Development of a Taxon-Specific Real-Time PCR Method Targeting the Bacillus subtilis Group to Strengthen the Control of Genetically Modified Bacteria in Fermentation Products

Abstract

Most of the bacteria that are used to produce fermentation products, such as enzymes, additives and flavorings, belong to the Bacillus subtilis group. Recently, unexpected contaminations with unauthorized genetically modified (GM) bacteria (viable cells and associated DNA) that were carrying antimicrobial resistance (AMR) genes was noticed in several microbial fermentation products that have been commercialized on the food and feed market. These contaminations consisted of GM Bacillus species belonging to the B. subtilis group. In order to screen for the potential presence of such contaminations, in this study we have developed a new real-time PCR method targeting the B. subtilis group, including B. subtilis, B. licheniformis, B. amyloliquefaciens and B. velezensis. The method’s performance was successfully assessed as specific and sensitive, complying with the Minimum Performance Requirements for Analytical Methods of GMO Testing that is used as a standard by the GMO enforcement laboratories. The method’s applicability was also tested on 25 commercial microbial fermentation products. In addition, this method was developed to be compatible with the PCR-based strategy that was recently developed for the detection of unauthorized GM bacteria. This taxon-specific method allows the strengthening of the set of screening markers that are targeting key sequences that are frequently found in GM bacteria (AMR genes and shuttle vector), rein-forcing control over the food and feed chain in order to guarantee its safety and traceability.