Kit for simultaneously detecting bluetongue virus, foot and mouth disease virus, schmallenberg virus, peste des petits ruminants virus and sheep pox virus

Abstract

The invention discloses a kit for simultaneously detecting bluetongue virus, foot and mouth disease virus, schmallenberg virus, peste des petits ruminants virus and sheep pox virus, and belongs to the technical field of virus detection. The kit comprises primers as shown in SEQ ID NO. 1 to SEQ ID NO. 18 and a probe as shown in SEQ ID NO. 18. After a single system of bluetongue virus, foot-and-mouth disease virus, schmallenberg virus, peste des petits ruminants virus and capripox virus is tested, a sextuple system containing an interior label is established, and through testing of indexes such as linearity, minimum detection limit, specificity, precision and repeatability, NTC repetition, comparison of multiple fluorescent RT-PCR and single fluorescent RT-PCR, the kit can be used for detecting the virus resistance of the peste des petits ruminants virus and the capripox virus. The currently developed fluorescent RT-PCR multiple detection kit for the sheep respiratory tract syndromes completely meets the development requirement, the specificity is high, the sensitivity is high, and the lowest detection limit can reach 10 Copi / mu L or below.

Description

Technical Field

[0001] The invention relates to the technical field of virus detection, in particular to a kit for simultaneously detecting bluetongue virus, foot-and-mouth disease virus, Schmallenberg virus, peste des petits ruminants virus and capripox virus. Background Art

[0002] Ovine Respiratory Disease Complex (ORDC) is one of the important diseases affecting the sheep and goat farming industry. Its etiology is complex and is usually caused by mixed or secondary infection of multiple pathogens, including but not limited to mycoplasma (such as Mycoplasma ovipneumoniae ), bacteria (such as Mannheimia haemolytica , Pasteurella multocida ), viruses (such as parainfluenza virus type 3, respiratory syncytial virus) and parasites. The disease is characterized by respiratory inflammation, high fever, dyspnea and high mortality, which seriously threatens animal health and causes huge economic losses to the breeding industry. Due to the diversity of pathogens, the high incidence of mixed infections and the difficulty of differential diagnosis, accurate testing is often not possible, and rapid clinical differential diagnosis faces great challenges.

[0003] Traditional detection methods (such as pathogen isolation and culture, serological antibody testing or single PCR technology) have significant limitations: ① Pathogen isolation and culture is time-consuming and labor-intensive, and has low sensitivity for pathogens that have demanding nutritional conditions or are difficult to proliferate in vitro (such as some mycoplasmas); ② Serological testing cannot distinguish between current infection and previous immune response, and there is a risk of cross-reaction; ③ Conventional single-plex PCR requires multiple independent tests to cover different pathogens, resulting in large sample consumption, long testing cycle, and high cost, which is difficult to meet the needs of clinical rapid screening.

[0004] In recent years, Multiplex Real-time PCR technology has been applied in human medicine and some animal disease diagnosis due to its advantages of high throughput, high sensitivity and simultaneous detection of multiple targets. Therefore, the development of a multiplex fluorescence PCR technology that can simultaneously detect the main pathogens of ORD and has both high sensitivity and specificity is of great value in achieving early accurate diagnosis, guiding targeted treatment and formulating regional prevention and control strategies. Summary of the invention

[0005] The purpose of the present invention is to provide a kit for simultaneously detecting bluetongue virus, foot-and-mouth disease virus, Schmallenberg virus, peste des petits ruminants virus and capripox virus, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions: One of the technical solutions of the present invention is a kit for simultaneously detecting bluetongue virus, foot-and-mouth disease virus, Schmallenberg virus, peste des petits ruminants virus and capripox virus, the kit comprising primers and probes as shown in SEQ ID NO.1 to SEQ ID NO.18.

[0007] The second technical solution of the present invention is a method for detecting bluetongue virus, foot-and-mouth disease virus, Schmallenberg virus, peste des petits ruminants virus and capripox virus for non-disease diagnosis or treatment purposes, comprising the following steps: (1) Using the extracted nucleic acid of the sample to be tested as a template, using the primers shown in SEQ ID NO.1~2, SEQ ID NO.4~5, SEQ ID NO.7~8, SEQ ID NO.10~11 and SEQ ID NO.13~14 to amplify the sample to be tested, and using the probes shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 and SEQ ID NO.15 to identify the amplified product; As shown in SEQ ID NO.3, the 5' end of the probe is provided with a CY5.5 fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; as shown in SEQ ID NO.6, the 5' end of the probe is provided with a FAM fluorescent label, and the 3' end is provided with a BHQ1 fluorescent label; as shown in SEQ ID NO.9, the 5' end of the probe is provided with a ROX fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; as shown in SEQ ID NO.12, the 5' end of the probe is provided with a CY5 fluorescent label, and the 3' end is provided with an MGB fluorescent label; as shown in SEQ ID NO.15, the 5' end of the probe is provided with a TAMRA fluorescent label, and the 3' end is provided with an MGB fluorescent label; (2) Simultaneously setting IPC internal standards including primers and probes as shown in SEQ ID NO.16 to SEQ ID NO.18, wherein the 5' end of the probe as shown in SEQ ID NO.18 is marked with HEX fluorescent marker, and the 3' end is marked with BHQ1 fluorescent marker; performing fluorescence quantitative detection, collecting the signal of the fluorescence channel; and adding the concentrations of 1.0×10 5 Copies / μL of the recombinant plasmid mixture of BTV, FMDV, SBV, PPRV, QrfV, and IPC internal standards were used as positive controls, and ultrapure water was used as negative controls; (3) Result determination: The negative control should have no Ct value and no specific amplification curve; the Ct value of all channels of the positive control should be ≤30 and a specific amplification curve should appear, and the test result is determined to be valid; If the negative control and positive control results do not meet the above conditions, the experiment will be considered invalid; On the premise that the test results are established, if the sample test result Ct value ≤ 30 and a specific amplification curve appears, it is judged as positive; if the sample 30<Ct value<37 and a specific amplification curve appears, it is judged as suspicious, and the suspicious sample must be retested and the result is judged after amplification. If the result is positive, it is judged as positive, and if the result is negative, it is judged as negative. If it is still suspicious, it can be judged as positive; if the sample Ct value is ≥37, it exceeds the detection sensitivity range of this method and is judged as negative; for some samples that do not present a specific amplification curve but have a high background, they should be judged as negative.

[0008] For the same sample, the following judgments were made: those with a positive result in the FAM channel were judged to be infected with the pathogen of foot-and-mouth disease, those with a positive result in the ROX channel were judged to be infected with the pathogen of Schmallenberg, those with a positive result in the CY5 channel were judged to be infected with the pathogen of peste des petits ruminants, those with a positive result in the TAMRA channel were judged to be infected with the pathogen of sheeppox, and those with a positive result in the CY5.5 channel were judged to be infected with the pathogen of bluetongue; if multiple channels were positive, it was judged that multiple pathogens corresponding to the multiple channels were mixedly infected.

[0009] Based on the above technical solution, the present invention has the following technical effects: After testing the single-plex system of bluetongue virus (BTV), foot-and-mouth disease virus (FMDV), Schmallenberg virus (SBV), peste des petits ruminants virus (PPRV), and sheep pox virus (QrfV), the present invention established a six-plex system including an internal standard. After testing indicators such as linearity, minimum detection limit, specificity, precision and repeatability, NTC repeat, comparison of multiple fluorescent RT-PCR and single fluorescent RT-PCR, the sheep respiratory syndrome fluorescent RT-PCR multiple detection kit of the present invention fully meets the development requirements, has strong specificity and high sensitivity, and the minimum detection limit can reach less than 10 Copies / μL. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 paying creative work.

[0011] Figure 1 It is a single fluorescence RT-PCR standard curve. Among them, A is the BTV-NS3 gene target linearity, B is the PPRV-M gene target linearity, C is the FMDV-3D gene target linearity, D is the QrfV-GPCR gene target linearity, and E is the SBV-N gene target linearity.

[0012] Figure 2This is the standard curve of multiplex system fluorescence RT-PCR.

[0013] Figure 3 The digital PCR value droplet diagram of the relevant target of the kit. Among them, A is the BTV-NS3 plasmid value result, B is the PPRV-M plasmid value result, C is the FMDV-3D plasmid value result, D is the QrfV-GPCR plasmid value result, and E is the SBV-N plasmid value result.

[0014] Figure 4 Comparison between multiplex fluorescence RT-PCR and each single fluorescence RT-PCR. A is the comparison of linear results between multiplex fluorescence RT-PCR and single fluorescence RT-PCR for bluetongue pathogen, B is the comparison of linear results between multiplex fluorescence RT-PCR and single fluorescence RT-PCR for peste des petits ruminants pathogen, C is the comparison of linear results between multiplex fluorescence RT-PCR and single fluorescence RT-PCR for foot-and-mouth disease pathogen, D is the comparison of linear results between multiplex fluorescence RT-PCR and single fluorescence RT-PCR for sheeppox pathogen, and E is the comparison of linear results between multiplex fluorescence RT-PCR and single fluorescence RT-PCR for Schmallenberg pathogen. Correlation coefficient R between single fluorescence RT-PCR and multiplex fluorescence RT-PCR 2 They were all greater than 0.99, indicating that the multiplex fluorescence RT-PCR and each single fluorescence RT-PCR were in good agreement, the multiplex fluorescence RT-PCR did not interfere with each other, and the amplification efficiency was not affected. DETAILED DESCRIPTION

[0015] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0016] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0017] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0018] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0019] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0020] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0021] An embodiment of the present invention provides a kit for simultaneously detecting bluetongue virus, foot-and-mouth disease virus, Schmallenberg virus, peste des petits ruminants virus and capripox virus, wherein the kit comprises primers and probes as shown in SEQ ID NO.1 to SEQ ID NO.18.

[0022] The embodiment of the present invention also provides a method for detecting bluetongue virus, foot-and-mouth disease virus, Schmallenberg virus, peste des petits ruminants virus and capripox virus for non-disease diagnosis or treatment purposes, comprising the following steps: (1) Using the extracted nucleic acid of the sample to be tested as a template, using the primers shown in SEQ ID NO.1~2, SEQ ID NO.4~5, SEQ ID NO.7~8, SEQ ID NO.10~11 and SEQ ID NO.13~14 to amplify the sample to be tested, and using the probes shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 and SEQ ID NO.15 to identify the amplified product; As shown in SEQ ID NO.3, the 5' end of the probe is provided with a CY5.5 fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; as shown in SEQ ID NO.6, the 5' end of the probe is provided with a FAM fluorescent label, and the 3' end is provided with a BHQ1 fluorescent label; as shown in SEQ ID NO.9, the 5' end of the probe is provided with a ROX fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; as shown in SEQ ID NO.12, the 5' end of the probe is provided with a CY5 fluorescent label, and the 3' end is provided with an MGB fluorescent label; as shown in SEQ ID NO.15, the 5' end of the probe is provided with a TAMRA fluorescent label, and the 3' end is provided with an MGB fluorescent label; (2) Simultaneously setting IPC internal standards including primers and probes as shown in SEQ ID NO.16 to SEQ ID NO.18, wherein the 5' end of the probe as shown in SEQ ID NO.18 is marked with HEX fluorescent marker, and the 3' end is marked with BHQ1 fluorescent marker; performing fluorescence quantitative detection, collecting the signal of the fluorescence channel; and adding the concentrations of 1.0×10 5 Copies / μL of the recombinant plasmid mixture of BTV, FMDV, SBV, PPRV, QrfV, and IPC internal standards were used as positive controls, and ultrapure water was used as negative controls; (3) Result determination: The negative control should have no Ct value and no specific amplification curve; the Ct value of all channels of the positive control should be ≤30 and a specific amplification curve should appear, and the test result is determined to be valid; If the negative control and positive control results do not meet the above conditions, the experiment will be considered invalid; On the premise that the test results are established, if the sample test result Ct value ≤ 30 and a specific amplification curve appears, it is judged as positive; if the sample 30<Ct value<37 and a specific amplification curve appears, it is judged as suspicious, and the suspicious sample must be retested and the result is judged after amplification. If the result is positive, it is judged as positive, and if the result is negative, it is judged as negative. If it is still suspicious, it can be judged as positive; if the sample Ct value is ≥37, it exceeds the detection sensitivity range of this method and is judged as negative; for some samples that do not present a specific amplification curve but have a high background, they should be judged as negative.

[0023] For the same sample, the following judgments were made: those with a positive result in the FAM channel were judged to be infected with the pathogen of foot-and-mouth disease, those with a positive result in the ROX channel were judged to be infected with the pathogen of Schmallenberg, those with a positive result in the CY5 channel were judged to be infected with the pathogen of peste des petits ruminants, those with a positive result in the TAMRA channel were judged to be infected with the pathogen of sheeppox, and those with a positive result in the CY5.5 channel were judged to be infected with the pathogen of bluetongue; if multiple channels were positive, it was judged that multiple pathogens corresponding to the multiple channels were mixedly infected.

[0024] In some specific embodiments, the reaction system of the amplification reaction includes: CoverAll Probe qRT-PCR Mix II (5×) 5 μL, Primer Mix 1 μL, Probe Mix 1 μL, Sample nucleic acid template 5 μL, dd H2O to 25 μL.

[0025] In some specific embodiments, the reaction conditions of the amplification reaction are: pre-denaturation at 95°C for 20 s, reverse transcription at 42°C for 30 min; denaturation at 95°C for 10 s, annealing and extension at 60°C for 20 s, 40 cycles, and collecting fluorescence signals of six channels, namely FAM, HEX, ROX, TAMRA, CY5, and CY5.5, at the end of each cycle. Example

[0026] The complete genome sequence or NS3 gene sequence of bluetongue virus was retrieved from the NCBI nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov) (GenBank numbers: OR917952.1, MT879210.1, OK018219.1, KY091906.1, MG255528.1, MG255668.1, JX889208.1, KP268823.1, MK893226.1, KY091901.1, FJ713323.1, AF044711.1, AF04478.1, AF044717.1). 4712.1, AF044374.1, AF044375.1, AY426597.1, etc.); the whole genome sequence or 3D gene sequence of foot-and-mouth disease virus (GenBank numbers: MT495499.1, MH053314.1, MT431607.1, HM854022.1, JN006719.1, MT495495.1, MT495497.1, JF749851.1, EF149010.1, AY593765.1, AY593763.1, X85493.1, etc.); the whole genome sequence or N gene sequence of Schmallenberg virus (GenBank numbers: MT495499.1, MH053314.1, MT431607.1, HM854022.1, JN006719.1, MT495495.1, MT495497.1, JF749851.1, EF149010.1, AY593765.1, AY593763.1, X85493.1, etc.); nk number: NC_043582.1, KT795133.1, KT795145.1, LC309158.1, KC139376.1, KX384876.1, LC309149.1, KC355456.1, KC108874.1, KT795141.1, LC309162.1, LC309151.1, LC309165.1, etc.); the whole genome sequence or M gene sequence of peste des petits ruminants virus (GenBank number: OR419686.1, MT109379.1, OK274205.1, KP789375.1, FJ9 05304.1, NC_006383.2, etc.); the whole genome sequence or GRT-PCR gene sequence of capripoxvirus (GenBank No.: KF661979.1, MW020571.1, MW167070.1, AY077833.1, ON961657.1, ON961655.1, OQ434239.1, MG731222.1, MK607165.1, FJ869349.1, JQ310666.1, FJ869345.1, KF495237.1, FJ869360.1, MK607162.1, KF495250.1, etc.); the IPC internal standard is based on the conservative gene sequence of the 16S rRNA gene in the sheep mitochondrial genome. After comparing the target gene sequences with SnapGene software, the relatively conservative nucleotide sequences of each pathogen were selected. According to the design principles of primers and TaqMan probes, combined with relevant national standards, the relevant primer and probe sequences were designed. The target and sequence of the kit are summarized in Table 1. .

[0027] Table 1 Primers and probes of multiplex fluorescent RT-PCR detection kit for ovine respiratory syndrome .

[0028] 2.1 Establishment of multiplex fluorescence RT-PCR reaction system The primer and probe stock solutions were diluted 10-fold in a gradient manner, and the primers were fully mixed to prepare a primer mixture. The final concentration of each target primer is shown in Table 1, and the configuration of each reaction system is shown in Table 2. The fluorescent RT-PCR reaction was performed according to the procedure shown in Table 3. The equipment used was JLM QX600 of JLM Corporation.

[0029] Table 2 Preparation of fluorescent RT-PCR reaction system .

[0030] Table 3 Fluorescence RT-PCR reaction parameter settings .

[0031] 2.2 Standard curve drawing The positive plasmids of Bluetongue Virus (BTV), Foot-and-mouthdisease virus (FMDV), Schmallenberg virus (SBV), Peste des petits ruminants virus (PPRV) and QrfV were diluted 10 times and then subjected to fluorescent RT-PCR. The concentration standard curve was made using the fluorescent Ct value of the template concentration.

[0032] Then, the positive plasmids of BTV, FMDV, SBV, PPRV and QrfV were mixed in equal amounts and diluted 10-fold in a gradient manner, and multiple fluorescent RT-PCR was performed, and the concentration standard curve was made using the template concentration fluorescent Ct value.

[0033] The concentration-Ct value standard curves were made using BTV, FMDV, SBV, PPRV, and QrfV positive plasmids. Figure 1 ).from Figure 1 and Figure 2It can be seen that no matter it is a single-plex system or a multiplex multi-positive system, the standard curve RT-PCR amplification efficiency E value, R and curve slope are all within the normal range, indicating that the amplification efficiency is good.

[0034] In the present invention, the concentrations are 1.0×10 5 A mixture of BTV, FMDV, SBV, PPRV, QrfV, and IPC internal standard recombinant plasmids at 10 copies / μL was used as a positive control, and ultrapure water was used as a negative control.

[0035] Result determination: The negative control should have no Ct value and no specific amplification curve; the Ct value of all channels of the positive control should be ≤30, and a specific amplification curve should appear, and the test result is determined to be valid; If the negative control and positive control results do not meet the above conditions, the experiment will be considered invalid; On the premise that the test results are established, if the sample test result Ct value ≤ 30 and a specific amplification curve appears, it is judged as positive; if the sample 30<Ct value<37 and a specific amplification curve appears, it is judged as suspicious, and the suspicious sample must be retested and the result is judged after amplification. If the result is positive, it is judged as positive, and if the result is negative, it is judged as negative. If it is still suspicious, it can be judged as positive; if the sample Ct value is ≥37, it exceeds the detection sensitivity range of this method and is judged as negative; for some samples that do not present a specific amplification curve but have a high background, they should be judged as negative.

[0036] For the same sample, the following judgments were made: those with a positive result in the FAM channel were judged to be infected with the pathogen of foot-and-mouth disease, those with a positive result in the ROX channel were judged to be infected with the pathogen of Schmallenberg, those with a positive result in the CY5 channel were judged to be infected with the pathogen of peste des petits ruminants, those with a positive result in the TAMRA channel were judged to be infected with the pathogen of sheeppox, and those with a positive result in the CY5.5 channel were judged to be infected with the pathogen of bluetongue; if multiple channels were positive, it was judged that multiple pathogens corresponding to the multiple channels were mixedly infected.

[0037] 2.3 Determination of the minimum detection limit The theoretical values ​​of BTV, FMDV, SBV, PPRV and QrfV are set to 10 6 The positive plasmid with a copy / μL was linearized and digested with two enzymes, QuickCutSac I and QuickCut Kpn I. The digestion system is shown in Table 4. The digestion was completed by keeping the temperature at 37°C for 15 minutes. 6 After linearization of the positive plasmid at 10 copies / μL, dilute to 10 3Copies / μL were then determined using JLM Digital Matrix-5000 digital RT-PCR from JLM. The determination system is shown in Table 5, and the digital PCR reaction was performed according to the procedure shown in Table 6.

[0038] Sample concentration (Copies / μL) = fixed value result (Copies / μL) × 20 (total reaction system) ÷ 2 (sample volume).

[0039] Table 4 Plasmid linearization restriction enzyme system .

[0040] Table 5 Preparation of digital PCR fixed value reaction system .

[0041] Table 6 Digital PCR reaction parameter settings .

[0042] The fixed plasmid was diluted in series, and then the minimum detection limit of the diluted plasmid was tested using BTV, FMDV, SBV, PPRV, and QrfV single-plex fluorescent RT-PCR. The Ct value that could be detected at the highest dilution multiple was the minimum detection limit.

[0043] Then, the fixed BTV, FMDV, SBV, PPRV, and QrfV positive plasmids were mixed in equal amounts and diluted in series for multiple fluorescent RT-PCR. The concentration corresponding to the Ct value that could be detected at the highest dilution multiple was the minimum detection limit.

[0044] Digital PCR was used to determine the value of each target plasmid of this kit diluted a certain multiple. The microdroplet diagram of the determination result is as follows: Figure 3 The digital PCR determination results of each target and the converted plasmid concentration are shown in Table 7. The plasmid after determination was diluted with TE buffer at a certain dilution multiple for minimum detection limit test. In order to further test the minimum detection limit of the multiplex multi-positive system, one more plasmid intermediate concentration was diluted for testing compared with the single-plex system. The minimum detection limit test results of the single-plex and multiplex multi-positive of this kit are shown in Table 8. The Ct value of the multiplex system is slightly larger than that of the single-plex system, but the minimum detection limits of both the single-plex system and the multiplex system can reach less than 10 Copies / μL.

[0045] Table 7 Statistics of target plasmid determination results of this kit .

[0046] Table 8 The minimum detection limit test results of single-plex and multiplex mixed systems for each target in this kit .

[0047] 2.4 Specificity test A total of eight sheep disease-positive plasmids were selected, including border virus (BDV), Brucella (Bru), toxoplasmosis (Tox), ovine endemic abortion (OEA), caprine arthritis-encephalitis virus (CAEV), bovine lumpy dermatosis (LSDV-ORF101, LSDV-ORF126), and peste des petits ruminants virus (PPRV). Single fluorescent RT-PCR was performed using primers and probes of BTV, FMDV, SBV, PPRV, and QrfV to verify the specificity of primers and probes.

[0048] In order to verify whether the system of this kit would cause non-specific amplification of other sheep disease-related plasmids, the single-plex system of the detection target involved in this kit was used to amplify 10 6 Copies / μL of other sheep disease template plasmids are specifically tested. If suspected nonspecific amplification is found, the plasmid mother liquor is further tested using the dye method. If the suspected nonspecific amplification Tm value is consistent with the target Tm value, it means that the plasmid mother liquor may be contaminated with the target plasmid or sample. If the suspected nonspecific amplification Tm value is inconsistent with the target Tm value, it means that it may be due to nonspecific amplification of primers and other templates.

[0049] The results showed that the SBV-N primer probe system of the kit of the present invention was found to be amplified with the Tox-B1 plasmid. Further testing using the dye method found that the melting temperature Tm value of the Tox-B1 plasmid was consistent with the target plasmid, indicating that there was contamination of the SBV-N plasmid or sample in the Tox-B1 plasmid mother solution. The results are shown in Tables 9 and 10.

[0050] Table 9 Specificity test of each target single-plex system of this kit .

[0051] Table 10 Dye method verification of suspected contamination of each target in this kit .

[0052] 2.5 Precision and repeatability test BTV, FMDV, SBV, PPRV, and QrfV positive plasmids were mixed in equal amounts to a final concentration of 10 5 Copies / μL and 10 3 Copies / μL of two plasmid mixtures were used for multiplex fluorescence RT-PCR test. The test was carried out for 5 consecutive days with 6 replicates each time, and the precision and repeatability of the multiplex system were statistically analyzed.

[0053] This kit multiplex system is for 10 5 Copies / μL and 10 3 Copies / μL mixed plasmid was tested for precision and repeatability for 5 days, and the test results are shown in Table 11 and Table 12. The results show that under the same day, 10 5 Copies / μL and 10 3 Copies / μL, the Ct value range of each target was less than 1, and the coefficient of variation was less than 1%; the Cq values ​​of five days were counted, 10 5 Copies / μL and 10 3 At the two concentrations of copies / μL, the range of Ct values ​​for each target was less than 1.5, and the coefficient of variation was less than 1.5%, both meeting the precision and repeatability standards of coefficient of variation (CV) ≤ 5% required for kit development.

[0054] Table 11 Multiplex system of this kit for 10 5 Copies / μL mixed plasmid precision and repeatability statistics .

[0055] Table 12 Multiplex system of this kit for 10 3 Copies / μL mixed plasmid precision and repeatability statistics .

[0056] 2.6 NTC Repeat Test The prepared multiplex system was subjected to NTC test to evaluate whether there was nonspecific amplification between the primer probes in the system. The test was performed for 5 consecutive days, with 6 NTC repeats each time, and the NTC test results of the multiplex system were statistically analyzed.

[0057] The prepared multiplex system was subjected to NTC testing for multiple days to evaluate whether there was nonspecific amplification between the primer probes in the system. The statistical results are shown in Table 13. The results showed that after repeated NTC testing for multiple days, there was no nonspecific amplification in each channel.

[0058] Table 13 NTC repeated tests on different days in multiple systems of this kit .

[0059] 2.7 Comparison of multiplex fluorescent RT-PCR and single fluorescent RT-PCR Equal amounts of BTV, FMDV, SBV, PPRV, and QrfV positive plasmids were mixed and diluted 10-fold. Multiple system fluorescent RT-PCR and single fluorescent RT-PCR of each target of BTV, FMDV, SBV, PPRV, and QrfV were performed respectively. The fluorescent Ct values ​​corresponding to the same template concentration were compared, the regression equation was solved, and the consistency of the multiple fluorescent RT-PCR and each single fluorescent RT-PCR was compared.

[0060] The results of the consistency test of multiple fluorescent RT-PCR and single fluorescent RT-PCR using the same template show the correlation coefficient between single fluorescent RT-PCR and multiple fluorescent RT-PCR for BTV, FMDV, SBV, PPRV, and QrfV R 2 The results are 0.9994, 0.9991, 0.9996, 0.9989 and 0.9996 respectively, indicating that the multiplex fluorescence RT-PCR and the single fluorescence RT-PCR are in good agreement, the multiplex fluorescence RT-PCR does not interfere with each other, and the amplification efficiency is not affected. Figure 4 .

[0061] After testing the single-plex system of bluetongue virus (BTV), foot-and-mouth disease virus (FMDV), Schmallenberg virus (SBV), peste des petits ruminants virus (PPRV), and sheep pox virus (QrfV), a six-plex system including internal standards was established. After testing indicators such as linearity, minimum detection limit, specificity, precision and repeatability, NTC repeat, and comparison of multiple fluorescence RT-PCR and single fluorescence RT-PCR, the currently developed sheep respiratory syndrome multiple fluorescence RT-PCR detection kit fully meets the development needs.

[0062] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A kit for simultaneously detecting bluetongue virus, foot-and-mouth disease virus, Schmallenberg virus, peste des petits ruminants virus and capripox virus, characterized in that: The kit includes primers and probes as shown in SEQ ID NO.1 to SEQ ID NO.

18.

2. A method for detecting bluetongue virus, foot-and-mouth disease virus, Schmallenberg virus, peste des petits ruminants virus and capripox virus for non-disease diagnosis or treatment purposes, characterized in that: The following steps are involved: (1) Using the extracted nucleic acid of the sample to be tested as a template, using the primers shown in SEQ ID NO.1~2, SEQ ID NO.4~5, SEQ ID NO.7~8, SEQ ID NO.10~11 and SEQ ID NO.13~14 to amplify the sample to be tested, and using the probes shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 and SEQ ID NO.15 to identify the amplified product; As shown in SEQ ID NO.3, the 5' end of the probe is provided with a CY5.5 fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; as shown in SEQ ID NO.6, the 5' end of the probe is provided with a FAM fluorescent label, and the 3' end is provided with a BHQ1 fluorescent label; as shown in SEQ ID NO.9, the 5' end of the probe is provided with a ROX fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; as shown in SEQ ID NO.12, the 5' end of the probe is provided with a CY5 fluorescent label, and the 3' end is provided with an MGB fluorescent label; as shown in SEQ ID NO.15, the 5' end of the probe is provided with a TAMRA fluorescent label, and the 3' end is provided with an MGB fluorescent label; (2) setting an IPC internal standard including primers and probes as shown in SEQ ID NO.16 to SEQ ID NO.18 at the same time, wherein the 5' end of the probe as shown in SEQ ID NO.18 is provided with a HEX fluorescent label, and the 3' end is provided with a BHQ1 fluorescent label; Perform fluorescence quantitative detection and collect the signal of the fluorescence channel; the concentrations are 1.0×10 5 Copies / μL of the recombinant plasmid mixture of BTV, FMDV, SBV, PPRV, QrfV, and IPC internal standards were used as positive controls, and ultrapure water was used as negative controls; (3) Result determination: The negative control should have no Ct value and no specific amplification curve; the Ct value of all channels of the positive control should be ≤30 and a specific amplification curve should appear, and the test result is determined to be valid; If the negative control and positive control results do not meet the above conditions, the experiment will be considered invalid; On the premise that the test results are established, if the Ct value of the sample test result is ≤30 and a specific amplification curve appears, it is judged as positive; If the sample has a Ct value of 30 < 37 and a specific amplification curve appears, it is judged as suspicious and the suspicious sample must be retested. The result is judged after amplification. If the result is positive, it is judged as positive, and if the result is negative, it is judged as negative. If it is still suspicious, it can be judged as positive; if the sample Ct value is ≥37, it exceeds the detection sensitivity range of this method and is judged as negative; for some samples that do not present a specific amplification curve but have a high background, they should be judged as negative.

3. Judgment of the same sample: FAM channel positive is judged as foot-and-mouth disease pathogen infection, ROX channel positive is judged as Schmallenberg pathogen infection, CY5 channel positive is judged as small ruminant peste pathogen infection, TAMRA channel positive is judged as sheeppox pathogen infection, CY5.5 channel positive is judged as bluetongue pathogen infection; if multiple channels are positive, it is judged that multiple pathogens corresponding to multiple channels are mixedly infected.

4. The method according to claim 2, characterized in that: The reaction system of the amplification reaction includes: CoverAllProbe qRT-PCR Mix II (5×) 5 μL, Primer Mix 1 μL, Probe Mix 1 μL, Sample nucleic acid template 5 μL, dd H2O to 25 μL.

5. The method according to claim 2, characterized in that: The amplification reaction was as follows: pre-denaturation at 95°C for 20s, reverse transcription at 42°C for 30min; denaturation at 95°C for 10s, annealing and extension at 60°C for 20s, 40 cycles, and at the end of each cycle, six channels of fluorescence signals, namely, FAM, HEX, ROX, TAMRA, CY5, and CY5.5, were collected.

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