A lyophilized PCR reagent and kit for monkeypox virus nucleic acid detection

Abstract

This invention provides a lyophilized PCR reagent and kit for detecting monkeypox virus nucleic acid. Specifically, through extensive research, this invention has obtained a highly sensitive PCR amplification reagent for detecting monkeypox virus nucleic acid. This PCR amplification reagent can maintain the performance stability of the PCR reagent before and after lyophilization, and is therefore suitable for preparing lyophilized PCR reagents. This solves the problems of cumbersome preparation process, large preparation error, poor reaction repeatability, and high cost of product storage and transportation for fluorescent PCR reaction reagents.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to a lyophilized PCR reagent for the detection of monkeypox virus nucleic acid. Background Technology

[0002] Monkeypox virus (MPXV) is a double-stranded DNA virus approximately 197 kb in length, belonging to the genus Orthopoxvirus in the family Poxviridae. It causes a viral zoonotic disease called monkeypox, with clinical manifestations mainly including fever, rash, and lymphadenopathy. Historically, it primarily occurred in Central and West Africa, with a mortality rate of 1%-10%.

[0003] Monkeypox virus was discovered in green monkeys in 1958, and the first confirmed case of human monkeypox was reported in the Democratic Republic of Congo in 1970. Since then, cases have been reported in 11 other African countries, including the Central African Republic, the Republic of Congo, and Nigeria. Human monkeypox cases are mainly distributed in the tropical rainforests of central and western Africa; outbreaks are less frequent outside of Africa, and local transmission is limited. Since 2022, five African countries—the Democratic Republic of Congo, the Republic of Congo, the Central African Republic, Nigeria, and Cameroon—have reported monkeypox cases. Furthermore, since May 2022, multiple countries worldwide have reported confirmed and suspected cases of monkeypox, affecting Europe, North America, South America, Oceania (Australia), and Asia (UAE), and human-to-human transmission has occurred. Research on monkeypox quarantine methods is of great significance.

[0004] Currently, laboratory diagnostic methods for MPXV are mainly divided into three categories:

[0005] 1. Isolation, Culture, and Identification: Monkeypox virus was isolated through cell culture. Due to the handling of live virus, extremely high biosafety requirements are placed on the laboratory, and MPXV culture takes a certain amount of time, which is not conducive to rapid diagnosis.

[0006] 2. Electron microscopy: This method involves examining the sample under an electron microscope to determine if a virus with the same morphology as orthopoxvirus is present. This method requires an expensive electron microscope, is extremely complex to operate, has low detection sensitivity, and involves complex sample preparation and a long processing time.

[0007] 3. Nucleic acid analysis: PCR is used to detect whether the sample contains monkeypox virus nucleic acid. Real-time quantitative PCR, in particular, uses specific primers and probes designed based on conserved sequences on the monkeypox virus genome to directly detect monkeypox virus nucleic acid. This method is more time-saving, specific, reduces cross-contamination, and can accurately quantify multiple samples.

[0008] Conventional PCR reagents include PCR reaction buffer, primers, probes, Taq enzyme, dNTPs, etc. They are mostly liquid reagents and contain temperature-sensitive components such as Taq enzyme, primers, and probes. They need to be transported and stored at low temperatures to prevent their properties from changing and becoming ineffective. In addition, the process of use is cumbersome and the cost of product storage and transportation is high. Summary of the Invention

[0009] The purpose of this invention is to provide a lyophilized PCR reagent and kit for monkeypox virus nucleic acid detection, so as to solve the problems of complicated preparation process of fluorescent PCR reaction reagents, large preparation error, poor reaction repeatability, and high product storage and transportation costs.

[0010] In a first aspect, the present invention provides a lyophilized PCR reagent for detecting monkeypox virus nucleic acid, the lyophilized PCR reagent comprising an upstream primer, a downstream primer, and a probe; wherein the upstream primer sequence is shown in SEQ ID NO.1, the downstream primer sequence is shown in SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.3.

[0011] In another preferred embodiment, the probe is labeled with a fluorescent group and a quenching group at both ends, respectively; preferably, the fluorescent group is selected from the group consisting of FAM, VIC, CY5, and Texas Red; and the quenching group is selected from the group consisting of BHQ1, BHQ2, BHQ3, TAMRA, and MGB.

[0012] In another preferred embodiment, the lyophilized PCR reagent further includes a lyophilization molding agent.

[0013] In another preferred embodiment, the freeze-drying forming agent comprises one or more components selected from the group consisting of: trehalose, sucrose, bovine serum albumin, raffinose, dextran, glycerol, DTT, Proclin 300, formamide, and hydroxypropyl-β-cyclodextrin.

[0014] In another preferred embodiment, the freeze-drying molding agent is composed of sucrose, raffinose, and hydroxypropyl-β-cyclodextrin.

[0015] In another preferred embodiment, the freeze-drying forming agent comprises:

[0016] 2-10 parts by weight of sucrose

[0017] 2-10 parts by weight of raffinose, and

[0018] Hydroxypropyl-β-cyclodextrin 2-10 parts by weight.

[0019] In another preferred embodiment, the freeze-drying forming agent comprises:

[0020] Trehalose 3 parts by weight

[0021] 3 parts by weight of raffinose, and

[0022] 3 parts by weight of sucrose.

[0023] In another preferred embodiment, the lyophilized PCR reagent further includes one or more components selected from the group consisting of: Taq DNA polymerase and dNTPs.

[0024] In another preferred embodiment, when the lyophilized PCR reagent is used to prepare the PCR reaction system, the content of each component in the PCR reaction system is as follows:

[0025]

[0026] The volume of the PCR reaction system is 25 μl.

[0027] In another preferred embodiment, when the lyophilized PCR reagent is used to prepare the PCR reaction system, the content of each component in the PCR reaction system is as follows:

[0028]

[0029] The volume of the PCR reaction system is 25 μl.

[0030] Furthermore, the lyophilized PCR reagent is prepared by the following lyophilization procedure: -50℃, 3h; -50℃, 0.2mbar, 2h; 0℃, 0.2mbar, 1h; 10℃, 0.2mbar, 1h; 20℃, 0.2mbar, 1h; 30℃, 2h.

[0031] In a second aspect, the present invention provides a kit for detecting monkeypox virus nucleic acid, the kit comprising the lyophilized PCR reagent described in the first aspect of the present invention.

[0032] In another preferred embodiment, the kit further includes a reconstituter for reconstituted the lyophilized PCR reagent.

[0033] In another preferred embodiment, the resolvent comprises: magnesium ions, potassium chloride, a surfactant, and Tris-HCl.

[0034] In another preferred embodiment, the resolvent comprises: 1-5 mM magnesium ions, 10-100 mM potassium chloride, 0-1% (w / v) surfactant and 10-100 Mm Tris-HCl.

[0035] In another preferred embodiment, the resolvent comprises: 3 mM magnesium ions, 50 mM potassium chloride, 0.05% (w / v) surfactant and 50 mM Tris-HCl.

[0036] In another preferred embodiment, the surfactant is selected from one or more of the following: Triton X-100, Tween 20, NP40.

[0037] In another preferred embodiment, the surfactant is Tween 20.

[0038] A third aspect of the present invention provides a method for preparing lyophilized PCR reagents for monkeypox virus nucleic acid detection, the method comprising the steps of:

[0039] (1) Preparation of liquid PCR reagents

[0040]

[0041] (2) Freeze-drying

[0042] The liquid PCR reagent provided in step (1) is freeze-dried to obtain the freeze-dried PCR reagent.

[0043] In another preferred embodiment, the freeze-drying process in step (2) is as follows: -50℃, 3h; -50℃, 0.2mbar, 2h; 0℃, 0.2mbar, 1h; 10℃, 0.2mbar, 1h; 20℃, 0.2mbar, 1h; 30℃, 2h.

[0044] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0045] Figure 1 This is a lyophilized PCR reagent for the detection of monkeypox virus nucleic acid.

[0046] Figure 2 The detection sensitivity results of the lyophilized PCR reagent used for monkeypox virus nucleic acid detection are shown;

[0047] Figure 3 The results show a comparison of the detection performance of lyophilized PCR reagents and liquid PCR reagents.

[0048] Figure 4 The results of the stability test for the lyophilized PCR reagent used for monkeypox virus nucleic acid detection are shown.

[0049] Figure 5 The results of using lyophilized PCR reagents for monkeypox virus nucleic acid detection in clinical samples are shown. Detailed Implementation

[0050] This invention provides a lyophilized PCR reagent and kit for detecting monkeypox virus nucleic acid. Specifically, through extensive research, this invention has obtained a highly sensitive PCR amplification reagent (primer and probe combination) for detecting monkeypox virus nucleic acid. This PCR amplification reagent can maintain the performance stability of the PCR reagent before and after lyophilization, and is therefore suitable for preparing lyophilized PCR reagents. This solves the problems of cumbersome preparation process, large preparation error, poor reaction reproducibility, and high cost of product storage and transportation for fluorescent PCR reaction reagents.

[0051] In one embodiment of the present invention, the present invention provides a lyophilized PCR reagent for monkeypox virus nucleic acid detection, so as to solve at least one of the above-mentioned technical problems.

[0052] This invention provides a lyophilized PCR reagent for detecting monkeypox virus nucleic acid, comprising Taq DNA polymerase, dNTPs, a lyophilization agent, an oligonucleotide upstream primer and an oligonucleotide downstream primer, and an oligonucleotide fluorescent probe. The oligonucleotide upstream primer sequence is 5'-CTCCAGAACCAGCATCACCT-3' (SEQ ID NO: 1), the oligonucleotide downstream primer sequence is 5'-TGGTGGTTCGTCGTTCCTAT-3' (SEQ ID NO: 2), and the oligonucleotide fluorescent probe sequence is 5'-AGATGACGGGTTAATCAGAGC-3' (SEQ ID NO: 3).

[0053] Furthermore, the oligonucleotide fluorescent probe is labeled with a fluorescent group and a quenching group at both ends. The fluorescent group is one of FAM, VIC, CY5, Texas Red or other fluorescent groups with luminescent properties, and the quenching group is one of BHQ1, BHQ2, BHQ3, TAMRA, MGB or other chemical groups with fluorescence quenching function.

[0054] Furthermore, each tube of lyophilized PCR reagent includes: 0.1 μM to 1 μM oligonucleotide upstream primer, 0.1 μM to 1 μM oligonucleotide downstream primer, 0.1 μM to 1 μM oligonucleotide fluorescent probe, 0.1 to 1 unit of Taq DNA polymerase, and 1% to 10% (m / v) of lyophilization agent and 0.1 to 1 mM dNTP.

[0055] Furthermore, each tube of lyophilized PCR reagent includes: 0.2 μM oligonucleotide upstream primer, 0.2 μM oligonucleotide downstream primer, 0.1 μM oligonucleotide fluorescent probe, 0.2 units of Taq DNA polymerase, lyophilization agent at 3% m / v, and 0.2 mM dNTP.

[0056] Furthermore, the freeze-drying forming agent is one or more of the following: trehalose, sucrose, bovine serum albumin, raffinose, dextran, glycerol, DTT, Proclin 300, formamide, hydroxypropyl-β-cyclodextrin, etc.

[0057] Furthermore, the freeze-drying forming agent is 1%-10% sucrose, 1%-10% raffinose, and 1%-10% hydroxypropyl-β-cyclodextrin.

[0058] Furthermore, the freeze-drying forming agent is 3% sucrose, 3% raffinose, and 3% hydroxypropyl-β-cyclodextrin.

[0059] Furthermore, the lyophilized PCR reagent for monkeypox virus nucleic acid detection was prepared by the following lyophilization procedure: -50℃, 3h; -50℃, 0.2mbar, 2h; 0℃, 0.2mbar, 1h; 10℃, 0.2mbar, 1h; 20℃, 0.2mbar, 1h; 30℃, 2h.

[0060] According to another aspect of the present invention, a kit for detecting monkeypox virus nucleic acid is provided, comprising the above-mentioned lyophilized PCR reagent and a reconstituter for reconstituted lyophilized PCR reagent, the reconstituter comprising the following components: 1-5 mM magnesium ions, 10-100 mM potassium chloride, 0-1% (w / v) surfactant and 10-100 Mm Tris-HCl.

[0061] Furthermore, the resolvent comprises the following components: 3 mM magnesium ions, 50 mM potassium chloride, 0.05% (w / v) surfactant and 50 mM Tris-HCl.

[0062] Furthermore, the surfactant is one or more of Triton X-100, Tween 20, NP40, etc.

[0063] Furthermore, the surfactant is Tween 20.

[0064] Furthermore, the quantitative real-time PCR procedure is as follows: 95℃ pre-denaturation for 20s, (95℃ denaturation for 5s, 58℃ annealing and extension for 10s), repeat this for 10 cycles, (95℃ denaturation for 5s, 58℃ annealing and extension for 30s fluorescence collection), repeat this for 30 cycles. Fluorescence is detected using the FAM channel. If a typical "S"-shaped amplification curve is observed, it indicates that the sample contains monkeypox virus; otherwise, it does not contain monkeypox virus.

[0065] This invention designs specific oligonucleotide primers and oligonucleotide fluorescent probes targeting conserved sequences on the monkeypox virus genome. These primers can specifically amplify conserved target sequences on the monkeypox virus genome, and the operation is simple, efficient, and rapid. It can accurately quantify multiple samples, with a detection limit of 1×10⁻⁶. 2With a sample count of [copy / mL], sample testing can be completed in approximately 50 minutes. This rapid screening method is of great significance for screening imported and exported goods, animals, and humans, and can prevent the introduction of monkeypox virus into my country, thus meeting the current needs for monkeypox quarantine.

[0066] The lyophilized PCR reagent prepared in this invention allows oligonucleotide primers and probes, Taq DNA polymerase, and dNTPs to coexist stably at room temperature and even high temperatures. The prepared lyophilized PCR reagent retains its detection efficacy after transportation at room temperature. This significantly reduces the production, transportation, and storage costs of fluorescent PCR reagents, while also reducing operational steps, improving reagent stability, extending shelf life, increasing sample volume flexibility, and enhancing detection sensitivity. This facilitates the global sales and transportation of fluorescent PCR reagents for monkeypox virus nucleic acid detection.

[0067] The main advantages of this invention are:

[0068] (1) Rapid detection: The Taq DNA polymerase used in this invention is an antibody-modified DNA polymerase that can release enzyme activity in a short time at 95°C. Furthermore, the fluorescence is not collected in the first 10 cycles of the PCR program, which can greatly shorten the detection time. The sample detection can be completed in about 50 minutes.

[0069] (2) High sensitivity: This invention uses lyophilized PCR reagents, and all volumes except the reconstitution solvent can be added as samples, increasing the sample usage to 20 μL, while achieving a detection limit of 1 × 10⁻⁶. 2 Copy / mL.

[0070] (3) The lyophilized PCR reagent for monkeypox virus nucleic acid detection prepared in this invention allows oligonucleotide primers, oligonucleotide probes, Taq DNA polymerase, and dNTPs to coexist stably at room temperature or even high temperature. The prepared lyophilized PCR reagent retains its detection efficacy even after transportation at room temperature. This significantly reduces production, transportation, and storage costs while minimizing operational steps, improving reagent stability, extending shelf life, increasing sample volume flexibility, and enhancing detection sensitivity. It facilitates the global sales and transportation of fluorescent PCR reagents for monkeypox virus nucleic acid detection.

[0071] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Manual* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents used in the following embodiments are commercially available.

[0072] Example 1

[0073] Preparation of lyophilized PCR reagents for detecting monkeypox virus nucleic acid

[0074] Prepare a 30% (m / v) freeze-drying molding agent stock solution: sucrose + raffinose + hydroxypropyl-β-cyclodextrin, where m / v refers to mass concentration, indicating the mass per unit volume, expressed in "kg / L" or "kg / m". 3 The weight ratio of sucrose, raffinose, and hydroxypropyl-β-cyclodextrin in the freeze-drying molding agent mother liquor is 1:1:1.

[0075] The lyophilized PCR reagent for monkeypox virus nucleic acid detection is prepared as follows:

[0076] Components Dosage (μL) Final concentration Oligonucleotide upstream primer (10 μM) 0.5 0.2μM Oligonucleotide downstream primer (10 μM) 0.5 0.2μM Oligonucleotide fluorescent probe (10 μM) 0.25 0.1μM Taq DNA polymerase (5 units) 1 0.2 units Freeze-drying molding agent masterbatch (30%) 2.5 3％ dNTPs (10mM) 0.5 0.2mM <![CDATA[ddH2O]]> 19.75 / Total 25 /

[0077] Mix the above reagents to prepare a final reaction volume of 25 μl of PCR reagent. Prepare the required volume of mixed solution (25 μl × N) according to the needs, mix thoroughly, and aliquot into 8-tube PCR tubes. Place the tubes uncapped in a lyophilizer to prepare the lyophilized PCR reagent, following the procedure below: -50℃, 3 h; -50℃, 0.2 mbar, 2 h; 0℃, 0.2 mbar, 1 h; 10℃, 0.2 mbar, 1 h; 20℃, 0.2 mbar, 1 h; 30℃, 2 h. After lyophilization, encapsulate the lyophilized PCR reagent under nitrogen atmosphere to obtain the lyophilized PCR reagent for monkeypox virus nucleic acid detection (e.g., ...). Figure 1 (As shown).

[0078] Example 2

[0079] Screening of high-sensitivity lyophilized PCR reagents for monkeypox virus nucleic acid detection

[0080] Based on in-depth analysis of the target gene of monkeypox virus, twenty sets of primer pairs and probe combinations targeting the target gene were designed, and lyophilized reagents were prepared according to the method in Example 1.

[0081] The pUC57 plasmid containing a monkeypox virus-specific gene fragment was used as the test sample to test the sensitivity of the lyophilized PCR reagent prepared for monkeypox virus nucleic acid detection.

[0082] The experiment also included ddH2O as a negative control for the test samples.

[0083] The specific steps are as follows:

[0084] 1. Sample preparation: Take pUC57 plasmid containing a monkeypox virus-specific gene fragment of known concentration and dilute it to the following concentration series: 1×10 1 1×10 2 2×10 22×10 3 2×10 4 2×10 5 2×10 6 Copy / mL, using this series of concentration samples as templates.

[0085] 2. Reconstitution of lyophilized PCR reagents: Add 5 μL of 5× reconstitution solution (reconstitution solution: 3 mM magnesium ions, 50 mM potassium chloride, 0.05% (w / v) Tween 20 and 50 mM Tris-HCl, prepare the reconstitution solution to 5× solution) to each tube of lyophilized PCR reagents to restore the lyophilized powder to a solution state.

[0086] 3. Sample addition: Take 20 μL of the series of samples prepared above and ddH2O respectively, and add them to the reconstituted lyophilized PCR reagent reaction tubes, with a total reaction volume of 25 μL.

[0087] 4. Rapid PCR amplification: The quantitative PCR program is as follows: 95℃ pre-denaturation for 20s, (95℃ denaturation for 5s, 58℃ annealing and extension for 10s), repeat for 10 cycles, (95℃ denaturation for 5s, 58℃ annealing and extension to collect fluorescence for 30s), repeat for 30 cycles, and detect fluorescence using the FAM channel.

[0088] 5. Test Results:

[0089] Lyophilized PCR reagents containing different primer and probe combinations were tested, and three sets of detection limits were obtained that reached 2×10⁻⁶. 2 For lyophilized PCR reagents at a concentration of [copy / mL], the optimal primer and probe combinations are as follows:

[0090]

[0091] The representative detection results of lyophilized PCR reagent #6 are as follows: Figure 2 As shown: The copy number of pUC57 plasmid containing a monkeypox virus-specific gene fragment is 1×10⁻⁶. 2 At concentrations above 10 copies / mL, a typical "S"-shaped amplification curve was observed, indicating that the detection limit of the prepared lyophilized PCR reagent for monkeypox virus nucleic acid detection can reach 1×10⁻⁶. 2 Copy / mL. At the detection limit concentration, the detection rate was 100% with 20 replicates.

[0092] Example 3

[0093] Comparing the detection performance of lyophilized PCR reagents and liquid PCR reagents

[0094] This embodiment uses lyophilized PCR reagents and liquid PCR reagents with the same formulation as lyophilized PCR reagents for comparative testing.

[0095] 1. Reconstitution of lyophilized PCR reagents: Prepare the reconstitution solvent according to the following formula: 3mM magnesium ions, 50mM potassium chloride, 0.05% (w / v) Tween 20 and 50mM Tris-HCl, and prepare a 5× solution.

[0096] Add 5 μL of 5× reconstitution solution and 5.25 μL of ddH2O to each tube of lyophilized PCR reagent to restore the lyophilized powder to a solution state.

[0097] 2. Preparation of liquid control PCR reagent: Prepare the liquid PCR reaction reagent according to the table below:

[0098] Components Dosage (μL) Final concentration Oligonucleotide upstream primer (10 μM) 0.5 0.2μM Oligonucleotide downstream primer (10 μM) 0.5 0.2μM Oligonucleotide fluorescent probe (10 μM) 0.25 0.1μM Taq DNA polymerase (5 units) 1 0.2 units Freeze-drying molding agent masterbatch (30%) 2.5 3％ dNTPs (10mM) 0.5 0.2mM 5× Resolvent Solution 5 1× Total 10.25 /

[0099] 3. Sample loading: The template is pUC57 plasmid containing a monkeypox virus-specific gene fragment. Add 14.75 μL to each of the reconstituted lyophilized PCR reagent wells and liquid control reagent wells as reaction templates, for a total reaction volume of 25 μL.

[0100] 4. Rapid PCR amplification: The quantitative PCR program is as follows: 95℃ pre-denaturation for 20s, (95℃ denaturation for 5s, 58℃ annealing and extension for 10s), repeat for 10 cycles, (95℃ denaturation for 5s, 58℃ annealing and extension to collect fluorescence for 30s), repeat for 30 cycles, and detect fluorescence using the FAM channel.

[0101] 5. Test Results:

[0102] The results showed that the lyophilized PCR reagent for monkeypox virus nucleic acid detection prepared using the method of this invention had essentially no change in detection performance compared to the liquid PCR reagent with the same formulation. Representative test results are shown below. Figure 3 As shown (lyophilized PCR reagent #6).

[0103] Example 4

[0104] Stability testing of lyophilized PCR reagents for monkeypox virus nucleic acid detection

[0105] After storing lyophilized PCR reagents 6#, 11#, and 15# at room temperature and 37°C for 6 months, respectively, they were combined with freshly prepared lyophilized PCR reagents. 5 μL of the reconstitution solution prepared according to Example 2 was added to each reagent, followed by 20 μL of pUC57 plasmid containing a monkeypox virus-specific gene fragment as the test sample. PCR amplification was performed using the following quantitative real-time PCR program: 95°C pre-denaturation for 20 s, (95°C denaturation for 5 s, 58°C annealing and extension for 10 s), repeated 10 times; (95°C denaturation for 5 s, 58°C annealing and extension for 30 s fluorescence collection), repeated 30 times. Fluorescence was detected using the FAM channel.

[0106] serial number Detection limit (room temperature) Detection limit (37℃) 6# <![CDATA[1×10 2 copies / mL]]> <![CDATA[1×10 2 copies / mL]]> 11# <![CDATA[2×10 4 copies / mL]]> <![CDATA[2×10 4 copies / mL]]> 15# <![CDATA[2×10 2 copies / mL]]> <![CDATA[2×10 4 copies / mL]]>

[0107] Test results showed that the detection limit of lyophilized PCR reagent #6 remained unchanged after 6 months of storage. However, the detection limits of lyophilized PCR reagents #11 and #15 increased significantly after 6 months of storage, rendering them unusable for the detection of monkeypox virus nucleic acid.

[0108] The test results of representative lyophilized PCR reagent #6 are as follows: Figure 4 As shown, the amplification curves after 6 months of storage at room temperature and 37°C showed a high degree of agreement with the amplification curves obtained from the control group using freshly prepared lyophilized PCR reagents.

[0109] Therefore, the lyophilized PCR reagent for monkeypox virus nucleic acid detection prepared in this invention can be stored at room temperature and 37°C for more than 6 months.

[0110] Example 5

[0111] Application of lyophilized PCR reagents for monkeypox virus nucleic acid detection in clinical samples

[0112] Samples to be tested: Serum from 10 febrile patients collected from the hospital outpatient department. DNA was extracted using a human serum DNA extraction kit as a template. ddH2O and pUC57 plasmid containing a monkeypox virus-specific gene fragment were set up as negative and positive controls for the samples to be tested.

[0113] The detection method is the same as in the above embodiments, and the detection results are as follows: Figure 5 As shown:

[0114] The positive control wells showed a typical "S" amplification curve; the negative control wells showed no detection of Ct and the amplification curve was horizontal, indicating that the prepared lyophilized PCR reagent for monkeypox virus nucleic acid detection has good performance.

[0115] Ct was not detected in any of the serum samples from the 10 febrile patients, and the amplification curves were horizontal. The results of monkeypox virus testing were all negative.

[0116] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A freeze-dried reagent for fluorescent quantitative PCR for monkeypox virus nucleic acid detection, characterized by, The lyophilized PCR reagent includes an upstream primer, a downstream primer, and a probe; wherein the upstream primer sequence is shown in SEQ ID NO.1, the downstream primer sequence is shown in SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.3; The lyophilized PCR reagent also includes a lyophilization forming agent, which is composed of sucrose, raffinose, and hydroxypropyl-β-cyclodextrin, and the weight proportions of each component are as follows: 2-10 parts by weight of sucrose 2-10 parts by weight of raffinose, and Hydroxypropyl-β-cyclodextrin 2-10 parts by weight.

2. The lyophilized reagent as described in claim 1, characterized in that, The probe is labeled with a fluorescent group and a quenching group at its two ends, respectively.

3. The lyophilized reagent as described in claim 2, characterized in that, The fluorescent group is selected from the group consisting of FAM, VIC, CY5, and Texas Red; the quenching group is selected from the group consisting of BHQ1, BHQ2, BHQ3, TAMRA, and MGB.

4. The lyophilized reagent as described in claim 1, characterized in that, The freeze-drying molding agent is: 2-3 parts by weight of sucrose 2-3 parts by weight of raffinose, and 2-3 parts by weight of hydroxypropyl-β-cyclodextrin.

5. The lyophilized reagent as described in claim 1, characterized in that, The freeze-drying molding agent is: 3 parts by weight of sucrose 3 parts by weight of raffinose, and 3 parts by weight of hydroxypropyl-β-cyclodextrin.

6. The lyophilized reagent according to claim 1, characterized in that, The lyophilized PCR reagent also includes one or more components selected from the group consisting of Taq DNA polymerase and dNTPs.

7. The lyophilized reagent according to claim 1, characterized in that, When the lyophilized PCR reagent is used to prepare the PCR reaction system, the content of each component in the PCR reaction system is as follows: The volume of the PCR reaction system is 25 μl.

8. A kit for detecting monkeypox virus nucleic acid, characterized in that, The kit includes the lyophilized reagent as described in claim 1.

9. The kit as described in claim 8, characterized in that, The kit also includes a reconstitution solvent for reconstituted lyophilized PCR reagents; the reconstitution solvent includes: magnesium ions, potassium chloride, surfactant and Tris-HCl.

10. A method for preparing the real-time PCR lyophilized reagent for monkeypox virus nucleic acid detection as described in claim 1, characterized in that, The method includes the following steps: (1) Preparation of liquid PCR reagents (2) Freeze-drying The liquid PCR reagent provided in step (1) is lyophilized to obtain the lyophilized reagent.

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