Development of the Saltatory Rolling Circle Amplification Assay for Rapid and Visual Detection of <i>Alicyclobacillus acidoterrestris</i> in Apple Juice

Abstract

A novel nucleic acid isothermal amplification method based on saltatory rolling circle amplification (SRCA) for rapid and visual detection of <i>Alicyclobacillus acidoterrestris</i> in apple juice was established. Fourteen <i>A. acidoterrestris</i> strains and 44 non-<i>A. acidoterrestris</i> strains were used to confirm the specificity. The sensitivity of SRCA was 4.5 × 10¹ CFU/mL by observing the white precipitate with the naked eye, while it was 4.5 × 10⁰ CFU/mL by fluorescence visualization. The detection limit of SRCA in artificially inoculated apple juice was 7.1 × 10¹ and 7.1 × 10⁰ CFU/mL via visualization of the white precipitate and fluorescence, respectively. Compared with the traditional PCR method, SRCA exhibited at least a 100-fold higher sensitivity and 100-fold lower detection limit. Seventy samples were investigated for <i>A. acidoterrestris</i> contamination, and the results showed 100% sensitivity, 97.01% specificity, and 97.14% accuracy compared with those by the conventional microbiological cultivation method. Overall, this method is a potentially useful tool for visual and rapid detection of <i>A. acidoterrestris</i>.