Avian mycoplasmos is one of the economically significant disease in commercial poultry industry. This study aimed to develop and optimized a novel tPCR approach to detect Mycoplasma gallispeticum, Mycoplasma synoviae and nonpathogenic mycoplasmas at the same in a single PCR reaction collected from diseased chicken. Direct PCR from the clinical samples produced false negative results. Both culture and PCR were combined as culture-enhanced PCR approach. Three different DNA crude preparation methods were used and broth dilution method found simpler and most efficient for positive PCR. Primers were selected for 16s rRNA and it could detect up to 100 cfu and 250 cfu from MS and MG respectively in a samples. We have tested pure DNA of other mycoplasmas (5 species from avian origin and 3 other mycoplasmas species) but it produced only the genus specific band. The optimized ratio of tPCR primers were 1: 1: 10: 1. For Outer F, Inner R, and Inner F and Outer R, respectively. MgCl2 concentration did not affect and added at 2.0 mM. Four different culture methods were compared for their efficiency for avian mycoplasmas culture. The B method (parts of trachea) were found most efficient for producing growth within 36 h while method A produced within 48 h nevertheless both had the same positivity (70%). While other two methods C (filtrate into broth) and D (filtrate onto agar) produced just 40% and 31%. Moreover, the C methods is the most time consuming method for growth production. The former two methods were superior in their productivity 30% and 39% over C and D methods, respectively. The tPCR approach could also be utilized for avian mcyoplasmas contamination on cell lines or suspect fertile eggs for virus vaccine production.
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