Development of an on-spot and rapid recombinase polymerase amplification assay for Aspergillus flavus detection in grains

Abstract

Aspergillus flavus is one of the main contaminants in grains, and most strains of A. flavus can produce aflatoxins (AFs), which poses a great threat to food safety and human health. Early detection of food contaminated by A. flavus can effectively reduce the harm of AFs to human health and prevent further spread of A. flavus. In this study, a sensitive and rapid method for on-spot detection of A. flavus in grains, based on recombinase polymerase amplification (RPA), has been developed. Three RPA primer pairs were designed, and then a suitable primer pair was selected. A simplified and efficient DNA extraction method for on-spot detection was developed and applied. Two temperatures, ambient temperature (AT, 26 ± 1 °C) and body temperature (BT, 36 ± 1 °C), were provided for the detection. The reaction time and primer concentration were optimized at AT and BT, respectively. And a fluorescent DNA dye was used for the visual detection. The whole detection-assay can be completed within 20 min at BT and 30 min at AT, respectively. The sensitivity evaluation proved that the limit of detection (LOD) of the RPA-assay was 101 conidia/g. The specificity of the assay was equal to the real-time PCR. The new RPA-assay also had great potential in other fields′ applications.