Product, method and application for simultaneously detecting mycoplasma gallisepticum and mycoplasma synoviae

Abstract

The invention discloses a product, method and application for simultaneously detecting mycoplasma gallisepticum and mycoplasma synoviae. According to the product and the method, a common specific protein antigen of mycoplasma gallisepticum and mycoplasma synoviae is preferably an outer membrane protein WP-041352022.1 as a detection object, a PCR primer pair S1 is designed and screened on the genome DNA level of the protein, and the PCR primer pair has the advantages of strong specificity, high sensitivity and no primer dimer generation. The invention provides a product and a method for simultaneously detecting two pathogens by using one pair of PCR primers, mutual interference between multiple PCR primers and the product is avoided, the detection rate is improved, and the method has the advantages of economy, simplicity, convenience and high efficiency.

Description

technical field

[0001] The invention relates to the field of animal pathogen detection, in particular to a product, method and application for simultaneously detecting mycoplasma gallisepticum and mycoplasma gallisepticum. Background technique

[0002] Mycoplasma gallisepticum and Mycoplasma gallisepticum are two important pathogenic pathogens of chickens. At present, the infection pressure in the environment is relatively high. Collecting laryngotracheal swabs from 7-14-day-old chicks can be used to detect whether the chicks are in the latent infection state of Mycoplasma gallisepticum and Mycoplasma gallisepticum, so as to determine drug delivery and vaccine immunity. program. In the prior art, the method for simultaneous detection of Mycoplasma gallisepticum and Mycoplasma gallisepticum is generally multiplex PCR, which needs to add at least 4 primers. The increase of primers will cause primer dimers, interfere with the normal amplification of PCR, and affect PCR detecti...

Examples

Embodiment 1

[0043] Embodiment 1, screening and primer design of Mycoplasma gallisepticum and Mycoplasma gallisepticum common antigen

[0044] 1. Screening of common antigens

[0045] Obtain all membrane protein data of Mycoplasma gallisepticum and Mycoplasma gallisepticum through the online protein database, and further screen for strong antigenicity (according to the website http: / / www.violinet.org / vaxign / The outer membrane protein whose antigen parameter is above 0.5) compares the shared proteins of the two pathogens and analyzes the homology of the proteins. For example, the following shared proteins are obtained: WP_041352022.1, WP_041352020.1, WP_041351882.1, WP_011283540. 1. The homology of WP_020003021.1, WP_081420552.1, and WP_041352050.1 is 97.21%, 94.72%, 93.82%, 63.2%, 60%, 40%, and 35.89% respectively; select the homology at 96% The outer membrane protein of the above WP_041352022.1 (as shown in SEQ ID No.4 in its amino acid sequence) was used as the research object, and it...

Embodiment 2

[0051] Embodiment 2, optimization of PCR reaction conditions

[0052] Method: The optimal annealing temperature was optimized with the DNA template of Mycoplasma gallisepticum, the DNA template of Mycoplasma gallisepticum, the DNA template of Mycoplasma gallisepticum+the DNA template of Mycoplasma gallisepticum, among which,

[0053] The PCR reaction system is as follows:

[0054] GENESTAR 2×PCR mix (including DNA polymerase and dNTP) 10 μL, template 0.1 μL (concentration 30 ng / μL), upstream primer F 1 μL (concentration 0.15 μg / μL), downstream primer R 1 μL (concentration 0.15 μg / μL), water supplement to 20 μL.

[0055] The PCR reaction conditions are as follows:

[0056] Pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 40s, annealing at 50°C-60°C for 40s, extension at 72°C for 30s, 30 cycles, and final extension at 72°C for 10 minutes;

[0057] The product was detected by agarose gel electrophoresis.

[0058] Result: if figure 1 , figure 2 and image 3...

Embodiment 3

[0059] Embodiment 3, specific detection

[0060] Method: Mycoplasma gallisepticum vaccine strain F36, st-11 strain and 6 / 85 strain, Mycoplasma gallisepticum vaccine strain H strain and Mycoplasma synovial bursa ATCC25204, Mycoplasma gallisepticum vaccine strain (F36) + chicken synovial bursa Mycoplasma vaccine strain (H strain), Mycoplasma bovis, Mycoplasma hyopneumoniae vaccine strain (RM48), Mycoplasma ovis, Escherichia coli, Salmonella, Staphylococcus, Streptococcus, Pasteurella, chicken infectious bronchial virus, chicken infection Laryngotracheitis virus, Newcastle disease virus, chicken influenza virus, and Haemophilus paragallinarum were used as templates, and PCR amplification was performed according to the reaction conditions and reaction system in Example 2, and the products were detected by agarose gel electrophoresis.

[0061] Results: Mycoplasma gallisepticum, Mycoplasma gallisepticum, and Mycoplasma gallisepticum + Mycoplasma gallisepticum can all amplify the tar...

technical field

[0001] The invention relates to the field of animal pathogen detection, in particular to a product, method and application for simultaneously detecting mycoplasma gallisepticum and mycoplasma gallisepticum. Background technique

[0002] Mycoplasma gallisepticum and Mycoplasma gallisepticum are two important pathogenic pathogens of chickens. At present, the infection pressure in the environment is relatively high. Collecting laryngotracheal swabs from 7-14-day-old chicks can be used to detect whether the chicks are in the latent infection state of Mycoplasma gallisepticum and Mycoplasma gallisepticum, so as to determine drug delivery and vaccine immunity. program. In the prior art, the method for simultaneous detection of Mycoplasma gallisepticum and Mycoplasma gallisepticum is generally multiplex PCR, which needs to add at least 4 primers. The increase of primers will cause primer dimers, interfere with the normal amplification of PCR, and affect PCR detecti...

Examples

Embodiment 1

[0043] Embodiment 1, screening and primer design of Mycoplasma gallisepticum and Mycoplasma gallisepticum common antigen

[0044] 1. Screening of common antigens

[0045] Obtain all membrane protein data of Mycoplasma gallisepticum and Mycoplasma gallisepticum through the online protein database, and further screen for strong antigenicity (according to the website http: / / www.violinet.org / vaxign / The outer membrane protein whose antigen parameter is above 0.5) compares the shared proteins of the two pathogens and analyzes the homology of the proteins. For example, the following shared proteins are obtained: WP_041352022.1, WP_041352020.1, WP_041351882.1, WP_011283540. 1. The homology of WP_020003021.1, WP_081420552.1, and WP_041352050.1 is 97.21%, 94.72%, 93.82%, 63.2%, 60%, 40%, and 35.89% respectively; select the homology at 96% The outer membrane protein of the above WP_041352022.1 (as shown in SEQ ID No.4 in its amino acid sequence) was used as the research object, and it...

Embodiment 2

[0051] Embodiment 2, optimization of PCR reaction conditions

[0052] Method: The optimal annealing temperature was optimized with the DNA template of Mycoplasma gallisepticum, the DNA template of Mycoplasma gallisepticum, the DNA template of Mycoplasma gallisepticum+the DNA template of Mycoplasma gallisepticum, among which,

[0053] The PCR reaction system is as follows:

[0054] GENESTAR 2×PCR mix (including DNA polymerase and dNTP) 10 μL, template 0.1 μL (concentration 30 ng / μL), upstream primer F 1 μL (concentration 0.15 μg / μL), downstream primer R 1 μL (concentration 0.15 μg / μL), water supplement to 20 μL.

[0055] The PCR reaction conditions are as follows:

[0056] Pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 40s, annealing at 50°C-60°C for 40s, extension at 72°C for 30s, 30 cycles, and final extension at 72°C for 10 minutes;

[0057] The product was detected by agarose gel electrophoresis.

[0058] Result: if figure 1 , figure 2 and image 3...

Embodiment 3

[0059] Embodiment 3, specific detection

[0060] Method: Mycoplasma gallisepticum vaccine strain F36, st-11 strain and 6 / 85 strain, Mycoplasma gallisepticum vaccine strain H strain and Mycoplasma synovial bursa ATCC25204, Mycoplasma gallisepticum vaccine strain (F36) + chicken synovial bursa Mycoplasma vaccine strain (H strain), Mycoplasma bovis, Mycoplasma hyopneumoniae vaccine strain (RM48), Mycoplasma ovis, Escherichia coli, Salmonella, Staphylococcus, Streptococcus, Pasteurella, chicken infectious bronchial virus, chicken infection Laryngotracheitis virus, Newcastle disease virus, chicken influenza virus, and Haemophilus paragallinarum were used as templates, and PCR amplification was performed according to the reaction conditions and reaction system in Example 2, and the products were detected by agarose gel electrophoresis.

[0061] Results: Mycoplasma gallisepticum, Mycoplasma gallisepticum, and Mycoplasma gallisepticum + Mycoplasma gallisepticum can all amplify the tar...

Images