Purpose: The purpose of this study was to compare the effectiveness of three real-time PCR platforms in detection of Salmonella in allspice, cinnamon, and oregano and to evaluate the efficiency of different DNA extraction methods.
Methods: Eighteen separate trials were conducted using two different cultivars from each spices (allspice, cinnamon, and oregano), inoculated with three Salmonella serotypes, Montevideo, Typhimurium, and Weltevreden. Inoculation levels ranged from 1.7 to 3.5 log CFU/25g. Overnight pre-enrichment cultures were used to extract DNA for PCR, using two different methods (boil lysis and the standard methods) for each PCR platforms. The three real-time PCR methods evaluated in the study were ABI-MicroSEQ®, FDA-PRLSW, and GeneDisc®.
Results: The detection rate of Salmonella by PCR methods from culture positive samples for all trials was 99.8% for MicroSEQ® (n=509) and 97.6% for both PRLSW (n=510) and GeneDisc® (n=509). The un-detected samples were mostly from cinnamon, indicating possible PCR inhibitors in cinnamon. Mean CT values for cinnamon (26.86) were significantly higher than those for allspice (22.26) and oregano (20.15) (P<0.0001). The difference of mean CT values between boil lysis and standard extraction was not significant for allspice (P=0.573) and oregano (P=0.064), but was significant for cinnamon (P<0.0001), with the standard extraction having lower Ct values.
Significance: The detection of Salmonella by all three PCR platforms was comparable with culture assay results. Selection between standard DNA extraction methods combined with different PCR methods or boil methods depended on the spice tested. Although the boil method is simple, when facing inhibitory problems, the standard DNA extraction protocol suggested by PCR kit manufacturers could perform better.