Nucleic acid detection kit for monkey pox virus and preparation method thereof

Abstract

The invention provides a monkey pox virus nucleic acid detection kit and a preparation method thereof. The kit is used for qualitatively detecting monkey pox virus nucleic acid in vitro. Specific primers and probes are designed for the target F3L gene and the internal standard RNase P gene respectively, and the primer and probe combination is excellent in specificity, stability, compatibility, anti-interference performance and instrument adaptability, high in accuracy and sensitivity, easy to operate and high in detection speed. According to the kit disclosed by the invention, a freeze-drying reagent is prepared by utilizing the primer probe, so that the kit can completely get rid of the limitation of a cold chain, convenience is provided for remote areas lacking cold chain transportation and long-distance transportation, and the transportation and storage cost is greatly reduced. The monkey pox virus nucleic acid detection kit is simple in preparation method, good in reproducibility and easy to realize large-scale production and clinical application.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a monkeypox virus nucleic acid detection kit and a preparation method thereof. Background Art

[0002] Monkeypox virus (MPXV) is classified in the genus Orthopoxvirus of the family Poxviridae. It is one of the four orthopoxviruses that are pathogenic to humans, the other three being smallpox virus, vaccinia virus, and cowpox virus. Under an electron microscope, monkeypox virus particles are brick-shaped or oval, 200nm×250nm in size, and have an envelope. Viruses contain structural proteins and DNA-dependent RNA polymerases. The genome is double-stranded DNA with a length of about 197kb. The genome has a highly conserved central region that encodes replication and its assembly mechanisms. In addition, the genome has more variable regions containing inverted terminal repeat (ITR) sequences, which contain genes involved in determining host range and pathogenesis. The inverted terminal repeat region of MPXV contains at least four open reading frames (ORFs).

[0003] Monkeypox virus invades the human body mainly through mucous membranes and broken skin. It is mainly transmitted by direct contact with the diseased skin or mucous membranes of cases. It can also be transmitted by contact with objects contaminated with the virus, inhalation of respiratory droplets from cases at close range for a long time, contact with respiratory secretions, disease exudates, blood and other body fluids of infected animals, or infection by bites or scratches of infected animals.

[0004] The incubation period of monkeypox virus is 5-21 days, mostly 6-13 days. Chills and fever appear in the early stage of the disease, and the body temperature is mostly above 38.5℃, which may be accompanied by symptoms such as headache, drowsiness, fatigue, back pain and myalgia. Most patients have swollen lymph nodes in the neck, armpits, groin and other parts. Rash appears 1-3 days after onset. The rash first appears on the face and gradually spreads to the limbs and other parts. The rash is mostly centrifugal. The rash on the face and limbs is more common than on the trunk. The rash can appear on the palms and soles of the feet, and the number of rashes ranges from a few to thousands; it can also affect the oral mucosa, digestive tract, genitals, conjunctiva and cornea. The rash goes through several stages of changes from macules, papules, herpes, pustules to scabs. Herpes and pustules are mostly spherical, about 0.5-1 cm in diameter, hard in texture, and may be accompanied by obvious itching and pain. It takes about 2-4 weeks from onset to scab shedding. After the scab falls off, erythema or pigmentation, or even scars, may remain, and the scars may last for several years.

[0005] Monkeypox is a self-limited disease with a generally good prognosis. Severe cases occur more often in young children and immunocompromised individuals, and the prognosis is related to the clade of virus that infects, the degree of exposure to the virus, previous health conditions, and the severity of complications. Laboratory tests used to diagnose monkeypox virus include immunohistochemistry, electron microscopy, real-time polymerase chain reaction (RT-PCR), and virus isolation.

[0006] 1. Immunohistochemistry: Using the principle of specific binding between antibodies and antigens, specific antibodies are labeled to detect whether monkeypox virus antigens exist in tissues or cells. Specific antibodies against monkeypox virus proteins are usually used. After binding to the antigens in the sample, the location of the virus is displayed through color development or fluorescent labeling.

[0007] 2. Electron microscope: It uses electron beams instead of light beams and focuses images through electron lenses to observe very small structures of objects. For monkeypox virus, the morphology, size and structural characteristics of the virus can be directly observed for diagnosis.

[0008] 3. Real-time polymerase chain reaction (RT-PCR): PCR technology is used to amplify specific monkeypox virus nucleic acid sequences in vitro. By detecting the presence and quantity of the amplified product, it can be determined whether the sample contains monkeypox virus nucleic acid.

[0009] 4. Virus isolation: Inoculate the sample into a suitable cell culture system or animal model to observe whether there are signs of viral infection. By isolating and culturing live viruses, the virus can be further identified and characterized.

[0010] WHO recommends confirmation of MPXV infection based on nucleic acid amplification testing (NAAT), using real-time or conventional polymerase chain reaction (PCR) on lesion material to detect unique sequences of viral DNA.

[0011] Currently, most of the monkeypox virus nucleic acid detection kits used in the market are in liquid form and require low-temperature cold chain transportation. The transportation cost is relatively high, especially for long-distance transportation. If there is a problem with the cold chain temperature during transportation, it may affect the detection accuracy and sensitivity of the kit, and thus affect the detection results of monkeypox virus nucleic acid. Summary of the invention

[0012] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a monkeypox virus nucleic acid detection kit and a preparation method thereof. The monkeypox virus nucleic acid detection kit provided by the present invention uses real-time fluorescence PCR technology to perform qualitative detection of monkeypox virus nucleic acid. This kit is designed with specific primer probes for the target F3L gene and the internal standard RNase P gene, respectively. The 5' end of the F3L gene fluorescent probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a TAMRA quenching group. The 5' end of the RNaseP gene fluorescent probe is labeled with a CY5 fluorescent group, and the 3' end is labeled with a TAMRA quenching group. Compared with simple PCR quantitative detection, this product has high accuracy and strong specificity, and has greatly improved and improved repeatability, stability, sensitivity and precision, and the detection time is also greatly shortened, which greatly improves the detection efficiency and reduces the detection cost.

[0013] The monkeypox virus nucleic acid detection reagent kit provided by the present invention prepares the nucleic acid amplification reagent used for monkeypox virus nucleic acid detection into a lyophilized reagent by vacuum freeze drying technology. The lyophilized reagent is stored in a sterile lyophilized cake shape, which can not only significantly improve the stability of the nucleic acid amplification reagent, but also completely get rid of the limitation of the cold chain, and is used for room temperature transportation and long-term storage at room temperature, which greatly reduces the transportation cost and the cost of low-temperature storage. After the lyophilized reagent is redissolved, the extracted nucleic acid can be added for detection. Compared with liquid reagents, there is no cumbersome mixing and packaging process, and the operation is simpler, which can fundamentally save time and increase the detection amount. The preparation method of the monkeypox virus nucleic acid detection kit is simple, reproducible, and easy to achieve large-scale production and clinical application.

[0014] The purpose of the present invention can be achieved by the following scheme:

[0015] In a first aspect, the present invention provides a primer-probe combination for detecting monkeypox virus nucleic acid, comprising the following sequence:

[0016] The upstream primer sequence for amplifying the F3L gene is shown in SEQ ID NO.1, and its specific sequence is: 5′-TGGAGAAGCGAGAAGTTA-3′,

[0017] The downstream primer sequence for amplifying the F3L gene is shown in SEQ ID NO.2, and its specific sequence is: 5′-ATCTGTAGGCCGTGTATCAG-3′,

[0018] The fluorescent probe sequence used to amplify the F3L gene is shown in SEQ ID NO.3, and its specific sequence is: 5′-GTTCCGACGATACTCCTCCT-3′,

[0019] The upstream primer sequence used to amplify the RNaseP gene is shown in SEQ ID NO.4, and its specific sequence is: 5'-AGATTTGGACCTGCGAGCG-3',

[0020] The downstream primer sequence for amplifying the RNaseP gene is shown in SEQ ID NO.5, and its specific sequence is: 5'-GAGCGGCTGTCTCCACAAGT-3',

[0021] The fluorescent probe sequence used to amplify the RNaseP gene is shown in SEQ ID NO.6, and its specific sequence is: 5'-TTCTGACCTGAAGGCTCTGCGCG-3'.

[0022] As an embodiment of the present invention, the nucleotide sequences of the target sequences of the F3L gene and the RNaseP gene are shown as SEQ ID NOs. 7-8, respectively.

[0023] In a second aspect, the present invention provides a detection kit comprising the primer-probe combination.

[0024] As an embodiment of the present invention, the detection kit comprises a lyophilized reagent, a reconstitution buffer, a positive control and a blank control, wherein the lyophilized reagent is a lyophilized reagent in 8-link PCR reaction tubes.

[0025] Further, the lyophilized reagent includes the following components: 50mM to 100mM Tris-HCl, 0.01wt.% to 0.05wt.% sodium azide, 0.1U to 5U Taq DNA polymerase, 0.4mM to 2mM dNTPs, 200nM to 400nM primer probe combination, 3wt.% to 10wt.% lyophilized protective agent. Wherein, the dNTPs include one or more of dATP, dGTP, dCTP, and dTTP.

[0026] In some embodiments, the pH of the tris(hydroxymethyl)aminomethane is 8.2.

[0027] Furthermore, the lyoprotectant includes one or more of sucrose, trehalose, mannitol, dextran, bovine serum albumin, and Tween20.

[0028] Further, the reconstitution buffer comprises: 50mM to 200mM Tris-HCl, 1mM to 10mM ammonium sulfate, 0.01wt.% to 0.5wt.% Tween20, 1mM to 5mM magnesium chloride, 40mM to 100mM potassium chloride, 0.01wt.% to 0.05wt.% sodium azide. In some embodiments, the pH of the Tris-HCl is 8.2.

[0029] Furthermore, the positive control is a plasmid comprising the F3L gene (target gene of monkeypox virus) and an internal standard RNase P gene-specific sequence.

[0030] Furthermore, the blank control comprises RNase-free water. The blank control does not contain the detection target sequence.

[0031] The monkeypox virus nucleic acid detection kit provided by the present invention adopts real-time fluorescence PCR technology to perform qualitative detection of monkeypox virus nucleic acid. This kit is designed with specific primer probes for the target F3L gene and the internal standard RNase P gene, respectively. The 5' end of the F3L gene fluorescent probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a TAMRA quenching group. The 5' end of the RNaseP gene fluorescent probe is labeled with a CY5 fluorescent group, and the 3' end is labeled with a TAMRA quenching group. The internal standard RNase P system monitors the collection and transportation of the sample to be tested, the extraction and purification of nucleic acid, the amplification of nucleic acid and the detection through the internal standard to avoid false negatives in the test results.

[0032] The test kit of the present invention is prepared into a freeze-dried test kit through a freeze-drying process. The test kit can solve the problems of missed detection, long detection time, and the need for cold chain transportation of monkeypox virus nucleic acid detection test kits currently on the market. The test kit can be used for auxiliary diagnosis and efficacy monitoring of monkeypox virus infection. Preferably, the nucleic acid amplification freeze-dried reagent only needs to be added to a re-solution solution to quickly re-dissolve the reagent. The re-dissolution process is simple and fast, and no complicated operating steps are required. The performance of the reagent after re-dissolution is equivalent to that of a liquid test kit, and the accuracy and reliability of the test results can be guaranteed. It also reduces the possibility of human operation errors, can fundamentally save time, and increase the amount of detection.

[0033] As an embodiment of the present invention, the lyophilized reagent is prepared by the following steps:

[0034] S1. Mix the components to prepare a freeze-dried reagent mixture;

[0035] S2, pre-freezing, sublimation drying, and analytical drying of the lyophilized reagent mixture to obtain.

[0036] Furthermore, in step S2, the pre-freezing setting is as follows: the pre-freezing temperature reaches -35°C to -55°C within 20min to 60min and is maintained for 1h to 5h.

[0037] Furthermore, the pre-freezing setting also includes annealing treatment (annealing temperature is set at -25°C to -35°C, time is 60min). Adding annealing operation in the pre-freezing process can change the crystal form and size of ice crystal particles, strengthen crystallization, make the pore size left after water vapor sublimation larger, and reduce resistance, thereby increasing the primary drying rate, alleviating the mass transfer control problem, and improving the uniformity of the product. Annealing can increase the glass transition temperature of the amorphous phase frozen concentrate, which is of great significance to the sublimation drying stage and the analytical drying stage, and also directly affects the quality and appearance of the freeze-dried product.

[0038] Furthermore, in step S2, the sublimation drying is set as follows: the sublimation drying temperature reaches -45°C to -25°C within 20min to 60min, is maintained for 5h to 25h, and the vacuum degree is 0 to 30Pa.

[0039] Furthermore, in step S2, the analytical drying is set as follows: the analytical drying temperature reaches 20°C to 40°C within 1h to 5h, is maintained for 2h to 10h, and the vacuum degree is the ultimate vacuum.

[0040] Furthermore, in step S2, after the analytical drying is completed, an inert gas is injected under vacuum, and the box is plugged and then taken out of the box. Preferably, the inert gas is one of nitrogen, argon and carbon dioxide.

[0041] Preferably, inert gas is flushed into the preparation process to prevent the entry of moisture and oxidation of active ingredients by oxygen in the air, thereby reducing the moisture content in the freeze-dried reagent to a certain extent, ensuring that the moisture content of the product is qualified, and keeping the moisture content of the freeze-dried reagent at a low level during long-term storage.

[0042] Preferably, the freeze-dried reagent obtained by the present invention is in the shape of a cake with good appearance, and has a good appearance: full shape, flat surface, no shrinkage, uniform color, good porosity, qualified moisture content, can be quickly re-dissolved by adding water, and can be stored for a long time.

[0043] In a third aspect, the present invention provides a method for detecting monkeypox virus nucleic acid in vitro for non-diagnostic purposes, comprising the following steps:

[0044] A1. Extract or release nucleic acid from the sample to be tested;

[0045] A2, using the detection kit to perform fluorescent quantitative PCR analysis on the nucleic acid;

[0046] A3. Obtain and interpret the results.

[0047] In the present invention (when the PCR instrument is normal), the results of the monkeypox virus nucleic acid test samples are interpreted according to the amplification results of the positive control and the blank control in the detection kit.

[0048] Furthermore, the judgment results are shown in the following table:

[0049]

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1. Compared with the existing F3L gene target sequence, the present invention selects the specific F3L gene and the internal standard RNase P gene target sequence (SEQ ID NO.7-8) to design a specific primer probe combination with high sensitivity (the minimum detection limit is as low as 5×10 2 copies / mL), excellent instrument adaptability, high anti-interference; and excellent specificity, stability and compatibility, strong accuracy, simple operation and fast detection speed.

[0052] 2. The monkeypox virus nucleic acid detection reagent kit provided by the present invention adopts vacuum freeze drying technology to prepare the nucleic acid amplification reagent used for monkeypox virus nucleic acid detection into a freeze-dried reagent. The freeze-dried reagent is stored in a sterile freeze-dried cake shape, which can significantly improve the stability of the nucleic acid amplification reagent, and solve the problem that the traditional liquid nucleic acid detection kit needs cold chain transportation and storage, so that it can be completely free from the limitation of the cold chain, and is used for room temperature transportation and long-term storage at room temperature, which greatly reduces the transportation cost and the cost of low-temperature storage. During the monkeypox virus nucleic acid detection, only the freeze-dried reagent needs to be redissolved, and the nucleic acid extracted from the sample to be tested is added, and PCR fluorescence qualitative detection can be performed, which reduces the error caused by human operation. And the freeze-drying process and preparation method provided by the present invention are simple and feasible, mild conditions, good reproducibility, strong controllability, suitable for large-scale production, and more convenient for clinical use and storage.

[0053] 3. After the freeze-dried reagent is reconstituted, the extracted nucleic acid can be added for detection. Compared with liquid reagents, there is no cumbersome mixing and packaging process, the operation is simpler, which can fundamentally save time and increase the detection volume. The preparation method of the monkeypox virus nucleic acid detection kit is simple, reproducible, and easy to achieve large-scale production and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0055] Figure 1 The 8-linked reaction tube freeze-dried reagent for detecting monkeypox virus nucleic acid provided in the embodiment; wherein, Figure A is an unpackaged 8-linked reaction tube freeze-dried reagent, and Figure B is a sealed packaged 8-linked reaction tube freeze-dried reagent;

[0056] Figure 2The monkeypox virus nucleic acid amplification vial lyophilized reagent provided in the embodiment;

[0057] Figure 3 The PCR amplification curves of the monkeypox F3L gene and the internal standard RNase P gene in the monkeypox virus nucleic acid detection kit and the commercially available kit are compared in the example; wherein, Figure A is the PCR amplification curve of the monkeypox F3L gene, and Figure B is the PCR amplification curve of the internal standard RNase P gene. DETAILED DESCRIPTION

[0058] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, provide detailed implementation methods and specific operation processes, and will help those skilled in the art to further understand the present invention. It should be pointed out that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made under the premise of the concept of the present invention all belong to the protection scope of the present invention.

[0059] Example 1

[0060] The present embodiment provides a monkeypox virus nucleic acid detection kit, which includes: 8-link PCR reaction tube freeze-dried reagents, reconstitution buffer, positive control and blank control.

[0061] 1. Design of specific primers and probes

[0062] In this example, specific primers and probes were designed for the F3L gene and the internal standard RNase P gene involved in the monkeypox virus nucleic acid detection kit.

[0063] According to the public information of WHO and China CDC, primers and probes for the F3L gene region of monkeypox virus were selected. The genome sequence of monkeypox virus was downloaded from Virologic.org, GISAID and the GenBank platform of NCBI in the United States. The F3L gene and the internal standard RNase P gene (as shown in SEQ ID NO.7-8) were compared and analyzed using BioEdit, and the conserved regions of the gene were selected. The Primer Express 3.0 software was used to design specific primers and probes, as shown in Table 1 below (as shown in SEQ ID NO.1-6). All primers and probes used in this example were synthesized by Shanghai Shuoying Biotechnology Co., Ltd.

[0064] Table 1 Primer and probe sequences for F3L-specific gene and internal standard RNase P gene

[0065] F3L-F 5'-TGGAGAAGCGAGAAGTTA-3'(SEQ ID NO.1) F3L-R 5'-ATCTGTAGGCCGTGTATCAG-3'(SEQ ID NO.2) F3L-P 5'-GTTCCGACGATACTCCTCCT-3'(SEQ ID NO.3) RNase PF 5'-AGATTTGGACCTGCGAGCG-3'(SEQ ID NO.4) RNase PR 5'-GAGCGGCTGTCTCCACAAGT-3'(SEQ ID NO.5) RNase PP 5'-TTCTGACCTGAAGGCTCTGCGCG-3'(SEQ ID NO.6)

[0066] 2. Preparation of 8-link PCR reaction tube freeze-dried reagents is shown in Table 2 below: (single test: 20 μL)

[0067] Table 2 Preparation of lyophilized reagents for 8-link PCR reaction tubes

[0068] 8-plex PCR reaction tube freeze-dried reagent concentration Tris-HCl PH = 8.2 70mM Sodium Azide 0.01％ Taq DNA polymerase 2U Primer probe mixture 200nM dATP 0.4mM dGTP 0.4mM dCTP 0.4mM dTTP 0.4mM Trehalose 2％ Mannitol 3％ BSA 0.5％ Dextran 3％

[0069] 3. Preparation of reconstitution buffer is shown in Table 3 below: (single test: 20 μL)

[0070] Table 3 Preparation of reconstitution buffer

[0071] Reconstitution Buffer concentration Tris-HCl PH = 8.2 50mM Ammonium sulfate 4mM Magnesium chloride 3mM Potassium chloride 70mM Sodium Azide 0.01％ Tween20 0.1％

[0072] 4. The components of the human papillomavirus nucleic acid detection kit are shown in Table 4 below:

[0073] Table 4 Kit components

[0074]

[0075] The positive control is a plasmid containing the specific sequence of the target gene F3L gene of the monkeypox tumor virus and the specific sequence of the internal standard RNase P gene (the plasmid was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.). During the experiment, the positive control was extracted and added to the lyophilized preparation PCR reaction tube after reconstitution for fluorescent PCR amplification, which was used for quality control of the correct preparation of reagents and correct addition of nucleic acids.

[0076] The blank control is RNase-free water, without amplification and without detection target sequence, which meets the acceptance criteria and can be used for quality control of reagent preparation, sampling and detection processes to avoid false positives caused by reagents or contamination.

[0077] Example 2

[0078] This embodiment provides a method for preparing a freeze-dried reagent for 8-plex PCR reaction tubes for detecting monkeypox virus nucleic acid, comprising the following steps:

[0079] 1. Reagent preparation: prepare monkeypox virus nucleic acid detection and amplification reagent according to the preparation method in Example 1.

[0080] 2. Use a precise dispensing system to dispense the nucleic acid amplification reagent into 8-link PCR reaction tubes.

[0081] 3. Place the 8-plex PCR reaction tubes in a vacuum freeze dryer for freeze drying.

[0082] 4. When the vacuum freeze dryer performs normally, the freeze-drying program parameters are set as shown in Table 5 below.

[0083] Table 5 Freeze-drying program parameter settings for freeze-dried reagents in 8-plex PCR reaction tubes

[0084]

[0085] 5. After freeze-drying, strictly control the temperature and humidity of the environment where the freeze dryer is located. When the temperature and humidity of the environment meet the requirements, open the freeze dryer door and take out the freeze-dried reagent of the 8-link PCR reaction tube for monkeypox virus nucleic acid detection.

[0086] 6. Observe the appearance of the freeze-dried reagent. The appearance of a qualified freeze-dried reagent should be: full in shape, smooth in surface, not shrinking, and uniform in color.

[0087] 7. Use a Karl Fischer moisture meter to measure the moisture content of the freeze-dried reagent (a moisture content of 1% to 3% is considered acceptable).

[0088] 8. Put the qualified monkeypox virus nucleic acid detection 8-link nucleic acid amplification freeze-dried reagent reaction tube and desiccant into an aluminum foil bag, evacuate and seal, and store at room temperature. The monkeypox virus 8-link nucleic acid amplification reaction tube freeze-dried reagent provided in the embodiment of the present invention is as shown in the attached Figure 1 shown.

[0089] Example 3

[0090] This embodiment provides a method for preparing a monkeypox virus nucleic acid detection vial freeze-dried reagent, comprising the following steps:

[0091] 1. Reagent preparation: prepare monkeypox virus nucleic acid detection and amplification reagent according to the preparation method in Example 1.

[0092] 3. Use a precise dispensing system to dispense the nucleic acid amplification reagent into vials. Each vial contains 50 servings of freeze-dried reagent.

[0093] 4. Place the vial in a vacuum freeze dryer for freeze drying.

[0094] 5. When the vacuum freeze dryer performs normally, the freeze-drying program parameters are set as shown in Table 6 below.

[0095] Table 6 Parameters for freeze drying of vials

[0096]

[0097] 6. After freeze-drying is completed, strictly control the temperature and humidity of the environment where the freeze dryer is located. When the temperature and humidity of the environment meet the requirements, open the freeze dryer door and take out the monkeypox virus nucleic acid detection vial freeze-dried reagent.

[0098] 7. Observe the appearance of the freeze-dried reagent. The appearance of a qualified freeze-dried reagent should be: full in shape, smooth in surface, not shrinking, uniform in color, etc.

[0099] 8. Observe the appearance of the freeze-dried reagent and use a Karl Fischer moisture meter to measure the moisture content of the freeze-dried reagent (a moisture content of 1% to 3% is considered acceptable).

[0100] 9. Seal the qualified vials of freeze-dried preparations and store the sealed vials at room temperature. Figure 2 shown.

[0101] Example 4

[0102] This embodiment provides a monkeypox virus nucleic acid detection kit for detecting monkeypox virus nucleic acid in a sample.

[0103] 1. Preparation (preparation preparation area)

[0104] Take out the kit, calculate the number of reaction tubes N (N = number of samples + 2 tubes of control), tear off the PCR tube cover or aluminum film, centrifuge the reconstitution buffer briefly, and add 20 μL of reconstitution buffer to each reaction tube.

[0105] 2. Sample preparation (sample preparation area)

[0106] 2.1 Nucleic acid extraction

[0107] It is recommended to use a quality-assured nucleic acid extraction kit for nucleic acid extraction, preferably a commercial magnetic bead method or membrane adsorption column nucleic acid extraction kit, and extract nucleic acid from the sample to be tested according to its instructions.

[0108] 2.2 Reconstitution of positive control

[0109] The positive control needs to be reconstituted when used for the first time. Add 50 μL of RNase-free water to each tube of positive control and shake and centrifuge.

[0110] 2.3 Sample addition

[0111] Add 5 μL of the processed sample nucleic acid, blank control, and positive control to the prepared PCR reaction tubes, cover the tubes tightly, tap the bottom of the tubes until clear, and centrifuge at low speed. To prevent interference with the fluorescent signal, try to avoid direct contact with the PCR reaction tubes with your hands and do not mark the tubes.

[0112] 3.PCR amplification detection (nucleic acid amplification area)

[0113] 3.1 Place the reaction tube into the fluorescent PCR amplification instrument for amplification detection.

[0114] 3.2 Take ABI7500 operation instructions as an example

[0115] 3.2.1 The PCR amplification program settings are shown in Table 7 below:

[0116] Table 7 PCR amplification program

[0117]

[0118] 3.2.2 Result analysis

[0119] In this embodiment, a method for analyzing the results of monkeypox virus nucleic acid detection is also provided. When the instrument is normal, the results of the monkeypox virus nucleic acid detection samples are interpreted according to the amplification results of the positive control and the blank control in the monkeypox virus nucleic acid detection kit, as shown in Table 8 below:

[0120] Table 8 Results Interpretation

[0121]

[0122] 3.2.3 Interpretation of test results:

[0123] Blank control: no typical S-type amplification.

[0124] Positive control: FAM showed a typical S-shaped amplification curve and the Ct value was ≤35.

[0125] The Ct of the internal standard (VIC) was ≤35.

[0126] The above requirements must be met in the same experiment, otherwise this experiment will be invalid.

[0127] Example 5

[0128] This example is a test comparison between the monkeypox virus nucleic acid detection kit of the present invention and a commercially available kit.

[0129] 1. Sample and commercial kit information

[0130] The sample was a commercially available monkeypox virus nucleic acid detection reagent national reference product (purchased from Jingliang Gene Technology (Shenzhen) Co., Ltd.). DNA was extracted using a nucleic acid extraction kit (magnetic bead method), which was purchased from Jiangsu Shuoshi Biotechnology Co., Ltd., item number: SDK60104.

[0131] The commercially available kit selected was the Monkeypox Virus Nucleic Acid Assay Kit (Fluorescence PCR Method), item number: YJC70115NW, purchased from Jiangsu ShuoShi Biotechnology Co., Ltd.

[0132] 2. Kit testing

[0133] The monkeypox virus nucleic acid detection kit (lyophilized type) provided by the present invention and the commercially available kit (YJC70115NW) are used in QuantStudio TM 5 Real-time PCR instruments were used to test the positive reference materials in the national reference materials of monkeypox virus nucleic acid detection reagents.

[0134] 3. Test results

[0135] The monkeypox virus nucleic acid detection kit in the present invention is compared with the commercially available kit using QuantStudio TM 5. Real-time PCR amplification results are shown in the attached Figure 3 shown.

[0136] Example 6

[0137] This example is an evaluation of the minimum detection limit of a monkeypox virus nucleic acid detection kit.

[0138] 1. Test on the minimum detection limit of monkeypox virus nucleic acid detection kit prepared by primer probes of SEQ ID NO.1-3 of the present invention

[0139] The monkeypox virus nucleic acid detection reagent national reference (positive reference) was mixed with human tonsil swabs, nasopharyngeal swabs, serum, and whole blood negative matrix to prepare simulated positive samples, and then diluted to 5×10 4 copies / mL,5×10 3 copies / mL,5×10 2 copies / mL,5×10 1 Each type of simulated sample was tested 20 times per dilution, and the limit of detection (LoD) was defined as a 95% positive detection level. TM 5 Real-time PCR instrument was used to test 3 batches (Lot 1, Lot 2, Lot 3) of monkeypox virus nucleic acid detection kit. The results showed that the positive detection rate was 95%, which was determined to be LoD. The results are shown in Table 9 below.

[0140] Table 9 Minimum detection limit test results

[0141]

[0142] Through the above minimum detection limit test evaluation, it is confirmed that the minimum detection limit of the monkeypox virus nucleic acid detection kit provided by the present invention is 5×10 2 copies / mL.

[0143] 2. Comparative test of the minimum detection limit of monkeypox virus F3L lyophilized reagent prepared with other primer probes designed with F3L target gene

[0144] 2.1 Synthesis of other primer probes designed based on F3L target gene

[0145] The sequences of other primers and probes designed with F3L target gene (primers and probes were synthesized by Shanghai Shuoying Biotechnology Co., Ltd.) are as follows:

[0146] F3L-F(SEQ ID NO.9):5'-TTCCATCTGCCTTATCGAAT-3'

[0147] F3L-R (SEQ ID NO.10): 5'-TGTTAACCCTGTCACCGT-3'

[0148] F3L-P (SEQ ID NO.11): 5'-CTCTTCCGTCAATG-3'

[0149] 2.2 Lyophilization of monkeypox virus nucleic acid detection reagent

[0150] 2.2.1 Prepare the monkeypox virus F3L freeze-dried reagent according to the method in Example 1.

[0151] 2.2.2 Prepare the monkeypox virus F3L freeze-dried reagent in 8-link PCR reaction tubes according to the method in Example 2.

[0152] 2.3 Comparative test of the minimum detection limit of the monkeypox virus F3L freeze-dried reagent prepared with the primer probes of SEQ ID NO.1-3 (experimental group) and the monkeypox virus F3L freeze-dried reagent prepared with other primer probes of SEQ ID NO.9-11 (comparative group)

[0153] The monkeypox virus nucleic acid detection reagent national reference (positive reference) was mixed with human tonsil swabs, nasopharyngeal swabs, serum, and whole blood negative matrix to prepare simulated positive samples, and then diluted to 5×10 4 copies / mL,5×10 3 copies / mL,5×10 2 copies / mL,5×10 1 Each type of simulated sample was tested 20 times per dilution. TM 5 Real-time PCR was used to detect the monkeypox virus F3L freeze-dried reagent in the experimental group and the control group. The results showed that the positive detection rate of the F3L target gene in the experimental group was higher than that in the control group, and the results are shown in Table 10 below.

[0154] Table 10 Comparison results of minimum detection limit test of monkeypox virus F3L lyophilized reagent

[0155]

[0156] The comparison results of the minimum detection limit test of the above-mentioned monkeypox virus F3L lyophilized reagent show that the positive detection rate of the primer probe (SEQ ID NO.1-3) designed by the present invention for the F3L target gene is higher than the positive detection rate of other primer probes (SEQ ID NO.9-11 designed with the F3L target gene) for the F3L target gene, and the sensitivity is higher.

[0157] Example 7

[0158] This example is a specificity evaluation of a monkeypox virus nucleic acid detection kit.

[0159] The monkeypox virus nucleic acid detection kit provided by the present invention was used to detect measles virus, varicella-zoster virus, Epstein-Barr virus, rubella virus, herpes simplex virus, cytomegalovirus, human herpes virus type 6, human herpes virus type 7, and human herpes virus type 8, and there was no cross reaction. The results are shown in Table 11 below.

[0160] Table 11 Specificity evaluation test results

[0161]

[0162] Through the above-mentioned specific test evaluation, it is confirmed that the monkeypox virus nucleic acid detection kit provided in the present invention has no cross reaction with the pathogens in the above table that infect the same part, cause similar symptoms or have similar sequences to the monkeypox virus.

[0163] Example 8

[0164] This example is a verification of the PCR instrument applicable to the monkeypox virus nucleic acid detection kit.

[0165] Using Bioperfectus STC-96S, SLAN-96P, Applied Biosystems 7500, QuantStudio TM 5. Using 12 simulated positive samples, 10 simulated negative samples and reference products (LoD, J1 and J2), PCR amplification was performed using the monkeypox virus 8-link nucleic acid amplification reaction tube freeze-dried reagent. The evaluation indicators of this example include accuracy, LoD and precision. The test results are as follows.

[0166] 1. Accuracy and LoD

[0167] Twelve simulated positive samples, 10 simulated negative samples and the lowest detection limit LoD were used to evaluate the suitability of PCR instruments using the monkeypox virus F3L freeze-dried reagent of the experimental group and the control group in Example 6, using Bioperfectus STC-96S, SLAN-96P, Applied Biosystems 7500, QuantStudio TM 5. The accuracy and LOD performance were evaluated. The results are shown in Table 12 below.

[0168] Table 12 Accuracy and LoD of 4 different real-time PCR instruments

[0169]

[0170]

[0171] 2. Precision

[0172] The monkeypox virus nucleic acid detection kit was used to detect the presence of Bioperfectus STC-96A, STC-96APLUS, AppliedBiosystems 7500, and QuantStudio TM 5 The precision reference products J1 and J2 (prepared by mixing equal volumes of monkeypox virus F3L and internal standard RNaseP plasmid) were evaluated, and the precision test data statistics are shown in Table 13. The experimental results are shown in Table 14 below. The coefficient of variation (CV%) between the four different PCR instruments is less than 5%. It can be seen that Bioperfectus STC-96A, STC-96APLUS, Applied Biosystems 7500, QuantStudio TM 5 4 different PCR instruments are all suitable for this monkeypox virus nucleic acid detection kit.

[0173] Table 13 Statistics of precision test data of 4 different real-time PCR instruments

[0174]

[0175]

[0176] Table 14 Precision evaluation results of different PCR instruments

[0177]

[0178] Through the verification of the above-mentioned applicable PCR instruments, it is confirmed that the monkeypox virus nucleic acid detection kit provided in the present invention is suitable for Bioperfectus STC-96S, SLAN-96P, Applied Biosystems 7500, QuantStudio TM 5 models of PCR instruments. And the PCR instrument applicability of the monkeypox virus F3L freeze-dried reagent (experimental group) in the present invention is slightly higher than that of the monkeypox virus F3L freeze-dried reagent in the control group.

[0179] Example 9

[0180] This example is a precision assessment of a monkeypox virus nucleic acid detection kit.

[0181] The precision of the monkeypox virus nucleic acid detection kit was evaluated using the 2×2×20 method with precision reference products J1 and J2 (prepared by mixing equal volumes of monkeypox virus F3L and internal standard RNase P plasmid). The results showed that for precision reference products J1 and J2, the coefficient of variation CV% of repeatability and intra-laboratory precision were less than 5%, as shown in Table 15 below.

[0182] Table 15 Precision evaluation results

[0183]

[0184] Through the above precision test evaluation, it is confirmed that the coefficient of variation CV% of the repeatability and laboratory precision of the monkeypox virus nucleic acid detection kit provided in the present invention is less than 5%.

[0185] Example 10

[0186] This example is an evaluation of the anti-interference ability of a monkeypox virus nucleic acid detection kit.

[0187] 1. Endogenous interfering substances test

[0188] 3% hemoglobin, 5% leukocytes, and 2g / dL mucin were selected for endogenous interfering substance testing. The monkeypox virus F3L lyophilized reagents in the experimental group and the control group in Example 6 were tested for nucleic acid detection according to the method in Example 4, and amplified according to the PCR amplification program. A positive control substance with a certain dilution multiple was added to each endogenous interfering substance, and the test was repeated 20 times. The test results are shown in Table 16. The monkeypox virus F3L lyophilized reagent provided by the experimental group of the present invention has good anti-interference ability against 3% hemoglobin, 5% leukocytes, and 2g / dL mucin.

[0189] Table 16 Evaluation of the monkeypox virus nucleic acid detection kit against endogenous interfering substances

[0190]

[0191] 2. Test for exogenous interfering substances

[0192] 60 mg / mL of Tecovir, 40 mg / mL of Bucinovovir, and 160 mg / mL of Cidofovir were selected for exogenous interfering substance testing. The monkeypox virus F3L lyophilized reagent of the experimental group and the control group of the present invention was tested for nucleic acid detection according to the method in Example 4, and amplified according to the PCR amplification program. A positive control substance with a certain dilution multiple was added to each concentration of exogenous interfering substance, and the test was repeated 20 times. The test results are shown in Table 17. The monkeypox virus F3L lyophilized reagent provided by the experimental group of the present invention has good anti-interference ability to 60 mg / mL of Tecovir, 40 mg / mL of Bucinovovir, and 60 mg / mL of Cidofovir.

[0193] Table 17 Evaluation of the monkeypox virus nucleic acid detection kit against exogenous interfering substances

[0194]

[0195] Through the above anti-interference ability test evaluation, it is confirmed that endogenous interfering substances such as fresh healthy human hemoglobin, leukocytes, mucin and exogenous interfering substances such as tecovir, bucinofovir and cidofovir have no interference with the monkeypox virus nucleic acid detection kit provided in the present invention. And the anti-interference ability of the monkeypox virus F3L freeze-dried reagent provided by the experimental group of the present invention to endogenous interfering substances and exogenous interfering substances is higher than that of the monkeypox virus F3L freeze-dried reagent of the control group.

[0196] Embodiment 11

[0197] This example is a real-time stability evaluation of a monkeypox virus nucleic acid detection kit.

[0198] Three batches of monkeypox virus nucleic acid detection kits were stored at room temperature and taken out at 0 days, 3 months, 6 months, 9 months, 12 months, and 18 months. The real-time stability of the kits was evaluated using positive simulation samples, negative simulation samples, minimum detection limit LOD, and precision reference materials (J1, J2), including positive reaction consistency, negative reaction consistency, LoD, and precision. The test results are shown in Tables 18-20 below:

[0199] Table 18 Real-time stability test results

[0200]

[0201]

[0202] Table 19 Real-time stability test results

[0203]

[0204]

[0205]

[0206] Table 20 Real-time stability test results

[0207]

[0208]

[0209] Through the above-mentioned real-time stability evaluation, it was confirmed that after the monkeypox virus nucleic acid detection kit provided in the present invention was placed at room temperature for 18 months, the real-time stability of the kit was evaluated using clinical positive simulation samples, negative simulation samples, minimum detection limit LOD and precision reference materials (J1, J2), and the test results were still within an acceptable range.

[0210] Example 12

[0211] This example is an accelerated stability assessment of a monkeypox virus nucleic acid detection kit at 37°C.

[0212] Three batches of monkeypox virus nucleic acid detection kits were stored at 37°C and taken out at 0 days, 3 months, 6 months, 9 months, 12 months, and 18 months. Positive simulation samples, negative simulation samples, minimum detection limit LOD, and precision reference materials (J1, J2) were used to evaluate the 37°C accelerated stability of the kits, including positive reaction consistency, negative reaction consistency, LoD, and precision. The test results are shown in Tables 21-23 below:

[0213] Table 21 37℃ accelerated stability test results

[0214]

[0215]

[0216] Table 22 Results of 37℃ accelerated stability test

[0217]

[0218]

[0219] Table 23 Results of 37℃ accelerated stability test

[0220]

[0221]

[0222] Through the above-mentioned 37°C accelerated stability evaluation, it was confirmed that after the monkeypox virus nucleic acid detection kit provided in the present invention was placed at 37°C for 18 months, the real-time stability of the kit was evaluated using clinical positive simulation samples, negative simulation samples, minimum detection limit LOD and precision reference materials (J1, J2), and the test results were still within an acceptable range.

[0223] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A primer-probe combination for detecting monkeypox virus nucleic acid, characterized in that: The following sequences are included: The upstream and downstream primer sequences used to amplify the F3L gene are shown in SEQ ID NO. 1-2 respectively; The fluorescent probe sequence used to amplify the F3L gene is shown in SEQ ID NO. 3; The upstream and downstream primer sequences used to amplify the RNaseP gene are shown in SEQ ID NO.4-5; The fluorescent probe sequence used to amplify the RNaseP gene is shown in SEQ ID NO.

6.

2. The primer-probe combination according to claim 1, characterized in that: The nucleotide sequences of the target sequences of the F3L gene and RNaseP gene are shown in SEQ ID NOs. 7-8, respectively.

3. A detection kit comprising the primer-probe combination according to claim 1 or 2, characterized in that: The detection kit comprises a lyophilized reagent, a reconstitution buffer, a positive control and a blank control.

4. The detection kit according to claim 3, characterized in that The freeze-dried reagent includes the following components: 50mM-100mM tris(hydroxymethyl)aminomethane, 0.01wt.%-0.05wt.% sodium azide, 0.1U-5U Taq DNA polymerase, 0.4mM-2mM dNTPs, 200nM-400nM primer-probe combination, 3wt.%-10wt.% freeze-dried protective agent; wherein the dNTPs include one or more of dATP, dGTP, dCTP, and dTTP.

5. The detection kit according to claim 4, characterized in that The lyophilization protective agent includes one or more of sucrose, trehalose, mannitol, dextran, bovine serum albumin, and Tween20.

6. The detection kit according to claim 3, characterized in that The reconstitution buffer comprises: 50mM to 200mM tris(hydroxymethyl)aminomethane, 1mM to 10mM ammonium sulfate, 0.01wt.% to 0.5wt.% Tween20, 1mM to 5mM magnesium chloride, 40mM to 100mM potassium chloride, and 0.01wt.% to 0.05wt.% sodium azide.

7. The detection kit according to claim 3, characterized in that The positive control is a plasmid containing the F3L gene and the internal standard RNase P gene specific sequence; the blank control includes RNase-free water.

8. The detection kit according to claim 3, characterized in that The lyophilized reagent is prepared by the following steps: S1. Mix the components to prepare a freeze-dried reagent mixture; S2, pre-freezing, sublimation drying, and analytical drying of the lyophilized reagent mixture to obtain.

9. The detection kit according to claim 8, characterized in that In step S2, the pre-freezing setting is as follows: the pre-freezing temperature reaches -35°C to -55°C within 20min to 60min and is maintained for 1h to 5h; The sublimation drying is set as follows: the sublimation drying temperature reaches -45°C to -25°C within 20min to 60min, is maintained for 5h to 25h, and the vacuum degree is 0 to 30Pa; The analytical drying is set as follows: the analytical drying temperature reaches 20° C. to 40° C. within 1 hour to 5 hours, is maintained for 2 hours to 10 hours, and the vacuum degree is the ultimate vacuum.

10. A method for detecting monkeypox virus nucleic acid in vitro for non-diagnostic purposes, characterized in that: The following steps are involved: A1. Extract or release nucleic acid from the sample to be tested; A2. Performing fluorescent quantitative PCR analysis on nucleic acid using the detection kit described in claim 3; A3. Obtain and interpret the results.

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