Real-Time PCR Detection of Alicyclobacillus acidoterrestris in Fruit Juice: Method Validation and Implications for Guaiacol-Related Spoilage

In this study, a SYBR Green-based real-time PCR workflow targeting the vdcC gene was optimized and validated for rapid detection of Alicyclobacillus acidoterrestris in fruit juice. A commercial DNA extraction kit showed the best performance, achieving a limit of detection (LOD) of 2 Log CFU/mL, whereas microwave-based extraction (30 s) provided a rapid alternative with an LOD of 3 Log CFU/mL. The vdcC primer set enabled clear discrimination of A. acidoterrestris from closely related species based on melting curve analysis. The method was successfully applied to orange, apple, and grape juice matrices, as well as to 30 commercial juice samples. Guaiacol analysis further indicated that the presence of A. acidoterrestris DNA did not necessarily correspond to active spoilage. Overall, this study provides a systematically optimized and practically validated workflow for monitoring A. acidoterrestris in fruit juice systems.