Primer probe combination, kit and application thereof for monkeypox virus detection

Abstract

The present invention relates to a primer-probe combination for detecting monkeypox virus, the primer-probe combination comprising: an upstream primer, a downstream primer and a probe, the upstream primer being: MPV3-F3L-F1 having a sequence as shown in SEQ ID NO:4, the downstream primer being: MPV3-F3L-nR2 having a sequence as shown in SEQ ID NO:11, and the probe being MPV-F3L-nP3 having a sequence as shown in SEQ ID NO:14. The primer-probe combination screened by the present invention has higher detection sensitivity, the detection result of the monkeypox virus detection kit has good accuracy and stability, is applicable to various types of samples to be tested, is simple and rapid to operate, and provides the possibility for preparing a monkeypox virus nucleic acid detection product suitable for clinical use.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology and relates to the field of immunochromatographic detection technology, and specifically relates to a primer-probe combination for monkeypox virus detection and an application thereof. Background Art

[0002] Monkeypox virus (MPXV) is an enveloped double-stranded DNA virus that belongs to the Poxviridae family and the Orthopoxvirus genus, along with Variola virus (VARV). The symptoms of infection are similar to those of smallpox, and it is commonly known as "monkey pox". Monkeypox can be transmitted from animals to humans, and cases usually occur near tropical rainforests. Although MPXV has limited human-to-human transmission, it can be infected through direct contact with the skin or lesions of monkeypox patients. Historically, the incubation period of monkeypox ranges from 5 to 21 days. Typically, the prophase of clinical illness lasts 1-5 days, during which patients may experience fever, headache, back pain, muscle pain, and lymphadenopathy. The second stage usually occurs after the fever subsides and is characterized by a rash that may involve the oral mucosa, conjunctiva, cornea, and / or genitals.

[0003] For MPXV test confirmation, the best diagnostic specimens are taken from skin (mucosal) lesions, including lesion surface swabs, acne exudate swabs, acne scab swabs, etc. With the development of molecular biology technology, rapid amplification based on gene targets has become a commonly used method in laboratories. At present, the use of quantitative polymerase chain reaction (qPCR) to detect viral DNA is the preferred laboratory detection method for monkeypox. However, it requires sophisticated temperature control instruments and well-trained staff, which limits its widespread application in resource-limited settings.

[0004] Based on the understanding of the principles of nucleic acid amplification, isothermal nucleic acid amplification has gradually become an alternative option. Combined with the understanding of immunochromatography technology, researchers are paying more and more attention to the development of instant detection technology. Isothermal amplification test strip technology, with its high sensitivity, low cost, fast speed, no need for instruments and on-site results, shows greater detection advantages in resource-poor laboratories and outdoor testing. Traditional molecular detection technology lacks these advantages due to the need for instruments, a higher experimental environment and trained professional technicians.

[0005] In view of this, it is very necessary to establish a simple, fast, and highly sensitive method for rapid detection of monkeypox virus and to develop rapid detection products for monkeypox that are suitable for current clinical and scientific research use. Summary of the invention

[0006] Based on this, the purpose of the present invention is to provide a primer-probe combination for detecting monkeypox virus, and its use in a reaction solution and a kit for detecting monkeypox virus. The primer-probe combination and the corresponding reaction solution can realize rapid, highly sensitive and characteristic detection of monkeypox virus.

[0007] The technical solutions for achieving the above-mentioned purpose include the following.

[0008] The first aspect of the present invention is to provide a primer-probe combination for detecting monkeypox virus, the primer-probe combination for detecting monkeypox virus comprising: an upstream primer, a downstream primer and a probe; the upstream primer is: MPV3-F3L-F1 having a sequence as shown in SEQ ID NO:4, the downstream primer is: MPV3-F3L-nR2 having a sequence as shown in SEQ ID NO:11, and the probe is MPV-F3L-nP3 having a sequence as shown in SEQ ID NO:14.

[0009] The second aspect of the present invention is to provide the use of the above primer-probe combination in the preparation of a monkeypox virus isothermal detection kit.

[0010] The third aspect of the present invention is to provide a monkeypox virus detection kit, which comprises any one of the above primer-probe combinations.

[0011] In some embodiments, the kit further comprises a dry powder reaction tube, wherein the reaction tube contains a lyophilized powder prepared from a reaction solution, wherein the reaction solution comprises 520-725 ng / μL of a recombinant enzyme; 100-200 ng / μL of a single-stranded DNA binding protein; 350-450 ng / μL of a DNA polymerase; and 300-400 ng / μL of a nuclease.

[0012] In some embodiments, the preparation method of the lyophilized powder includes the following steps: pre-freezing the reaction solution at -80°C for 1 to 1.5 hours to obtain a pre-frozen reagent, first placing the pre-frozen reagent in an environment of -35 to -45°C and drying it for 2 to 10 hours, and then placing it in an environment of 10 to 18°C ​​and drying it for 1 to 2 hours to obtain a lyophilized powder.

[0013] In some embodiments, the kit further comprises a reaction solution, comprising Abuffer, Bbuffer and ddH2O; the Abuffer comprises: 15mM~25mM Tris-HCl, 130mM~170mM KCl, 4v / v%~6v / v% PEG, 0.5v / v%~1.5v / v% TRITON-X100, the pH of the Abuffer is 8.0~9.0, and the Bbuffer comprises: 180mM~220mM magnesium acetate.

[0014] In some embodiments, the kit further comprises a test strip, which is prepared by a PVC backboard and a sample pad, a conjugate pad, a nitrocellulose membrane and absorbent paper sequentially arranged on the PVC backboard, and the control line and the test line are coated with BSA-biotin and anti-fluorescein antibody, respectively.

[0015] In some embodiments, the kit further includes a reaction chamber, which includes a constant temperature module and the above-mentioned test strip.

[0016] In some embodiments, the kit further comprises a double-balloon dropper, which can be used as a substitute for a pipette.

[0017] In some embodiments, the kit further comprises a diluent comprising ddH2O.

[0018] In some embodiments, the kit further comprises: Abuffer, B buffer and ddH2O; the Abuffer comprises: 15mM-25mM Tris-HCl, 130mM-170mM KCl, 4v / v%-6v / v% PEG, 0.5v / v%-1.5v / v% TRITON-X100, and the pH of the Abuffer is 8.0-9.0.

[0019] The B buffer includes: 180mM to 220mM magnesium acetate.

[0020] The fourth aspect of the present invention is to provide a reaction system for detecting monkeypox virus, which comprises: Abuffer, Bbuffer, a primer-probe combination and ddH2O,

[0021] The primer-probe combination includes: an upstream primer, a downstream primer and a probe, the upstream primer is: MPV3-F3L-F1 whose sequence is shown in SEQ ID NO:4, the downstream primer is: MPV3-F3L-nR2 whose sequence is shown in SEQ ID NO:11, and the probe is: MPV-F3L-nP3 whose sequence is shown in SEQ ID NO:14.

[0022] In some of the embodiments, in the reaction system, the concentration of the upstream primer is 10 μM, the concentration of the downstream primer is 10 μM, and the concentration of the probe is 10 μM.

[0023] In some embodiments, the concentration of magnesium ions in the reaction system is 200 mM.

[0024] The fifth aspect of the present invention is to provide a method for detecting monkeypox virus nucleic acid for non-diagnostic purposes using any of the above-mentioned kits or any of the above-mentioned reaction systems.

[0025] A method for detecting monkeypox virus nucleic acid for non-disease diagnosis purposes, comprising the following steps:

[0026] S1. Extracting nucleic acid from the sample to be tested;

[0027] S2. Add the nucleic acid to the reaction system containing Abuffer, primer probe combination, ddH2O, and B Buffer, mix thoroughly, and incubate in a constant temperature device at 39°C to 42°C for 20 minutes;

[0028] S3, adding ddH2O, mixing, and detecting with colloidal gold test paper for monkeypox virus nucleic acid and observing the results;

[0029] The primer-probe combination in step S2 includes: an upstream primer, a downstream primer and a probe, the upstream primer is: MPV3-F3L-F1 whose sequence is shown in SEQ ID NO:4, the downstream primer is: MPV3-F3L-nR2 whose sequence is shown in SEQ ID NO:11, and the probe is: MPV-F3L-nP3 whose sequence is shown in SEQ ID NO:14.

[0030] In some embodiments, the sample to be tested is selected from at least one of body fluids such as throat swabs, rash exudate swabs, acne fluid, acne epidermis, acne scabs, whole blood, serum, etc.

[0031] The present invention, through a large number of studies on monkeypox virus by the inventors, explores and designs primers and probes based on the F3L gene, and screens out a primer-probe combination with higher detection sensitivity. The detection results of the monkeypox virus detection kit have good accuracy and stability, are suitable for various types of samples to be tested, and have fewer detection operation steps, simpler amplification reaction conditions, and shorter time consumption, and do not rely on expensive instruments and equipment, thereby providing the possibility of preparing a simple, rapid, and highly sensitive monkeypox virus nucleic acid detection product suitable for clinical use and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram showing the gene comparison results based on the F3L gene primer probe in Example 1.

[0033] Figure 2 This is a diagram of the screening combination based on the F3L gene primer probe MPV-F3L-nP2 in Example 1.

[0034] Figure 3 This is a diagram of the screening combination based on the F3L gene primer probe MPV-F3L-nP3 in Example 1.

[0035] Figure 4 This is a graph showing the electrophoresis results of the P2 concentration sample based on the F3L gene primer screening combination in Example 1.

[0036] Figure 5 This is a graph showing the electrophoresis results of the P3 concentration sample based on the F3L gene primer screening combination in Example 1.

[0037] Figure 6 This is a diagram showing the optimization of isothermal amplification reaction conditions based on the F3L gene primer probe in Example 1.

[0038] Figure 7 This is a graph showing the detection sensitivity results of the monkeypox virus isothermal detection kit based on the F3L gene primer probe in Example 2.

[0039] Figure 8 This is a graph showing the sensitivity results of the Jiangsu ShuoShi Monkeypox Virus Nucleic Acid Detection Kit based on the F3L gene primer probe in Example 2.

[0040] Fig. 9 This is a graph showing the detection results of the monkeypox virus isothermal detection kit based on the F3L gene primer probe in Example 3.

[0041] Fig.10 This is the result diagram of the Jiangsu ShuoShi Monkeypox Virus Nucleic Acid Detection Kit based on the F3L gene primer probe in Example 3.

[0042] Fig.11 This is a graph showing the experimental results of the inter-batch and intra-batch repeatability and stability of the monkeypox virus isothermal detection kit based on the F3L gene primer probe in Example 3.

[0043] Fig.12 This is a result diagram of the monkeypox virus isothermal detection kit based on the F3L gene primer probe in Example 4.

[0044] Fig.13 This is a result diagram of the monkeypox virus isothermal detection kit based on the F3L gene primer probe in Example 5. DETAILED DESCRIPTION

[0045] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0046] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0047] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0048] The present invention is further described in detail below with reference to specific embodiments.

[0049] Example 1: Design and screening of primer and probe combinations for F3L gene

[0050] 1. Primer and probe design

[0051] The present invention compares and analyzes the gene sequence of monkeypox F3L. Figure 1 The comparison results showed that the monkeypox F3L gene sequence was conservative and could be used as a target gene for primer probe design. At the same time, the upstream and downstream primer combinations (as shown in Table 1) of the two primer probes based on the F3L gene (MPV-F3L-nP2 and MPV-F3L-nP3) were compared. The comparison results were as follows: Figure 2 and Figure 3 It was shown that these primers were able to form 27 screening combinations (as shown in Table 2).

[0052] Table 1 Primer probe sequences based on F3L gene

[0053]

[0054] *Note: "biotion" is modified biotin; "FAM" is a fluorescent group; "THF" is a tetrahydrofuran site. "3'C3spacer".

[0055] Table 2 Primer combinations

[0056]

[0057]

[0058] 2. Primer combination screening

[0059] Screening of primer combinations for the F3L gene, the reaction system and operation procedures are as follows:

[0060] 1. Screening primer probe reaction system combination:

[0061] The reaction system is: 29.4 μL Abuffer, 2 μL upstream primer, 2 μL downstream primer, 4.1 μL ddH2O, 10 μL nucleic acid template, 2.5 μL B buffer; the total volume of the reaction is 50 μL. The Abuffer includes: 20 mM Tris-HCl (pH=8.5, 25°C), 150 mM KCl, 5 v / v% PEG, 1 v / v% TRITON-X100; the B buffer is 200 mM magnesium acetate; the concentration of the primer in the reaction is: 10 μM.

[0062] 2. Prepare the sample:

[0063] The monkeypox positive sample nucleic acid was selected for dilution, and the dilution process is shown in Table 3.

[0064] Table 3 Dilution process of monkeypox virus nucleic acid template

[0065]

[0066] 3. The operation process includes:

[0067] (1) Add 29.4 μL Abuffer to each dry powder reaction tube, and then add 2 μL upstream primer, 2 μL downstream primer and 0.6 μL probe (the concentration of probe and primer is 10 μM) to each reaction tube; the dry powder reaction tube contains lyophilized powder, and the lyophilized powder components are 700 ng / μL recombinant enzyme; 120 ng / μL single-stranded DNA binding protein; 400 ng / μL DNA polymerase; 480 ng / μL reverse transcriptase; 300 ng / μL nuclease to make a reaction solution, pre-freeze the reaction solution at -80°C for 1.2 hours to obtain a pre-frozen reagent, first dry the pre-frozen reagent at -40°C for 7 hours, and then dry it at 15°C for 1.5 hours to obtain a lyophilized powder.

[0068] (2) Add 3.5 μL ddH2O and 10 μL nucleic acid template (concentration P2, P3) to the reaction tube in sequence, then add 2.5 μL B Buffer to the reaction tube and mix thoroughly; after mixing, shake the reaction solution to the bottom of the reaction tube, and immediately place the reaction tube in a constant temperature device at 39-42°C for incubation for 20 min;

[0069] (3) After the reaction is completed, 50 μL of phenol / chloroform is added to extract the reaction solution, centrifuged at 12,000 rpm for 5 min, and 5-8 μL of the supernatant is taken for electrophoresis detection.

[0070] 4. Primer screening results:

[0071] Select P2 / P3 concentration for basic electrophoresis amplification screening. At P2 concentration, 12 primer combinations can have visible target bands ( Figure 4 ); At P3 concentration, 7 combinations have visible target bands ( Figure 5 ), and selected 7 selected primer combinations and probes for subsequent experiments.

[0072] 3. Primer-probe combination screening

[0073] 1. After screening out primers with good amplification efficiency and matching probes, a total of 7 sets of primer-probe combinations can be obtained as shown in Table 4. Table 4 Statistics of primer sets with good amplification efficiency and matching probes

[0074]

[0075] 2. Screening primer probe reaction system combination:

[0076] The reaction system is: 29.4 μL Abuffer, 2 μL upstream primer, 2 μL downstream primer, 0.6 μL probe, 3.5 μL ddH2O, 10 μL nucleic acid template, 2.5 μL B buffer; the total volume of the reaction is 50 μL. The Abuffer includes: 20 mM Tris-HCl (pH=8.5, 25°C), 150 mM KCl, 5 v / v% PEG, 1 v / v% TRITON-X100; the B buffer is 200 mM magnesium acetate; the concentration of the primer and probe in the reaction is: 10 μM.

[0077] 3. Prepare the sample:

[0078] The monkeypox positive sample nucleic acid was selected for dilution, and the dilution process was the same as that in Table 3.

[0079] 4. The operation process includes:

[0080] (1) Add 29.4 μL Abuffer to each dry powder reaction tube, and then add 2 μL upstream primer, 2 μL downstream primer and 0.6 μL probe to each reaction tube (the concentration of probe and primer is 10 μM);

[0081] (2) 3.5 μL ddH2O and 10 μL nucleic acid template were added to the reaction tube in sequence, wherein the nucleic acid concentration was 0 (blank control, i.e., ddH2O) and P2-P4 (Table 3), and then 2.5 μL B Buffer was added to the reaction tube and mixed thoroughly; after mixing, the reaction solution was shaken to the bottom of the reaction tube, and the reaction tube was immediately placed in a reaction chamber and incubated at 39°C for 20 min;

[0082] (3) Prepare colloidal gold test strips for monkeypox virus nucleic acid according to conventional methods and set aside. The test strips are prepared from a PVC backing plate and a sample pad, a conjugate pad, a nitrocellulose membrane and absorbent paper arranged on the PVC backing plate in sequence, and the control line and the test line are coated with BSA-biotin and anti-fluorescein antibody, respectively.

[0083] (4) After the reaction is completed, take 10 μL of the reaction solution and add it to a centrifuge tube containing 190 μL of ddH2O. After mixing evenly, use a pipette to take 50 μL and drop it into the sample well of the test strip in (3) above. Wait for 2-3 minutes and observe the quality control line and the test line to read the results.

[0084] 5. Primer probe screening results:

[0085] The blank control test was performed on 7 sets of primer-probe combinations. The 4th and 6th sets were abnormal and were eliminated. The remaining 5 sets were compared for positive sensitivity. The MPV3-F3L-F1 / MPV3-F3L-nR2 / MPV-F3L-nP3 combination could stably detect the P4 concentration. The optimal combination was MPV3-F3L-F1 / MPV3-F3L-nR2 / MPV-F3L-nP3( Figure 6 ).

[0086] Note: a.1-7: 7 sets of primer-probe combinations, refer to Table 4;

[0087] b.1-4 are the results of the MPV2-F3L-F1 / MPV2-F3L-nR1 / MPV-F3L-nP2 combination, 5-8 are the results of the MPV2-F3L-F1 / MPV2-F3L-nR3 / MPV-F3L-nP2 combination;

[0088] c.1-4 are the results of the MPV2-F3L-F2 / MPV2-F3L-nR1 / MPV-F3L-nP2 combination, 5-8 are the results of the MPV3-F3L-F1 / MPV3-F3L-nR2 / MPV-F3L-nP3 combination;

[0089] d. MPV3-F3L-F3 / MPV3-F3L-nR2 / MPV-F3L-nP3 combination results.

[0090] bd concentration: 1, 5: blank control, 2, 6: P2 concentration, 3, 7: P3 concentration, 4, 8: P4 concentration.

[0091] Example 2: Comparison of sensitivity with Jiangsu Shuoshi Monkeypox Virus Nucleic Acid Detection Kit

[0092] The sensitivity of the monkeypox virus isothermal detection kit described in Example 1 of the present invention was compared with the monkeypox virus nucleic acid detection kit (fluorescence PCR method) commercially available from Jiangsu ShuoShi.

[0093] The monkeypox virus isothermal detection kit provided by the present invention at least includes an upstream primer, a downstream primer, a probe, and a nucleic acid detection test strip. For ease of use, the reagents of the kit include Abuffer, B buffer, an upstream primer, a downstream primer, a probe, and ddH2O. The nucleic acid detection test strip is prepared from a conventional nitrocellulose membrane, a sample pad, a conjugate pad, and a blotting paper.

[0094] The primer-probe combination in the monkeypox virus isothermal detection kit used in this example is the optimal combination selected in Example 1: upstream primer MPV3-F3L-F1, downstream primer MPV3-F3L-nR2, and probe MPV-F3L-nP3.

[0095] 1. The detection operation steps of the monkeypox virus constant temperature detection kit of the present invention are as follows:

[0096] (1) Select monkeypox positive sample nucleic acid for dilution. The dilution process is the same as Table 3. The volume of nucleic acid template should not be less than 60 μL.

[0097] (2) Use a double-balloon dropper, press the upper balloon to absorb enough nucleic acid (the nucleic acid is filled in the dropper tube as the standard, and the excess liquid will flow into the lower balloon), absorb 50 μL of nucleic acid, the nucleic acid concentration is 0 (blank control, i.e., ddH2O) and P3-P4, add the nucleic acid into the reaction chamber through the opening on one side of the test strip reaction chamber assembly, and drip all the absorbed nucleic acid into the reaction chamber through the opening on one side of the test strip assembly (it is normal for liquid to remain in the lower balloon, and there is no need to squeeze out the liquid in the lower balloon).

[0098] (3) Amplify in the reaction chamber at 39-42°C for 20 min.

[0099] (4) After the reaction is completed, add diluent (ddH2O) to the reaction chamber to dilute the amplified product 10-20 times. After sufficient mixing, the test strip is automatically developed in the reaction chamber. The quality control line and the detection line are observed within 5 minutes to read the results.

[0100] (5) Interpretation of results:

[0101] Positive result: There are bands on both the quality control line and the test line, indicating that the amplified product contains the target nucleic acid fragment to be detected, and its content is greater than or equal to the minimum detection amount of the test strip.

[0102] Negative result: Only the quality control line has a band, and the test line has no band, indicating that the amplified product does not contain the target nucleic acid fragment to be detected.

[0103] Invalid result: No bands appear on the control line or the test line. This result indicates that the test strip used is invalid, damaged, or operated incorrectly. At this time, you should read the instructions carefully and re-amplify and test strictly according to the instructions.

[0104] 2. The reaction system and procedure of Jiangsu ShuoShi Monkeypox Virus Nucleic Acid Detection Kit are as follows:

[0105] The reaction system consists of 12.5 μL nucleic acid amplification reaction solution, 7.5 μL monkeypox virus detection solution, and 5 μL template, and the total system is 25 μL. The reaction procedure is 95°C, 5 min, 1 cycle of pre-denaturation and 95°C, 10s+60°C, 30s, 45 cycles of cyclic reaction. The nucleic acid is the same as the nucleic acid used for the monkeypox virus constant temperature detection kit of the present invention, and the nucleic acid concentration is 0 (blank control, i.e. ddH2O) and P1-P5.

[0106] The sensitivity test results of the monkeypox virus constant temperature detection kit of the present invention and the monkeypox virus nucleic acid detection kit of Jiangsu Shuoshi are as follows: Figure 7 and Figure 8 The sensitivity test results are summarized in Table 5.

[0107] Table 5 Comparison of detection sensitivity results of kits based on F3L gene primer probe

[0108]

[0109] According to the sensitivity test comparison results (see Figure 7 and Figure 8 ), the sensitivity of the monkeypox virus constant temperature detection kit is P3 concentration sample nucleic acid, which is consistent with the Jiangsu ShuoShi monkeypox virus nucleic acid detection kit, and the corresponding qPCR CT value is about 37.

[0110] The sensitivity of clinical samples was compared and verified with qPCR. The experimental results showed that the clinical sample sensitivity of the monkeypox virus isothermal detection kit we developed (primer probe combination: MPV3-F3L-F1 / MPV3-F3L-nR2 / MPV-F3L-nP3) was P3 concentration, and the corresponding CT value of qPCR was about 36-37, and the sensitivity of the two was consistent.

[0111] Example 3: Verification of monkeypox positive sample nucleic acid conformity and batch-to-batch repeatability and stability experiments

[0112] The present invention verifies the nucleic acid conformity of monkeypox positive samples, and also conducts batch-to-batch repeatability and stability experiments.

[0113] 1. Stability test, the steps include the following:

[0114] In the stability experiment of monkeypox positive sample nucleic acid conformity verification, the Jiangsu Shuoshi monkeypox virus nucleic acid detection kit and the monkeypox virus constant temperature detection kit of the present invention were respectively subjected to multiple clinically verified positive sample nucleic acid detection. The two detection kits were respectively based on the same template, sample type and template dilution concentration, and the CT value results of the detection were compared, and the operation steps were the same as in Example 2.

[0115] The test results of the monkeypox virus constant temperature detection kit of the present invention and the test results of the Jiangsu Shuoshi monkeypox virus nucleic acid detection kit are as follows: Fig. 9 and Fig.10 , as shown in Table 6 and Table 7.

[0116] Table 6 Detection results of the monkeypox virus isothermal detection kit based on the F3L gene primer probe

[0117] serial number Sample Type Template concentration Shuoshi Ct Results of the present invention 1 Acne fluid Dilute 10 times 32.09 + 2 Acne fluid Dilute 10 times 27.26 + 3 Acne epidermis Dilute 10 times 22.72 + 4 Acne fluid Dilute 10 times 23.05 + 5 Swab Dilute 10 times 34.72 + 6 serum Original concentration 37.28 / 35.56 + / + 7 Rash exudate swab Original concentration 35.17 + / + 8 Acne scab Original concentration 38.83 / 35.82 + / - 9 Acne fluid Dilute 10 times 26.43 + 10 Acne scab Dilute 10 times 23.45 + 11 Acne fluid Original concentration 33.44 + 12 Acne epidermis Dilute 10 times 35.03 + BC Blank control - - -

[0118] Note: Corresponding Fig. 9 The numbers shown are sample numbers.

[0119] Table 7 Results of Jiangsu Shuoshi Monkeypox Virus Nucleic Acid Detection Kit

[0120] serial number Sample Type Template concentration Shuoshi Ct Customer Provided Ct 1 Acne fluid Dilute 10 times 32.09 23.79 2 Acne fluid Dilute 10 times 27.26 24.9 3 Acne epidermis Dilute 10 times 22.72 18.23 4 Acne fluid Dilute 10 times 23.05 18.56 5 Swab Dilute 10 times 34.72 28.26 6 serum Original concentration 37.28 / 35.56 39.43 7 Rash, exudate swab Original concentration 35.17 30.16 8 Acne scab Original concentration 38.83 / 35.82 34.25 9 Acne fluid Dilute 10 times 26.43 20.03 10 Acne scab Dilute 10 times 23.45 21.89 11 Acne fluid Original concentration 33.44 32.3 12 Acne epidermis Dilute 10 times 35.03 26.66 Comparison - - - -

[0121] Note: Corresponding Fig.10 , red experiment number 1 diluted 10 times, blue experiment number 2 diluted 10 times, green experiment number 3 diluted 10 times, light orange experiment number 4 diluted 10 times, pink experiment number 5 diluted 10 times, purple experiment number 6 original concentration, gray experiment number 7 original concentration, fluorescent blue experiment number 8 original concentration, orange experiment number 9 diluted 10 times, light blue experiment number 10 diluted 10 times, grass green experiment number 11 original concentration, yellow experiment number 12 diluted 10 times;

[0122] like Fig. 9 and Fig.10 As shown in Tables 6 and 7, 11 of the 12 groups of clinical sample nucleic acids could be stably detected, among which the nucleic acid of sample No. 8 was unstable, with a compliance rate of about 100%.

[0123] The other 23 samples were tested. The operation steps were the same as those in Example 2, and the dilution steps of some samples were the same as those in Table 3. The results are shown in Table 8.

[0124] Table 8 Results of nucleic acid stability test of monkeypox samples based on F3L gene primer probe

[0125] Sample name Sample Type Jiangsu Shuoshi Ct Results of the present invention Mixed template 1 / 36.64 / 39.01 + / + / + Mixed Template 2 / 39.01 / - + / + / - Sample 5 was diluted 10 times Throat swab 36.93 / 37.99 + / + / + Sample 6 was diluted 10 times Throat swab 35.29 / 34.55 + / + / + Mixed Template 3 / - / - + / + / - Sample 14 was diluted 10 times Throat swab 36.67 / - - / - / + Sample 19 diluted 10 times Whole blood 37.40 / - + / + / + Mixed Template 4 / 35.79 / 34.46 / 34.40 + / + / + Sample 24 Acne fluid - / 36.84 + / + / + 1 Acne fluid 32.09 / 32.02 / 32.10 + / + / + 2 Acne fluid 27.26 / 27.25 / 27.23 + / + / + 3 Acne epidermis 22.72 / 22.95 + / + / + 4 Acne fluid 23.05 / 23.00 + / + / + 5 Swab 34.72 / 35.96 / 36.19 + / + / + / + 6 serum 37.28 / 35.56 / 35.02 / 35.55 + / + / + / + 7 Rash exudate swab 35.17 / 34.04 / 33.75 + / + / + / + 8 Acne scab 38.83 / 35.82 / 36.57 / 36.56 + / - / + / + 9 Acne fluid 26.43 / 27.16 / 27.14 + / + / + 10 Acne scab 23.45 / 24.32 + / + / + 11 Acne fluid 33.44 / 31.97 / 32.15 + / + / + 12 Acne epidermis 35.03 / 35.15 / 35.67 + / + / + / + 2-P2 Acne fluid 35.02 / 34.96 + / + / + 2-P3 Acne fluid 37.36 / 38.04 + / + / +

[0126] Table 8 shows that among the 23 groups of conformity tests, a total of 22 groups of qPCR and the present invention stably detected positive results; 4 groups of qPCR were unstable, corresponding to the present invention, there were 2 groups of stable detection and 2 groups of unstable detection; 1 group of qPCR was detected, and the present invention was unstable; 1 group of qPCR was not detected, and the present invention was stable; the corresponding qPCR reagent Ct38-39 suspected interval limit sensitivity can be detected. The positive conformity rate is about 100%, and the comprehensive judgment of the clinical sample nucleic acid conformity rate meets the requirements for conformity rate in the quality evaluation requirements of the monkeypox virus constant temperature detection kit.

[0127] 2. Inter-batch and intra-batch repeatability experiments

[0128] According to the experimental steps described in 1, the inter-batch and intra-batch repeatability experiments were also carried out on the monkeypox virus isothermal detection kit of the present invention.

[0129] The blue line is the C line (Control line), that is, the quality control line. A positive quality control line indicates that the experimental test results are valid, otherwise it is invalid; the red line is the T line (Test line), that is, the test line. A stripe on the test line indicates that the test result is positive, otherwise it is negative.

[0130] The results of five repeated tests are as follows Fig.11 The results show that when the experiment was conducted with the same template concentration, the color development within the batch was not very different, and the difference between batches was also not large, which was consistent.

[0131] The types of nucleic acid samples currently verified include throat swabs, whole blood, serum, acne scabs, acne fluid, surface swabs, rash exudate swabs and other types of samples, and the suspected concentration range of nucleic acid (CT37-40) corresponding to the Jiangsu Shuo Shi Monkeypox Virus Nucleic Acid Detection Kit can be detected. The monkeypox virus constant temperature detection kit has been tested on a large number of clinical sample nucleic acid, and its detection sensitivity, accuracy, and clinical sample nucleic acid detection compliance rate can reach the level of the Jiangsu Shuo Shi Monkeypox Virus Nucleic Acid Detection Kit currently used in clinical practice. The monkeypox virus constant temperature detection kit can complete amplification in 20 minutes at a constant temperature of 39-42°C, and the operation is simple and fast.

[0132] Example 4: Comparison of the sensitivity of the present invention and the Jiangsu ShuoShi Monkeypox Virus Nucleic Acid Detection Kit for extracting nucleic acid using magnetic bead extraction

[0133] The sensitivity of the monkeypox virus isothermal detection kit described in Example 1 of the present invention and the monkeypox virus nucleic acid detection kit (fluorescence PCR method) commercially available from Jiangsu Shuoshi was compared after nucleic acid was extracted from clinical samples using the magnetic bead extraction method.

[0134] The components of the monkeypox virus isothermal detection kit provided by the present invention are consistent with those in Example 2.

[0135] The primer-probe combination in the monkeypox virus isothermal detection kit used in this example is the optimal combination selected in Example 1: upstream primer MPV3-F3L-F1, downstream primer MPV3-F3L-nR2, and probe MPV-F3L-nP3.

[0136] (1) Three clinical samples (provided by Guangzhou Center for Disease Control and Prevention) were serially diluted using ddH2O to obtain 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 times the sample concentration.

[0137] (2) A nucleic acid extraction or purification kit (magnetic bead extraction method) (purchased from Jiangsu ShuoShi Biotechnology Co., Ltd.) was used to extract and purify the sample in (1) and its original concentration to obtain nucleic acid. The operation steps were carried out according to the instructions.

[0138] (3) The detection operation steps of the monkeypox virus isothermal detection kit of the present invention are consistent with those of Example 2.

[0139] (4) The detection procedure of Jiangsu ShuoShi Monkeypox Virus Nucleic Acid Detection Kit is the same as that of Example 2.

[0140] The test results are shown in Table 9 and Fig.12 shown.

[0141] Table 9 Comparison of detection sensitivity results of kits for extracting nucleic acid based on magnetic bead extraction

[0142]

[0143] The sensitivity of the clinical sample nucleic acid extracted by the magnetic bead extraction method was compared between the present invention and the Jiangsu ShuoShi monkeypox virus nucleic acid detection kit. The experimental results showed that the monkeypox virus constant temperature detection kit of the present invention has higher sensitivity.

[0144] Example 5: Comparison of sensitivity between the quick release solution of the present invention and the Jiangsu ShuoShi Monkeypox Virus Nucleic Acid Detection Kit

[0145] The monkeypox virus constant temperature detection kit described in Example 1 of the present invention uses a matching rapid release liquid to process clinical samples, and the sensitivity is compared with the monkeypox virus nucleic acid detection kit (fluorescence PCR method) commercially available from Jiangsu Shuo Shi (using a matching magnetic bead extraction method to extract clinical sample nucleic acid).

[0146] The components of the monkeypox virus isothermal detection kit provided by the present invention are consistent with those in Example 2.

[0147] The primer-probe combination in the monkeypox virus isothermal detection kit used in this example is the optimal combination selected in Example 1: upstream primer MPV3-F3L-F1, downstream primer MPV3-F3L-nR2, and probe MPV-F3L-nP3.

[0148] (1) Two clinical samples (provided by Guangzhou Center for Disease Control and Prevention) were serially diluted using ddH2O to obtain 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 times the sample concentration.

[0149] (2) The nucleic acid was obtained by treating the sample (1) and its original concentration sample with a nucleic acid release agent (Nucleic Acid Release Liquid Type II, purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd.).

[0150] (3) A nucleic acid extraction or purification kit (magnetic bead extraction method) (purchased from Jiangsu ShuoShi Biotechnology Co., Ltd.) was used to extract and purify the above (1) sample and its original concentration sample to obtain nucleic acid. The operation steps were consistent with Example 4.

[0151] (4) The detection operation steps of the monkeypox virus isothermal detection kit of the present invention are the same as those of Example 2, using the nucleic acid sample treated in (2) above.

[0152] (5) The detection procedure of Jiangsu ShuoShi Monkeypox Virus Nucleic Acid Detection Kit is the same as that of Example 2, using the nucleic acid sample treated in (3) above.

[0153] The test results are shown in Table 10 and Fig.13 shown.

[0154] Table 10 Comparison of detection sensitivity results of kits based on different methods for extracting nucleic acids

[0155]

[0156]

[0157] Note: Some samples were not tested at lower concentrations because they tested negative at two consecutive gradient concentrations.

[0158] The sensitivity of the detection kit of the present invention was compared with the commercially available nucleic acid releaser and the monkeypox detection kit (combined magnetic bead extraction method) sold by Jiangsu Shuoshi. The experimental results showed that the monkeypox virus isothermal detection kit of the present invention has higher sensitivity.

[0159] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A primer-probe combination for detecting monkeypox virus, characterized in that: The primer-probe combination comprises: an upstream primer, a downstream primer and a probe, and the sequence compositions thereof are shown in SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:14 respectively.

2. Use of the primer-probe combination according to claim 1 in preparing a kit for isothermal detection of monkeypox virus.

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

4. The kit according to claim 3, characterized in that The kit also includes a test strip, which is prepared by a PVC backboard and a sample pad, a conjugation pad, a nitrocellulose membrane and absorbent paper arranged on the PVC backboard in sequence, and the control line and the test line are respectively coated with BSA-biotin and anti-fluorescein antibody.

5. The kit according to any one of claims 3 to 4, characterized in that The kit also includes a reaction solution, which includes A buffer, B buffer and ddH2O; the A buffer includes: 15 mM to 25 mM Tris-HCl, 130 mM to 170 mM KCl, 4 v / v% to 6 v / v% PEG, 0.5 v / v% to 1.5 v / v% TRITON-X100, the pH of the Abuffer is 8.0 to 9.0, and the B buffer includes: 180 mM to 220 mM magnesium acetate.

6. The kit according to any one of claims 3 to 4, characterized in that It also includes a dry powder reaction tube, which contains lyophilized powder. The lyophilized powder is freeze-dried from a solution including 520-725 ng / µL of recombinant enzyme; 100-200 ng / µL of single-stranded DNA binding protein; 350-450 ng / µL of DNA polymerase; 400-500 ng / µL of reverse transcriptase; and 300-400 ng / µL of nuclease.

7. The kit according to claim 6, characterized in that The preparation method of the freeze-dried powder comprises the following steps: pre-freezing the reaction solution at -80°C for 1 to 1.5 hours to obtain a pre-frozen reagent, first drying the pre-frozen reagent at -35 to -45°C for 2 to 10 hours, and then drying the pre-frozen reagent at 10 to 18°C ​​for 1 to 2 hours to obtain the freeze-dried powder.

8. A reaction system for detecting monkeypox virus, characterized in that: The reaction liquid system comprises: A buffer, Bbuffer, upstream primer, downstream primer, probe and ddH2O, the upstream primer is: MPV3-F3L-F1 with a sequence as shown in SEQ ID NO:4, the downstream primer is: MPV3-F3L-nR2 with a sequence as shown in SEQ ID NO:11, the probe is: MPV-F3L-nP3 with a sequence as shown in SEQ ID NO:14, the concentrations of the primers and the probe in the reaction are: 10 μM, and the concentration of magnesium ions in the reaction system is 200 mM.

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