Methods to improve molecular detection of Salmonella in complex herbal matrices containing inhibitors

Salmonella is one of the main causes of foodborne diseases worldwide. Molecular tests such as the polymerase chain reaction (PCR) are rapid and sensitive and are increasingly becoming a preferred method for pathogen detection. However, the presence of PCR inhibiting substances in the analyzed samples could reduce the sensitivity or totally inhibit PCR amplification, which might result in failure of detection of the pathogen. Using multiplex real-time PCR, I investigated the detection of Salmonella enterica serovar Typhimurium in three herbal matrices containing inhibiting substances: i) chamomile (Matricaria recutita); ii) sage (Salvia officinalis) and iii) mint (Menthae piperitae). Internal positive controls (IPC) in the multiplex PCR reactions indicated the degree of inhibition. All of the three herbs inhibited PCR amplification at standard concentration of the matrix (10% suspensions). I applied and compared four approaches to overcome the negative effect of the matrices on the PCR detection of Salmonella. The efficiency strongly depended on the matrix and the method used for removing the inhibiting substances. By a series of centrifugation steps combined with direct PCR, I managed to remove the PCR inhibitors and successfully detected the pathogen in each of the tested matrices. Moreover, this approach did not decrease significantly the PCR sensitivity and the detection of the pathogen was with Cq delay of only 1.48 ± 1.05 cycle, compared to the control. Hence, by the proposed method, PCR detection of Salmonella became possible in matrices with strong inhibitory effect on the PCR reaction. In summary, a simple, efficient, reliable, quick and cost-effective generic approach for removal of PCR inhibitors, and detection of foodborne bacterial pathogens in complex matrices containing PCR inhibitors, was proposed.