Establishment Of The Polymerase Chain Reaction To Detect Bovine Mycoplasma And Its Clinical Application

The main agent causing of pneumonia, arthritis, mastitis, keratoconjunctivitis etc is bovine mycoplasma, but also caused a range diseases of reproductive system. Mycoplasma pneumonia in calves suffered from higher mortality rates, serious impacted on milk production and quality.In some areas of Shandong, cows emerged the case with the main feature of fever, difficulty in breathing in the 2007. In the past two years, a total of 30 head of cattle performed different degrees of clinical symptoms, the incidence rate of which had 20 adult cattle is 39.2%; of which 4 bred cattle is 57.1%; of which had 6 calf is 75%, the mortality rate 1.5%, the rejection rate of 4.5%. Combined performance of clinical symptoms and pathological anatomy and treatment of conditions suspected to be mycoplasma.We Collected lungs, liver, pleural effusion etc. inoculated broth medium of mycoplasma, at the same time, they was Shredded small materials or pleural effusion crossed in 20% horse serum agar medium in box culture of 5% CO2, 37℃, after 5 ~ 7d observed results. in the begging, showed a slightly cloudy or white point-like and filamentous growth in broth, then gradually evenly turbid and translucent Shaodai opalescent, non-producing strains film or precipitation, and no particle suspension. In 20% horse serum agar medium, it growth retardation, it is the tiny teardrop-shaped micro-colonies round slightly gray and the Central papillae., in microscopic observation, colony is multiple patterns, the most common shape is the ball which size is 125 ~ 250 nm, as Giemsa stain.Template is distilled from lung organizations, and mycoplasma, Mycoplasma agalactiae, Mycoplasma mycoides etc were positive controls, after, using universal primers amplified Mycoplasma its 16S rRNA, has established detection methods by extended 16S rRNA sequences of Mycoplasma, amplification of the 16S rRNA gene fragment, the size was 1.5 kb, after sequencing, and Mycoplasma sequence homology is 97~100% by comparing with GenBank published by Mycoplasma 16S rRNA gene sequences.It is very difficult to culture Mycoplasma, and the growth cycle of Mycoplasma is long, the nutritional conditions of Mycoplasma is harsh. In this study, four pairs of specific primers were designed for detecting the cattle mycoplasma disease Mycoplasma mycoides, Mycoplasma bovis, Mycoplasma agalactiae and Mycoplasma ovipneumoniae which caused the cattle mycoplasma disease. A multi-PCR method with rapid detection, test specificity and sensitivity characteristics had been established, which provide technical support for the clinical detection of the cattle mycoplasma disease.