Robustness of Loop-mediated Isothermal Amplification Assays for Salmonella Detection

Introduction:  Salmonella is a leading cause of foodborne illnesses and deaths in the United States and worldwide. Molecular-based methods such as PCR and more recently, loop-mediated isothermal amplification, have gained wide applications in Salmonelladetection, due to their rapidity, specificity and sensitivity. However, there is a paucity of data regarding the robustness of these assays. 

Purpose:  This study aimed at evaluating the robustness of two recently developed loop-mediated isothermal amplification (LAMP) assays for Salmonella detection, using PCR as the comparison method. 

Methods:  Performances of the LAMP and PCR assays were examined under various abusive assay preparation and running temperatures, pH and in the presence of potential inhibitors in food applications (media used for enrichment and dilution, plant polysaccharide, humic acid) and actual food rinses (chicken and ground beef).

Results:  The LAMP assays achieved robust detection of Salmonellacells under abusive assay preparation (25 and 37°C with holding up to 30 min) and running temperatures (60-68°C). By using a hot start DNA polymerase, PCR obtained comparable results under these temperature ranges. However, PCR performed markedly poorer under abusive pH values. In the presence of inhibitors, LAMP assays also demonstrated greater tolerance than PCR. When chicken and ground beef rinses were added at 20% into the reaction mix, PCR amplifications were completely inhibited, but LAMP reactions were not. 

Significance:  LAMP assays were demonstrated to be a robust alternative to PCR in detecting Salmonella under abusive reaction conditions and with inhibitors. Therefore LAMP could be adopted for routine Salmonella testing in food samples with rapidity and robustness.