Primer probe group and kit for specific detection of highly pathogenic monkey pox virus subtype

Abstract

The invention relates to a primer probe group and a kit for specific detection of a highly pathogenic monkey pox virus subtype. The preferable combination of the primer probe group is as follows: a forward primer: SEQ ID NO: 3, a reverse primer: SEQ ID NO: 4 and a probe: SEQ ID NO: 5. The primer probe group and the kit disclosed by the invention have specific amplification capacity on the monkey pox virus Ib type and do not have cross reaction on non-monkey pox virus Ib type.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a preparation and application of a fluorescent PCR kit for detecting high pathogenic monkeypox virus subtypes, which involves the search for specific target points of high pathogenic monkeypox virus subtypes, the design and screening of primer probe sets, and the performance evaluation of the detection kit based on the primer probe set, including the minimum detection limit, the linear range and the subtype specificity identification. BACKGROUND

[0002] Monkeypox is a viral zoonosis caused by monkeypox virus, which has similar clinical manifestations to smallpox, but the clinical symptoms are relatively mild.

[0003] There are multiple subtypes of monkeypox virus, among which monkeypox virus Clade Ib (type Ib) has unique genetic characteristics and epidemiological characteristics. Compared with other subtypes, monkeypox virus type Ib may differ in terms of transmission speed and pathogenicity. Accurate and rapid detection and identification of high pathogenic monkeypox virus subtypes is crucial for the prevention and control of the epidemic, diagnosis and treatment of patients. Timely identification of this subtype virus helps to take targeted prevention and control measures to prevent further spread of the epidemic.

[0004] Currently, the detection methods for monkeypox virus mainly include nucleic acid detection and immunological detection. Nucleic acid detection methods such as polymerase chain reaction (PCR) technology have high sensitivity and specificity, but most of the existing primers and probes are designed for general sequences of monkeypox virus, lacking specific recognition ability for high pathogenic monkeypox virus subtypes, which can easily lead to false negative or false positive results. Although immunological detection methods are relatively simple to operate, they have low sensitivity and cannot accurately distinguish different subtypes of monkeypox virus.

[0005] In addition, the existing monkeypox virus detection kits have problems such as low detection limit and narrow linear range when detecting high pathogenic monkeypox virus subtypes, which cannot meet the actual needs of clinical and epidemic prevention. SUMMARY

[0006] The purpose of the present application is to provide a primer probe set and kit that can specifically detect and identify high pathogenic monkeypox virus subtypes, providing a reliable and convenient detection tool for medical detection institutions, disease control centers and other organizations, meeting the urgent need for precise detection of high pathogenic monkeypox virus subtypes in the market, and promoting the development of monkeypox virus detection technology.

[0007] The above-mentioned purposes are achieved by the following technical solutions.

[0008] A primer probe set for specific detection of high pathogenic monkeypox virus subtypes, characterized in that it comprises:

[0009] Forward primer: one selected from the nucleotide sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12;

[0010] Reverse primer: one selected from the nucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13;

[0011] Probe: one selected from the nucleotide sequences shown in SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 14.

[0012] Further, the probe is modified with a fluorescent label (including FAM, HEX, ROX, CY5) and a quenching group (including BHQ1, BHQ2, MGB).

[0013] Further, the combination is preferably:

[0014] Forward primer: SEQ ID NO: 3;

[0015] Reverse primer: SEQ ID NO: 4;

[0016] Probe: SEQ ID NO: 5;

[0017] The combination has specific amplification ability for monkeypox virus type Ib, and no cross-reaction for non-type Ib (see Figure 1-2 ).

[0018] To achieve the above-mentioned purpose, the present application further provides a hormone or scheme of a high-pathogenic monkeypox virus subtype-specific detection kit, comprising:

[0019] (a) the primer probe set described above;

[0020] (b) PCR premix, comprising hot-start DNA polymerase, dNTPs, MgCl2, PCR buffer, and uracil glycosylase (UNG);

[0021] (c) positive control: gradient dilution plasmid comprising the sequence shown in SEQ ID NO: 1;

[0022] (d) negative control: nuclease-free water;

[0023] Wherein, the reaction system of the kit is: 20 μL PCR premix + 5 μL nucleic acid extractive of the sample to be tested (see Table 4.1).

[0024] Further, the composition of the PCR premix is:

[0025] 2x FastAmpli Premix-UNG IV: 12.5 μL;

[0026] Forward primer (10 μM): 0.5 μL;

[0027] Reverse primer (10 μM): 0.5 μL;

[0028] Probe (10 μM): 0.25 μL;

[0029] Nuclease-free water: up to 20 μL.

[0030] The detection sensitivity of the kit is ≤500 copies / mL, the linear range correlation coefficient R 2 ≥0.99, and the amplification efficiency is 90%-110% (see Figure 5-6 ).

[0031] The kit has no cross-reaction with cross-pathogens, including varicella virus, herpes virus, measles virus, HPV, and bacterial samples, as shown in Table 4.3.

[0032] The present application also provides a detection method for high pathogenic monkeypox virus subtypes, using the above-mentioned kit, comprising the following steps:

[0033] (a) extracting nucleic acid from the sample to be tested;

[0034] (b) mixing the nucleic acid extraction solution with the PCR premix solution and amplifying under the following conditions:

[0035] - 50°C for 2 minutes → 95°C for 5 minutes → 40 cycles (95°C for 10 seconds, 60°C for 20 seconds);

[0036] (c) collecting the fluorescence signal through the FAM channel, and the determination criteria are:

[0037] - Ct≤35 and S-shaped amplification curve: monkeypox virus Ib positive;

[0038] - No amplification signal: negative (see Table 4.4).

[0039] According to the above technical solution, the inventors have carried out the following work during the research process,

[0040] 1. First, the high pathogenic monkeypox virus subtype target is found

[0041] The whole genome sequence of the highly pathogenic monkeypox virus subtype is analyzed by bioinformatics technology, and is compared with the sequences of other monkeypox virus subtypes to find the gene region specific to the highly pathogenic monkeypox virus subtype as a detection target. These targets have high conservation and specificity, and can effectively distinguish the highly pathogenic monkeypox virus subtype from other subtypes. The target gene finally determined by the inventors is the monkeypox virus A36R gene, the nucleotide sequence of which is shown in SEQ ID NO: 1 (monkeypox virus Ib type) or SEQ ID NO: 2 (monkeypox virus non-Ib type); wherein SEQ ID NO: 1 comprises a 48-base deletion region specific to Ib type in the A36R gene.

[0042] 2. Design of primer probe set for highly pathogenic monkeypox virus subtype

[0043] According to the determined detection target and the design principles of primers and probes, a series of primers and probes are designed. The length of the primer is controlled between 18-25 nucleotides, the Tm value is in the range of 55-65℃, and the GC content is between 40%-60%. The probe is labeled with fluorescence, and the marker is selected from common fluorescent dyes such as FAM, HEX, ROX, CY5, etc.

[0044] 3. Specific detection of highly pathogenic monkeypox virus subtype primer probe set

[0045] Using the nucleic acids of monkeypox virus Ib and non-Ib as amplification templates, real-time fluorescent PCR amplification is performed using the designed primer probe set. Only the primer probe set that can specifically amplify the nucleic acid of monkeypox virus Ib, but has no amplification signal for the nucleic acid of non-Ib, is retained for the next step of screening.

[0046] Using the specific primer probe set of the highly pathogenic monkeypox virus subtype as the core detection system, monkeypox virus negative samples are used as the detection object, and the sample range covers healthy human population basic samples and cross-reaction verification sample set meeting the requirements of “Monkeypox Virus Nucleic Acid Detection Reagent Registration and Examination Guiding Principles”. By optimizing the sequence specificity of the primer probe and the reaction system parameters, it is ensured that under the standardized fluorescent PCR detection conditions, all detection samples do not present specific amplification signals, thereby meeting the strict specificity requirements of the kit for the target pathogen, and effectively excluding the interference reactions that may be caused by closely related virus species and human genome.

[0047] 4. Sensitivity detection of highly pathogenic monkeypox virus subtype primer probe set

[0048] The amplification efficiency of the high pathogenic monkeypox virus subtype primer probe set is detected. Real-time fluorescent PCR amplification is carried out with different concentrations of monkeypox virus Ib type nucleic acid as a template, a standard curve is drawn, and the amplification efficiency of the primer probe set is calculated. The primer probe set with an amplification efficiency of 90%-110% is selected as the final detection primer probe set.

[0049] A series of different concentrations of high pathogenic monkeypox virus subtype nucleic acid samples are prepared, and the detection kit is used for detection, and the Ct value is recorded. The standard curve is drawn with the logarithmic value of the nucleic acid concentration as the abscissa and the Ct value as the ordinate. The correlation coefficient (R 2 ) of the standard curve is calculated to evaluate the linearity of the kit, and R 2 ≥0.99.

[0050] The known concentration of high pathogenic monkeypox virus nucleic acid is gradiently diluted, and real-time fluorescent PCR detection is carried out using the primer probe set and the matching reaction system. The lowest nucleic acid concentration that can produce a positive amplification signal is used as the minimum detection limit of the kit.

[0051] The primer probe set and the kit of the present application aim to solve the above-mentioned problems existing in the prior art, and improve the detection specificity, sensitivity and accuracy of the high pathogenic monkeypox virus subtype by optimizing the target selection, primer probe design and screening method, and provide strong technical support for the prevention and control of monkeypox epidemic.

[0052] In summary, through a series of rigorous experimental steps, including target finding, primer probe set design and screening, and determination of the minimum detection limit, linearity and specificity of the detection kit, the product can quickly and accurately detect the high pathogenic monkeypox virus subtype in clinical diagnosis, epidemic monitoring and other scenes.

[0053] The primer probe set and the kit of the present application have the following beneficial effects:

[0054] High specificity: by detecting the specific gene target of the high pathogenic monkeypox virus subtype, the high pathogenic monkeypox virus can be accurately distinguished from other subtypes, and misdiagnosis and missed diagnosis are avoided.

[0055] High sensitivity: the minimum detection limit can reach a low nucleic acid concentration, which can detect early infection and low viral load samples, and improve the accuracy of detection.

[0056] Good linearity: the kit has good linearity in a wide range of nucleic acid concentrations, which can accurately reflect the content of viral nucleic acid in the sample.

[0057] The application value is high: the primer probe set and the kit can be widely applied to clinical diagnosis, epidemic monitoring and scientific research of the high pathogenic monkeypox virus subtype, and provide strong technical support for prevention and control of the monkeypox virus. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 The figure is a specific detection result graph of the high pathogenic monkeypox virus subtype primer probe set combination 1.

[0059] Figure 2 The figure is a specific detection result graph of the high pathogenic monkeypox virus subtype primer probe set combination 2.

[0060] Figure 3 The figure is a specific detection result graph of the high pathogenic monkeypox virus subtype primer probe set combination 3.

[0061] Figure 4 The figure is a specific detection result graph of the high pathogenic monkeypox virus subtype primer probe set combination 4.

[0062] Figure 5 The figure is a fluorescence PCR amplification curve graph of the high pathogenic monkeypox virus subtype primer probe set for detecting the target gene gradient dilution plasmid.

[0063] Figure 6 The figure is a linear graph of the high pathogenic monkeypox virus subtype primer probe set for detecting the target gene gradient dilution plasmid. DETAILED DESCRIPTION

[0064] In order to facilitate understanding of the present application, the embodiments of the present application are described below, but the present application is not limited thereto. The present application is not limited to each of the configurations described below, and various modifications can be made within the scope of the present application, and embodiments obtained by appropriately combining the technical means disclosed in each of the embodiments are also included in the technical scope of the present application.

[0065] <DEFINITIONS>

[0066] In the present application, the numerical range represented by "numerical value A to numerical value B" or "numerical value A-numerical value B" means a range including the end point values A and B.

[0067] In the present application, the numerical range represented by "above" or "below" means a numerical range including the number.

[0068] In the present application, the meaning represented by "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process. In the present specification, "optional" or "optionally" means that the event or circumstance described next can occur or can not occur, and the description includes the case where the event occurs and the case where the event does not occur.

[0069] In the present application, the term "a" or "an" or "the" can mean "one", "one or more", "at least one", and "one or more than one".

[0070] In the present application, the term "comprise", "have", "include" or "contain" can mean inclusive or open-ended and does not exclude additional, unrecited elements or method steps. At the same time, "comprise", "have", "include" or "contain" can also mean closed, excluding additional, unrecited elements or method steps.

[0071] In the present application, the term "about" can mean that a value includes the standard deviation of error of the device or method used to determine the value. The numerical ranges and parameters regarding the present application are approximate values, and the related values in the specific examples have been presented as accurately as possible. However, any numerical value inherently cannot avoid the standard deviation caused by the aforementioned testing device or method. Therefore, unless otherwise explicitly stated, all ranges, quantities, numerical values and percentages used in the present application should be understood as being modified by "about". Herein, "about" generally means that the actual value is within ±10%, ±5%, ±1% or ±0.5% of a certain value or range.

[0072] In the present application, "target gene of interest" refers to the gene targeted for nucleic acid detection. For example, when PCR is used for nucleic acid detection, the target gene of interest includes the gene fragment to be amplified by PCR.

[0073] Unless otherwise defined, other technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0074] Target gene of interest

[0075] In some embodiments, the gene of the high pathogenic monkeypox virus subtype described in the present application is the A36R gene.

[0076] Further, in some specific embodiments, the target gene of interest for nucleic acid detection of the high pathogenic monkeypox virus subtype described in the present application comprises the A36R gene of monkeypox virus.

[0077] The present application does not make special limitations on other reagents contained in the kit, and for example, the kit can further comprise nucleic acid extraction reagents, reagents required for polymerase chain reaction, etc.

[0078] <Nucleotide sequences involved in the present application>

[0079] Nucleotide sequence of the target gene A36R of monkeypox virus type 1b (SEQ ID NO: 1);

[0080] Nucleotide sequence of the target gene of monkeypox virus non-1b type (SEQ ID NO: 2);

[0081] A36R forward primer sequence 1 (SEQ ID NO: 3);

[0082] A36R reverse primer sequence 1 (SEQ ID NO: 4);

[0083] A36R probe sequence 1 (SEQ ID NO: 5);

[0084] A36R forward primer sequence 2 (SEQ ID NO: 6);

[0085] A36R reverse primer sequence 2 (SEQ ID NO: 7);

[0086] A36R probe sequence 2 (SEQ ID NO: 8);

[0087] A36R forward primer sequence 3 (SEQ ID NO: 9);

[0088] A36R reverse primer sequence 3 (SEQ ID NO: 10);

[0089] A36R probe sequence 3 (SEQ ID NO: 11);

[0090] A36R forward primer sequence 4 (SEQ ID NO: 12);

[0091] A36R reverse primer sequence 4 (SEQ ID NO: 13);

[0092] A36R probe sequence 4 (SEQ ID NO: 14).

[0093] Embodiments of the present application will be described in detail with reference to the drawings, but it will be understood that the present application is not limited to the embodiments described below, and the following examples are intended to illustrate the present application and should not be construed as limiting the scope of the present application. In the examples, unless otherwise specified, the conditions were carried out under conventional conditions or conditions recommended by the manufacturer. The materials or instruments used were commercially available conventional products unless otherwise specified.

[0094] Example 1: High pathogenic monkeypox virus subtype target search

[0095] 1. Collection and screening of sequence data

[0096] Data source selection: A large number of monkeypox pathogen genome sequences were collected from the authoritative database GISAID. As a professional and authoritative database, GISAID covers a wealth of monkeypox virus sequence information globally, providing a sufficient data basis for subsequent analysis.

[0097] Sequence filtering and cleaning: Remove redundant sequences, low-quality sequences, and highly incomplete sequences. Redundant sequences increase the complexity and computational load of analysis, low-quality sequences may contain incorrect information affecting the analysis results, and highly incomplete sequences cannot provide comprehensive and accurate genetic information. Keep high-coverage and representative sequences to ensure the accuracy and comprehensiveness of the analysis. The data processed in this way can more truly reflect the genetic characteristics of monkeypox virus.

[0098] Sample regional and temporal coverage: Ensure that the collected sequences have diversity in geographical location and time. Different regions of monkeypox virus may have certain genetic differences, and viruses at different times may also evolve. Avoid regional bias and enhance the widespread applicability of conserved sequences.

[0099] 2. Phylogenetic analysis and feature region selection

[0100] Construction of phylogenetic tree: Based on the screened monkeypox virus genome, use Maximum Likelihood (Maximum Likelihood) and other phylogenetic analysis tools (such as RAxML or IQ-TREE) to construct a phylogenetic tree. Maximum likelihood method can calculate the most likely evolutionary tree topology according to the given evolutionary model and sequence data. Verify the reliability of the phylogenetic tree through Bootstrap (Bootstrap value), the higher the Bootstrap value, the more reliable the evolutionary relationship of the branch, ensuring the accuracy of the evolutionary relationship.

[0101] Evolutionary relationship analysis: Combine the phylogenetic tree topology to analyze the branch relationship of different types or subtypes of monkeypox virus. By observing the distribution and distance of the branches, we can identify the hot spot regions of variation, which are often places where viruses evolve faster and genes change more. At the same time, identify the representative characteristics of each type or subtype to provide a reference for the subsequent search for target points.

[0102] Feature region selection: Through comparison and phylogenetic analysis results, screen highly conserved gene regions. These regions have high sequence consistency and GC content among different types or subtypes, high sequence consistency ensures the coverage of detection, i.e. different types of monkeypox virus can be detected, and appropriate GC content helps to ensure the specificity of detection, reducing non-specific binding, ensuring the coverage and specificity of the detection sequence.

[0103] 3. Search and verification of A36R target point

[0104] Sequence alignment search for A36R gene: The screened sequences are aligned with the known A36R gene sequences, focusing on the sequences of highly pathogenic monkeypox viruses during the alignment process. The specific location and situation of the 48-base deletion of the A36R gene of the highly pathogenic monkeypox virus are determined through the alignment results.

[0105] Through the above steps, the specific characteristics of the A36R gene of the highly pathogenic monkeypox virus are determined, including its sequence information after deleting 48 bases (see Table 1), as well as its position in phylogenetics and its relationship with other genes. The feasibility of A36R gene as a target is verified, providing a new target and theoretical basis for the research and prevention and control of monkeypox virus.

[0106] Table 1 Target sequence

[0107]

[0108] Example 2: Design of highly pathogenic monkeypox virus subtype primer probe set

[0109] Obtain the specific gene sequences of the highly pathogenic monkeypox virus subtype from authoritative databases (such as NCBI). Select a highly conserved and specific region as the target sequence to ensure that the primers and probes can accurately identify the highly pathogenic monkeypox virus subtype. Use professional primer design software (Oligo7.0) to preliminarily design primers and probes for the target sequence. The software will automatically generate a large number of primer and probe candidate sequences based on the input sequence. According to the design principles, the preliminarily generated candidate sequences are screened. The specific screening conditions are as follows:

[0110] PCR product size: Select primer pairs that can amplify 50-150bp products. Such product size helps to improve the sensitivity and amplification efficiency of the reaction.

[0111] GC content: Screen primer and probe sequences with GC content between 30-80%. Suitable GC content can ensure the stability of primer and probe binding to the template.

[0112] Repeat sequence: Eliminate primers and probes containing more than 4 consecutive G repeats. Continuous G repeats can cause primers or probes to form secondary structures, affecting amplification efficiency.

[0113] TM value: Select sequences with probe TM values of 68-70°C and primer TM values of 58-60°C. The appropriate range of TM values helps to ensure the specific binding of primers and probes in PCR reactions.

[0114] Length requirement: Select sequences with probe lengths of 13-30bp and primer lengths of 20bp. Suitable length can ensure the specificity and binding ability of primers and probes.

[0115] Base distribution:

[0116] The probe avoids 6 consecutive A, and the 5' end does not start with G. Among the first 4 sequences of the 3' end of the probe, the number of G is not more than 3. Among the first 5 sequences of the 3' end of the primer, the number of C and G is not more than 2.

[0117] After the above screening process, the high pathogenic monkeypox virus subtype primer probe combination (see Table 2) is obtained.

[0118] Table 2 High pathogenic monkeypox virus subtype primer probe combination

[0119]

[0120] Example 3: Kit for detecting high pathogenic monkeypox virus subtype

[0121] The kit for detecting high pathogenic monkeypox virus subtype provided in this embodiment is composed of PCR reaction solution (including PCR buffer, MgCl2, dNTPs and primer probe group of Example 2), enzyme mixture, positive control and negative control; wherein the PCR reaction solution is composed of 2x FastAmpli Premix-UNG IV (Probe qPCR) (12.5 μL), MPV-1b-A36R-F1 (10 μM, 0.5 μL), MPV-1b-A36R-R1 (10 μM, 0.5 μL), MPV-1b-A36R-PB1 (10 μM, 0.25 μL) and DNase / RNase-free Water, as shown in Table 3.

[0122] Table 3 Composition of PCR reaction solution of high pathogenic monkeypox virus subtype detection kit

[0123] PCR reaction solution component Amount added 2x FastAmpli Premix-UNG IV (Probe qPCR) 12.5 μL MPV-1b-A36R-F1 (10 μM) 0.5 μL MPV-1b-A36R-R1 (10 μM) 0.5 μL MPV-1b-A36R-PB1 (10 μM) 0.25 μL DNase / RNase-free Water Added to 19 μL

[0124] The enzyme mixture contains uracil-N-glycosylase (UNG) and thermostable DNA polymerase (Taq enzyme).

[0125] The positive control is composed of high pathogenic monkeypox virus subtype detection target plasmid. The specific preparation method is as follows: take the plasmid stock solution with an initial concentration of 1 ng / μL, dilute it by 10 times for 4 gradients, and use it as the positive control working solution.

[0126] The negative control is DNase / RNase-free Water.

[0127] Example 4: A method for detecting high pathogenic monkeypox virus subtype

[0128] This embodiment provides a method for detecting highly pathogenic monkeypox virus subtypes. The method uses the kit from Example 2 and includes the following steps:

[0129] Step 1: Extract the nucleic acid from the sample using a viral DNA / RNA purification kit to obtain the nucleic acid extract;

[0130] Step 2: Mix 5 μL of nucleic acid extract, 5 μL of negative control, and 5 μL of positive control with 19 μL of PCR reaction solution and 1 μL of enzyme mixture, respectively, and perform fluorescent PCR using a fluorescent PCR instrument (reaction system and conditions are shown in Tables 4.1 and 4.2). Select the FAM channel to collect real-time fluorescence signals. Based on the collected real-time fluorescence signals, refer to the positive and negative controls to determine whether monkeypox virus is present in the test sample (when analyzing the results, the baseline fluorescence signal is taken as 3-10 or 6-15 cycles, and the threshold is set so that the threshold line just exceeds the highest point of the amplification curve of the normal negative control). The normal results of the positive and negative controls are shown in Table 4.3, and the criteria for judging whether monkeypox virus is present in the test sample are shown in Table 4.4.

[0131] Table 4.1 PCR reaction system

[0132] Component Amount added PCR reaction solution 20 μL Enzyme mixture 1 μL Sample 5 μL

[0133] Table 4.2 PCR reaction conditions

[0134]

[0135] Table 4.3 Normal results for positive and negative controls

[0136] Group Channel Normal result Negative control FAM No value Positive control FAM CT≤35 and with S-shaped amplification curve

[0137] Table 4.4 Criteria for determining the presence of highly pathogenic monkeypox virus subtypes in the test samples

[0138] FAM channel Experimental result judgment CT≤35 Highly pathogenic monkeypox virus subtype positive

[0139] Example 5: Specific detection of highly pathogenic monkeypox virus subtypes using primer and probe sets

[0140] Experiment 1: Using the monkeypox virus Clade Ib and non-Ib target fragment plasmids provided in Example 1 as amplification templates, real-time fluorescence PCR detection was performed using the primer and probe set from Example 2 (each set was repeated twice; the Q-PCR reaction system and conditions were the same as in Example 3). Results are as follows: Figure 1-4As shown: combination 1 and combination 2 produce specific amplification curves for monkeypox virus Ib type target fragment plasmid, and do not produce non-specific amplification curves for monkeypox virus non-Ib type target fragment plasmid; combination 3 and combination 4 produce non-specific amplification curves for non-Ib type target fragment plasmid. This shows that combination 1 and combination 2 have good specificity and can meet the needs of identifying 1b and non-1b. At the same time, the CT value of combination 1 is smaller than that of combination 2, indicating that its amplification efficiency is higher. Therefore, combination 1 is selected for subsequent experiments.

[0141] Experiment two: In the present application, the specificity verification of the kit is realized by the following scheme: select monkeypox virus related samples (including healthy population samples and cross reactivity verification samples selected according to “Monkeypox virus nucleic acid detection reagent registration and examination guiding principles”), and use specific primer probe groups designed for high pathogenic monkeypox virus subtypes for real-time fluorescent PCR detection, each 2 repeats. The experimental results are shown in Table 5, and no positive amplification signal is observed for all test samples (n = 20), and the verification results show that the kit has excellent specificity for detecting high pathogenic monkeypox virus subtypes, and has no cross reaction with related pathogenic organisms and human genome DNA.

[0142] Table 5 Detection results of high pathogenic monkeypox virus negative samples

[0143]

[0144]

[0145] Example 6: Sensitivity detection of high pathogenic monkeypox virus subtype primer probe group

[0146] Experiment one: The high pathogenic monkeypox virus subtype target fragment plasmid was diluted by 10 times at a starting concentration of 5E+08 copies / mL for 6 gradients, and a series of gradient dilution products were obtained. After nucleic acid extraction, Q-PCR detection (QPCR reaction conditions are the same as in Example 3) was performed, and each concentration was repeated twice. The results are shown in Figure 5-6 and Table 6, according to “Performance evaluation requirements for biotechnology nucleic acid target sequence quantification methods qPCR method and dPCR method” (GB / T 42077-2022), the linear correlation coefficient and amplification efficiency of the high pathogenic monkeypox virus subtype kit meet the standards (R2> 0.99, and the amplification efficiency E is in the range of 0.9-1.1). It shows that the high pathogenic monkeypox virus subtype kit of the present application has good linearity and amplification efficiency.

[0147] Table 6 Linear and amplification efficiency of high pathogenic monkeypox virus subtype primer probe group

[0148] Dilution factor Correlation coefficient Amplification efficiency 5E8-5E3 -0.99889 0.99141

[0149] Experiment 2: The target fragment plasmid of high pathogenic monkeypox virus subtype with the concentration of 2000, 1000, 200 and 4 copies / mL was taken, and after nucleic acid extraction, Q-PCR detection (QPCR reaction conditions were the same as those in Example 3) was carried out. Each concentration was tested 5 times, and the 95% detection level was taken as the minimum detection limit of the kit. The results showed that the minimum detection limit of the high pathogenic monkeypox virus subtype kit was less than 500 copies / mL.

Claims

1. A primer and probe set for the specific detection of highly pathogenic monkeypox virus subtypes, characterized in that, Include: Forward primer: Selected from one of the nucleotide sequences shown in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:12; Reverse primer: selected from one of the nucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:13; Probe: Selected from one of the nucleotide sequences shown in SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, and SEQ ID NO:

14.

2. The primer-probe set according to claim 1, characterized in that, The probe is modified with fluorescent markers and quenching groups.

3. The primer-probe set according to claim 1, characterized in that, The preferred combination is: Forward primer: SEQ ID NO:3; Reverse primer: SEQ ID NO:4; Probe: SEQ ID NO:

5.

4. A highly pathogenic monkeypox virus subtype-specific detection kit, characterized in that, Include: (a) The primer-probe set according to claim 1, 2 or 3; (b) PCR premix containing hot-start DNA polymerase, dNTPs, MgCl2, PCR buffer and uracil glycosylase (UNG); (c) Positive control: Gradual dilution plasmid containing the sequence shown in SEQ ID NO:1; (d) Negative control: Nuclease-free water; The reaction system of the kit is: 20 μL PCR premix + 5 μL nucleic acid extraction solution of the sample to be tested.

5. The reagent kit according to claim 4, characterized in that, The composition of the PCR premix is ​​as follows: 2×FastAmpli Premix-UNG IV: 12.5μL; Forward primer (10 μM): 0.5 μL; Reverse primer (10 μM): 0.5 μL; Probe (10μM): 0.25μL; Nuclease-free water: Add to a final volume of 20 μL.

6. The kit according to claim 4 or 5, characterized in that, The detection sensitivity of the kit is ≤500 copies / mL, and the linear range correlation coefficient R0 is [value missing]. 2 ≥0.99, amplification efficiency of 90%–110%.

7. The kit according to claim 4 or 5, characterized in that, The kit does not react with cross-pathogens, including varicella virus, herpes virus, measles virus, HPV, and bacterial samples.

8. A method for detecting a highly pathogenic monkeypox virus subtype, characterized in that, Using the kit according to claim 5 or 6, the steps include: (a) Extracting nucleic acid from the sample to be tested; (b) Mix the nucleic acid extract with the PCR premix and amplify under the following conditions: -50℃ for 2 minutes → 95℃ for 5 minutes → 40 cycles (95℃ for 10 seconds, 60℃ for 20 seconds); (c) Fluorescence signals are acquired via the FAM channel, and the judgment criteria are as follows: -Ct≤35 and exhibiting an S-shaped amplification curve: positive for monkeypox virus type Ib; - No amplification signal: negative.

Images