Primer group and kit for detecting four bacterial abortion diseases of sheep

Abstract

The invention provides a primer group and a kit for detecting four bacterial abortion diseases of sheep, belongs to the technical field of biological detection, establishes a multiplex PCR (polymerase chain reaction) detection method for sheep brucellosis, sheep abortion salmonellosis, listeriosis and chlamydia disease and a kit thereof, and has the characteristics of high sensitivity, strong specificity, simplicity and convenience in operation, short time consumption and the like. The rapid differential diagnosis of four sheep bacterial abortion diseases can be realized through one-time PCR amplification, and the method has important advantages in the field of sheep disease diagnosis and identification.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to primer sets and kits for detecting four bacterial abortion diseases in sheep. Background Technology

[0002] Brucella ( Brucella, Bru Salmonella in sheep abortion ( Salmonella Abortus ovis, Sal Listeria monocytogenes ( ) Listeria monocytogenes, Li ), Chlamydia psittaci ( Chlamydia psittaci, Chl All of these diseases are characterized by symptoms such as abortion, stillbirth, and weak lambs in pregnant ewes. Among them, *Brucella melitensis*, *Listeria monocytogenes*, and *Chlamydia trachomatis* are all zoonotic diseases, causing infection and illness in various animals and humans. Brucellosis (… Brucellosis Chlamydia is a disease caused by Brucella bacteria, characterized by infertility, miscarriage, retained placenta, and inflammation of the reproductive organs and fetal membranes. Chlamydiosis Chlamydia psittaci is an infectious disease characterized by fever, abortion, conjunctivitis, and arthritis, caused by Chlamydia psittaci. It mainly affects adult goats over 2 years old; rams are not infected. Listeria monocytogenes is a small, Gram-positive bacillus, most susceptible to lambs and pregnant ewes, exhibiting significant seasonal epidemicity. Typical symptoms include meningoencephalitis, septicemia, and abortion in pregnant ewes. Salmonella ovis is a Gram-negative, short bacillus with blunt ends, which can cause abortion or stillbirth in ewes during late pregnancy, with abortion and mortality rates reaching up to 60%.

[0003] In sheep reproductive disorders, mixed infections of multiple bacteria are common, and it is difficult to make a diagnosis based solely on clinical symptoms. Traditional diagnostic methods such as bacterial isolation and identification, serological and immunological methods have drawbacks such as cumbersome operation, long detection cycle, low sensitivity, poor specificity, and easy spread of the virus, and therefore cannot meet the requirements of large-scale clinical testing.

[0004] Molecular biology detection techniques, with their advantages of high sensitivity and specificity, have seen rapid development and widespread application in clinical testing. Multiplex PCR, also known as multiplex primer PCR or complex PCR, involves adding two or more pairs of primers to the same reaction system to simultaneously amplify multiple gene fragments, enabling the diagnosis of various pathogens or genotypes. Compared to singlex PCR and quantitative real-time PCR, this method offers advantages such as simple operation, shorter processing time, high sensitivity, high specificity, and lower detection costs, making it more suitable for clinical use and promotion. Currently, a quadruple PCR detection method that can simultaneously detect ovine brucellosis, ovine aborted salmonellosis, listeriosis, and chlamydia has not been reported. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a primer set and kit for detecting four bacterial abortion diseases in sheep.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a primer set for detecting four bacterial abortion diseases in sheep, including Bru-F, Bru-R, Sal-F, Sal-R, Li-F, Li-R, Chl-F, and Chl-R; The nucleotide sequence of the Bru-F is shown in SEQ ID NO.1; The nucleotide sequence of the Bru-R is shown in SEQ ID NO.2; The nucleotide sequence of the Sal-F is shown in SEQ ID NO.3; The nucleotide sequence of the Sal-R is shown in SEQ ID NO.4; The nucleotide sequence of the Li-F is shown in SEQ ID NO.5; The nucleotide sequence of the Li-R is shown in SEQ ID NO.6; The nucleotide sequence of Chl-F is shown in SEQ ID NO.7; The nucleotide sequence of Chl-R is shown in SEQ ID NO.8.

[0007] This invention provides the application of the primer set described herein in the preparation of a kit for detecting Brucella, Salmonella abortus, Listeria, and Chlamydia psittaci.

[0008] This invention provides a kit for detecting Brucella, Salmonella abortus, Listeria, or Chlamydia psittaci, respectively, comprising the aforementioned primer set; The primers for detecting Brucella include Bru-F and Bru-R; The primers for detecting Salmonella abortus in sheep include Sal-F and Sal-R; The primers for detecting Listeria include Li-F and Li-R; The primers for detecting Chlamydia psittaci include Chl-F and Chl-R.

[0009] This invention provides a kit for the simultaneous detection of Brucella, Salmonella abortus, Listeria, and Chlamydia psittaci, comprising the aforementioned primer set.

[0010] Preferably, the kit further includes bacterial DNA extraction reagent, PCR amplification reagent, positive control and negative control; The positive control comprises nucleic acids with nucleotide sequences as shown in SEQ ID NO. 9-12; the negative control comprises DEPC water.

[0011] Preferably, the method of using the kit includes the following steps: 1) Using the genomic DNA of the sample to be tested as a template, perform multiplex PCR amplification using the primer set or the kit to obtain PCR products; 2) The PCR products were electrophoresed on an agarose gel. When the amplified product fragment contained 713 bp of nucleic acid, Brucella was positive in the sample; when the amplified product fragment contained 206 bp of nucleic acid, Salmonella abortus was positive in the sample; when the amplified fragment contained 356 bp of nucleic acid, Listeria was positive in the sample; and when the amplified fragment contained 566 bp of nucleic acid, Chlamydia psittaci was positive in the sample.

[0012] Preferably, the reaction program for the multiplex PCR amplification is as follows: 95 ℃ for 3 min, 94 ℃ for 30 s, 52 ℃~56.3 ℃ for 30 s, 72 ℃ for 30 s, 35 cycles, followed by an extension at 72 ℃ for 10 min.

[0013] Preferably, the reaction system for the multiplex PCR amplification consists of 8-12 μL of 2x Taq mix, 1-4 μL of the primer set, 1-3 μL of genomic DNA from the sample to be tested, and 4-8 μL of DEPC water.

[0014] Preferably, the final concentration of Bru-F in the reaction system is 0.1~0.2 μM; The final concentration of Bru-R in the reaction system was 0.1–0.2 μM; The final concentration of Sal-F in the reaction system is 0.3~1.0 μM; The final concentration of Sal-F in the reaction system is 0.3~1.0 μM; The final concentration of Chl-F in the reaction system was 0.03~0.1 μM; The final concentration of Chl-R in the reaction system was 0.03–0.1 μM; The final concentration of Li-F in the reaction system was 0.03~0.1 μM; The final concentration of Li-R in the reaction system is 0.03~0.1 μM.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The multiplex PCR detection method and kit for ovine brucellosis, ovine abortive salmonellosis, listeriosis and chlamydia are established in this invention. They have the characteristics of high sensitivity, strong specificity, simple operation and short time consumption. They can realize the rapid identification and diagnosis of four ovine bacterial abortion diseases with a single PCR amplification, which has important advantages in the field of ovine disease diagnosis and identification. Attached Figure Description

[0016] Figure 1 This represents the singlet PCR reaction of Brucella mesenteriae, where A is the determination of the optimal annealing temperature for Bruce singlet PCR; B is the determination of the optimal primer concentration for Bruce singlet PCR; C is the determination of the sensitivity of Bruce singlet PCR; D is the determination of the optimal specificity of Bruce singlet PCR; M: DNA marker, with values ​​of 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp, respectively.

[0017] Figure 2 This represents the singlet PCR reaction of Listeria monocytogenes, where A is the determination of the optimal annealing temperature for Li singlet PCR; B is the determination of the optimal primer concentration for Li singlet PCR; C is the determination of the sensitivity of Li singlet PCR; D is the determination of the optimal specificity of Li singlet PCR; M: DNA marker, with values ​​of 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp, respectively.

[0018] Figure 3 This is a singleton PCR reaction for Chlamydia psittaci. A represents the determination of the optimal annealing temperature for Chl singleton PCR; B represents the determination of the optimal primer concentration for Chl singleton PCR; C represents the sensitivity determination for Chl singleton PCR; D represents the optimal specificity determination for Chl singleton PCR; M: DNA marker, with values ​​of 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

[0019] Figure 4 This section describes the singlet PCR reaction for Salmonella abortion in sheep. A represents the determination of the optimal annealing temperature for Sal singlet PCR; B represents the determination of the optimal primer concentration for Sal singlet PCR; C represents the sensitivity determination for Sal singlet PCR; D represents the optimal specificity determination for Sal singlet PCR; M represents the DNA marker, with values ​​of 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

[0020] Figure 5 Multiplex PCR reactions were performed for Brucella mesenteriae, Listeria monocytogenes, Chlamydia psittaci, and Salmonella abortus. A represents the determination of the optimal annealing temperature for multiplex PCR; B represents the determination of the optimal primer concentration for multiplex PCR; C represents the determination of the sensitivity of multiplex PCR; D represents the determination of the optimal specificity of multiplex PCR; M represents the DNA marker, with values ​​of 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. Detailed Implementation

[0021] This invention provides a primer set for detecting four bacterial abortion diseases in sheep, including Bru-F, Bru-R, Sal-F, Sal-R, Li-F, Li-R, Chl-F, and Chl-R; The nucleotide sequence of the Bru-F is shown in SEQ ID NO.1; SEQ ID NO.1: 5'-GCAATTTTCTCGCAGCCTCAT-3'; The nucleotide sequence of the Bru-R is shown in SEQ ID NO.2; SEQ ID NO.2: 5'-GACCGATACGTTATAGCTGTT-3'; The nucleotide sequence of the Sal-F is shown in SEQ ID NO.3; SEQ ID NO.3: 5'-CCGCCCGTAACTATATTT-3'; The nucleotide sequence of the Sal-R is shown in SEQ ID NO.4; SEQ ID NO.4: 5'-CTAATGGTATACACCGGCG-3'; The nucleotide sequence of the Li-F is shown in SEQ ID NO.5; SEQ ID NO.5: 5'-AGACCTTCCAGATTTTTCGG-3'; The nucleotide sequence of the Li-R is shown in SEQ ID NO.6; SEQ ID NO.6: 5'-ACTCCTGGTGTTCTCGATTAA-3'; The nucleotide sequence of Chl-F is shown in SEQ ID NO.7; SEQ ID NO.7: 5'-CGATGGCACTATGTGGGAAGG-3'; The nucleotide sequence of Chl-R is shown in SEQ ID NO.8; SEQ ID NO. 8: 5'-TAGATTGAGCGTATTGGAACT-3'.

[0022] This invention provides the application of the primer set described herein in the preparation of a kit for detecting Brucella, Salmonella abortus, Listeria, and Chlamydia psittaci.

[0023] This invention provides a kit for detecting Brucella, Salmonella abortus, Listeria, or Chlamydia psittaci, respectively, comprising the aforementioned primer set; The primers for detecting Brucella include Bru-F and Bru-R; The primers for detecting Salmonella abortus in sheep include Sal-F and Sal-R; The primers for detecting Listeria include Li-F and Li-R; The primers for detecting Chlamydia psittaci include Chl-F and Chl-R.

[0024] This invention provides a kit for the simultaneous detection of Brucella, Salmonella abortus, Listeria, and Chlamydia psittaci, comprising the aforementioned primer set.

[0025] In this invention, the kit preferably further includes bacterial DNA extraction reagent, PCR amplification reagent, positive control and negative control; The positive control comprises nucleic acids with nucleotide sequences as shown in SEQ ID NO. 9-12; the negative control comprises DEPC water.

[0026] SEQ ID NO.9 (Brucella positive control DNA sequence 713 bp): GCAATTTTCTCGCAGCCTCATTTTCCACAATCATGCTCGTCGGCGCTTTCAGCCTGCCCGCTTTCGCACAGGAGAATCAGATGACGACGCAGCCCGCGCGCATCGCCGTCACCGGGGAAGGCATGATGACGGCCTCGCCCGATATGGCCATTCTCAATCTCTCGGTGCTACGCCAGGCAAAGACCGCGCGCGAAGCCATGACCGCGAATAATGAAGCCATGACAAAAGTGCTCGATGCCATGAAGAAGGCCGGCATCGAAGATCGCGATCTCCAGACAGGCGGCATCAATATCCAGCCGATTTATGTCTATCCTGACGACAAGAACAACCTGAAAGAGCCTACCATCACCGGCTATTCTGTATCCACCAGTCTCACGGTTCGCGTGCGCGAACTGGCCAATGTTGGAAAAATTTTGGATGAATCCGTCACGCTCGGTGTTAATCAGGGCGGTGATTTGAACCTGGTCAATGATAATCCCTCTGCCGTGATCAACGAGGCGCGCAAGCGCGCAGTGGCCAATGCCATTGCCAAGGCGAAGACGCTTGCCGACGCTGCAGGCGTGGGGCTTGGCCGTGTGGTGGAAATCAGTGAACTGAGCCGCCCGCCCATGCCGATGCCAATTGCGCGCGGACAGTTCAGAACCATGCTAGCAGCCGCACCGGACAATTCCGTGCCGATTGCCGCAGGCGAAAACAGCTATAACGTATCGGTC SEQ ID NO.10 (Salmonella abortusovis positive control DNA sequence, 430 bp, the amplified fragment of 206 bp is underlined): AATGACTACCTTCTGGGCGCGATGAGCATCACCGATAGTGTCGCTGGCGTATTTTAT CCGCCCCGTAAC TATATTTGCATGGGCGTCGACTCTAACGTGTCGCAGCAAAAGCCGTTTGGCGTGCAGGACTCAAAGCTGGTTTTTAA ATTAAAAGTGATACGGCCTTTTATTAATATGGTGACGATCCCCCGCCAGACAATGTTTACCGTCTATGTGACGACCT CTACCGGCGACGCGTTGAGCACGCCGGTGTATACCATTAGCTACAGCGGCAAAGTGGAAGTACCGCAAAACTGTGAAGTGAATGCCGGACAGGTCGTGGAGTTTGATTTCGGCGATATCGGCGCGTCGTTATTTAGTCAGGCGGGAGCGGGTAATCGTCCGCAAGGCGTCACGCCGCAAGCGAAAACTATCGCTATCAAATGTACCA SEQ ID NO.11 (Listeria monocytogenes positive control DNA sequence 420 bp, underlined position is the 356 bp amplified fragment): AATGAACCTACA AGACCTTCCAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGA GTAAATGCAGAAAATCCTCCTGCATATATCTCAAGTGTGGCATACGGCCGTCAAGTTTATTTGAAATTATCGACTAA TTCCCATAGTACTAAAGTAAAAGCTGCTTTTGATGCTGCCGTAAGTGGGAAATCTGTCTCAGGTGATGTAGAATTAA CAAATATCATCAAAAATTCTTCCTTCAAAGCCGTAATTTACGGTGGTTCCGCAAAAGATGAAGTTCAAATCATCGAT GGCAACCTCGGAGACTTACGAGATATTTTGAAAAAAGGTGCTACTTTTAATCGAGAAACACCAGGAGT TCCCATTGCTTATACAACAAATTTCTTAAAAGACAATGAATTAGCTGTTATT SEQ ID NO.12 (580 bp DNA sequence of Chlamydia psittaci positive control, with the underlined position being the amplified fragment at 566 bp): TTATTAAT CGATGGCACTATGTGGGAAGGTGCTTCAGGAGATCCTTGCGATCCTTGCGCTACTTGGTGT GACGCCATTAGCATCCGCGCAGGATACTACGGAGATTATGTTTTCGATCGTGTATTAAAAGTTGATGTGAATAAAAC TTTTAGCGGCATGGCTGCAACTCCTACGCAGGCTACAGGTAACGCAAGTAATACTAATCAGCCAGAAGCAAATGGCA GACCGAACATCGCTTACGGAAGGCATATGCAAGATGCAGAGTGGTTTTCAAATGCAGCCTTCCTAGCCTTAAACATT TGGGATCGCTTCGACATTTTCTGCACCTTAGGGGCATCCAATGGATACTTCAAATCAAGTTCGGCTGCATTCAACTT GGTTGGGTTAATAGGGTTTTCAGCTACCAGCTCAACCTCTACCGAGCTTCCAATGCAACTTCCTAACGTAGGCATTA CCCAAGGTGTTGTGGAATTTTATACAGACACATCATTTTCTTGGAGCGTAGGTGCACGTGGAGCTTTATGGGAATGT GGTTGTGCAACTTTAGGAGCTGAGTTCCAATACGCTCAATCTA ATCCTA In this invention, the method of using the reagent kit includes the following steps: 1) Using the genomic DNA of the sample to be tested as a template, perform multiplex PCR amplification using the primer set or the kit to obtain PCR products; 2) The PCR products were electrophoresed on an agarose gel. When the amplified product fragment contained 713 bp of nucleic acid, Brucella was positive in the sample; when the amplified product fragment contained 206 bp of nucleic acid, Salmonella abortus was positive in the sample; when the amplified fragment contained 356 bp of nucleic acid, Listeria was positive in the sample; and when the amplified fragment contained 566 bp of nucleic acid, Chlamydia psittaci was positive in the sample.

[0027] In this invention, the preferred reaction program for the multiplex PCR amplification is: 95 ℃ for 3 min, 94 ℃ for 30 s, 52 ℃~56.3 ℃ for 30 s, 72 ℃ for 30 s, followed by 35 cycles, and then an extension at 72 ℃ for 10 min. The preferred annealing temperature is 52 ℃~56.3 ℃, and more preferably 52 ℃.

[0028] In this invention, the reaction system for the multiplex PCR amplification is as follows: The preferred amount of 2x Taq mix added is 8~12 μL, more preferably 9~11 μL, and even more preferably 10 μL; The primer set is preferably 1~4 μL, more preferably 1.5~3.5 μL, and even more preferably 2 μL; The genomic DNA of the sample to be tested is preferably 1~3 μL, more preferably 1.5~2.5 μL, and even more preferably 2 μL; The preferred amount of DEPC water added is 4~8 μL, more preferably 5~7 μL, and even more preferably 6 μL.

[0029] In this invention, the final concentration of Bru-F in the reaction system is preferably 0.1~0.2 μM, more preferably 0.11~0.15 μM, and even more preferably 0.125 μM; The final concentration of Bru-R in the reaction system is preferably 0.1~0.2 μM, more preferably 0.11~0.15 μM, and even more preferably 0.125 μM; The final concentration of Sal-F in the reaction system is preferably 0.3~1.0 μM, more preferably 0.5~0.8 μM, and even more preferably 0.625 μM; The final concentration of Sal-F in the reaction system is preferably 0.3~1.0 μM, more preferably 0.5~0.8 μM, and even more preferably 0.625 μM; The final concentration of Chl-F in the reaction system is preferably 0.03~0.1 μM, more preferably 0.05~0.08 μM, and even more preferably 0.0625 μM; The final concentration of Chl-R in the reaction system is preferably 0.03~0.1 μM, more preferably 0.05~0.08 μM, and even more preferably 0.0625 μM; The final concentration of Li-F in the reaction system is preferably 0.03~0.1 μM, more preferably 0.05~0.08 μM, and even more preferably 0.0625 μM; The final concentration of Li-R in the reaction system is preferably 0.03~0.1 μM, more preferably 0.05~0.08 μM, and even more preferably 0.0625 μM.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Source of materials

[0032] The bacterial genome extraction kit was manufactured by Tiangen Biotech Co., Ltd. DNA molecular weight standard and 2x Taq mix were purchased from Takara Bio Engineering (Dalian) Co., Ltd. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0033] Example 1

[0034] Brucella, Listeria, Chlamydia psittaci, and Salmonella abortus in sheep singleton PCR reaction

[0035] Using a bacterial genome extraction kit, DNA was extracted from inactivated Brucella, Listeria, Chlamydia psittaci, and Salmonella abortus. The DNA concentrations were determined to be 330 ng / μL for Brucella, 488 ng / μL for Listeria, 517 ng / μL for Chlamydia psittaci, and 492 ng / μL for Salmonella abortus. The samples were stored at -20 ℃ for later use.

[0036] Using the primers shown in Table 1, singlet PCR amplification was performed with the whole genomes of Brucella, Listeria, Chlamydia psittaci, and Salmonella abortus as templates, respectively. The obtained PCR products were electrophoresed using a 1.5% agarose gel, and the PCR amplification results were observed using a PCR gel imaging system.

[0037] Table 1 Primer Sequences

[0038] (1.1) Bru singlet PCR

[0039] The amount of upstream and downstream primers added was studied. The reaction system was set at 20 μL, including 10 μL of 2x Taq mix, 0.25~1 μL (10 μM) of upstream and downstream primers, 0.5 μL of DNA, and sterile water added to a final volume of 20 μL. The results showed that ( Figure 1 The optimal reaction system for Bru singlet PCR is 10 μL of 2x Taq mix, 0.5 μL (10 μM) of upstream and downstream primers, 0.5 μL of DNA, and 20 μL of sterile water. The effect of annealing temperature on amplification efficiency was investigated. The reaction program was set as follows: 95 ℃ for 3 min, 94 ℃ for 30 s, 52–60 ℃ for 30 s, 72 ℃ for 1 min, 35 cycles, followed by a 10-min extension at 72 ℃, and storage at 4 ℃. The target fragment was approximately 713 bp. The results showed that the optimal reaction program for Bru singlet PCR was 95 ℃ for 3 min, 94 ℃ for 30 s, 54 ℃ for 30 s, 72 ℃ for 1 min, 35 cycles, followed by a 10-min extension at 72 ℃.

[0040] Sensitivity testing results showed that the minimum detectable concentration for this reaction was 1 pg / μL.

[0041] Specific detection results showed that Pasteurella multocida (Pas), Escherichia coli (coli), Salmonella, Listeria, Chlamydia psittaci, and the negative control all showed no bands, while Brucella showed a clear target band, indicating that the PCR had good specificity.

[0042] (1.2) Sal single PCR

[0043] The amount of upstream and downstream primers added was studied. The reaction system was 20 μL, including 10 μL of 2x Taq mix, 0.5–1.5 μL (10 μM) of upstream and downstream primers, 0.5 μL of DNA, and sterile water added to a final volume of 20 μL. The results showed that ( Figure 4 The optimal reaction system for Sal singlet PCR is 10 μL of 2x Taq mix, 1.5 μL (10 μM) of upstream and downstream primers, 0.5 μL of DNA, and 20 μL of sterile water.

[0044] The effect of annealing temperature on amplification efficiency was investigated. The optimal reaction program was: 95℃ for 3 min, 94℃ for 30 s, 52℃~60℃ for 30 s, 72℃ for 1 min, 35 cycles, followed by a 10-min extension at 72℃, and storage at 4℃. The target fragment was approximately 206 bp. The results showed that the optimal reaction program for Sal singlet PCR was 95℃ for 3 min, 94℃ for 30 s, 56.3℃ for 30 s, 72℃ for 1 min, 35 cycles, followed by a 10-min extension at 72℃.

[0045] Sensitivity testing results showed that the minimum detectable concentration for this reaction was 10 pg / μL.

[0046] Specific detection results showed that Pasteurella multocida (Pas), Escherichia coli (coli), Chlamydia psittaci, Brucella, Listeria, and the negative control all showed no bands, while Salmonella showed a clear target band, indicating that the PCR had good specificity.

[0047] (1.3) Li singlet PCR

[0048] The study investigated the amount of upstream and downstream primers added. The reaction system was set at 20 μL, including 10 μL of 2x Taq mix, 0.5–1 μL (10 μM) of upstream and downstream primers, 0.5 μL of DNA, and sterile water added to a final volume of 20 μL. The results showed that ( Figure 2 The optimal reaction system for Li singlet PCR is 10 μL of 2x Taq mix, 1 μL (10 μM) of upstream and downstream primers, 0.5 μL of DNA, and 20 μL of sterile water.

[0049] The effect of annealing temperature on amplification efficiency was investigated. The reaction program was set as follows: 95 ℃ for 3 min, 94 ℃ for 30 s, 52–61 ℃ for 30 s, 72 ℃ for 1 min, 35 cycles, followed by a 10-min extension at 72 ℃, and storage at 4 ℃. The target fragment was approximately 356 bp. The results showed that the optimal reaction program for Li singlet PCR was 95 ℃ for 3 min, 94 ℃ for 30 s, 54 ℃ for 30 s, 72 ℃ for 1 min, 35 cycles, followed by a 10-min extension at 72 ℃.

[0050] Sensitivity testing results showed that the minimum detectable concentration for this reaction was 1 pg / μL.

[0051] (1.4) Chl singleton PCR

[0052] The reaction mixture was 20 μL, containing 10 μL of 2x Taq mix, 0.5–1 μL (10 μM) of upstream and downstream primers, 0.5 μL of DNA, and sterile water to a final volume of 20 μL. Results showed that ( Figure 3 The optimal reaction system for Chl singlet PCR is 10 μL of 2x Taq mix, 0.5 μL (10 μM) of upstream and downstream primers, 0.5 μL of DNA, and 20 μL of sterile water.

[0053] The optimal reaction program was: 95 ℃ for 3 min, 94 ℃ for 30 s, 52 ℃~58.3 ℃ for 30 s, 72 ℃ for 1 min, 35 cycles, followed by a 10-min extension at 72 ℃, and storage at 4 ℃. The target fragment was approximately 566 bp. The results showed that the optimal reaction program was: 95 ℃ for 3 min, 94 ℃ for 30 s, 56.3 ℃ for 30 s, 72 ℃ for 1 min, 35 cycles, followed by a 10-min extension at 72 ℃, and storage at 4 ℃.

[0054] Sensitivity testing results showed that the minimum detectable concentration for this reaction was 0.1 pg / μL.

[0055] Specific detection results showed that Pasteurella multocida (Pas), Escherichia coli (coli), Salmonella, Brucella, Listeria, and the negative control all showed no bands, while Chlamydia psittaci showed a clear target band, indicating that the PCR had good specificity.

[0056] Example 2

[0057] The above four primer pairs were mixed, and the genomes of Brucella, Listeria, Chlamydia psittaci, and Salmonella abortus were extracted by mixing equal amounts of each. The genomes were then used as templates for multiplex PCR amplification. The obtained PCR products were electrophoresed using a 1.5% agarose gel, and the PCR amplification results were observed using a PCR gel imaging system.

[0058] (2.1) Primer preparation

[0059] According to Example 1, four pairs of primers were mixed, and the optimal primer concentrations were Bru 2.5 μM, Sal 12.5 μM, Chl 1.25 μM, and Li 1.25 μM.

[0060] (2.2) Reaction system

[0061] The reaction mixture was 20 μL, containing 10 μL of 2x Taq mix, 0.5–2 μL of forward and reverse primers, 2 μL of DNA, and sterile water to a final volume of 20 μL. Results showed ( Figure 5 The optimal reaction system for multiplex PCR is 10 μL of 2x Taq mix, 0.5 μL of upstream and downstream primers, 2 μL of DNA, and sterile water to a final volume of 20 μL.

[0062] (2.3) Optimal response procedure

[0063] The optimal reaction program for multiplex PCR was 95 ℃ for 3 min, 94 ℃ for 30 s, 52 ℃~56.3 ℃ for 30 s, and 72 ℃ for 30 s, followed by a 10-min extension at 72 ℃ and storage at 4 ℃. The target fragments for Brucella, Chlamydia psittaci, Listeria, and Salmonella were 713 bp, 566 bp, 356 bp, and 206 bp, respectively. The results indicate that the optimal reaction program for multiplex PCR is 95 ℃ for 3 min, 94 ℃ for 30 s, 52 ℃ for 30 s, and 72 ℃ for 30 s, followed by a 10-min extension at 72 ℃.

[0064] (2.4) Sensitivity detection of multiplex PCR method

[0065] The mixed sample was serially diluted, and the sensitivity of the reaction was determined according to the reaction conditions in (2.2) and (2.3). The results showed that the minimum detectable concentration for this reaction was 10 pg / μL.

[0066] (2.5) Specificity detection of multiplex PCR detection method

[0067] Specific assay results showed that Pasteurella multocida ( Pasteurella multocida,Pas ), Escherichia coli ( Escherichia coli,coli No bands were observed in either the positive control or the negative control; clear target bands were visible in Salmonella, Chlamydia psittaci, Brucella, and Listeria, indicating that the PCR had good specificity.

[0068] Using the optimal reaction procedure: 95 ℃ for 3 min, 94 ℃ for 30 s, 52 ℃ for 30 s, 72 ℃ for 30 s, for 35 cycles, followed by an extension at 72 ℃ for 10 min to obtain the PCR product. The PCR product was immediately subjected to 1.5% agarose gel electrophoresis. When the amplified product fragment was 713 bp, the sample was positive for Brucella; when the amplified fragment was 566 bp, the sample was positive for Chlamydia psittaci; when the amplified fragment was 356 bp, the sample was positive for Listeria; and when the amplified product fragment was 206 bp, the sample was positive for Salmonella abortus. The PCR product can be stored at 4 ℃ overnight; for long-term storage, it should be stored below -20 ℃. When using this PCR for detection, both negative and positive controls should be included.

[0069] The negative control was DEPC water. The positive control was prepared by synthesizing plasmids containing the corresponding gene sequences (SEQ ID NO. 9~SEQ ID NO. 12) of *Rhus chinensis*, *Chlamydia psittaci*, *Salmonella abortus*, and *Listeria* from Gene Bank (synthesized by General Biosystems (Anhui) Co., Ltd.), and then diluting and mixing them. The concentration of the four bacterial plasmids was 100 pg / μL.

[0070] Example 3

[0071] Thirty-five samples of aborted fetuses, such as placenta and amniotic fluid, stored at -40 °C were randomly selected and tested using the optimal detection methods from Examples 1 and 2. The results showed that 5 samples were Bru-positive (14.3% positivity rate); 3 samples were Chl-positive (8.6% positivity rate); 3 samples were Sal-positive (8.6% positivity rate); and 2 samples were Li-positive (5.7% positivity rate). The concordance rate between the two methods was 100%. One case of mixed Bru and Sal infection was detected using singleton PCR of Bru and Sal as described in Example 1. The results were consistent with the multiplex PCR results, with a concordance rate of 100% (Table 2).

[0072] Table 2. Detection of clinical samples

[0073] Comparative Example 1

[0074] Results of amplification using other non-optimal primers

[0075] The primers used in Example 2 were replaced with the following non-optimal primers, while other steps remained the same as in Example 2. The PCR detection sensitivity was then tested after the primer replacement. The results showed that the minimum sample detection concentration for this reaction was 50 pg / μL, significantly lower than the detection sensitivity of the optimal primers.

[0076] The replacement primers are shown below: Bru-F' (SEQ ID NO. 13): 5'-CCTCATTTTCCACAATCATGC-3'; Bru-R' (SEQ ID NO. 14): 5'-TCAACAACGACATCGACCGA-3'; Sal-F' (SEQ ID NO. 15): 5'-TCGACTCTAAACGTGTCGCA-3'; Sal-R' (SEQ ID NO. 16): 5'-TTGCGATACTTCCACTTTG-3'; Li-F' (SEQ ID NO. 17): 5'-GGCAAAGCTGTCACTAAAGA-3'; Li-R' (SEQ ID NO. 18): 5'-GCTGTATAAGCAATGGGAACT-3'; Chl-F' (SEQ ID NO. 19): 5'-GGATGGTGCTTCAGGAGAT-3'; Chl-R' (SEQ ID NO. 20): 5'-TCATTCTTAGGATTAGATCG-3'.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer set for detecting four bacterial abortion diseases in sheep, characterized in that, Including Bru-F, Bru-R, Sal-F, Sal-R, Li-F, Li-R, Chl-F, and Chl-R; The nucleotide sequence of the Bru-F is shown in SEQ ID NO.1; The nucleotide sequence of the Bru-R is shown in SEQ ID NO.2; The nucleotide sequence of the Sal-F is shown in SEQ ID NO.3; The nucleotide sequence of the Sal-R is shown in SEQ ID NO.4; The nucleotide sequence of the Li-F is shown in SEQ ID NO.5; The nucleotide sequence of the Li-R is shown in SEQ ID NO.6; The nucleotide sequence of Chl-F is shown in SEQ ID NO.7; The nucleotide sequence of Chl-R is shown in SEQ ID NO.

8.

2. The use of the primer set according to claim 1 in the preparation of a kit for detecting Brucella, Salmonella abortus, Listeria, and Chlamydia psittaci.

3. A kit for detecting Brucella, Salmonella abortus, Listeria, or Chlamydia psittaci, respectively, characterized in that, Includes the primer set as described in claim 1; The primers for detecting Brucella include Bru-F and Bru-R; The primers for detecting Salmonella abortus in sheep include Sal-F and Sal-R; The primers for detecting Listeria include Li-F and Li-R; The primers for detecting Chlamydia psittaci include Chl-F and Chl-R.

4. A kit for simultaneous detection of Brucella, Salmonella abortus, Listeria, and Chlamydia psittaci, characterized in that, Includes the primer set as described in claim 1.

5. The reagent kit according to claim 4, characterized in that, The kit also includes bacterial DNA extraction reagents, PCR amplification reagents, positive controls, and negative controls; The positive control comprises nucleic acids with nucleotide sequences as shown in SEQ ID NO. 9-12; the negative control comprises DEPC water.

6. The kit according to claim 4 or 5, characterized in that, The method of using the kit includes the following steps: 1) Using the genomic DNA of the sample to be tested as a template, perform multiplex PCR amplification using the primer set described in claim 1 or the kit described in claim 4 or 5 to obtain PCR products; 2) The PCR products were electrophoresed on an agarose gel. When the amplified product fragment contained 713 bp of nucleic acid, Brucella was positive in the sample; when the amplified product fragment contained 206 bp of nucleic acid, Salmonella abortus was positive in the sample; when the amplified fragment contained 356 bp of nucleic acid, Listeria was positive in the sample; and when the amplified fragment contained 566 bp of nucleic acid, Chlamydia psittaci was positive in the sample.

7. The reagent kit according to claim 6, characterized in that, The reaction program for the multiplex PCR amplification was as follows: 95 ℃ for 3 min, 94 ℃ for 30 s, 52 ℃~56.3 ℃ for 30 s, 72 ℃ for 30 s, 35 cycles, followed by a 10 min extension at 72 ℃.

8. The kit according to claim 6 or 7, characterized in that, The reaction system for the multiplex PCR amplification consists of 8-12 μL of 2xTaq mix, 0.5-4 μL of the primer set described in claim 1, 1-3 μL of genomic DNA from the sample to be tested, and 4-8 μL of DEPC water.

9. The reagent kit according to claim 8, characterized in that, The final concentration of Bru-F in the reaction system is 0.1~0.2 μM; The final concentration of Bru-R in the reaction system was 0.1–0.2 μM; The final concentration of Sal-F in the reaction system is 0.3~1.0 μM; The final concentration of Sal-F in the reaction system is 0.3~1.0 μM; The final concentration of Chl-F in the reaction system was 0.03~0.1 μM; The final concentration of Chl-R in the reaction system was 0.03–0.1 μM; The final concentration of Li-F in the reaction system was 0.03~0.1 μM; The final concentration of Li-R in the reaction system is 0.03~0.1 μM.

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