Multiplex SYBR Green Real-Time PCR Assay for Detection of Common Respiratory Pathogens of Poultry

Abstract

Due to intensive farming practises and coinfection with bacteria (MG, MS, E. coli, and Avibacterium spp.) and viruses (IBV and ILTV), mixed respiratory infections are widespread in poultry.The conventional methods are laborious and time-consuming, so this study was conducted with the following goals in mind to address the challenges associated with the detection of mixed respiratory pathogens: Multiplex SYBR Green Real-Time PCR assay for detection of common respiratory pathogens of poultry by targeting IGSR gene for MG, vlhAgene 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\(^{\circ}\)C for 3.59 mins, followed by 40 cycles of denaturation at 94\(^{\circ}\)C for 20 seconds, annealing at 55\(^{\circ}\)C for 45 seconds and extension at 72\(^{\circ}\)C for 1 min and final extension step was at 72\(^{\circ}\)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 were deemed positive if the recorded threshold cycle value was lower than 35. 19 farms were checked, and 2 tested positive for ILT, 10 for MG, 7 for MS, and 5 for both MG and MS. All 19 tested negative for IB. In  screened 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. Rapid, accurate and simultaneous detection of the respiratory pathogens and adoption of appropriate control strategies in the poultry farms could greatly help in combating the economic losses in the poultry industry.