African horse sickness virus and west nile virus detection primer and nucleic acid mass spectrometry detection method

Abstract

The present application relates to the technical field of biological detection, and in particular to a detection primer and nucleic acid mass spectrometry detection method for African horse sickness virus and West Nile virus. Based on the MALDI-TOF nucleic acid mass spectrometry detection method, the present application selects two relatively conservative genes, VP7 and NS2, as target genes for African horse sickness virus; and for West Nile virus, two equally conservative genes, NS2A and NS5, are selected. Based on these target genes, the present application designs four pairs of amplification primers and four extension primers. Through optimization of the selection of amplification sites and addition of modified bases, the molecular weight of the extension probe is effectively distinguished within the detection range, thereby achieving the goal of multiplex detection, and at the same time ensuring high accuracy, specificity and sensitivity of the detection. The present application 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.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a detection primer for African horse sickness virus and West Nile virus and a nucleic acid mass spectrometry detection method. BACKGROUND

[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 and Aedes, but it is not a zoonosis and cannot be transmitted to humans through mosquitoes. West Nile virus can be transmitted to humans and other animals, including horses, birds and other mammals, through the bites of mosquitoes.

[0003] African horse sickness virus is a double-stranded RNA virus, which belongs to the orbivirus genus of the Reoviridae family together with bluetongue disease, and is mainly prevalent in Africa. In recent years, it has spread to Asia and other regions. African horse sickness mainly infects equine animals, and the mortality rate after infection is as high as 95%, which often leads to internal bleeding, pulmonary edema and other symptoms.

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

[0005] At present, with the increasing frequency of international exchanges, the risk of infection with African horse sickness virus and West Nile virus in China is gradually increasing. And equine animals are relatively high-value economic animals, and once infected, it will bring great economic losses, so there is an urgent need for a highly efficient and accurate detection method to carry out quarantine work on imported horses.

[0006] Virus isolation is the most classical method for identifying and detecting pathogens, but this method is time-consuming and labor-intensive, and requires high bio-safety conditions. At present, PCR technology is mainly used to detect and identify equine diseases. PCR technology is a highly sensitive molecular biology technique 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 of pathogen detection. Taking GB / T 21675-2022 “African Horse Sickness Diagnostic Techniques” 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 universal or typing identification is performed, the reaction system needs to be prepared separately, and multiple RT-PCR experiments need to be repeated for a sample to achieve differential diagnosis of pathogens. If it is expected to perform differential diagnosis on multiple target genes of two pathogens, more experimental operations need to be performed, which limits its universality in practical application. And because RNA viruses have a high mutation rate, a double-target identification strategy for a single pathogen can significantly improve the accuracy and reliability of detection and effectively avoid missed detection. Therefore, developing a nucleic acid mass spectrometry-based detection kit for the identification of foreign equine diseases and achieving rapid and accurate detection is an important problem faced by current technology. SUMMARY

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

[0008] The technical solution of the present application is as follows:

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

[0010] a specific primer sequence SEQ ID NO. 1 and SEQ ID NO. 2 for amplifying the NS2 segment of the African horse sickness virus,

[0011] a specific primer sequence SEQ ID NO. 3 and SEQ ID NO. 4 for amplifying the VP7 segment of the African horse sickness virus,

[0012] Specific primer sequence SEQ ID NO. 5 and SEQ ID NO. 6 for amplifying NS2A segment of West Nile virus,

[0013] Specific primer sequence SEQ ID NO. 7 and SEQ ID NO. 8 for amplifying NS5 segment of West Nile virus,

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

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

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

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

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

[0019] The present application 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 the four target genes are downloaded and collected from NCBI, and after sequence alignment and phylogenetic tree analysis, the conserved regions on each conserved gene are confirmed.

[0020] The primer is from the conserved sequence of the target fragment, and optionally a tag sequence is added as needed, so that the molecular weight of the RT-PCR multiplex amplification primer can be easily distinguished by MALDI-TOF MS. Therefore, the length of the primer is adjusted to avoid its molecular weight falling within the conventional detection range (4000-10000 Da) 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 single base extension primer is designed in the conserved sequence region of the amplification region, and a designed and determined base is allowed to be extended at the 3' end of the single base extension primer, as a specific sequence marker of different targets; thereby realizing precise detection of multiple targets of African horse sickness virus and West Nile virus.

[0021] As Figure 1As shown, taking the selection of the conserved region of WNV-NS2A gene as an example, it can be obviously seen that the sequence is more conserved in the range of 30-145 bp at the 5' end relative to the whole gene fragment. Since the design of the extension primer does not allow the existence of degenerate bases, we select the most conserved 60-80 bp region as the binding region of the extension primer. When designing the extension primer, some basic primer design principles also need to be followed, for example, it is necessary to avoid the occurrence of adverse conditions such as the formation of dimers between primers or primers themselves.

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

[0023] Table 1

[0024]

[0025] In the sequence of the extension product, the extension base part that may exist is marked with a shadow. In the interpretation rule, A / T and G / C represent different types of extension bases. In the sequence of Table 1, the lowercase letters represent artificially added bases that are not complementary to the template DNA. These additional bases aim to optimize the molecular weight difference between each extension primer and the extension product, so as to more accurately distinguish them according to the detection accuracy of MALDI-TOF MS (requiring the peak spacing between each primer and the extension product to be not less than 16 Da).

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

[0027] Further, in some preferred embodiments, the product comprises a unique reagent, a chip or a kit.

[0028] In a third aspect, the present application provides a product, the components of which comprise: the primer composition SEQ ID NO 1-12; the product comprises a unique reagent, a chip or a kit; and the product is used for detecting African horse sickness virus, and / or, West Nile virus.

[0029] Further, in some preferred embodiments, the components of the product further comprise: reaction reagents for RT-PCR multiplex amplification; the reagents comprise: enzymes, and / or, reaction Buffer. In some embodiments, the enzymes comprise at least one of reverse transcriptase and Taq enzyme. The reaction Buffer comprises: Mg 2+ , dNTPs and H2O in combination.

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

[0031] Further, in some preferred embodiments, the components of the product further comprise: a single base extension reagent; the reagent comprises: an enzyme, and / or, a reaction buffer. In some embodiments, the reaction buffer comprises: a combination of one or more of Mg 2+ , dNTPs, and H2O.

[0032] Further, in some preferred embodiments, the components of the product further comprise: a negative control, and / or, a positive control.

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

[0034] (S1) performing RT-PCR multiplex amplification reaction on the nucleic acid sample to be tested by taking SEQ ID NO: 1-8 as amplification primers, to obtain an amplification product;

[0035] (S2) performing dephosphorylation treatment on the amplification product by using alkaline phosphatase, to obtain a dephosphorylated product;

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

[0037] (S4) purifying the extension product;

[0038] (S5) performing MALDI-TOF mass spectrometry detection on the molecular weight of the purified product obtained in step (S4), and identifying African horse sickness virus and West Nile virus according to 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] Further, in some preferred embodiments, the detection method comprises:

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

[0043]

[0044] The reaction procedure of the dephosphorylation in step (S2) comprises: 37℃ incubation for 40 min; then 85℃, 5 min to deactivate the phosphatase; and finally 4℃ low-temperature preservation;

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

[0046]

[0047] In some preferred embodiments, in the step S(5), the result is judged according to whether the known single-base extension primer is extended and specific extension peaks and bases appear. There are two peak positions of the single-base extension primer and the single-base extension product in the mass spectrum analysis result of each target, 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 the target and the signal-to-noise ratio (SNR) > 6, it is judged that the target is positive: otherwise, it is judged to be negative. The judgment rule is shown in Table 1 above.

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

[0049] Based on the nucleic acid mass spectrum detection method, the present application selects two relatively conservative genes VP7 and NS2 as target genes for African horse sickness virus (AHSV), and selects two also conservative genes NS2A and NS5 for West Nile virus (WNV). Based on these target genes, the present application designs four pairs of amplification primers and four extension primers. Through optimization of the selection of amplification sites and addition of modified bases, the molecular weight of the extension probe is effectively distinguished within the detection range, so as to realize the goal of multiplex detection, and at the same time ensure the high accuracy, specificity and sensitivity of the detection. Through the above design, in one experiment, only the peak graph of the extension product needs to be observed, and whether the corresponding pathogen infection exists can be reported.

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

[0051] The application fully considers the evolution characteristics of the RNA virus genome in the construction of the detection system. By selecting the double-conserved genes for differential diagnosis, the stability and reliability of the detection results are ensured. Meanwhile, the MALDI-TOF technology is adopted to get rid of the limitation of the fluorescence channel, and the flexibility and accuracy of the detection are improved. Compared with the low-throughput fluorescent quantitative PCR, the reagent cost of the application is equivalent, but the primer system used does not need to be modified by a fluorescent group, and only simple PAGE purification can meet the experimental and instrument requirements, so that the detection cost is significantly reduced. Therefore, the application can not only efficiently, sensitively and accurately identify the infection conditions of the African horse sickness virus and the West Nile virus, but also can be flexibly adjusted and optimized according to the actual needs, and is expected to play an important role in the future virus detection and differential diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0052] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0053] Figure 1 Schematic diagram for selecting the conserved region of the WNV-NS2A gene;

[0054] Figure 2 Mass spectrum detection result of the AHSV-NS2 target gene site in the AHSV nucleic acid sample;

[0055] Figure 3 Mass spectrum detection result of the AHSV-VP7 target gene site in the AHSV nucleic acid sample;

[0056] Figure 4 Mass spectrum detection result of the WNV-NS2A target gene site in the WNV nucleic acid sample;

[0057] Figure 5 Mass spectrum detection result of the WNV-NS5 target gene site in the WNV nucleic acid sample;

[0058] Figure 6 Mass spectrum detection result of the mixed nucleic acid sample of mixed infection of the two viruses (AHSV+WNV);

[0059] Figure 7 Mass spectrum detection result of the blue tongue virus BTV nucleic acid;

[0060] Figure 8 Detection result of the AHSV-NS2 gene site when the nucleic acid load is 10 copies / mL;

[0061] Figure 9 Detection results of AHSV-VP7 gene site when the nucleic acid load was 10 copies / mL;

[0062] Figure 10 Detection results of WNV-NS2A gene site when the nucleic acid load was 10 copies / mL;

[0063] Figure 11 Detection results of WNV-NS5 gene site when the nucleic acid load was 10 copies / mL. DETAILED DESCRIPTION

[0064] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work under the premise that the present application falls within the scope of protection. The specific conditions are not specified in the embodiments, which are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used are not specified by the manufacturer, which are all conventional products that can be purchased in the market.

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

[0066] The obtaining routes of various biological materials described in the embodiments are only provided to achieve the specific disclosed purposes, and should not be regarded as a limitation on the sources of biological materials of the present application. In fact, the sources of biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the embodiments.

[0067] The terms “comprising” and “having” and any variations thereof in the present invention are intended to cover a non-exclusive inclusion. For example, a process, method, device, product, or equipment that includes a series of steps is not limited to the listed steps or modules, but can optionally also include steps not listed, or can optionally also include other steps inherent to these processes, methods, products, or equipment. “Multiple” mentioned in the present invention refers to two or more. “And / or”, which describes the relationship between the associated objects, means that there can be three relationships, for example, A and / or B can represent the following three cases: A exists alone, A and B exist together, and B exists alone. The character “ / ” generally represents an “or” relationship between the associated objects before and after it.

[0068] Application of the detection method, primer composition and kit provided by the present invention in detecting African horse sickness virus and West Nile virus

[0069] 1. Selection of African horse sickness virus (AHSV) target:

[0070] The VP7 gene is one of the core structural protein genes of 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 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 pathogenesis of African horse sickness virus. Although its sequence may have certain variations compared to the structural protein genes, there is still a certain degree of conservation among different virus strains. Due to the functional importance of NS2 protein, using it as a target gene for detection helps to further understand the replication mechanism and pathogenesis of the virus. At the same time, the detection method based on the NS2 gene can also be used to monitor the variation of the virus.

[0072] 2. Selection of West Nile virus (WNV) target:

[0073] The non-structural protein encoded by the NS2A gene has multiple functions, including participating in virus replication, virion assembly, inducing host cell death, etc. Although there is a certain sequence variation in the NS2A gene, there are still certain conserved regions among different virus strains. These conserved regions can be used 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 as an RNA polymerase in the replication process of the virus. Due to the high conservation of NS5 protein and its importance in virus replication, the detection method based on the NS5 gene generally has high sensitivity and specificity.

[0075] Therefore, in combination with a large number of literature research reports, four target genes AHSV-VP7, AHSV-NS2, WNV-NS2A and WNV-NS5 of African horse sickness virus (AHSV) and West Nile virus (WNV) are finally selected for primer design. Then the sequences of the 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. The primers are derived from the conserved sequences of the target fragments, and optionally tag sequences are added as needed, so that the size of the RT-PCR multiplex amplification product can be easily distinguished by MALDI-TOF MS, and all are within the mass spectrometry detection range (4000-10000 Da).

[0076] Meanwhile, a single base extension primer is designed in the conserved sequence region in the amplification region, and a designed and determined base is allowed to be extended at the 3' end of the single base extension primer as a specific sequence marker of different targets.

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

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

[0079] II. Sample nucleic acid extraction

[0080] In order to simulate the reverse transcription process in RT-PCR, each target gene is entrusted to Zhenjiang Aibimeng Biotechnology Co., Ltd. to synthesize lentivirus reference, using the vector Lentiviral Vector (CMV) (pLenti-GIII-CMV-CBH-GFP-2A-Puro) (item number DNAFor LV002-b) and using the virus DNA / RNA extraction kit 4.0 of Xi'an Tianlong Technology Co., Ltd. to extract nucleic acid with a full-automatic nucleic acid extractor. After extraction, 60μL of nucleic acid eluent in the deep well plate is transferred to a clean EP tube and labeled.

[0081] III. Detection steps

[0082] 1. RT-PCR multiplex amplification reaction

[0083] The RT-PCR multiplex amplification reaction was carried out on the test sample using amplification primers SEQ ID NO: 1-8, 4 target nucleic acids as positive control, and ultrapure water as negative control. The RT-PCR multiplex amplification was carried out to obtain the amplification product, and the reaction system is shown in Table 2:

[0084] Table 2

[0085] Reagent Manufacturer Item / Size Amount RT-PCR reaction mixture Zhejiang Dips Diagnostics Technology Co., Ltd. DPGPM-V 2.5 μL RT-PCR enzyme solution Zhejiang Dips Diagnostics Technology Co., Ltd. DPGPM-V 0.3 μL Multiplex amplification primer mixture - 5 μM of each 0.2 μL

[0086] Among them, the multiplex amplification primer mixture is amplification primer 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] According to the actual test results of the RT-PCR multiplex amplification reaction, the effect of the 2-step cycle method can meet the amplification requirements and can effectively amplify the target fragment.

[0091] 2, dephosphorylation reaction

[0092] In order to eliminate the excess dNTPs after the multiplex PCR amplification reaction and ensure the accuracy of the single base extension 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] Reagent Ultra-pure water SAP reaction buffer SAP enzyme mixture Multiplex PCR product Amount 1.53 μL 0.17 μL 0.3 μL 5 μL

[0095] Among them, the SAP enzyme and the reaction buffer were purchased from Zhejiang Disu Diagnostics Technology Co., Ltd.

[0096] The reaction program was set as 37°C incubation for 40 min, and the SAP was removed to remove the remaining dNTPs; then the SAP enzyme was inactivated at 85°C for 5 min, and after completion, it was stored at 4°C.

[0097] 3, single base extension reaction

[0098] Each component was added in turn according to the reaction system sequence shown in Table 5, and the prepared reaction system was subjected to single base extension according to the reaction program shown in Table 6.

[0099] Table 5

[0100]

[0101] The extension primer mixture contained amplification primers SEQ ID NOs: 9-12, and the extension enzyme and reaction buffer were purchased from Zhejiang Dipu Diagnostics Technology Co., Ltd. The concentration of each primer was 10 μM.

[0102] Table 6

[0103]

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

[0105] 18 μL of DEPC water was pre-added to each well of a 384-well reaction plate. The single-base extension product (the actual volume may be slightly less than the theoretical 9 μL due to losses during the reaction) was added and distributed to each well containing DEPC water. After mixing, the product was sealed with a film sealant and centrifuged to evenly distribute the mixture across the bottom of the wells. Ensure that the assay file accurately contains the molecular weight information for all single-base extension primers and their extension products and import it into the operating software of a DP-TOF time-of-flight nucleic acid mass spectrometer (Zhejiang Dipu Diagnostics Technology Co., Ltd.). The nucleic acid time-of-flight mass spectrometer deck was opened, the 384-well plate sealant was removed, and the plate was placed in the designated position. 10 μL of resin was added to each well. The instrument automatically performed the product desalting and purification steps, followed by automatic sample placement and mass spectrometry detection.

[0106] The results are determined based on whether a known single-base extension primer undergoes extension and the appearance of a specific extension peak and base. Mass spectrometry analysis results for each target show 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 corresponding to any extension product of a target and the signal-to-noise ratio (SNR) is greater than 6, the target is considered positive; otherwise, it is considered negative. The interpretation rules are shown in Table 1 above.

[0107] 4. Test results:

[0108] 1. The mass spectrometry detection 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 (6173.0 Da) disappears and the corresponding T base extension product peak (6500.2 Da) appears.

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

[0110] 3. Mass spectrum detection results of mixed nucleic acid samples simulating mixed infection of two viruses (AHSV+WNV) are shown in Table 3. Figure 6

[0111] The above results show that the extension product (the extension base corresponding to the detected peak value) of each sample and the molecular weight (Da) value of the product are as shown in the following table, which are consistent with the original sample information:

[0112]

[0113] Example 2 Verification of specificity of the detection method, primer combination and kit provided by the present application

[0114] The information of the samples to be tested used in this example is as follows:

[0115]

[0116]

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

[0118] The detection results show that the Mass value statistical information of the extension product of each sample is as shown in the following table, which is consistent with the original sample information (for example Figure 7 Mass spectrum detection results of blue tongue disease virus BTV nucleic acid, each site is negative):

[0119]

[0120] It can be seen that for each pathogen sample not within the detection range, no extension product peak is observed at the AHSV-NS2, AHSV-VP7, WNV-NS2A and WNV-NS5 sites, which fully indicates that the detection method, primer combination and kit provided by the present application all have good specificity.

[0121] Example 3 Verification of sensitivity of the detection method, primer combination and kit provided by the present application

[0122] ​In this embodiment, qPCR was used to quantify the copy number of the 4-gene pseudovirus nucleic acid samples (AHSV-NS2 / AHSV-VP7 / WNV-NS2A and WNV-NS5, respectively). Each positive nucleic acid was initially quantified at 1 x 10 5 copies / mL, which was then diluted by 10-fold, with the dilution load ranging from 1 x 10 5 copies / mL to 1 copy / mL, for a total of 6 dilutions. Each gradient was tested in triplicate according to the methods, procedures, etc. described in Embodiment 1.

[0123] The detection results of the AHSV-NS2 gene site at a nucleic acid load of 10 copies / mL are shown in Table 1. Figure 8

[0124] The detection results of the AHSV-VP7 gene site at a nucleic acid load of 10 copies / mL are shown in Table 2. Figure 9

[0125] The detection results of the WNV-NS2A gene site at a nucleic acid load of 10 copies / mL are shown in Table 3. Figure 10

[0126] The detection results of the WNV-NS5 gene site at a nucleic acid load of 10 copies / mL are shown in Table 4. Figure 11

[0127] The detection results show that when the 4-gene pseudovirus nucleic acid samples are diluted to 10 copies / mL, the extension primers for the AHSV-VP7 and WNV-NS2A genes are not completely consumed, indicating that the detection method has reached the critical detection limit. At this time, the extension primers for the AHSV-NS2 and WNV-NS5 genes are completely consumed, suggesting that the detection sensitivity of the AHSV-NS2 and WNV-NS5 genes is slightly higher. However, when the nucleic acid is diluted to 1 copy / mL, no extension product is detected in each well of the 4 genes. Therefore, the detection method, primer combination, and kit provided by the present application have a minimum detection limit of between 1 copy / mL and 10 copies / mL.

[0128] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​​​​

Claims

1. Use of a primer combination in preparing a product for detecting African horse sickness virus and West Nile virus, characterized in that: The primer composition comprises: Specific primer sequences for amplifying the NS2 segment of African horse sickness virus are SEQ ID NO.1 and SEQ ID NO.2, Specific primer sequences for amplifying the VP7 segment of African horse sickness virus are SEQ ID NO.3 and SEQ ID NO.4, Specific primer sequences for amplifying the NS2A fragment of West Nile virus are SEQ ID NO.5 and SEQ ID NO.6, 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 segment of African horse sickness virus, Single base extension primer SEQ ID NO.10 for the VP7 segment 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; The products include independent reagents, chips or kits; The components of the product include: the primer composition, RT-PCR multiple amplification reaction reagents, dephosphorylation reaction reagents, single base extension reaction reagents, negative control and positive control; the reagents include: enzymes and / or reaction buffers.

2. A MALDI-TOF mass spectrometry method for detecting African horse sickness virus and West Nile virus, characterized in that: The detection method is for non-disease treatment and / or diagnosis, and comprises the following steps: (S1) performing RT-PCR multiplex amplification reaction on the nucleic acid sample to be tested using SEQ ID NOs: 1-8 as amplification primers to obtain amplified products; (S2) dephosphorylating the amplified product using alkaline phosphatase to obtain a dephosphorylated product; (S3) using SEQ ID NOs: 9-12 as single-base extension primers, 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.

3. The detection method according to claim 2, 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: incubation at 37°C for 40 minutes; then inactivation of the dephosphatase at 85°C for 5 minutes; and finally storage at 4°C. The reaction procedure of the single base extension in step (S3) is as follows:

Images