TaqMan real-time fluorescent quantitative PCR (Polymerase Chain Reaction) primer, probe, detection method and kit for detecting oviductus ovatus and application of TaqMan real-time fluorescent quantitative PCR primer and probe

Abstract

The invention discloses TaqMan real-time fluorescent quantitative PCR (polymerase chain reaction) primers, a probe, a detection method and a kit for detecting oviductus ovatus and application of the TaqMan real-time fluorescent quantitative PCR primers and the probe. The primer comprises an upstream primer HO-F and a downstream primer HO-R, and the nucleotide sequences of the upstream primer HO-F and the downstream primer HO-R are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2. The nucleotide sequence of the probe HO-P is shown as SEQ ID NO.3, the 5'end of the probe HO-P is marked with a fluorescence reporter group FAM, and the 3 'end of the probe HO-P is marked with a quenching group BHQ1. The TaqMan real-time fluorescent quantitative PCR method established by using the primer and the probe can specifically detect the ovulus ovatus of sheep, and has no cross reaction with other bacteria of ovulus ovatus and 47 common bacteria of cattle and sheep. The method or the kit not only can be applied to rapid identification of oviductus ovatus, but also can be applied to qualitative, relative quantitative and absolute quantitative detection of oviductus ovatus in cattle and sheep clinical samples, has the advantages of strong specificity, high sensitivity, good repeatability and the like, provides a reliable method for rapid detection of oviductus ovatus infection of cattle, sheep and sheep, and has a wide application prospect. Good popularization and application prospects are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary microbiological detection, and particularly relates to a TaqMan real-time fluorescence quantitative PCR primer, probe, detection method, kit and application for detecting Helcococcus ovis. Background Art

[0002] Helcococcus ovis is a bacterial species taxonomically named by Collins et al. in the UK in [1] 1999, and is an important opportunistic pathogen of various animals and humans. This bacterium mainly infects cattle and sheep, and can cause pneumonia, endometritis [2] , mastitis [3] , abortion [4] , endocarditis [5] and suppurative arthritis [6] in cattle, as well as pneumonia [7] in goats and pneumonia [8] in sheep. This bacterium can also infect horses and humans, causing lung abscess [9] in horses and ophthalmia

[10] in humans. This bacterium endangers the health of various animals and humans. Therefore, establishing a rapid detection method for Helcococcus ovis is of great significance for the prevention and treatment of Helcococcus ovis infections in cattle, sheep and humans.

[0003] In terms of the detection of the pathogen of Helcococcus ovis, there is no report on the real-time fluorescence quantitative PCR method and other nucleic acid detection methods for Helcococcus ovis at home and abroad. The real-time fluorescence quantitative PCR detection method is divided into two types. One is the dye-based real-time fluorescence quantitative PCR method, and the other is the probe-based real-time fluorescence quantitative PCR method. The dye-based real-time fluorescence quantitative PCR method has the advantages of simple operation and relatively low cost. However, since the dye-based real-time fluorescence quantitative PCR method detects all double-stranded DNA in the reaction system, the appearance of some non-specific amplification or primer dimers will lead to false positives in the detection results, and its specificity is poor, which limits its application in clinical detection. The probe-based real-time fluorescence quantitative PCR detection method has the following advantages: (1) The probe-based real-time fluorescence quantitative PCR method detects the target nucleotide sequence in the reaction system (this nucleotide sequence must be complementary to both the primer and the probe nucleotide sequences), and is not affected by non-specific amplification and primer dimers, overcoming the disadvantage of poor specificity of the dye-based real-time fluorescence quantitative PCR method; (2) The probe-based real-time fluorescence quantitative PCR detection method takes a short time (about 50 minutes), and the detection result can be determined by observing the amplification curve after the reaction ends, overcoming the disadvantages of the long time-consuming (90 minutes) of the ordinary PCR detection method and the need for cumbersome agarose gel electrophoresis and gel imaging analysis (50 minutes) to determine the PCR detection result after the reaction ends. In this invention, the 16S rDNA gene sequences of all 3 species of bacteria in the genus Helcococcus (including Helcococcus ovis, Helcococcus kunzii, and Helcococcus sueciensis) were downloaded from the NCBI GenBank database, and gene similarity alignment was performed using the MegAlign software. The specific conserved gene fragment of Helcococcus ovis was selected, and the specific upstream primer HO-F, downstream primer HO-R, and probe HO-P of Helcococcus ovis were designed using the Beacon Designer 7.7 software. By optimizing the reaction system and amplification program and establishing a standard curve, a specific TaqMan real-time fluorescence quantitative PCR detection method for Helcococcus ovis was established. When the established TaqMan real-time fluorescence quantitative PCR detection method was used to detect the recombinant plasmid standard pMD-HO with a concentration of 4.1×10 5 ~4.1×10 0 copies / μL, there was a good linear relationship between the initial copy number of the recombinant plasmid and the cycle number (Ct value). The standard curve equation was Y = 39.154 - 3.132×logX (Y is the Ct value of the tested sample, and X is the initial copy number of the sample DNA). The C t value of the sample tSubstituting the value into the equation can calculate the initial copy number of Helcococcus ovis 16S rDNA in the clinical sample. Then, based on the weight of the bovine and ovine clinical samples used for DNA extraction, the absolute content of Helcococcus ovis in the bovine and ovine clinical samples can be calculated, thus realizing the absolute quantitative detection of Helcococcus ovis in the bovine and ovine clinical samples.

[0004] References:

[0005] [1] Collins MD, Falsen E, Foster G, et al. Helcococcus ovis sp. nov., a gram - positive organism from sheep. Int J Syst Bacteriol, 1999, 49(Pt 4):1429 - 1432;

[0006] [2] Locatelli C, Scaccabarozzi L, Pisoni G, et al. Helcococcus kunzii and Helcococcus ovis isolated in dairy cows with puerperal metritis. J Gen Appl Microbiol, 2013, 59(5):371 - 374;

[0007] [3] Liu K, Deng Z, Zhang L, et al. Biological characteristics and pathogenicity of Helcococcus ovis isolated from clinical bovine mastitis in a Chinese dairy herd. Front Vet Sci, 2022, 8:756438;

[0008] [4] Bovine abortions caused by Helcococcus ovis. Vet Rec, 2014, 175(2):38 - 41;

[0009] [5] Kutzer P, Schulze C, Engelhardt A, et al. Helcococcus ovis, an emerging pathogen in bovine valvular endocarditis. J Clin Microbiol, 2008, 46(10):3291 - 3295;

[0010] [6] Jost A, Sickinger M. Helcococcus ovis associated with septic arthritis and bursitis in calves - a case report. BMC Vet Res, 2021, 17(1):291;

[0011] [7] García A, Risco D, Benítez JM, et al. Helcococcus ovis isolated from a goat with purulent bronchopneumonia and pulmonary abscesses. J Vet Diagn Invest, 2012, 24(1):235 - 237;

[0012] [8] Zhang Y, Cui J, Parkinson A, et al. Isolation of Helcococcus ovis from sheep with pleuritis and bronchopneumonia. J Vet Diagn Invest, 2009, 21(1):164 - 166;

[0013] [9] Rothschild CM, Oaks JL, Schaupp JK, et al. Helcococcus ovis isolated from a pulmonary abscess in a horse. J Clin Microbiol, 2004, 42(5):2224 - 2226;

[0014]

[10] Mao L, Chen Z, Lu Y, et al. Helcococcus ovis in a patient with an artificial eye: a case report and literature review. BMC Infect Dis, 2018, 18(1):401. Summary of the Invention

[0015] The technical problem to be solved by the present invention is to overcome the deficiencies of the lack of TaqMan real-time fluorescence quantitative PCR detection methods and other nucleic acid detection methods for Staphylococcus ovis in China and abroad, and to provide a TaqMan real-time fluorescence quantitative PCR detection method capable of qualitatively, relatively quantitatively, and absolutely quantitatively detecting Staphylococcus ovis. This method has the advantages of strong specificity, high sensitivity, good repeatability, simple operation, rapidity, high throughput, and low cost.

[0016] The first object of the present invention is to provide a pair of TaqMan real-time fluorescence quantitative PCR primers and a probe specific for Staphylococcus ovis.

[0017] The primers include an upstream primer and a downstream primer. The upstream primer is HO-F, and the downstream primer is HO-R. Their nucleotide sequences are 5'-TGTGGCTCAACCATAGTA-3' (as shown in SEQ ID NO.1) and 5'-GTATCTAATCCTGTTTGCTC-3' (as shown in SEQ ID NO.2) respectively; the probe is HO-P, and its nucleotide sequence is 5'-FAM-CCTCAGCGTCAGTTAGATTCCA-BHQ1-3' (as shown in SEQ ID NO.3). The 5' end of the probe HO-P is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a quenching group BHQ1.

[0018] The second object of the present invention is to provide a TaqMan real-time fluorescence quantitative PCR detection method and kit specific for Staphylococcus ovis based on the above primers and probe.

[0019] Among them, the detection method includes the following steps:

[0020] Extract the bacterial genomic DNA of the sample to be tested as a template; perform real-time fluorescence quantitative PCR reaction using the above detection primers and probe; after the reaction, it can be accurately determined whether Staphylococcus ovis exists in the sample to be tested according to whether an amplification curve appears in the sample to be tested (qualitative detection), or the relative content of Staphylococcus ovis in the sample to be tested can be accurately determined by comparing the Ct values of the sample to be tested (or the early or late appearance of the amplification curve peak) (relative quantitative detection). It is also possible to substitute the Ct value of the DNA of the sample to be tested into the standard curve equation, and then the absolute content of the 16S rDNA of Staphylococcus ovis in the DNA sample to be tested can be calculated. Further, according to the weight of the clinical sample used for DNA extraction, the absolute content of Staphylococcus ovis in the clinical sample can be deduced (absolute quantitative detection). The TaqMan real-time fluorescence quantitative PCR detection method specific for Staphylococcus ovis can not only be applied to the rapid identification of Staphylococcus ovis, but also to the qualitative, relative quantitative, and absolute quantitative detection of Staphylococcus ovis in clinical samples of cattle and sheep.

[0021] Furthermore, the optimal reaction system of 20 μL includes:

[0022] 7.8 μL of ddH2O, 10 μL of 2×SuperReal PreMix (Probe), 0.2 μL of 50×ROX Preference Dye, 0.4 μL of the upstream primer HO-F (10 μmol / L), 0.4 μL of the downstream primer HO-R (10 μmol / L), 0.2 μL of the probe HO-P (10 μmol / L), and 1 μL of the DNA template. Among them, the TaqMan real-time fluorescence quantitative PCR premix reagent SuperReal PreMix (Probe) contains the buffer, DNA polymerase, and dNTP required for real-time fluorescence quantitative PCR reaction.

[0023] Furthermore, the optimal amplification program includes:

[0024] 15 min at 95°C; 5 s at 95°C, 30 s at 58°C, and collect fluorescence signals for 40 cycles.

[0025] Furthermore, the criteria for judging the qualitative detection results include:

[0026] If the TaqMan real-time fluorescence quantitative PCR amplification curve (the curve shows a peak) appears in the tested sample, it is judged as positive, indicating the presence of 16S rDNA of Pseudomonas aeruginosa in sheep (or Pseudomonas aeruginosa); if no amplification curve appears (the curve does not show a peak, approximately a horizontal straight line), it is judged as negative, indicating the absence of 16S rDNA of Pseudomonas aeruginosa in sheep (or Pseudomonas aeruginosa) in the tested sample.

[0027] Furthermore, the criteria for judging the relative quantitative detection results include:

[0028] Compare the Ct values of the tested samples (or the early or late appearance of the amplification curve peaks). The smaller the Ct value (or the earlier the amplification curve appears), the higher the relative content of 16S rDNA of Pseudomonas aeruginosa in sheep (or Pseudomonas aeruginosa) in the tested sample.

[0029] Furthermore, the criteria for judging the absolute quantitative detection results include:

[0030] The standard curve equation of the TaqMan real-time fluorescence quantitative PCR method for Pseudomonas aeruginosa is Y = 39.154 - 3.132×logX (Y is the Ct value of the tested sample, X is the initial copy number of the sample DNA). Substitute the Ct value of the tested sample DNA into the equation to calculate the initial copy number of 16S rDNA of Pseudomonas aeruginosa in the tested sample DNA, and then based on the weight of the bovine and sheep clinical samples used for DNA extraction, the absolute content of Pseudomonas aeruginosa in the bovine and sheep clinical samples can be further calculated. t value, X is the initial copy number of the sample DNA), substituting the Ct t value of the tested sample DNA into the equation can calculate the initial copy number of 16S rDNA of Pseudomonas aeruginosa in the tested sample DNA, and then based on the weight of the bovine and sheep clinical samples used for DNA extraction, the absolute content of Pseudomonas aeruginosa in the bovine and sheep clinical samples can be further calculated.

[0031] The TaqMan real-time fluorescence quantitative PCR kit for detecting Coccus ovis in wounds of sheep, which includes the primers, probes, reaction system, amplification program of the TaqMan real-time fluorescence quantitative PCR method for detecting Coccus ovis in wounds of sheep, and the DNA positive control of Coccus ovis CCUG 37441 T and the ddH2O negative control.

[0032] The present invention also provides an application of the TaqMan real-time fluorescence quantitative PCR method or kit for detecting Coccus ovis in wounds of sheep in the rapid identification of Coccus ovis in wounds of sheep, or in the qualitative, relative quantitative, and absolute quantitative detection of Coccus ovis in clinical samples of cattle and sheep.

[0033] Beneficial effects of the present invention:

[0034] The present invention has established a TaqMan real-time fluorescence quantitative PCR method or kit for specifically detecting Coccus ovis in wounds of sheep. This detection method or kit has the advantages of strong specificity, high sensitivity, good repeatability, rapid detection, and low cost, providing a reliable method for the rapid identification of Coccus ovis in wounds of sheep and the rapid qualitative, relative quantitative, and absolute quantitative detection of Coccus ovis infection in cattle and sheep, which is of great significance for the prevention and control of Coccus ovis infection in cattle and sheep. Description of the drawings

[0035] Figure 1 It is the amplification curve of the TaqMan real-time fluorescence quantitative PCR method under the optimal reaction system and amplification conditions.

[0036] Figure 2 It is the standard curve of the TaqMan real-time fluorescence quantitative PCR method or kit.

[0037] Figure 3 It is the specific evaluation result of the TaqMan real-time fluorescence quantitative PCR method or kit.

[0038] Figure 4 It is the sensitivity evaluation result of the TaqMan real-time fluorescence quantitative PCR method or kit.

[0039] Figure 5 It is the result of the rapid identification of Coccus ovis in wounds of sheep by the TaqMan real-time fluorescence quantitative PCR method or kit.

[0040] Figure 6 It is the result of the qualitative, relative quantitative, and absolute quantitative detection of clinical samples of cattle and sheep by the TaqMan real-time fluorescence quantitative PCR method or kit. Detailed implementation manners

[0041] The following examples are for further illustration of the present invention rather than limitations thereof. Unless otherwise specified, the reagents, reagent kits, consumables, instruments and equipment used in the present invention are conventional reagents, reagent kits, consumables and instruments in the technical field of the present invention.

[0042] Example 1 Primer Design and Synthesis

[0043] Download the type strains of all three species of bacteria in the genus Kytococcus and the 16S rDNA gene sequences of other reference strains from the NCBI GenBank database. The type strains of all three species of bacteria in the genus Kytococcus and their 16S rDNA gene accession numbers are Kytococcus ovis CCUG 37441 T (gene accession number Y16279), Kytococcus kocurii CCUG 32213 T (gene accession number JH601088), and Kytococcus swedicus CCUG 47334 T(GenBank accession number AJ579914). The other reference strains of all three species of the genus Kytococcus and their 16S rDNA GenBank accession numbers are Kytococcus ovis 823 (GenBank accession number LC367055.1), KG38 (GenBank accession number CP121192), YNAU-HB24 (GenBank accession number MT758192.1), and Tongji (GenBank accession number MG188744); Kytococcus kuehnii 22 (GenBank accession number NR_029237.1), ID1 (GenBank accession number MW391555.1), and Tongji (GenBank accession number PQ727373.1); and Kytococcus sedentarius 604224 / 2010 (GenBank accession number HQ215522.1).The gene similarity was analyzed using the MegAlign software in the DNAstar 7.0.1 software package, and a specific conserved gene fragment of *Kocuria ovis* was selected. Its sequence is: CAGCAGCCGCGGTAATACGTATGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGTACGTAGGCGGTAATTTAAGTCTGAATTTAAAGGCTGTGGCTCAACCATAGTAAGGTTCAGATACTGGATTACTTGAGTAGATGAGGGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCTGTGGCGAAGGCGACTTTCTGGAATCTAACTGACGCTGAGGTACGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCAGTAAACGATGAGTGCTAGTTGTCGGGAGTCAAATCTCGGTGACGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGTACGCAAGTATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGGCTTGACATATACAGGGATATACTAGAGATAGTATAGTTTTTTCGGAAACTTGTATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTATCTTTAGTTACCAGCATTTCGGATGGGGACTCTAGAGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCTATATGTCTTGGGCTACACACGTGCTACAATGGTCTGAACAAAGCGCAGCTACCTCGTGAGAGCAAGCGAATCGCATAAAACAGATCTCAGTTCGGAT (as shown in SEQ ID NO.4);

[0044] The specific upstream primer HO-F, downstream primer HO-R, and specific probe HO-P for *Kocuria ovis* were designed using the Beacon Designer 7.7 software. The nucleotide sequence of the upstream primer HO-F is:

[0045] 5'-TGTGGCTCAACCATAGTA-3' (as shown in SEQ ID NO.1);

[0046] The nucleotide sequence of the downstream primer HO-R is:

[0047] 5'-GTATCTAATCCTGTTTGCTC-3' (as shown in SEQ ID NO.2);

[0048] The nucleotide sequence of the probe HO-P is:

[0049] 5'-FAM-CCTCAGCGTCAGTTAGATTCCA-BHQ1-3' (as shown in SEQ ID NO.3);

[0050] The 5' end of the probe HO-P is labeled with the fluorescent reporter group FAM, and the 3' end is labeled with the quenching group BHQ1;

[0051] The primers and probes were all synthesized by Invitrogen.

[0052] Example 2 Optimization of the TaqMan real-time fluorescence quantitative PCR reaction system and amplification program and composition of the kit

[0053] 1. Strains

[0054] The type strain of Kocuria ovis CCUG 37441 T Stored in the Key Laboratory of Tropical and Subtropical Animal Viral Diseases, Yunnan Academy of Animal Husbandry and Veterinary Sciences.

[0055] 2. Extraction of bacterial genomic DNA

[0056] The genomic DNA of the type strain of Kocuria ovis CCUG 37441 was extracted using a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) T as the template for the TaqMan real-time fluorescence quantitative PCR reaction. The method for extracting bacterial genomic DNA was carried out according to the operation instructions of the kit, and the obtained Kocuria ovis DNA was stored at -20°C for later use. The present invention has no special limitation on the method for extracting the bacterial genomic DNA, and conventional methods can be used.

[0057] 3. Optimization of the TaqMan real-time fluorescence quantitative PCR reaction system

[0058] Under the condition that the basic amplification program (95°C for 15 min; 95°C for 5 s, 60°C for 30 s, 40 cycles) remained unchanged, the square matrix method was used to optimize the volumes of primer HO-F (0.2 μL, 0.3 μL, 0.4 μL, and 0.5 μL), primer HO-R (0.2 μL, 0.3 μL, 0.4 μL, and 0.5 μL), and probe HO-P (0.1 μL, 0.2 μL, 0.3 μL, 0.4 μL, and 0.5 μL) in a 20-μL basic reaction system (8.2 μL of ddH2O, 10 μL of 2×SuperReal PreMix (Probe), 0.2 μL of 50×ROX Preference Dye, 0.2 μL each of primers HO-F (10 μmol / L) and HO-R (10 μmol / L), 0.2 μL of probe HO-P (10 μmol / L), and 1 μL of DNA template). The optimal 20-μL reaction system was obtained: 7.8 μL of ddH2O, 10 μL of 2×SuperReal PreMix (Probe), 0.2 μL of 50×ROX Preference Dye, 0.4 μL of primer HO-F (10 μmol / L), 0.4 μL of primer HO-R (10 μmol / L), 0.2 μL of probe HO-P (10 μmol / L), and 1 μL of DNA template.

[0059] 4. Optimization of TaqMan real-time fluorescence quantitative PCR amplification program

[0060] See Figure 1 as shown Figure 1 in which: 1 is Streptococcus ovis CCUG 37441 T ; 2 is the negative control (ddH2O). Under the condition that the above optimal 20-μL reaction system remained unchanged, the annealing and extension temperatures (54°C, 56°C, 58°C, 60°C, and 62°C) in the basic amplification program (95°C for 15 min; 95°C for 5 s, 60°C for 30 s, 40 cycles) were optimized, and the optimal annealing and extension temperature was 58°C, that is, the optimal amplification program was 95°C for 15 min; 95°C for 5 s, 58°C for 30 s, 40 cycles. Under the optimal reaction system and amplification program, the amplification curve of Streptococcus ovis CCUG 37441

[0061] DNA was better (the curve started to peak early and the peak was higher), and the negative control (ddH2O) had no amplification curve (the curve did not peak and was approximately a horizontal straight line) ( T )), indicating that the optimized reaction system and amplification program were good. Figure 1 )

[0062] 5. Positive control and negative control

[0063] The positive control was DNA of *Micrococcus ovis* CCUG 37441 T , and the negative control was ddH2O.

[0064] 6. Composition of the kit

[0065] The primers, probes, reaction system, amplification program of the TaqMan real-time fluorescence quantitative PCR method described in Example 1, and DNA positive control of *Micrococcus ovis* CCUG 37441 T and ddH2O negative control were combined to obtain a TaqMan real-time fluorescence quantitative PCR kit for detecting *Micrococcus ovis*.

[0066] 7. Criteria for judging qualitative and relative quantitative detection results

[0067] In qualitative detection: After the TaqMan real-time fluorescence quantitative PCR detection, if an amplification curve (the curve shows a peak) appears in the sample to be detected, it is judged as positive, indicating that the 16S rDNA of *Micrococcus ovis* (or *Micrococcus ovis*) exists in the sample to be detected; if no amplification curve appears (the curve does not show a peak, approximately a horizontal straight line), it is judged as negative, indicating that the 16S rDNA of *Micrococcus ovis* (or *Micrococcus ovis*) does not exist in the sample to be detected.

[0068] In relative quantitative detection: After the TaqMan real-time fluorescence quantitative PCR detection, the smaller the Ct value of the sample to be detected (or the earlier the amplification curve shows a peak), the higher the relative content of the 16S rDNA of *Micrococcus ovis* (or *Micrococcus ovis*) in the sample to be detected.

[0069] Establishment of the standard curve of the TaqMan real-time fluorescence quantitative PCR method or kit in Example 3

[0070] To achieve the absolute quantitative detection of the TaqMan real-time fluorescence quantitative PCR method, it is also necessary to establish its standard curve. The method steps are as follows:

[0071] 1. Preparation of TaqMan real-time fluorescence quantitative PCR recombinant plasmid standard

[0072] The preparation method of the TaqMan real-time fluorescence quantitative PCR standard is as follows:

[0073] (1) PCR amplification of the target gene fragment: Using the extracted *Micrococcus ovis* CCUG 37441 mentioned above TUsing the DNA as a template, 2×EasyTaq PCR SuperMix (TransGen Biotech Co., Ltd., Beijing) as the PCR premix, and primers HO-F and HO-R to amplify the target gene fragment (182 bp in size) by ordinary PCR. The PCR amplification program is as follows: 94°C for 3 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, for 35 cycles; 72°C for 5 min;

[0074] (2) Gel recovery of the target gene fragment: Perform 1.2% agarose gel electrophoresis on the above PCR product, and use an agarose gel DNA recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to recover the target gene band;

[0075] (3) Ligation of the target gene fragment with the vector: Use the pMD19-T vector cloning kit (Takara Bio Inc., Dalian) to clone the target gene fragment into the pMD19-T vector to construct a recombinant plasmid;

[0076] (4) Transformation of the recombinant plasmid into Escherichia coli: Transform the recombinant plasmid into Escherichia coli DH5α competent cells, pick a single colony into LB liquid medium for expanded culture to obtain a bacterial solution;

[0077] (5) Extraction of the recombinant plasmid: Use a plasmid miniprep kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the recombinant plasmid from the bacterial solution;

[0078] (6) Sequencing and identification of the recombinant plasmid: Send the recombinant plasmid to Beijing Tsingke Biotechnology Co., Ltd. for sequencing. The inserted gene sequence obtained by sequencing is:

[0079] TGTGGCTCAACCATAGTAAGGTTCAGATACTGGATTACTTGAGTAGATGAGGGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCTGTGGCGAAGGCGACTTTCTGGAATCTAACTGACGCTGAGGTACGAAGGCGTGGGGAGCAAACAGGATTAGATAC (as shown in SEQ ID NO.5);

[0080] The similarity between the inserted gene sequence analyzed by MegAlign software and the target gene sequence shown in SEQ ID NO.6: TGTGGCTCAACCATAGTAAGGTTCAGATACTGGATTACTTGAGTAGATGAGGGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCTGTGGCGAAGGCGACTTTCTGGAATCTAACTGACGCTGAGGTACGAAGGCGTGGGGAGCAAACAGGATTAGATAC is 100%, indicating that the recombinant plasmid construction was successful. It was numbered pMD-HO and used as the standard for TaqMan real-time fluorescence quantitative PCR;

[0081] (7) Determination and conversion of the concentration of the recombinant plasmid standard pMD-HO: The mass concentration of the recombinant plasmid pMD-HO was determined by ultraviolet spectrophotometry to be 13 ng / μL. Using the formula (copy number concentration (copies / μL) = (6.02×10 23 )×(mass concentration (ng / μL)×10 -9 ) / (DNA sequence size × 660)), the mass concentration was converted to a copy number concentration of 4.1×10 9 copies / μL. There are no special limitations on the PCR amplification, gel recovery of the target gene fragment, ligation of the target gene fragment with the vector, plasmid transformation of Escherichia coli, plasmid extraction, sequencing identification of the recombinant plasmid, plasmid concentration determination, and plasmid concentration conversion methods in the present invention, and conventional methods can be used.

[0082] 2. Establishment of the standard curve for the TaqMan real-time fluorescence quantitative PCR method or kit

[0083] See Figure 2 as shown.

[0084] The recombinant plasmid standard pMD-HO with a concentration of 4.1×10 9 copies / μL was serially diluted 10-fold with TE buffer as the template, and the TaqMan real-time fluorescence quantitative PCR method optimized in Example 2 was used to detect the recombinant plasmid standard pMD-HO with a concentration of 4.1×10 5 ~4.1×10 0 copies / μL. Three replicates were set for each concentration and one detection was performed. After the detection, the ABI 7500 Software v 2.0.1 was used to automatically generate the standard curve ( Figure 2 ). The results showed that the concentration of the standard pMD-HO was in the range of 4.1×10 5 ~4.1×10 0In the range of copies / μL, the starting copy number and Ct value have a good linear relationship, and the standard curve equation is Y=39.154-3.132×logX (Y is the Ct value of the sample being tested). t value, X is the starting copy number of sample DNA), correlation coefficient R 2 The value was 0.988. The standard curve equation can be used for the absolute quantitative detection of Ovis vulnificus in clinical samples.

[0085] 3. Criteria for determining absolute quantitative test results

[0086] In the absolute quantitative detection of clinical samples, the Ct value of the sample to be tested is substituted into the standard curve equation Y = 39.154-3.132×logX (Y is the Ct value of the sample to be tested). t value, X is the starting copy number of the sample DNA), the absolute content of 16S rDNA of Ovis vulnificus in the tested DNA sample can be calculated, and then according to the weight of the clinical sample used to extract the DNA sample and the volume of the extracted DNA sample, the absolute content of 16S rDNA of Ovis vulnificus in the clinical sample can be further calculated. Since the copy number of the 16S rDNA gene in the genomic DNA of Ovis vulnificus is 2 (https: / / www.ncbi.nlm.nih.gov / nuccore / NZ_CP121192.1), the absolute content of the above 16S rDNA is divided by 2 to obtain the absolute content of Ovis vulnificus in the clinical sample (in cfu / g), wherein cfu is colony forming units (cfu).

[0087] Example 4 Performance evaluation of TaqMan real-time fluorescence quantitative PCR method or kit

[0088] 1. Specificity evaluation

[0089] (1)Strain

[0090] Ovis vulnificus Yunnan isolate YN250330, Cushing's vulnificus model strain CCUG 32213 T and Swedish Coccus vulnificus type strain CCUG 47334 T A total of 50 bacteria, as well as 47 common bacteria in cattle and sheep, and their sources and 16S rDNA gene GenBank accession numbers are shown in Table 1. All 50 bacteria were deposited by the Yunnan Key Laboratory of Tropical and Subtropical Animal Viral Diseases, Yunnan Academy of Animal Husbandry and Veterinary Medicine.

[0091] (2) Extraction of bacterial genomic DNA

[0092] Extract the genomic DNA of the above 50 bacteria using the bacterial genomic DNA extraction method described in Example 2, and store it at -20°C for TaqMan real-time fluorescence quantitative PCR detection.

[0093] (3) Specificity evaluation

[0094] See Figure 3 as shown in Figure 3 : 1 is the positive control (Stomatococcus ovis CCUG 37441 T ); 2 is Stomatococcus ovis YN250330; 3 - 52 are Cutibacterium curtisii CCUG 32213 T , Stomatococcus suecicus CCUG 47334 T , Mannheimia haemolytica NCTC 9380 T , Mannheimia ruminalis CCUG 38470 T , Mannheimia bovis KCTC 25018 T , Mannheimia ovis CGMCC 1.13620 T , Acinetobacter lwoffii 20083, Acinetobacter pseudolwoffii 211144, Aerococcus equinus ASV210852, Bacillus safensis ASV21102, Bergeyella thalassae ASV21108, Clostridium welchii ASV200849, Corynebacterium pseudotuberculosis ASV220610, Enterococcus marimammalium ASV220416, Enterococcus faecalis ASV210624, Escherichia coli ASV210723, Escherichia fergusonii ASV220419, Escherichia ruegeri ASV210736, Lactococcus lactis ASV22069, Listeria monocytogenes DSM20600 T , Listeria innocua ASV201080, Moraxella bovis ASV211134, Moraxella ovis ASV210756, Moraxella bovis CCUG 75921 T , Moraxella caprae NBRC 115473 T , Moraxella ovis CCUG 75922 T , Moraxella haemolytica CCUG 75920 T, Pasteurella aerogenes ASV20097, Pasteurella multocida ASV201160, Proteus mirabilis ASV210740, Pseudomonas aeruginosa 200921, Rothia nasimurium ASV210634, Salmonella enterica ASV201158, Serratia plymuthica ASV201241, Shigella flexneri ASV211126, Staphylococcus aureus ASV201020, Staphylococcus epidermidis ASV220412, Staphylococcus chromogenes ASV210921, Streptococcus dysgalactiae ASV201139, Streptococcus paris ASV200845, Streptococcus suis subsp. parasuis ASV211143, Streptococcus gallolyticus subsp. gallolyticus YN220769, Streptococcus equinus ASV210759, Yersinia enterocolitica ASV211212, Corynebacterium amycolatum YN21088, Histophilus somni YN24102, Arcanobacterium pyogenes YN170843, Mycoplasma bovis YN13077, Mycoplasma capricolum subsp. capricolum YN191154 and negative control (ddH2O).

[0095] The DNA of the above 50 kinds of bacteria was detected by the optimized TaqMan real-time fluorescence quantitative PCR method in Example 2. At the same time, a positive control (Streptococcus ovis CCUG 37441 T ) and a negative control (ddH2O) were established. The results showed that an amplification curve (the curve started to peak) appeared in the positive control, and no amplification curve (the curve was approximately horizontal) appeared in the negative control, indicating that the control was established and the detection was effective. An amplification curve appeared for the DNA of Streptococcus ovis YN250330, and the detection was positive; no amplification curve appeared for the DNA of the other 49 kinds of bacteria, and the detection was negative ( Figure 3 , Table 1). It was shown that the TaqMan real-time fluorescence quantitative PCR method could specifically detect Streptococcus ovis, and there was no cross-reaction with other bacteria in the genus Streptococcus and common bacteria of cattle and sheep, indicating strong specificity.

[0096] Table 1. Strains used for specificity evaluation and specificity detection results

[0097]

[0098]

[0099] Note: + indicates positive; - indicates negative.

[0100] 2. Sensitivity evaluation

[0101] As shown in Figure 4 : 1-7 are respectively concentrations of 4.1×10 Figure 4 ~4.1×10 5 ~4.1×10 -1Recombinant plasmid standard pMD-HO at copies / μL; 8 was the negative control (ddH2O).

[0102] Use TE buffer to perform a ten-fold serial dilution on the recombinant plasmid standard pMD-HO at 4.1×10 9 copies / μL, and use the TaqMan real-time fluorescence quantitative PCR method optimized in Example 2 to detect the recombinant plasmid standard pMD-HO at a concentration of 4.1×10 5 ~4.1×10 -1 copies / μL. At the same time, set up a negative control (ddH2O) to determine the lowest detectable concentration of the standard pMD-HO. The detection results showed that only the recombinant plasmid standard pMD-HO at a concentration of 4.1×10 5 ~4.1×10 0 copies / μL showed an amplification curve. Therefore, the lowest concentration of the standard pMD-HO detected as positive was 4.1 copies / μL( Figure 4 ), indicating that the TaqMan real-time fluorescence quantitative PCR method has high sensitivity.

[0103] 3. Repeatability evaluation

[0104] Intra-assay repeatability test: Use the TaqMan real-time fluorescence quantitative PCR method optimized in Example 2 to detect the recombinant plasmid standard at the above concentration of 4.1×10 5 ~4.1×10 2 copies / μL. Set up 3 replicates for each concentration and perform 1 detection. Calculate the coefficient of variation within the batch according to the Ct value. Inter-assay repeatability test: Use the TaqMan real-time fluorescence quantitative PCR method optimized in Example 2 to detect the recombinant plasmid standard at the above concentration of 4.1×10 5 ~4.1×10 2 copies / μL, and perform 3 detections in total. Calculate the coefficient of variation between batches according to the Ct value. The results showed that the coefficients of variation in both intra-assay and inter-assay repeatability tests were less than 3% (Table 2), indicating that the method has good repeatability.

[0105] Table 2. Results of the repeatability test of the TaqMan real-time fluorescence quantitative PCR method or kit

[0106]

[0107] Note: represents the average; SD represents the standard deviation; CV represents the coefficient of variation.

[0108] Example 5 Application of the TaqMan real-time fluorescence quantitative PCR method or kit in the rapid identification of Staphylococcus ovis in sheep wounds

[0109] 1. Strains

[0110] The strains YN250336, YN250310, and YN250210 were isolated from dairy cows in a cattle farm in Yunnan Province, and the strains YN25038, YN240862, and YN241225 were isolated from goats in a sheep farm in Yunnan Province. These 6 strains of bacteria were isolated, identified, and preserved by the Key Laboratory of Tropical and Subtropical Animal Viral Diseases of Yunnan Academy of Animal Husbandry and Veterinary Sciences, Yunnan Province.

[0111] 2. Extraction of Bacterial Genomic DNA

[0112] The genomic DNA of the above 6 strains of bacteria was extracted using the bacterial genomic DNA extraction method described in Example 2 and stored at -20°C for TaqMan real-time fluorescence quantitative PCR detection.

[0113] 3. Rapid Identification of Aerococcus ovis by TaqMan Real-Time Fluorescence Quantitative PCR Method or Kit

[0114] See Figure 5 shown in Figure 5 : 1 is the positive control (Aerococcus ovis CCUG 37441 T ); 2 is the strain YN250336; 3-8 are the strains YN250310, YN250210, YN25038, YN240862, YN241225, and the negative control (ddH2O), respectively.

[0115] The DNA of the above 6 strains of bacteria was detected using the TaqMan real-time fluorescence quantitative PCR method optimized in Example 2. At the same time, a positive control (Aerococcus ovis CCUG 37441 T ) and a negative control (ddH2O) were set up. The results showed that the positive control showed an amplification curve (the curve started to peak), and the negative control did not show an amplification curve (the curve was approximately horizontal), indicating that the control was valid and the detection was effective. The strain YN250336 showed an amplification curve and was identified as Aerococcus ovis( Figure 5 , Table 3); the strains YN250310, YN250210, YN25038, YN240862, and YN241225 did not show an amplification curve and were identified as non-Aerococcus ovis( Figure 5 , Table 3).

[0116] To confirm the accuracy of the TaqMan real-time fluorescence quantitative PCR method optimized in Example 2 for identifying Aerococcus ovis, the above 6 strains of bacteria were identified using the 16S rDNA gene similarity alignment identification method at the same time. The specific steps of the 16S rDNA gene similarity alignment identification method for bacteria are as follows:

[0117] (1) PCR amplification of 16S rDNA gene: Using the DNA of the above 6 strains of bacteria as templates respectively, the bacterial universal primers 27F (whose nucleotide sequence is 5'-AGAGTTTGATCATGGCTCAG-3', as shown in SEQ ID NO.7) and 1492R (whose nucleotide sequence is: 5'-GGTTACCTTGTTACGACTT-3', (as shown in SEQ ID NO.8)) were used for PCR amplification of the 16S rDNA gene;

[0118] (2) Sequencing of 16S rDNA gene: The PCR amplification products of the 6 strains of bacteria were sequenced using primers 27F and 1492R respectively, and the gene sequences were submitted to the NCBI GenBank database. The GenBank accession numbers of the 16S rDNA genes of strains YN250336, YN250310, YN250210, YN25038, YN240862 and YN241225 are PV533819, PV533820, PV533822, PV533821, PQ273271 and PV533823 respectively;

[0119] (3) Identification of bacteria by gene sequence similarity alignment: The 16S rDNA gene sequences of the 6 strains of bacteria were respectively aligned and identified in the EzBioCloud EzTaxon-e database (https: / / www.ezbiocloud.net / ). The results showed that the similarity between strain YN250336 and Helcococcus ovis CCUG 37441 T was the highest at 99.9%, and it was identified as Helcococcus ovis; the similarity between strain YN250310 and Streptococcus lutetiensis CIP 106849 T was the highest at 99.9%, and it was identified as Streptococcus lutetiensis (not Helcococcus ovis); the similarity between strain YN250210 and Mannheimia haemolytica NCTC 9380 T was the highest at 100%, and it was identified as Mannheimia haemolytica (not Helcococcus ovis); the similarity between strain YN25038 and Escherichia coli ATCC 11775 T was the highest at 99.5%, and it was identified as Escherichia coli (not Helcococcus ovis); the similarity between strain YN240862 and Histophilus somni ATCC 43625 T was the highest at 99.9%, and it was identified as Histophilus somni (not Helcococcus ovis); the similarity between strain YN241225 and Enterococcus faecium LMG 11423 TThe highest similarity was 99.9%, and it was identified as Enterococcus faecalis (not Pseudomonas aeruginosa subsp. aeruginosa) (Table 3). Comparing the identification results of the TaqMan real-time fluorescence quantitative PCR method and the 16S rDNA gene similarity alignment method for identifying bacteria, the results showed that the identification results of the two methods were completely consistent, that is, both identification methods identified strain YN250336 as Pseudomonas aeruginosa subsp. aeruginosa, and strains YN250310, YN250210, YN25038, YN240862, and YN241225 were identified as non-Pseudomonas aeruginosa subsp. aeruginosa (Table 3), indicating that the TaqMan real-time fluorescence quantitative PCR method optimized in Example 2 had high accuracy in identifying Pseudomonas aeruginosa subsp. aeruginosa. However, there was a significant difference in the time required for the two methods to identify bacteria. The time required for the 16S rDNA gene similarity alignment method to identify bacteria was about 24 hours, while the time required for the TaqMan real-time fluorescence quantitative PCR method optimized in Example 2 to identify Pseudomonas aeruginosa subsp. aeruginosa was about 2 hours. Therefore, the TaqMan real-time fluorescence quantitative PCR method optimized in Example 2 also had the advantage of being fast in identifying Pseudomonas aeruginosa subsp. aeruginosa.

[0120] Table 3. Results of identifying bacteria by two methods

[0121]

[0122]

[0123] Application of the TaqMan real-time fluorescence quantitative PCR method or kit in qualitative, relative quantitative, and absolute quantitative detection of clinical samples in Example 6

[0124] 1. Clinical samples

[0125] Three dairy cow lung samples DLN25101, DLN25102, and DLN25103 were from dairy cows with respiratory diseases in a cattle farm in Yunnan Province, and three goat lung samples LQY25021, LQY25022, and LQY25023 were from goats with respiratory diseases in a sheep farm in Yunnan Province. The six clinical samples were all stored in the Key Laboratory of Tropical and Subtropical Animal Viral Diseases, Yunnan Academy of Animal Husbandry and Veterinary Sciences, Yunnan Province.

[0126] 2. Extraction of genomic DNA from clinical samples

[0127] If absolute quantitative detection of Pseudomonas aeruginosa subsp. aeruginosa in clinical samples is required, the weight of the clinical samples used for DNA extraction, the volume of the clinical sample homogenate, and the volume of the TE buffer used for eluting DNA must be quantitatively used. The specific steps are as follows:

[0128] (1) Sample pretreatment: Take 1 g of lung tissue and place it in a mortar. Then add 2 mL of sterile normal saline and grind the lung tissue thoroughly into a slurry. Adjust the volume of the slurry to 10 mL with sterile normal saline. Take 500 μL of the slurry (equivalent to 1 / 20 g of lung tissue) and centrifuge it at 12,000 rpm for 3 min. Discard the supernatant and take the precipitate for the extraction of bacterial genomic DNA.

[0129] (2) Extraction of genomic DNA: Use the method for extracting bacterial genomic DNA described in Example 2 to extract the genomic DNA of the above 6 lung tissue samples respectively. The volume of each obtained DNA sample is 100 μL, which is used for TaqMan real-time fluorescence quantitative PCR detection. That is, during the extraction of genomic DNA from clinical samples, 100 μL of genomic DNA can be extracted from every 1 / 20 g of lung tissue sample.

[0130] 3. Absolute quantification of clinical samples by TaqMan real-time fluorescence quantitative PCR method

[0131] See Figure 6 as shown in Figure 6 : 1 is the positive control (Streptococcus ovis CCUG 37441 T ); 2 is bovine lung DLN25101; 3 is ovine lung LQY25022; 4 - 8 are bovine lungs DLN25102 and DLN25103, ovine lungs LQY25021 and LQY25023, and the negative control (ddH2O) respectively.

[0132] Use the optimized TaqMan real-time fluorescence quantitative PCR method obtained in Example 2 to detect the genomic DNA of the above clinical samples. Substitute the Ct value of the tested DNA into the standard curve equation Y = 39.154 - 3.132×logX (Y is the C of the tested sample tvalue, where X is the initial copy number of the sample DNA), the copy number X of the 16S rDNA gene of C. ovis in 1 μL of the DNA sample (the volume of the DNA template in the reaction system is 1 μL) can be obtained. It can be deduced that the copy number of the 16S rDNA gene of C. ovis in 100 μL of the DNA sample (equivalent to 1 / 20 g of the lung sample) is 100×X, and further deduced that the copy number of the 16S rDNA gene of C. ovis in 1 g of the lung sample is 100×X×20. Since the copy number of the 16S rDNA gene in the genome of C. ovis is 2 (https: / / www.ncbi.nlm.nih.gov / nuccore / NZ_CP121192.1), the number of C. ovis in 1 g of the lung sample is 100×X×20÷2 (unit: cfu), that is, the content of C. ovis in the lung sample is 100×X×20÷2 (unit: cfu / g), where the cfu is colony forming units (cfu).

[0133] In this absolute quantitative detection experiment, the DNA of the above 6 lung samples was detected by the TaqMan real-time fluorescence quantitative PCR method optimized in Example 2, and a positive control (C. ovis CCUG 37441 T ) and a negative control (ddH2O) were established at the same time. The results showed that an amplification curve (the curve started to peak) appeared in the positive control, and no amplification curve (the curve was approximately horizontal) appeared in the negative control, indicating that the control was valid and the detection was effective. The amplification curves of the bovine lung sample DLN25101 and the ovine lung sample LQY25022 were good, and their Ct values were 29.277 and 33.567( Figure 6 , Table 4); there were no amplification curves and no Ct values for the bovine lung samples DLN25102 and DLN25103 and the ovine lung samples LQY25021 and LQY25023. Substituting the Ct value of 29.277 of the bovine lung sample DLN25101 into the standard curve equation Y = 39.154 - 3.132×logX (Y is the C t value of the sample to be detected, and X is the initial copy number of the sample DNA), the copy number of the 16S rDNA gene of C. ovis in 1 μL of the DNA sample can be obtained as 10 3.154 , and then it can be deduced that the copy number of the 16S rDNA gene of C. ovis in 100 μL of the DNA sample (equivalent to 1 / 20 g of the lung sample) is 10 5.154 , and then it can be deduced that the copy number of the 16S rDNA gene of C. ovis in 1 g of the lung sample is 2×10 6.154 , and finally it can be deduced that the absolute content of C. ovis in 1 g of the lung sample is 10 6.154cfu / g. Using the POWER function POWER(number, power) in Microsoft Office Excel 2007 (where the parameter number represents the base; the parameter power represents the exponent), convert 10 6.154 to 1425608 = 1.43×10 6 , that is, the absolute content of Staphylococcus succinus in the bovine lung sample DLN25101 is 1.43×10 6 cfu / g (Table 4). Using the same method, the absolute content of Staphylococcus succinus in the ovine lung sample LQY25022 can be calculated as 6.08×10 4 cfu / g (Table 4). For the bovine lung samples DLN25102 and DLN25103 and the ovine lung samples LQY25021 and LQY25023, there are no amplification curves and no Ct values, and their absolute contents are all 0 cfu / g.

[0134] Table 4. Qualitative, relative quantitative, and absolute quantitative detection results of clinical samples

[0135]

[0136] Note: + indicates positive; - indicates negative; ND indicates not detected.

[0137] Sequence list information:

[0138] DTD version: V1_3

[0139] File name: Teacher Wang.xml

[0140] Software name: WIPOSequence

[0141] Software version: 2.1.0

[0142] Generation date: 2025-04-25

[0143] Basic information:

[0144] Current application / Applicant file name: 125300004312022453

[0145] Applicant name: Yunnan Academy of Animal Husbandry and Veterinary Sciences

[0146] Applicant name / Language: zh

[0147] Applicant name / Latin name: Yunnan Academy of Animal Husbandry and Veterinary Sciences

[0148] Inventor's Name: Li Fuxiang

[0149] Inventor's Name / Language: zh

[0150] Inventor's Name / Latin Name: Fu xiang LI

[0151] Title of the Invention: TaqMan Real-Time Fluorescent Quantitative PCR Primers, Probes, Detection Methods, Kits and Their Applications for Detecting Staphylococcus in Sheep Wounds (zh)

[0152] Total Number of Sequences: 8

[0153] Sequence:

[0154] Sequence Number (ID): 1

[0155] Length: 18

[0156] Molecular Type: DNA

[0157] Feature Location / Qualifier:

[0158] -source,1..18

[0159] >mol_type,other DNA

[0160] >organism,synthetic construct

[0161] Residue:

[0162] tgtggctcaa ccatagta 18 Sequence Number (ID): 2

[0163] Length: 20

[0164] Molecular Type: DNA

[0165] Feature Location / Qualifier:

[0166] -source,1..20

[0167] >mol_type,other DNA

[0168] >organism,synthetic construct

[0169] Residue:

[0170] gtatctaatc ctgtttgctc 20 Sequence Number (ID): 3

[0171] Length: 22

[0172] Molecular Type: DNA

[0173] Feature position / qualifier:

[0174] -source,1..22

[0175] >mol_type,other DNA

[0176] >organism,synthetic construct

[0177] Residue:

[0178] cctcagcgtc agttagattc ca 22 Sequence number (ID): 4

[0179] Length: 786

[0180] Molecular type: DNA

[0181] Feature position / qualifier:

[0182] -source,1..786

[0183] >mol_type,genomic DNA

[0184] >organism,Helcococcus ovis Residue:

[0185]

[0186] Sequence number (ID): 5

[0187] Length: 182

[0188] Molecular type: DNA

[0189] Feature position / qualifier:

[0190] -source,1..182

[0191] >mol_type,other DNA

[0192] >organism,synthetic construct Residue:

[0193]

[0194] Sequence number (ID): 6

[0195] Length: 182

[0196] Molecular type: DNA

[0197] Feature position / qualifier:

[0198] -source, 1..182

[0199] >mol_type, other DNA

[0200] >organism, synthetic construct

[0201] Residues:

[0202]

[0203] Sequence number (ID): 7

[0204] Length: 20

[0205] Molecular type: DNA

[0206] Feature position / qualifier:

[0207] -source, 1..20

[0208] >mol_type, other DNA

[0209] >organism, synthetic construct

[0210] Residues:

[0211] agagtttgat catggctcag 20

[0212] Sequence number (ID): 8

[0213] Length: 19

[0214] Molecular type: DNA

[0215] Feature position / qualifier:

[0216] -source, 1..19

[0217] >mol_type, other DNA

[0218] >organism, synthetic construct

[0219] Residues:

[0220] ggttaccttg ttacgactt 19

[0221] END.

Claims

1. A TaqMan real-time fluorescence quantitative PCR primer for detecting coccus in sheep wounds, characterized in that, The primers include an upstream primer HO-F and a downstream primer HO-R, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

2. A TaqMan real-time fluorescence quantitative PCR probe for detecting coccus in sheep wounds, characterized in that, The probe is HO-P, and its nucleotide sequence is shown in SEQ ID NO.

3. The 5' end of the probe HO-P is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a quenching group BHQ1.

3. A TaqMan real-time fluorescence quantitative PCR method for detecting Staphylococcus ovis for non-diagnostic purposes, characterized in that, The primers and probe used in the TaqMan real-time fluorescence quantitative PCR reaction are the TaqMan real-time fluorescence quantitative PCR primers described in claim 1 and the TaqMan real-time fluorescence quantitative PCR probe described in claim 2 respectively.

4. The TaqMan real-time fluorescence quantitative PCR method according to claim 3, characterized in that, The reaction system of TaqMan real-time fluorescence quantitative PCR includes: 7.8 μL of ddH2O, 10 μL of 2×SuperReal PreMix, 0.2 μL of 50×ROX Preference Dye, 0.4 μL of the upstream primer HO-F with a molar concentration of 10 μmol / L, 0.4 μL of the downstream primer HO-R with a molar concentration of 10 μmol / L, 0.2 μL of the probe HO-P with a molar concentration of 10 μmol / L, and 1 μL of DNA template.

5. The TaqMan real-time fluorescence quantitative PCR method according to claim 4, characterized in that, The amplification program of TaqMan real-time fluorescence quantitative PCR is: 15 min at 95°C; 5 s at 95°C, 30 s at 58°C, for 40 cycles.

6. According to the TaqMan real-time fluorescence quantitative PCR method described in claim 3, it is characterized in that: The standard curve equation is Y = 39.154 - 3.132×logX, where Y is the C value of the sample to be tested, and X is the initial copy number of the DNA of the sample to be tested; t value, and X is the starting copy number of the DNA of the sample to be tested; Substitute the C value of the DNA of the sample to be tested t into the equation to calculate the initial copy number of 16S rDNA of Staphylococcus succinus in the DNA of the sample to be tested, and then calculate the absolute content of Staphylococcus succinus in the clinical samples of cattle and sheep based on the weight of the clinical samples of cattle and sheep used for extracting the DNA sample.

7. The TaqMan real-time fluorescence quantitative PCR method according to any one of claims 3-6, characterized in that, The TaqMan real-time fluorescence quantitative PCR method can specifically detect Stomatococcus ovis, and has no cross-reaction with other species of bacteria in the genus Stomatococcus and common bacteria of cattle and sheep; The other species of bacteria in the genus Stomatococcus are Stomatococcus kocurii and Stomatococcus suecicus; The common bacteria of cattle and sheep are: Mannheimia haemolytica, Mannheimia ruminalis, Mannheimia bovis, Mannheimia ovis, Acinetobacter lwoffii, Acinetobacter pseudolwoffii, Aerococcus viridans, Bacillus safensis, Bergeyella zoohelcum, Clostridium welchii, Corynebacterium pseudotuberculosis, Enterococcus marimammalium, Enterococcus faecalis, Escherichia coli, Escherichia fergusonii, Escherichia ruegeri, Lactococcus lactis, Listeria monocytogenes, Listeria innocua, Moraxella bovoculi, Moraxella ovis, Moraxella bovis, Moraxella caprae, Moraxella ovis, Moraxella haemolytica, Pasteurella multocida, Pasteurella pneumotropica, Proteus mirabilis, Pseudomonas aeruginosa, Rothia nasimurium, Salmonella enterica, Serratia plymuthica, Shigella flexneri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus chromogenes, Streptococcus dysgalactiae, Streptococcus parauberis, Streptococcus gallolyticus, Streptococcus equi subsp. zooepidemicus, Streptococcus suis, Yersinia enterocolitica, Corynebacterium amycolatum, Histophilus somni, Arcanobacterium pyogenes, Mycoplasma bovis, and Mycoplasma capricolum.

8. The TaqMan real-time fluorescence quantitative PCR method according to any one of claims 3-6, characterized in that, The lowest concentration of the recombinant plasmid standard pMD-HO that can be detected as positive is 4.1 copies / μL.

9. Use of the TaqMan real-time fluorescence quantitative PCR method for detecting *C. ovis* for non-diagnostic purposes according to any one of claims 3-6 in the rapid identification of *C. ovis*, or in the qualitative, relative quantitative, and absolute quantitative detection of *C. ovis* in clinical samples of cattle and sheep.

10. A TaqMan real-time fluorescence quantitative PCR kit for detecting coccus in sheep wounds, characterized in that, Comprising the TaqMan real-time fluorescence quantitative PCR primers for detecting *C. ovis* according to claim 1 and the TaqMan real-time fluorescence quantitative PCR probe for detecting *C. ovis* according to claim 2.

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