Evaluation of Real-time PCR Combined with Immunomagnetic Separation or Centrifugation for Detection of Low Levels of Healthy and Sanitizer-Injured Salmonella spp. on Mung Bean Sprouts

Introduction: Mung bean sprouts, which are used extensively in Asian cuisine, are now well recognized to harbor of Salmonella spp., and are implicated in salmonellosis outbreaks. To ensure the safety of bean sprouts consumption, thus the presence of Salmonella spp. needs to be screened using an accurate and rapid method.

Purpose: The aim of this study was to develop a rapid and accurate detection methodology for low levels of healthy and sanitizer-injured Salmonella on mung bean sprouts using real-time PCR coupled with either immunomagnetic separation (PCR-IMS) or centrifugation (PCR-cen).

Methods: The parameters for IMS including specificity/sensitivity, bacterial concentration and bead incubation time were optimized. Limit of detection (LOD) was also determined for the optimized PCR-IMS and PCR-cen. Both methods were compared against PCR alone (PCR) and the standard culture method (ISO) for their ability to detect Salmonella using inoculated and uninoculated sprouts. Mean values were compared using ANOVA.

Results: Under optimum IMS conditions (10⁵ CFU/ml for 30 min), capture efficiency of Salmonella in sprout suspensions was lower than 40%, most probably due to the non-specific binding of the background microbiota. PCR-IMS and PCR-cen had a similar LOD at 10³ CFU/ml, which was one log unit lower than PCR. Enrichment of 10 h was sufficient to detect 100% of the inoculated sprouts with both PCR-IMS and PCR-cen, which was significantly faster compared to PCR and the ISO method. Moreover, the validation study using uninoculated sprouts revealed that PCR-IMS and PCR-cen were equally effective on Salmonella detection, showing 98.3% accuracy.

Significance: These results suggest that PCR-cen would be the effective and less costly method for the detection of low levels of healthy and sanitizer-injured Salmonella on mung bean sprouts.