Out of the 25, 10 samples were of trachea, 6 of air sac, 4 of oral swab and 5 of lungs tissues. These characterize on the basis of biochemical and cultural studies.

The biochemical profile of Mycoplasma gallisepticum including glucose fermentation and phosphatase activity were given in Table 3. Optimization of multiplex polymerase chain reaction was done by varying the annealing temperature varying the MgCl₂ concentrations in the reaction mixture and varying the concentration of template DNA.

Firstly, optimizing of annealing temperature of the thermal cycler ranging from 48°C to 58°C and its effect on the amplicon was noted. In electrophoresis the intensity of the band revelead its significance. At 48°C, no amplification was observed, at 50°C amplification was observed in (Mycoplasma gallisepticum). DNA amplified well. At 54°C Mycoplasma gallisepticum DNA amplified very well. The results are shown in table 4.

Secondly, the effect of decreasing and increasing the concentration of MgCl₂ was observed and optimized at better result. A total of 25 samples were processed through multiplex polymerase chain reaction. These samples were categorized into 5 groups including 5 samples each. The concentration of MgCl₂ lies between 1.5 m Mol gave 2+ results, 2.5 m Mol gave 3+ results and 3.0 m Mol gave better results. From the above data it was concluded that the concentration of 2.5 m Mol gave better results and the concentration of better results of MgCl₂ was optimized at 2. 5 m Mol.

Thirdly, optimizing of multiplex PCR was carried out by using different amount of template DNA or target DNA after visualizing in gel documentation system. Three different quantities of 5 µl, 8 µl and 10 µl of the template DNA were used and the band intensity was noted. Table 5 is showing the results for this parameter.

In Pakistan, poultry has emerged as a viable alternative to beef and mutton. A drop in egg production of 10-20%, an increase in embryo mortality and chick mortality of 5-10%, and a fall in weight gain and feed conversion efficiency of 10-20% are all losses caused by Mycoplasma infection. Only four of the 22 identified species recovered from avian sources were established pathogens for domestic poultry, namely Mycoplasma Gallisepticum (Ley and Yoder, 1997)

Chronic Respiratory Condition (CRD) is the name for the disease caused by Mycoplasma. Mycoplasma gallisepticum is the most important pathogenic and commercial pathogen, and the disease caused by it has been declared as notifiable by the Office International des Epizooties (OIE). It is a big concern in the poultry business around the world, because sinusitis causes infection in turkeys. Losses related to Mycoplasmosis, specifically MG infection, include decreased egg productivity and egg quality, poor hatchability (high rate of embryonic mortality and culling of day-old birds), low feed efficiency, increased mortality, and carcass condemnation, in addition to medicine costs 4.

Various specimen samples were collected from clinically chronic respiratory disease cases of broiler and layer chickens, including trachea, air sac swab, oral swab, and lungs tissues, for the successful isolation of Mycoplasma species, but the trachea and air sac samples were the sample of choice because it is the predilection site of manipulation of Mycoplasma species. Beef heart infusion, peptones, yeast extract, and serum are the most important nutrients for the growth of mycoplasma spp. Along with NAD and cysteine, serum is a critical need for Mycoplasma growth. Additionally, because Mycoplasmas are absolute resistant to these medications, penicillin and relative insusceptible thallium acetate are utilized to produce selective 2.

Mycoplasma species obtained in Brain Heart Infusion broth had a morphology that was tiny, smooth, circular, and had a fried egg look with a central opaque and outer translucent area. Several researchers have observed similar traits in mycoplasma species colonies.

From the total of 25 samples, 25 (100%) had a positive Mycoplasma isolation result. The biochemical test yielded the highest number of isolations, with MG accounting for 10/25. Commercial DNA-based PCR kits have been developed as a result of recent advancements in diagnostic techniques for Mycoplasma infections. However, the reaction cost for clinical samples increased by a factor of two. The multi-species PCR allowed for the detection of multiple Mycoplasma in a single sample. The methods for detecting pathogenic avian mycoplasma using multiplex PCR described above are specific, sensitive, and consistent. It assisted us in enhancing the specific and accurate diagnosis of Mycoplasmosis in our commercial poultry sector by employing specific primer sequences and contributed to the elimination and reduction of losses due to Mycoplasmosis 6.