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To develop a real-time PCR-based rapid detection method for spoilage Alicyclobacillus spp. in juice products.The squalene-hopene cyclase-encoding gene was targeted for primer-and-probe development. Gene fragments from representative strains were cloned, and PCR primers and probe were designed by DNA sequence comparison. Selected bacteria were examined for cross-reactivity by the new method. Cells were serially diluted in apple juice and saline, and examined by the new method to establish detection sensitivity. Using the newly developed Taqman® real-time PCR-based method, strains of Alicyclobacillus acidocaldarius and A. acidoterrestris were detected without cross reactivity with other common food-borne micro-organisms. Detection of <10 cells per PCR reaction from juice samples was accomplished within 3–5 h.This is the first reported real-time PCR-based detection method for Alicyclobacillus spp. and its application in juice products is demonstrated.As a favourable alternative for the laborious and time-consuming culture- or biochemical characterization-based techniques, the system has great potential for industrial applications from raw material screening to final product quality control.