Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples.

Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection.

Rapid detection of Salmonella enterica in food samples by a novel approach with combination of sample concentration and direct PCR.

Foodborne salmonellosis remains a major economic burden worldwide and particularly for food industries. The diverse and complexity of food matrices pose great challenges for rapid and ultra-sensitive detection of Salmonella in food samples. In this study, combination of pathogen pre-concentration with rapid molecular identification is presented to overcome these challenges. This combination enabled effective real-time PCR detection of low levels of Salmonella enterica serovar Typhimurium without culture enrichment. Anti-salmonella antibody, immobilized on protein AG-magnetic beads, could efficiently concentrate Salmonella Typhimurium with a capturing efficiency of 95%. In the direct PCR, a strong linear relationship between bacteria concentration and the number of cycles was observed with a relative PCR efficiency of ∼92% resulting in a limit of detection (LoD) of ∼2 CFU/mL. Analysis of spiked food samples that include vegetable salad, egg yolk, egg white, whole egg and minced pork meat has validated the precision of the method. A relative accuracy of 98.3% with a sensitivity of 91.6% and specificity of 100% was achieved in the Salmonella spiked food samples. The use of a Phusion hot start DNA polymerase with a high tolerance to possible PCR inhibitors allowed the integration of direct PCR, and thereby reducing the duration of analysis to less than 3 h. The Cohen's kappa index showed excellent agreement (0.88) signifying the capability of this method to overcome the food matrix effects in rapid and ultra-sensitive detection of Salmonella in food. This approach may lay a future platform for the integration into a Lab-on-a-chip system for online monitoring of foodborne pathogens.