Rapid Single-Tube Detection of Peach (Prunus persica)-Derived Ingredients by Recombinase-Aided Amplification Combined with Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-Associated Proteins (Cas)12a

Abstract

A rapid single-tube method for the detection of peach-derived ingredients was established using recombinase-aided amplification (RAA) combined with clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas)12a. Specific primers for RAA and CRISPR RNA (crRNA) were designed. The specificity, sensitivity and detection limit of the developed method were analyzed, and it was applied to commercial samples. The results showed that the method enabled rapid detection of peach-derived ingredients without the need for large-scale instruments in less than 30 minutes. No cross-contamination with other common fruit species was observed, indicating good specificity. The sensitivity of this method was 1.0 × 10-1 ng/µL, and the limit of detection was 1%. The results of detection of 20 commercial fruit juice samples by this method were consistent with those of real-time polymerase chain reaction (PCR). Therefore, the single-tube integrated RAA-CRISPR/Cas12a method has the advantages of high specificity and sensitivity, providing a new technology for rapid detection of peach-derived ingredients.

Keywords

• recombinase-aided amplification; clustered regularly interspaced short palindromic repeat (crispr)/crispr-associated proteins 12a; single-tube; peach; rapid detection