A Multiplex Liquid Array Assay Based on Luminex xTAG Technology for the Detection of Five Respiratory Pathogens in SPF Chickens.

A multiplex liquid array assay based on Luminex xTAG technology was developed for the first time for the simultaneous detection of five respiratory bacterial pathogens in Specific Pathogen-Free (SPF) chickens-Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), Pasteurella multocida (Pm), Avibacterium paragallinarum (Apg), and Mycobacterium avium (Mav)-filling the gap that these five pathogens could not be simultaneously detected previously. Specific primers were designed targeting conserved genes of each pathogen, with a TAG sequence attached to the 5' end of each forward primer and biotin to the 5' end of each reverse primer. After multiplex PCR amplification, the products were hybridized with MagPlex-TAG microspheres and analyzed using the Luminex system. The method was validated for sensitivity, specificity, and repeatability, and tested with simulated positive samples (a mixture of genomic DNA from the five target pathogens) and SPF chicken samples. The LOD was 10 copies/μL for Pm and 100 copies/μL for the other four pathogens. No cross-reactivity was observed with 18 non-target bacterial species. Intra- and Inter-assay coefficients of variation (CV) ranged from 0.8% to 12.2% and 2.3% to 14.4%, respectively, both below 15%. All simulated mixed positive samples yielded results consistent with expectations, and none of the 114 nasopharyngeal swab samples from SPF chickens tested positive for any of the five targets. The developed assay offers high throughput, high sensitivity, high specificity, and good repeatability, providing a robust tool for routine quality control in SPF chickens.