A novel rapid detection method for chicken adulteration based on recombinant polymerase amplification and multicomponent nuclease (MNAzyme)

Abstract

The identification of meat adulteration is of great significance to food safety. This work reports a novel rapid detection platform for chicken adulteration based on recombinase polymerase amplification (RPA) and multicomponent nuclease (MNAzyme). The conserved sequence target of the Cytb gene was used to design RPA primers and MNAzyme. First, the RPA amplification products were introduced into the detection system, which included MNAzyme and DNA-RNA composite probes. When the chicken-derived RPA amplification product was present in the sample, it generated an “input” signal that triggered MNAzyme's catalytic core to cleave the DNA-RNA complex probe, leading to a fluorescence signal. The entire procedure, from nucleic acid extraction to detection, took just 60 min, and the results could be easily interpreted using a portable fluorescent thermostatic amplifier. The RPA-MNAzyme assay detected 8 copies/µL of chicken DNA in 60 min, which is consistent with the results of the RPA assay, demonstrating that the method is highly accurate and specific (does not cross-react with nucleic acids from other animal sources). And it could detect as low as 0.5% chicken in beef and lamb. By harnessing MNAzyme to specifically identify target genes in RPA amplification products, the platform eliminates the need for RPA primer screening and RPA probe design, reducing experimental costs.