Development of multiplex PCR for rapid and simultaneous detection of E. coli (APEC), Salmonella, Mycoplasma gallisepticum and Mycoplasma Synoviae

The Escherichia coli (E. coli), Salmonella, Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) are the major avian bacterial pathogens closely related to avian colibacillosis, salmonellosis, and mycoplasmosis respectively. In present study a single-tube multiplex PCR (m-PCR) assay was developed to allow the rapid, specific and simultaneous detection of these pathogenic species of major avian bacterial diseases. For this purpose, species specific genes of the relevant pathogens were first identified and utilized to obtain similar thermal profile conditions in singleplex PCR against the species-specific target genes “vat” of E. coli, “FimH” of Salmonella, “DNA polymerase III PolC” of MS and “mgc2” gene of MG. The primer pairs amplified specifically DNA fragment of the target genes in singleplex PCR, of 200 bp (vat), 300 bp (fimH), 380 bp (DNA polymerase III PolC) and 450 bp (mgc2). The validated species-specific primers from the similar thermal profile of singleplex PCR were then simultaneously subjected to optimize multiplex PCR condition at Tm 63 °C and amplified the same size products on agarose gel. The m-PCR was able to detect and differentiate simultaneously the aforementioned four major pathogenic species. The assay was corroborated for validation against field trial at the laboratory. The developed multiplex PCR for simultaneous identification of the aforementioned avian bacterial pathogens will help to enhance the diagnostic procedures of different poultry laboratories for field samples.