Detection primer and nucleic acid mass spectrometry detection method for African horse pestivirus and west nile virus

Abstract

The invention relates to the technical field of biological detection, in particular to a detection primer for African horse pestivirus and west nile virus and a nucleic acid mass spectrometry detection method. On the basis of an MALDI-TOF nucleic acid mass spectrum detection method, two relatively conservative genes of VP7 and NS2 are selected as target genes for the African horse pestivirus; and for the West Nile virus, two same conservative genes NS2A and NS5 are selected. Based on the target genes, four pairs of amplification primers and four extension primers are designed. By optimizing selection of amplification sites and adding of modified bases, it is ensured that the molecular weight of the extension probe is effectively distinguished within the detection range, so that the multiple detection target is achieved, and meanwhile high accuracy, specificity and sensitivity of detection are ensured. The kit can efficiently, sensitively and accurately identify the infection conditions of the African horse pestivirus and the west nile virus, can be flexibly adjusted and optimized according to actual requirements, and is expected to play an important role in virus detection and differential diagnosis in the future.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a detection primer and a nucleic acid mass spectrometry detection method for African horse sickness virus and West Nile virus. Background Art

[0002] African Horse Sickness Virus (AHSV) and West Nile Virus (WNV) can both infect equine animals. Among them, African Horse Sickness Virus mainly infects equine animals and is transmitted through the bites of insects such as mosquitoes (such as Culicoides and Aedes), but it is not a zoonosis and will not be transmitted to humans through mosquitoes. West Nile Virus can be transmitted to humans and many other animals, including horses, birds and other mammals, through mosquito bites.

[0003] African horse sickness virus is a double-stranded RNA virus that belongs to the genus Orbivirus of the family Reoviridae, the same as bluetongue. It is mainly prevalent in Africa and has spread to Asia and other regions in recent years. African horse sickness mainly infects equine animals, and the symptoms after infection are severe, with a mortality rate of up to 95%, and often leads to internal bleeding, pulmonary edema, etc.

[0004] West Nile virus is a single-stranded positive-sense RNA virus that belongs to the genus Flavivirus, which belongs to the same genus as Japanese encephalitis, St. Louis encephalitis, yellow fever, dengue fever and other viruses. West Nile virus has appeared in many countries and regions. It was initially prevalent in Africa and later spread to other regions, including Asia, Europe, North America and Australia. West Nile virus is one of the most widely spread mosquito-borne flaviviruses. Humans, horses and other mammals can be infected, and severe cases can cause neurological diseases such as encephalitis or meningitis, and even death. In about 80% of infections, people have few or no symptoms. About 20% of people will experience fever, headache, vomiting or rash, and less than 1% will develop encephalitis or meningitis, with associated neck stiffness, confusion or seizures, and recovery may take weeks to months.

[0005] At present, with the increasing frequency of international exchanges, my country is facing a greater risk of infection from African horse sickness virus and West Nile virus. As horses are economic animals of relatively high value, once infected, they will bring great economic losses. Therefore, an efficient and accurate detection method is urgently needed to quarantine imported horses.

[0006] Virus isolation is the most classic method for pathogen detection, but this method is time-consuming and labor-intensive and requires high biosafety conditions. At present, PCR technology is mainly used to detect and identify horse diseases. PCR technology is a highly sensitive molecular biology technology that can detect extremely small amounts of target nucleic acids. Compared with serological detection methods, PCR detection greatly improves the sensitivity of detection and shortens the window period for pathogen detection. Taking GB / T 21675-2022 "Diagnostic Technology for African Horse Sickness" as an example, the national standard introduces virus culture, serological methods and RT-PCR methods for identifying African horse sickness. Among them, the RT-PCR method is limited by the fluorescence channel of the instrument platform. If a universal or typing identification is performed, the reaction system needs to be prepared separately. A sample needs to repeat the RT-PCR experiment multiple times to achieve the differential diagnosis of the pathogen. If it is expected to perform differential diagnosis on multiple target genes of two pathogens, more experimental operations are required, which limits its extensiveness in practical applications. And because RNA viruses have a high mutation rate, a dual-target identification strategy for a single pathogen can significantly improve the accuracy and reliability of detection and effectively avoid the occurrence of missed detection. Therefore, the development of a nucleic acid mass spectrometry-based detection kit to focus on the identification of exotic equine diseases and achieve rapid and accurate detection is an important issue facing current technology. Summary of the invention

[0007] In view of this, the present invention proposes a detection primer, a detection kit and a nucleic acid mass spectrometry detection method based on matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF) for African horse sickness virus (AHSV) and West Nile virus (WNV). The method combines RT-PCR multiple amplification technology, a dual-gene multi-target strategy and a single base extension reaction. Therefore, the present invention provides a method for simultaneously detecting African horse sickness virus and West Nile virus efficiently, accurately and sensitively.

[0008] The technical solution of the present invention is achieved in this way:

[0009] In a first aspect, the present invention provides a primer composition comprising:

[0010] The specific primer sequences for amplifying the NS2 fragment of African horse sickness virus are SEQ ID NO.1 and SEQ ID NO.2,

[0011] The specific primer sequences for amplifying the VP7 fragment of African horse sickness virus are SEQ ID NO.3 and SEQ ID NO.4,

[0012] The specific primer sequences for amplifying the NS2A fragment of West Nile virus are SEQ ID NO.5 and SEQ ID NO.6,

[0013] The specific primer sequences for amplifying the NS5 fragment of West Nile virus are SEQ ID NO.7 and SEQ ID NO.8,

[0014] Single base extension primer SEQ ID NO.9 for the NS2 fragment of African horse sickness virus,

[0015] Single base extension primer SEQ ID NO.10 for the VP7 fragment of African horse sickness virus,

[0016] Single base extension primer SEQ ID NO.11 of the NS2A fragment of West Nile virus, and

[0017] Single base extension primer for the NS5 fragment of West Nile virus SEQ ID NO.12.

[0018] The selection of targets and primer design ideas include the following:

[0019] The present invention combines a large number of literature research reports, and finally selects four target genes AHSV-VP7, AHSV-NS2, WNV-NS2A and WNV-NS5 of African horse sickness virus (AHSV) and West Nile virus (WNV) for primer design. The sequences of these four target genes are downloaded and collected from NCBI respectively, and after sequence alignment and phylogenetic tree analysis, the conserved regions on each conserved gene are confirmed.

[0020] The primers are derived from the conservative sequence of the target fragment, and a tag sequence is optionally added as needed, so that the molecular weight of the RT-PCR multiple amplification primer can be easily distinguished by MALDI-TOF MS. Therefore, the present invention adjusts the length of the primer to avoid its molecular weight falling within the conventional detection range (4000-10000Da) of MALDI-TOF MS. When the molecular weight of the amplification primer exceeds this detection range, the amplification product will also exceed the detection range. At this time, only the extension primer and the extension product are within the detection range, thereby avoiding interference with the interpretation after the extension reaction. At the same time, the present invention designs a single-base extension primer in the conservative sequence region in the amplification region, and allows a designed and determined base to be extended at the 3' end of the single-base extension primer as a specific sequence marker for different targets; thereby achieving accurate detection of multiple targets of African horse sickness virus and West Nile virus.

[0021] like Figure 1As shown in the figure, taking the conservative region selection of WNV-NS2A gene as an example, it can be clearly seen that within the 30-145bp range at the 5' end, the sequence is more conservative relative to the entire gene fragment. Since degenerate bases are not allowed in the design of extension primers, we choose the most conservative 60-80bp region as the binding region of the extension primer. When designing extension primers, some basic primer design principles need to be followed, for example, it is necessary to avoid the occurrence of adverse situations such as the formation of dimers between primers or primers themselves.

[0022] The information related to primers, products and molecular weight is shown in Table 1:

[0023] Table 1

[0024]

[0025] Among them, the extended base parts that may exist in the extended product sequence have been shaded. In the interpretation rules, A / T and G / C represent different types of extended bases. In the sequences in Table 1, lowercase letters represent artificially added bases that are not complementary to the template DNA. These additionally added bases are intended to optimize the molecular weight difference between each extended primer and the extended product, so as to more accurately distinguish them based on the detection accuracy of MALDI-TOF MS (the distance between each primer and the extension product peak is required to be no less than 16Da).

[0026] In a second aspect, the present invention provides the use of the primer composition in preparing a product for detecting African horse sickness virus and / or West Nile virus.

[0027] Furthermore, in some preferred embodiments, the product includes independent reagents, chips or kits.

[0028] In a third aspect, the present invention provides a product, the components of which include: the primer composition SEQ ID NO1-12; the product includes independent reagents, chips or kits; the product is used to detect African horse sickness virus and / or West Nile virus.

[0029] Furthermore, in some preferred embodiments, the components of the product also include: reaction reagents for RT-PCR multiplex amplification; the reagents include: enzymes, and / or reaction buffers. In some embodiments, the enzymes include at least one of reverse transcriptase and Taq enzyme. The reaction buffer includes: Mg 2+ , dNTPs and H2O or a combination of one or more thereof.

[0030] Furthermore, in some preferred embodiments, the components of the product also include: a dephosphorylation reaction reagent; the reagent includes: an enzyme, and / or a reaction buffer. In some embodiments, the enzyme is shrimp alkaline phosphatase (SAP enzyme). The reaction buffer includes: Mg 2+ , dNTPs and H2O. The reaction buffer includes: Mg 2+ , Zn2+, dNTPs, Bis-Tris HCl and H2O.

[0031] Furthermore, in some preferred embodiments, the components of the product also include: single base extension reaction reagents; the reagents include: enzymes, and / or reaction buffers. In some embodiments, the reaction buffer includes: Mg 2+ , dNTPs and H2O or a combination of one or more thereof.

[0032] Furthermore, in some preferred embodiments, the components of the product also include: a negative control, and / or a positive control.

[0033] In a fourth aspect, the present invention further provides a MALDI-TOF mass spectrometry detection method for African horse sickness virus and West Nile virus, wherein the detection method is for non-disease treatment and / or diagnosis, and comprises the following steps:

[0034] (S1) using SEQ ID NO: 1-8 as amplification primers, performing RT-PCR multiple amplification reaction on the nucleic acid sample to be tested to obtain amplification products;

[0035] (S2) dephosphorylating the amplified product using alkaline phosphatase to obtain a dephosphorylated product;

[0036] (S3) using SEQ ID NO: 9-12 as a single base extension primer, performing single base extension on the dephosphorylated product to obtain an extension product;

[0037] (S4) purifying the extension product;

[0038] (S5) MALDI-TOF mass spectrometry is used to detect the molecular weight of the purified product obtained in step (S4), and African horse sickness virus and West Nile virus are identified based on the difference in molecular weight.

[0039] In the step (S1), the sample to be tested is an extracted nucleic acid sample.

[0040] Further, in some preferred embodiments, in the step (S4), the purification comprises one of column chromatography purification, PAGE purification, magnetic bead purification and HPLC purification. The column chromatography purification comprises resin desalting purification.

[0041] Furthermore, in some preferred embodiments, the detection method comprises:

[0042] The reaction procedure of RT-PCR multiplex amplification in step (S1) is as follows:

[0043]

[0044] The dephosphorylation reaction procedure in step (S2) includes: incubating at 37°C for 40 minutes; then inactivating the dephosphatase at 85°C for 5 minutes; and finally storing at 4°C.

[0045] The reaction procedure of the single base extension in step (S3) is as follows:

[0046]

[0047] In some preferred embodiments, in step S(5), the result is determined based on whether the known single-base extension primer is extended and a specific extension peak and base appear. The mass spectrometry analysis results of each target correspond to two peak positions, the single-base extension primer and the single-base extension product. If the primer extension conversion rate is 100%, the single-base extension primer peak disappears. If a single peak appears at the molecular weight position corresponding to any extension product of a target and the signal-to-noise ratio (SNR) is greater than 6, the target is judged to be positive: otherwise, it is judged to be negative. The judgment rules are shown in Table 1 above.

[0048] The beneficial effects of the present invention include at least the following:

[0049] The present invention is based on nucleic acid mass spectrometry detection. For African horse sickness virus (AHSV), two relatively conservative genes, VP7 and NS2, are selected as target genes; and for West Nile virus (WNV), two equally conservative genes, NS2A and NS5, are selected. Based on these target genes, the present invention designs four pairs of amplification primers and four extension primers. By optimizing the selection of amplification sites and the addition of modified bases, it is ensured that the molecular weight of the extension probe is effectively distinguished within the detection range, thereby achieving the goal of multiple detection, and at the same time ensuring high accuracy, specificity and sensitivity of the detection. Through the above design, in an experiment, only the peak graph of the extension product needs to be observed to clearly report whether there is infection with the corresponding pathogen.

[0050] In addition, the present invention is also compatible with high-density 384-well chips, so that single-well reactions can complete all experiments and data analysis in a relatively short time, which is very suitable for rapid screening of large-scale samples. The sample loading process is flexible and convenient, and the chip can be used multiple times, which further reduces the detection cost and improves the efficiency of resource utilization.

[0051] In the construction of the detection system, the present invention fully considers the evolutionary characteristics of the RNA virus genome. By selecting double conserved genes for differential diagnosis, we ensure the stability and reliability of the test results. At the same time, the use of MALDI-TOF technology gets rid of the limitations of the fluorescence channel and improves the flexibility and accuracy of the detection. Compared with low-throughput fluorescence quantitative PCR, the reagent cost of the present invention is comparable, but the primer system used does not require fluorescent group modification, and only simple PAGE purification is required to meet the needs of experiments and instruments, thereby significantly reducing the detection cost. Therefore, the present invention can not only efficiently, sensitively and accurately identify the infection of African horse sickness virus and West Nile virus, but also can be flexibly adjusted and optimized according to actual needs, and is expected to play an important role in future virus detection and differential diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0053] Figure 1 Schematic diagram of the selected conserved regions of the WNV-NS2A gene;

[0054] Figure 2 It is the mass spectrometry detection result of AHSV-NS2 target gene site in AHSV nucleic acid sample;

[0055] Figure 3 It is the mass spectrometry detection result of AHSV-VP7 target gene site in AHSV nucleic acid sample;

[0056] Figure 4 It is the mass spectrometry detection result of WNV-NS2A target gene site in WNV nucleic acid sample;

[0057] Figure 5 It is the mass spectrometry detection result of WNV-NS5 target gene site in WNV nucleic acid sample;

[0058] Figure 6 This is the mass spectrometry detection result of a mixed nucleic acid sample infected with two viruses (AHSV+WNV);

[0059] Figure 7 This is the mass spectrometry test result of bluetongue virus BTV nucleic acid;

[0060] Figure 8 It is the AHSV-NS2 gene locus detection result when the nucleic acid load is 10 copies / mL;

[0061] Fig. 9 It is the AHSV-VP7 gene locus detection result when the nucleic acid load is 10 copies / mL;

[0062] Fig.10 This is the WNV-NS2A gene locus detection result when the nucleic acid load is 10 copies / mL;

[0063] Fig.11 This is the WNV-NS5 gene locus detection result when the nucleic acid load is 10 copies / mL. DETAILED DESCRIPTION

[0064] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0065] The methods used in the following examples are all conventional methods unless otherwise specified. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0066] The methods for obtaining various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0067] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps that are not listed, or may optionally include other steps that are inherent to these processes, methods, products or equipment. The "plurality" mentioned in the present invention refers to two or more. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the previously associated objects are in an "or" relationship.

[0068] Example 1 Application of the detection method, primer combination and kit provided by the present invention in detecting African horse sickness virus and West Nile virus I. Target selection, specific amplification primer and single base extension primer design

[0069] 1. Selection of African Horse Sickness Virus (AHSV) Targets:

[0070] The VP7 gene is one of the core structural protein genes of the African horse sickness virus, and its sequence is relatively conserved among different virus strains. The VP7 protein has good immunogenicity and can stimulate the body to produce a specific immune response. Therefore, the detection method based on the VP7 gene has high sensitivity and specificity.

[0071] The non-structural protein encoded by the NS2 gene plays an important role in the replication and pathogenicity of African horse sickness virus. Although its sequence may vary to a certain extent relative to the structural protein gene, it is still conservative to a certain extent between different virus strains. Due to the functional importance of the NS2 protein, detecting it as a target gene helps to gain a deeper understanding of the virus's replication and pathogenic mechanisms. At the same time, the detection method based on the NS2 gene can also be used to monitor the mutation of the virus.

[0072] 2. Selection of West Nile Virus (WNV) Target:

[0073] The nonstructural protein encoded by the NS2A gene has multiple functions, including participating in viral replication, viral particle assembly, and inducing host cell death. Although there are certain sequence variations in the NS2A gene, there are still certain conserved regions between different virus strains. These conserved regions can serve as the basis for designing detection primers and probes.

[0074] The protein encoded by the NS5 gene is one of the largest and most conserved proteins of West Nile virus. It plays a key role in the replication of the virus as an RNA polymerase. Due to the high conservatism of the NS5 protein and its importance in viral replication, detection methods based on the NS5 gene usually have high sensitivity and specificity.

[0075] Therefore, combined with a large number of literature research reports, the four target genes AHSV-VP7, AHSV-NS2, WNV-NS2A and WNV-NS5 of African horse sickness virus (AHSV) and West Nile virus (WNV) were finally selected for primer design. Then the sequences of these four target genes were downloaded and collected from NCBI respectively, and the conserved regions on each conserved gene were confirmed after sequence alignment and phylogenetic tree analysis. The primers are derived from the conserved sequences of the target fragments, and tag sequences can be added as needed, so that the size of the RT-PCR multiple amplification products can be easily distinguished by MALDI-TOF MS, and they are all within the mass spectrometry detection range (4000-10000Da).

[0076] At the same time, the present invention designs a single base extension primer in the conserved sequence region in the amplification region, and allows a designed and determined base to be extended at the 3' end of the single base extension primer as a specific sequence marker for different targets.

[0077] like Figure 1 As shown in the figure, taking the conservative region selection of WNV-NS2A gene as an example, it can be clearly seen that within the 30-145bp range at the 5' end, the sequence is more conservative relative to the entire gene fragment. Since degenerate bases are not allowed in the design of extension primers, we choose the most conservative 60-80bp region as the binding region of the extension primer. When designing extension primers, some basic primer design principles need to be followed, for example, it is necessary to avoid the occurrence of adverse situations such as the formation of dimers between primers or primers themselves.

[0078] The sequence and molecular weight information of the amplification primers, extension primers and products are specifically shown in Table 1 above.

[0079] 2. Sample Nucleic Acid Extraction

[0080] To simulate the reverse transcription process in RT-PCR, each target gene was commissioned to Zhenjiang Aibimeng Biotechnology Co., Ltd. to synthesize lentiviral reference products, using the vector Lentiviral Vector (CMV) (pLenti-GIII-CMV-CBH-GFP-2A-Puro) (Cat. No. DNAFor LV002-b) and the viral DNA / RNA extraction kit 4.0 of Xi'an Tianlong Technology Co., Ltd., with a fully automatic nucleic acid extractor for nucleic acid extraction. After extraction, 60 μL of nucleic acid eluate in the deep-well plate was transferred to a clean EP tube and marked.

[0081] 3. Testing steps

[0082] 1. RT-PCR multiple amplification reaction

[0083] Amplification primers SEQ ID NO: 1-8 were used to perform RT-PCR multiple amplification reaction on the sample to be tested, and 4 target nucleic acids were used as positive controls and ultrapure water was used as negative controls to perform RT-PCR multiple amplification to obtain amplification products, wherein the reaction system is shown in Table 2:

[0084] Table 2

[0085] Reagents factory Item No. / Specification Dosage RT-PCR reaction mixture Zhejiang Dipu Diagnostics Technology Co., Ltd. DPGPM-V 2.5μL RT-PCR enzyme solution Zhejiang Dipu Diagnostics Technology Co., Ltd. DPGPM-V 0.3μL Multiplex Amplification Primer Mix - 5 μM of each 0.2μL

[0086] The multiple amplification primer mixture solution contains amplification primers SEQ ID NO: 1-8; the concentration of each primer is 5 μM.

[0087] The reaction procedure is shown in Table 3:

[0088] Table 3

[0089]

[0090] The actual test results of RT-PCR multiple amplification reactions showed that the two-step cycle method can meet the amplification requirements and can effectively amplify the target fragments.

[0091] 2. Dephosphorylation reaction

[0092] In order to eliminate excess dNTPs after multiple PCR amplification reactions and ensure the accuracy of single-base extension detection by mass spectrometry, shrimp alkaline phosphatase (SAP) was used for treatment. The SAP digestion enzyme reaction system is shown in Table 4:

[0093] Table 4

[0094] Reagents Ultrapure water SAP Reaction Buffer SAP enzyme mixture Multiplex PCR products Dosage 1.53μL 0.17μL 0.3μL 5μL

[0095] Among them, SAP enzyme and reaction buffer were purchased from Zhejiang Dipu Diagnostic Technology Co., Ltd.

[0096] The reaction program was set to incubate at 37°C for 40 minutes to allow SAP to remove excess dNTPs; then the SAP enzyme was inactivated at 85°C for 5 minutes, and then stored at 4°C after completion.

[0097] 3. Single base extension reaction

[0098] The components were added in sequence according to the reaction system sequence in Table 5, and the configured reaction system was used for single base extension according to the reaction procedure shown in Table 6.

[0099] Table 5

[0100]

[0101] The extension primer mixture is amplification primers SEQ ID NO: 9-12; the extension enzyme and reaction buffer are purchased from Zhejiang Dipu Diagnostic Technology Co., Ltd. The concentration of each primer is 10 μM.

[0102] Table 6

[0103]

[0104] 4. Resin desalting purification and MALDI-TOF mass spectrometry detection

[0105] 18 μL DEPC water was pre-added to each well at the set position in the 384 reaction plate, and all the above single base extension products (the actual volume may be slightly less than the theoretical value of 9 μL, and there is a certain loss during the reaction) were added and distributed to each well containing DEPC water, mixed and sealed with a sealing film, and centrifuged to evenly distribute the mixture at the bottom of the well. Ensure that the Assay file accurately contains the molecular weight information of all single base extension primers and their extension products, and import it into the operating software of the DP-TOF time-of-flight nucleic acid mass spectrometer (Zhejiang Dipu Diagnostic Technology Co., Ltd.). Open the nucleic acid flight mass spectrometer deck, remove the sealing film of the 384 reaction plate, and place it in the designated position. Set each well to add 10 μL of resin, and the instrument will automatically run the product desalting and purification steps, and perform automatic spotting and mass spectrometry detection.

[0106] The result is determined based on whether the known single-base extension primer is extended and a specific extension peak and base appear. The mass spectrometry analysis results of each target correspond to two peak positions, the single-base extension primer and the single-base extension product. If the primer extension conversion rate is 100%, the single-base extension primer peak disappears. If a single peak appears at the molecular weight position corresponding to any extension product of a target and the signal-to-noise ratio (SNR) is >6, the target is judged to be positive: otherwise, it is judged to be negative. The interpretation rules are shown in Table 1 above.

[0107] 4. Test results:

[0108] 1. The mass spectrometry results of AHSV nucleic acid samples are as follows: Figure 2 and Figure 3 As shown. Figure 2 This is the mass spectrometry detection result of the AHSV-NS2 target gene site. At this time, the AHSV-NS2 extension primer peak (5265.4Da) disappears, and the corresponding A base extension product peak (5536.6Da) appears; Figure 3 This is the mass spectrometry detection result of the AHSV-VP7 target gene site. At this time, the AHSV-VP7 extension primer peak (6173.0Da) disappears and the corresponding T base extension product peak (6500.2Da) appears.

[0109] 2. Mass spectrometry results of WNV nucleic acid samples are as follows: Figure 4and Figure 5 As shown. Figure 4 This is the mass spectrometry detection result of the WNV-NS2A target gene site. At this time, the WNV-NS2A extension primer peak (5490.6Da) disappears, and the corresponding G base extension product peak (5777.8Da) appears; Figure 5 This is the mass spectrometry detection result of the WNV-NS5 target gene site. At this time, the WNV-NS5 extension primer peak (5810.8Da) disappears, and the corresponding A base extension product peak (6082.0Da) appears.

[0110] 3. The mass spectrometry results of mixed nucleic acid samples simulating mixed infection of two viruses (AHSV+WNV) are as follows: Figure 6 shown.

[0111] The above results show that the statistical information of the extension products (extended bases corresponding to the detected peaks) and the molecular weight (Da) values ​​of each sample are shown in the following table, which are consistent with the original sample information:

[0112]

[0113] Example 2 Specificity verification of the detection method, primer combination and kit provided by the present invention

[0114] The sample information to be tested used in this embodiment is as follows:

[0115]

[0116]

[0117] The nucleic acid extraction and detection steps of the samples are the same as those in Example 1.

[0118] The test results show that the statistical information of the extension product Mass value of each sample is shown in the following table, which is consistent with the original sample information (for example Figure 7 The results of mass spectrometry detection of bluetongue virus BTV nucleic acid, all points are negative):

[0119]

[0120] It can be seen that for samples of pathogens that are not within the detection range, no extension product peaks were observed at the AHSV-NS2, AHSV-VP7, WNV-NS2A and WNV-NS5 sites, which fully demonstrates that the detection method, primer combination and kit provided by the present invention have good specificity.

[0121] Example 3 Sensitivity verification of the detection method, primer combination and kit provided by the present invention

[0122] In this example, qPCR was used to quantify the copy number of pseudovirus nucleic acid samples of four genes (AHSV-NS2 / AHSV-VP7 / WNV-NS2A and WNV-NS5). The initial quantification of each positive nucleic acid was 1×10 5 copies / mL, and then dilute it 10 times, with the dilution loading ranging from 1×10 5 The dilutions ranged from 1 copy / mL to 1 copy / mL, with a total of 6 dilutions. The tests were performed according to the methods and steps in Example 1, and each gradient was repeated 3 times.

[0123] The results of AHSV-NS2 gene locus detection when the nucleic acid load was 10 copies / mL were as follows Figure 8 shown.

[0124] The results of AHSV-VP7 gene locus detection when the nucleic acid load was 10 copies / mL were as follows Fig. 9 shown.

[0125] When the nucleic acid load was 10 copies / mL, the WNV-NS2A gene locus detection results were as follows: Fig.10 shown.

[0126] When the nucleic acid load was 10 copies / mL, the WNV-NS5 gene locus detection results were as follows: Fig.11 shown.

[0127] The test results show that when the pseudovirus nucleic acid samples of the four genes are diluted to 10 copies / mL, the extension primers of the AHSV-VP7 and WNV-NS2A genes are partially not completely consumed, indicating that the critical detection limit of the detection method has been reached at this time, and the extension primers of the AHSV-NS2 gene and the WNV-NS5 gene are completely consumed at this time. It is speculated that the detection sensitivity of the AHSV-NS2 gene and the WNV-NS5 gene is slightly higher, but when the nucleic acid is diluted to 1 copy / mL, no extension products are detected in each duplicate well of the four genes. Therefore, the minimum detection limit of the detection method, primer combination and kit provided by the present invention is between 1 copy / mL and 10 copies / mL.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer composition, characterized in that include: Specific primer sequences for amplifying the NS2 fragment of African horse sickness virus are SEQ ID NO.1 and SEQ ID NO.2, The specific primer sequences for amplifying the VP7 fragment of African horse sickness virus are SEQ ID NO.3 and SEQ ID NO.4, The specific primer sequences for amplifying the NS2A fragment of West Nile virus are SEQ ID NO.5 and SEQ ID NO.6, The specific primer sequences for amplifying the NS5 fragment of West Nile virus are SEQ ID NO.7 and SEQ ID NO.8, Single base extension primer SEQ ID NO.9 for the NS2 fragment of African horse sickness virus, Single base extension primer SEQ ID NO.10 for the VP7 fragment of African horse sickness virus, Single base extension primer SEQ ID NO.11 of the NS2A fragment of West Nile virus, and Single base extension primer for the NS5 fragment of West Nile virus SEQ ID NO.

12.

2. The use of the primer composition according to claim 1, characterized in that: Application in the preparation of products for detecting African horse sickness virus and / or West Nile virus.

3. The use according to claim 2, characterized in that: The products include independent reagents, chips or kits.

4. A product, characterized in that The components of the product include: the primer composition described in claim 1; the product includes independent reagents, chips or kits; the product is used to detect African horse sickness virus and / or West Nile virus.

5. The product according to claim 4, characterized in that: The components of the product also include: reaction reagents for RT-PCR multiple amplification; the reagents include: enzymes, and / or reaction buffers.

6. The product according to claim 4, characterized in that: The components of the product also include: dephosphorylation reaction reagent; the reagent includes: enzyme, and / or reaction buffer.

7. The product according to claim 4, characterized in that: The components of the product also include: single base extension reaction reagents; the reagents include: enzymes, and / or reaction buffers.

8. The product according to claim 4, characterized in that: The components of the product also include: a negative control, and / or a positive control.

9. A MALDI-TOF mass spectrometry method for detecting African horse sickness virus and West Nile virus, characterized in that: The detection method is non-disease treatment and / or diagnosis, and comprises the following steps: (S1) using SEQ ID NO: 1-8 as amplification primers, performing RT-PCR multiple amplification reaction on the nucleic acid sample to be tested to obtain amplification products; (S2) dephosphorylating the amplified product using alkaline phosphatase to obtain a dephosphorylated product; (S3) using SEQ ID NO: 9-12 as a single base extension primer, performing single base extension on the dephosphorylated product to obtain an extension product; (S4) purifying the extension product; (S5) MALDI-TOF mass spectrometry is used to detect the molecular weight of the purified product obtained in step (S4), and African horse sickness virus and West Nile virus are identified based on the difference in molecular weight.

10. The detection method according to claim 9, characterized in that: include: The reaction procedure of RT-PCR multiplex amplification in step (S1) is as follows: The dephosphorylation reaction procedure in step (S2) includes: incubating at 37°C for 40 minutes; then inactivating the dephosphatase at 85°C for 5 minutes; and finally storing at 4°C. The reaction procedure of the single base extension in step (S3) is as follows:

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