in the Molecular Cloning Laboratory Manual (Sambrook, et al. New York: Cold Spring Harbor Laboratory Press, 1989) or those provided by the reagent manufacturers.

[0057] This invention first selects and determines the specific gene loci of the universal, Ia, Ib, and II branches of monkeypox virus. Specific primer and probe sequences are designed for these gene loci using the Primer 3Plus online website. The optimal reaction system and PCR amplification procedure are determined, and experiments are conducted to verify sensitivity, specificity, and stability. A rapid multiplex real-time fluorescence PCR typing method for the universal, Ia, Ib, and II branches of monkeypox virus has been successfully established.

[0058] Example 1: Design and synthesis of primers and probes for specific sites of monkeypox virus universal, Ia, Ib and II clades.

[0059] 1.1 Selection of specific sites in monkeypox virus universal, Ia, Ib and II clades

[0060] The sequences used in this invention were obtained from the GISAID (https: / / gisaid.org / ) public database. The main selection criteria were: (a) sequences from different prevalent clades with recent sampling times; (b) sequences with high coverage and full-length genome sequences; (c) sequences with high evaluation scores on the Nextclade (https: / / clades.nextstrain.org / ) website; and (d) representative viral sequences selected after alignment of sequences from different clades. The multiple sequence ratio results of representative strains from the universal, Ia, Ib, and II clades of monkeypox virus selected in this invention are as follows: Figure 1-4 As shown.

[0061] Specifically, in this invention, the genome sequences of representative sequences from the selected universal serotype, Ia, Ib, and II branches of monkeypox virus were input into MAFFT software for multiple sequence alignment. For the universal serotype, conserved gene sites were screened as target genes for the design of specific primers and probes. For different branches of the virus, differentially expressed sites between branches were screened as target genes for the design of specific real-time fluorescent PCR primers and probes. Ultimately, the gene selected by this invention for detecting the universal serotype of monkeypox virus is the OPG210 gene, and the genes used to distinguish between branches Ia, Ib, and II of monkeypox virus are the OPG032 gene, ΔOPG032 gene, and OPG002 gene, respectively.

[0062] 1.2 Design and Synthesis of Primers and Probes

[0063] Primers and probes were designed using the online primer design website Primer3plus (https: / / www.primer3plus.com) to target universal conserved gene sites of monkeypox virus and genomically different sites among different branches of the virus. Parameters such as GC content and melting temperature of the primers and probes were evaluated. The designed universal, Ia, Ib, and II branch-specific primer and probe sequences for monkeypox virus were synthesized by Qingke Biotechnology Co., Ltd. All primers and probes synthesized in this invention were purified using HPLC methods to achieve higher purity. The primers and probes provided in this invention for detecting monkeypox virus and its branches are shown in Table 1.

[0064] Table 1. Primer and probe sequences and labeled fluorescent groups for detecting monkeypox virus and its branches.

[0065] ;

[0066] Note: Unless otherwise specified, “F” in the table represents the upstream primer; “R” represents the downstream primer; and “P” represents the probe.

[0067] Example 2: A method for detecting monkeypox virus and its branches based on multiplex real-time fluorescence PCR

[0068] (1) Extracting nucleic acid from the sample

[0069] Nucleic acid was extracted from the sample using the DNeasy Blood & Tissue Kit from Qiagen, Germany.

[0070] (2) Preparation of PCR reaction system

[0071] .

[0072] The PCR premix used was ABI's TaqMan™ Fast Advanced premix (P / N: 4444557).

[0073] The primer-probe mixture was prepared by the following method: the primers and probes provided in Table 1 were mixed at a volume ratio of 2:1 to form the primer-probe mixture. 7.5 μL of the mixture was added to the PCR reaction system to make the final concentration of the primers 300 nM and the final concentration of the probes 150 nM.

[0074] The final concentration of the DNA template was 1 pg - 4 ng.

[0075] The positive template control was prepared by adding 5 μL of a mixture of four positive templates in equal volumes to the PCR reaction system, with each template being added after a 2-fold serial dilution.

[0076] The negative template control was RNase-free water.

[0077] (3) PCR amplification reaction

[0078] Amplification was performed using an ABI Q7 real-time quantitative PCR instrument, and the procedure is as follows:

[0079] First, set up four fluorescence acquisition channels: FAM, VIC, ROX, and CY5.

[0080] UNG enzyme incubation: 50℃, 2 min;

[0081] Polymerase activation: 95℃, 20 s;

[0082] There are 40 cycles in total.

[0083] (4) The result judgment criteria are: Ct value ≤ 38, judged as positive;

[0084] A Ct value ≥ 38 or no obvious amplification curve indicates a negative result.

[0085] Example 3 Specificity detection of a one-step multiplex fluorescent PCR detection method

[0086] The multiplex real-time fluorescence PCR reaction system established in Example 2 was used to detect positive plasmid templates for monkeypox virus universal type Ia, Ib, and II branches, as well as positive samples from seven other common pathogens (SARS-CoV-2, varicella-zoster virus, dengue virus, influenza A virus, influenza B virus, enterovirus, and respiratory syncytial virus). The detection results are as follows: Figure 5As shown, the results indicate that only the universal type, Ia, Ib, and II branches of monkeypox virus showed corresponding specific fluorescence amplification curves in the FAM, VIC, ROX, and CY5 detection channels, respectively, and no cross-reaction was observed between the branches. In contrast, no amplification curves were observed for the other seven virus samples, indicating that the method has good specificity.

[0087] Example 4 Sensitivity detection of a one-step multiplex fluorescent PCR method

[0088] The corresponding PCR amplification region sequences of the universal, Ia, Ib, and II branches of monkeypox virus designed in this invention were artificially synthesized. These sequence fragments were inserted into the pUC57 E. coli cloning vector plasmid using gene cloning methods. After amplification culture, the plasmids were purified and extracted, and named pUC57-universal, pUC57-Ia, pUC57-Ib, and pUC57-II, respectively. The plasmids were sent to Qingke Biotechnology Co., Ltd. for sequencing confirmation. The correctly sequenced plasmids were used to establish a standard curve for the multiplex real-time fluorescence PCR detection method.

[0089] All positive plasmids were quantified using a NanoDrop micro spectrophotometer (model: ND-1000). After quantification, the plasmid mass was converted to copy number based on the base number, and 1.25 × 10⁻⁶ copies were taken from each. 2 2.5×10 2 5×10 2 1×10 3 2×10 3 4×10 3 8×10 3 1.6×10 4 3.2×10 4 6.4×10 4 1.28×10 5 2.56×10 5 5.12×10 5 1.024×10 6 and 2.048×10 6 Recombinant plasmids of 1 copy / μL were used as standard templates and added to the one-step multiplex real-time fluorescence PCR reaction system established in Example 2 for amplification. Standard curves were established based on the Ct value and the logarithm of the template concentration.

[0090] Figure 6 The results showed that the copy number logarithmic values ​​of pUC57-general, pUC57-Ia, pUC57-Ib, and pUC57-II positive plasmids had a good linear relationship with the Ct value, and the detection limit was approximately 250 copies / mL after conversion, indicating that the one-step multiplex real-time fluorescence PCR detection method established in this invention has high sensitivity.

[0091] Example 5: Clinical Sample Validation of a One-Step Multiplex Real-Time Fluorescence PCR Method

[0092] Clinical monkeypox virus samples of different types and viral concentrations, whose genotypes were determined by genome sequencing, were selected as sample test trays. These trays covered common sample types and samples with different viral concentrations encountered in actual epidemic prevention and control. The multiplex real-time fluorescence PCR method established in Example 2 was used to detect the universal and genotyped monkeypox virus in the sample trays, and the results are shown in Table 2.

[0093] Table 2 Results of clinical sample testing for monkeypox virus

[0094]

[0095] The results show that the multiplex real-time fluorescence PCR method established in this invention can correctly detect the universal type of monkeypox virus, with 100% specificity and consistency compared to gene sequencing results. Furthermore, the method provided in this invention can successfully identify specific branches of monkeypox virus, with 100% consistency with standard genotyping methods based on genome sequencing.

[0096] Example 6: Optimization process of primers and probes for detecting monkeypox virus and its branches

[0097] Those skilled in the art will understand that the core key to designing universal, Ia, Ib, and II branch-specific primers and probes for monkeypox virus lies in the selection of specific gene loci. The process of screening conserved gene loci and differentially expressed loci among different branches of monkeypox virus in this invention is as follows: Figure 1-4 As shown, the conserved gene finally obtained for detecting monkeypox virus is the OPG210 gene, and the genes used to distinguish between branches Ia, Ib and II are the OPG032 gene, ΔOPG032 gene and OPG002 gene, respectively.

[0098] Typically, after identifying the target gene, those skilled in the art use primer design software to design multiple pairs of backup primers for all target genes or DNA fragments within target genes. All backup primers are subjected to BLAST analysis to screen for primer schemes with high specificity as experimental validation schemes. Usually, 2-3 primer pairs are retained for each target gene for subsequent experiments. Each experimental validation scheme is validated using singleton PCR to determine primer usability, including specificity and sensitivity assessments, and unsuccessful backup primers are eliminated. All validated primers are then mixed for multiplex PCR to verify their usability during co-amplification, and primer pairs that form primer dimers or fail to correctly detect positive samples are eliminated. Based on the above process, the present invention ultimately screens the optimal primer-probe combinations shown in Table 1. Exemplarily, the present invention will provide the following eliminated or optimized primer probes to illustrate the process of obtaining the primer-probe combinations shown in Table 1.

[0099] (1) Screening process for target genes

[0100] Example 1: Screening and optimization process of target genes and primers / probes for universal detection of monkeypox virus: The candidate target genes initially designed for universal detection of monkeypox virus in this invention include OPG002, OPG210, OPG065 and OPG195, and multiple pairs of primers and probes are designed for each target gene, with representative sequences shown in Table 3.

[0101] Table 3. Candidate primer and probe sequences for MPXV universal detection

[0102] .

[0103] During the specific screening process, the inventors found that primer-probe combinations targeting the target genes OPG002, OPG065, and OPG195 all failed to correctly detect positive samples (Table 4). Figure 7 Ultimately, the present invention preferably uses the MPXVOPG210 primer-probe combination as the best universal primer-probe combination.

[0104] Table 4. Screening Results of MPXV Universal Detection Primers and Probes

[0105]

[0106] Example 2, regarding the screening and optimization process of primers and probes for detecting monkeypox virus branch II: The target genes initially designed for the detection of monkeypox virus branch II in this invention include OPG002 and OPG031-034. Multiple pairs of primers and probes were designed for these target genes, and the representative sequences are shown in Table 5.

[0107] Table 5 Candidate sequences for II branch detection primers and probes

[0108] .

[0109] The screening results are shown in Table 6 and Figure 8 As shown, the primer-probe combination targeting the OPG031-034 failed to detect positive samples correctly. Ultimately, the II_OPG002 primer-probe combination was selected as the best primer-probe combination for the II branch detection.

[0110] Table 6. Screening results of candidate sequences for II-branch detection primers and probes.

[0111] .

[0112] Example 3, regarding the screening and optimization process of primers and probes for detecting monkeypox virus Ia branch: The target genes initially designed for the detection of monkeypox virus Ia branch in this invention include OPG037-038, OPG032 and OPG032-1, and multiple pairs of primers and probes are designed for each target gene, with representative sequences shown in Table 7.

[0113] Table 7 Candidate sequences for the Ia branch primer and probe

[0114] .

[0115] The screening results are shown in Table 8 and Figure 9 As shown, both primer-probe combinations targeting the target genes OPG037-038 and OPG032-1 resulted in false positives due to misclassification, indicating low specificity. Ultimately, the Ia_OPG032 primer-probe combination was selected as the optimal primer-probe combination for Ia branch detection.

[0116] Table 8. Screening results of candidate sequences for Ia branch detection primers and probes

[0117] .

[0118] Example 4, regarding the screening and optimization process of primers and probes for detecting monkeypox virus Ib: The target genes initially designed for the detection of monkeypox virus Ib in this invention include ΔOPG032, OPG045 and OPG190-191, and the specific primer and probe sequences are shown in Table 9.

[0119] Table 9 Candidate sequences for Ib branched primers and probes

[0120] .

[0121] The screening results are shown in Table 10. Figure 10As shown, during the screening of optimal primers and probes for detecting monkeypox virus Ib branch, the primer and probe combination targeting the target genes OPG045 and OPG190-191 showed low sensitivity in failing to correctly genotype all four Ib-positive samples, while Ib_ΔOPG032 was correctly genotyped in all cases. Ultimately, the ΔOPG032 primer and probe combination was selected as the optimal primer and probe combination for detecting Ib branch.

[0122] Table 10. Screening results of candidate sequences for Ib branched primers and probes

[0123] .

[0124] (2) The process of screening the best primers and probes

[0125] After determining that the optimal detection gene locus for the MPXV universal type is OPG210, the optimal detection gene locus for the II branch is OPG002, the optimal detection locus for the Ia branch is OPG032, and the optimal detection locus for the Ib branch is ΔOPG032, technicians usually further optimize the primers and probes for these gene loci. This includes performing singleton PCR validation on each primer pair to determine primer availability, specificity assessment, and sensitivity assessment, and eliminating unsatisfactory backup primers; and performing multiplex PCR on all validated primers to verify their availability during co-amplification, and eliminating primer pairs that form primer dimers or fail to correctly detect positive samples.

[0126] This invention designs multiple primer-probe pairs for each of the above-mentioned optimal gene loci. Blast analysis is performed on all designed primer pairs to screen for 2-3 primer pairs with high specificity for experimental validation. Specifically, the primer-probe combinations retained for experimental validation in this invention are shown in the table below:

[0127] Table 11 Candidate primer and probe sequences

[0128] .

[0129] First, technicians performed singleton PCR verification on each primer-probe pair provided in the table above. They found that each primer-probe pair could amplify the target gene; however, some primer-probe signals were poor, and these primer-probe combinations, including those shown in SEQ ID NO:37-39, SEQ ID NO:46-48, SEQ ID NO:52-54, and SEQ ID NO:55-60, were deleted. The remaining verified primer-probe combinations were then mixed according to their detection sites and subjected to multiplex PCR to verify their usability during co-amplification. Combinations that formed primer dimers or failed to correctly detect positive samples were eliminated. After comprehensive verification, the optimal combination of this invention is shown in Table 1. This combination does not form primer dimers, and the detection results are completely consistent with the gold standard gene sequencing results. Therefore, the primers and probes screened by this invention for detecting monkeypox virus and its branches consist of SEQ ID NO:1-12.

[0130] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify or make equivalent substitutions to the technical solutions described in the foregoing embodiments. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer-probe combination for detecting monkeypox virus and its branches, characterized in that, The primer-probe combination consists of the primer-probe groups shown in (1)-(4): (1) A first primer and probe set for detecting a universal type of monkeypox virus, including an upstream primer as shown in SEQ ID NO:1, a downstream primer as shown in SEQ ID NO:2, and a probe as shown in SEQ ID NO:3; (2) A second primer and probe set for detecting monkeypox virus Ia branch, including an upstream primer as shown in SEQ ID NO:4, a downstream primer as shown in SEQ ID NO:5, and a probe as shown in SEQ ID NO:6; (3) A third primer and probe set for detecting monkeypox virus Ib branch, including an upstream primer as shown in SEQ ID NO:7, a downstream primer as shown in SEQ ID NO:8, and a probe as shown in SEQ ID NO:9; (4) A fourth primer and probe set for detecting monkeypox virus branch II, including an upstream primer as shown in SEQ ID NO:10, a downstream primer as shown in SEQ ID NO:11, and a probe as shown in SEQ ID NO:

12.

2. The primer-probe combination according to claim 1, characterized in that, The probe has a fluorescent reporter group labeled at its 5' end, and the fluorescent reporter signals of the probe do not interfere with each other. The probe has a fluorescent quencher group labeled at its 3' end.

3. The primer-probe combination according to claim 2, characterized in that, The fluorescent reporter group labeled at the 5' end of the probe is selected from one of ROX, CY5, FAM, and VIC.

4. The primer-probe combination according to claim 3, characterized in that, The probe 5' end is marked ROX as shown in SEQ NO:3; the probe 5' end is marked CY5 as shown in SEQ NO:6; the probe 5' end is marked FAM as shown in SEQ NO:9; and the probe 5' end is marked VIC as shown in SEQ NO:

12.

5. A kit for detecting monkeypox virus and its branches, characterized in that, The kit comprises the primer-probe combination as described in any one of claims 1-4.

6. The reagent kit according to claim 5, characterized in that, The kit also includes a positive template control, a negative template control, and reagents required for real-time quantitative PCR detection.

7. The reagent kit according to claim 6, characterized in that, The reagents include at least one of high-fidelity Taq enzyme, UDG enzyme, magnesium ions, and dNTPs.

8. The use of the primer-probe combination according to any one of claims 1-4 and / or the kit according to any one of claims 5-7 in at least one of the following: a) Application in the preparation of products for detecting monkeypox virus; b) Application in the preparation of products for identifying monkeypox virus branches; The monkeypox virus branches include monkeypox virus branch Ia, monkeypox virus branch Ib, and monkeypox virus branch II.

9. A method for detecting monkeypox virus and its branches based on multiplex real-time fluorescence PCR, wherein the method is not intended for disease diagnosis, characterized in that... The method includes the following steps: (1) Extract DNA or RNA from the sample to be tested; (2) Configure a PCR amplification system containing the primer-probe combination according to any one of claims 1-4; (3) Set up four fluorescence channels for PCR amplification reaction; (4) Determine whether the sample to be tested contains monkeypox virus and its branches based on the Ct value and / or amplification curve.

10. The method according to claim 9, characterized in that, In the PCR amplification system described in step (2), the final concentration of the primers is selected from 200-300 nM, and the final concentration of the probe is selected from 100-150 nM.

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