Development of a multiplex PCR-based test kit for the rapid diagnosis of common viral and bacterial pathogens in poultry

The rapid diagnosis of poultry diseases is important in terms of treatment, control, and biosecurity. In this thesis study, the aim was to develop a multiplex PCR-based diagnostic kit capable of detecting multiple pathogens simultaneously. The relevant pathogens were identified as Infectious Laryngotracheitis (ILT – GaHV-1), Newcastle Disease Virus (NDV), Avian Influenza Virus subtypes (AIV's - A/H5N1, A/H9N2, A/H7N9), E. coli and E. coli O157:H7, Salmonella spp., and Pasteurella multocida, and specific primer-probe sets were designed for each. The complete genome sequences of the pathogens were obtained from databases, and genes were selected to design pathogen-specific primers based on these sequences. The Eurofins Genomics in-silico tool was used for primer-probe design. The obtained data (GC content, Tm values, and targeted amplicon length) were verified using BLAST and Primer3 in-silico software tools. The designed primer-probe sequences were compared with all known viral and bacterial genomes in the NCBI database to ensure specificity and that they did not match any other viral or bacterial genomes. Primer-probe characterizations, optimizations, and minimum detection limits (LOD) were determined and used in the tests. To measure probe emissions for different pathogens simultaneously, different fluorophore channels were used, and pathogen-specific sigmoidal curves were obtained. In the probe design, different dyes were used with a fluorophore at the 5' end and a quencher at the 3' end. The following combinations were preferred: Texas Red – BHQ1 for ILT, FAM – MGB for AIV A/H5N1, HEX – MGB for AIV A/H9N2, Texas Red – BHQ1 for AIV A/H7N2, Cy-2 – BHQ2 for NDV, FAM – MGB for E. coli and E. coli O157:H7, HEX – MGB for Salmonella spp., and Cy5 – BHQ2 for P. multocida. In contrast, the detection channels used to monitor the fluorescent signals were as follows: ROX channel for ILT, FAM channel for AIV subtype A/H5N1, ROX channel for A/H7N2, HEX channel for A/H9N2, Cy5 channel for NDV, FAM channel for E. coli and E. coli O157:H7, HEX channel for Salmonella spp., and Cy5 channel for P. multocida. The findings indicated that all agents could be detected using pathogen-specific primers. It was also demonstrated that all positive samples could be successfully identified even in the presence of multiple pathogen genomes in the test environment. By optimizing the amounts of master mix and primer-probe used, the per-test reagent consumption was reduced, lowering the per-test cost by approximately 25%. In the study, the LOD values based on nucleic acid concentration were evaluated, and it was determined that this value was 0.5 ng for P. multocida and 0.05 ng for all other pathogens. In conclusion, a real-time PCR-based multiplex rapid diagnostic panel kit was developed that enables the simultaneous detection of ILT (GaHV-1), NDV, AIV's (A/H5N1, A/H9N2, A/H7N9), E. coli and E. coli O157:H7, Salmonella spp., and P. multocida nucleic acids, allowing the concurrent diagnosis of the diseases caused by these pathogens with a single diagnostic kit. Keywords: Poultry, real-time PCR, diagnostic kit