Production of RPSL12-TetR fusion and development of two multiplex methods for simultaneous detection of tetracyclines and aminoglycosides in pork

Nowadays, multiplex immunoassays capable of simultaneously detecting two classes of drugs are highly desirable. In this study, the genes of TetR and RPSL₁₂ were fused and expressed to produce a novel fusion protein that recognizes 10 tetracyclines (TCs) and 12 aminoglycosides (AGs) simultaneously. A multiplex direct competition fluorescence assay and a multiplex direct competition chemiluminescence assay were established for the simultaneous detection of these two drug classes in pork samples. The multiplex direct competition fluorescence assay required only a single sample addition step and could be completed within 35 min, with detection limits ranging from 1.1 to 10.2 ng/mL. The multiplex direct competition chemiluminescence assay could be completed within 30 min and achieved significantly lower detection limits (0.0016-1.8 ng/mL). Based on a comprehensive evaluation of assay time, operational complexity, and sensitivity, both methods demonstrated superior performance compared to previously reported immunoassays for these drug classes.