Anti-idiotypic nanobody-phage display-mediated real-time immuno-PCR for sensitive, simultaneous and quantitative detection of total aflatoxins and zearalenone in grains

Abstract

An anti-idiotypic nanobody-phage display-mediated immuno-polymerase chain reaction (PD-IPCR) method was developed for simultaneous quantitative detection of total aflatoxins and zearalenone in cereals. Two phages, displaying the variable domain of the heavy chain anti-idiotypic nanobody that binds aflatoxin- or zearalenone-specific monoclonal antibody (1C11 or 2D3), were used as competitors for corresponding analytes. Specific DNA sequences encoding anti-idiotypic nanobodies were used to design the primers for PCR amplification. The results indicated that detection limits for total aflatoxins and zearalenone in a sample were 0.03 and 0.09 ng mL^-1, respectively. Recoveries of spiked aflatoxins and zearalenone were 80-118% and 76.7-111%, respectively. Validation results were in good agreement with the gold-standard high-performance liquid chromatography method. This report is the first to describe PD-IPCR for simultaneous quantitative detection of total aflatoxins and zearalenone in cereals.

Keywords: Aflatoxin; Aflatoxin B1 (PubChem CID: 186907); Aflatoxin B2 (PubChem CID: 2724360); Aflatoxin G1 (PubChem CID: 14421); Aflatoxin G2 (PubChem CID: 2724362); Nanobody; Phage display mediated immuno-polymerase chain reaction (PD-IPCR); Zearalenone; Zearalenone (PubChem CID: 5281576).