Simultaneous and rapid detection of avian respiratory diseases of small poultry using multiplex reverse transcription-Polymerase Chain Reaction assay

Major viral infections, such as Newcastle disease virus, infectious bronchitis virus, avian influenza virus, and infectious bursal disease virus, inflict significant injury to small poultry and tremendous economic damage to the poultry sector. This research aims to develop a multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) approach to simultaneously determine these important viral pathogens. The conserved segment of various viral genetic sequences was used to design and synthesize specific primers. Moreover, as positive controls, recombinant vectors were synthesized in this investigation. The d-optimal approach was used to improve PCR conditions in this investigation. Positive controls and clinical samples were used to assess the m-PCR assay's specificity, sensitivity, repeatability, and reproducibility. According to the sensitivity test findings, the m-PCR technique could generate the 8 target genes from viral genomes using 1 x 102. In addition, 8 viral pathogens were detected from the infected samples. The findings also suggest that live animal oral swabs were not significantly different from tissue sampling of a dead animal (P < 0.05), and this kit had a high sensitivity for analyzing both types of samples. The suggested m-PCR test may detect and evaluate viral infection in birds with excellent speci-ficity, sensitivity, and throughput.

Automated summary

This study aimed to develop a multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) method for simultaneous detection of important avian viral pathogens. Specific primers were designed and synthesized using conserved segments of viral genetic sequences, and recombinant vectors were synthesized as positive controls. The d-optimal approach was used to optimize PCR conditions. The m-PCR assay showed high specificity, sensitivity, and reproducibility in detecting and evaluating viral infection in birds, with the ability to detect 8 target genes and identify 8 viral pathogens from infected samples. The test also demonstrated that live animal oral swabs were as effective as tissue sampling from dead animals, making it a valuable tool for viral infection analysis in poultry.