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- Title
Response surface methodology to design a selective enrichment broth for rapid detection of Salmonella spp. by SYBR Green Ι real-time PCR.
- Authors
Zhang, Qiaoyan; Chen, Tingting; Yang, Shengli; Wang, Xiaofu; Guo, Hui
- 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 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.
- Subjects
SALMONELLA; PATHOGENIC microorganisms; ACARBOSE; BILE salts; RESPONSE surfaces (Statistics); QUANTITATIVE research
- Publication
Applied Microbiology & Biotechnology, 2013, Vol 97, Issue 9, p4149
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-013-4780-6