Purpose: This study compared the time needed to detect A. acidoterrestris spores in juice concentrates using culture-based methods and the GeneDisc System—a real time, quantitative, PCR-based system.
Methods: Apple and orange juice concentrates were inoculated in triplicate with A. acidoterrestris spores at 0.5 spores/ml and 0.1 spores/ml. Concentrates were diluted 1:10 with water (apple) or YSG broth (orange) and heat shocked at 80°C for 10 min. Orange juice was incubated at 43°C and sampled daily. Apple juice was filtered through a 0.45 µm pore-size filter. The filter was placed in 10 ml YSG broth, incubated at 43°C and sampled daily. For analysis by culturing, samples were plated on YSG agar and incubated at 43°C. For analysis, DNA was extracted from samples and amplified per the system protocol.
Results: Samples spiked with 0.5 spores/ml were detected positive by the system after a 24-hour enrichment. Cycle threshold (Ct) values were 13.43 ± 0.96 for apple juice and 27.67 ± 3.45 for orange juice (mean ± sd). Using culturing, A. acidoterrestris was not detectable until at least 48 hrs. Samples spiked with 0.1 spores/ml were detected positive by the system after a 48-hour enrichment. Ct values were 15.50 ± 1.23 for apple juice and 31.27 ± 6.15 for orange juice. Detection by culturing required 48 hrs for apple juice and up to 5 days for orange juice.
Significance: The system can substantially reduce the time needed to detect A. acidoterrestris contamination of fruit juice concentrates.