Phage display-mediated immuno-polymerase chain reaction(PD-IPCR)is an ultrasensitive detection technology that combines the advantages of immuno-PCR and phage display.The phage particle,which displayed antibody fragments including single-chain fragment variable(scFv),variable domain of heavy-chain antibodies(VHH),and antigen-binding fragment(Fab)on the surface can be directly used in IPCR,supplying both the detection antibody and deoxyribonucleic acid(DNA)template.In this work,we used ochratoxin A(OTA)as a model system to study the capacity of PD-IPCR in the detection of toxic small molecular weight compounds,especially mycotoxins.An alpaca-derived VHH library was constructed and subjected to four cycles of panning.In total,16 clones with four unique sequences were selected by competitive binding with OTA.The clone VHH-28 resulted in the lowest 50%inhibitory concentration of 0.31 ng/mL in the phage enzyme-linked immunosorbent assay(ELISA)and was selected to develop the VHH phage-based real-time immuno-PCR(RT-IPCR).The detection limit of the VHH phage-based RT-IPCR was 3.7 pg/L,with a linear range of 0.01-1000 pg/mL.This method was compared with conventional ELISA,and validation results indicated the reliability of VHH phage-based RT-IPCR in the detection of OTA in cereal samples.This study provides a new idea for the ultrasensitive detection of mycotoxins and other toxic small molecular weight compounds.