期刊
INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY
卷 137, 期 2-3, 页码 168-174出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.ijfoodmicro.2009.12.004
关键词
5 '-nuclease real-time PCR; Bioinformatics; Pathogen detection; Salmonella; Target mining
资金
- National Natural Science Foundation of China [30771792]
- Science and Technology Commission of Shanghai Municipality [08391911000, 08142200700, 08DZ0504200]
- US Department of Agriculture
- Agricultural Research Service
- Eastern Regional Research Center
- Shanghai Jiao Tong University
- USDA
- ARS
- ERRC
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.
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