A kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, ovine enzootic abortion, and caprine arthritis encephalitis virus

Abstract

The present invention discloses a kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, ovine enzootic abortion, and caprine arthritis encephalitis virus, belonging to the technical field of virus detection. The kit comprises primers and probes shown as SEQ ID NO.1 to SEQ ID NO.18. After singleplex system tests on border disease virus, brucellosis, toxoplasmosis, ovine enzootic abortion, and caprine arthritis encephalitis virus, the present invention established a six-plex system including an internal standard. After tests on indicators such as linearity, lowest detection limit, specificity, precision and repeatability, NTC repeat, comparison between multiplex fluorescence RT-PCR and single fluorescence RT-PCR, etc., the currently developed multiplex fluorescence RT-PCR kit for sheep reproductive disorder syndrome fully meets the development requirements, and the lowest detection limit can reach below 10 Copies / μL.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and particularly relates to a kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, enzootic abortion of ewes and caprine arthritis encephalitis virus. Background Art

[0002] Sheep reproductive disorder syndrome is an important problem affecting the economic benefits of the sheep industry. Its etiology is complex and is often caused by the combined action 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 detection, etc. have defects such as long time consumption, low sensitivity, and limited single detection targets, and it is difficult to meet the needs of clinical rapid diagnosis and precise prevention and control. For example, the culture method takes several days to several weeks, and some pathogens are difficult to culture in vitro; serological detection is easily interfered by cross-reactions and cannot distinguish between past infections and current infections.

[0003] In recent years, real-time fluorescence quantitative PCR technology has been gradually applied to the detection of animal diseases due to its high sensitivity and specificity. However, conventional singleplex PCR can only detect a single pathogen at a time, resulting in low detection efficiency and high cost. Although there have been studies on developing multiplex fluorescence PCR technology in animals such as pigs and cattle (such as a kit for simultaneously detecting 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 disorder syndrome.

[0004] Therefore, developing a multiplex fluorescence RT-PCR simultaneous detection technology for sheep reproductive disorder syndrome, and realizing the rapid and precise identification of multiple pathogens by optimizing primer-probe design, reaction system and amplification conditions, is of great significance for improving the health management level of sheep flocks and reducing economic losses, and also provides technical support for subsequent molecular epidemiology research and vaccine development. [[ID=;16]]Summary of the Invention

[0005] The purpose of the present invention is to provide a kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, enzootic abortion of ewes and caprine arthritis encephalitis virus to solve the problems existing in the above-mentioned prior art.

[0006] [[ID=;22]]To achieve the above purpose, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, enzootic abortion of ewes and caprine arthritis encephalitis virus, and the kit includes primers and probes shown in SEQ ID NO.1 to SEQ ID NO.18.

[0008] The second technical solution of the present invention is a method for detecting border disease virus, brucellosis, toxoplasmosis, ovine enzootic abortion, and caprine arthritis encephalitis virus for non-diagnostic or therapeutic purposes, comprising the following steps:

[0009] (1) Using the extracted nucleic acid of the sample to be tested as a template, amplify the sample to be tested with 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, and identify the amplification products with probes shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, and SEQ ID NO.15;

[0010] The 5' end of the probe shown in SEQ ID NO.3 is provided with a CY5.5 fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; the 5' end of the probe shown in SEQ ID NO.6 is provided with a FAM fluorescent label, and the 3' end is provided with a BHQ1 fluorescent label; the 5' end of the probe shown in SEQ ID NO.9 is provided with a ROX fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; the 5' end of the probe shown in SEQ ID NO.12 is provided with a CY5 fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; the 5' end of the probe shown in SEQ ID NO.15 is provided with a TAMRA fluorescent label, and the 3' end is provided with an MGB fluorescent label;

[0011] (2) Simultaneously set the IPC internal standard including primers and probes shown in SEQ ID NO.16~SEQ ID NO.18. The 5' end of the probe 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 signals of the fluorescence channels. At the same time, use a recombinant plasmid mixture of BDV, Bru, Tox, OEA, CAEV, and IPC internal standard with concentrations of 1.0×10 5 copies / μL as a positive control, and use ultrapure water as a negative control;

[0012] (3) Result determination: The negative control should have no Ct value and no specific amplification curve. The Ct values of all channels of the positive control should be ≤30, and a specific amplification curve should appear, and the detection result is determined to be valid;

[0013] If the results of the negative control and the positive control do not meet the above conditions, this experiment is regarded as invalid;

[0014] On the premise that the test result is valid, if the Ct value of the sample test result is ≤ 30 and a specific amplification curve appears, it is determined as positive; if the Ct value of the sample is 30 < Ct value < 37 and a specific amplification curve appears, it is determined as suspicious. The suspicious sample must be retested, and the result is determined after amplification. Those with a positive result are determined as positive, and those with a negative result are determined as negative. If it is still suspicious, it can be determined as positive; when the Ct value of the sample is ≥ 37, it exceeds the detection sensitivity range of this method and is determined as negative; for some samples that do not show a specific amplification curve but have a high background, they should be determined as negative.

[0015] Determination of the same sample: Those positive in the FAM channel are determined to be infected with Brucella pathogen, those positive in the ROX channel are determined to be infected with Toxoplasma pathogen, those positive in the CY5 channel are determined to be infected with the pathogen of ovine enzootic abortion, those positive in the TAMRA channel are determined to be infected with Caprine arthritis pathogen, and those positive in the CY5.5 channel are determined to be infected with Border disease pathogen; if there are multiple channels showing positive, it is determined as a mixed infection of multiple pathogens corresponding to the corresponding channels.

[0016] Based on the above technical solutions, the present invention has the following technical effects:

[0017] After single - system testing of Border disease virus (BDV), Brucellosis (Bru), Toxoplasmosis (Tox), Ovine enzootic abortion (OEA), and Caprine arthritis virus (CAEV) of the present invention, a six - plex system including an internal standard was established. After testing with indicators such as linearity, minimum detection limit, specificity, precision and repeatability, NTC repeat, comparison between multiplex fluorescence RT - PCR and single - fluorescence RT - PCR, the currently developed RT - PCR multiplex fluorescence RT - PCR detection kit for sheep reproductive disorder syndrome fully meets the development requirements, and the minimum detection limit can reach below 10 Copies / μL. Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0019] Figure 1 It is the standard curve of single - fluorescence RT - PCR. Among them, A is the linearity of the BDV - UTR gene target, B is the linearity of the OEA - ompA gene target, C is the linearity of the Bru - IS711 gene target, D is the linearity of the CAEV - gag gene target, and E is the linearity of the Tox - B1 gene target.

[0020] Figure 2 It is a standard curve for multiplex fluorescent RT-PCR.

[0021] Figure 3 It is the digital droplet graph of the target quantitative value of this kit. Among them, A is the quantitative value result of BDV-UTR plasmid, B is the quantitative value result of OEA-ompA plasmid, C is the quantitative value result of Bru-IS711 plasmid, D is the quantitative value result of CAEV-gag plasmid, and E is the quantitative value result of Tox-B1 plasmid.

[0022] Figure 4 It is the comparison between multiplex fluorescent RT-PCR and each single fluorescent RT-PCR, and the compliance test of multiplex fluorescent RT-PCR and single fluorescent RT-PCR using the same template. Among them, A is the comparison of the linear results of multiplex fluorescent RT-PCR and single fluorescent RT-PCR for the border disease pathogen target, B is the comparison of the linear results of multiplex fluorescent RT-PCR and single fluorescent RT-PCR for the enzootic abortion of ewes pathogen target, C is the comparison of the linear results of multiplex fluorescent RT-PCR and single fluorescent RT-PCR for the Brucella pathogen target, D is the comparison of the linear results of multiplex fluorescent RT-PCR and single fluorescent RT-PCR for the caprine arthritis pathogen target, and E is the comparison of the linear results of multiplex fluorescent RT-PCR and single fluorescent RT-PCR for the toxoplasmosis pathogen target. The correlation coefficient R between single fluorescent RT-PCR and multiplex fluorescent RT-PCR 2 is greater than 0.99, indicating that the compliance between multiplex fluorescent RT-PCR and each single fluorescent RT-PCR is good, multiplex fluorescent RT-PCR does not produce mutual interference, and the amplification efficiency is not affected. Detailed implementation manners

[0023] Now, various exemplary implementation manners of the present invention will be described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although only preferred methods and materials are described in this invention, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0026] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this application are merely exemplary.

[0027] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0028] The technical solutions described in this invention are all conventional solutions in the art unless otherwise specified. The reagents or raw materials used are all purchased from commercial channels or have been made public unless otherwise specified.

[0029] An embodiment of this invention provides a kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, enzootic abortion of ewes and caprine arthritis virus. The kit includes primers and probes as shown in SEQ ID NO.1 to SEQ ID NO.18.

[0030] An embodiment of this invention also provides a method for detecting border disease virus, brucellosis, toxoplasmosis, enzootic abortion of ewes and caprine arthritis virus for non-disease diagnosis or treatment purposes, including the following steps:

[0031] (1) Using the extracted nucleic acid of the sample to be tested as a template, amplifying the sample to be tested with primers as shown in SEQ ID NO.1 to 2, SEQ ID NO.4 to 5, SEQ ID NO.7 to 8, SEQ ID NO.10 to 11 and SEQ ID NO.13 to 14, and identifying the amplification product with probes as shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 and SEQ ID NO.15;

[0032] The 5'-end of the probe shown in SEQ ID NO.3 is set with a CY5.5 fluorescent label, and the 3'-end is set with a BHQ2 fluorescent label; the 5'-end of the probe shown in SEQ ID NO.6 is set with a FAM fluorescent label, and the 3'-end is set with a BHQ1 fluorescent label; the 5'-end of the probe shown in SEQ ID NO.9 is set with a ROX fluorescent label, and the 3'-end is set with a BHQ2 fluorescent label; the 5'-end of the probe shown in SEQ ID NO.12 is set with a CY5 fluorescent label, and the 3'-end is set with a BHQ2 fluorescent label; the 5'-end of the probe shown in SEQ ID NO.15 is set with a TAMRA fluorescent label, and the 3'-end is set with an MGB fluorescent label;

[0033] (2)Simultaneously set the IPC internal standard including the primers and probes shown in SEQ ID NO.16~SEQ ID NO.18. The 5'-end of the probe shown in SEQ ID NO.18 is set with a HEX fluorescent label, and the 3'-end is set with a BHQ1 fluorescent label; perform fluorescence quantitative detection and collect the signals of the fluorescence channels; at the same time, use the recombinant plasmid mixtures of BDV, Bru, Tox, OEA, CAEV, and IPC internal standard with concentrations of 1.0×10 5 copies / μL as the positive control, and use ultrapure water as the negative control;

[0034] (3)Result determination: The negative control should have no Ct value and no specific amplification curve. The Ct values of all channels of the positive control should be ≤30, and a specific amplification curve should appear, and the detection result is determined to be valid;

[0035] If the results of the negative control and the positive control do not meet the above conditions, this experiment is regarded as invalid;

[0036] On the premise that the detection result is valid, if the Ct value of the sample detection result is ≤30 and a specific amplification curve appears, it is determined to be positive; if 30 < Ct value < 37 for the sample and a specific amplification curve appears, it is determined to be suspicious. The suspicious sample must be retested, and the result is determined after amplification. Those with a positive result are determined to be positive, and those with a negative result are determined to be negative. If it is still suspicious, it can be determined to be positive; if the Ct value of the sample is ≥37, it exceeds the detection sensitivity range of this method and is determined to be negative; for some samples with no specific amplification curve but a high background, it should be determined to be negative.

[0037] Determination of the same sample: If it is positive in the FAM channel, it is determined to be infected with Brucella pathogen; if it is positive in the ROX channel, it is determined to be infected with Toxoplasma pathogen; if it is positive in the CY5 channel, it is determined to be infected with the pathogen of enzootic abortion of ewes; if it is positive in the TAMRA channel, it is determined to be infected with Caprine arthritis pathogen; if it is positive in the CY5.5 channel, it is determined to be infected with Border disease pathogen; if there are multiple positive channels, it is determined to be a mixed infection of multiple pathogens corresponding to the corresponding channels.

[0038] In some specific embodiments, the reaction system of the amplification reaction comprises: 5 μL of CoverAll Probe qRT-PCR Mix Ⅱ (5×), 1 μL of primer Mix, 1 μL of probe Mix, 5 μL of sample nucleic acid template, and supplemented with dd H2O to 25 μL.

[0039] In some specific embodiments, the amplification reaction is 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, 40 cycles, and fluorescence signals of six channels, namely FAM, HEX, ROX, TAMRA, CY5, and CY5.5, are collected at the end of each cycle. Example 1

[0040] Retrieve the complete genome sequence or 5'UTR gene sequence of border disease virus (GenBank accession 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.) from the nucleic acid database GenBank of NCBI (http: / / www.ncbi.nlm.nih.gov); the complete genome sequence of Brucella (GenBank accession numbers: 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 complete genome sequence or B1 gene sequence of the pathogen of toxoplasmosis (GenBank accession numbers: AB703301.1, AF179871.1, KC607827.1, KX270366.1, LN714499.1, MK521885.1, MN542678.1, MZ717192.1, OR547658.1, etc.); the complete genome sequence or ompA gene sequence of the pathogen of ovine enzootic abortion (GenBank accession 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 complete genome sequence or gag gene sequence of caprine arthritis virus (GenBank accession 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.). The IPC internal standard gene is based on the conserved gene sequence of the 16S rRNA gene in the ovine mitochondrial genome. After aligning the sequences of each target gene with SnapGene software, relatively conserved nucleotide sequences of each pathogen are selected respectively. According to the primer and TaqMan probe design principles and in combination with the relevant national standard requirements, the relevant primer and probe sequences are designed.

[0041] The targets and sequences detected by the kit are summarized in Table 1.

[0042] Table 1 Primers and Probes of Multiplex Fluorescent RT-PCR Detection Kit for Ovine Reproductive Disorder Syndrome

[0043] 。

[0044] 2.1 Establishment of Multiplex Fluorescent RT-PCR Reaction System

[0045] The primer and probe stock solutions were diluted 10-fold in gradient. The primers were fully mixed to prepare a primer mixture. The final concentrations of the primers for each target are shown in Table 1. Each reaction system was prepared as shown in Table 2, and the fluorescent RT-PCR reaction was carried out according to the procedure shown in Table 3. The equipment used was JLM QX600 from Jielaimai Company.

[0046] Table 2 Preparation of Fluorescent RT-PCR Reaction System

[0047] 。

[0048] Table 3 Fluorescent RT-PCR Reaction Parameter Settings

[0049] 。

[0050] 2.2 Standard Curve Drawing

[0051] The positive plasmids of Border disease virus (BDV), Brucella (Bru), Toxoplasmosis (Tox), Ovine enzootic abortion (OEA), and Caprine arthritis-encephalitis virus (CAEV) were diluted 10-fold in gradient and then subjected to fluorescent RT-PCR. The concentration standard curve was made with the fluorescence Ct value of the template concentration.

[0052] Then, the positive plasmids of BDV, Bru, Tox, OEA, and CAEV were mixed in equal amounts and diluted 10-fold in gradient, and multiplex fluorescent RT-PCR was carried out. The concentration standard curve was made with the fluorescence Ct value of the template concentration.

[0053] The concentration-Ct value standard curves were made respectively with the positive plasmids of BDV, Bru, Tox, OEA, and CAEV ( Figure 1 ). From Figure 1 and Figure 2It can be seen that whether it is a single system or a multiple multi-positive system, the E value, R, and curve slope of the standard curve RT-PCR amplification efficiency are all within the normal range, indicating good amplification efficiency.

[0054] In the present invention, a mixture of recombinant plasmids of BDV, Bru, Tox, OEA, CAEV, and IPC internal standards with concentrations of 1.0×10 5 copies / μL was used as the positive control, and ultrapure water was used as the negative control.

[0055] 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;

[0056] If the results of the negative control and the positive control do not meet the above conditions, this experiment is regarded as invalid;

[0057] On the premise that the test result is valid, if the Ct value of the sample test result is ≤30 and a specific amplification curve appears, it is determined to be positive; if the sample 30 < Ct value < 37 and a specific amplification curve appears, it is determined to be suspicious, and the suspicious sample must be retested. After amplification, the result is determined. If the result is positive, it is determined to be positive; if the result is negative, it is determined to be negative. If it is still suspicious, it can be determined to be positive; if the Ct value of the sample is ≥37, it exceeds the detection sensitivity range of this method and is determined to be negative; for some samples that do not show a specific amplification curve but have a high background, they should be determined to be negative.

[0058] Determination of the same sample: If the FAM channel is positive, it is determined to be infected with Brucella pathogen; if the ROX channel is positive, it is determined to be infected with Toxoplasma pathogen; if the CY5 channel is positive, it is determined to be infected with the pathogen of enzootic abortion of ewes; if the TAMRA channel is positive, it is determined to be infected with the pathogen of caprine arthritis; if the CY5.5 channel is positive, it is determined to be infected with the border disease pathogen; if there are multiple channels showing positive, it is determined to be a mixed infection of multiple pathogens corresponding to the corresponding channels.

[0059] 2.3 Determination of the lowest detection limit

[0060] The positive plasmids with a theoretical value of 10 6 Copies / μL of BDV, Bru, Tox, OEA, and CAEV were subjected to plasmid linearization digestion using two restriction enzymes, QuickCut SacI and QuickCut Kpn I. The digestion system is shown in Table 4, and the digestion can be completed by incubating at a constant temperature of 37°C for 15 minutes. After linearizing and digesting the positive plasmids with a theoretical value of 10 6 Copies / μL, they were diluted to 10 3After [Copies / μL], it was quantified using the JLM Digital Matrix-5000 digital PCR from Jielaimi Company. The quantification system is shown in Table 5, and the digital PCR reaction was carried out according to the procedure shown in Table 6.

[0061] Sample concentration (Copies / μL) = Quantification result (Copies / μL) × 20 (total reaction system) ÷ 2 (sample loading volume).

[0062] Table 4 Plasmid linearization digestion system

[0063] 。

[0064] Table 5 Preparation of digital PCR quantification reaction system

[0065] 。

[0066] Table 6 Digital PCR reaction parameter settings

[0067] 。

[0068] The quantified plasmid was serially diluted, and then the minimum detection limits of the diluted plasmid were tested using single fluorescence RT-PCR for BDV, Bru, Tox, OEA, and CAEV respectively. The Ct value that could be detected at the highest dilution factor was the minimum detection limit.

[0069] Then, the quantified positive plasmids of BDV, Bru, Tox, OEA, and CAEV were mixed in equal amounts and serially diluted for multiplex fluorescence RT-PCR. The concentration corresponding to the Ct value that could be detected at the highest dilution factor was the minimum detection limit.

[0070] Digital PCR was used to quantify each target plasmid of this kit diluted by a certain factor. The microdroplet image of the quantification result is as Figure 3 shown. The digital PCR quantification results of each target and the converted plasmid concentrations are shown in Table 7. The quantified plasmid was diluted with TE buffer by a certain dilution factor to perform the minimum detection limit test. In order to further test the minimum detection limit of the multiplex multi-positive system, an intermediate plasmid concentration was diluted one more time for comparison with the single system. The minimum detection limit test results of the single 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 system, but the minimum detection limits of both the single and multiplex systems can reach below 10 Copies / μL.

[0071] Table 7 Statistics of quantification results of related target plasmids of this kit

[0072] 。

[0073] Table 8 Test results of the lowest detection limits of single and multiplex mixed systems of each target in this kit

[0074] 。

[0075] 2.4 Specificity test

[0076] Eight positive plasmids of sheep diseases, namely Bluetongue virus (BTV), Foot-and-mouth disease virus (FMDV), Peste des petits ruminants virus (PPRV-M, PPRV-N), Sheep pox virus (QrfV), Lumpy skin disease virus (LSDV-ORF101, LSDV-ORF126), Schmallenberg virus (SBV), were selected and single fluorescence RT-PCR was performed respectively using the primers and probes of BDV, Bru, Tox, OEA, and CAEV to verify the specificity of the primers and probes.

[0077] To verify whether the system of this kit would have non-specific amplification for other sheep disease-related plasmids, the singleplex systems of the detection targets involved in this kit were used to perform specificity tests on other sheep disease template plasmids at 10 6 Copies / μL. If suspected non-specific amplification was found, the plasmid mother liquor was further tested using the dye method. If the Tm value of the suspected non-specific amplification was consistent with the Tm value of the target, it indicated that there might be contamination of the target plasmid or sample in the plasmid mother liquor. If the Tm value of the suspected non-specific amplification was inconsistent with the Tm value of the target, it indicated that it might be due to non-specific amplification of the primer with other templates.

[0078] The results showed that through testing, it was found that the non-specific amplification of other templates in several systems such as BDV-UTR, Bru-IS711, OEA-ompA, and CAEV-gag was basically consistent with the melting temperature Tm value of the target template, indicating that it could be determined that there were contamination problems in other templates, mainly including the following types of contamination: 1) There was contamination of the BDV-UTR plasmid or sample in the FMDV-3D plasmid mother liquor; 2) There was contamination of the Bru-IS711 plasmid or sample in the Tox-B1 plasmid mother liquor; 3) There was contamination of the OEA-ompA plasmid or sample in the PPRV-M plasmid mother liquor; 4) There was contamination of the CAEV-gag plasmid or sample in the Bru-IS711 plasmid mother liquor. The results are shown in Table 9 and Table 10.

[0079] Table 9 Specificity test of the singleplex systems of each target in this kit

[0080] 。

[0081] Table 10 Verification of suspected contamination of each target in this kit by the dye method

[0082] 。

[0083] 2.5 Precision and Repeatability Tests

[0084] Mix the positive plasmids of BDV, Bru, Tox, OEA, and CAEV in equal amounts to form two plasmid mixtures with final concentrations of 10 5 Copies / μL and 10 3 Copies / μL, and perform multiplex fluorescent RT-PCR tests. Test continuously for 5 days, with 6 replicates each time, and count the precision and repeatability of the multiplex system.

[0085] The multiplex system of this kit was subjected to precision and repeatability tests on the 10 5 Copies / μL and 10 3 Copies / μL mixed plasmids for 5 days. The test results are shown in Tables 11 and 12. The results show that under the same-day conditions, at the two concentrations of 10 5 Copies / μL and 10 3 Copies / μL, the extreme differences in Ct values for each target are less than 1, and the coefficients of variation are less than 1%; when counting the Cq values for five days, at the two concentrations of 10 5 Copies / μL and 10 3 Copies / μL, the extreme differences in Ct values for each target are less than 1, and the coefficients of variation are less than 1%, both meeting the precision and repeatability standards with a coefficient of variation (CV) ≤ 5% required for kit development.

[0086] Table 11 Statistical Results of Precision and Repeatability of the Multiplex System of this Kit for 10 5 Copies / μL Mixed Plasmids

[0087] 。

[0088] Table 12 Statistical Results of Precision and Repeatability of the Multiplex System of this Kit for 10 3 Copies / μL Mixed Plasmids

[0089] 。

[0090] 2.6 NTC Repeated Tests

[0091] Perform NTC tests on the prepared multiplex system to evaluate whether there is non-specific amplification between the primers and probes in the system. Test continuously for 5 days, with 6 NTC replicates each time, and count the NTC test results of the multiplex system.

[0092] The prepared multiplex system was subjected to NTC testing for multiple days to evaluate whether there was non-specific amplification between the primers and probes in the system. The statistical results are shown in Table 13. The results indicate that after multiple days of NTC repeated testing, there was no non-specific amplification in each channel.

[0093] Table 13 Repeated NTC testing of the multiplex system of this kit for different days

[0094] 。

[0095] 2.7 Comparison between multiplex fluorescence RT-PCR and single fluorescence RT-PCR

[0096] After equally mixing the positive plasmids of BDV, Bru, Tox, OEA, and CAEV and performing 10-fold serial dilutions, multiplex fluorescence RT-PCR of the multiplex system and single fluorescence RT-PCR of each target of BDV, Bru, Tox, OEA, and CAEV were respectively carried out. The fluorescence Ct values corresponding to the same template concentration were compared to solve the regression equation and compare the compliance between multiplex fluorescence RT-PCR and each single fluorescence RT-PCR.

[0097] The results of the compliance test of multiplex fluorescence RT-PCR and single fluorescence RT-PCR using the same template show that the correlation coefficients between single fluorescence RT-PCR of BDV, Bru, Tox, OEA, and CAEV and multiplex fluorescence RT-PCR R 2 were 0.9995, 0.9960, 0.998, 0.9992, and 0.9997 respectively, indicating good compliance between multiplex fluorescence RT-PCR and each single fluorescence RT-PCR. Multiplex fluorescence RT-PCR did not cause mutual interference and the amplification efficiency was not affected. The results are shown in Figure 4 。

[0098] After single system testing of border disease virus (BDV), brucellosis (Bru), toxoplasmosis (Tox), ovine enzootic abortion (OEA), and caprine arthritis encephalitis virus (CAEV) of the present invention, a six-plex system including an internal standard was established. After testing indicators such as linearity, minimum detection limit, specificity, precision and repeatability, NTC repeat, and comparison between multiplex fluorescence RT-PCR and single fluorescence RT-PCR, the developed multiplex fluorescence RT-PCR detection kit for sheep reproductive disorder syndrome fully meets the development requirements.

[0099] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A kit for simultaneously detecting border disease virus, brucellosis, toxoplasmosis, enzootic abortion of ewes and caprine arthritis encephalitis virus, characterized in that, The kit includes primers and probes shown in SEQ ID NO.1 to SEQ ID NO.18; The 5' end of the probe shown in SEQ ID NO.3 is provided with a CY5.5 fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; the 5' end of the probe shown in SEQ ID NO.6 is provided with a FAM fluorescent label, and the 3' end is provided with a BHQ1 fluorescent label; the 5' end of the probe shown in SEQ ID NO.9 is provided with a ROX fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; the 5' end of the probe shown in SEQ ID NO.12 is provided with a CY5 fluorescent label, and the 3' end is provided with a BHQ2 fluorescent label; the 5' end of the probe shown in SEQ ID NO.15 is provided with a TAMRA fluorescent label, and the 3' end is provided with an MGB fluorescent label; the 5' end of the probe shown in SEQ ID NO.18 is provided with a HEX fluorescent label, and the 3' end is provided with a BHQ1 fluorescent label.

Images