Primer probe set and kit for monkeypox virus detection

Abstract

The present invention relates to a primer probe group and a kit for detecting monkeypox virus, and belongs to the technical field of molecular biology. The present invention provides a primer probe group for detecting monkeypox virus, and the primer probe group includes a primer probe group targeting F3L gene, a primer probe group targeting B6R gene, and a primer probe group targeting RNase P gene. The primer probe group effectively avoids mutual interference between multiple primer probe groups through sequence design, and the primer probe group uses RNase P gene as an internal standard to monitor the collection, transportation and extraction process of the sample to be tested, effectively avoiding false negatives in the test results. Therefore, the use of the primer probe group to perform monkeypox virus quantitative detection on the sample to be tested has the advantages of high accuracy, strong specificity and high sensitivity, and has great application prospects.

Description

Technical Field

[0001] The invention relates to a primer probe set and a kit for detecting monkeypox virus, and belongs to the technical field of molecular biology. Background Art

[0002] Monkeypox is a viral zoonosis caused by infection with the monkeypox virus (MPXV). In the early stages of monkeypox, prodromal symptoms include fever, chills, headache, drowsiness, fatigue, back pain, and myalgia. 90% of monkeypox patients have obvious superficial lymphadenopathy, such as in the neck, axillae, and groin. A rash appears 1 to 3 days after the onset of monkeypox. The rash first appears on the face and gradually spreads to the limbs. The rash may appear on the palms and soles of the feet. The rash goes through several stages from macules, papules, herpes, pustules, and crusts, and different forms of rashes may exist at the same time. Among them, herpes and pustules are mostly spherical, about 0.5 to 1 cm in diameter, hard in texture, and may be accompanied by obvious itching and pain. The number of herpes varies from a few to thousands, and they are mostly centrifugally distributed, which can affect the oral mucosa, digestive tract, genitals, conjunctiva and cornea. The course of the disease is about 2 to 4 weeks. After the scab falls off, erythema or pigmentation may remain, or even scars may last for several years. Some monkeypox patients may develop complications, including secondary bacterial infection of the skin lesions, severe dehydration caused by diarrhea, bronchopneumonia, encephalitis and corneal infection.

[0003] Timely detection of monkeypox virus is critical for the diagnosis and subsequent treatment of patients. The positive result obtained by using fluorescent quantitative PCR detection technology to detect patients is the gold standard for confirming monkeypox. For fluorescent quantitative PCR detection technology, the selection of targets to be amplified is very critical. After selecting the appropriate target to be amplified, the design of the corresponding primer probe set is also very demanding. The design of the primer probe set is one of the key factors affecting the success of fluorescent quantitative PCR and whether its results are reliable. It is urgent to design more primer probe sets with better amplification effects to improve the accuracy, specificity and sensitivity of monkeypox virus detection using fluorescent quantitative PCR detection technology. Summary of the invention

[0004] To solve the above problems, the present invention provides a primer probe set for monkeypox virus detection, wherein the primer probe set includes a primer probe set targeting the F3L gene, a primer probe set targeting the B6R gene, and a primer probe set targeting the RNase P gene.

[0005] In one embodiment of the present invention, the primer probe set consists of a primer probe set targeting the F3L gene, a primer probe set targeting the B6R gene, and a primer probe set targeting the RNase P gene.

[0006] In one embodiment of the present invention, the primer probe set targeting the F3L gene includes a forward primer F3L-F having a nucleotide sequence as shown in SEQ ID NO.1, a reverse primer F3L-R having a nucleotide sequence as shown in SEQ ID NO.2, and a probe F3L-P having a nucleotide sequence as shown in SEQ ID NO.3.

[0007] In one embodiment of the present invention, the primer probe set targeting the B6R gene includes a forward primer B6R-F having a nucleotide sequence as shown in SEQ ID NO.4, a reverse primer B6R-R having a nucleotide sequence as shown in SEQ ID NO.5, and a probe B6R-P having a nucleotide sequence as shown in SEQ ID NO.6.

[0008] In one embodiment of the present invention, the primer probe set targeting RNase P gene includes a forward primer RNase PF with a nucleotide sequence as shown in SEQ ID NO.7, a reverse primer RNase PR with a nucleotide sequence as shown in SEQ ID NO.8, and a probe RNase PP with a nucleotide sequence as shown in SEQ ID NO.9.

[0009] In one embodiment of the present invention, the probe F3L-P, the probe B6R-P and the probe RNase PP are labeled with a fluorescent group and a fluorescent quenching group.

[0010] In one embodiment of the present invention, the 5' end of the probe F3L-P, the probe B6R-P and the probe RNase PP is labeled with a fluorescent group, and the 3' end is labeled with a fluorescent quenching group.

[0011] In one embodiment of the present invention, the fluorescent group includes one or more of FAM, VIC, CY5, ROX, HEX, JOE, NED, Texas Red and CY3.

[0012] In one embodiment of the present invention, the fluorescence quenching group includes one or more of BHQ-1, BHQ-2 and BHQ-3.

[0013] The present invention also provides a kit for detecting monkeypox virus, wherein the kit comprises the above primer probe set.

[0014] In one embodiment of the present invention, the kit further comprises a DNA polymerase, dNTPs and a buffer.

[0015] In one embodiment of the present invention, the pH of the buffer solution is 7.0-9.0.

[0016] In one embodiment of the present invention, the kit further comprises a negative control; the negative control is sterile water.

[0017] In one embodiment of the present invention, the kit further comprises a positive control; the positive control is a monkeypox pseudovirus or a quality control product; the quality control product comprises an F3L gene, a B6R gene and an RNase P gene.

[0018] In one embodiment of the present invention, the nucleotide sequence of the F3L gene is shown as SEQ ID NO.10; the nucleotide sequence of the B6R gene is shown as SEQ ID NO.11; and the nucleotide sequence of the RNase P gene is shown as SEQ ID NO.12.

[0019] In one embodiment of the present invention, the kit further comprises a sample collection solution; the components of the sample collection solution include water, sodium bicarbonate, sodium carbonate, Tween-20, cresol red and yeast ribonucleic acid.

[0020] In one embodiment of the present invention, the water is sterile water.

[0021] The present invention also provides a method for quantitatively detecting monkeypox virus, which is not intended for diagnosis and treatment of diseases. The method comprises: using the above-mentioned kit to detect a sample to be detected.

[0022] In one embodiment of the present invention, the method comprises the following steps:

[0023] Step 1: Perform virus lysis and nucleic acid purification on the sample to be tested to obtain a nucleic acid extract;

[0024] Step 2: Use the above kit to perform fluorescence quantitative PCR on the nucleic acid extract, and determine whether monkeypox virus exists in the sample to be tested based on the fluorescence signal data obtained by fluorescence quantitative PCR.

[0025] The present invention also provides the use of the primer probe set or the kit or the method in the quantitative detection of monkeypox virus, and the use is not for the purpose of disease diagnosis and treatment.

[0026] The technical solution of the present invention has the following advantages:

[0027] 1. The present invention provides a primer probe group for detecting monkeypox virus, wherein the primer probe group includes a primer probe group targeting F3L gene, a primer probe group targeting B6R gene and a primer probe group targeting RNase P gene; the primer probe group targeting F3L gene includes a forward primer F3L-F having a nucleotide sequence as shown in SEQ ID NO.1, a reverse primer F3L-R having a nucleotide sequence as shown in SEQ ID NO.2 and a probe F3L-P having a nucleotide sequence as shown in SEQ ID NO.3; the primer probe group targeting B6R gene includes a forward primer B6R-F having a nucleotide sequence as shown in SEQ ID NO.4, a reverse primer B6R-R having a nucleotide sequence as shown in SEQ ID NO.5 and a probe B6R-P having a nucleotide sequence as shown in SEQ ID NO.6; the primer probe group targeting RNase P gene includes a forward primer RNase PF having a nucleotide sequence as shown in SEQ ID NO.7, a reverse primer RNase PR having a nucleotide sequence as shown in SEQ ID NO.8 and a probe RNase P having a nucleotide sequence as shown in SEQ ID NO.9 PP. The primer probe set effectively avoids mutual interference between multiple primer probe sets through sequence design, and the primer probe set uses RNase P gene as an internal standard to monitor the collection, transportation and extraction process of the sample to be tested, effectively avoiding false negative test results. Therefore, the use of the primer probe set for quantitative detection of monkeypox virus in the sample to be tested has the advantages of high accuracy, strong specificity and high sensitivity, and has great application prospects.

[0028] Further, the 5' end of the probe F3L-P, the probe B6R-P and the probe RNase PP is labeled with a fluorescent group, and the 3' end is labeled with a fluorescent quenching group. When the primer probe set is used to perform quantitative detection of monkeypox virus on the sample to be tested, when the probe remains intact, the fluorescent quenching group will significantly reduce the fluorescence emitted by the fluorescent group through fluorescence resonance energy transfer (FRET), when PCR amplification, the primer and the probe will bind to the template at the same time, and the binding position of the probe is located between the upstream and downstream primers. When the amplification extends to the position where the probe is bound, the Taq enzyme will use the 5' exonuclease activity to cut the fluorescent group connected to the 5' end of the probe from the probe, so that it emits fluorescence, therefore, the number of fluorescent molecules detected is proportional to the number of PCR products, the number of initial DNA templates can be calculated according to the fluorescence intensity in the PCR reaction system, and a real-time amplification curve can be drawn according to the detected fluorescent signal, thereby realizing quantitative detection of monkeypox virus at the nucleic acid level, with intuitive, simple operation, and low professional requirements for personnel and equipment.

[0029] 2. The present invention provides a kit for detecting monkeypox virus, the kit comprising the above-mentioned primer probe set. The primer probe set used in the kit effectively avoids mutual interference between multiple primer probe sets through sequence design, and the primer probe set used in the kit uses RNase P gene as an internal standard to monitor the collection, transportation and extraction process of the sample to be tested, effectively avoiding false negatives in the test results. Therefore, the use of the kit for quantitative detection of monkeypox virus in the sample to be tested has the advantages of high accuracy, strong specificity and high sensitivity, and has great application prospects.

[0030] Further, in the primer probe group used in the kit, the 5' end of probe F3L-P, probe B6R-P and probe RNaseP-P is labeled with a fluorescent group, and the 3' end is labeled with a fluorescent quenching group. When the kit is used to perform monkeypox virus quantitative detection on the sample to be tested, when the probe remains intact, the fluorescent quenching group can significantly reduce the fluorescence emitted by the fluorescent group through fluorescence resonance energy transfer (FRET), when PCR amplification, the primer and the probe can be simultaneously bound to the template, and the binding position of the probe is located between the upstream and downstream primers, when the amplification extends to the position where the probe is bound, the Taq enzyme can utilize 5' exonuclease activity to cut the fluorescent group connected to the 5' end of the probe from the probe, so that it emits fluorescence, therefore, the number of fluorescent molecules detected is proportional to the number of PCR products, the number of initial DNA templates can be calculated according to the fluorescence intensity in the PCR reaction system, and a real-time amplification curve can be drawn according to the detected fluorescent signal, thereby realizing the quantitative detection of monkeypox virus at the nucleic acid level, with intuitive, simple operation, and low professional requirements for personnel and equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Fluorescence quantitative PCR amplification curve of the primer probe set for monkeypox virus detection in Example 1 for detecting F3L gene gradient dilution samples.

[0032] Figure 2 : Fluorescence quantitative PCR amplification curve of the primer probe set for monkeypox virus detection in Example 1 for detecting B6R gene gradient dilution samples.

[0033] Figure 3 : Fluorescence quantitative PCR amplification curve of the primer probe set for monkeypox virus detection in Example 1 for detecting RNase P gene gradient dilution samples.

[0034] Figure 4 : Fluorescence quantitative PCR amplification curve of repeated detection of F3L gene diluted samples by the primer probe set for monkeypox virus detection in Example 1.

[0035] Figure 5: Fluorescence quantitative PCR amplification curve of repeated detection of B6R gene diluted samples by the primer probe set for monkeypox virus detection in Example 1.

[0036] Figure 6 : Fluorescence quantitative PCR amplification curve of repeated detection of RNase P gene diluted samples by the primer probe set for monkeypox virus detection in Example 1.

[0037] Figure 7 : Fluorescence quantitative PCR amplification curve of 25 negative samples detected by the primer probe set for monkeypox virus detection in Example 1.

[0038] Figure 8 : Fluorescence quantitative PCR amplification curves of F3L gene diluted samples detected by different primer probe sets for monkeypox virus detection.

[0039] Fig. 9 : Fluorescence quantitative PCR amplification curves of B6R gene diluted samples detected by different primer probe sets for monkeypox virus detection.

[0040] Fig.10 : Fluorescence quantitative PCR amplification curves of different primer probe sets for monkeypox virus detection in detecting RNase P gene diluted samples. DETAILED DESCRIPTION

[0041] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0042] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0043] The DNase / RNase-free Water (CAT: 10601ES76), 4×UniversalTaqMan qPCR buffer, and Taq (50U / μL) involved in the following examples were purchased from Yishen Biotechnology Co., Ltd.; the positive reference product L1 (monkeypox virus nucleic acid detection performance verification reference product) involved in the following examples was purchased from Guangzhou Bondeson Biotechnology Co., Ltd. (the positive reference product L1 was obtained by mixing the B6R pseudovirus sequence shown in SEQ ID No. 37 and the F3L pseudovirus sequence shown in SEQ ID No. 38).

[0044] Example 1: Primer probe set for monkeypox virus detection

[0045] The present embodiment provides a primer probe set for detecting monkeypox virus, wherein the primer probe set comprises a primer probe set targeting F3L gene, a primer probe set targeting B6R gene and a primer probe set targeting RNase P gene; the primer probe set targeting F3L gene comprises a forward primer F3L-F-1 having a nucleotide sequence as shown in SEQ ID NO.1, a reverse primer F3L-R-1 having a nucleotide sequence as shown in SEQ ID NO.2 and a probe F3L-P-1 having a nucleotide sequence as shown in SEQ ID NO.3; the primer probe set targeting B6R gene comprises a forward primer B6R-F-1 having a nucleotide sequence as shown in SEQ ID NO.4, a reverse primer B6R-R-1 having a nucleotide sequence as shown in SEQ ID NO.5 and a probe B6R-P-1 having a nucleotide sequence as shown in SEQ ID NO.6; the primer probe set targeting RNase P gene comprises a forward primer RNase PF-1 having a nucleotide sequence as shown in SEQ ID NO.7, a reverse primer RNase P-1 having a nucleotide sequence as shown in SEQ ID NO.8 PR-1 and a probe RNase PP-1 whose nucleotide sequence is shown in SEQ ID NO.9; the 5' ends of the probes F3L-P-1, B6R-P-1 and RNase PP-1 are respectively labeled with fluorescent groups FAM, CY5 and VIC, and the 3' ends are respectively labeled with fluorescent quenching groups BHQ1, BHQ3 and BHQ2 (the sequences of the primer probe sets are shown in Table 1, and the target sequences are shown in Table 2).

[0046] Table 1 Primer probe set sequences

[0047]

[0048] Table 2 Target sequences

[0049]

[0050]

[0051] Example 2: Kit for monkeypox virus detection

[0052] This embodiment provides a kit for detecting monkeypox virus, the kit comprising a sample collection solution, a premix solution (including the primer probe set of Example 1), a positive control and a negative control group;

[0053] The sample collection solution is DNase / RNase-free water containing 6.4 g / L sodium bicarbonate, 12.6 g / L sodium carbonate, 0.02% (v / v) Tween-20, 0.01% (m / v, g / 100 mL) cresol red and 4 M guanidine hydrochloride;

[0054] The master mix was prepared by 4×Universal TaqMan qPCR buffer (6μL), Taq (0.1μL), F3L-F-1 (10μM, 0.6μL), F3L-R-1 (10μM, 0.6μL), F3L-P-1 (10μL M, 0.4μL), B6R-F-1 (10μM, 0.6μL), B6R-R-1 (10μM, 0.6μL), B6R-P-1 (10μM, 0.4μL), RNase Composed of PF-1 (10μM, 0.4μL), RNase PR-1 (10μM, 0.4μL), RNase PP-1 (10μM, 0.2μL) and DNase / RNase-free Water (9.7μL);

[0055] The positive control is the positive reference substance L1, and the preparation concentration is 500 copies / mL;

[0056] The negative control is DNase / RNase-free Water.

[0057] Example 3: A method for quantitative detection of monkeypox virus

[0058] This embodiment provides a method for quantitatively detecting monkeypox virus, the method using the kit of embodiment 2, comprising the following steps:

[0059] Step 1: Add the sample to be tested (such as a throat swab sample or 5 μL of blood sample) into a collection tube containing 600 μL of sample collection solution, mix well, and obtain a nucleic acid extract;

[0060] Step 2: After 5 μL of nucleic acid extract, 5 μL of negative control, and 5 μL of positive control were mixed with 20 μL of premixed solution, fluorescent quantitative PCR was performed using a fluorescent quantitative PCR instrument, and three channels of FAM, VIC, and CY5 were selected to collect real-time fluorescent signals; according to the real-time fluorescent signals collected, with reference to the positive control and the negative control, it was determined whether monkeypox virus existed in the sample to be tested (when analyzing the results, the baseline was taken as the fluorescent signal of 3 to 10 or 6 to 15 cycles, and the threshold setting principle was that the threshold line just exceeded the highest point of the amplification curve of the normal negative control);

[0061] Among them, the amplification parameters of fluorescent quantitative PCR are shown in Table 3, the normal results of positive control and negative control are shown in Table 4, and the judgment criteria for whether monkeypox virus exists in the sample to be tested are shown in Table 5.

[0062] Table 3 Amplification parameters

[0063] Cycle Type Temperature(℃) Time (seconds) Number of cycles Pre-denaturation 95 180 1 transsexual 95 5 40 Annealing, extension 58 20 40

[0064] Table 4 Normal results of positive control and negative control

[0065]

[0066] Table 5 Criteria for judging whether monkeypox virus exists in the sample to be tested

[0067]

[0068] Comparative Example 1: Primer probe set for monkeypox virus detection

[0069] This comparative example provides a primer probe set for detecting monkeypox virus. The primer probe set, based on Example 1, uses a primer probe set targeting the F3L gene consisting of a forward primer F3L-F-2 having a nucleotide sequence as shown in SEQ ID NO.13, a reverse primer F3L-R-2 having a nucleotide sequence as shown in SEQ ID NO.14, and a probe F3L-P-2 having a nucleotide sequence as shown in SEQ ID NO.15 (the primer probe set sequence is shown in Table 6, and the target sequence is shown in Table 12).

[0070] Table 6 Primer probe set sequences

[0071]

[0072] Comparative Example 2: Primer probe set for monkeypox virus detection

[0073] This comparative example provides a primer probe set for detecting monkeypox virus. The primer probe set, based on Example 1, uses a primer probe set targeting the F3L gene consisting of a forward primer F3L-F-3 having a nucleotide sequence as shown in SEQ ID NO.16, a reverse primer F3L-R-3 having a nucleotide sequence as shown in SEQ ID NO.17, and a probe F3L-P-3 having a nucleotide sequence as shown in SEQ ID NO.18 (the primer probe set sequence is shown in Table 7, and the target sequence is shown in Table 13).

[0074] Table 7 Primer probe set sequences

[0075]

[0076] Comparative Example 3: Kit for monkeypox virus detection

[0077] This comparative example provides a kit for detecting monkeypox virus. The primer probe set, based on Example 1, uses a primer probe set targeting the B6R gene consisting of a forward primer B6R-F-2 having a nucleotide sequence as shown in SEQ ID NO.19, a reverse primer B6R-R-2 having a nucleotide sequence as shown in SEQ ID NO.20, and a probe B6R-P-2 having a nucleotide sequence as shown in SEQ ID NO.21 (the primer probe set sequence is shown in Table 8, and the target sequence is shown in Table 12).

[0078] Table 8 Primer probe set sequences

[0079]

[0080] Comparative Example 4: Kit for monkeypox virus detection

[0081] This comparative example provides a kit for detecting monkeypox virus. The primer probe set, based on Example 1, uses a primer probe set targeting the B6R gene consisting of a forward primer B6R-F-3 having a nucleotide sequence as shown in SEQ ID NO.22, a reverse primer B6R-R-3 having a nucleotide sequence as shown in SEQ ID NO.23, and a probe B6R-P-3 having a nucleotide sequence as shown in SEQ ID NO.24 (the primer probe set sequence is shown in Table 9, and the target sequence is shown in Table 13).

[0082] Table 9 Primer probe set sequences

[0083]

[0084] Comparative Example 5: Kit for monkeypox virus detection

[0085] This comparative example provides a kit for detecting monkeypox virus. The primer probe set is based on Example 1, and uses a primer probe set targeting the RNase P gene composed of a forward primer RNase PF-2 having a nucleotide sequence as shown in SEQ ID NO.25, a reverse primer RNase PR-2 having a nucleotide sequence as shown in SEQ ID NO.26, and a probe RNase PP-2 having a nucleotide sequence as shown in SEQ ID NO.27 (the primer probe set sequence is shown in Table 10, and the target sequence is shown in Table 12).

[0086] Table 10 Primer probe set sequences

[0087]

[0088] Comparative Example 6: Kit for monkeypox virus detection

[0089] This comparative example provides a kit for detecting monkeypox virus. The primer probe set is based on Example 1, and uses a primer probe set targeting the RNase P gene composed of a forward primer RNase PF-3 having a nucleotide sequence as shown in SEQ ID NO.28, a reverse primer RNase PR-3 having a nucleotide sequence as shown in SEQ ID NO.29, and a probe RNase PP-3 having a nucleotide sequence as shown in SEQ ID NO.30 (the primer probe set sequence is shown in Table 11, and the target sequence is shown in Table 13).

[0090] Table 11 Primer probe set sequences

[0091]

[0092] Table 12 Target sequences

[0093]

[0094] Table 13 Target sequences

[0095]

[0096]

[0097] Comparative Examples 7-12: Kits for monkeypox virus detection

[0098] This comparative example provides a kit for detecting monkeypox virus. The kit is based on Example 2, except that the primer probe set of Example 1 is replaced by the primer probe sets of Comparative Examples 1 to 6, respectively.

[0099] Comparative Examples 13-18: A method for quantitative detection of monkeypox virus

[0100] This comparative example provides a method for quantitatively detecting monkeypox virus. The method is based on Example 3, except that the kit of Example 2 is replaced by the kits of Comparative Examples 7 to 12 respectively.

[0101] Experimental Example 1: Performance Verification of Primer Probe Set

[0102] In this experimental example, the performance of the primer probe sets in Example 1 and Comparative Examples 1 to 6 was verified using the positive reference L1 (prepared at a concentration of 500 copies / mL) and the negative monkeypox virus sample as the test samples, and the verification process was as follows:

[0103] Experiment 1: Using the positive reference L1 as the sample to be tested, the sample to be tested was diluted 10 times with DNase / RNase-free Water to a concentration of 1×10 9 , 1×10 8 , 1×107 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 copies / mL, and obtain the sample solution to be tested with a gradient concentration; the sample solution to be tested is used as the nucleic acid extract, and the nucleic acid extract is subjected to fluorescent quantitative PCR using the method of Example 3. The test results are shown in Figures 1 to 3 .

[0104] Experiment 2: Using the positive reference L1 as the sample to be tested, the sample to be tested was diluted 10 times with DNase / RNase-free Water to a concentration of 1×10 3 copies / mL, and obtain the sample solution to be tested; the sample solution to be tested is used as the nucleic acid extract, and the nucleic acid extract is subjected to fluorescent quantitative PCR using the method of Example 3, and the test is repeated 10 times. The test results are shown in Figures 4 to 6 .

[0105] Experiment 3: 25 negative monkeypox virus samples (throat swab samples from people without the disease) were used as test samples, and fluorescent quantitative PCR was performed on the test samples using the method of Example 3. The test results are shown in 7.

[0106] Experiment 4: Using the positive reference L1 as the sample to be tested, the sample to be tested was diluted 10 times with DNase / RNase-free Water to a concentration of 1×10 3 copies / mL, and obtain the sample solution to be tested; the sample solution to be tested is used as the nucleic acid extract, and the nucleic acid extract is subjected to fluorescent quantitative PCR using the methods of Example 3 and Comparative Examples 13 to 18, respectively. The test results are shown in Figures 8 to 10 .

[0107] Depend on Figures 1 to 3 It can be seen that when the primer probe set of Example 1 is used to perform nucleic acid detection on the sample to be tested, the detection results are all positive and the gradient data show a good arithmetic progression relationship.

[0108] Depend on Figures 4 to 6 It can be seen that when the primer probe set of Example 1 was used to detect monkeypox virus in the sample to be tested, the test was repeated 10 times, and the F3L and B6R gene tests were both positive, with a detection rate of 100%, high accuracy, strong specificity and high sensitivity.

[0109] Depend on Figure 7 It can be seen that among the 25 negative monkeypox virus samples tested, the F3L and B6R gene tests were all negative, with a detection rate of 100%.

[0110] Depend on Figures 8 to 10It can be seen that the sensitivity of detecting F3L, B6R, and RNase P genes using the primer probe set in Comparative Examples 1 to 6 is far lower than the sensitivity of detecting F3L, B6R, and RNase P genes using the primer probe set in Example 1.

[0111] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A primer probe set for detecting monkeypox virus, characterized in that: The primer probe set includes a primer probe set targeting the F3L gene, a primer probe set targeting the B6R gene, and a primer probe set targeting the RNase P gene; The primer probe set targeting the F3L gene includes a forward primer F3L-F having a nucleotide sequence as shown in SEQ ID NO.1, a reverse primer F3L-R having a nucleotide sequence as shown in SEQ ID NO.2, and a probe F3L-P having a nucleotide sequence as shown in SEQ ID NO.3; The primer probe set targeting the B6R gene includes a forward primer B6R-F having a nucleotide sequence as shown in SEQ ID NO.4, a reverse primer B6R-R having a nucleotide sequence as shown in SEQ ID NO.5, and a probe B6R-P having a nucleotide sequence as shown in SEQ ID NO.6; The primer probe set targeting the RNase P gene includes a forward primer RNase PF with a nucleotide sequence as shown in SEQ ID NO.7, a reverse primer RNase PR with a nucleotide sequence as shown in SEQ ID NO.8, and a probe RNase PP with a nucleotide sequence as shown in SEQ ID NO.

9.

2. The primer probe set according to claim 1, characterized in that: The probe F3L-P, the probe B6R-P and the probe RNase PP are labeled with a fluorescent group and a fluorescent quenching group.

3. A kit for detecting monkeypox virus, characterized in that: The kit comprises the primer probe set according to claim 1 or 2.

4. The kit according to claim 3, characterized in that The kit also includes DNA polymerase, dNTPs and a buffer.

5. The kit according to claim 3 or 4, characterized in that The kit also includes quality control products; the quality control products are F3L gene, B6R gene and RNase P gene.

6. A method for quantitatively detecting monkeypox virus, wherein the method is not for the purpose of disease diagnosis and treatment, characterized in that: The method comprises: using the kit described in any one of claims 3 to 5 to detect the sample to be tested.

7. Use of the primer probe set according to claim 1 or 2, the kit according to any one of claims 3 to 5, or the method according to claim 6 in the quantitative detection of monkeypox virus, wherein the use is for non-disease diagnosis and treatment purposes.

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