H Luo1, A E Yousef, H H Wang. 1. Department of Food Science and Technology, The Ohio State University, Columbus, OH 43210, USA.
Abstract
AIMS: To develop a real-time PCR-based rapid detection method for spoilage Alicyclobacillus spp. in juice products. METHODS AND RESULTS: 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. CONCLUSION: This is the first reported real-time PCR-based detection method for Alicyclobacillus spp. and its application in juice products is demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.
AIMS: To develop a real-time PCR-based rapid detection method for spoilage Alicyclobacillus spp. in juice products. METHODS AND RESULTS: 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. CONCLUSION: This is the first reported real-time PCR-based detection method for Alicyclobacillus spp. and its application in juice products is demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.
Authors: Matthew L Ranieri; Reid A Ivy; W Robert Mitchell; Emma Call; Stephanie N Masiello; Martin Wiedmann; Kathryn J Boor Journal: Appl Environ Microbiol Date: 2012-06-08 Impact factor: 4.792