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Method for rapidly and qualitatively detecting mycoplasma pneumoniae of sheep

A Mycoplasma pneumoniae, qualitative detection technology, applied in the field of animal infectious disease diagnosis, can solve the problems of low G+C content, low sensitivity, slow growth, etc., and achieve the effect of simple detection procedure, high detection efficiency and high sensitivity

Inactive Publication Date: 2013-05-15
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high nutritional requirements for the growth of mycoplasma and the slow growth, it is difficult to isolate and identify the pathogen
With the rapid development of modern molecular biology, the PCR method has become a routine technique for detecting pathogens due to its advantages of rapidity, specificity and sensitivity. The only detection method reported was the PCR method established by McAuliffe L based on 16S rRNA ([5] McAuliffe L, Hatchell FM, Ayling RD, et al. Detection of Mycoplasma ovipneumoni ae in Pasteurella- vaccinated sheep flocks with respiratory disease in England. Vet Rec, 2003, 153(22):687~688.), this method has good specificity, but it is found that the sensitivity is not high when it is actually applied to the detection of clinical samples
In addition, a large number of studies have shown that Mycoplasma ovis is highly heterogeneous at the nucleic acid level and protein level, and its genome has low G+C content, which makes it difficult to select molecular detection targets ([6] Ionas G , Norman NG, Clarke JK, Marshall RB. A study of the heterogeneity of isolates of Mycoplasma ovipneumoniae from sheep in New Zealand. Vet Microbiol. 1991 Nov;29(3-4):339-47;[7] Parham K, Churchward CP, McAuliffe L, Nicholas RA, Ayling RD. A high level of strain variation within the Mycoplasma ovipneumoniae population of the UK has implications for disease diagnosis and management. Vet Microbiol. 2006 Nov 26;118(1-2):83-90 .)

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  • Method for rapidly and qualitatively detecting mycoplasma pneumoniae of sheep
  • Method for rapidly and qualitatively detecting mycoplasma pneumoniae of sheep
  • Method for rapidly and qualitatively detecting mycoplasma pneumoniae of sheep

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 specific detection

[0050] This example uses the established rapid detection method for Mycoplasma pneumoniae to detect Mycoplasma mycoides filamentous subsp. LC type Y-goat strain (M. mycoides subsp. mycoides. LC), Mycoplasma mycoides subsp. .Capri), Mycoplasma bovis (M.bovis), Mycoplasma agalactiae (M.agalactiae), Mycoplasma arginini (M.arginini), Mannheimia haemolytica, Escherichia coil 013, Staphylococcus aureus strain SW07 (Staphylococcus aureus), Pasteurella (Pasteurella) and Salmonella (Salmonella), the reference strain Y98 was used as a positive control, and no template control was used as a negative control to evaluate the specificity of the method.

[0051] a. PCR detection:

[0052] The DNA extraction method refers to the preparation of template DNA in [Technical Scheme]. Use the extracted DNA as a template for PCR detection, reaction system 25μL: 10×PCR buffer 2.5μL, 25mM Mg 2+ 1.5μL, 2.5mM dNTPs 2μL, 10μM upstream and downstream primers 1μL,...

Embodiment 2

[0055] Example 2 Detection of known positive samples

[0056] In this example, the established rapid detection method for Mycoplasma ovis was used to detect 35 known clinical isolates of Mycoplasma ovis (based on 16S rRNA PCR identification), and the reference strain Y98 was used as a positive control, and the no-template control was used as a negative control. Evaluate the method's detection of known positive samples.

[0057] a. PCR detection:

[0058] The DNA extraction method refers to the preparation of template DNA in [Technical Scheme]. Use the extracted DNA as a template for PCR detection, reaction system 25μL: 10×PCR buffer 2.5μL, 25mM Mg 2+ 1.5μL, 2.5mM dNTPs 2μL, 10μM upstream and downstream primers 1μL, 5U / μL Taq enzyme 0.125μL, 50ng / μL DNA template 2μL, sterilized double distilled water to make up to 25μL; reaction conditions: 95℃ pre-denaturation for 5min; 95℃ Denaturation for 30s, annealing at 60°C for 30s, extension at 72°C for 30s, a total of 30 cycles; f...

Embodiment 3

[0061] Example 3 Sensitivity evaluation of the rapid detection method for Mycoplasma pneumoniae in sheep

[0062] In this embodiment, after carrying out 10-fold serial dilution of the reference bacterial strain Y98, the rapid detection method for Mycoplasma ovis pneumonia established by the present invention is compared with the only PCR method established by McAuliffe L based on 16S rRNA at present, and the evaluation method established by the present invention is evaluated. method sensitivity.

[0063] a. PCR detection:

[0064] The DNA extraction method refers to the preparation of template DNA in [Technical Scheme]. The concentration of the reference strain Y98 DNA measured by a nucleic acid and protein detector was 220 ng / μL, which was serially diluted 10 times (2.2×10 1 ~2.2×10 -8 ng / μL), amplified according to the PCR reaction system and reaction conditions established in the present invention and the method established by McAuliffe L, respectively.

[0065] b. Res...

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Abstract

The invention discloses a method for rapidly and qualitatively detecting mycoplasma pneumoniae of sheep. The method comprises the following steps of: carrying out species specificity polymerase chain reaction (PCR) by employing a self-designed species specificity primer of a sheep mycoplasma pneumoniae heat shock protein 70 (HSP70) gene sequence, and establishing a simple, rapid and accurate qualitative detection method for the sheep mycoplasma pneumoniae through agarose gel electrophoresis atlas analysis. The method can be used for fast diagnosis on the sheep mycoplasma pneumonia, and has the advantages of being strong in specificity, good in repeatability, and high in sensitivity, and can facilitate the standardization.

Description

technical field [0001] The invention relates to the diagnosis of animal infectious diseases, in particular to the technical field of a PCR method for detecting sheep mycoplasma pneumoniae. Background technique [0002] Mycoplasma ovis pneumoniae can cause atypical pneumonia in sheep and goats. Clinically, it is characterized by cough, runny nose, progressive emaciation and proliferative interstitial pneumonia ([1] Besser T E, Cassirer E F, Potter K A, et al. Association of Mycoplasma ovipneumoniae infection with population-limiting respiratory disease in free -ranging Rocky Mountain bighorn sheep ( Ovis Canadensis canadensis).J Clin Microbiol,2008,4 6:423~430. [2] Parham K,Churchward C P,McAuliffe L, et al. A high level of strain variation within Mycoplasma ovipneumoniae population of the UK has implications for disease diagnosis and management. Vet Microbiol, 2006, 118:83~90.). At present, Mycoplasma ovis pneumoniae is distributed all over the world, and it is very preval...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 岳华汤承杨发龙
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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