Akabane disease pseudovirus particle as well as preparation method and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to akabane disease pseudovirus particles and a preparation method and application thereof.The akabane disease pseudovirus particles comprise recombinant lentiviral vectors and packaging plasmids, the recombinant lentiviral vectors comprise lentiviral shuttle plasmids and S whole-genome fragments of akabane disease viruses, the nucleotide sequence of the S whole-genome fragments is SEQ ID NO: 1, and the nucleotide sequence of the S whole-genome fragments is SEQ ID NO: 2. The preparation method comprises the following steps: (1) preparing a recombinant lentiviral vector; (2) preparing a mixture of a recombinant lentiviral vector and a transfection reagent; (3) culturing an HEK293T cell; (4) transfecting an HEK293T cell; and (5) obtaining the pseudovirus, the pseudovirus solution containing the AKAV S gene prepared by the invention can be used as a positive quality control product for AKAV nucleic acid detection, plays an effective monitoring role in the whole process from nucleic acid extraction to nucleic acid detection, and lays a foundation for preparing akabane disease nucleic acid diagnostic reagents.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to Akabane disease pseudovirus particles, a preparation method and application thereof. Background Art

[0002] Akabane disease, also known as Akabane spot disease, is an insect-borne infectious disease caused by the Akabane virus (AKAV) that infects cattle and sheep. It is characterized by miscarriage, premature birth, stillbirth in female animals, fetal malformations or mummification, and joint curvature and hydrocephalus in newborn animals. Mosquitoes and Culicoides are the main transmission vectors.

[0003] Akabane disease virus AKAV virus particles are generally spherical, with capsule and fibrils, and the diameter of the virus particles is about 80-120nm. It belongs to Bunyavirales, Pan-Bunyaviridae, Orthobunyavirus, Simbu serogroup. AKAV is relatively stable in the pH range of 6-10, sensitive to ether, chloroform, and acid (pH3), not heat-resistant, and quickly inactivated at 56°C. AKAV is more sensitive to Hmlu-1, Vero, BHK-21 and other passage cells, and can produce obvious cytopathic effect (CPE) after infecting cells, which can be used for virus isolation, identification and virus purification.

[0004] AKAV has only one serotype, which is a single-stranded negative-strand RNA virus. Its genome can be divided into three RNA segments: L gene, M gene, and S gene. Based on the sequence-specific analysis of AKAV's S gene, AKAV strains can be divided into five genotypes, namely genotype I, genotype II, genotype III, genotype IV, and genotype V. Genotype I is further divided into genotype I a subgroup and genotype I b subgroup. Different AKAV strains differ greatly in gene sequence, virulence, and antigenicity. Genotype I strains of AKAV are more neurotoxic than genotype II strains.

[0005] The S gene of AKAV can encode nucleocapsid protein (N) and non-structural protein (NS). The nucleotide sequence of the nucleocapsid (N) protein is 702 bp, and the N protein is composed of 233 amino acids. Its amino acid sequence is highly conserved and group-specific, and can induce the body to produce antibodies. N protein antibodies are of great significance in serological diagnosis.

[0006] AKAV is difficult to isolate and can be obtained from the blood of cattle and sheep infected with AKAV and producing viremia. Virus isolation is difficult. At the same time, AKAV is an RNA virus. The extracted nucleic acid is easily degraded and difficult to store for a long time. The use of AKAV nucleic acid diagnostic kits is limited. Therefore, when conducting AKAV molecular biology detection, there is a lack of corresponding standard substances and quality control cannot be performed. A pseudovirus particle containing the full S gene fragment of Akabane disease virus that needs to be provided can be used as a standard substance for AKAV to solve the problem of lack of Akabane disease standard substances and the problem of short shelf life of AKAV nucleic acid diagnostic kits. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides an Akabane disease pseudovirus particle, a preparation method and application thereof, which are used to solve the problem of lack of standard substances for Akabane disease virus nucleic acid detection.

[0008] To solve the above problems, the technical solution of the present invention is as follows: an Akabane disease pseudovirus particle, comprising a recombinant lentiviral vector and a packaging plasmid, the recombinant lentiviral vector and the packaging plasmid are co-transfected into HEK293T cells at the same time, the recombinant lentiviral vector comprises a lentiviral shuttle plasmid, and the lentiviral shuttle plasmid is provided with an S full gene segment of the Akabane disease virus used as an Akabane disease virus detection target, and the nucleic acid sequence of the S full gene segment is SEQ ID NO: 1.

[0009] Furthermore, the lentiviral shuttle plasmid is a pLenti-GIII-CMV-CBH-GFP-2A-Puro vector.

[0010] Furthermore, the packaging plasmid is a third generation packaging system mixture, 3rd Generation Packaging System Mix.

[0011] A method for preparing the above-mentioned Akabane disease virus pseudoviral particles comprises the following steps: (1) Preparation of recombinant lentiviral vectors; (2) Preparation of a mixture of recombinant lentiviral vector and transfection reagent; (3) Cultivate HEK293T cells; (4) Transfection of HEK293T cells; (5) Obtain pseudovirus. 45-50 h after transfection, collect the cell culture supernatant, filter the culture medium with a 0.4-0.5 μm low protein binding PES filter membrane to obtain a purified virus solution, and collect the pseudovirus by centrifugation at 10,000 r / min for 4 h. Harvest the purified pseudovirus-containing solution, add DNase I to remove residual nucleic acid plasmids, and store it at -80°C for later use after aliquoting.

[0012] Furthermore, the step (1) includes inserting the S full gene fragment into the lentiviral shuttle plasmid pLenti-GIII-CMV-CBH-GFP-2A-Puro.

[0013] Furthermore, the step (2) comprises: adding 9.5-10.5 μg of the recombinant lentiviral vector and 9.5-10.5 μg of the packaging plasmid to 1 mL of serum-free culture medium, mixing, and incubating at room temperature for 4-6 min to obtain a vector mixed solution; diluting 75-85 μL of the transfection reagent Lentifectin™ into 1 mL of serum-free culture medium, mixing, and incubating at room temperature for 4-6 min to obtain a transfection reagent dilution solution; adding the transfection reagent dilution solution to the vector mixed solution, mixing, incubating at room temperature for 15-25 min, and then adding 4.0-5.0 mL of serum-free culture medium to obtain a mixed vector and transfection reagent mixture.

[0014] Furthermore, the step (3) includes transferring the HEK293T cells into a 10 cm cell plate and culturing them for 46 to 50 h, adding 8 to 12 mL of DMEM medium containing 8 to 12% heat-inactivated fetal bovine serum into the cell plate, culturing the cells at 35 to 40° C. under 5% CO2 conditions, and performing transfection when the cells reach a fusion rate of 70% to 80%.

[0015] Furthermore, the step (4) includes first discarding the culture medium in the HEK293T cells, then adding the vector and transfection reagent mixture, gently vortexing the plate to disperse the complex, culturing overnight at 35-40°C under 5% CO2 for 8-14 h, and adding fresh DMEM culture medium containing 5% heat-inactivated fetal bovine serum, penicillin and streptomycin to replace the old culture medium.

[0016] The invention comprises the use of the Akabane disease pseudovirus particles in a standard substance, and the standard substance is used as an Akabane disease virus nucleic acid diagnostic reagent.

[0017] Compared with the prior art, the present invention has the following beneficial effects: a pseudovirus containing the full gene fragment of AKAV S is successfully constructed, which can be used as a positive control for AKAV nucleic acid detection, laying a foundation for the subsequent development of AKAV nucleic acid diagnostic kits.

[0018] Quality control products in the form of pseudoviruses only contain partial RNA fragments of the virus, do not have the ability to replicate and proliferate, have no biological infectivity, and are relatively safe. Pseudoviruses can also simulate the characteristics of natural viruses to the greatest extent, and can achieve full-process quality control of nucleic acid extraction and amplification of clinical samples, ensuring the accuracy of clinical test results. In addition, compared with cDNA, plasmid DNA, naked RNA, and in vitro transcribed RNA, pseudoviruses have better stability and are easier to preserve and promote as positive controls in nucleic acid diagnostic reagents.

[0019] The lentiviral shuttle plasmid pLenti-GIII-CMV-CBH-GFP-2A-Puro contains a fluorescent gene, which can be used to determine the expression and transport of the protein by observing fluorescence. At the same time, the vector carries a kanamycin resistance gene, which can be used to detect the residual nucleic acid plasmid by transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the construction of the recombinant lentiviral vector of the present invention; Figure 2 The sequencing verification results of the recombinant lentiviral vector pLenti-GIII-CMV-CBH-GFP-2A-Puro-AKAV in the results and analysis (A) of the present invention; Figure 3 Results and analysis (B) of the present invention, fluorescence expression, cell morphology and fluorescence expression 72 h after infection, indicating the cell morphology of the pseudovirus solution infected cell group; Figure 4 The fluorescence expression in the results and analysis (B) of the present invention, the cell morphology and fluorescence expression 72 h after infection, represents the cell morphology of the blank control group; Figure 5 The fluorescence expression in the results and analysis (B) of the present invention, the cell morphology and fluorescence expression 72 h after infection, represents the fluorescence expression of the pseudovirus solution infected cell group; Figure 6 In the results and analysis (B) of the present invention, the fluorescence expression, the cell morphology and fluorescence expression after 72 h of infection, and the fluorescence expression of the blank control group; Figure 7 The results of the nucleic acid plasmid residue detection in the results and analysis (C) of the present invention; Figure 8 The results and analysis (D) of the present invention are RT-PCR identification results, wherein M. DL2000 DNA Marker, 1. Pseudovirus solution, 2. Blank control; Fig. 9 The results and analysis (E) of the present invention show the results of fluorescence quantitative RT-PCR identification, wherein the X-axis represents the cycle number and the Y-axis represents the fluorescence value; Fig.10 This is the result and analysis (F) of the present invention, the digital RT-PCR value identification result, wherein the X-axis represents 8 repeated tests, and the Y-axis represents the number of labeled fluorescent positive nucleic acid copies. DETAILED DESCRIPTION

[0021] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0022] Example 1. Materials and methods Main reagents and materials: pLenti-GIII-CMV-CBH-GFP-2A-Puro, 3rd Generation Packaging System Mix, transfection reagent Lentifectin™, DNase I (Jiangsu Fuan Biotechnology Co., Ltd.); viral DNA / RNA extraction kit (Xi'an Tianlong Technology Co., Ltd.); Evo M-MLV One stepRT-qPCR Kit (Hunan Aikerui Biotechnology Co., Ltd.); Low-Profile PCR Tubes 8-tube strip (Bio-Rad Biomedical Products (Shanghai) Co., Ltd.); One-step ddPCR supermix (Sinafo (Suzhou) Life Science Co., Ltd.); DMEM, FBS (Thermo Fisher Scientific); isopropanol, anhydrous ethanol, RNaseA, etc. are all domestic analytical grade reagents (Sangon Biotechnology (Shanghai) Co., Ltd.).

[0023] Main instruments and equipment: Class II biological safety cabinet, carbon dioxide incubator (Likang Biomedical Technology Holding Co., Ltd.), automated nucleic acid extractor (Xi'an Tianlong Technology Co., Ltd.), high-speed refrigerated centrifuge, PCR instrument, fluorescence quantitative PCR instrument (Thermo Fisher Scientific), fluorescence microscope (Carl Zeiss Optics (China) Co., Ltd.); digital PCR instrument (Sinafo (Suzhou) Life Science Technology Co., Ltd.), clean bench (Suzhou Antai Air Technology Co., Ltd.), pipette (Germany Ebende AG), etc.

[0024] The method for preparing Akabane pseudovirus particles comprises the following steps: (1) Preparation of recombinant lentiviral vector; The AKAV S full gene fragment was derived from the strain AKAV-TJ2016 (GenBank accession number MT755621.1). The S full gene fragment was 856 bp in total. The S full gene was synthesized and inserted into the lentiviral shuttle plasmid pLenti-GIII-CMV-CBH-GFP-2A-Puro. The AKAV S full gene sequence was inserted as shown in the figure. Figure 1 As shown in FIG. 1 , the lentiviral shuttle plasmid contains the green fluorescent protein (GFP) gene, which can be analyzed by sequencing the recombinant lentiviral shuttle vector. Figure 2As shown, verify whether the target gene has been successfully inserted into the vector, and compare and analyze whether the inserted target gene is complete and accurate compared with the full gene sequence of AKAV-TJ2016 strain S.

[0025] (2) Preparation of the mixture of recombinant lentiviral vector and transfection reagent: add 10 μg of recombinant lentiviral vector and 10 μg of packaging plasmid into 1 mL of serum-free culture medium, mix well, and incubate at room temperature for 5 min to obtain a vector mixed solution; dilute 80 μL of transfection reagent Lentifectin™ into 1 mL of serum-free culture medium, mix well, and incubate at room temperature for 5 min to obtain a transfection reagent dilution solution; add the transfection reagent dilution solution to the vector mixed solution, mix well, incubate at room temperature for 20 min, and then add 4.5 mL of serum-free culture medium to obtain a mixed vector and transfection reagent mixture.

[0026] (3) Cultivate HEK293T cells: Move HEK293T cells into a 10 cm cell plate and culture for 48 h. Add 10 mL of DMEM medium containing 10% heat-inactivated fetal bovine serum to the cell plate. Culture the cells at 35-40°C under 5% CO2. Transfect the cells when the cell fusion rate reaches 70%-80%.

[0027] (4) Transfection of HEK293T cells: First, discard the culture medium in the HEK293T cells, then add the vector and transfection reagent mixture, gently vortex the plate to disperse the complex, and culture it overnight at 35-40℃ under 5% CO2 for 8-14 h. Add fresh DMEM culture medium containing 5% heat-inactivated fetal bovine serum, penicillin and streptomycin to replace the old culture medium.

[0028] (5) Obtaining pseudoviruses: 48 h after transfection, collect the cell culture supernatant, filter the culture medium with a 0.45 μm low protein binding PES filter membrane to obtain a purified virus solution, and collect the pseudoviruses by centrifugation at 10,000 r / min for 4 h. Harvest the purified pseudovirus-containing solution, add DNase I to remove residual nucleic acid plasmids, and store the solution at -80°C for later use after aliquoting.

[0029] Detection of prepared pseudovirus particles 1. Detection of nucleic acid plasmid residues Take the purified pseudovirus solution prepared in step (5), introduce 5 μL of the solution into DH5a competent cells by heat shock, add 800 μL of culture medium, and activate in a 37°C shaker for 45 min; evenly spread the activated competent cells on a culture dish containing kanamycin resistance, culture at 37°C overnight, and observe the growth of plaques on the plate to determine whether there is any nucleic acid plasmid residue.

[0030] 2. Identification of fluorescence expression In step (iii), 2 μL of purified cell culture supernatant was used to infect HEK293T cells with a co-confluence of 70% to 80%, and cultured for 72 h. Uninfected HEK293T cells were used as blank controls, and fluorescence microscopy was used to observe whether there was fluorescence expression in the infected HEK293T cells.

[0031] 3. RT-PCR identification The total nucleic acid of the purified cell culture supernatant prepared in step (V) was extracted using a viral DNA / RNA extraction kit. Sterile water was used as a blank control. The extracted nucleic acid was detected using the RT-PCR method with the AKAV S gene as the target. The reaction procedure was as follows: reverse transcription at 50 °C for 30 min; pre-denaturation at 95 °C for 15 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 40 s, extension at 72 °C for 1 min, 35 cycles; extension at 72 °C for 10 min. After the reaction was completed, gel electrophoresis was performed to observe the results to determine whether the AKAV S gene fragment could be amplified using the RT-PCR method. The primer information is shown in Table 1.

[0032] (IV) Fluorescence quantitative RT-PCR identification The total nucleic acid of the purified cell culture supernatant prepared in step (V) was extracted using a viral DNA / RNA extraction kit. Sterile water was used as a blank control. The extracted nucleic acid was detected using a fluorescent quantitative RT-PCR method targeting the AKAV S gene. The reaction procedure was as follows: 42°C for 5 min; 95°C for 30 s; 95°C for 5 s, 57°C for 30 s; 72°C for 30 s, 40 cycles; fluorescence was collected at 72°C to observe whether an "S-shaped" amplification curve was generated, and the Ct value was used to determine whether the S gene fragment could be amplified using the fluorescent quantitative RT-PCR method. The primer and probe information is shown in Table 1.

[0033] 5. Digital RT-PCR test value determination The total nucleic acid from the purified cell culture supernatant prepared in step (V) was extracted using a viral DNA / RNA extraction kit. The AKAV digital RT-PCR method was used to perform absolute determination of the extracted nucleic acid according to the reaction procedure of “60°C for 5 min; 95°C for 15 min; 95°C for 20 s, 60°C for 30 s, 40 cycles”. The test was repeated 8 times, and the arithmetic mean was taken as the final determination result. The primer and probe information was the same as that of the fluorescence quantitative RT-PCR test, as shown in Table 1.

[0034] Table 1 Primer and probe information in RT-PCR identification

[0035] In Table 1, the nucleotide sequence of primer AKAV-Sgene-F is SEQ ID NO: 2, the nucleotide sequence of primer AKAV-Sgene-R is SEQ ID NO: 3, the nucleotide sequence of primer AKAV23-Sgene-F is SEQ ID NO: 4, the nucleotide sequence of primer AKAV23-Sgene-R is SEQ ID NO: 5, and the nucleotide sequence of probe AKAV-Sgene-P is SEQ ID NO: 6.

[0036] 2. Results and Analysis (A) Identification of the recombinant lentiviral vector prepared in step (1) The synthetic gene was sequenced and confirmed that the full length of the S gene fragment of AKAV-TJ2016 strain (GenBank No. MT755621.1) was 856 bp, which can completely cover the primer and probe regions of the fluorescent quantitative RT-PCR method and the loop-mediated isothermal amplification method in the current AKAV detection technical standard. The sequencing verification results of the lentiviral recombinant shuttle vector are shown in the sequence table. The full S gene of AKAV-TJ2016 strain has been successfully inserted into the lentiviral vector, and the inserted target gene is complete and accurate. The identification results show that the recombinant lentiviral vector pLenti-GIII-CMV-CBH-GFP-2A-Puro-AKAV was successfully constructed.

[0037] (B) Fluorescence expression The results of fluorescence microscopy observations were as follows Figure 3-6 As shown, green fluorescence was observed in HEK293T cells 72 h after infection with the purified cell culture supernatant. Since the recombinant lentiviral vector contains the GFP gene, the pseudovirus infects HEK293T cells to express the GFP gene, while no fluorescence signal was observed in the blank control. Therefore, the GFP gene can be used as a marker gene to identify whether the pseudovirus is successfully obtained. The results show that the pseudovirus is successfully obtained.

[0038] (C) Detection of nucleic acid plasmid residues After overnight incubation at 35-40°C, no plaques grew on the plate. Figure 7 As shown, the results showed that no nucleic acid plasmid residue was detected in the harvested and purified pseudovirus solution.

[0039] (D) RT-PCR identification The results are as follows Figure 8 As shown, a target fragment of about 355 bp was amplified from the pseudovirus solution, indicating that the AKAV S gene fragment can be successfully amplified from the purified pseudovirus solution by the RT-PCR method.

[0040] (E) Fluorescence quantitative RT-PCR identification The results are as follows Fig. 9 As shown, after fluorescence quantitative RT-PCR amplification, an "S-shaped" amplification curve was generated, and the Ct value was 16, while the blank control group did not generate an "S-shaped" amplification curve and had no Ct value. The results showed that the AKAV S gene fragment could be successfully amplified from the pseudovirus solution by the fluorescence quantitative RT-PCR method.

[0041] (F) Digital RT-PCR determination The prepared pseudovirus solution was tested 8 times by digital RT-PCR test, and the arithmetic mean and coefficient of variation (CV) of the repeatability test were calculated. The results are shown in Table 2 and Fig.10 As shown in the figure, the repeatability test CV is 3.62%, and the copy number concentration of the prepared pseudovirus solution is 1.34×10 5 copies / μL.

[0042] Table 2 Results of digital RT-PCR test

[0043] The present application selected the AKAV S gene as the packaging sequence, adopted the lentiviral packaging technology, co-transfected 293T cells with the recombinant lentiviral vector and the packaging vector, prepared a pseudovirus solution containing the AKAV S gene, and identified and preliminarily applied it. Subsequently, the digital RT-PCR method was used to determine the copy number concentration of the pseudovirus solution prepared in the present application to be 1.34×10 5 In summary, the pseudovirus solution containing the AKAV S gene prepared in this application can be used as a positive quality control product for AKAV nucleic acid detection, and will play an effective monitoring role in the entire process from nucleic acid extraction to detection, laying the foundation for the preparation of Akabane disease nucleic acid diagnostic reagents.

[0044] The above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, a person skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A Akabane disease pseudoviral particle, characterized in that: It comprises a recombinant lentiviral vector and a packaging plasmid. The recombinant lentiviral vector and the packaging plasmid are co-transfected into HEK293T cells simultaneously. The recombinant lentiviral vector comprises a lentiviral shuttle plasmid. The lentiviral shuttle plasmid is provided with an S full gene segment of Akabane disease virus used as an Akabane disease virus detection target. The nucleic acid sequence of the S full gene segment is SEQ ID NO:

1.

2. The Akabane disease pseudovirus particle according to claim 1, characterized in that: The lentiviral shuttle plasmid is a pLenti-GIII-CMV-CBH-GFP-2A-Puro vector.

3. The Akabane disease pseudovirus particle according to claim 2, characterized in that: The packaging plasmid is a third generation packaging system mixture.

4. A method for preparing the Akabane disease pseudovirus particles according to any one of claims 1 to 3, characterized in that: The following steps are included: (1) Preparation of recombinant lentiviral vectors; (2) Preparation of a mixture of recombinant lentiviral vector and transfection reagent; (3) Cultivate HEK293T cells; (4) Transfection of HEK293T cells; (5) Obtain pseudovirus. 45-50 h after transfection, collect the cell culture supernatant, filter the culture medium with a 0.4-0.5 μm low protein binding PES filter membrane to obtain a purified virus solution, and collect the pseudovirus by centrifugation at 10,000 r / min for 4 h. Harvest the purified pseudovirus-containing solution, add DNase I to remove residual nucleic acid plasmids, and store it at -80°C for later use after aliquoting.

5. The preparation method according to claim 4, characterized in that: The step (1) includes inserting the S full gene fragment into the lentiviral shuttle plasmid pLenti-GIII-CMV-CBH-GFP-2A-Puro.

6. The preparation method according to claim 5, characterized in that: The step (2) comprises adding 9.5-10.5 μg of the recombinant lentiviral vector and 9.5-10.5 μg of the packaging plasmid to 1 mL of serum-free culture medium, mixing, and incubating at room temperature for 4-6 min to obtain a vector mixed solution; diluting 75-85 μL of the transfection reagent into 1 mL of serum-free culture medium, mixing, and incubating at room temperature for 4-6 min to obtain a transfection reagent dilution solution; adding the transfection reagent dilution solution to the vector mixed solution, mixing, incubating at room temperature for 15-25 min, and then adding 4.0-5.0 mL of serum-free culture medium to obtain a mixed vector and transfection reagent mixture.

7. The preparation method according to claim 6, characterized in that: The step (3) comprises: transferring the HEK293T cells into a 10 cm cell plate and culturing them for 46-50 h, adding 8-12 mL of DMEM culture medium containing 8-12% heat-inactivated fetal bovine serum into the cell plate, culturing the cells at 35-40° C. under 5% CO2 conditions, and performing transfection when the cells reach a fusion rate of 70%-80%.

8. The preparation method according to claim 7, characterized in that: The step (4) comprises first discarding the culture medium in the HEK293T cells, then adding the vector and transfection reagent mixture in step (2), gently vortexing the plate to disperse the complex, culturing overnight at 35-40° C. under 5% CO2 for 8-14 h, and adding fresh DMEM culture medium containing 4-6% heat-inactivated fetal bovine serum, penicillin and streptomycin to replace the old culture medium.

9. A use of the Akabane disease pseudovirus particles according to any one of claims 1 to 3 in a standard substance, characterized in that: The standard substance is used for Akabane disease virus nucleic acid diagnostic reagent.

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