title: VHH phage-based competitive real-time immuno-polymerase chain reaction for ultrasensitive detection of ochratoxin A in cereal.
abstract: 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.
authors.affiliation: State Key Laboratory of Food Science and Technology and ‡Sino-Germany Joint Research Institute, Nanchang University , 235 Nanjing East Road, Nanchang 330047, People's Republic of China.
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.forename: Yong-hua
authors.initials: YH
authors.lastname: Xiong
authors.forename: Zhui
authors.initials: Z
authors.lastname: Tu
authors.forename: Yan-ping
authors.initials: YP
authors.lastname: Li
authors.forename: Zhen-yun
authors.initials: ZY
authors.lastname: He
authors.forename: Yu-lou
authors.initials: YL
authors.lastname: Qiu
authors.forename: Jin-heng
authors.initials: JH
authors.lastname: Fu
authors.forename: Shirley J
authors.initials: SJ
authors.lastname: Gee
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
mesh_terms: D001435:Bacteriophages / Q000737:chemistry*; D001483:Base Sequence; D017931:DNA Primers; D002523:Edible Grain / Q000737:chemistry*; D057230:Limit of Detection; D009793:Ochratoxins / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D009793:Ochratoxins; C025589:ochratoxin A