Sterility testing of shelf-stable pudding by PCR

Abstract

A PCR-based method for determining the sterility of shelf-stable pudding was developed. PCR primers were universal for conserved sequences of the bacterial ribosomal operon and resulting amplicons corresponded to the 16s–23s spacer region. Commercially sterile, aseptically filled pudding inoculated with Bacillus cereus was used to show the dilution needed to overcome PCR inhibition by the food menstruum and sensitivity of the method. A 60-fold dilution of the food was required to relieve PCR inhibition and a minimum of 106cells g−1were needed to detect the PCR product on ethidium bromide stained agarose gels. The specific growth rate of B. cereus in the pudding at 35°C was measured to be 0·148 h−1(4·68 h doubling time). The enrichment time needed to achieve 106cells g−1for detection of bacteria from a theoretical one cell per individual serving size (100 g) was determined to be 3·91 days. When fluorescence detection of the PCR product was performed using a dsDNA binding dye, the sensitivity was increased four-fold, allowing for detection of bacterial contamination after 3·52 days of enrichment incubation. The use of fluorescence detection reduced enrichment time and eliminated the need for agarose gel electrophoresis, making the method more user friendly. Routine sterility checks of shelf-stable pudding can be automated to a 96-well format and take 4 days to complete, including enrichment incubation, PCR and fluorescence detection.