Simultaneous Detection of Mycoplasma gallisepticum, Mycoplasma synoviae, Infectious bronchitis virus and Infectious laryngotracheitis virus in Poultry by Multiplex SYBR Green Real Time PCR

Quick Response Code Abstract: Multifactorial respiratory problems are very common in poultry due to intensive rearing methods and caused by coinfection with bacteria (MG, MS, E. coli and Avibacterium spp.) and viruses, such as IBV and ILTV. The conventional procedures are tedious and time taking, to overcome the difficulties in diagnosis of mixed respiratory pathogens, this study was undertaken with the following objective simultaneous detection of MG, MS, IBV and ILTV by Multiplex SYBR Green Real Time PCR. The SYBR Green Real time PCR assay was standardized for simultaneous detection of respiratory pathogens in poultry by targeting IGSR gene for MG, vlhA gene for MS, N gene for IBV and ICP4 gene for ILTV in single tube. The optimized conditions for all genes was initial denaturation at 94 o C for 3.59 mins, followed by 40 cycles of denaturation at 94 o C for 20 seconds, annealing at 55 o C for 45 seconds and extension at 72 o C for 1 min and final extension step was at 72 o C for 10 mins. The amplifications of all four genes was observed and recorded the ct values of 30.28 (IGSR gene), 24.16 (vlhA gene), 19.39 (ICP4 gene) and 11.81 (N gene) and also the recorded Tms were 74.0 (IGSR gene), 82.5 (vlhA gene), 85.5 (ICP4 gene) and 80.5 (N gene). The samples with below 35 recorded threshold cycles value were considered as positive. Out of 19 farms screened, 2 were positive for ILT, 10 for MG,7 for MS and 5 for MG and MS and all were negative for IB. In sreened samples, the recorded ct values for IGSR genes ranged between 11.81 to 30.28, product Tms ranged between 74 to 87, for vlhA gene ct values ranged between 17.39 to25.81, product Tms ranged between 81 to 87, for ICP4 gene ct value was 19.08 and 19.23,product Tms was 81 and 80.5.