Composition for detecting typing of type I and type II monkey pox viruses as well as typing detection kit and application thereof

Abstract

The invention discloses a composition for detecting type I and type II monkey pox virus typing and a typing detection kit and application thereof, the composition comprises an LNA-Taqman probe for detecting the monkey pox virus I, the nucleotide sequence of the LNA-Taqman probe is shown as SEQ ID NO. 1, the nucleotide sequence of the LNA-Taqman probe is shown as SEQ ID NO. 2, and the nucleotide sequence of the LNA-Taqman probe is shown as SEQ ID NO. The nucleotide sequence of the upstream primer for typing detection of the monkey pox virus is shown as SEQ ID NO. 2; and a monkey pox virus typing detection downstream primer, the nucleotide sequence of which is as shown in SEQ ID NO. 3. According to the invention, the characteristic that the hybridization stability is sharply reduced when a locked nucleic acid (LNA) probe is mismatched with a single base is utilized, and an LNA monomer is accurately designed at a monkey pox virus typing SNP key site of the probe. By means of the design, typing signal differences can be remarkably amplified, and highly homologous virus branches can be effectively distinguished. The problems of cross reaction and false positive caused by high mismatching tolerance of a traditional DNA probe are avoided, and the accuracy of typing identification is ensured.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a composition for detecting type I and II monkeypox virus typing, its typing detection kit, and its application. Background Technology

[0002] Monkeypox is a zoonotic disease caused by the monkeypox virus, which belongs to the Poxviridae family (…). Poxviridae ) genus of poxvirus ( Orthopoxvirus The World Health Organization (WHO) has declared it a potential public health emergency. Based on significant differences in genomics and pathogenicity, monkeypoxviruses are mainly divided into two distinct evolutionary branches: Branch I (Central African Basin Branch) and Branch II (West African Branch).

[0003] Branch I is highly pathogenic. Historical data shows its case fatality rate (CFR) can be as high as 10%, especially in unvaccinated children and immunocompromised individuals. Patients infected with this virus typically develop high fever, severe headache, generalized lymphadenopathy, and a widespread, dense rash. Severe cases are often accompanied by serious complications such as secondary bacterial infections, pneumonia, and encephalitis. Branch II, on the other hand, is relatively less virulent. Its case fatality rate is usually less than 1%. Clinical manifestations are generally milder, with possible prodromal fever or mild symptoms. The rash is fewer in number and often localized to the genital, perianal, or oral regions.

[0004] Currently, most detection methods for monkeypox virus are general-purpose methods. Guidelines and standards such as the "Technical Guidelines for Monkeypox Prevention and Control (2022)" are general-purpose monkeypox virus detection methods and cannot further distinguish specific types of monkeypox virus.

[0005] Currently, detection methods for monkeypox virus typing include real-time quantitative PCR (qPCR), which is the gold standard for routine laboratory testing and has the advantages of speed and sensitivity; however, its detection effect is highly dependent on the specificity of primers and probes, and false negatives may occur when the virus undergoes genetic mutations. Furthermore, its relative quantification method based on standard curves is insufficient for precise typing and distinguishing between minute differences. Loop-mediated isothermal amplification (LAMP) requires simple equipment and is suitable for rapid on-site screening; however, its reaction is prone to non-specific amplification, which may lead to false positive results, and it is currently mostly used for qualitative or semi-quantitative detection, limiting its application in complex scenarios requiring precise typing. Droplet digital PCR (ddPCR) possesses absolute quantification capability and high tolerance to inhibitors, making it particularly suitable for precise quantification of low viral loads; however, its instrument and consumable costs are high, the operation procedure is relatively complex, and the throughput is low, making it difficult to use as a routine method for large-scale typing screening. High-throughput whole genome sequencing (NGS) technology can provide the most comprehensive genomic information and is the ultimate tool for typing, tracing, and mutation research; however, it is extremely expensive, data analysis is complex and time-consuming, and it has high requirements for sample quality and viral load. It is usually used for key cases or scientific research analysis and cannot meet the needs of rapid, high-throughput routine typing diagnosis.

[0006] Given the significant differences in virulence and clinical manifestations among different branches of monkeypox virus, developing a genotyping detection method can be used for precise clinical diagnosis and treatment, disease prevention and risk assessment, and can help relevant departments formulate differentiated isolation, vaccination and resource allocation strategies.

[0007] Therefore, developing a typing detection method that can quickly and accurately distinguish between monkeypox virus branches I and II is of great significance for achieving individualized precision treatment in clinical practice and improving the effectiveness of public health prevention and control. Summary of the Invention

[0008] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0009] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0010] Therefore, the object of the present invention is to overcome the shortcomings of the prior art and provide a composition for detecting monkeypox virus type I and II.

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a composition for detecting monkeypox virus type I and II, comprising an LNA-Taqman probe for detecting monkeypox virus I, the nucleic acid sequence of which is shown in SEQ ID NO. 1; The upstream primer for monkeypox virus typing detection has the nucleic acid sequence shown in SEQ ID NO. 2; Additionally, the downstream primers for monkeypox virus typing detection have the nucleic acid sequence shown in SEQ ID NO. 3.

[0012] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of the composition in the preparation of a kit for detecting monkeypox virus types I and II.

[0013] Another object of the present invention is to overcome the shortcomings of the prior art and provide a kit for detecting monkeypox virus types I and II, comprising the aforementioned composition.

[0014] As a preferred embodiment of the kit described in this invention, it further includes a real-time PCR premix, a positive control, a negative control, and water.

[0015] In a preferred embodiment of the kit described in this invention, the positive control comprises monkeypox virus I plasmid with a copy number of 6.90 × 10⁻⁶. 7 The negative control includes a monkeypox virus type II negative detection plasmid with a copy number of 6.57 × 10⁻⁶ μL. 7 copies / μL.

[0016] As a preferred embodiment of the kit described in this invention, the following components are included: the premixed solution for real-time PCR is 12.5 μL, the upstream primer concentration is 10 μM, the upstream primer volume is 1 μL, the downstream primer concentration is 10 μM, the downstream primer volume is 1 μL, the LNA-Taqman probe concentration for detecting monkeypox virus I is 10 μM, the probe volume is 1 μL, the positive control volume is 5 μL, the water volume is 4.5 μL, and the total volume is 25 μL.

[0017] As a preferred embodiment of the reagent kit described in this invention, the detection limit of the reagent kit reaches 6.90 copies / μL.

[0018] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for using a kit for detecting monkeypox virus types I and II, comprising assembling the raw materials into a kit and performing a real-time PCR reaction, wherein the reaction program is 95°C for 20s; 95°C for 15s; 62°C for 45s, for a total of 40 cycles.

[0019] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for detecting monkeypox virus types I and II based on LNA for non-therapeutic diseases, including detecting monkeypox virus types I and II by quantitative real-time PCR technology. The reaction system consisted of: 12.5 μL of 2×Probe qPCR Mix MultiPlus, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 1 μL of LNA-Taqman probe (10 μM), 5 μL of DNA template, 0.5 μL of ROX Reference Dye II (100×), and 4 μL of sterile enzyme-free water, for a total reaction volume of 25 μL. The reaction program was: 95℃, 20s; 95℃, 15s; 62℃, 45s, for a total of 40 cycles.

[0020] In a preferred embodiment of the typing detection method of the present invention, the DNA template includes monkeypox virus I plasmid and monkeypox virus II plasmid, with copy numbers of 6.90 × 10⁻⁶ and 6.90 × 10⁻⁶, respectively. 7 copies / μL, 6.57×10 7 copies / μL.

[0021] Beneficial effects of this invention: (1) High detection specificity and accurate and reliable typing results: This invention utilizes the characteristic that the hybridization stability of locked nucleic acid (LNA) probes decreases sharply when there is a single base mismatch, and precisely designs LNA monomers at the key SNP sites of monkeypox virus typing on the probe. This design can significantly amplify the differences in typing signals and effectively distinguish highly homologous viral branches. It avoids the cross-reaction and false positive problems caused by the high mismatch tolerance of traditional DNA probes, and ensures the accuracy of typing identification.

[0022] (2) High detection sensitivity, suitable for low viral load samples: The introduction of LNA significantly increases the hybridization affinity Tm value between the probe and the target sequence, so that the probe used in this invention can achieve stable and efficient hybridization and signal detection even when the viral template concentration is low, thereby greatly improving the sensitivity of the detection method and reducing the risk of missed detection.

[0023] (3) The method is rapid, simple, and compatible with conventional technologies: The detection system established by this invention is based on mature real-time fluorescence PCR technology; no complicated sample pretreatment or additional operation steps are required. The entire detection process can be completed in 1.5 to 2 hours, realizing high-throughput and rapid detection from sample to result, which is very suitable for rapid clinical diagnosis and large-scale epidemiological screening.

[0024] (4) Overcoming secondary structure interference and strong stability: The strong hybridization ability of LNA probes enables them to effectively compete for and open complex secondary structures such as stem loops that may exist in the target DNA sequence, thereby ensuring the smooth binding of the probe to the target sequence, improving the robustness of detection, and reducing detection failure or sensitivity reduction caused by template structure problems. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a map of SNP detection sites for branches I and II of monkeypox virus in an embodiment of the present invention, wherein the bases of SNPs (Ia and Ib) in branch I are G, and the bases of SNPs in branch II are A.

[0026] Figure 2 This is a schematic diagram showing the sequence information of monkeypox virus branches I and II, as well as the positions of the locking nucleic acid probes and primers in an embodiment of the present invention.

[0027] Figure 3 Figure A shows the verification results of locked nucleic acid probes and primers for monkeypox virus branches I and II in this embodiment of the invention. Figure A is an electrophoresis diagram verifying the annealing temperature range of the primers for monkeypox virus branches I and II. Lane 1 is annealing temperature 62℃; lane 2 is annealing temperature 63℃; lane 3 is annealing temperature 64℃; lane 4 is annealing temperature 65℃; lane 5 is annealing temperature 66℃; lane 6 is annealing temperature 67℃; lane 7 is annealing temperature 68℃; lane 8 is the negative control. Figure B is an electrophoresis diagram verifying the annealing temperature range of the locked nucleic acid probes for monkeypox virus branches I and II. Lane 2 is annealing temperature 62℃; lane 3 is annealing temperature 63℃; lane 4 is annealing temperature 64℃; lane 5 is annealing temperature 65℃; lane 6 is annealing temperature 66℃; lane 7 is annealing temperature 67℃; lane 8 is annealing temperature 68℃; lane 9 is the negative control.

[0028] Figure 4 This is a diagram showing the locations of digital PCR primers and probes for monkeypox virus branches I and II in this embodiment of the invention.

[0029] Figure 5 This is a scatter plot of digital PCR quantification of plasmids PUC57-Ⅰ and PUC57-Ⅱ in the embodiments of the present invention.

[0030] Figure 6These are amplification curves for monkeypox virus branches I and II based on locked nucleic acid probes in this embodiment of the invention. Figures A to E show the typing results of monkeypox virus branches I and II using locked nucleic acid probes under different annealing temperatures. The horizontal axis in each figure represents the cycle number, and the vertical axis represents the fluorescence signal intensity. Figure A shows the typing results at an annealing temperature of 60℃; Figure B shows the typing results at an annealing temperature of 62℃; Figure C shows the typing results at an annealing temperature of 64℃; Figure D shows the typing results at an annealing temperature of 66℃; and Figure E shows the typing results at an annealing temperature of 67℃.

[0031] Figure 7 The figure shows the amplification curve of the sensitivity test based on the LNA-Taqman monkeypox virus I typing method in the embodiments of the present invention. The horizontal axis is the cycle number and the vertical axis is the fluorescence signal intensity. Different colored curves in the figure represent samples with different copy numbers. The others not marked are negative controls and monkeypox virus II branches.

[0032] Figure 8 This is an amplification curve of the sensitivity test based on the LNA-Taqman monkeypox virus II typing method in an embodiment of the present invention.

[0033] Figure 9 This is a specific amplification curve based on the LNA-Taqman monkeypox virus I typing method in this embodiment of the invention. The horizontal axis represents the cycle number, and the vertical axis represents the fluorescence signal intensity. Different curves in the figure represent different samples: Branch I has been marked in the figure, and the remaining unmarked amplification curves are the negative control, monkeypox virus Branch II, DNA of 4 bacteria, cDNA of 4 viruses, monkey VERO cell nucleic acid, and human HEK 293 cell nucleic acid.

[0034] Figure 10 This is a diagram showing SNP site information in the comparative examples of this invention.

[0035] Figure 11 This is a sequence information diagram in the comparative example of the present invention.

[0036] Figure 12 This is a diagram showing the typing failure results of monkeypox virus type I and type II in the comparative examples of this invention. Detailed Implementation

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0038] Currently, several technologies used for monkeypox virus typing detection have technical drawbacks such as limited sample quantity, poor specificity, low sensitivity, high cost, and long processing time.

[0039] For some genotyping assays targeting highly homologous monkeypox viruses, traditional gold standard methods such as PCR and qPCR face significant challenges. The two branches of the monkeypox virus genome have highly conserved sequences, and genotyping relies on a few specific single nucleotide polymorphism (SNP) sites. When using conventional DNA or RNA oligonucleotide probes, it is often difficult to distinguish these minute sequence differences. Even when the target sequence and non-target sequence have only one or a few base mismatches, conventional probes may still undergo non-specific hybridization, leading to false positives or cross-reactions. This phenomenon severely affects the accuracy and reliability of genotyping results. Furthermore, the complex secondary structures that may exist in the viral genome can also hinder the effective binding of probes to target sequences, reducing detection sensitivity.

[0040] Example 1 LNA detection system established: (1) SNP detection site analysis and plasmid synthesis of monkeypox virus I and II branches Based on the whole-genome nucleic acid sequence information of the GISAID monkeypox virus database, 1550 whole-genome sequences of two branches, I (Ia, Ib) and II, were selected and downloaded. SNP analysis of monkeypox virus branches I and II was performed to obtain a set of SNPs that could distinguish monkeypox virus branches I and II across the entire genome. Following the design principles of primers and probes for locked nucleic acid detection methods, SNP position 46743 (GenBank: OZ310432.1) was selected as the optimal detection site, where branch I is base G and branch II is base A (…). Figure 1 ).

[0041] Based on the design principles of locked nucleic acid detection methods, the nucleic acid sequence of the detection region selected the relevant regions of monkeypox virus branches I and II (GenBank: OZ310432.1:46482-47018) as the detection template. Figure 2 The sequences were cloned into PUC57 using the KpnⅠ and HindⅢ restriction sites. Monkeypox virus I cloned PUC57-Ⅰ, and the detection sequence information is shown in Table 1 (MPXV-Ⅰ). Monkeypox virus II cloned PUC57-Ⅱ, and the detection sequence information is shown in Table 1 (MPXV-Ⅱ).

[0042] Table 1. Monkeypox Virus I / II Detection Sequence Information

[0043] The constructed PUC57-Ⅰ and PUC57-Ⅱ plasmids were transformed into DH5α using a chemical transformation method (thermal shock method). The specific steps are as follows: ① Mixing competent cells with plasmids: Remove the aliquoted DH5α competent cells from the -80℃ freezer and thaw them slowly on ice; in an aseptic environment, add 100 μL of DH5α competent cells to each pre-chilled 1.5 mL centrifuge tube, followed by 10 μL of pUC57-Ⅰ and pUC57-Ⅱ plasmid DNA solutions; mix gently, avoiding vigorous vortexing; The following controls were also set up: an equal volume of sterile, enzyme-free water was used instead of plasmid DNA as a negative control; and an intact plasmid with a known structure was used as a positive transformation control.

[0044] ② Ice bath and heat shock: Place the mixture on ice for 30 minutes to allow the plasmid DNA to fully bind to the competent cell membrane; then, quickly transfer the centrifuge tube to a water bath set to 42°C for 1 minute and 30 seconds of heat shock to induce the cells to absorb the exogenous DNA; after the heat shock, immediately return the centrifuge tube to the ice bath and let it cool for 1 minute.

[0045] ③ Resuscitation culture: Add 890 μL of antibiotic-free LB liquid medium preheated to 37°C to each tube of conversion mixture; place the centrifuge tube in a 37°C constant temperature shaker and gently shake at 200 rpm for 60 minutes to allow the cells to recover to normal growth state and express the plasmid-encoded ampicillin resistance gene.

[0046] ④ Spreading and screening: After resuscitation, take 100 μL of the resuscitation bacterial solution in a sterile operating table and spread it evenly on LB solid agar plates containing ampicillin. Invert the plates and incubate overnight at 37°C for 12-16 hours.

[0047] ⑤ Transformation verification: After the culture is completed, independent single colonies should grow on the plates of the positive control and experimental groups, while no colonies should grow on the negative control plates; Several single colonies were randomly picked from pUC57-Ⅰ and pUC57-Ⅱ transformation plates, respectively, and inoculated into LB liquid medium containing antibiotics for expansion culture. Subsequently, PCR was used to verify the integrity and accuracy of the plasmid sequence.

[0048] (2) Design of LNA-Taqman probes and primers for monkeypox virus branches I and II Based on the selected SNP sites from the alignment results, an LNA-Taqman probe was designed for branch I (see...). Figure 2 ).

[0049] Locked nucleic acid structures were inserted two bases apart on the left and right sides of the SNP site, for a total of 4 locked nucleic acid structures; A FAM fluorescent group was added to the 5' end of the sequence, and a BHQⅠ quencher group was added to the 3' end; The sequence was synthesized by Genscript Biotech (Nanjing, China), see Table 2, SEQ ID NO. 1.

[0050] Design universal upstream and downstream primers for three branches in selected conserved regions of the fragment (see [link]). Figure 2 To ensure that the Tm values ​​of the upstream and downstream primers are at the same level as the probe's Tm value, but lower than the probe's Tm value, the designed primer pairs are compared in NCBIblast to ensure that the primer pairs have good specificity and no overlap with other species.

[0051] The prepared sequences were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. Primer and locked nucleic acid probe sequence information is shown in Table 2, SEQ ID NO. 2 and SEQ ID NO. 3.

[0052] Table 2

[0053] Example 2 Optimal operating temperatures for primers and probes for monkeypox virus LNA detection: The detection method of locked nucleic acid is based on the special structure of locked nucleic acid, which can highly identify SNP sites. The introduction of LNA monomer can increase the Tm value of double strand by 2-8℃. Therefore, temperature is an important factor for the high recognition of locked nucleic acid detection. However, temperature is a double-edged sword in locked nucleic acid detection. While high temperatures can improve detection resolution, excessively high temperatures can reduce the binding affinity of primers and probes to the template. Conversely, low temperatures allow primers and probes to bind well to the template, but excessively low temperatures can reduce detection accuracy (i.e., make it impossible to distinguish single-base mutations). Therefore, exploring the optimal temperature range is a key focus in the development of locked nucleic acid detection methods, as explained below: (1) Verification of primer annealing temperature range: The powdered upstream and downstream primers synthesized in Example 1 were centrifuged and dissolved in sterile enzyme-free water. A gradient PCR experiment was designed, and the reaction system without template was used as a negative control.

[0054] PCR reactions were performed using Probe qPCR Mix MultiPlus (RR393A) manufactured by Takara, and all reactions were carried out according to the reaction system recommended in the product manual. Reaction system: 12.5 μL of 2×Probe qPCR Mix MultiPlus, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 5 μL of DNA template, 5.5 μL of sterile enzyme-free water, and a total reaction volume of 25 μL; Reaction program: 95℃ for 20s; 95℃ for 15s; 62℃ for 45s, for a total of 40 cycles, with the annealing temperature range set at 62-68℃; The amplified product was verified by agarose gel electrophoresis: the gel concentration was 1.5%, the current and voltage were 100V and 200mA respectively, and the reaction time was 35 min. This experiment confirms the annealing temperature range that primers can be used, avoiding false negative results caused by primers failing to bind to the template at high annealing temperatures, resulting in the inability to amplify the target fragment. Based on electrophoresis results, the optimal annealing temperature range for the primers was determined to be 66℃ and below. (See electrophoresis results for details.) Figure 3 A.

[0055] (2) Validation of the annealing temperature range of the locked nucleic acid probe: The LNA probe sequence without fluorescent group synthesized by Sangon Biotech (Shanghai) Co., Ltd. was used as the upstream primer and paired with the downstream primer. The primer information is shown in Table 3. The annealing temperature of the LNA probe was verified. The reaction system without template was used as the negative control. Table 3. Validation primer sequences for the LNA-Taqman probe annealing temperature range

[0056] The reaction system and reaction procedure are the same as those in the "Primer Annealing Temperature Range Verification" section above; the annealing temperature range of the reaction procedure is set to 62-68℃, and the amplified products are verified by agarose gel electrophoresis: the gel concentration is 1.5%, the current and voltage are 100V and 200mA respectively, and the reaction time is 35 min. Based on the electrophoresis results, the optimal binding temperature range for the locked nucleic acid probe is 66℃ and below. (See electrophoresis results below.) Figure 3 B.

[0057] By verifying the optimal annealing temperature range of the primers and locked nucleic acid probes, it was found that the optimal binding temperature range of the locked nucleic acid primers and probes is 66℃ and below. Further verification is needed to verify the typing of monkeypox virus I and II under different temperature conditions of the primer and LNA probe combination in order to determine the optimal temperature range of this locked nucleic acid detection system.

[0058] Example 3 Determination of temperature conditions for LNA-based monkeypox virus I and II typing detection: (1) Plasmid extraction: The bacterial cultures containing PUC57-Ⅰ and PUC57-Ⅱ plasmids were inoculated into LB medium at a volume of 1:100 and cultured overnight; Take 500 μL of the cultured bacterial solution and extract plasmids according to the instructions of the plasmid miniprep kit (DP103) from Tiangen Biotech (Beijing) Co., Ltd.

[0059] (2) Quantification of monkeypox virus I and II plasmid templates by digital PCR: After plasmid extraction, the copy number of plasmids PUC57-Ⅰ and PUC57-Ⅱ was quantified using digital PCR.

[0060] A universal real-time PCR detection method was developed for the detection plasmids (PUC57-I and PUC57-II) targeting monkeypox virus branches I and II. Sequence information is shown in Table 4, and primer and probe positions are listed below. Figure 4 .

[0061] Table 4

[0062] The specific method is as follows: Applied Biosystems™ Absolute Q™ one-step RT-dPCR digital PCR premix (4X, A55146) was used. All reactions were carried out according to the reaction system and procedure recommended in the product instructions. The digital PCR reaction system table is shown in Table 5.

[0063] Table 5. Digital PCR Reaction System

[0064] Reaction system: Absolute Q™ 1-Step RT-dPCR Master Mix (4×) 2.5 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, probe (300 nM) 1 μL, DNA template 2.5 μL, sterile enzyme-free water 2 μL, total reaction volume 10 μL. Reaction program: 96℃, 10 min; 96℃, 5 s; 60℃, 10 s, for a total of 40 cycles.

[0065] Table 6 Digital PCR Reaction Procedure Table

[0066] Note: The reaction systems in Table 6 use the reaction procedures recommended in the Applied Biosystems™ Absolute Q™ One-Step RT-dPCR Premix (4X, A55146) instruction manual.

[0067] For the digital PCR quantification results of the plasmid templates used for monkeypox virus I and II typing, please refer to [link to relevant documentation]. Figure 5 The copy number of the monkeypox virus I (PUC57-I) plasmid template was 6.90 × 10⁻⁶. 7 The copy number of the monkeypox virus II (PUC57-II) plasmid template was 6.57 × 10^6 copies / μL. 7 copies / μL.

[0068] (3) Real-time quantitative PCR detection Using the extracted plasmids as templates, a gradient real-time PCR experiment was designed. The reaction system without template was used as a negative control. The quantitative real-time PCR reaction was performed using Probe qPCR Mix MultiPlus (RR393A) manufactured by Takara, and all reactions were carried out according to the reaction system recommended in the product manual.

[0069] The reaction system consisted of: 12.5 μL of 2×Probe qPCR Mix MultiPlus, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 1 μL of LNA-Taqman probe (10 μM), 5 μL of DNA template, 0.5 μL of ROX Reference Dye II (100×), and 4 μL of sterile enzyme-free water, for a total reaction volume of 25 μL.

[0070] The reaction program was as follows: 95℃, 20s; 95℃, 15s; 62℃, 45s, for a total of 40 cycles; each temperature interval constituted a group, and each group was repeated 3 times, for a total of 4 groups. See Table 7 for the quantitative PCR reaction system table.

[0071] Table 7. Quantitative Real-Time PCR Reaction System

[0072] Note: The reaction systems in Table 7 use the reaction systems recommended in the Takara Probe qPCR Mix MultiPlus (RR393A) instruction manual.

[0073] Table 8. Quantitative Real-Time PCR Reaction Procedure

[0074] Note: The reaction systems in Table 8 use the reaction procedures recommended in the Takara Probe qPCR Mix MultiPlus (RR393A) manual.

[0075] (4) The annealing temperature is gradually increased from low to high to achieve the purpose of separating the two branches. Repeatability tests showed that an annealing temperature of 60℃ was insufficient for typing monkeypox virus branches I and II. Annealing temperatures between 62-67℃ were sufficient to detect monkeypox virus branch I, while results for monkeypox virus branch II were negative. Further analysis of CT and ΔRn values ​​is needed to determine the optimal temperature conditions for locking nucleic acid typing of monkeypox virus branches I and II. Amplification curves are shown below. Figure 6.

[0076] To determine the optimal annealing temperature for genotyping of monkeypox virus I and II branches, ANOVA analysis was used to analyze the CT and ΔR values ​​at annealing temperatures of 62℃, 64℃, 66℃, and 67℃.

[0077] result( Figure 6 The results showed that the CT values ​​of the annealing temperature groups at 62℃ and 64℃ were significantly lower than those at 66℃ and 67℃ (p<0.05), but there was no significant difference in CT values ​​between the annealing temperature groups at 62℃ and 64℃ (P>0.05). The ΔRn values ​​of the annealing temperature groups at 62℃ and 64℃ were significantly higher than those at 66℃ and 67℃ (p<0.05), but there was no significant difference in ΔRn values ​​between the annealing temperature groups at 62℃ and 64℃ (P>0.05). This indicates that 62℃ and 64℃ are the optimal annealing temperatures for monkeypox virus I and II branch locked nucleic acid typing. Ultimately, 62℃ was selected as the optimal reaction condition for PCR, as it allows primers and probes to bind more stably to the template.

[0078] Example 4 Validation of locked nucleic acid probe typing method for monkeypox virus branches I and II: (1) Sensitivity test The plasmid PUC57-Ⅰ (6.90×10⁻⁶) was quantified by digital PCR. 7 (copies / μL) and PUC57-II (6.57×10) 7 The sensitivity of this method was verified using the LNA-Taqman monkeypox virus I branch typing method (copies / μL) template.

[0079] The monkeypox virus I plasmid template was diluted as follows (6.90 × 10⁻⁶). 7 copies / μL, 6.90×10 6 copies / μL, 6.90×10 5 copies / μL, 6.90×10 4 copies / μL, 6.90×10 3 copies / μL, 6.90×10 2 copies / μL, 6.90×10 1 copies / μL, 6.90×10 0 copies / μL, 6.90×10 -1 (copies / μL) The monkeypox virus II plasmid template was diluted as follows (6.57 × 10⁻⁶). 7copies / μL, 6.57×10 6 copies / μL, 6.57×10 5 copies / μL, 6.57×10 4 copies / μL, 6.57×10 3 copies / μL, 6.57×10 2 copies / μL, 6.57×10 1 copies / μL, 6.57×10 0 copies / μL, 6.57×10 -1 (copies / μL) were used to verify sensitivity.

[0080] The quantitative real-time PCR reaction was performed using Probe qPCR Mix MultiPlus (RR393A) manufactured by Takara, and all reactions were carried out according to the reaction system recommended in the product manual.

[0081] The reaction system consisted of: 12.5 μL of 2×Probe qPCR Mix MultiPlus, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 1 μL of LNA-Taqman probe (10 μM), 5 μL of DNA template, 0.5 μL of ROX Reference Dye II (100×), and 4 μL of sterile enzyme-free water, for a total reaction volume of 25 μL.

[0082] The reaction program was: 95℃, 20s; 95℃, 15s; 62℃, 45s, for a total of 40 cycles.

[0083] The sensitivity of the reagent kit involves two aspects: ① Validation of the detection limit for monkeypox virus type I. The established LNA-Taqman-based detection method for monkeypox virus type I achieved a detection limit of 6.90 copies / μL (see...). Figure 7 ).

[0084] ② Validation of the detection limit for monkeypox virus type II. This method showed a negative result for monkeypox virus type II, with a limit of 6.57 × 10⁻⁶. 7 The test result was still negative at a concentration of copies / μL (high concentration) (see Figure 8 No false positive results were found.

[0085] (2) Specificity test Four types of bacteria, four types of viruses, one type of monkey cell line, and one type of human cell line were selected from the sample library. The four types of bacteria were, in order: *Escherichia coli*, *Staphylococcus aureus*, *Candida albicans*, and *Pseudomonas aeruginosa*; the four types of viruses were, in order: Zika virus, influenza A virus H1N1, enterovirus 71, and poliovirus 1; the cells were monkey VERO cells and human HEK 293 cells. Nucleic acids were extracted using the BioFlux MagaBio Pathogen DNA / RNA Extraction Kit (BSC75S1E), and four RNA viruses were reverse transcribed into cDNA using the TaKaRa PrimeScript™ RT Master Mix Kit. To ensure specificity and accuracy, all exogenous reference materials were given at concentrations within 10-1. 7 More than copies / µL; (3) Real-time PCR experimental system and procedure: The quantitative real-time PCR reaction was performed using Probe qPCR Mix MultiPlus (RR393A) manufactured by Takara, and all reactions were carried out according to the reaction system recommended in the product manual. The reaction system consisted of: 12.5 μL of 2×Probe qPCR Mix MultiPlus, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 1 μL of LNA-Taqman probe (10 μM), 5 μL of DNA template, 0.5 μL of ROX Reference Dye II (100×), and 4 μL of sterile enzyme-free water, for a total reaction volume of 25 μL. The reaction program was: 95℃, 20s; 95℃, 15s; 62℃, 45s, for a total of 40 cycles; The reaction system without template was used as a negative control.

[0086] Amplification curves are shown below. Figure 9 According to the final amplification curve, only branch I of monkeypox virus was detected, which was a positive result, while the other pathogens were not detected, which was a negative result.

[0087] Example 5 This embodiment provides a kit for detecting monkeypox virus I and II genotyping and its usage method: (1) Components of the kit LNA-Taqman probes: Primer and locked nucleic acid probe sequence information is shown in Table 2.

[0088] Positive control: A plasmid for detecting monkeypox virus type I using this method (6.90 × 10⁻⁶). 7 (copies / μL).

[0089] Negative control: sterile, enzyme-free water; monkeypox virus type II negative detection plasmid for this method (6.57 × 10⁻⁶). 7 (copies / μL).

[0090] Quantitative real-time PCR premix: Taq DNA polymerase 5U, dNTPs 0.2mM, Mg 2+ 3.0 mM, Tris-HCl buffer 30 mM, and K + 30mM, UNG enzyme 2U.

[0091] (2) Sample requirements This kit is suitable for monkeypox virus I and II typing detection in the following human samples and contaminated environmental samples: Skin / mucosal lesion swabs, oropharyngeal / nasopharyngeal swabs, whole blood / serum / plasma, environmental samples; DNA samples extracted from these types of samples and their cultures can all be detected using this kit.

[0092] (3) Operating steps Sample preparation: Extract DNA from the sample according to the sample type; the extracted DNA can be used directly for detection; if the sample is not to be detected immediately after extraction, it can also be stored at -20℃ for later use, and repeated freeze-thaw cycles should be avoided.

[0093] System preparation: Remove the reagent from the kit and thaw it at room temperature. After the reagent is completely thawed, invert and mix well, then centrifuge briefly. If the number of samples to be tested is n (n = number of samples + positive control + negative control), then the reaction system is prepared according to n+1 reactions, and the reaction system is prepared as shown in Table 9 below.

[0094] The reaction system for quantitative real-time PCR is shown in Table 9.

[0095] Table 9

[0096] Note: Sample type can be any one of a / b / c / d. a. Sample extraction, b. Negative control (type II monkeypox virus detection plasmid), c. Positive control (type I monkeypox virus detection plasmid), d. Sterile enzyme-free water.

[0097] System aliquoting: After mixing and centrifuging the above reaction solution, aliquot 25 μL into PCR tubes suitable for the fluorescence PCR instrument.

[0098] Fluorescent PCR cycling conditions: See Table 10 for the fluorescent quantitative PCR reaction program.

[0099] Table 10

[0100] Threshold setting: The principle for setting the threshold is to use the highest point of fluorescence signal that just exceeds that of the normal negative control as the threshold line, or to adjust it according to the specific situation of the instrument.

[0101] Quality control criteria: The experiment is considered valid if the negative control shows no amplification curve and the positive control shows an S-shaped amplification curve in the FAM detection channel. Otherwise, the experimental results are considered invalid.

[0102] Results Analysis and Judgment: If, under annealing temperature of 62℃, the sample exhibits an S-shaped amplification curve in the detection channel and the Ct value is lower than the preset threshold (CT value ≤ 36), it is identified as monkeypox virus type I; if the sample does not exhibit an S-shaped amplification curve in the detection channel and the Ct value is higher than the preset threshold (CT value ≥ 36), it is identified as monkeypox virus type II. (The above determination is based on the premise that the sample tests positive for monkeypox virus).

[0103] (4) Product performance indicators Limit of detection: 6.90 copis / μL Cross-reactivity: No cross-reactivity was observed with any of the following bacteria that may cross-react with monkeypox virus (Escherichia coli, Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa, Zika virus, influenza A virus H1N1, enterovirus 71 and poliovirus 1, monkey VERO cells and human HEK 293 cells).

[0104] Comparative Example 1 When selecting genotyping SNP sites for monkeypox virus types I and II, as well as designing primers and LNA probes, the first step is to obtain a set of SNPs that can distinguish between monkeypox virus branches I and II across the entire genome through multiple sequence alignment. Then, specific primers and LNA probes are designed for candidate SNP sites. The genotyping capabilities of these primers and LNA probes are systematically compared through experiments, with the aim of selecting primers and LNA probes that can accurately and stably genotype target monkeypox virus types I and II.

[0105] The information provided includes: SNP locus information ( Figure 10 ), sequence information ( Figure 11 Primer and LNA probe location information ( Figure 11 Primer and LNA probe sequence information table (Table 11), and graph of monkeypox virus type I and II typing failure results ( Figure 12 ).

[0106] Table 11

[0107] A real-time PCR assay was designed for monkeypox virus type I and II typing. The reaction system and reaction procedure used in the assay were the same as those in the kit of this invention. The annealing temperature range set in the reaction procedure was still 62-67℃. Based on the typing results, within this annealing temperature range, primers and LNA probes designed for this candidate SNP site cannot achieve typing of monkeypox virus types I and II.

[0108] Based on the use of digital PCR precise quantitative detection template, this invention achieves a detection limit of 6.90 copies / μL for monkeypox virus branch I typing, while also exhibiting good specificity.

[0109] This invention employs TaqMan probes with locked nucleic acid (LNA) structures to construct a highly specific method for genotyping detection of monkeypox virus branches I and II. Leveraging the superior properties of LNA, its application in monkeypox virus genotyping probe design effectively addresses the lack of specificity in traditional methods due to high sequence homology. By precisely introducing LNA monomers into key positions in the probe sequence corresponding to genotyping SNP sites, highly specific and sensitive locked nucleic acid probes can be constructed. These probes can stably hybridize and generate strong fluorescence signals only with perfectly matching viral genotyping target sequences during real-time quantitative PCR. For non-target genotypes with only one or a few base differences, almost no signal is generated, thus achieving rapid, accurate, and reliable identification and genotyping of monkeypox virus.

[0110] It should be noted that 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A composition for detecting monkeypox virus types I and II, characterized in that: include, The LNA-Taqman probe for detecting monkeypox virus I has the nucleic acid sequence shown in SEQ ID NO. 1; The upstream primer for monkeypox virus typing detection has the nucleic acid sequence shown in SEQ ID NO. 2; Additionally, the downstream primers for monkeypox virus typing detection have the nucleic acid sequence shown in SEQ ID NO.

3.

2. The use of the composition of claim 1 in the preparation of a kit for detecting monkeypox virus types I and II.

3. A kit for detecting monkeypox virus types I and II, characterized in that: The kit includes the composition of claim 1.

4. The kit as described in claim 3, characterized in that: It also includes a premixed solution for quantitative PCR, a positive control, a negative control, and water.

5. The kit according to claim 4, characterized in that: The positive control included monkeypox virus I plasmid with a copy number of 6.90 × 10⁻⁶. 7 The negative control includes a monkeypox virus type II negative detection plasmid with a copy number of 6.57 × 10⁻⁶ μL. 7 copies / μL.

6. The kit as described in claim 5, characterized in that: The premixed solution for quantitative real-time PCR was 12.5 μL, the upstream primer concentration was 10 μM and the upstream primer volume was 1 μL, the downstream primer concentration was 10 μM and the downstream primer volume was 1 μL, the LNA-Taqman probe concentration for detecting monkeypox virus I was 10 μM and the probe volume was 1 μL, the positive control volume was 5 μL, the water volume was 4.5 μL, and the total volume was 25 μL.

7. The kit according to claim 5, characterized in that: The detection limit of the kit is 6.90 copies / μL.

8. A method for using a kit for detecting monkeypox virus types I and II, characterized in that: This includes assembling the raw materials into a kit and performing a real-time PCR reaction at 95°C for 20 seconds. 95℃, 15s; 62℃, 45s, for a total of 40 cycles.

9. A method for LNA-based genotyping of monkeypox virus types I and II targeting non-therapeutic diseases, characterized in that: include, Monkeypox virus types I and II were detected by quantitative real-time PCR. The reaction system consisted of: 12.5 μL of 2×Probe qPCR Mix MultiPlus, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 1 μL of 10 μM LNA-Taqman probe, 5 μL of DNA template, 0.5 μL of ROX Reference Dye II (100×), and 4 μL of sterile enzyme-free water, for a total reaction volume of 25 μL. The reaction program was: 95℃, 20s; 95℃, 15s; 62℃, 45s, for a total of 40 cycles.

10. The typing detection method as described in claim 9, characterized in that: The DNA template includes monkeypoxvirus I plasmid and monkeypoxvirus II plasmid, with copy numbers of 6.90 × 10⁻⁶ and 6.90 × 10⁻⁶, respectively. 7 copies / μL, 6.57×10 7 copies / μL.

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