Multiplex PCR (Polymerase Chain Reaction) detection primer group, kit and detection method for mastitis pathogenic bacteria
Through multiple PCR technology and specific primer sets, a five-fold PCR system was established for detecting pathogenic bacteria of mastitis, solving the problems of low detection efficiency and false positive false negatives in the prior art, and achieving rapid, accurate and high sensitivity detection effects.
Patent Information
- Application Number
- CN202510036677.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-04-25
AI Technical Summary
The prior art is difficult to detect mastitis pathogenic bacteria quickly and accurately, especially in the early diagnosis and identification of pathogenic bacteria types and severity of infection, and conventional culture methods are time-consuming, expensive and labor-intensive.
Multiple PCR technology combined with specific primer sets was used to design primer pairs for amplifying Staphylococcus aureus, Streptococcus agaricus, Pseudomonas, Klebsiella pneumoniae and E. coli, establish a five-fold PCR system, and detect it through real-time fluorescence PCR and agarose gel electrophoresis analysis.
It realizes fast, accurate and high-sensitivity detection, reduces the occurrence of false positive and false negative results, replaces traditional conventional cultivation methods, and improves detection efficiency and accuracy.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of biological detection, and in particular to a multiplex PCR detection primer set, a kit and a detection method for mastitis pathogenic bacteria. Background Art
[0002] In recent years, with the development of the dairy industry, the safety of milk has received keen attention from the society. In addition to the routine safety testing of dairy products, people are increasingly concerned about the source of milk and the safety of raw milk, such as mastitis.
[0003] The main basis for the diagnosis of mastitis is visible physiological symptoms, such as swelling and inflammation of the mammary gland or obvious changes in milk. Since these symptoms only appear in the chronic or clinical state of mastitis, its early diagnosis relies on a variety of simple diagnostic methods. The most commonly used of these methods are measuring somatic cell counts and enzyme analysis, and the California Mastitis Test is also used. However, there are many differences in these simple methods, which may increase false positive or false negative results. In addition, these simple methods cannot directly identify the pathogenic bacteria and the severity of infection in sick animals. This information is usually obtained through conventional culture methods. However, bacterial culture is very expensive, time-consuming and labor-intensive. The development of PCP technology has provided a favorable method for rapid identification of bacteria. Using this method, the identification of bacterial pathogens can be completed in a few hours, which is much shorter than bacterial culture, and the high sensitivity of PCR can improve the detection level.
[0004] Of particular relevance to mastitis diagnosis, multiplex PCR in a single test allows for simultaneous screening of multiple pathogens that may cause the disease. In multiplex PCR, multiple pairs of primers specific for different DNA fragments amplify multiple target sequences in the same assay tube with the same reaction program. In many cases, more than four pairs of PCR primers may be used. The primers used for multiplex PCR and amplification should have similar melting temperatures (Tm), because a difference of more than 10 °C in the Tm of two sets of primers may result in differential yields of amplified products. The target DNAs in multiplex PCR should also be of similar length, because a larger difference in target DNA length will favor the amplification of longer target fragments, resulting in differentially large amounts of amplified products. The main advantage of multiplex PCR over conventional PCR is cost-effectiveness. It can reduce the number of reagents (such as Taq DNA polymerase) used for each diagnostic. In addition, it requires less preparation and analysis time compared to systems that use multiple tubes of monoclonal PCR. Multiplex PCR has now been used to detect and type several bacteria and viruses simultaneously.
[0005] In summary, rapid and efficient detection is a time-saving and labor-saving method for food safety issues. The development and application of multiplex PCR technology provides an efficient, convenient and economical method for milk quality detection. Therefore, the use of multiplex PCR to detect mastitis pathogens is of great significance. Summary of the invention
[0006] In order to solve the above technical problems, the present application provides a multiplex PCR detection primer set, a kit and a detection method for mastitis pathogens.
[0007] In a first aspect, the present application provides a multiplex PCR detection primer set for mastitis pathogens, comprising the following primer pairs: a primer pair for amplifying a Staphylococcus aureus gene, the primer nucleotide sequence being shown in SEQ ID NO.1-2; A primer pair for amplifying a gene of Streptococcus agalactiae, wherein the nucleotide sequence of the primers is shown in SEQ ID NO.3-4; A primer pair for amplifying a Pseudomonas gene, the primer nucleotide sequences are shown in SEQ ID NO.5-6; A primer pair for amplifying Klebsiella pneumoniae genes, the primer nucleotide sequences are shown in SEQ ID NO.7-8; The primer pair is used to amplify the Escherichia coli gene, and the primer nucleotide sequences are shown in SEQ ID NO.9-10.
[0008] In this experiment, some dairy farms in Shaanxi Province were sampled and tested, and it was found that the main pathogens of mastitis in dairy cows in Shaanxi Province were Escherichia coli, Klebsiella pneumoniae, and Pseudomonas. The research significance of this application is to use multiplex PCR technology to establish a method that can quickly, accurately and sensitively detect and identify Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, Pseudomonas, and Klebsiella pneumoniae, thereby replacing conventional diagnostic methods.
[0009] Preferably, the size of the Staphylococcus aureus gene amplified fragment is 894 bp, the size of the Streptococcus agalactiae gene amplified fragment is 315 bp, the size of the Pseudomonas gene amplified fragment is 617 bp, the size of the Klebsiella pneumoniae gene amplified fragment is 428 bp, and the size of the Escherichia coli gene amplified fragment is 186 bp.
[0010] Preferably, the 5' end of at least one primer in the primer pair is fluorescently labeled.
[0011] In a second aspect, the present application provides a multiplex PCR detection kit for mastitis pathogenic bacteria, comprising the above-mentioned multiplex PCR detection primer set for mastitis pathogenic bacteria.
[0012] Preferably, the mastitis pathogenic bacteria multiplex PCR detection kit comprises a DNA template and ddH2O.
[0013] In a third aspect, the present application provides a multiplex PCR detection method for mastitis pathogenic bacteria, which uses the above-mentioned multiplex PCR detection primer set for mastitis pathogenic bacteria or the above-mentioned multiplex PCR detection kit for mastitis pathogenic bacteria for detection.
[0014] Preferably, the multiplex PCR detection method for mastitis pathogens specifically comprises the following steps in sequence: Extracting DNA from samples to be tested; Take specific primers and the DNA template of the sample to be tested to form an amplification reaction system; Placing the amplification reaction system on a real-time fluorescence PCR instrument to perform a real-time fluorescence PCR reaction; The PCR products were analyzed by 2% agarose gel electrophoresis.
[0015] Preferably, the PCR reaction system is as follows: the concentration of each primer is: 0.5 Umol / L for Staphylococcus aureus, Pseudomonas, Streptococcus agalactiae and Escherichia coli, and 1 Umol / L for Klebsiella pneumoniae; The concentration of each pathogenic bacteria DNA template is 100 pg / μl for Staphylococcus aureus, Pseudomonas and Escherichia coli, and 1 ng / μl for Streptococcus agalactiae and Klebsiella pneumoniae, and 1 μl of each template is added.
[0016] Preferably, the PCR reaction conditions are: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 29 s, 30 cycles, and extension at 72°C for another 10 min.
[0017] In summary, the technical solution of this application has the following effects: The present application establishes a five-plex PCR system that can rapidly detect and identify Staphylococcus aureus, Pseudomonas, Klebsiella pneumoniae, Streptococcus agalactiae and Escherichia coli, and the PCR system has good specificity and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is a graph showing the amplification results of the primer concentration selection for Staphylococcus aureus in Example 1.
[0019] Figure 2 This is a graph showing the amplification results of the primer concentration selection for Pseudomonas in Example 1.
[0020] Figure 3 This is a graph showing the amplification results of the Klebsiella pneumoniae primer concentration selection in Example 1.
[0021] Figure 4This is a graph showing the amplification results of the primer concentration selection for Streptococcus agalactiae in Example 1.
[0022] Figure 5 This is a graph showing the amplification results of the primer concentration selection for E. coli in Example 1.
[0023] Figure 6 This is a graph showing the amplification results of the annealing temperature selection for Staphylococcus aureus in Example 1.
[0024] Figure 7 This is a graph showing the amplification results of the Pseudomonas annealing temperature selection in Example 1.
[0025] Figure 8 This is a graph showing the amplification results of Klebsiella pneumoniae annealing temperature selection in Example 1.
[0026] Fig. 9 This is a graph showing the amplification results of the annealing temperature selection for Streptococcus agalactiae in Example 1.
[0027] Fig.10 This is a graph showing the amplification results of the E. coli annealing temperature selection in Example 1.
[0028] Fig.11 This is a diagram of the sensitivity amplification results of Escherichia coli in Example 1.
[0029] Fig.12 This is a diagram showing the sensitivity amplification results of Klebsiella pneumoniae in Example 1.
[0030] Fig.13 This is a graph showing the sensitivity amplification results of Pseudomonas and Streptococcus agalactiae in Example 1.
[0031] Fig.14 This is a diagram showing the sensitivity amplification results of Staphylococcus aureus in Example 1.
[0032] Fig.15 This is a graph showing the multiplex PCR amplification results at different primer concentrations in Example 1.
[0033] Fig.16 This is a graph showing the results of multiplex PCR amplification at different temperatures in Example 1.
[0034] Fig.17 This is a diagram of the multiplex PCR specific detection amplification results in Example 1.
[0035] Fig.18 This is a graph showing the amplification results of the multiplex PCR sensitivity detection in Example 1. DETAILED DESCRIPTION
[0036] The present application is further described in detail below in conjunction with embodiments and performance test applications. These embodiments should not be construed as limiting the scope of protection claimed in the present application. Example Example 1
[0037] This embodiment provides a multiplex PCR detection primer set, a kit and a detection method for mastitis pathogens.
[0038] 1 Experimental Materials 1.1 Strains Staphylococcus aureus and Streptococcus agalactiae were from strains preserved in our laboratory, and Klebsiella pneumoniae, Pseudomonas and Escherichia coli were from 50 milk samples from dairy farms around Shaanxi Province.
[0039] 1.2 Reagents The reagents required for the experiment are shown in Table 1.
[0040] Table 1 Reagents required for the experiment
[0041] 1.3 Preparation of main solutions 1×TAE electrophoresis buffer: 12.1 g Tris and 18.6 g Na2EDTA·2H2O were dissolved in 400 mL of deionized water. After fully dissolved, 28.55 mL of acetic acid was added. Stir slowly to dissolve the mixture and then add the fixed volume to 500 mL. Store at 4°C.
[0042] 1×TAE electrophoresis fluid: dilute 50×TAE electrophoresis buffer and use immediately after preparation.
[0043] LB liquid culture medium: 1g of tryptone, 0.5g of yeast powder, 1g of sodium chloride, add water to make up to 100mL, adjust the pH to 7.0~7.2. If using solid culture medium, add 15g of agar powder.
[0044] MacConkey medium: 2 g peptone, 2 g agar, 0.5 g sodium chloride, 0.5 g bile salt, 0.5 g lactose, 0.5 mL 1% neutral red aqueous solution, 100 mL distilled water. Add each component into water, heat to dissolve, adjust the pH to 7.2, boil, and filter with gauze absorbent cotton.
[0045] 2% agarose gel: weigh 0.4 g agarose, take 20 mL 1×TAE buffer, add the weighed agarose, shake well, heat until dissolved, add 0.2 μl GelStain nucleic acid dye, shake well, pour into a mold, and cool until solidified.
[0046] 1.4 Collection of milk samples A total of 50 non-clinical mastitis milk samples were collected from two dairy farms in Shaanxi Province. Before collection, the nipples and surrounding areas were disinfected with 70% alcohol cotton balls. The first 2 to 3 handfuls of milk were discarded during milking, and about 15 mL of milk was collected. The milk samples were placed in 50 mL sterile centrifuge tubes and stored at -20°C within 12 hours after collection.
[0047] 2 Experimental methods 2.1 Activation of bacteria After streaking the glycerol-preserved strain on MacConkey agar plates for 24 h, select a single colony and streak it again on a new MacConkey agar plate for 24 h. Select a single colony on the second streaking MacConkey agar plate and inoculate it into a 5 mL LB liquid culture medium test tube and culture it in a constant temperature shaker for 8-10 h to activate and proliferate the colonies.
[0048] 2.2 Extraction of bacterial DNA There are three methods for extracting bacterial DNA: direct extraction, high temperature boiling and kit extraction. In this application, the genomes of Gram-negative bacteria and Gram-positive bacteria are extracted using the Polymerase Bacterial Genome Extraction Kit.
[0049] 2.3 Primer design Primers for Staphylococcus aureus S.aureus, Streptococcus agalactiae S.agalactiae, Pseudomonas Pseudomonas, and Klebsiella pneumoniae K.pneumonia were designed according to the literature, and specific primers for Escherichia coli Escherichia coil were designed based on the phoA gene using DNA Star software, Primer 5.0, and Oligo6.0 software, and the annealing temperature of each primer was calculated using a network calculator. Among them, the gene amplification of Staphylococcus aureus was 894bp, the gene amplification of Streptococcus agalactiae was 315bp, the gene amplification of Pseudomonas was 617bp, the gene amplification of Klebsiella pneumoniae was 428bp, and the gene amplification of Escherichia coli was 186bp. The primer nucleic acid sequences are shown in Table 2.
[0050] Table 2 Primer sequences
[0051] 2.4 Optimization of singleplex PCR conditions First, the PCR reaction conditions of the five bacteria were optimized to determine the optimal annealing temperature and primer concentration. The primer concentration was 0.2-1.5 μmol / L; the annealing temperature was 49-54°C, with 6 temperature gradients. The specific reaction process is shown in Table 3.
[0052] Table 3 Optimization of singleplex PCR conditions
[0053] 2.5 Sensitivity of singleplex PCR The determination of single-plex PCR sensitivity is of great significance for the selection of primer concentration in multiplex PCR. According to the results of single-plex PCR optimization, the most suitable annealing temperature and primer concentration are selected. The DNA template of each bacterium is diluted 10 times from 10 ng and the sensitivity of single-plex PCR is determined according to the above reaction procedure.
[0054] 2.6 Specificity of singleplex PCR Testing the specificity of single-plex PCR is an important guarantee for the correctness of multiplex PCR. According to the results of single-plex PCR optimization, the most suitable annealing temperature and primer concentration were selected, and some common pathogens and the pathogens in this study were selected to test the specificity of each primer in the single-plex according to the above reaction procedure.
[0055] 2.7 Establishment and optimization of multiplex PCR conditions Combined with the results of single-plex PCR optimization, the most suitable annealing temperature and reaction program were selected to establish a multiplex PCR system. After determining the single-plex PCR system, each primer was mixed with the same volume and the appropriate template concentration to perform multiplex PCR amplification. The amplified products were detected by electrophoresis on 2% agarose gel. The possible results and solutions are shown in Table 4.
[0056] Table 4 Optimization of multiplex PCR conditions
[0057] 2.8 Specificity and sensitivity of multiplex PCR In order to ensure that there is no non-specific pairing between primers or between primers and other bacterial DNA templates in multiplex PCR, some common pathogenic bacteria such as Streptococcus bovis, Tetracoccus, Staphylococcus saprophyticus, Staphylococcus squirrel, Bacillus cereus and the pathogenic bacteria in this application were selected, and appropriate primer concentrations and annealing temperatures were selected according to the optimized reaction system for amplification, and then multiple specificity was detected by 2% agarose gel electrophoresis.
[0058] The extracted pathogenic bacteria DNA was diluted in a 10-fold gradient starting from 1 ng / μl, amplified according to the established five-fold system, and tested for multiple sensitivities.
[0059] 2.9 Clinical testing of quintuplex PCR The clinical effect of the five-plex PCR was established by purchasing three bags of milk (Mengniu, Yili, and Duoxian) from supermarkets and milk sold by free-range cattle households in the surrounding areas. 10 μl of milk sample was added to 5 mL of LB culture medium for overnight culture, and the bacterial solution was used for amplification according to the established multiplex system. The amplified products were observed after 2% agarose gel electrophoresis.
[0060] 3 Experimental Results 3.1 Optimization results of singleplex PCR conditions After analyzing the electrophoresis results of PCR amplification products under different primers and different annealing temperatures, it was determined that the optimal primer concentration was 0.5 μmol / L and the optimal annealing temperature was 54°C. At this concentration and annealing temperature, the amplified product had no nonspecific bands, no mixed bands, and had high sensitivity. The amplification results of the primer concentration selection for Staphylococcus aureus, Pseudomonas, Klebsiella pneumoniae, Streptococcus agalactiae, and Escherichia coli (Note: M is 1000 DNA Marker; 1 is 1.5 μmol / L; 2 is 1.2 μmol / L; 3 is 1.0 μmol / L; 4 is 0.5 μmol / L; 5 is 0.2 μmol / L) are as follows: Figure 1-5 As shown; the annealing temperature selection for Staphylococcus aureus, Pseudomonas, Klebsiella pneumoniae, Streptococcus agalactiae, and Escherichia coli (Note: M is 1000 DNA Marker; 1 is 49°C; 2 is 50°C; 3 is 51°C; 4 is 52°C; 5 is 53°C; 6 is 54°C) are as shown. Figure 6-10 shown.
[0061] Staphylococcus aureus, Pseudomonas, and Escherichia coli can all amplify obvious bands at different primer concentrations. The band of Staphylococcus aureus is darker, which may be caused by repeated freezing and thawing of the template DNA or too low template DNA concentration; Klebsiella pneumoniae produces nonspecific bands when the primer concentration is 1.5μmol / L, 1.2μmol / L, and 1.0μmol / L; Streptococcus agalactiae has darker bands when the primer concentration is 1.5μmol / L and 1.0μmol / L, and brighter bands when the primer concentration is 1.2μmol / L and 0.5μmol / L.
[0062] All five pathogens can amplify obvious bands at different temperatures. Klebsiella pneumoniae shows brighter bands at 54℃ than at other temperatures. Different temperatures have little effect on Staphylococcus aureus and Pseudomonas. The amplification product of Streptococcus agalactiae shows an increasing trend with the increase of temperature. Based on the optimal annealing temperature of primers for each pathogen and the results of PCR amplification products, 54℃ was selected as the more suitable annealing temperature.
[0063] 3.2 Sensitivity of singleplex PCR In the single-plex sensitivity test, the five pathogens showed different sensitivities and showed good amplification effects when the template DNA concentration was very low. The results are as follows: Escherichia coli can be detected at a minimum concentration of 1pg / μl, Klebsiella pneumoniae can be detected at a minimum concentration of 10pg / μl, Pseudomonas can be detected at a minimum concentration of 1pg / μl, Streptococcus agalactiae can be detected at a minimum concentration of 100pg / μl, and Staphylococcus aureus can be detected at a minimum concentration of 1fg / μl. The sensitivity tests of each bacteria are as follows: Escherichia coli sensitivity results are as follows Fig.11 As shown in (Note: M is 1000 DNA Marker; 1 is 10 ng / μl; 2 is 1 ng / μl; 3 is 100 pg / μl; 4 is 10 pg / μl; 5 is 1 pg / μl; 6 is 100 fg / μl; 7 is negative control; 8 is positive control), the sensitivity results of Klebsiella pneumoniae are as follows Fig.12 As shown in (M is 1000 DNA Marker; 1 is 10 ng / μl; 2 is 1 ng / μl; 3 is 100 pg / μl; 4 is 10 pg / μl; 5 is 1 pg / μl; 6 is 100 fg / μl; 7 is 10 fg / μl; 8 is negative control; 9 is positive control), Pseudomonas and Streptococcus agalactiae sensitivity results are as shown in Fig.13 As shown (Note: M.1000DNAMarker; Pseudomonas: 1 is 10ng / μl; 2 is 1ng / μl; 3 is 100pg / μl; 4 is 10pg / μl; 5 is 1pg / μl; 6 is 100fg / μl; 7 is 10fg / μl; 8 is negative control; 9 is positive control; Streptococcus agalactiae: 10 is 10ng / μl; 11 is 1ng / μl; 12 is 100pg / μl; 13 is 10pg / μl; 14 is 1pg / μl; 15 is 100fg / μl; 16 is negative control; 17 is positive control), the sensitivity results of Staphylococcus aureus are as shown Fig.14 As shown (Note: M is 1000 DNA Marker; 1 is 1 ng / μl; 2 is 100 pg / μl; 3 is 10 pg / μl; 4 is 1 pg / μl; 5 is 100 fg / μl; 6 is 10 fg / μl; 7 is 1 fg / μl; 8 is 10 -1 fg / μl; 9 = 10 -2 fg / μl; 10 is 10 - 3 fg / μl; 11 is negative control; 12 is positive control).
[0064] 3.3 Multiplex PCR primer concentration optimization results According to the results of multiplex PCR amplification at different primer concentrations, it can be concluded that the optimal primer concentration for Staphylococcus aureus, Pseudomonas, Streptococcus agalactiae and Escherichia coli is 0.5 μmol / L, and the optimal primer concentration for Klebsiella pneumoniae is 1 μmol / L. Fig.15 As shown (Note: M is 1000 DNA Marker; 1 is the primer concentration of 2.0μmol / L; 2 is the primer concentration of 1.5μmol / L; 3 is the primer concentration of 1μmol / L; 4 is the primer concentration of 0.5μmol / L; 5 is the primer concentration of 0.1μmol / L).
[0065] 3.4 Multiplex PCR annealing temperature optimization results like Fig.16 As shown in the figure, according to the results of electrophoresis of multiple PCR amplification products at different temperatures (Note: M is 1000 DNAMarker; 1. Annealing temperature is 51℃; 2. Annealing temperature is 52℃; 3. Annealing temperature is 53℃; 4. Annealing temperature is 54℃; 5. Annealing temperature is 56℃), it can be concluded that 53℃ or 54℃ is a more suitable annealing temperature. At 51℃ and 52℃, the amplification product has non-specific bands, the band of Staphylococcus aureus amplification product at 53℃ is darker than that at 54℃, the band of Klebsiella pneumoniae amplification product at 54℃ is darker than that at 53℃, and the band of Staphylococcus aureus is tailing at 55 and 56℃.
[0066] To summarize, the final reaction system of multiplex PCR is: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 29s, 30 cycles, and 72℃ extension for 10min. The concentration of each primer is 0.5Umol / L for Staphylococcus aureus, Pseudomonas, Streptococcus agalactiae and Escherichia coli, and 1Umol / L for Klebsiella pneumoniae. The concentration of each pathogenic bacterial DNA template is 100pg / μl for Staphylococcus aureus, Pseudomonas and Escherichia coli, 1ng / μl for Streptococcus agalactiae and Klebsiella pneumoniae, and 1μl is added to each template.
[0067] 3.5 Multiplex PCR specificity detection Multiple PCR detection using overnight cultured bacterial liquid of bovine Streptococcus, tetra-streptococcus, Staphylococcus saprophyticus, Staphylococcus sciuri and Bacillus cereus and the main pathogens in this study showed high specificity. Escherichia coli 186bp, Streptococcus agalactiae 315bp, Klebsiella pneumoniae 428bp, Pseudomonas 617bp, Staphylococcus aureus 894bp could amplify relatively obvious target bands. The multiple PCR specific detection results are shown in Fig.17As shown (Note: M is 1000 DNA Marker; 1 is Escherichia coli, Streptococcus bovis, Tetracoccus, Staphylococcus saprophyticus and Staphylococcus sciuri; 2 is Streptococcus agalactiae, Streptococcus bovis, Tetracoccus, Staphylococcus saprophyticus and Staphylococcus sciuri; 3 is Klebsiella pneumoniae, Streptococcus bovis, Tetracoccus, Staphylococcus saprophyticus and Staphylococcus sciuri; 4 is Pseudomonas, Streptococcus bovis, Tetracoccus, Staphylococcus saprophyticus and Staphylococcus sciuri; 5 is Staphylococcus aureus, Streptococcus bovis, Tetracoccus, Staphylococcus saprophyticus and Staphylococcus sciuri; 6 is Escherichia coli, Streptococcus agalactiae, Streptococcus bovis, Tetracoccus and Staphylococcus sciuri Staphylococcus aureus; 7 is Escherichia coli, Klebsiella pneumoniae, Streptococcus bovis, Tetragenous Staphylococcus and Staphylococcus sciuri; 8 is Escherichia coli, Streptococcus bovis, Pseudomonas, Staphylococcus saprophyticus and Bacillus cereus; 9 is Escherichia coli, Staphylococcus aureus, Tetragenous Staphylococcus saprophyticus and Bacillus cereus; 10 is Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus bovis, Staphylococcus saprophyticus; 11 is Streptococcus agalactiae, Pseudomonas, Streptococcus bovis, Staphylococcus sciuri and Bacillus cereus; 12 is Streptococcus agalactiae, Staphylococcus aureus, Tetragenous Streptococcus, Staphylococcus sciuri and Bacillus cereus Bacillus; 13 is Klebsiella pneumoniae, Pseudomonas, Streptococcus tetras, Streptococcus bovis and Staphylococcus sciuri; 14 is Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus bovis, Streptococcus tetras and Bacillus cereus; 15 is Pseudomonas, Staphylococcus aureus, Streptococcus bovis, Tetras and Bacillus cereus; 16 is Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus bovis and Streptococcus tetras; 17 is Escherichia coli, Pseudomonas, Staphylococcus aureus, Staphylococcus sciuri, Staphylococcus saprophyticus; 18 is Streptococcus agalactiae, Klebsiella pneumoniae, Pseudomonas, Streptococcus bovis, Streptococcus sciuri, Staphylococcus saprophyticus. cereus; 19 is Streptococcus agalactiae, Pseudomonas, Staphylococcus aureus, Tetragenococcus and Staphylococcus saprophyticus; 20 is Klebsiella pneumoniae, Pseudomonas, Staphylococcus aureus, Staphylococcus sciuri and Bacillus cereus; 21 is Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Pseudomonas and Streptococcus bovis; 22 is Streptococcus agalactiae, Klebsiella pneumoniae, Pseudomonas, Staphylococcus aureus and Tetragenococcus; 23 is Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Pseudomonas, Staphylococcus aureus, Staphylococcus saprophyticus and Streptococcus bovis).
[0068] 3.6 Multiplex PCR sensitivity testing The five-fold system was diluted 10 times from 1 ng / μl for PCR amplification. The amplified product was electrophoresed ( Fig.18As shown; Note: M is 1000 DNA Marker; 1 is 1ng / μl; 2 is 100pg / μl; 3 is 10pg / μl; 4 is 1pg / μl; 5 is 100fg / μl). It can be observed that the lowest concentration that can be detected is 10pg / μl of each template DNA concentration. Compared with the single-sensitivity test results of each pathogen, the sensitivity of Staphylococcus aureus is reduced, and the sensitivity of Klebsiella pneumoniae is enhanced. Overall, the established five-fold system has a higher sensitivity. 4. Conclusion This application includes primer design, extraction of template DNA, establishment of multiplex PCR system and clinical application of multiplex detection technology.
[0069] In the primer design process, first the specificity of the primer is crucial, it is closely related to whether a certain bacterium can be efficiently detected and there are no other non-specific bands, and the establishment of the subsequent multiplex PCR system. Secondly, non-specific binding cannot occur between primers, otherwise false positive results are very likely to occur in the experimental process, affecting the experimental results. Finally, the size of each primer amplification target band should differ by at least 40bp from each other, so that it can be clearly observed in the electrophoresis result whether the target pathogenic bacteria are detected. The primers selected in this application amplify the target bands, which differ by at least 113bp between each other, and the detection result is accurate and easy to observe.
[0070] Before extracting pathogenic bacteria DNA, in order to ensure that the selected pathogens are correct and purified, 16sRNA is first used to perform PCR amplification on the overnight cultured bacterial solution, and the amplified product is sent to Bioengineering for sequencing. After that, the bacteria are re-inoculated in new LB culture medium and cultured overnight. The bacterial solution can be used for DNA extraction after microscopic examination. There are many methods for extracting pathogenic bacteria DNA. This study uses a kit for extraction, which is easy to operate and can be used for both Gram-negative and Gram-positive bacteria. In order to ensure that the bacteria are not contaminated by environmental pathogens during the operation, which affects the extraction results, the extraction process is carried out in a sterile operation room after being sterilized by ultraviolet light for half an hour.
[0071] During the establishment of the multiplex PCR system, the primer concentration and annealing temperature of a single bacterium are first optimized, so that data can be relied upon in the establishment of the multiplex reaction process. Adjustments can be made based on the results to effectively improve the accuracy of the establishment of the multiplex system. Detecting single-plex sensitivity provides a reference for the selection of template concentration in the multiplex system. In the multiplex system, a template concentration with medium sensitivity to pathogenic bacteria is selected. If the concentration is too low, the amplification results are not obvious, and if the concentration is too high, other target bands or miscellaneous bands are difficult to observe. According to the multiplex PCR amplification results, the template concentration or the volume of the template in the reaction system can be appropriately changed.
[0072] In the multiple specific detection, bacterial cultures of bovine Streptococcus, tetra-streptococci, saprophytic Staphylococcus, squirrel Staphylococcus and Bacillus cereus were selected from our laboratory and PCR amplified with the main pathogenic bacteria in this study. After 2% agarose gel electrophoresis, obvious target bands were observed, and no other non-specific bands were seen, indicating that the quintuple PCR detection established in this application has high accuracy and high specificity of primers.
[0073] This application mainly studies the pathogenic bacteria that cause mastitis in dairy cows in Shaanxi Province. By sampling the main dairy farms in Shaanxi Province, the positive milk was isolated and identified for bacteria. The study found that the main pathogenic bacteria include Klebsiella pneumoniae, Escherichia coli and Pseudomonas. In the establishment of multiple PCR, common pathogenic bacteria Staphylococcus aureus and Streptococcus agalactiae were added to improve its detection efficiency. The multiple PCR established in this application is not only for the Shaanxi region, but also can be used for the detection of mastitis pathogens in Shaanxi dairy farms, and the detection of Klebsiella pneumoniae and Pseudomonas has been added, which has been innovated in clinical application.
[0074] In summary, the present application has established a five-plex PCR system that can rapidly detect and identify Staphylococcus aureus, Pseudomonas, Klebsiella pneumoniae, Streptococcus agalactiae and Escherichia coli. The sensitivity and specificity test results show that the five-plex PCR system has good specificity and sensitivity.
[0075] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.
Claims
1. A multiplex PCR primer set for detecting mastitis pathogens, characterized in that: The primer pair includes the following: a primer pair for amplifying a Staphylococcus aureus gene, the primer nucleotide sequence is shown in SEQ ID NO.1-2; A primer pair for amplifying a gene of Streptococcus agalactiae, wherein the nucleotide sequence of the primers is shown in SEQ ID NO.3-4; A primer pair for amplifying a Pseudomonas gene, the primer nucleotide sequences are shown in SEQ ID NO.5-6; A primer pair for amplifying Klebsiella pneumoniae genes, the primer nucleotide sequences are shown in SEQ ID NO.7-8; The primer pair is used to amplify the Escherichia coli gene, and the primer nucleotide sequences are shown in SEQ ID NO.9-10.
2. The multiplex PCR detection primer set for mastitis pathogens according to claim 1, characterized in that: The size of the Staphylococcus aureus gene amplification fragment is 894 bp, the size of the Streptococcus agalactiae gene amplification fragment is 315 bp, the size of the Pseudomonas gene amplification fragment is 617 bp, the size of the Klebsiella pneumoniae gene amplification fragment is 428 bp, and the size of the Escherichia coli gene amplification fragment is 186 bp.
3. The multiplex PCR detection primer set for mastitis pathogens according to claim 1, characterized in that: The 5' end of at least one primer in the primer pair is fluorescently labeled.
4. A multiplex PCR detection kit for mastitis pathogens, characterized in that: The invention comprises the multiplex PCR detection primer set for mastitis pathogenic bacteria according to any one of claims 1 to 3.
5. The multiplex PCR detection kit for mastitis pathogens according to claim 4, characterized in that: Including DNA template, ddH2O.
6. A multiplex PCR detection method for mastitis pathogens, characterized in that: The detection is performed using the mastitis pathogenic bacteria multiple PCR detection primer set described in any one of claims 1 to 3 or the mastitis pathogenic bacteria multiple PCR detection kit described in any one of claims 4 to 5.
7. The multiplex PCR detection method for mastitis pathogens according to claim 6, characterized in that: Specifically, the following steps are performed in sequence: Extracting DNA from samples to be tested; Take specific primers and the DNA template of the sample to be tested to form an amplification reaction system; Placing the amplification reaction system on a real-time fluorescence PCR instrument to perform a real-time fluorescence PCR reaction; The PCR products were analyzed by 2% agarose gel electrophoresis.
8. The multiplex PCR detection method for mastitis pathogens according to claim 7, characterized in that: The PCR reaction system was as follows: the concentration of each primer was 0.5 Umol / L for Staphylococcus aureus, Pseudomonas, Streptococcus agalactiae and Escherichia coli, and 1 Umol / L for Klebsiella pneumoniae; The concentration of each pathogenic bacteria DNA template is 100 pg / μl for Staphylococcus aureus, Pseudomonas and Escherichia coli, and 1 ng / μl for Streptococcus agalactiae and Klebsiella pneumoniae, and 1 μl of each template is added.
9. The multiplex PCR detection method for mastitis pathogens according to claim 8, characterized in that: The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 29 s, 30 cycles, and extension at 72°C for another 10 min.