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Updated: May 15, 2026
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Development of a specific real-time PCR assay targeting the poly-γ-glutamic acid synthesis gene, pgsB, for the
Xiaoyu Yong1, Ruifu Zhang, Nan Zhang
1Agricultural Ministry Key Lab of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Jiangsu Key Lab and Engineering Center for Solid Organic Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China.
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.
