Unlock instant, AI-driven research and patent intelligence for your innovation.

Product, method and application for simultaneous detection of Mycoplasma gallisepticum and Mycoplasma gallisepticum

A technology for Mycoplasma synovialis and Mycoplasma gallisepticum is applied in biochemical equipment and methods, measuring devices, and microbial determination/inspection, etc., and can solve the problems of interfering with normal PCR amplification, affecting PCR sensitivity and specificity, etc. Improve the detection rate, eliminate mutual interference, and have the effect of high sensitivity

Active Publication Date: 2022-07-05
SHANDONG LVDU BIO SICIENCE & TECH
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 detection. sensitivity and specificity

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Product, method and application for simultaneous detection of Mycoplasma gallisepticum and Mycoplasma gallisepticum
  • Product, method and application for simultaneous detection of Mycoplasma gallisepticum and Mycoplasma gallisepticum
  • Product, method and application for simultaneous detection of Mycoplasma gallisepticum and Mycoplasma gallisepticum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1. Screening and primer design of common antigens of Mycoplasma gallisepticum and Mycoplasma gallis Synovium

[0044] 1. Screening of common antigens

[0045] Obtain all membrane protein data of Mycoplasma gallisepticum and Mycoplasma synovialis through the online protein database, and further screen for strong antigenicity (by website http: / / www.violinet.org / vaxign / The outer membrane proteins with antigen parameters above 0.5) were compared, and the shared proteins of the two pathogens were compared, and the homology of the proteins was analyzed. 1. WP_020003021.1, WP_081420552.1, and WP_041352050.1, their homology is 97.21%, 94.72%, 93.82%, 63.2%, 60%, 40%, and 35.89% respectively; the homology is selected at 96% The outer membrane protein of the above WP_041352022.1 (its amino acid sequence is shown in SEQ ID No. 4) was taken as the research object, and its gene sequence (shown in SEQ ID No. 1) was obtained from the NCBI database. BLAST analysis showed ...

Embodiment 2

[0051] Embodiment 2. Optimization of PCR reaction conditions

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

[0053] The PCR reaction system is as follows:

[0054] GENESTAR’s 2×PCR mix (containing DNA polymerase and dNTP) 10 μL, template 0.1 μL (concentration 30 ng / μL), upstream primer F1 μL (concentration 0.15 μg / μL), downstream primer R 1 μL (concentration 0.15 μg / μL), water supplement to 20 μL.

[0055] PCR reaction conditions are as follows:

[0056] Pre-denaturation at 95°C for 5 min, 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 10min;

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

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

Embodiment 3

[0059] Embodiment 3, specific detection

[0060] Methods: Mycoplasma gallisepticum vaccine strains F36, st-11 and 6 / 85 strains, Mycoplasma gallisepticum vaccine strain H strain and Mycoplasma synovium ATCC25204, Mycoplasma gallisepticum vaccine strain (F36) + chicken synovial sac Mycoplasma vaccine strain (H strain), Mycoplasma bovis, Mycoplasma hyopneumoniae vaccine strain (RM48), Mycoplasma capricolum, Escherichia coli, Salmonella, Staphylococcus, Streptococcus, Pasteurella, Infectious bronchial virus in chickens, Chicken sensing Using laryngotracheitis virus, Newcastle disease virus, chicken influenza virus, and Haemophilus paragallinarum as templates, 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] Result: Mycoplasma gallisepticum, Mycoplasma gallisepticum, and Mycoplasma gallisepticum + Mycoplasma gallis Synovium can amplify the target fragment of...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present application discloses products, methods and applications for the simultaneous detection of Mycoplasma gallisepticum and Mycoplasma Galli synovium. The product and method take the common specific protein antigen of Mycoplasma gallisepticum and Mycoplasma gallis Synovium as the detection object, preferably the outer membrane protein WP_041352022.1, and design and screen out PCR primer pairs on the level of genomic DNA of this protein. S1, the PCR primer pair has the advantages of strong specificity, high sensitivity, and no primer-dimer generation. The present application provides a product and method that can simultaneously detect two pathogens by using a pair of PCR primers, eliminating the need for multiple PCR primers and The mutual interference between the products improves the detection rate, and has the advantages of economy, simplicity 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 simultaneous detection of Mycoplasma gallisepticum and Mycoplasma Gallinarum. Background technique [0002] Mycoplasma gallisepticum and Mycoplasma gallinarum are two important pathogenic pathogens in chickens. At present, the infection pressure in the environment is relatively high. Collect laryngotracheal swabs of 7-14-day-old chicks, and use PCR method to detect whether the chicks are in the latent infection state of Mycoplasma gallisepticum and Mycoplasma gallis synovium to determine drug delivery and vaccine immunization. program. In the prior art, the method for simultaneously detecting Mycoplasma gallisepticum and Mycoplasma gallis Synovium is generally multiplex PCR, and at least 4 primers need to be added. The increase of primers will cause primer dimers, and interfere with the normal amplification of PCR, affecting PCR dete...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11G01N33/569
CPCC12Q1/689C12Q1/686G01N33/56933C12Q2600/16G01N2333/30C12Q2537/143Y02A50/30
Inventor 李书光沈志强程立坤曲光刚王文秀杨立芳赵家磊
Owner SHANDONG LVDU BIO SICIENCE & TECH