Authentication of the Apis cerana honey with the proofreading enzyme-mediated probe cleavage combined with ladder-shape melting temperature isothermal amplification (Proofman-LMTIA) method

Abstract

Honey adulteration has been on the rise with the increasing demand for high-quality honey, and the authentication of A. cerana honey is a major problem that the current bee industry needs to solve. The aim of this research was to establish the proofreading enzyme-mediated probe cleavage combined with ladder-shape melting temperature isothermal amplification (Proofman-LMTIA) method for the authentication of Apis cerana honey. The LMTIA primers were designed to target the specific 18S rRNA sequence of A. cerana, and the conditions for Proofman-LMTIA amplification were optimized. The specificity and sensitivity of the method were determined and compared with that of the droplet digital polymerase chain reaction (PCR). The optimal temperature of the established Proofman-LMTIA method was 62 °C. The method can specially to detect the A. cerana gene without cross-reactivity with the genomes from other animal and plant species, and the amplification reaction can be finished within 20 min with the sensitivity of 10 pg A. cerana genomic DNA, which was more sensitive than the droplet digital PCR method. The obtained results demonstrate that the established Proofman-LMTIA procedure was specific, sensitive, and rapid, and can be used for authentication of A. cerana honey.

Keywords

• Apis cerana honey
• authentication
• ladder-shape melting temperature isothermal amplification of nucleic acids
• Proofman probe