Co-culture method and quadruple PCR (Polymerase Chain Reaction) detection method aiming at swine infectious arthritis related pathogenic bacteria

Abstract

The invention discloses a general culture medium and a quadruple PCR (Polymerase Chain Reaction) detection method aiming at swine infectious arthritis pathogenic bacteria such as streptococcus suis, graisseria parasuis, swine erysipelothrix erysipelas and mycoplasma hyorhinis, and belongs to the field of molecular biology. Aiming at the problem that culture media are different, the invention optimizes a culture medium, realizes co-culture of four pathogenic bacteria and is used for subsequent multiple PCR bacterium enrichment, discloses a specific primer group aiming at pathogenic bacteria conserved genes and establishes a quadruple PCR detection method for synchronously identifying the four pathogenic bacteria. According to the co-culture and detection method disclosed by the invention, the problem of complexity caused by using multiple culture mediums and the problem of low efficiency of single PCR are solved, and the method has relatively high sensitivity and specificity and has a wide application prospect in clinical mixed infection detection.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and relates to a co-culture method of porcine infectious arthritis pathogens Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae and Mycoplasma hyorhinis and a quadruple PCR detection method. Background Art

[0002] Porcine infectious arthritis is a serious disease that severely impacts the pig farming industry. Its typical clinical symptoms include joint swelling, lameness, and even inability to stand, which not only impairs animal welfare but also severely impacts pig production performance. The etiology of porcine infectious arthritis is complex, primarily caused by pathogens such as Streptococcus suis (SS), Glaesserella parasuis (GPS), Erysipelothrix rhusiopathiae (ER), and Mycoplasma hyorhinis (Mhr). These pathogens can cause single infections or, more often, mixed infections. Their identification is challenging due to the highly similar clinical symptoms they induce.

[0003] Due to the differences in nutritional requirements of different pathogens and the different growth patterns of different pathogens, the culture media for various pathogens are not the same. Traditional methods require the use of multiple culture media for separate cultivation, resulting in a cumbersome process for pathogen enrichment in the early stages. Developing a culture medium that can simultaneously support the growth of four culture media will simplify the early pathogen enrichment steps and save resources and manpower.

[0004] Currently, there are few studies on detection methods for porcine arthritis pathogens. PCR technology is currently the most commonly used molecular diagnostic technology, which identifies pathogens by amplifying specific DNA fragments. In the case of mixed clinical infections, the use of single-plex PCR requires separate PCR testing for each pathogen, resulting in high sample consumption, increased reagent costs, and time-consuming and labor-intensive testing. Multiplex PCR is a reaction that simultaneously amplifies multiple DNA fragments in the same system. Compared with traditional PCR, multiplex PCR has higher throughput and efficiency, and can detect multiple pathogens simultaneously in complex samples to complete synchronous detection, greatly improving diagnostic efficiency.

[0005] Establishing a co-culture system and a matching quadruple PCR for the above four porcine arthritis pathogens can not only improve detection efficiency, but also has important application value in ensuring the healthy development of the pig industry. Summary of the Invention

[0006] The present invention aims to provide a primer set and a quadruple PCR detection method, as well as a universal culture medium for enrichment, that can simultaneously detect the pathogens of porcine infectious arthritis, including Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, in a short time. This enables the simultaneous detection of multiple pathogens in a short period of time. This invention has promising application prospects in the diagnosis of porcine infectious arthritis.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides a KM2-PN culture medium that can be used to co-cultivate Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis. The culture medium is composed of a mycoplasma basal culture medium, porcine serum, and nicotinamide adenine dinucleotide. The culture medium comprises the following steps:

[0009] (1) Prepare conventional KM2 mycoplasma basal culture medium, autoclave at 121°C for 30 min, and cool to room temperature;

[0010] (2) Aseptically adding filter-sterilized nicotinamide adenine dinucleotide (NAD) to the KM2 mycoplasma basal medium to control the final concentration of nicotinamide adenine dinucleotide (NAD) to 5 μg / mL-20 μg / mL, and then adding sterile porcine serum to control the volume proportion of sterile porcine serum in the finished medium to 5%-50%;

[0011] (3) Aseptically adjust the pH to 7.4-7.6 to obtain KM2-PN medium;

[0012] (4) Streptococcus suis, Glaseria parasuis, and Erysipelothrix rhusiopathiae were inoculated into KM2-PN medium at a volume ratio (V:V) of 1:100, mixed, and cultured in a shaking incubator at 37°C for 12-24 hours. Mycoplasma hyorhinis was inoculated into KM2-PN medium at a volume ratio (V:V) of 1:5, mixed, and cultured in a 37°C bacterial incubator for 48 hours.

[0013] The KM2-PN culture medium can be used to culture pathogens of porcine infectious arthritis, such as Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis.

[0014] In the KM2-PN medium, the volume of the porcine serum accounts for 20% of the finished medium, and the final concentration of nicotinamide adenine dinucleotide is 10 μg / mL.

[0015] The method of co-culturing pathogens using KM2-PN medium can be used for bacterial enrichment before quadruple PCR detection of Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, and high-quality templates can be obtained by the above method.

[0016] The present invention also provides a multiplex PCR primer set for simultaneously detecting multiple pathogens, wherein the multiple pathogens include Streptococcus suis, Glasseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis. The multiplex PCR primer set includes a primer pair A, a primer pair B, a primer pair C, and a primer pair D. The primer pair A includes primer 1 and primer 2 for detecting Streptococcus suis, the primer pair B includes primer 3 and primer 4 for detecting Glasseria parasuis, the primer pair C includes primer 5 and primer 6 for detecting Erysipelothrix rhusiopathiae, and the primer pair D includes primer 7 and primer 8 for detecting Mycoplasma hyorhinis. The nucleotide sequences of primers 1 to 8 are shown in SEQ ID NO.1 to SEQ ID NO.8, respectively.

[0017] The present invention also provides a kit for detecting Streptococcus suis, Glasseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, comprising primers 1 and 2 for detecting Streptococcus suis, primers 3 and 4 for detecting Glasseria parasuis, primers 5 and 6 for detecting Erysipelothrix rhusiopathiae, and primers 7 and 8 for detecting Mycoplasma hyorhinis. The nucleotide sequences of primers 1 to 8 are shown in SEQ ID NO.1 to SEQ ID NO.8, respectively.

[0018] The primer set consists of the following primers:

[0019] SS-F: 5'-GCAGCGTATTCTGTCAAACG-3' (SEQ ID NO.1),

[0020] SS-R: 5'-CCATGGACAGATAAAGATGG-3' (SEQ ID NO. 2);

[0021] GPS-F: 5'-CCTTCTTAGATACACCAGGACA-3'(SEQ ID NO.3),

[0022] GPS-R: 5'-GCCATATTCAAAGCCACAA-3' (SEQ ID NO.4);

[0023] ER-F: 5'-CGATTATATTCTTAGCACGCACCGCAACG-3' (SEQ ID NO.5),

[0024] ER-R: 5'-TGCTTGTGTTGTTGATTTCTTGACG-3' (SEQ ID NO. 6);

[0025] Mhr-F: 5'-CACTCCAACATCATACGGAA-3' (SEQ ID NO.7),

[0026] Mhr-R: 5'-GGAACTTTAGATGATTTCCAT-3' (SEQ ID NO. 8).

[0027] The kit further comprises Multiplex buffer, Multiplex DNA Polymerase, and DNA template standards of Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis.

[0028] The concentrations of primers 1 to 8 in the kit are all 10 μmol / L, the volume of primers 1 to 4 is 1.25 μL, the volume of primers 5 and 6 is 0.25 μL, and the volume of primers 7 and 8 is 1.75 μL.

[0029] The present invention also provides a method for performing multiplex PCR detection using the above kit, which is for non-diagnostic purposes and comprises the following steps:

[0030] (1) extracting nucleic acid from the sample to be tested;

[0031] (2) using the nucleic acid of the sample to be tested as a template and using primers 1 to 8 to perform a quadruple PCR reaction;

[0032] (3) The amplified product is subjected to electrophoresis. If a 689 bp band appears in the amplified product, Streptococcus suis is present in the sample to be tested; if a 491 bp band appears in the amplified product, Glaseria parasuis is present in the sample to be tested; if a 938 bp band appears in the amplified product, Erysipelothrix rhusiopathiae is present in the sample to be tested; if a 153 bp band appears in the amplified product, Mycoplasma hyorhinis is present in the sample to be tested.

[0033] In the detection method, the reaction system of the quadruple PCR reaction is: 2 μL of the sample template to be tested, 12.5 μL of 2×Multiplex Buffer, 1 μL of Multiplex DNA Polymerase, 1.25 μL each of the upstream and downstream primers of Streptococcus suis, 1.25 μL each of the upstream and downstream primers of Glaseria parasuis, 0.25 μL each of the upstream and downstream primers of Erysipelothrix rhusiopathiae, 1.75 μL each of the upstream and downstream primers of Mycoplasma hyorhinis, and 1.5 μL of ddH2O, with a total volume of 25 μL.

[0034] In the detection method, the reaction procedure of the quadruple PCR reaction is: pre-denaturation at 95°C for 30s; denaturation at 95°C for 30s, annealing at 63°C for 90s, extension at 72°C for 60s, 35 cycles; final extension at 72°C for 10min; and storage at 4°C.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The present invention optimizes the KM2 mycoplasma culture medium, adds porcine serum and nicotinamide adenine dinucleotide, and realizes the co-culture of Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, thereby reducing the cultivation steps of pathogens and lowering labor costs.

[0037] (2) The results of the quadruple PCR detection method provided by the present invention showed that the quadruple PCR had good sensitivity and good specificity, and no amplified bands were found for Escherichia coli, Staphylococcus suis, Pasteurella multocida, and Actinobacillus pleuropneumoniae.

[0038] (3) The quadruple PCR system of the present invention has a high amplification efficiency for a total of 15 pathogen DNA combinations, including quadruple (simultaneous detection of four pathogens), triple (combination of any three pathogens), double (combination of any two pathogens), and single (detection of a single pathogen). The established quadruple PCR detection system can stably amplify specific bands of the expected size under all combination conditions, and no nonspecific amplification products appear, indicating that the system has good amplification efficiency and can meet the detection needs of different pathogen combinations in clinical samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the growth of Streptococcus suis, Glassella parasuis, and Erysipelothrix rhusiopathiae in KM2-PN medium; 1: negative control, 2: Streptococcus suis, 3: Glassella parasuis, 4: Erysipelothrix rhusiopathiae.

[0040] Figure 2 Schematic diagram of the growth of Mycoplasma hyorhinis in KM2-PN medium; 1-4: Mycoplasma hyorhinis, 5: negative control.

[0041] Figure 3 This is a schematic diagram of the growth of Streptococcus suis in KM2-PN solid culture medium.

[0042] Figure 4 This is a schematic diagram of the growth of Glassera parasuis in KM2-PN solid culture medium.

[0043] Figure 5 This is a schematic diagram of the growth of Erysipelothrix rhusiopathiae in KM2-PN solid culture medium.

[0044] Figure 6 Schematic diagram of the growth of Mycoplasma hyorhinis in KM2-PN solid medium (observed under a microscope);

[0045] Figure 7Schematic diagram of the results of the Streptococcus suis primer specificity test; M: Marker, 1: Streptococcus suis, 2: Glasserella parasuis, 3: Erysipelothrix rhusiopathiae, 4: Mycoplasma hyorhinis, 5: Escherichia coli, 6: Staphylococcus suis, 7: Pasteurella multocida, 8: Actinobacillus pleuropneumoniae, 9: negative control.

[0046] Figure 8 Schematic diagram of the primer specificity test results for Glassera parasuis; M: Marker, 1: Glassera parasuis, 2: Streptococcus suis, 3: Erysipelothrix rhusiopathiae, 4: Mycoplasma hyorhinis, 5: Escherichia coli, 6: Staphylococcus suis, 7: Pasteurella multocida, 8: Actinobacillus pleuropneumoniae, 9: negative control.

[0047] Figure 9 Schematic diagram of the primer specificity test results for Erysipelothrix rhusiopathiae; M: Marker, 1: Erysipelothrix rhusiopathiae, 2: Streptococcus suis, 3: Glasseria parasuis, 4: Mycoplasma hyorhinis, 5: Escherichia coli, 6: Staphylococcus suis, 7: Pasteurella multocida, 8: Actinobacillus pleuropneumoniae, 9: negative control.

[0048] Figure 10 Schematic diagram of the results of the primer specificity test for Mycoplasma hyorhinis; M: Marker, 1: Mycoplasma hyorhinis, 2: Streptococcus suis, 3: Glasseria parasuis, 4: Erysipelothrix rhusiopathiae, 5: Escherichia coli, 6: Staphylococcus suis, 7: Pasteurella multocida, 8: Actinobacillus pleuropneumoniae, 9: negative control.

[0049] Figure 11 Schematic diagram of the specificity test results of primers for Streptococcus suis, Glasseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis in the quadruple system; M: Marker, 1: mixed DNA of Streptococcus suis, Glasseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, 2: Escherichia coli, 3: Staphylococcus suis, 4: Pasteurella multocida, 5: Actinobacillus pleuropneumoniae, 6: negative control.

[0050] Figure 12Schematic diagram of amplification of different template combinations under the quadruple system; M: Marker, 1: Mixed DNA of Streptococcus suis, Glasseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, 2: Mixed DNA of Streptococcus suis, Glasseria parasuis, and Erysipelothrix rhusiopathiae, 3: Mixed DNA of Streptococcus suis, Glasseria parasuis, and Mycoplasma hyorhinis, 4: Mixed DNA of Streptococcus suis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, 5: Mixed DNA of Glasseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, 6: Mixed DNA of Streptococcus suis, Mixed DNA of Glassera parasuis, 7: Mixed DNA of Streptococcus suis and Erysipelothrix rhusiopathiae, 8: Mixed DNA of Streptococcus suis and Mycoplasma hyorhinis, 9: Mixed DNA of Glassera parasuis and Erysipelothrix rhusiopathiae, 10: Mixed DNA of Glassera parasuis and Mycoplasma hyorhinis, 11: Mixed DNA of Erysipelothrix rhusiopathiae and Mycoplasma hyorhinis, 12: Streptococcus suis DNA, 13: Glassera parasuis DNA, 14: Erysipelothrix rhusiopathiae DNA, 15: Mycoplasma hyorhinis DNA, 16: negative control.

[0051] Figure 13 Schematic diagram of the sensitivity results of Streptococcus suis, Glasseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis in multiple systems; M: Marker, 1: Genome concentration: 10 6 CFU / μL, 2: Genome concentration: 10 5 CFU / μL, 3: Genome concentration: 10 4 CFU / μL, 4: genome concentration: 10 3 CFU / μL, 5: genome concentration: 10 2 CFU / μL, 6: genome concentration: 10 1 CFU / μL, 7: genome concentration: 10 0 CFU / μL, 8: negative control. DETAILED DESCRIPTION

[0052] The following is a clear and complete description of the technical solutions of the present invention in conjunction with the embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0053] Example 1: Cultivation of porcine infectious arthritis pathogens Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis in KM2-PN medium

[0054] This embodiment provides a universal culture medium KM2-PN for pathogens related to porcine infectious arthritis. The culture medium consists of a KM2 mycoplasma basal culture medium and auxiliary components, wherein the auxiliary components consist of porcine serum and nicotinamide adenine dinucleotide.

[0055] Preparation of KM2-PN medium: Weigh 23.9 g of KM2 Mycoplasma Basal Medium powder into 800 ml of pure water, dissolve at room temperature, and autoclave at 121°C for 30 minutes. Add filter-sterilized NAD to a final concentration of 10 μg / mL. Aseptically add 200 ml of sterile healthy porcine serum and adjust the pH to 7.4-7.6. Add 1.5% agar powder to the KM2 liquid medium to create a solid medium for bacterial and mycoplasma culture.

[0056] Streptococcus suis, Glaseria parasuis, and Erysipelothrix rhusiopathiae were inoculated at a ratio of 1:100 into KM2 medium (KM2-PN) supplemented with 20% sterile porcine serum and a final concentration of 10 μg / mL of sterile nicotinamide adenine dinucleotide and cultured in a 37°C shaker. Mycoplasma hyorhinis was inoculated at a ratio of 1:5 into KM2-PN liquid medium and cultured in a 37°C incubator. Solid culture: Streak Streptococcus suis, Glaseria parasuis, and Erysipelothrix rhusiopathiae on solid culture medium, invert and place in a 37°C incubator, and let it stand overnight. 100 μL of the mycoplasma liquid was spread on KM2-PN solid culture medium and inverted and placed in a 37°C incubator for culture.

[0057] The results are as follows Figure 1 As shown in the figure, after overnight culture of Streptococcus suis, Glaseria parasuis, and Erysipelothrix rhusiopathiae in KM2-PN medium, the color of the medium changed from red to yellow and the liquid became turbid. Figure 2 As shown in the figure, the culture medium inoculated with Mycoplasma hyorhinis turned from red to yellow, and the liquid was clear and not turbid. Figure 3 (Streptococcus suis), Figure 4 (Glaserella parasuis), Figure 5 (Erysipelothrix rhusiopathiae) is shown in Figure 2. When Streptococcus suis, Glaseria parasuis, and Erysipelothrix rhusiopathiae are inoculated on KM2-PN solid medium and cultured overnight, the corresponding bacterial colonies can be observed on the plate, and the color of the medium changes from red to yellow where the colonies grow. Figure 6 As shown, after Mycoplasma hyorhinis was cultured on KM2-PN solid medium, "poached egg"-like colonies were observed under a microscope. The above experimental results show that the four pathogens can be co-cultured in KM2-PN medium.

[0058] Example 2: PCR primer design

[0059] (1) Design of PCR primers targeting Streptococcus suis genes: Primers were designed using Primer5 based on the gdh gene sequence of Streptococcus suis published in the NCBI database as the target gene. The primer sequences were as follows:

[0060] SS-F: 5'-GCAGCGTATTCTGTCAAACG-3' (SEQ ID NO.1),

[0061] SS-R: 5'-CCATGGACAGATAAAGATGG-3' (SEQ ID NO. 2).

[0062] (2) PCR primers were designed targeting the G. parasuis gene. The infB gene sequence of G. parasuis published in the NCBI database was used as the target gene. Primer5 was used to design primers. The primer sequences were:

[0063] GPS-F: 5'-CCTTCTTAGATACACCAGGACA-3'(SEQ ID NO.3),

[0064] GPS-R: 5'-GCCATATTCAAAGCCACAA-3' (SEQ ID NO. 4).

[0065] (3) PCR primers were designed targeting the Erysipelothrix rhusiopathiae gene. The epsG gene sequence of Erysipelothrix rhusiopathiae published in the NCBI database was used as the target gene. Primer5 was used to design primers. The primer sequences were as follows:

[0066] ER-F: 5'-CGATTATATTCTTAGCACGCACCGCAACG-3' (SEQ ID NO.5),

[0067] ER-R: 5'-TGCTTGTGTTGTGATTTCTTGACG-3' (SEQ ID NO. 6).

[0068] (4) PCR primers were designed targeting the Mycoplasma hyorhinis gene. The Mycoplasma hyorhinis P37 gene sequence published in the NCBI database was used as the target gene. Primer5 was used to design primers. The primer sequences were:

[0069] Mhr-F: 5'-CACTCCAACATCATACGGAA-3' (SEQ ID NO.7),

[0070] Mhr-R: 5'-GGAACTTTAGATGATTTCCAT-3' (SEQ ID NO. 8).

[0071] The above primers were synthesized by Youkang Biotechnology Co., Ltd.

[0072] Example 3: Primer specificity detection

[0073] Bacterial DNA was extracted using a commercial bacterial DNA kit.

[0074] Use Streptococcus suis primers SS-F and SS-R to specifically detect common pathogenic bacteria of pigs (list removed). The specificity of the detection primers is:

[0075] Table 1 Strains used for PCR specificity detection

[0076] Serial number Strain name Strain source 1 Streptococcus suis This laboratory keeps 2 Glassera parasuis This laboratory keeps 3 Erysipelothrix rhusiopathiae This laboratory keeps 4 Mycoplasma hyorhinis This laboratory keeps 5 Escherichia coli This laboratory keeps 6 Staphylococcus hyosus This laboratory keeps 7 Pasteurella This laboratory keeps 8 Actinobacillus pleuropneumoniae This laboratory keeps

[0077] (1) Specific detection of Streptococcus suis primers SS-F and SS-R.

[0078] The specificity of primers was tested by using DNA of Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, Mycoplasma hyorhinis, Escherichia coli, Staphylococcus suis, Pasteurella multocida and Actinobacillus pleuropneumoniae as templates and Streptococcus suis primers SS-F and SS-R as primers.

[0079] The PCR amplification system was as follows: 25 μL of the mix, 1 μL each of 10 μmol / L upstream and downstream primers, 1 μL of DNA template, and 22 μL of ddH2O. The PCR program was as follows: 94°C for 1 minute; 30 cycles of 98°C for 10 seconds, 63°C for 15 seconds, and 68°C for 30 seconds. After the PCR program was completed, the PCR product was added to 2.5 μL of 10× Loading Buffer and 2.5 μL of 10× Gelred, and the target band was observed by electrophoresis on a 3% agarose gel. The results were as follows: Figure 7 As shown, the Streptococcus suis primers only produce specific target bands for the genome of Streptococcus suis, and the band size of Streptococcus suis is 689 bp. The DNA amplification results of the other 7 strains are negative, which proves that the Streptococcus suis amplification primers SS-F and SS-R of the present invention have good specificity.

[0080] (2) Specific detection of Glassera parasuis primers GPS-F and GPS-R.

[0081] The specificity of primers was tested using DNAs of Glaseria parasuis, Streptococcus suis, Erysipelothrix rhusiopathiae, Mycoplasma hyorhinis, Escherichia coli, Staphylococcus suis, Pasteurella multocida, and Actinobacillus pleuropneumoniae as templates and Glaseria parasuis primers GPS-F and GPS-R as primers.

[0082] The PCR amplification system consisted of 25 μL of the mix, 1 μL of each 10 μmol / L upstream and downstream primers, 1 μL of DNA template, and 22 μL of ddH2O. The PCR program was as follows: 94°C for 1 minute; 98°C for 10 seconds, 63°C for 15 seconds, and 68°C for 30 seconds, for a total of 30 cycles. After the PCR program was completed, the PCR product was added to 2.5 μL of 10× Loading Buffer and 2.5 μL of 10× Gelred, and the target band was observed by electrophoresis on a 3% agarose gel. The results were as follows: Figure 8 As shown, the primers for G. parasuis only produce specific target bands for the genome of G. parasuis. The band size of G. parasuis is 491bp, and the DNA amplification results for the other 7 strains are negative, proving that the G. parasuis amplification primers GPS-F and GPS-R of the present invention have good specificity.

[0083] (3) Specific detection of Erysipelothrix rhusiopathiae primers ER-F and ER-R.

[0084] The specificity of primers was tested by using DNA of Erysipelothrix rhusiopathiae, Streptococcus suis, Glasseria parasuis, Mycoplasma hyorhinis, Escherichia coli, Staphylococcus suis, Pasteurella multocida and Actinobacillus pleuropneumoniae as templates and Erysipelothrix rhusiopathiae primers ER-F and ER-R as primers.

[0085] The PCR amplification system consisted of 25 μL of the mix, 1 μL of each 10 μmol / L upstream and downstream primers, 1 μL of DNA template, and 22 μL of ddH2O. The PCR program was as follows: 94°C for 1 minute; 98°C for 10 seconds, 63°C for 15 seconds, and 68°C for 30 seconds, for a total of 30 cycles. After the PCR program was completed, the PCR product was added to 2.5 μL of 10× Loading Buffer and 2.5 μL of 10× Gelred, and the target band was observed by electrophoresis on a 3% agarose gel. The results were as follows: Figure 9 As shown, the Erysipelothrix rhusiopathiae primers only produce specific target bands for the genome of Erysipelothrix rhusiopathiae. The band size of Erysipelothrix rhusiopathiae is 938 bp, and the DNA amplification results for the other 7 strains are negative, proving that the Erysipelothrix rhusiopathiae amplification primers ER-F and ER-R of the present invention have good specificity.

[0086] (4) Specific detection of Mycoplasma hyorhinis primers Mhr-F and Mhr-R.

[0087] The specificity of primers was tested by using DNA of Mycoplasma hyorhinis, Streptococcus suis, Glasseria parasuis, Erysipelothrix rhusiopathiae, Escherichia coli, Staphylococcus suis, Pasteurella multocida and Actinobacillus pleuropneumoniae as templates and Mycoplasma hyorhinis primers Mhr-F and Mhr-R as primers.

[0088] The PCR amplification system consisted of 25 μL of the mix, 1 μL of each 10 μmol / L upstream and downstream primers, 1 μL of DNA template, and 22 μL of ddH2O. The PCR program was as follows: 94°C for 1 minute; 98°C for 10 seconds, 63°C for 15 seconds, and 68°C for 30 seconds, for a total of 30 cycles. After the PCR program was completed, the PCR product was added to 2.5 μL of 10× Loading Buffer and 2.5 μL of 10× Gelred, and the target band was observed by electrophoresis on a 3% agarose gel. The results were as follows: Figure 10 As shown, the primers for Mycoplasma hyorhinis only produce a specific target band for the genome of Mycoplasma hyorhinis, and the band size of Mycoplasma hyorhinis is 153 bp. The amplification results for the DNA of the other seven strains are negative, demonstrating that the Mycoplasma hyorhinis amplification primers Mhr-F and Mhr-R of the present invention have good specificity.

[0089] (5) Specific detection of a mixture of Streptococcus suis primers SS-F, SS-R, Glasneria parasuis primers GPS-F, GPS-R, Erysipelothrix rhusiopathiae ER-F, ER-R, and Mycoplasma hyorhinis primers Mhr-F, Mhr-R.

[0090] A mixed template of DNA from Streptococcus suis, Glassella parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis was selected, and DNA from Escherichia coli, Staphylococcus suis, Pasteurella multocida, and Actinobacillus pleuropneumoniae was selected as templates, respectively. Using Streptococcus suis primers SS-F and SS-R, Glassella parasuis primers GPS-F and GPS-R, Erysipelothrix rhusiopathiae ER-F and ER-R, and Mycoplasma hyorhinis primers Mhr-F and Mhr-R as primers, a multiplex PCR system was used for amplification and specificity was detected.

[0091] The PCR amplification system is 12.5μL of Multiplex buffer, 1μL of Multiplex DNA Polymerase, 1.25μL of 10μmol / L primers, 1.25μL of upstream and downstream primers for Streptococcus suis, 1.25μL of upstream and downstream primers for Glassera parasuis, 0.25μL of upstream and downstream primers for Erysipelothrix rhusiopathiae, 1.75μL of upstream and downstream primers for Mycoplasma hyorhinis, 2μL of DNA, 1.5μL of ddH2O, and a total volume of 25μL. The reaction program is 95℃ for 30s; cycle 95℃ for 30s, 63℃ for 90s, 72℃ for 60s, for a total of 35 cycles, and 72℃ for 10min. After the PCR program is completed, take the PCR product and add 2.5μL of 10× Loading Buffer and 2.5μL of 10× Gelred, and detect it by 3% agarose gel electrophoresis to observe the target band. The results are as follows Figure 11As shown, the quadruple PCR assay produced specific bands in a mixed positive sample containing Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, but no amplified bands were detected for non-target genomes. These results demonstrate that the quadruple PCR assay has excellent specificity and can simultaneously detect and accurately differentiate the target pathogens from other common swine pathogens.

[0092] Example 4: Amplification of different template combinations in a quadruple system

[0093] DNA of Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathogenes, and Mycoplasma hyorhinis were selected as templates to evaluate the effectiveness of the quadruple PCR system for simultaneous detection of four pathogens, such as Figure 12 Group 1), triple (any combination of three pathogens, such as Figure 12 Groups 2 to 5 of the ), dual (any combination of two pathogens, such as Figure 12 Groups 6 to 11), single (single pathogen detection, such as Figure 12 The amplification efficiency of a total of 15 pathogen DNA combinations was measured (Groups 12 to 15). Figure 12 As shown in the figure, the established quadruple PCR detection system can stably amplify specific bands of the expected size under all combination conditions, and no non-specific amplification products appear, indicating that the system has good amplification efficiency and can meet the detection needs of different pathogen combinations in clinical samples.

[0094] Example 5: Sensitivity of quadruple PCR system

[0095] Select the initial concentration of 10 6 The DNA templates of Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae and Mycoplasma hyorhinis were diluted tenfold to obtain 10 CFU / μL, respectively. 6 CFU / μL, 10 5 CFU / μL, 10 4 CFU / μL, 10 3 CFU / μL, 10 2 CFU / μL, 10 1 CFU / μL, 10 0 The genome concentration of CFU / μL, each group of genome concentrations includes a mixed DNA template of Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, and the detection sensitivity of the quadruple PCR system.

[0096] The PCR amplification system consisted of 12.5 μL of Multiplex buffer, 1 μL of Multiplex DNA Polymerase, 1.25 μL each of the upstream and downstream primers for Streptococcus suis (10 μmol / L), 1.25 μL each of the upstream and downstream primers for Glassera parasuis (10 μmol / L), 0.25 μL each of the upstream and downstream primers for Erysipelothrix rhusiopathiae (0.25 μL), 1.75 μL each of the upstream and downstream primers for Mycoplasma hyorhinis (1.75 μL), 2 μL of DNA, and 1.5 μL of ddH2O, for a total volume of 25 μL. The reaction program was 95°C for 30 s, followed by 35 cycles of 95°C for 30 s, 63°C for 90 s, and 72°C for 60 s, and 72°C for 10 min. The PCR product was added to 2.5 μL of 10× Loading Buffer and 2.5 μL of 10× Gelred and analyzed by electrophoresis on a 3% agarose gel.

[0097] The results are as follows Figure 13 As shown, the detection limit of Streptococcus suis was 10 3 The detection limit of CFU / μL and G. parasuis was 10 2 The detection limit of Erysipelothrix rhusiopathiae was 10 5 The detection limit of Mycoplasma hyorhinis was 10 2 CFU / μL.

Claims

1. A universal culture medium (KM2-PN) for the pathogens of porcine infectious arthritis, including Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathogenes, and Mycoplasma hyorhinis, characterized by: The KM2-PN culture medium includes a KM2 basal culture medium and auxiliary components, wherein the auxiliary components include porcine serum and nicotinamide adenine dinucleotide; the final concentration of the nicotinamide adenine dinucleotide is 5 μg / mL-20 μg / mL, and the volume of the porcine serum in the finished culture medium accounts for 5%-50%.

2. The universal culture medium (KM2-PN) for pathogens related to porcine infectious arthritis, such as Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, according to claim 1, characterized in that: The volume of the porcine serum in the finished culture medium accounts for 20%.

3. The universal culture medium (KM2-PN) for pathogens related to porcine infectious arthritis, such as Streptococcus suis, Glaseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, according to claim 1, characterized in that: The final concentration of the nicotinamide adenine dinucleotide was 10 μg / mL.

4. A multiplex PCR primer set for simultaneous detection of multiple pathogens, characterized in that: The multiple pathogens include Streptococcus suis, Glassella parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis. The multiple PCR primer set includes primer pair A, primer pair B, primer pair C, and primer pair D. The primer pair A includes primer 1 and primer 2 for detecting Streptococcus suis, the primer pair B includes primer 3 and primer 4 for detecting Glassella parasuis, the primer pair C includes primer 5 and primer 6 for detecting Erysipelothrix rhusiopathiae, and the primer pair D includes primer 7 and primer 8 for detecting Mycoplasma hyorhinis. The nucleotide sequences of primers 1 to 8 are shown in SEQ ID NO.1 to SEQ ID NO.8, respectively.

5. A kit for detecting Streptococcus suis, Glasseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis, characterized in that: The method comprises primers 1 and 2 for detecting Streptococcus suis according to claim 5, primers 3 and 4 for detecting Glassera parasuis, primers 5 and 6 for detecting Erysipelothrix rhusiopathiae, and primers 7 and 8 for detecting Mycoplasma hyorhinis, wherein the nucleotide sequences of primers 1 to 8 are shown in SEQ ID NO.1 to SEQ ID NO.8, respectively.

6. The kit according to claim 5, characterized in that The kit further comprises Multiplex buffer, Multiplex DNA Polymerase, and DNA template standards of Streptococcus suis, Glasseria parasuis, Erysipelothrix rhusiopathiae, and Mycoplasma hyorhinis.

7. The kit according to claim 5, wherein The concentrations of primers 1 to 8 in the kit are all 10 μmol / L, the volume of primers 1 to 4 is 1.25 μL, the volume of primers 5 and 6 is 0.25 μL, and the volume of primers 7 and 8 is 1.75 μL.

8. A method for performing multiplex PCR detection using the kit according to any one of claims 5 to 7, wherein the method is for non-diagnostic purposes, characterized in that: The following steps are included: (1) extracting nucleic acid from the sample to be tested; (2) using the nucleic acid of the sample to be tested as a template and using primers 1 to 8 to perform a quadruple PCR reaction; (3) The amplified product is subjected to electrophoresis. If a 689 bp band appears in the amplified product, Streptococcus suis is present in the sample to be tested; if a 491 bp band appears in the amplified product, Glasneria parasuis is present in the sample to be tested; If a 938 bp band appears in the amplified product, Erysipelothrix rhusiopathiae is present in the sample to be tested. If a 153 bp band appears in the amplified product, Mycoplasma hyorhinis is present in the sample to be tested.

9. The method according to claim 8, characterized in that The system of the quadruple PCR reaction: Multiplex buffer 12.5 μL, Multiplex DNA Polymerase 1 μL, 1.25 μL each of 10 μmol / L primers of Streptococcus suis upstream and downstream primers, 1.25 μL each of upstream and downstream primers of Glaseria parasuis, 0.25 μL each of upstream and downstream primers of Erysipelothrix rhusiopathiae, 1.75 μL each of upstream and downstream primers of Mycoplasma hyorhinis, 2 μL of DNA, 1.5 μL of ddH2O, and a total volume of 25 μL.

10. The method according to claim 8, characterized in that: The program of the quadruple PCR reaction was as follows: 95°C for 30s; 35 cycles of 95°C for 30s, 63°C for 90s, and 72°C for 60s, for a total of 35 cycles; and 72°C for 10min.

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