Bioresource Technology, Vol.129, 477-484, 2013
Development of a specific real-time PCR assay targeting the poly-gamma-glutamic acid synthesis gene, pgsB, for the quantification of Bacillus amyloliquefaciens in solid-state fermentation
A TaqMan real-tithe PCR procedure was developed for specific detection and quantification of strains belonging to Bacillus amyloliquefaciens group. The primer pair pgsB726-f/pgsB791-r and the pgsB-probe were designed from one of the poly-gamma-glutamic acid synthesis gene (pgsB) of B. amyloliquefaciens. The detection limit was approximately between 10(2)-10(3) cells/mL. A linear correlation between the logo input pMD-pgsB plasmid DNA copies and the threshold cycle values were observed with a magnitude of linearity in the range of 9.415 x 10(3)-10(7) copies/mL for the standard curve, which exhibited a slope of -3.35 and an R-2 value of 99.8%. Results of validation of this method with artificially contaminated and natural solid-state fermentation samples showed that it was suitable for specific and sensitive detection and quantification for the target strains in solid-state fermentation samples. This could be more useful to understand the fermentation starting strain and the final microbiological properties of fermentation products. (C) 2012 Elsevier Ltd. All rights reserved.
Keywords:TaqMan real-time PCR;Poly-gamma-glutamic acid synthesis gene (pgsB);Bacillus amyloliquefaciens;Detection;Solid-state fermentation