title: Response surface methodology to design a selective enrichment broth for rapid detection of Salmonella spp. by SYBR Green Ι real-time PCR.
abstract: In order to meet dominant growth of Salmonella spp. in a composed system of five pathogens for accurate detection, designing an appropriate selective enrichment broth was clearly needed. First, we built a high-throughput assay procedure based on SYBR Green Ι real-time PCR, which possessed the necessary specificity for Salmonella spp., a good linear standard curve with typical R (2) value (0.9984) and high amplification efficiency (99.0 %). Further, for the larger target biomass in the mixed microflora, acarbose, LiCl and bile salt were selected to optimize their concentrations using response surface methodology (RSM). A central composite design was employed to collect the data and fit the response. A quadratic polynomial model was derived by computer simulation. Statistical analysis was carried out to explore the action and interaction of the variables on the response. In the end, a novel broth (Sal-5) was formulated to allow the efficient enrichment of Salmonella spp. and inhibit the growth of other tested strains. A detection platform was developed, including selective enrichment in Sal-5, DNA extraction by the boiling lysis method and real-time PCR test based on SYBR Green Ι. This work could extend the application of RSM and real-time PCR in the design of other selective enrichment media for common pathogens.
authors: Zhejiang Province Key Lab for Food Safety, Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China. yanyan0014@sina.com
authors: Qiaoyan
authors: Q
authors: Zhang
authors: Tingting
authors: T
authors: Chen
authors: Shengli
authors: S
authors: Yang
authors: Xiaofu
authors: X
authors: Wang
authors: Hui
authors: H
authors: Guo
mesh_terms: D003198:Computer Simulation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000235:genetics* / Q000254:growth & development*