Rapid detection of Geobacillus and Anoxybacillus species by quantitative qPCR (qPCR) in commercial dairy products

Thermophilic spore-forming bacteria, especially Anoxybacillus and Geobacillus species, are a major problem for dairy industry. The presence of these thermophilic bacilli in the dairy products is considered a poor hygiene indicator during product processing. The commonly preferred culture-dependent detection methods are time consuming. Therefore, the development of rapid and reliable molecular approaches for the detection of these problematic microorganisms in food processing is essential. In this context, specific primers for the gene (spo0A) that initiates sporulation were designed for Anoxybacillus kamchatkensis subsp. asaccharedens (F81) and Geobacillus thermodenitrificans (DSM 465(T)), which have been shown to be strong biofilm producers in dairy products. For this purpose, a quantitative polymerase chain reaction (qPCR) assay was developed. The qPCR standard curves obtained with these primers allowed a total viable cell count in the range of 10(1)-10(8) CFU/ml for G. thermodenitrificans, while the spore count was in the range of 10(3)-10(8) CFU/ml. The primer developed for A. kamchatkensis subsp. asaccharedens was able to detect viable total cells and spores with much higher sensitivity (10(1)-10(8) CFU/ml viable total cells, 10(2)-10(8) CFU/ml spores). The primers developed in this study allowed separate detection of Anoxybacillus and Geobacillus in dual culture experiments. The primers and method developed in this study can also be used for rapid detection of Anoxybacillus and Geobacillus contamination in mixed cultures.