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PCR (Polymerase Chain Reaction) amplification primer for quickly detecting mycoplasma ovipneumoniae, and application thereof

A technology of mycoplasma pneumoniae and amplification primers, which is applied in the field of animal bacteriology and molecular biology, can solve the problems of difficult acquisition, difficult artificial culture, cumbersome and time-consuming separation and culture methods, and achieve high sensitivity, low cost, and less time-consuming Effect

Active Publication Date: 2018-05-04
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Mycoplasma ovis pneumoniae is a microorganism without a cell wall, and it is difficult to cultivate artificially. In addition, the isolation and cultivation methods are cumbersome and time-consuming.
Serological diagnostic methods are commonly used to detect mycoplasma infection, however, the existence of cross-reactions and non-specific reactions between species greatly hinders the application of serological methods
In addition, the identification of mycoplasma isolates requires corresponding specific hyperimmune serum, which is not easy to obtain, and it is very difficult to detect and identify Mycoplasma pneumoniae by using the methods of pathogen isolation and identification and serological identification

Method used

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  • PCR (Polymerase Chain Reaction) amplification primer for quickly detecting mycoplasma ovipneumoniae, and application thereof
  • PCR (Polymerase Chain Reaction) amplification primer for quickly detecting mycoplasma ovipneumoniae, and application thereof
  • PCR (Polymerase Chain Reaction) amplification primer for quickly detecting mycoplasma ovipneumoniae, and application thereof

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Embodiment 1

[0077] The optimum annealing temperature test of embodiment 1 ovine mycoplasma pneumoniae PCR amplification primer

[0078] Using ultrapure water as a control, PCR amplification was performed at annealing temperatures of 45°C, 47°C, 49°C, 51°C, 53°C, 55°C, and 57°C to determine the optimal annealing temperature. The results showed that the designed primers had a high tolerance for annealing temperature, and could amplify well under the reaction programs with annealing temperatures of 45°C, 47°C, 49°C, 51°C, 53°C, 55°C, and 57°C. Add a single destination strip ( figure 1 ). For this reason, the reaction program used in this PCR method is: 95°C for 5 min; followed by 35 cycles of 95°C for 45 s, 49°C for 45 s, and 72°C for 45 s; and finally 72°C for 7 min.

Embodiment 2

[0079] The specific detection result of embodiment 2 ovine mycoplasma pneumoniae PCR amplification primers

[0080] Extract the genomic DNA of Mycoplasma ovis pneumoniae, Mycoplasma goat subsp., Mycobacterium hemolyticus, and Pasteurella, perform PCR amplification using the optimized reaction system and reaction program, and detect the specificity of the detection method of the present invention , the results showed that only the target fragment band was amplified in the Mycoplasma ovis pneumoniae sample, which was a positive result, and no amplification occurred in the 4 control strain reaction tubes and the water control reaction tube, which was a negative result ( figure 2 ), indicating that the method has good specificity.

Embodiment 3

[0081] Example 3 The Sensitivity Detection Result of Ovis Mycoplasma Pneumoniae PCR Amplification Primer

[0082] The initial concentration of the positive recombinant plasmid was 2.12×10 ng / μL, and it was serially diluted 10 times with RNA-Free Water. The optimized reaction conditions were used for PCR amplification and sensitivity testing. The results showed that the established PCR detection method The lowest detection limit is 2.12×10 -3 ng / μL ( image 3 ).

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) amplification primer for quickly detecting mycoplasma ovipneumoniae, and application thereof, which belong to the field of animal bacteriologyand molecular biology. The PCR amplification primer is formed by an upstream primer with a nucleotide sequence as shown in an SEQ ID NO:1 and a downstream primer with a nucleotide sequence as shown in an SEQ ID NO:2; the primer is used for preparing a PCR amplification kit for detecting the mycoplasma ovipneumoniae. A PCR detection method provided by the kit has high specificity and sensibility,good repeatability, and high reliability, can specifically detect the mycoplasma ovipneumoniae and quickly and accurately obtain a detection result, is low in price, simple and convenient to operate,and suitable for grass roots at the same time, and can be used as a quick, accurate, simple and convenient detection tool for quick identification in a mycoplasma ovipneumoniae laboratory and large-scale epidemiological investigation.

Description

technical field [0001] The invention relates to the technical fields of animal bacteriology and molecular biology, in particular to a rapid, simple and low-cost PCR amplification primer for detecting ovine mycoplasma pneumoniae and its application. Background technique [0002] Mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae) is one of the important pathogens that cause respiratory diseases in sheep, and can cause atypical pneumonia in goats, sheep and wild small ruminants. The main clinical manifestations of affected sheep are cough, runny nose, emaciation, anemia, and growth retardation. At the same time, the susceptibility of sheep to pathogens such as Pasteurella multocida, Mannella hemolyticus and parainfluenza virus increased after infection with Mycoplasma ovis pneumoniae. At present, the pathogen is distributed all over the world, and it is also widely distributed in China, causing huge economic losses and becoming one of the important pathogens threatening the s...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12Q1/6895C12Q1/04C12N15/11C12R1/35
CPCC12Q1/686C12Q1/6895C12Q2565/125
Inventor 马春霞李军陶立潘艳冯世文彭昊吴翠兰谢永平钟舒红胡帅杨威陈泽祥贺会利李常挺
Owner GUANGXI VETERINARY RES INST
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