A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis

作者：Chen, Jing; Zhang, Lida; Paoli, George C; Shi, Chunlei; Tu, Shu I; Shi, Xianming^*

来源：International Journal of Food Microbiology, 2010, 137(2-3): 168-174.

DOI：10.1016/j.ijfoodmicro.2009.12.004

• 5 '-nuclease real-time PCR
• Bioinformatics
• Pathogen detection
• Salmonella
• Target mining

A 5'-nuclease real-time PCR assay using a minor groove binding probe was developed for the detection of Salmonella enterica from artificially contaminated foods. S. entenica-specific sequences were identified by a comparative genomic approach. Several species-specific target sequences were evaluated for specificity. A real-time PCR assay was developed targeting a nucleotide sequence within the putative type III secretion ATP synthase gene (ssaN). An internal amplification control (IAC) probe was designed by randomly shuffling the target probe sequence and a single-stranded oligonucleotide was synthesized to serve as an IAC. The assay demonstrated 100% inclusivity for the 40 Salmonella strains tested and 100% exclusivity for 24 non-Salmonella strains. The detection limit of the real-time PCR assay was 41.2 fg/PCR with Salmonella Typhimurium genomic DNA and 18.6 fg/PCR using Salmonella Enteritidis genomic DNA: 8 and 4 genome equivalents, respectively. In the presence of a natural background flora derived from chicken meat enrichment cultures. the sample preparation and PCR method were capable of detecting as few as 130 Salmonella cfu/mL. Using the developed real-time PCR method to detect Salmonella in artificially contaminated chicken, liquid egg and peanut butter samples, as few as 1 cfu/10 g of sample was detectable after a brief (6 h) non-selective culture enrichment. Published by Elsevier B.V.

• 出版日期2010-2-28
• 单位上海交通大学