Development of a specific real-time PCR assay targeting the poly-γ-glutamic acid synthesis gene, pgsB, for the quantification of Bacillus amyloliquefaciens in solid-state fermentation

Abstract

A TaqMan real-time 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-γ-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 log10 input pMD-pgsB plasmid DNA copies and the threshold cycle values were observed with a magnitude of linearity in the range of 9.415×10(3)-10(7) copies/mL for the standard curve, which exhibited a slope of -3.35 and an R2 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.