Bacillus subtilis is a widely used bacterial strain in probiotic production. The strains belong to this species are endosporogenic, tolerant to acidic pH conditions in the stomach, and many have been shown to produce enzymes that aid in food digestion and inhibit pathogenic microorganisms. In this study, a detection and qualitification method was developed based on amplification of aprE gene ultilising realtime PCR. The aprE target gene encodes subtilisin toxin precursor and is a highly conservative and specific gene of B. subtilis. The method yielded high sensitivity with LOD is 102 (CFU/mL), LOQ is 102 (CFU/mL), specificity, and accuracy are both 100%. The preparatory tools were constructed using cycle threshold (Ct) compare with B. subtilis concentration, which showed linear correlation (R2 = 1, slope = - 3.584).
realtime PCR, Bacillus subtilis, aprE, probiotic
. R. D. M. T. S. Batista, J. D. Paccez,; W. B. Luiz, E. L. Ferreira, R. C. Cavalcante, R. C. Ferreira, and L. C. Ferreira, , "Gut Adhesive Bacillus subtilis Spores as a Platform for Mucosal Delivery of Antigens," Infection and Immunity, vol. 82, pp. 1414-1423, 2014.
. S. M. R. Lefevre, G. Ripert, B. Housez, M. Cazaubiel, C. Maudet, P. Jüsten, P. Marteau, and M. C. Urdaci, "Probiotic strain Bacillus subtilis CU1 stimulates immune system of elderly during common infectious disease period: a randomized, doubleblind placebo-controlled study," Immunity & Ageing, vol. 12, p. 24, 2015.
. S. M. M. R. Lefevre, M. Denayrolles, G. Ripert, T. Desfougères, A. R. Lobach, R. Simon,; F. Pélerin, and M. C. P. U. Jüsten, "Safety assessment of Bacillus subtilis CU1 for use as a probiotic in humans.," Regulatory Toxicology and Pharmacology: RTP, vol. 83, pp. 54-65, 2017.
. A. T. A. Hosoi, K. Kiuchi, and S. Kaminogawa, "Improved growth and viability of lactobacilli in the presence of Bacillus subtilis (natto), catalase, or subtilisin," Canadian Journal of Microbiology, vol. 46, pp. 892-897, 2000.
. S. M. M. R. Lefevre, G. Ripert, B. Housez, M. Cazaubiel, C. Maudet, P. Jüsten, P. Marteau, and M. C. Urdaci, "Probiotic strain Bacillus subtilis CU1 stimulates immune system of elderly during common infectious disease period: a randomized, doubleblind placebo-controlled study," Immunity & Ageing, vol. 12, p. 24, 2015.
. I. M. C. P. Urdaci, "Antimicrobial activity of Bacillus probiotics.," In Bacterial Spore Formers-Probiotics and Emerging Applications; Horizon Bioscience: Norfolk, UK,, pp. 171-182, 2004.
. S. N. S. Khochamit, P. Sukon, W. Siripornadulsil, "Antibacterial activity and genotypic-phenotypic characteristics of bacteriocin-producing Bacillus subtilis KKU213: Potential as a probiotic strain," Microbiolical Research, vol. 170, pp. 36-50, 2015.
. Y. P. Z. Piewngam, T. H. Nguyen, S. W. Dickey, H. S. Joo, A. E. Villaruz, J. Chiou, K. A. Glose, and R. L. E. L. H. Fisher, et al., "Pathogen elimination by probiotic Bacillus via signalling interference," Nature, vol. 562, pp. 532, 2018.
. L. H. Y. Zhao, X. Xu, C. Jiang, J. Shi, Y. Zhang, L. Liu, S. Lei, D. Shao, and Q. Huang, "Potential of Bacillus subtilis lipopeptides in anti-cancer I: induction of apoptosis and paraptosis and inhibition of autophagy in K562 cells," AMB Express, vol. 8, p. 78, 2018.
. S. M. S. K. Marcial-Coba, and D. S. Nielsen, "Low-moisture food matrices as probiotic carriers," FEMS Microbiol. Lett., vol. 366, 2019.
. S. A. M. Alireza Sadeghi, A. R. Bahrami, B. Sadeghi, and M. M. Matin, "Designing a SYBR Green Absolute Real time PCR Assay for Specific Detection and Quantification of Bacillus subtilis in Dough Used for Bread Making," Journal of Cell and Molecular Research, vol. 6, pp. 84-93, 2014.
. T. C. Sơn, Method validation in chemical and microbiological analysis, 2010.
. Accreditation Office, Additional requirements for accreditation of biofield laboratories, 2016.
. C. A. Commission, "Guidelines on performance criteria and validation of methods for detection, identification and quantification of specific DNA sequences and specific protiens in foods," pp. 74, 2010.