A primer pair and probe set for detecting animal Q fever, abortive chlamydia and brucellosis, a kit and its application

Abstract

The present invention relates to a primer pair and a probe group, a kit and an application thereof for detecting animal Q fever, abortion chlamydia and brucellosis, and belongs to the technical field of pathogenic microorganism detection. The primer pair and the probe group and the kit of the present invention adopt triple fluorescence PCR detection, and can simultaneously detect and identify animal Q fever, abortion chlamydia and brucellosis that cause abortion, and have the advantages of high accuracy, high sensitivity, strong specificity, rapid high throughput, simple operation, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogenic microorganism detection, and in particular to a primer pair and a probe group, a kit and applications thereof for detecting animal Q fever, abortive chlamydia and brucellosis. Background Art

[0002] Animal Q fever is a natural zoonosis caused by Coxiella burnetii. Coxiella burnetii can infect humans, ruminants, birds, arthropods, etc. through aerosols. So far, animal Q fever has become one of the most widespread zoonoses in the world.

[0003] Chlamydophila abortus is a zoonosis caused by Chlamydophila abortus. Chlamydophila abortus can cause endemic abortion in ruminants (such as sheep, goats, cattle, pigs, etc.), and can also infect pregnant women, causing miscarriage or other diseases. Animals infected with Chlamydophila abortus usually do not show typical clinical symptoms in the early stages, so they are often ignored, resulting in abortion, stillbirth, and weak piglets in the late pregnancy of infected animals, causing serious economic losses.

[0004] Brucellosis, also known as undulant fever, Mediterranean fever, and Malta fever, is a zoonotic infectious disease caused by Brucella. The main symptoms of human infection with brucellosis include fever, sweating, fatigue, joint pain, etc.; the clinical symptoms of animal infection with brucellosis are serious damage to the reproductive system, among which female animals show miscarriage and infertility, and male animals show orchitis and epididymitis. Brucellosis seriously restricts the sustainable development of animal husbandry and directly threatens food safety and the safety of people's lives and property.

[0005] Coxiella burnetii, Chlamydia abortus, and Brucella can all survive, latently infect, and escape immunity in host cells. The reproductive disorder symptoms they cause are very similar and difficult to distinguish clinically. As far as animal Q fever is concerned, the existing technology lacks effective laboratory diagnostic identification technology and epidemiological data verification, and misdiagnosis and missed diagnosis are serious; as far as Chlamydia abortus disease is concerned, current epidemiological surveys mostly use indirect hemagglutination tests, lack effective pathogen detection methods, resulting in large deviations in test results; as far as brucellosis is concerned, there are currently no special unique clinical symptoms indicating the disease, so it is difficult to distinguish it from diseases caused by other pathogenic microorganisms based on clinical symptoms alone. There is also no integrated research on the detection methods of animal Q fever, Chlamydia abortus disease, and Brucellosis in the existing technology. When animals have miscarriages, it is difficult to quickly identify the cause. Summary of the invention

[0006] The purpose of the present invention is to provide a primer pair and a probe group, a kit and applications thereof for detecting animal Q fever, abortion chlamydia and brucellosis, so as to solve the problems of large deviation in the detection results of animal Q fever, abortion chlamydia and brucellosis, low effectiveness, difficulty in determining the cause, serious misdiagnosis and missed diagnosis in the prior art, and fill the technical gap in the prior art for simultaneously detecting and identifying three common animal Q fever, abortion chlamydia and brucellosis that can cause abortion in my country.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a primer pair and a probe group for detecting animal Q fever, chlamydia aborti and brucellosis. The primer pair and the probe group comprise: an animal Q fever detection forward primer Q forward, an animal Q fever detection reverse primer Q reverse, an animal Q fever detection probe Q probe, an abortion chlamydia detection forward primer AB forward, an abortion chlamydia detection reverse primer AB reverse, an abortion chlamydia detection probe AB probe, a brucellosis detection forward primer BR forward, a brucellosis detection reverse primer BR reverse and a brucellosis detection probe BR probe;

[0009] The nucleotide sequence of the animal Q fever detection forward primer Q forward is shown in SEQ ID NO.2, the nucleotide sequence of the animal Q fever detection reverse primer Q reverse is shown in SEQ ID NO.3, the nucleotide sequence of the animal Q fever detection probe Qprobe is shown in SEQ ID NO.4, the nucleotide sequence of the abortion chlamydia disease detection forward primer AB forward is shown in SEQ ID NO.5, the nucleotide sequence of the abortion chlamydia disease detection reverse primer AB reverse is shown in SEQ ID NO.6, the nucleotide sequence of the abortion chlamydia disease detection probe AB probe is shown in SEQ ID NO.7, the nucleotide sequence of the brucellosis detection forward primer BR forward is shown in SEQ ID NO.8, the nucleotide sequence of the brucellosis detection reverse primer BR reverse is shown in SEQ ID NO.9, and the nucleotide sequence of the brucellosis detection probe BRprobe is shown in SEQ ID NO.10.

[0010] Preferably, the 5' end of the animal Q fever detection probe Q probe is labeled with FAM, the 5' end of the abortion chlamydia disease detection probe AB probe is labeled with VIC, the 5' end of the brucellosis detection probe BR probe is labeled with CY5, and the 3' ends of the animal Q fever detection probe Q probe, the abortion chlamydia disease detection probe AB probe, and the brucellosis detection probe BRprobe are labeled with MGB.

[0011] The present invention also provides an application of the primer pair and probe group in preparing a product for detecting animal Q fever, abortive chlamydia and brucellosis.

[0012] The present invention also provides a kit for detecting animal Q fever, abortive chlamydia and brucellosis, comprising the primer pair and probe group.

[0013] Preferably, the detection method of the kit is triple fluorescence PCR detection.

[0014] Preferably, the concentrations of the animal Q fever detection forward primer Q forward, animal Q fever detection reverse primer Q reverse, animal Q fever detection probe Q probe, abortion chlamydia detection forward primer AB forward, abortion chlamydia detection reverse primer AB reverse, abortion chlamydia detection probe AB probe, brucellosis detection forward primer BR forward, brucellosis detection reverse primer BR reverse, and brucellosis detection probe BR probe in the kit are independently 5 to 15 μmol / L.

[0015] The present invention also provides the use of the kit in detecting animal Q fever, abortive chlamydia and brucellosis for non-diagnostic or therapeutic purposes.

[0016] The present invention has the following technical effects and advantages:

[0017] (1) High sensitivity: The minimum detection limit for Coxiella burnetii nucleic acid, Chlamydia abortus nucleic acid and Brucella nucleic acid can reach 10.3 copies / μL.

[0018] (2) Strong specificity: It can specifically generate corresponding fluorescent amplification signals for the genomes of Coxiella burnetii, Chlamydia abortus and Brucella. It has been verified that it has no cross-reaction with relevant ruminant disease bacteria such as Mycobacterium tuberculosis, Francisella tularensis, Staphylococcus aureus, Listeria monocytogenes, and nucleic acid samples such as tissues and blood from healthy animals.

[0019] (3) Rapid and high-throughput: The detection speed is fast. The entire Taqman MGB triple fluorescence PCR detection process can be completed within 1 hour after adding the nucleic acid sample. It can simultaneously detect and identify Coxiella burnetii, Chlamydia abortus and Brucella, and can simultaneously detect up to 96 to 384 nucleic acid samples.

[0020] (4) Easy operation: All Taqman MGB triple fluorescence PCR tests and result determination can be completed using only a fluorescence quantitative PCR instrument, without the need for other technical means such as agarose gel electrophoresis to assist in identification. At the same time, the primer pairs and probe groups used are optimized based on sequence site differences, with low detection background values, making it easier to determine the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The sensitivity test results of the detection method for animal Q fever, abortion chlamydia and brucellosis are shown in Figure 1 to 7, which are the Taqman MGB triple fluorescence PCR amplification curves of plasmid pUC57-Q-AB-BR working standards 1 to 7, and 8 is the Taqman MGB triple fluorescence PCR amplification curve of the negative control nuclease-free water;

[0022] Figure 2 These are the specificity test results of the detection method for animal Q fever, abortive chlamydia and brucellosis. Figure 1 is the Taqman MGB triple fluorescence PCR amplification curve of the positive control plasmid pUC57-Q-AB-BR working standard 1, and Figures 2 to 7 are the Taqman MGB triple fluorescence PCR amplification curves of bovine tuberculosis Mycobacterium, Francisella tularensis, Staphylococcus aureus, Listeria nucleic acid, healthy bovine tissue, and healthy sheep whole blood nucleic acid samples. DETAILED DESCRIPTION

[0023] The invention provides a primer pair and a probe group for detecting animal Q fever, chlamydia aborti and brucellosis. The primer pair and the probe group comprise: an animal Q fever detection forward primer Q forward, an animal Q fever detection reverse primer Q reverse, an animal Q fever detection probe Q probe, an abortion chlamydia detection forward primer AB forward, an abortion chlamydia detection reverse primer AB reverse, an abortion chlamydia detection probe AB probe, a brucellosis detection forward primer BR forward, a brucellosis detection reverse primer BR reverse and a brucellosis detection probe BR probe;

[0024] The nucleotide sequence of the animal Q fever detection forward primer Q forward is shown in SEQ ID NO.2, the nucleotide sequence of the animal Q fever detection reverse primer Q reverse is shown in SEQ ID NO.3, the nucleotide sequence of the animal Q fever detection probe Qprobe is shown in SEQ ID NO.4, the nucleotide sequence of the abortion chlamydia disease detection forward primer AB forward is shown in SEQ ID NO.5, the nucleotide sequence of the abortion chlamydia disease detection reverse primer AB reverse is shown in SEQ ID NO.6, the nucleotide sequence of the abortion chlamydia disease detection probe AB probe is shown in SEQ ID NO.7, the nucleotide sequence of the brucellosis detection forward primer BR forward is shown in SEQ ID NO.8, the nucleotide sequence of the brucellosis detection reverse primer BR reverse is shown in SEQ ID NO.9, and the nucleotide sequence of the brucellosis detection probe BRprobe is shown in SEQ ID NO.10.

[0025] In the present invention, the 5' end of the animal Q fever detection probe Q probe is labeled with carboxyfluorescein FAM, the 5' end of the abortion chlamydia disease detection probe AB probe is labeled with tetrachlorohexacarboxyfluorescein VIC, the 5' end of the brucellosis detection probe BRprobe is labeled with cyanine dye CY5, and the 3' ends of the animal Q fever detection probe Q probe, the abortion chlamydia disease detection probe ABprobe, and the brucellosis detection probe BRprobe are labeled with a quenching group MGB.

[0026] The present invention also provides an application of the primer pair and probe group in preparing a product for detecting animal Q fever, abortive chlamydia and brucellosis.

[0027] The present invention also provides a kit for detecting animal Q fever, abortive chlamydia and brucellosis, comprising the primer pair and probe group.

[0028] In the present invention, the detection method of the kit is triple fluorescence PCR detection, preferably Taqman MGB triple fluorescence PCR detection.

[0029] In the present invention, the Taqman MGB triple fluorescence PCR detection comprises the following steps:

[0030] (1) constructing a detection system, wherein the detection system is 24.4 to 25.3 μL, preferably 25 μL, and comprises 12.5 μL of PrimxEx Taq fluorescent PCR reaction solution, 0.4 to 0.5 μL, preferably 0.5 μL, of each of an animal Q fever detection forward primer Qforward, an animal Q fever detection reverse primer Qreverse, an abortion chlamydia detection forward primer AB forward, an abortion chlamydia detection reverse primer ABreverse, a brucellosis detection forward primer BR forward, and a brucellosis detection reverse primer BR reverse, an animal Q fever detection probe Q probe, an abortion chlamydia detection probe AB probe, and a brucellosis detection probe BRprobe, preferably 0.4 to 0.5 μL, preferably 0.4 μL, a sample DNA template 2 μL, and a balance of ddH2O;

[0031] (2) performing Taqman MGB triple fluorescence PCR detection, wherein the reaction conditions of the Taqman MGB triple fluorescence PCR are: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and 40 cycles;

[0032] (3) signal collection, wherein the signal collection is to collect the carboxyfluorescein FAM, tetrachlorohexacarboxyfluorescein VIC and cyanine dye CY5 signals of the detection system of step (1) during the annealing of step (2);

[0033] (4) The test result is determined according to the threshold setting principle, wherein the threshold setting principle is that the threshold line just exceeds the highest point of the amplification curve of the negative control, the nucleic acid sample has no Ct value and no amplification curve, and is determined to be a negative result; the nucleic acid sample Ct value is ≤36.0, and a typical amplification curve appears, and is determined to be a positive result; the positive result is preferably when a carboxyfluorescein FAM signal appears, it is determined to be Coxiella burnetii nucleic acid positive, and the test result is determined to be animal Q fever; when a tetrachlorohexacarboxyfluorescein VIC signal appears, it is determined to be Chlamydia abortus nucleic acid positive, and the test result is determined to be Chlamydia abortus disease; when a cyanine dye CY5 signal appears, it is determined to be Brucella nucleic acid positive, and the test result is determined to be brucellosis.

[0034] In the present invention, the concentrations of the animal Q fever detection forward primer Q forward, animal Q fever detection reverse primer Q reverse, animal Q fever detection probe Q probe, abortion chlamydia detection forward primer AB forward, abortion chlamydia detection reverse primer AB reverse, abortion chlamydia detection probe AB probe, brucellosis detection forward primer BR forward, brucellosis detection reverse primer BR reverse, and brucellosis detection probe BR probe in the kit are independently 5 to 15 μmol / L, preferably 10 μmol / L.

[0035] The present invention also provides the use of the kit in detecting animal Q fever, abortive chlamydia and brucellosis for non-diagnostic or therapeutic purposes.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1: Design of nucleotide sequences of primer pairs and probe sets for detecting Q fever, Chlamydia abortus and Brucellosis in animals

[0038] The genome sequences of Coxiella burnetii, Chlamydia abortus, and Brucella were downloaded from NCBI. After sequence analysis using MEGA7 (version 7.0.21), the pUC57 plasmid vector was selected and the plasmid pUC57-Q-AB-BR was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The nucleotide sequence of the plasmid is shown in SEQ ID NO.1.

[0039] Based on the nucleotide sequence of plasmid pUC57-Q-AB-BR, primer pairs and probe sets for detecting animal Q fever, abortive chlamydia and brucellosis were designed using primer design software DNASTAR Lasergene, and the nucleotide sequences of each primer and probe are shown in Table 1. Among them, the 5' end of the animal Q fever detection probe Q probe is labeled with carboxyfluorescein FAM, the 5' end of the abortive chlamydia detection probe AB probe is labeled with tetrachlorohexacarboxyfluorescein VIC, the 5' end of the brucellosis detection probe BRprobe is labeled with cyanine dye CY5, and the 3' end of the animal Q fever detection probe Q probe, the abortive chlamydia detection probe AB probe, and the brucellosis detection probe BRprobe is labeled with a quenching group MGB.

[0040] Table 1 Primer pairs and probe set sequences for detecting animal Q fever, Chlamydia abortus and Brucellosis

[0041]

[0042]

[0043] Example 2: Detection Methods for Animal Q Fever, Chlamydia Abortus and Brucellosis

[0044] Construction of detection system: The detection system, in 25 μL, includes 12.5 μL of Primx Ex Taq fluorescent PCR reaction solution, 0.5 μL of animal Q fever detection forward primer Q forward at a concentration of 10 μmol / L, 0.5 μL of animal Q fever detection reverse primer Q reverse at a concentration of 10 μmol / L, 0.5 μL of abortion chlamydia detection forward primer ABforward at a concentration of 10 μmol / L, 0.5 μL of abortion chlamydia detection reverse primer AB reverse at a concentration of 10 μmol / L, 0.5 μL of brucellosis detection forward primer BR forward at a concentration of 10 μmol / L, 0.5 μL of brucellosis detection reverse primer BRreverse at a concentration of 10 μmol / L, 0.4 μL of animal Q fever detection probe Q probe at a concentration of 10 μmol / L, and 10 μmol / L abortion chlamydia detection probe AB probe 0.4μL, 10μmol / L Brucellosis detection probe BR probe 0.4μL, sample DNA template 2μL and the balance ddH2O.

[0045] The reaction conditions of Taqman MGB triple fluorescence PCR were set as follows: 95℃ pre-denaturation for 5min, followed by 95℃ denaturation for 10s, 60℃ annealing for 30s, and 40 cycles in total. The fluorescence signal of the detection system was collected during annealing. The Taqman MGB triple fluorescence PCR reaction was performed in a QuantStudio5 fluorescence quantitative PCR instrument (Applied Biosystems), and the fluorescence channels were set as follows: Channel 1: carboxyfluorescein FAM signal fluorescence channel, Channel 2: tetrachlorohexacarboxyfluorescein VIC signal fluorescence channel, and Channel 3: cyanine dye CY5 signal fluorescence channel.

[0046] Determination of test results: The test results were determined according to the threshold setting principle. The threshold setting principle was that the threshold line just exceeded the highest point of the negative control amplification curve. The nucleic acid sample had no Ct value and no amplification curve, and was determined to be a negative result. The nucleic acid sample had a Ct value ≤ 36.0 and a typical amplification curve, and was determined to be a positive result. When the carboxyfluorescein FAM signal appeared, it was determined to be Coxiella burnetii nucleic acid positive, and the test result was determined to be animal Q fever. When the tetrachlorohexacarboxyfluorescein VIC signal appeared, it was determined to be Chlamydia abortus nucleic acid positive, and the test result was determined to be Chlamydia abortus disease. When the cyanine dye CY5 signal appeared, it was determined to be Brucella nucleic acid positive, and the test result was determined to be brucellosis.

[0047] Example 3: Sensitivity test of the detection method for animal Q fever, abortive chlamydia and brucellosis

[0048] Plasmid pUC57-Q-AB-BR working standards 1 to 7 and negative control (nuclease-free water) were tested. The specific steps are as follows:

[0049] Preparation of plasmid pUC57-Q-AB-BR working standard: The initial concentration of plasmid pUC57-Q-AB-BR was determined to be 45.01 ng / μL, and the number of positive plasmid copies was 1.03×10 10 The plasmid pUC57-Q-AB-BR was gradiently diluted to obtain 7 plasmid pUC57-Q-AB-BR working standards, namely plasmid pUC57-Q-AB-BR working standard 1: the positive plasmid copy number was 1.03×10 6 copies / μL; plasmid pUC57-Q-AB-BR working standard 2: the number of positive plasmid copies is 1.03×10 5 copies / μL; plasmid pUC57-Q-AB-BR working standard 3: the number of positive plasmid copies is 1.03×10 4 copies / μL; plasmid pUC57-Q-AB-BR working standard 4: the number of positive plasmid copies is 1.03×10 3 copies / μL; plasmid pUC57-Q-AB-BR working standard 5: the number of positive plasmid copies is 1.03×10 2 copies / μL; plasmid pUC57-Q-AB-BR working standard 6: the positive plasmid copy number is 1.03×10 copies / μL; plasmid pUC57-Q-AB-BR working standard 7: the positive plasmid copy number is 1.03 copies / μL.

[0050] Sensitivity test: Plasmid pUC57-Q-AB-BR working standard products 1 to 7 were used as sample DNA templates, and nuclease-free water was used as a negative control. The method of Example 2 was used for detection. The results were as follows: Figure 1 shown.

[0051] The results showed that plasmid pUC57-Q-AB-BR working standards 1 to 6 showed typical amplification curves of FAM, VIC and CY5 signals, while plasmid pUC57-Q-AB-BR working standard 7 and negative control had no amplification curve. The results showed that the minimum detection limit of the detection method of the present invention for animal Q fever, abortion chlamydia and brucellosis was plasmid pUC57-Q-AB-BR working standard 6, and the sensitivity was 10.3 copies / μL.

[0052] Example 4: Specificity test of the detection method for animal Q fever, abortion chlamydia and brucellosis

[0053] The nucleic acid samples of bovine tuberculosis, Francisella tularensis, Staphylococcus aureus, Listeria monocytogenes, healthy bovine tissue, healthy sheep whole blood and the positive control (plasmid pUC57-Q-AB-BR working standard 1 described in Example 3) were tested in the following specific steps:

[0054] Specific sample preparation: Nucleic acid samples of bovine tuberculosis, Francisella tularensis, Staphylococcus aureus, Listeria monocytogenes, healthy bovine tissues, and healthy sheep whole blood were provided by the Zoonotic Disease Monitoring Office of the China Animal Health and Epidemiology Center. Tissue samples were first homogenized, and then nucleic acids were extracted according to the commercial tissue DNA extraction method; whole blood, serum, and plasma samples were lysed, magnetic bead enriched, washed, eluted, and other steps to extract nucleic acids; nucleic acid samples were stored at -20°C for later use.

[0055] Specificity test: The nucleic acid samples of bovine tuberculosis, Francisella tularensis, Staphylococcus aureus, Listeria monocytogenes, healthy bovine tissues, and healthy sheep whole blood were used as sample DNA templates, and the plasmid pUC57-Q-AB-BR working standard 1 was used as a positive control. The method of Example 2 was used for detection. The results are as follows: Figure 2 shown.

[0056] The results showed that the positive control showed typical amplification curves of FAM, VIC and CY5 signals, and no amplification signals were found in the nucleic acid samples of bovine tuberculosis, Francisella tularensis, Staphylococcus aureus and Listeria, and no amplification signals were found in the nucleic acid samples of healthy bovine tissues and healthy sheep whole blood. The results showed that the detection method of the present invention had no cross-reaction to the nucleic acid samples of the relevant ruminant disease bacteria and healthy animals, and had good specificity.

[0057] Example 5: Repeatability test of the detection method for animal Q fever, abortion chlamydia and brucellosis

[0058] The plasmid pUC57-Q-AB-BR working standards 1 to 6 described in Example 3 were tested three times using the method of Example 2, and the inter-batch coefficient of variation was calculated; the same batch of Taqman MGB triple fluorescence PCR detection was tested three times, and the intra-batch coefficient of variation was calculated. The results are shown in Table 2.

[0059] The coefficient of variation CV% is calculated as follows: CV%=(standard deviation SD / mean value Mean)×100%.

[0060] Table 2 Results of repeatability tests for detection methods of animal Q fever, abortive chlamydia and brucellosis

[0061]

[0062] The results showed that the inter-batch repeatability test result was 0.15%≤inter-batch variation coefficient≤10.21%, and the intra-batch repeatability test result was 0.35%≤intra-batch variation coefficient≤8.88%. The results showed that the detection method of the present invention has good repeatability.

[0063] Example 6: Clinical sample testing

[0064] The method of Example 2 was used to detect clinical samples such as whole blood, saliva, and tissue collected from cattle and sheep suspected of being infected with Q fever, abortive chlamydia, and / or brucellosis. The plasmid pUC57-Q-AB-BR working standard 1 described in Example 3 was used as a positive control, and nuclease-free water was used as a negative control. The Taqman MGB triple fluorescent PCR reaction products were sequenced and identified. The specific steps are as follows:

[0065] Clinical sample preparation: A total of 81 clinical samples of whole blood, saliva, tissues, etc. from cattle and sheep suspected of being infected with animal Q fever, abortive chlamydia and / or brucellosis were collected and provided by the Inner Mongolia Autonomous Region Animal Disease Prevention and Control Center. Tissue samples were first homogenized and then nucleic acids were extracted according to the commercial tissue DNA extraction method; whole blood, serum, and plasma samples were lysed, magnetic bead enriched, washed, eluted, and other steps were used to extract nucleic acids; nucleic acid samples were stored at -20°C for future use.

[0066] Clinical sample detection: 81 nucleic acid samples of clinical specimens were used as sample DNA templates, the plasmid pUC57-Q-AB-BR working standard 1 described in Example 3 was used as a positive control, and nuclease-free water was used as a negative control. The method of Example 2 was used for detection, and the Taqman MGB triple fluorescence PCR reaction products were sequenced and identified.

[0067] The results showed that the detection method of the present invention detected 13 positive results of animal Q fever, 8 positive results of abortion chlamydia, 7 positive results of brucellosis, and 53 negative results. All Taqman MGB triple fluorescence PCR reaction products with positive results were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results were verified by BLAST comparison in GenBank. It was verified that the verification results of all nucleic acid samples were consistent with the judgment results of Taqman MGB triple fluorescence PCR detection, with an accuracy of 100%.

[0068] As can be seen from the above embodiments, the present invention provides a primer pair and probe set, a kit and application thereof for detecting animal Q fever, abortion chlamydia and brucellosis. According to the primer pair and probe set, the kit and the detection method of the present invention, animal Q fever, abortion chlamydia and brucellosis are detected, and the detection accuracy is 100%, with the advantages of high accuracy, high sensitivity, strong specificity, rapid high throughput, simple operation, etc., and can simultaneously detect and identify three common animal Q fever, abortion chlamydia and brucellosis that cause abortion in my country.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer pair and probe set for detecting Q fever, abortive chlamydia and brucellosis in animals, characterized in that: The primer pair and probe group include: animal Q fever detection forward primer Q forward, animal Q fever detection reverse primer Q reverse, animal Q fever detection probe Q probe, abortion chlamydia detection forward primer AB forward, abortion chlamydia detection reverse primer AB reverse, abortion chlamydia detection probe AB probe, brucellosis detection forward primer BR forward, brucellosis detection reverse primer BR reverse and brucellosis detection probe BR probe; The nucleotide sequence of the animal Q fever detection forward primer Q forward is shown in SEQ ID NO.2, the nucleotide sequence of the animal Q fever detection reverse primer Q reverse is shown in SEQ ID NO.3, the nucleotide sequence of the animal Q fever detection probe Q probe is shown in SEQ ID NO.4, the nucleotide sequence of the abortion chlamydia disease detection forward primer AB forward is shown in SEQ ID NO.5, the nucleotide sequence of the abortion chlamydia disease detection reverse primer AB reverse is shown in SEQ ID NO.6, the nucleotide sequence of the abortion chlamydia disease detection probe AB probe is shown in SEQ ID NO.7, the nucleotide sequence of the brucellosis detection forward primer BR forward is shown in SEQ ID NO.8, the nucleotide sequence of the brucellosis detection reverse primer BR reverse is shown in SEQ ID NO.9, and the nucleotide sequence of the brucellosis detection probe BRprobe is shown in SEQ ID NO.

10.

2. The primer pair and probe set according to claim 1, characterized in that: The 5' end of the animal Q fever detection probe Q probe is labeled with FAM, the 5' end of the abortion Chlamydia disease detection probe AB probe is labeled with VIC, the 5' end of the brucellosis detection probe BRprobe is labeled with CY5, and the 3' ends of the animal Q fever detection probe Q probe, the abortion Chlamydia disease detection probe ABprobe, and the brucellosis detection probe BRprobe are labeled with MGB.

3. Use of the primer pair and probe set according to claim 1 or 2 in the preparation of products for detecting animal Q fever, abortive chlamydia and brucellosis.

4. A kit for detecting animal Q fever, abortive chlamydia and brucellosis, characterized in that: Comprising the primer pair and probe set according to claim 1 or 2.

5. The kit according to claim 4, characterized in that The detection method of the kit is triple fluorescence PCR detection.

6. The kit according to claim 5, characterized in that The concentrations of the animal Q fever detection forward primer Qforward, the animal Q fever detection reverse primer Q reverse, the animal Q fever detection probe Q probe, the abortion chlamydia detection forward primer AB forward, the abortion chlamydia detection reverse primer AB reverse, the abortion chlamydia detection probe ABprobe, the brucellosis detection forward primer BR forward, the brucellosis detection reverse primer BRreverse, and the brucellosis detection probe BRprobe in the kit are independently 5 to 15 μmol / L.

7. Use of the kit according to any one of claims 4 to 6 for detecting Q fever, Chlamydia abortus and Brucellosis in animals for non-diagnostic or therapeutic purposes.

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