Kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, sheep endemic abortion and goat arthritis virus

Abstract

The invention discloses a kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, sheep endemic abortion and goat arthritis 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. According to the invention, after a single system of border disease virus, brucellosis, toxoplasmosis, sheep endemic abortion and goat arthritis virus is tested, a sextuple system containing an internal standard is established, and through tests 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 sheep endemic abortion and the goat arthritis. The currently developed fluorescent RT-PCR multiple detection kit for the sheep reproductive disorder syndrome completely meets the development requirement, and the lowest detection limit can reach 10 Copis / mu L or below.

Description

Technical Field

[0001] The invention relates to the technical field of virus detection, in particular to a test kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, sheep endemic abortion and goat arthritis virus. Background Art

[0002] Sheep reproductive disorder syndrome is an important issue that affects the economic benefits of the sheep industry. Its etiology is complex and is often caused by a combination of multiple pathogens (such as Brucella, Chlamydia, Toxoplasma, Q fever Rickettsia, etc.) or genetic and environmental factors. Traditional detection methods such as pathogen isolation and culture, serological testing, etc. have defects such as long time consumption, low sensitivity, and limited single detection targets, which are difficult to meet the needs of clinical rapid diagnosis and precise prevention and control67. For example, the culture method takes several days to weeks, and some pathogens are difficult to culture in vitro; serological testing is easily interfered by cross-reactions and cannot distinguish between previous infections and current infections.

[0003] In recent years, fluorescent quantitative PCR technology has been gradually applied to animal disease detection due to its high sensitivity and specificity. However, conventional single-plex PCR can only detect a single pathogen at a time, resulting in low detection efficiency and high cost. Although studies have developed multiple fluorescent PCR technologies in pigs, cattle and other animals (such as kits for simultaneous detection of pathogens such as porcine circovirus and pseudorabies virus), these technologies mostly focus on a single livestock species or specific diseases, and lack a systematic solution for the simultaneous detection of multiple pathogens in sheep reproductive syndrome.

[0004] Therefore, developing a multiplex fluorescent RT-PCR synchronous detection technology for sheep reproductive disorder syndrome and realizing rapid and accurate identification of multiple pathogens by optimizing primer probe design, reaction system and amplification conditions is of great significance to improving the health management level of sheep flocks and reducing economic losses. It also provides technical support for subsequent molecular epidemiological research and vaccine development. Summary of the invention

[0005] The purpose of the present invention is to provide a test kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, sheep endemic abortion and caprine arthritis 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 border disease virus, brucellosis, toxoplasmosis, ovine endemic abortion and caprine arthritis 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 border disease virus, brucellosis, toxoplasmosis, sheep endemic abortion and caprine arthritis 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 a BHQ2 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) IPC internal standards were set up simultaneously, including primers and probes as shown in SEQ ID NO.16 to SEQ ID NO.18. For example, the 5' end of the probe as shown in SEQ ID NO.18 was marked with HEX fluorescent marker, and the 3' end was marked with BHQ1 fluorescent marker. Fluorescence quantitative detection was performed, and the signal of the fluorescence channel was collected. At the same time, the concentrations of 1.0×10 5 Copies / μL of BDV, Bru, Tox, OEA, CAEV, and IPC internal standard recombinant plasmid mixtures 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 FAM channel were judged to be infected with Brucella pathogen, those with a positive result in ROX channel were judged to be infected with Toxoplasma pathogen, those with a positive result in CY5 channel were judged to be infected with ovine endemic abortion pathogen, those with a positive result in TAMRA channel were judged to be infected with goat arthritis pathogen, and those with a positive result in CY5.5 channel were judged to be infected with border disease pathogen; if multiple channels were positive, it was judged to be a mixed infection of multiple pathogens corresponding to the corresponding channels.

[0009] Based on the above technical solution, the present invention has the following technical effects: After testing the single-plex system of border disease virus (BDV), brucellosis (Bru), toxoplasmosis (Tox), ovine endemic abortion (OEA), and caprine arthritis virus (CAEV), 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 currently developed ovine reproductive disorder syndrome RT-PCR multiple fluorescent RT-PCR detection kit fully meets the development requirements, 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 BDV-UTR gene target linearity, B is the OEA-ompA gene target linearity, C is the Bru-IS711 gene target linearity, D is the CAEV-gag gene target linearity, and E is the Tox-B1 gene target linearity.

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

[0013] Figure 3 The following are the droplet diagrams of the digital PCR assays of the relevant targets of this kit. A is the assay result of the BDV-UTR plasmid, B is the assay result of the OEA-ompA plasmid, C is the assay result of the Bru-IS711 plasmid, D is the assay result of the CAEV-gag plasmid, and E is the assay result of the Tox-B1 plasmid.

[0014] Figure 4 This is a comparison between multiple fluorescent RT-PCR and each single fluorescent RT-PCR, and the consistency test of multiple fluorescent RT-PCR and single fluorescent RT-PCR was performed using the same template. Among them, A is a comparison of the linear results of multiple fluorescent RT-PCR and single fluorescent RT-PCR for border disease pathogen targets, B is a comparison of the linear results of multiple fluorescent RT-PCR and single fluorescent RT-PCR for sheep endemic abortion pathogen targets, C is a comparison of the linear results of multiple fluorescent RT-PCR and single fluorescent RT-PCR for Brucella pathogen targets, D is a comparison of the linear results of multiple fluorescent RT-PCR and single fluorescent RT-PCR for goat arthritis pathogen targets, and E is a comparison of the linear results of multiple fluorescent RT-PCR and single fluorescent RT-PCR for toxoplasmosis pathogen targets. The correlation coefficient R between single fluorescent RT-PCR and multiple fluorescent RT-PCR 2 All of them were 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] The embodiment of the present invention provides a kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, ovine endemic abortion and caprine arthritis 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 border disease virus, brucellosis, toxoplasmosis, sheep endemic abortion and caprine arthritis 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 a BHQ2 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) IPC internal standards were set up simultaneously, including primers and probes as shown in SEQ ID NO.16 to SEQ ID NO.18. For example, the 5' end of the probe as shown in SEQ ID NO.18 was marked with HEX fluorescent marker, and the 3' end was marked with BHQ1 fluorescent marker. Fluorescence quantitative detection was performed, and the signal of the fluorescence channel was collected. At the same time, the concentrations of 1.0×10 5 Copies / μL of BDV, Bru, Tox, OEA, CAEV, and IPC internal standard recombinant plasmid mixtures 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 FAM channel were judged to be infected with Brucella pathogen, those with a positive result in ROX channel were judged to be infected with Toxoplasma pathogen, those with a positive result in CY5 channel were judged to be infected with ovine endemic abortion pathogen, those with a positive result in TAMRA channel were judged to be infected with goat arthritis pathogen, and those with a positive result in CY5.5 channel were judged to be infected with border disease pathogen; if multiple channels were positive, it was judged to be a mixed infection of multiple pathogens corresponding to the corresponding channels.

[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 amplification reaction is: 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 at the end of each cycle, six channels of fluorescence signals, namely FAM, HEX, ROX, TAMRA, CY5, and CY5.5, are collected. Example 1

[0026] The whole genome sequence or 5'UTR gene sequence of border disease virus (GenBank numbers: GQ902940.1, GU270877.1, KF925348.1, KJ463422.1, LR824489.1, MF102262.1, MG649392.1, MW762535.1, MZ664275.1, NC_003679.1, U41112.1, etc.) and the whole genome sequence of Brucella (GenBank numbers: GQ902940.1, GU270877.1, KF925348.1, KJ463422.1, LR824489.1, MF102262.1, MG649392.1, MW762535.1, MZ664275.1, NC_003679.1, U41112.1, etc.) were retrieved from the NCBI nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov). Sequence number: CP018554.1, CP018566.1, CP025680.1, CP025820.1, CP025821.1, CP035797.1, CP044342.1, CP044985.1, CP044986.1, CP069385.1, LT962937.1, LT962945.1, LT963350.1, etc.); the whole genome sequence or B1 gene sequence of the pathogen of toxoplasmosis (GenBank sequence number: AB703301.1, AF179871.1, KC607827.1, KX270366.1, LN 714499.1, MK521885.1, MN542678.1, MZ717192.1, OR547658.1, etc.); the whole genome sequence or ompA gene sequence of sheep endemic abortion pathogens (GenBank numbers: AJ440239.1, AJ005617.1, CP018296.1, CR848038.1, EU531729.1, JF728984.1, KY399850.1, L39020.1, MF140909.1, KP984478.1, X51859.1, etc.); the whole genome sequence or ompA gene sequence of goat arthritis virus gag gene sequences (GenBank numbers: PP382771.1, AY900630.1, DQ190049.1, EU983109.1, AJ305041.1, MH827518.1, M33677.1, EU300978.1, GQ161211.1, MW199997.1, NC_001463.1, KC241941.1, FJ195346.1, GQ161214.1, KC241927.1, KT214469.1, MW711326.1, etc.); IPC internal standard genes are based on 16S The conservative gene sequence of the rRNA gene was compared with each target gene sequence using SnapGene software, and then the relatively conservative nucleotide sequences of each pathogen were selected. According to the design principles of primers and TaqMan probes and combined with the relevant national standards, the relevant primer and probe sequences were designed.

[0027] The targets and sequences detected by the kit are summarized in Table 1.

[0028] Table 1 Primers and probes of multiplex fluorescent RT-PCR detection kit for sheep reproductive failure syndrome .

[0029] 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.

[0030] Table 2 Preparation of fluorescent RT-PCR reaction system .

[0031] Table 3 Fluorescence RT-PCR reaction parameter settings .

[0032] 2.2 Standard curve drawing The positive plasmids of border disease virus (BDV), brucellosis (Bru), toxoplasmosis (Tox), ovine enzootic abortion (OEA) and caprine arthritis-encephalitis virus (CAEV) were diluted 10 times and then subjected to fluorescent RT-PCR. The template concentration fluorescence Ct value was used to make the concentration standard curve.

[0033] Then, BDV, Bru, Tox, OEA, and CAEV positive plasmids were mixed in equal amounts and diluted 10-fold in a gradient manner for multiple fluorescence RT-PCR, and the concentration standard curve was made using the template concentration fluorescence Ct value.

[0034] The concentration-Ct value standard curves were made using BDV, Bru, Tox, OEA, and CAEV positive plasmids. Figure 1 ).from Figure 1 and Figure 2 It 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.

[0035] In the present invention, the concentrations are 1.0×105 A mixture of BDV, Bru, Tox, OEA, CAEV, 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.

[0036] 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.

[0037] For the same sample, the following judgments were made: those with a positive result in FAM channel were judged to be infected with Brucella pathogen, those with a positive result in ROX channel were judged to be infected with Toxoplasma pathogen, those with a positive result in CY5 channel were judged to be infected with ovine endemic abortion pathogen, those with a positive result in TAMRA channel were judged to be infected with goat arthritis pathogen, and those with a positive result in CY5.5 channel were judged to be infected with border disease pathogen; if multiple channels were positive, it was judged to be a mixed infection of multiple pathogens corresponding to the corresponding channels.

[0038] 2.3 Determination of the minimum detection limit The theoretical values ​​of BDV, Bru, Tox, OEA and CAEV are set to 10 6 The positive plasmid with a copy / μL was linearized and digested with two enzymes, QuickCut SacI 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 3 Copies / μL were then determined using JLM Digital Matrix-5000 digital 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.

[0039] Sample concentration (Copies / μL) = fixed value result (Copies / μL) × 20 (total reaction system) ÷ 2 (sample volume).

[0040] Table 4 Plasmid linearization restriction enzyme system .

[0041] Table 5 Preparation of digital PCR fixed value reaction system .

[0042] Table 6 Digital PCR reaction parameter settings .

[0043] The fixed plasmid was diluted in series, and then the minimum detection limit of the diluted plasmid was tested using BDV, Bru, Tox, OEA, and CAEV single-plex fluorescent RT-PCR. The Ct value that can be detected at the highest dilution multiple is the minimum detection limit.

[0044] Then, the fixed BDV, Bru, Tox, OEA, and CAEV positive plasmids were mixed in equal amounts and diluted in gradients for multiple fluorescent RT-PCR. The concentration corresponding to the Ct value that can be detected at the highest dilution multiple is the minimum detection limit.

[0045] 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.

[0046] Table 7 Statistics of target plasmid determination results of this kit .

[0047] Table 8 The minimum detection limit test results of single-plex and multiplex mixed systems for each target in this kit .

[0048] 2.4 Specificity test A total of 8 sheep disease-positive plasmids were selected, including bluetongue virus (BTV), foot-and-mouth disease virus (FMDV), peste des petits ruminants virus (PPRV-M, PPRV-N), capripox virus (QrfV), bovine lumpy skin disease (LSDV-ORF101, LSDV-ORF126), and Schmallenberg virus (SBV). Single fluorescent RT-PCR was performed with primers and probes of BDV, Bru, Tox, OEA, and CAEV to verify the specificity of primers and probes.

[0049] 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.

[0050] The results showed that after testing, it was found that the nonspecific amplification of other templates in several systems such as BDV-UTR, Bru-IS711, OEA-ompA, CAEV-gag, etc. was basically consistent with the melting temperature Tm value of the target template, indicating that it can be determined that there are contamination problems in other templates. The main contaminations are as follows: 1) FMDV-3D plasmid mother liquor is contaminated with BDV-UTR plasmid or sample; 2) Tox-B1 plasmid mother liquor is contaminated with Bru-IS711 plasmid or sample; 3) PPRV-M plasmid mother liquor is contaminated with OEA-ompA plasmid or sample; 4) Bru-IS711 plasmid mother liquor is contaminated with CAEV-gag plasmid or sample. The results are shown in Tables 9 and 10.

[0051] Table 9 Specificity test of each target single-plex system of this kit .

[0052] Table 10 Dye method verification of suspected contamination of each target in this kit .

[0053] 2.5 Precision and repeatability test BDV, Bru, Tox, OEA, and CAEV positive plasmids were mixed in equal amounts to a final concentration of 10 5 Copies / μL and 10 3Copies / 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.

[0054] 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, and the coefficient of variation was less than 1%, both meeting the precision and repeatability standards of coefficient of variation (CV) ≤ 5% required for kit development.

[0055] Table 11 Multiplex system of this kit for 10 5 Copies / μL mixed plasmid precision and repeatability statistics .

[0056] Table 12 Multiplex system of this kit for 10 3 Copies / μL mixed plasmid precision and repeatability statistics .

[0057] 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.

[0058] 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.

[0059] Table 13 NTC repeated tests on different days in multiple systems of this kit .

[0060] 2.7 Comparison of multiplex fluorescent RT-PCR and single fluorescent RT-PCR Equal amounts of BDV, Bru, Tox, OEA, and CAEV positive plasmids were mixed and diluted 10-fold. Multiplex system fluorescent RT-PCR and single fluorescent RT-PCR of each target of BDV, Bru, Tox, OEA, and CAEV 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 multiplex fluorescent RT-PCR and each single fluorescent RT-PCR was compared.

[0061] The results of the compliance 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 BDV, Bru, Tox, OEA, and CAEV R 2 The results are 0.9995, 0.9960, 0.998, 0.9992 and 0.9997 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 .

[0062] After testing the single-plex system of border disease virus (BDV), brucellosis (Bru), toxoplasmosis (Tox), ovine endemic abortion (OEA), and caprine arthritis virus (CAEV), 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 developed ovine reproductive disorder syndrome multiple fluorescent RT-PCR detection kit fully meets the development needs.

[0063] 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 test kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, ovine endemic abortion and caprine arthritis 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 border disease virus, brucellosis, toxoplasmosis, ovine endemic abortion and caprine arthritis 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 a BHQ2 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; Fluorescence quantitative detection was performed to collect the signal of the fluorescence channel; at the same time, the concentrations of 1.0×10 5 Copies / μL of BDV, Bru, Tox, OEA, CAEV, and IPC internal standard recombinant plasmid mixture 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 Brucella pathogen infection, ROX channel positive is judged as Toxoplasma pathogen infection, CY5 channel positive is judged as ovine endemic abortion pathogen infection, TAMRA channel positive is judged as goat arthritis pathogen infection, CY5.5 channel positive is judged as border disease pathogen infection; if multiple channels are positive, it is judged as a mixed infection of multiple pathogens corresponding to the corresponding channels.

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 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, and 40 cycles. At the end of each cycle, fluorescence signals of six channels, namely, FAM, HEX, ROX, TAMRA, CY5, and CY5.5, were collected.

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