A Rapid RPA-CRISPR/Cas12a Detection Method for Adulteration of Goat Milk Powder

Because of the serious adulteration of goat milk, the rapid on-site detection of goat milk powder adulteration is needed. In this study, the CRISPR/Cas12a detection system combined with recombinase polymerase amplification (RPA) was employed to qualitatively detect the adulteration of goat milk powder with cattle-derived components. Specific primers and crRNA were designed and screened. After the optimization of RPA and the Cas system, the RPA-CRISPR/Cas12a detection method was established. The detection can complete the rapid identification of cattle-derived components in 45 min, without the assistant of large equipment. The absolute detectability of the RPA-CRISPR/Cas12a assay could reach 10^-2 ng/μL for cattle genomic DNA, and 1% (w/w) for cattle milk powder, which is suitable to meet the testing requirements for on-site detection. In total, 55 commercial goat milk powder products were collected for blind testing. The results showed that 27.3% of the samples were adulterated with cattle ingredients, revealing a serious adulteration situation in goat milk powder market. The RPA-CRISPR/Cas12a assay established in this research exhibited its potential for practical use of on-site detection to detect cow milk powder in goat milk powder and can provide reliable technical reference for combating food fraud of adulteration of goat milk products.