Development of a real-time PCR assay for rapid screening of acetic acid bacteria as a group in food products

AAB are Gram-negative, obligate aerobic, rod-shaped bacteria that produce acetic acid from various alcohols or monosaccharides. They play a multifaceted role in the vinegar, dairy, and wine industry. Currently, the detection of AAB involves laborious and time-consuming culture methods (> 3-5 days). To develop a low-cost, easy to use, and reliable assay for the detection of AAB in food products, we designed a novel real-time PCR primer set, K-AAB, targeting the 16S rRNA genes of all known AAB species (46 species across 11 genera). In in silico PCR analysis, all 46 AAB species tested positive, whereas all (34 out of 34) closely related microorganisms tested negative. In inclusivity and exclusivity analyses, the real-time PCR assay showed positive results with 16 AAB species, with a melting temperature of all amplicons of 81.5 degrees C, whereas negative results were observed in 20 species of unrelated food microorganisms. These results indicated 100% sensitivity and specificity of the assay. Furthermore, the novel K-AAB primer-based assay enabled linear detection of AAB in the range of 10(1)-10(5) colony-forming units (CFU) per reaction in yogurt and vinegar. The novel assay was successfully applied for the screening of food samples naturally harboring AAB, which was accomplished within 2 h. These findings demonstrate that the novel method can be used for effective and sensitive screening for the presence of AAB in food products, either to detect AAB contaminants or explore the presence of novel functional strains.