Reagent or kit for detecting bovine respiratory syndrome pathogenic bacteria and application of reagent or kit

Abstract

The invention discloses a reagent or a kit for detecting bovine respiratory syndrome pathogenic bacteria and application of the reagent or the kit, and belongs to the field of animal husbandry pathogenic bacteria detection. The technical problem to be solved by the invention is how to detect bovine respiratory syndrome pathogenic bacteria. The reagent or the kit disclosed by the invention contains primer pairs as shown in SEQ ID No.1 and 2, primer pairs as shown in SEQ ID No.4 and 5, primer pairs as shown in SEQ ID No.7 and 8, primer pairs as shown in SEQ ID No.10 and 11, probes as shown in SEQ ID No.3, 6, 9 and 12, a bovine mycoplasma positive reference substance, a bovine mycobacterium positive reference substance, a bovine pasteurella multocida positive reference substance and a bovine klebsiella pneumoniae positive reference substance and / or a bovine klebsiella pneumoniae negative reference substance. The reagent or the kit disclosed by the invention can be used for successfully detecting the mycoplasma bovis, the mycobacterium bovis, the bovine pasteurella multocida and the bovine klebsiella pneumoniae.

Description

Technical Field

[0001] The present invention belongs to the field of detection of pathogenic bacteria in animal husbandry, and particularly relates to a reagent or kit for detecting pathogenic bacteria of bovine respiratory syndrome and its application. Background Art

[0002] Bovine respiratory disease is an infectious disease caused by single / mixed infection of virus or bacteria. Its clinical features include persistent cough, serous to purulent nasal discharge, rapid breathing, and moist rales or increased bronchial sounds can be heard by auscultation. Pathologically, it mainly shows pneumonia and bronchitis. This disease is essentially the result of the interaction of multiple factors, which can be summarized into two categories: environmental stress factors and pathogenic microorganism infections. The former involves management factors such as host immune status, transportation stress, and climate change, while the latter includes infections by various pathogens such as viruses, bacteria, and parasites. The combined effects of various etiologies have led to it also being called bovine respiratory disease syndrome. The main bacterial pathogens of bovine respiratory disease include Mycoplasma bovis, Pasteurella multocida, Klebsiella pneumoniae, and Mycobacterium bovis, etc. These pathogens can infect alone, but more often act synergistically to cause disease.

[0003] The diagnosis of bacterial pathogens includes conventional identification methods, bacterial automatic identification instruments, serological diagnosis, and molecular biology techniques, etc. The conventional bacteriological identification method is to identify through the morphological characteristics, culture characteristics, and biochemical reactions of bacteria. Due to its cumbersome detection process and long cycle, it is difficult to meet the needs of modern clinical rapid and accurate identification. The automated identification system has become an important means for the identification of bovine respiratory pathogens due to its advantages of standardization, miniaturization, and easy operation. However, this method is easily affected by interference factors and produces false negative results. Therefore, it is crucial to select an appropriate rapid diagnosis technology for disease prevention and control. Serological detection is an important means for the diagnosis of bovine respiratory disease, mainly including ELISA and slide agglutination test. Among them, ELISA has high sensitivity and specificity and is suitable for rapid detection, but the antigen preparation process is relatively complex; although the slide agglutination test is easy to operate, it can only be used to detect convalescent bovine sera for strains isolated from the same farm, and its application range is limited. With the progress of molecular biology technology, various nucleic acid amplification technologies have been widely used in the field of pathogen detection. Real-time fluorescence quantitative PCR is the most mature and widely used technology in current molecular diagnosis, with significant advantages in the diagnosis of infectious diseases and tumors. The multiplex fluorescence quantitative PCR technology has the advantages of high accuracy and rapid detection, and is particularly suitable for large-scale clinical screening, and can simultaneously detect mixed infections of multiple pathogens.

[0004] At present, several domestic research institutions have successively developed multiplex fluorescence quantitative PCR detection methods for bovine bacterial respiratory infections. However, the research on multiplex qPCR detection of bacterial pathogens is relatively lacking, and a quadruple qPCR detection method for Mycoplasma bovis, Mycobacterium bovis, Klebsiella pneumoniae bovis, and Pasteurella multocida has not been reported. Establishing such a multiplex detection method will significantly improve the diagnostic efficiency of bovine respiratory diseases.

[0005] During the development of qPCR detection kits, there is a huge risk of decreased sensitivity and specificity when detecting clinical samples. The main reason is that during the establishment of the qPCR method, plasmids or nucleic acids extracted from purified pathogens are mostly used to complete various links of method establishment, including the screening of primers and probes, condition optimization, system establishment, and performance verification of the kit. Because pure and single target nucleic acids are used, the non-specific reaction problem between the reaction system and the background nucleic acids of the samples has never been evaluated or verified during the method establishment process, resulting in a decrease in the performance of the system when detecting clinical samples. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to detect four bovine respiratory syndrome pathogenic bacteria, namely Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida bovis, and Klebsiella pneumoniae bovis.

[0007] To solve the above technical problem, the present invention first provides a reagent or kit for detecting bovine respiratory syndrome pathogenic bacteria (denoted as reagent or kit 1), and the reagent or kit 1 contains the following 1)-5):

[0008] 1) Primers MPglyA-F and MPglyA-R for detecting Mycoplasma bovis and probe MPglyA-P, where MPglyA-F and MPglyA-R are single-stranded DNAs shown in SEQ ID No.1 and SEQ ID No.2 respectively, and the sequence of MPglyA-P is as shown in SEQ ID No.3;

[0009] 2) Primers MB229-F and MB229-R for detecting Mycobacterium bovis and probe MB229-P, where MB229-F and MB229-R are single-stranded DNAs shown in SEQ ID No.4 and SEQ ID No.5 respectively, and the sequence of MB229-P is as shown in SEQ ID No.6;

[0010] 3) Primers PMfecD-F and PMfecD-R for detecting Pasteurella multocida bovis and probe PMfecD-P, where PMfecD-F and PMfecD-R are single-stranded DNAs shown in SEQ ID No.7 and SEQ ID No.8 respectively, and the sequence of PMfecD-P is as shown in SEQ ID No.9;

[0011] 4) Primers KBuge-F and KBuge-R and probe KBuge-P for detecting Klebsiella pneumoniae in cattle, where KBuge-F and KBuge-R are single-stranded DNAs shown in SEQ ID No. 10 and SEQ ID No. 11 respectively, and the sequence of KBuge-P is as shown in SEQ ID No. 12;

[0012] 5) Positive control products for Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida in cattle, Klebsiella pneumoniae in cattle, and negative control products; the positive control products for Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida in cattle, and Klebsiella pneumoniae in cattle are obtained by adding background nucleic acid samples to Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida, and Klebsiella pneumoniae in cattle; the negative control product contains the background nucleic acid sample, and the background nucleic acid sample is prepared from healthy bovine respiratory tract swabs that are negative in the etiological examinations of Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida, and Klebsiella pneumoniae in cattle.

[0013] The specific preparation method of the background nucleic acid sample includes: mixing a healthy calf respiratory tract nasal swab sample (mixing a healthy calf respiratory tract nasal swab with an RNAstore sample preservation solution (Tiangen Biotech, DP408-02)), centrifuging at 1000 r / min for 20 min, and taking the supernatant as the background nucleic acid sample.

[0014] In the above reagent or kit 1, the negative control product can be obtained by adding PBS and a lyophilized reagent to the background nucleic acid sample. The lyophilized reagent consists of a solvent and a solute. The solvent is water, and the solute and its concentration in the lyophilized reagent are 3 g / 100 mL trehalose, 0.5 g / 100 mL glycine, and 0.5 g / 100 mL PEG6000 respectively.

[0015] In the above reagent or kit 1, the negative control product can be obtained by mixing the background nucleic acid sample, PBS, and the lyophilized reagent and then lyophilizing.

[0016] The positive control products for Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida in cattle, and Klebsiella pneumoniae in cattle are obtained by adding the background nucleic acid sample and the lyophilized reagent to Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida, and Klebsiella pneumoniae in cattle respectively and then lyophilizing.

[0017] When preparing the negative control product, the volume ratio of the background nucleic acid sample, PBS, and the lyophilized reagent can be 10:1:9.

[0018] When preparing the positive control products for each pathogen, the ratio of the pathogen (at a concentration of 2×10 7 CFU / mL), the background nucleic acid sample and the lyophilized reagent can be 1:10:9.

[0019] In the above reagent or kit 1, both ends of the MPglyA-P, the MB229-P, the PMfecD-P and the KBuge-P can be labeled with a fluorescence quenching group (such as MGB, VIC, Texas Red, CY5) and a fluorescence group (such as FAM). The fluorescence groups of the four probes are different.

[0020] In the reagent or kit 1, the molar ratio of the MPglyA-F, the MPglyA-R, the MPglyA-P, the MB229-F, the MB229-R, the MB229-P, the PMfecD-F, the PMfecD-R, the PMfecD-P, the KBuge-F, the KBuge-R, the KBuge-P can be 20:20:5:20:20:5:20:20:5:20:20:5.

[0021] The reagent or kit 1 can be composed of the above 1)-5).

[0022] The application of the reagent or kit 1 in the preparation of products for detecting bovine respiratory syndrome pathogens also belongs to the protection scope of the present invention, and the bovine respiratory syndrome pathogens are Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida bovis and Klebsiella pneumoniae bovis.

[0023] The present invention also provides a reagent or kit for detecting bovine respiratory syndrome pathogens (denoted as reagent or kit 2), and the reagent or kit 2 contains the MPglyA-F, the MPglyA-R, the MPglyA-P, the MB229-F, the MB229-R, the MB229-P, the PMfecD-F, the PMfecD-R, the PMfecD-P, the KBuge-F, the KBuge-R and the KBuge-P.

[0024] The reagent or kit 2 can be composed of the MPglyA-F, the MPglyA-R, the MPglyA-P, the MB229-F, the MB229-R, the MB229-P, the PMfecD-F, the PMfecD-R, the PMfecD-P, the KBuge-F, the KBuge-R and the KBuge-P.

[0025] The use of the reagent or kit 2 in the preparation of products for detecting bovine respiratory syndrome pathogens also falls within the scope of protection of the present invention. The bovine respiratory syndrome pathogens are Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida bovis, and Klebsiella pneumoniae bovis.

[0026] The present invention established a background nucleic acid database of bovine respiratory samples by determining the sequence composition of nucleic acids in healthy bovine respiratory swab samples, and designed a primer specificity analysis and evaluation software based on this database to evaluate the specificity of primers and probes for four pathogens, namely Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida bovis, and Klebsiella pneumoniae bovis. In addition, using bovine respiratory samples with clear background nucleic acid information as background nucleic acid samples, corresponding positive control products were prepared by adding four inactivated pathogens respectively, and were used for primer and probe screening, reaction condition optimization, detection method establishment, and kit development and evaluation. The multiplex qPCR detection kit for the four pathogens, namely Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida bovis, and Klebsiella pneumoniae bovis, described in the present invention utilizes the background nucleic acid database of bovine respiratory samples, primer and probe specificity evaluation software, and positive controls for the four pathogens to achieve quality control of the entire process of detection method establishment and kit development, and greatly reduces the risk of performance degradation when the final product detects clinical samples.

[0027] The following further describes the present invention in detail in conjunction with specific embodiments. The given embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way. Brief Description of the Drawings

[0028] Figure 1 . Background nucleic acid library construction process.

[0029] Figure 2 . Flow chart of primer specificity evaluation method.

[0030] Figure 3 . Standard curves of pathogens of calf respiratory syndrome. A, Standard curve of Mycoplasma bovis; B, Standard curve of Mycobacterium bovis; C, Standard curve of Pasteurella multocida bovis; D, Standard curve of Klebsiella pneumoniae bovis.

[0031] Figure 4 Sensitivity detection of pathogens of calf respiratory syndrome. A, Sensitivity detection of Mycoplasma bovis; B, Sensitivity detection of Mycobacterium bovis; C, Sensitivity detection of Pasteurella multocida bovis; D, Sensitivity detection of Klebsiella pneumoniae bovis. The numbers 0, 1, 2, 3, 4, 5, 6 in the figure respectively represent 10 0 copies / μL, 10 1 copies / μL, 10 2 copies / μL, 103 copies / μL, 10 4 copies / μL, 10 5 copies / μL, 10 6 copies / μL.

[0032] Figure 5 . Specific detection of pathogens causing calf respiratory syndrome.

[0033] Figure 6 . Melting curves of different primer pairs for different pathogens. A. Melting curves of two primer pairs for Mycoplasma bovis, MP F1 / R1 represents MPglyA-F and MPglyA-R, MP F2 / R2 represents MPglyA-F2 and MPglyA-R2; B. Melting curves of two primer pairs for Mycobacterium bovis, MB F1 / R1 represents MB229-F and MB229-R, MB F2 / R2 represents MB229-F2 and MB229-R2; C. Melting curves of two primer pairs for Pasteurella multocida, PM F1 / R1 represents PMfecD-F and PMfecD-R, PM F2 / R2 represents PMfecD-F2 and PMfecD-R2; D. Melting curves of two primer pairs for Klebsiella pneumoniae, KBF1 / R1 represents KBuge-F and KBuge-R, KB F2 / R2 represents KBuge-F2 and KBuge-R2.

[0034] Figure 7 . Amplification curves of different probes for different pathogens. A. Amplification curves of two probes for Mycoplasma bovis, MP represents Mycoplasma bovis, P1 represents MPglyA-P, P2 represents MPglyA-P2; B. Curves of two probes for Mycobacterium bovis, MB represents Mycobacterium bovis, P1 represents MB229-P, P2 represents MB229-P2; C. Amplification curves of two probes for Pasteurella multocida, PM represents Pasteurella multocida, P1 represents PMfecD-P, P2 represents PMfecD-P2; D. Amplification curve of Klebsiella pneumoniae, KB represents Klebsiella pneumoniae, P1 represents KBuge-P, P2 represents KBuge-P2. Specific embodiments

[0035] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. For the quantitative tests in the following examples, at least three repeated experiments are set up.

[0036] Mycoplasma bovis in the following examples (Wang Mengzhu, et al. Research progress on diagnostic techniques for Mycoplasma bovis [J]. Chinese Agricultural Science Bulletin, 2023, 39(16): 118-123.). The public can obtain this biological material from the applicant in accordance with the relevant regulations on national biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0037] Mycobacterium bovis in the following examples (Li Fanfei, He Xiaoli, Cheng Cheng, et al. Research progress on molecular detection techniques for Mycobacterium bovis based on PCR method [J]. Chinese Journal of Veterinary Medicine, 2018, 54(05): 70-73.). The public can obtain this biological material from the applicant in accordance with the relevant regulations on national biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0038] Pasteurella multocida in the following examples (Yin Yuanyuan, He Fang, Zhao Guangfu, et al. Research progress on main virulence factors of Pasteurella multocida [J]. Acta Veterinaria Sinica, 2021, 41(06): 1210-1218.). The public can obtain this biological material from the applicant in accordance with the relevant regulations on national biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0039] Klebsiella pneumoniae in the following examples (Sheng Xijing, Zhang Fan, Liu Fangjia, et al. Research progress on pathogenicity and drug resistance of Klebsiella pneumoniae from animals [J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(07): 2966-2973.). The public can obtain this biological material from the applicant in accordance with the relevant regulations on national biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0040] Example 1. Preparation of negative and positive control products for pathogens of calf respiratory disease syndrome

[0041] I. Preparation of background nucleic acid samples

[0042] 1.1 Screening of healthy calves and collection of respiratory samples

[0043] Healthy calves aged 2-3 months. The selected calves should have sound limbs and normal movement, and there should be no clinical symptoms such as loss of appetite, mental depression, cough, dyspnea, diarrhea, joint swelling, etc. in clinical observation, and the etiological examinations for Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida, Klebsiella pneumoniae, etc. should all be negative.

[0044] Wipe the nostrils with an alcohol cotton ball. Insert a sterile nasal swab deep into the nasal cavity and leave it for 10 seconds. Collect samples from both nostrils separately and place them in the same sterile centrifuge tube after collection. Break the swab tip into a 15 mL sterile centrifuge tube containing 1.0 mL of RNAstore sample preservation solution (Tiangen Biotech, DP408-02), tighten the tube cap, and label the cattle number.

[0045] 1.2 High-throughput sequencing

[0046] Entrust Shanghai Sangon Biotech Co., Ltd. to conduct high-throughput metagenomic sequencing.

[0047] 1.3 Construction of background nucleic acid library

[0048] 1.3.1 Methods and steps for constructing the background nucleic acid library

[0049] The method for constructing the background nucleic acid library is as Figure 1 shown and includes the following steps:

[0050] S01: Collect specific types of samples, such as healthy cattle nasal swab samples, ensuring that the samples have a wide time and geographical span.

[0051] S02: Based on high-throughput sequencing technology, conduct metagenomic sequencing on these samples.

[0052] S03: Independently perform quality control on the downloaded data (FASTQ format) to obtain Cleandata. Merge all the quality-controlled R1-end data into a total R1 file, and merge all the quality-controlled R2-end data into a total R2 file.

[0053] S04: On a high-performance server, use the SPADES and MEGAHIT programs to perform De novo assembly on the total R1 file and the total R2 file, and then use the CD-HIT program to remove redundancy from the assembled contigs under the parameters of -c 0.99 -aS 0.9 to finally obtain the background nucleic acid sequence library.

[0054] 1.3.2 Explanation of steps

[0055] In step S03, the downloaded data is a gzip-compressed file in FASTQ format, which is a sequence text file containing quality values. Here, Q stands for quality, and it is generally used to store raw sequencing data, with the extension usually being fastq or fq. The following is the common sequence format of the FASTQ format: The first line: starts with '@', which is the name of this short read sequence. This string is converted from the status information during sequencing and there are no spaces in the middle. It is the unique identifier of each short read sequence and will not appear repeatedly in the same FASTQ file, nor even in different FASTQ files. The second line: is the sequence of the sequencing short read, composed of the five letters A, C, G, T, and N. This is also the DNA sequence that we are really concerned about, and N represents the bases that cannot be recognized during sequencing. The third line: starts with '+'. In the old version of the FASTQ file, it would directly repeat the information in the first line, but now generally nothing is added (to save storage space). The fourth line: is the quality value of the sequencing short read, which is as important as the base information in the second line. It describes the reliability of each sequenced base and is represented by ASCII code. Currently, most software can directly process the compressed format, so the general FASTQ format is presented in the compressed format with the extension fq.gz, and compression or decompression can be performed if needed. The independent quality control is to use the fastp program to independently process the paired-end files (R1 and R2 files) of each sample: retain bases with a quality value greater than 15, retain sequencing reads with more than 60% of the bases qualified, remove sequencing reads with a length less than 60bp and trim the adapter sequences to obtain the quality-controlled sequencing data, that is, the so-called Cleandata. Use the zcat command to decompress, merge all quality-controlled R1 files (fastq.gz), and recompress them into a summary R1 file (fastq.gz), and obtain the summary R2 file in the same way;

[0056] In step S04, since the assembly effects of different assembly programs are different, in this exemplary embodiment, two mainstream assembly programs are used for De novo assembly to obtain two assembled sequence files. After summarization, the CD-HIT program is used for redundancy removal. Among them, it should be noted that the aS value represents the ratio of the alignment length to the total length of the query sequence during the alignment process. The -c 0.99 parameter delimits the sequence similarity threshold for clustering into a group to be above 99%, and the -aS0.9 parameter takes the query sequence with an aS value exceeding 0.9 as a necessary condition for being delimited as a subclass of the subject sequence. The contigs refer to a set of continuous nucleic acid sequences obtained through sequence assembly technology.

[0057] 1.4 Background nucleic acid samples

[0058] Mix the nasal swabs of healthy calves with clear background nucleic acid information, break the swab heads into a 15 mL sterile centrifuge tube containing 1.0 mL of RNAstore sample preservation solution (Tiangen Biotech, DP408-02), centrifuge at 1000 r / min for 20 min, and take the supernatant as the background nucleic acid sample.

[0059] II. Preparation of negative / positive control products for multiplex detection of pathogenic bacteria in calf respiratory disease syndrome

[0060] 2.1 Cultivation of pathogenic bacteria and viable bacteria counting

[0061] 2.1.1 Cultivation and counting of Mycoplasma bovis

[0062] 1) Main culture medium

[0063] PPLO liquid medium: 21.0 g of PPLO broth powder, 25.0 g of yeast extract powder, 200 ml of horse serum, penicillin with a final concentration of 200 IU / mL. Adjust the pH to 7.4 - 7.8 with 1 mol / L NaOH aqueous solution, autoclave at 120 °C for 30 minutes, and store at 2 - 8 °C for later use.

[0064] PPLO solid medium: 21.0 g of PPLO broth powder, 25.0 g of yeast extract powder, 12.0 g of agar powder, dissolve in 800 ml of ddH2O, autoclave, add 200 mL of horse serum in a super clean bench, add penicillin with a final concentration of 200 IU / mL, and adjust the pH to 7.4 - 7.8 with sterilized NaOH aqueous solution (1 mol / L).

[0065] 2) Resuscitation and enrichment culture of Mycoplasma bovis

[0066] Take out the cryopreserved Mycoplasma bovis from -80 °C and inoculate it into the PPLO liquid medium at a ratio of 4%. Incubate in a 5% CO2 incubator at 37 °C for 3 days to obtain Mycoplasma bovis bacterial liquid.

[0067] 3) Determine CFU

[0068] Dilute the Mycoplasma bovis bacterial liquid 10-fold into 12 concentration gradients. Take 10 μL from each of the 12 centrifuge tubes with serial dilutions, cover the surface of the Mycoplasma bovis solid medium (i.e., PPLO solid medium) without spreading, and make the inoculum completely visible within one microscope field of view. Incubate in a 37 °C 5% CO2 incubator, start counting from the highest dilution at which colonies can appear, and record the colony counts of the first two dilutions at the same time. Calculate the geometric mean of the colony counts of the three dilutions, which is the CFU / mL titer of this culture.

[0069] 2.1.2 Cultivation and counting of Mycobacterium bovis

[0070] 1) Main culture medium

[0071] Middle Brook 7H9 liquid medium: 4.7 g of 7H9 powder, 2 mL of glycerol, made up to 900 mL with distilled water, autoclaved at 121 °C for 10 min. After cooling to about 45 °C, add 100 mL of OADC and store at 4 °C.

[0072] MIddlle Brook 7H11 medium: 21 g of 7H11 powder, 5 mL of glycerol, made up to 900 mL with distilled water, autoclaved at 121 °C for 15 min. After cooling to about 50 - 55 °C, add 100 mL of OADC, mix well and pour 18 - 20 mL per plate into 90 mm petri dishes, let it dry and store at room temperature.

[0073] 2) Subculture

[0074] Thaw the Mycobacterium bovis strain stored at -80 °C, streak it on Middle brook 7H11 medium with an inoculation loop, and incubate it upside down at 37 °C in a 5% CO₂ incubator for about 4 weeks. Pick a single colony from Middle brook 7H11 medium and inoculate it into Middle brook 7H9 liquid medium, incubate it statically at 37 °C for about 2 weeks until the strain reaches the logarithmic growth phase. Collect the strain in the logarithmic growth phase, centrifuge it at 4000 r / min for 10 min, discard the supernatant, add an appropriate amount of Middle brook 7H9 liquid medium to resuspend the bacterial pellet, grind the bacterial suspension thoroughly with a mortar, then repeatedly pipette the suspension with a 1 mL syringe to disperse the bacteria, let it stand for 5 - 10 min and then collect the supernatant, aliquot it into cryotubes and store at -80 °C.

[0075] 3) Determination of CFU

[0076] Take an appropriate amount of the bacterial solution and perform serial dilution with sterile PBS, evenly spread it on the Middle brook 7H11 medium plate with a disposable spreader. After the liquid on the surface of the medium dries, incubate it upside down at 37 °C in a 5% CO₂ incubator for about 4 weeks. When the bacterial colonies can be clearly distinguished by the naked eye, calculate the colony forming unit number (CFU) and obtain the average value.

[0077] 2.1.3 Culture and counting of Pasteurella multocida and Klebsiella pneumoniae of cattle

[0078] Pasteurella multocida and Klebsiella pneumoniae of cattle are cultured under the same conditions as follows:

[0079] 1) Main culture medium

[0080] Tryptic Soytone Agar (TSA): Weigh 8 g of TSA powder and place it in a 500 mL conical flask containing 200 mL of ddH2O. Autoclave at 121 °C for 15 min. When the temperature of the autoclave drops to 60 °C, take out the liquid. Then add 10 mL of newborn bovine serum according to a ratio of 5% and mix well. Before the medium cools and solidifies, dispense it into sterile petri dishes that have been dried by heat. Take one and place it in a 37 °C incubator overnight. If it is sterile, it is qualified. The rest are packaged and stored in a 4 °C refrigerator to prepare the TSA solid medium.

[0081] Tryptic Soy Broth (TSB): Weigh 6 g of TSB powder and place it in a 500 mL conical flask containing 200 mL of ddH2O. Autoclave at 121 °C for 15 min. When the temperature of the autoclave drops to 60 °C, take out the liquid.

[0082] 2) Pathogen culture

[0083] Thaw the strains (Pasteurella multocida or Klebsiella pneumoniae) stored at -80 °C. Use an inoculation loop to streak on the TSA solid medium and incubate it upside down at 37 °C in a 5% CO2 incubator for 18 - 24 h. Pick a single colony on the TSA solid medium and inoculate it into the TSB liquid medium, and incubate it with shaking at 37 °C overnight. After the culture is completed, store it at -80 °C.

[0084] 3) Determination of CFU

[0085] Take an appropriate amount of the bacterial liquid and perform serial dilution with sterile PBS. Use a disposable spreader to evenly spread it on the TSB medium plate. After the liquid on the surface of the medium dries, incubate it upside down at 37 °C in a 5% CO2 incubator for 18 - 24 h. When the bacterial colonies can be clearly distinguished by the naked eye, calculate the number of colony-forming units (CFU) and obtain the average value.

[0086] 2.2 Inactivation

[0087] After counting, add saturated formaldehyde solution to the culture solutions of Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida, and Klebsiella pneumoniae according to a ratio of 0.2% (V / V) respectively, and place them at 4 °C for 48 h to obtain the inactivated bacterial solutions of Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida, and Klebsiella pneumoniae respectively.

[0088] 2.3 Preparation of positive / negative control products for quadruple detection of pathogens causing calf respiratory disease syndrome

[0089] 2.3.1 Preparation of control products

[0090] According to the results of viable count of pathogenic bacteria, the viable bacterial suspensions of Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida bovis, and Klebsiella pneumoniae bovis were diluted to the corresponding concentrations with PBS respectively to obtain pathogenic bacterial solutions, and the positive and negative controls of the four pathogenic bacteria were prepared according to Table 1 below.

[0091] Table 1 Composition of each control

[0092] Pathogenic bacteria solution Background nucleic acid sample Lyophilized reagent Positive control <![CDATA[0.05mL(2×10 7 CFU / mL)]]> 0.5 mL 0.45 mL Negative control 0.05 mL PBS 0.5 mL 0.45 mL

[0093] Among them, the freeze-dried reagent solvent is water, and the solute and its concentration are 3 g / 100 mL trehalose, 0.5 g / 100 mL glycine, and 0.5 g / 100 mL PEG 6000 respectively. There are four kinds of positive controls obtained, namely the positive control of Mycoplasma bovis, the positive control of Mycobacterium bovis, the positive control of Pasteurella multocida bovis, and the positive control of Klebsiella pneumoniae bovis. There is one kind of negative control.

[0094] 2.3.2 Freeze-drying

[0095] The positive and negative controls containing the four pathogenic bacteria obtained in step 2.3.1 were freeze-dried, and the freeze-drying procedure is shown in Table 2 below.

[0096] Table 2 Freeze-drying parameters of the control

[0097]

[0098] III. Performance verification of the control

[0099] 3.1 Digital PCR quantification

[0100] The nucleic acids of the positive control of Mycoplasma bovis, the positive control of Mycobacterium bovis, the positive control of Pasteurella multocida bovis, the positive control of Klebsiella pneumoniae bovis, and the negative control were extracted using a DNA / RNA co-extraction kit (Tiangen Biotech, DP422). The nucleic acids of the obtained positive controls of each pathogen were quantified and analyzed using digital PCR and qPCR.

[0101] 3.1.1 Primers and probes

[0102] The information of the primers and probes used is shown in Table 3.

[0103] Table 3 Information of primers and probes

[0104]

[0105] Note: All probes (labeled "P") are labeled with FAM at the 5' end and MGB at the 3' end.

[0106] 3.1.2 Digital PCR detection

[0107] Entrusted Shanghai Sangon Biotech Co., Ltd. to conduct digital PCR detection.

[0108] 3.1.3 qPCR Detection

[0109] 1) Prepare the reaction solution

[0110] Prepare the single - reaction solution for each pathogen according to the system composition in the following table, with a total volume of 20 μL. Among them, the concentrations of the corresponding primers and probes for each pathogen in the primer - probe mixture are as follows: the concentrations of both primers for Mycoplasma bovis are 200 nmol / L, and the probe concentration is 50 nmol / L; the concentrations of both primers for Mycobacterium bovis are 200 nmol / L, and the probe concentration is 50 nmol / L; the concentrations of both primers for Pasteurella multocida are 200 nmol / L, and the probe concentration is 50 nmol / L; the concentrations of both primers for Klebsiella pneumoniae are 200 nmol / L, and the probe concentration is 50 nmol / L.

[0111] Table 4 qPCR Reaction System

[0112] Composition Single reaction addition amount (μL) Primer-probe mixture 2 2×One Step U+Mix (Vazyme) 10 One Step U+Enzyme Mix (Vazyme) 1 DEPC water 5 Template 2

[0113] 2) Reaction program

[0114] Set the reaction conditions according to the following table, where 60 °C is used as the annealing temperature for each pathogen.

[0115] Table 5 qPCR Reaction Conditions

[0116]

[0117] 3.1.4 Quantification Results

[0118] Table 6 Comparison of Digital PCR and qPCR Results of Each Positive Control

[0119]

[0120] 3.2 Verification of the Homogeneity of Control Samples

[0121] To evaluate the homogeneity of each positive control, randomly select 5 samples from the prepared positive controls and detect them by qPCR detection method for the corresponding pathogen, with each sample detected 3 times. By analyzing the coefficient of variation, it can be seen that the homogeneity of each positive control is good. The experimental results are shown in Table 7 below.

[0122] Table 7 Homogeneity Detection Results of Each Positive Control

[0123]

[0124] 3.3 Stability Verification

[0125] Stability includes transportation stability, stability after reconstitution and storage, and stability after repeated freeze-thaw cycles after reconstitution. Randomly select the prepared positive control samples and detect them using the qPCR detection method for the corresponding pathogen, with each sample being detected 3 times. Determine whether the stability meets the requirements by calculating the coefficient of variation (CV) between groups.

[0126] 3.3.1 Verification of storage stability

[0127] Select 3 candidate storage temperatures, namely room temperature (25°C), 4°C, and -20°C. The simulated storage durations are: 0 days, 7 days, 14 days, 1 month, 3 months, 6 months, and 9 months. The results show that the CV values of each pathogen positive control sample are all <10% within 9 months, indicating good stability. As shown in Table 8 below.

[0128] Table 8 Stability results of Mycoplasma bovis positive control samples

[0129]

[0130] Table 9 Stability results of Mycobacterium bovis positive control samples

[0131]

[0132] Table 10 Stability results of Pasteurella multocida bovis positive control samples

[0133]

[0134] Table 11 Stability results of Klebsiella pneumoniae bovis positive control samples

[0135]

[0136] 3.3.2 Stability test of the control sample after reconstitution

[0137] After reconstituting the lyophilized positive control sample, place it at 2 - 8°C and take samples for detection on the 1st, 3rd, and 7th days respectively. The results are shown in Table 12 below. The positive control sample can be stored at 2 - 8°C for one week after reconstitution, and the detection results show no obvious changes.

[0138] Table 12 Stability detection results of the positive control sample after reconstitution

[0139]

[0140] 3.3.3 Repeated freeze-thaw test of the control sample after reconstitution

[0141] After reconstituting the lyophilized positive control sample, test its stability at -20°C with repeated freeze-thaw cycles of 1 time, 5 times, and 10 times. The results are shown in Table 13 below. The detection results of the positive control sample show no obvious changes after 10 repeated freeze-thaw cycles after reconstitution.

[0142] Detection Results of Repeated Freezing and Thawing after Reconstitution of Positive Control

[0143]

[0144] 3.3.4 Preservation Stability Test of Inactivated Bacterial Solution as Control

[0145] Taking the inactivated bacterial solution of each pathogen as a control for the stability test, it was found that at room temperature (25°C), as the storage period increased, the coefficient of variation CV value showed an upward trend. For some pathogens, the stability requirement (CV < 10%) was exceeded at 3 months. As shown in Table 14 below.

[0146] Table 14 Stability Results of Inactivated Bacterial Solution as Control at Room Temperature

[0147]

[0148] 3.3.5 Preservation Stability Test of Plasmid as Control

[0149] Taking the plasmid solution as a control for the stability test, it was found that at room temperature (25°C), the stability requirement was exceeded after 7 days. The plasmids used were p-1, p-2, p-3, and p-4. The results are shown in Table 15.

[0150] Inserting the target fragment of Mycoplasma bovis into the multiple cloning site between the puC57 vectors, the recombinant plasmid p-1 was obtained. The sequence of the inserted target fragment of Mycoplasma bovis is as follows:

[0151]

[0152]

[0153] Inserting the target fragment of Mycobacterium bovis into the multiple cloning site between the puC57 vectors, the recombinant plasmid p-2 was obtained. The sequence of the inserted target fragment of Mycobacterium bovis is as follows:

[0154]

[0155] Inserting the target fragment of Pasteurella multocida bovis into the multiple cloning site between the puC57 vectors, the recombinant plasmid p-3 was obtained. The sequence of the inserted target fragment of Pasteurella multocida bovis is as follows:

[0156]

[0157]

[0158] Inserting the target fragment of Klebsiella pneumoniae bovis into the multiple cloning site between the puC57 vectors, the recombinant plasmid p-4 was obtained. The sequence of the inserted target fragment of Klebsiella pneumoniae bovis is as follows:

[0159]

[0160] Table 15 Stability results of plasmid as a positive control at room temperature

[0161]

[0162] Example 2 Quadruple qPCR detection of pathogens causing calf respiratory disease syndrome

[0163] I. Establishment of a quadruple qPCR method for detecting pathogens causing calf respiratory syndrome The primers and probes for detecting pathogens causing calf respiratory syndrome are shown in Table 16.

[0164] Table 16 Primer and probe sequences

[0165]

[0166] The qPCR reaction system for detecting pathogens causing calf respiratory disease syndrome is shown in Table 17.

[0167] Table 17 Composition of the reaction system

[0168] Composition Volume (μL) Primer-probe mixture 2 2×One Step U+Mix (Vazyme) 10 One Step U+Enzyme Mix (Vazyme) 1 DEPC water 5 Template 2

[0169] Among them, in the primer-probe mixture, the concentrations of the two primers for Mycoplasma bovis are both 200 nmol / L, and the probe concentration is 50 nmol / L; the concentrations of the two primers for Mycobacterium bovis are both 200 nmol / L, and the probe concentration is 50 nmol / L; the concentrations of the two primers for Pasteurella multocida are both 200 nmol / L, and the probe concentration is 50 nmol / L; the concentrations of the two primers for Klebsiella pneumoniae are both 200 nmol / L, and the probe concentration is 50 nmol / L.

[0170] The qPCR reaction conditions for detecting pathogens causing calf respiratory disease syndrome are shown in Table 18.

[0171] Table 18 qPCR reaction conditions

[0172]

[0173] II. Standard curve of the quadruple qPCR method for detecting pathogens causing calf respiratory disease syndrome

[0174] Using the nucleic acids extracted from the positive control products of four pathogens, namely Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida, and Klebsiella pneumoniae, as templates, after 10-fold serial dilution, the quadruple qPCR was performed using the method in step I.

[0175] The results showed that the slope of the standard curves of Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida and Klebsiella pneumoniae were -2.96, -3.42, -3.18 and -3.00 respectively, the correlation coefficients were all ≥0.98, and the amplification efficiencies were 117.69%, 96.06%, 106.28% and 115.44% respectively, indicating that the standard curves had good linear relationships and the amplification efficiency of the quadruple qPCR method was good. Figure 3 )

[0176] III. Performance Verification of the Quadruple qPCR Detection Kit for Pathogens of Bovine Abdominal Respiratory Syndrome

[0177] 3.1 Sensitivity

[0178] According to the quantification results of digital PCR, the positive control products of the four pathogens of Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida and Klebsiella pneumoniae in Example 1 were diluted 10-fold serially, and quadruple qPCR was performed using the method of Step 1.

[0179] The results were as Figure 4 shown. The sensitivity amplification curves of each pathogen were as Figure 4 shown. The minimum copy number of the pathogen for which a Ct value could be obtained was the lowest detection limit. The lowest detection limits of Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida and Klebsiella pneumoniae were 10 copies / μL, 10 copies / μL, 10 copies / μL and 1 copy / μL respectively.

[0180] 3.2 Specificity

[0181] Samples to be tested: Positive and negative control products for the four pathogenic bacteria in Example 1; Bovine astrovirus BastV (Liu Mengyao et al., Establishment of a quadruple real-time fluorescence quantitative RT-PCR detection method for bovine astrovirus, bovine viral diarrhea virus type 1, bovine coronavirus and bovine rotavirus, Acta Veterinaria et Zootechnica Sinica 2021, 52(7): 1942-1952), Bovine viral diarrhea virus type 1 (BVDV-1) (Liu Mengyao et al., Establishment of a quadruple real-time fluorescence quantitative RT-PCR detection method for bovine astrovirus, bovine viral diarrhea virus type 1, bovine coronavirus and bovine rotavirus, Acta Veterinaria et Zootechnica Sinica 2021, 52(7): 1942-1952), Bovine coronavirus BCV (Liu Mengyao et al., Establishment of a quadruple real-time fluorescence quantitative RT-PCR detection method for bovine astrovirus, bovine viral diarrhea virus type 1, bovine coronavirus and bovine rotavirus, Acta Veterinaria et Zootechnica Sinica 2021, 52(7): 1942-1952); Bovine norovirus BNoV (Wang Yuelin, Molecular detection and genomic research of bovine norovirus, Master's thesis of Southwest Minzu University, 2020); Bovine adenovirus type 3 strain HLJ0955 (Shi Hongfei, Study on the pathogenicity of bovine adenovirus type 3 and bovine parainfluenza virus type 3 to experimental animals, Dissertation of Chinese Academy of Agricultural Sciences, May 2014); Bovine parainfluenza virus type 3 strain SD0835 (Shi Hongfei, Study on the pathogenicity of bovine adenovirus type 3 and bovine parainfluenza virus type 3 to experimental animals, Dissertation of Chinese Academy of Agricultural Sciences, May 2014); Bovine infectious rhinotracheitis virus strain BK1295, Salmonella enterica subsp. enterica serovar Typhimurium strain CVCC3949, Pasteurella multocida strain CVCC391. Among them, bovine infectious rhinotracheitis virus strain BK1295, Salmonella enterica subsp. enterica serovar Typhimurium strain CVCC3949, and Pasteurella multocida strain CVCC391 are all products of the China Veterinary Culture Collection Center (CVCC), and the strain numbers are CVCCAV252, CVCC3949, and CVCC391 respectively.

[0182] Perform quadruple qPCR using the method in Step 1. The specific detection results of the quadruple qPCR are as Figure 5 shown. Except for the positive reference products of Mycobacterium bovis, Mycoplasma bovis, Klebsiella pneumoniae bovis, and Pasteurella multocida having positive amplification curves, the nucleic acids of the remaining pathogens and the negative reference products have no fluorescence amplification curves, indicating that the established quadruple qPCR method has strong specificity.

[0183] 3.3 Repeatability

[0184] Samples to be tested: Positive control products for the four pathogenic bacteria in Example 1.

[0185] Perform quadruple qPCR using the method in Step 1, and the results are shown in Table 19 below. The coefficient of variation of Ct values within batches is 0.78% - 3.44%, and the coefficient of variation of Ct values between batches is 2.20% - 2.64%, both less than 10.0%, indicating that the repeatability of this qPCR method is good.

[0186] Table 19 Results of Repeatability Detection of Quadruple qPCR for Pathogens of Bovine Respiratory Syndrome

[0187]

[0188] 3.4 Detection of Clinical Samples

[0189] Perform quadruple qPCR using the method in Step 1, and successively detect 117 clinical samples from different regions. The results are shown in Table 20 below. The quadruple qPCR products of each sample were sequenced. The results show that all 20 positive samples of Mycoplasma bovis in Table 20 contain Mycoplasma bovis, all 17 positive samples of Mycobacterium bovis contain Mycobacterium bovis, all 8 positive samples of Pasteurella multocida contain Pasteurella multocida, and all 53 positive samples of Klebsiella pneumoniae contain Klebsiella pneumoniae.

[0190] Table 20 Detection Results of Pathogens in Bovine Respiratory Samples

[0191] Pathogen name Number of positive samples Positive detection rate Mycoplasma bovis 20 17.09％ Mycobacterium bovis 17 14.53％ Pasteurella multocida 8 6.84％ Klebsiella pneumoniae 53 45.30％

[0192] Comparative Example 1

[0193] According to the method in Step 1 of Example 2, serially dilute the positive control products of the four pathogens in Example 1 by 10-fold as templates, replace MPglyA-F, MPglyA-R, and MPglyA-P with MPglyA-F2, MPglyA-R2, and MPglyA-P2 respectively, replace MB229-F, MB229-R, and MB229-P with MB229-F2, MB229-R2, and MB229-P2 respectively, replace PMfecD-F, PMfecD-R, and PMfecD-P with PMfecD-F2, PMfecD-R2, and PMfecD-P2 respectively, and keep other steps unchanged. The results show that the minimum detection limits of Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida, and Klebsiella pneumoniae are 10 copies / μL, 10 copies / μL, 10 copies / μL, and 10 copies / μL respectively, and the detection sensitivity of Klebsiella pneumoniae has decreased.

[0194] The replaced primer-probes are as follows:

[0195]

[0196] Comparative Example 2

[0197] According to the method in Step 1 of Example 2, using the clinical samples of Example 2 as the detection objects, replace MPglyA-F, MPglyA-R, and MPglyA-P with MPglyA-F2, MPglyA-R2, and MPglyA-P2 in Comparative Example 1 respectively, replace MB229-F, MB229-R, and MB229-P with MB229-F2, MB229-R2, and MB229-P2 in Comparative Example 1 respectively, replace PMfecD-F, PMfecD-R, and PMfecD-P with PMfecD-F2, PMfecD-R2, and PMfecD-P2 in Comparative Example 1 respectively, replace KBuge-F, KBuge-R, and KBuge-P with KBuge-F2, KBuge-R2, and KBuge-P2 in Comparative Example 1 respectively, and keep other steps unchanged. The results show that compared with the detection results of 3.4 clinical samples in Example 2, the positive detection rates of each pathogen have significantly decreased.

[0198] Table 21 Detection Results of Pathogens in Bovine Respiratory Samples

[0199] Pathogen name Number of positive samples Positive detection rate Mycoplasma bovis 18 15.38％ Mycobacterium bovis 15 12.82％ Pasteurella multocida 8 6.84％ Klebsiella pneumoniae 37 31.62％

[0200] Comparative Example 3

[0201] Use a DNA / RNA co-extraction kit (Tiangen Biotech, DP422) to extract the nucleic acids of the Mycoplasma bovis positive control, Mycobacterium bovis positive control, Pasteurella multocida positive control, Klebsiella pneumoniae positive control, and negative control in Example 1 respectively.

[0202] Using the nucleic acids of the Mycoplasma bovis positive control and the negative control (NTC) as templates respectively, perform SYBR Green dye-based fluorescence quantitative PCR using the primer pairs MPglyA-F and MPglyA-R, and the primer pair MPglyA-F2 and MPglyA-R2 in Comparative Example 1 respectively. The copy number of Mycoplasma bovis is set to 10 7 copies / μL, 10 3 copies / μL. The results show that the melting curves of MPglyA-F and MPglyA-R present single peaks, with good specificity, Figure 6 in A.

[0203] Using the nucleic acids of Mycobacterium bovis positive control and negative control (NTC) as templates respectively, fluorescence quantitative PCR using SYBR Green dye method was performed with primer pairs MB229-F and MB229-R, and primer pairs MB229-F2 and MB229-R2 in Comparative Example 1. The copy number of Mycobacterium bovis was set to 10 7 copies / μL, 10 3 copies / μL. The results showed that the melting curves of MB229-F and MB229-R presented a single peak with good specificity, Figure 6 in B.

[0204] Using the nucleic acids of Pasteurella multocida bovis positive control and negative control (NTC) as templates respectively, fluorescence quantitative PCR using SYBR Green dye method was performed with primer pairs PMfecD-F and PMfecD-R, and primer pairs PMfecD-F2 and PMfecD-R2 in Comparative Example 1. The copy number of Pasteurella multocida bovis was set to 10 7 copies / μL, 10 3 copies / μL. The results showed that the melting curves of PMfecD-F and PMfecD-R presented a single peak with good specificity, Figure 6 in C.

[0205] Using the nucleic acids of Klebsiella pneumoniae bovis positive control and negative control (NTC) as templates respectively, fluorescence quantitative PCR using SYBR Green dye method was performed with primer pairs KBuge-F and KBuge-R, and primer pairs KBuge-F2 and KBuge-R2 in Comparative Example 1. The copy number of Klebsiella pneumoniae bovis was set to 10 7 copies / μL, 10 3 copies / μL. The results showed that the melting curves of KBuge-F and KBuge-R presented a single peak with good specificity, Figure 6 in D.

[0206] Comparative Example 4,

[0207] Using a DNA / RNA co-extraction kit (Tiangen Biotech, DP422), nucleic acids of Mycoplasma bovis positive control, Mycobacterium bovis positive control, Pasteurella multocida bovis positive control, Klebsiella pneumoniae bovis positive control, and negative control in Example 1 were extracted respectively.

[0208] Using the nucleic acid of Mycoplasma bovis positive control as the test sample, the amplification curves of MPglyA-P and MPglyA-P2 probe in Comparative Example 1 were detected by TaqMan TM qPCR method. The results showed that MPglyA-P had good detection effect, Figure 7 in A.

[0209] Using the nucleic acid of Mycobacterium bovis positive control as the sample to be tested, the amplification curves of MB229-P and the MB229-P2 probe in Comparative Example 1 were detected by TaqMan TM qPCR method. The results showed that MB229-P had a good detection effect. Figure 7 In B.

[0210] Using the nucleic acid of Pasteurella multocida bovis positive control as the sample to be tested, the amplification curves of PMfecD-P and the PMfecD-P2 probe in Comparative Example 1 were detected by TaqMan TM qPCR method. The results showed that PMfecD-P had a good detection effect. Figure 7 In C.

[0211] Using the nucleic acid of Klebsiella pneumoniae bovis positive control as the sample to be tested, the amplification curves of KBuge-P and the KBuge-P2 probe in Comparative Example 1 were detected by TaqMan TM qPCR method. The results showed that KBuge-P had a good detection effect. Figure 7 In D.

[0212] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A reagent or kit for detecting the pathogen of bovine respiratory syndrome, characterized in that: The reagent or kit contains the following 1)-5): 1) Primers MPglyA-F and MPglyA-R and probe MPglyA-P for detecting Mycoplasma bovis, wherein MPglyA-F and MPglyA-R are single-stranded DNAs shown in SEQ ID No.1 and SEQ ID No.2 respectively, and the sequence of MPglyA-P is as shown in SEQ ID No.3; 2) Primers MB229-F and MB229-R and probe MB229-P for detecting Mycobacterium bovis, wherein MB229-F and MB229-R are single-stranded DNAs shown in SEQ ID No.4 and SEQ ID No.5 respectively, and the sequence of MB229-P is as shown in SEQ ID No.6; 3) Primers PMfecD-F and PMfecD-R and probe PMfecD-P for detecting Pasteurella multocida in cattle, wherein PMfecD-F and PMfecD-R are single-stranded DNAs shown in SEQ ID No.7 and SEQ ID No.8 respectively, and the sequence of PMfecD-P is as shown in SEQ ID No.9; 4) Primers KBuge-F and KBuge-R and probe KBuge-P for detecting Klebsiella pneumoniae in cattle, wherein KBuge-F and KBuge-R are single-stranded DNAs shown in SEQ ID No.10 and SEQ ID No.11 respectively, and the sequence of KBuge-P is as shown in SEQ ID No.12; 5) Positive control products for Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida in cattle, Klebsiella pneumoniae in cattle and negative control; the positive control products for Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida in cattle and Klebsiella pneumoniae in cattle are obtained by adding background nucleic acid samples to Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida and Klebsiella pneumoniae in cattle; the negative control contains the background nucleic acid sample, and the background nucleic acid sample is prepared from healthy bovine respiratory tract swabs that are negative for the etiological examinations of Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida and Klebsiella pneumoniae in cattle.

2. The reagent or kit according to claim 1, characterized in that: The negative control is obtained by adding PBS and a freeze-dried reagent to the background nucleic acid sample. The freeze-dried reagent consists of a solvent and a solute. The solvent is water, and the solute and its concentration in the freeze-dried reagent are 3 g / 100 mL trehalose, 0.5 g / 100 mL glycine, and 0.5 g / 100 mL PEG6000 respectively.

3. The reagent or kit according to claim 1 or 2, characterized in that: The negative control is obtained by mixing the background nucleic acid sample, PBS and the freeze-dried reagent and then freeze-drying. The Mycoplasma bovis positive control, the Mycobacterium bovis positive control, the Pasteurella multocida bovis positive control, and the Klebsiella pneumoniae bovis positive control are obtained by freeze-drying after adding the background nucleic acid sample and the lyophilized reagent to Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida bovis, and Klebsiella pneumoniae bovis, respectively.

4. The reagent or kit according to claim 1 or 2, characterized in that: Both ends of the MPglyA-P, the MB229-P, the PMfecD-P, and the KBuge-P are labeled with a fluorescence quenching group and a fluorescence group; And / or, in the reagent or kit, the molar ratio of the MPglyA-F, the MPglyA-R, the MPglyA-P, the MB229-F, the MB229-R, the MB229-P, the PMfecD-F, the PMfecD-R, the PMfecD-P, the KBuge-F, the KBuge-R, and the KBuge-P is 20:20:5:20:20:5:20:20:5:20:20:

5.

5. Use of a reagent or kit in the preparation of a product for detecting bovine respiratory syndrome pathogenic bacteria, characterized in that: The reagent or kit is the reagent or kit according to any one of claims 1-4, and the bovine respiratory syndrome pathogens are Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida bovis, and Klebsiella pneumoniae bovis.

6. A reagent or kit for detecting the pathogen of bovine respiratory syndrome, characterized in that: The reagent or kit contains the MPglyA-F, the MPglyA-R, the MPglyA-P, the MB229-F, the MB229-R, the MB229-P, the PMfecD-F, the PMfecD-R, the PMfecD-P, the KBuge-F, the KBuge-R, and the KBuge-P according to claims 1-4.

7. Use of a reagent or kit in the preparation of a product for detecting bovine respiratory syndrome pathogens, characterized in that: The reagent or kit is the reagent or kit according to claim 6, and the bovine respiratory syndrome pathogens are Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida bovis, and Klebsiella pneumoniae bovis.

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