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.

Pneumocystis is an opportunistic fungal pathogen that causes severe Pneumocystis pneumonia (PCP) in immunocompromised individuals and laboratory animals. Three host-specific species—Pneumocystis murina (P. murina), Pneumocystis carinii (P. carinii), and Pneumocystis jirovecii (P. jirovecii)—are closely associated with infections in humans and laboratory animals. However, the conventional method, microscopic staining, suffers from low sensitivity, operator-dependent subjectivity, and inability to differentiate species, highlighting the urgent need for a multiplex qPCR assay. In this study, we established a multiplex qPCR method targeting the mtLSUrRNA gene of P. murina, the TS gene of P. carinii, and the mtSSUrRNA gene of P. jirovecii. Primers and probes were designed and optimized using a matrix approach. The method was systematically evaluated for sensitivity, specificity, and reproducibility using recombinant plasmid standards and laboratory animal samples. Validation was performed on 260 mouse lung samples, 30 P. murina-positive samples, 25 rat lung samples, 6 rat bronchoalveolar lavage fluid (BALF) samples, and 8 P. carinii-positive samples. Results were compared with single-plex qPCR and staining microscopy (performed on 68 mouse lung samples, 38 Pneumocystis-positive samples). The limits of detection (LOD) were 5 copies/μL for P. murina, 6 copies/μL for P. carinii, and 8 copies/μL for P. jirovecii. Standard curves showed excellent linearity (R² ≥ 0.999) with amplification efficiencies of 90–110%. No non-specific reactions were observed with 22 common pathogens, and intra-/inter-group coefficients of variation (CV%) were below 1%. Moreover, interference testing revealed minimal matrix effects on the amplification system and no mutual interference among the primers and probes. The multiplex qPCR detected all 38 positive samples (100%), showing 100% concordance with single-plex qPCR, whereas Giemsa staining detected none (0%) and toluidine blue staining only 60% (3/5) of the tested positives, suggesting that the multiplex qPCR achieved higher detection rates than staining microscopy. In conclusion, this novel multiplex qPCR method offers high sensitivity, specificity, and reproducibility, providing a sensitive and specific tool for laboratory animal health monitoring and epidemiological surveillance. Its clinical application for human PCP diagnosis requires further validation with authentic human specimens.