Methods To Improve Molecular Detection of Salmonella in Complex Herbal Matrices Containing Inhibitors.

Abstract: Salmonella is one of the main causes of foodborne diseases worldwide. Molecular tests such as the PCR assay are rapid and sensitive and are increasingly becoming the preferred method for pathogen detection. However, the presence in the analyzed samples of substances that reduce the sensitivity of the assay or totally inhibit PCR amplification might result in failure of pathogen detection. Using a multiplex real-time PCR assay, 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 in the multiplex PCR reactions indicated the degree of inhibition. All three herbs inhibited PCR amplification at the standard matrix concentration (10% suspension). I applied and compared four approaches for overcoming 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 inhibitory substances. By using a series of centrifugation steps combined with a direct PCR, I removed the PCR inhibitors and successfully detected the pathogen in each of the tested matrices. This approach did not significantly decrease the sensitivity of the PCR assay, and the detection of the pathogen was with a quantification cycle delay of only 1.48 ± 1.05 cycles compared with the control. Thus, the proposed simple, efficient, reliable, quick, and cost-effective method allowed for removal of PCR inhibitors and subsequent detection of foodborne bacterial pathogens in complex matrices containing PCR inhibitors.