AbstractUsing a recently developed genetically modified microorganisms (GMM) detection strategy, unexpected contaminations of unauthorized GMM in commercialized microbial fermentation products have been reported. A first-line real-time PCR screening analysis was initially performed to determine the presence of key targets frequently found in genetically modified (GM) bacteria. A second-line real-time PCR analysis was subsequently applied to identify specific GMM, including to date a GM Bacillus velezensis producing protease and a GM B. subtilis producing vitamin B₂. In this study, an additional real-time PCR method specific to a newly identified GMM producing alpha-amylase was developed to be integrated in such second-line real-time PCR analysis, allowing to strengthen the GMM detection strategy. This method was successfully validated based on the assessment of its specificity and sensitivity performance. In addition, its applicability was confirmed using several food enzyme products commercialized on the market. Finally, via its transfer to an external laboratory, the transferability of the in-house validated method was positively evaluated, allowing its easy implementation in enforcement laboratories.