Title: A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis Authors
Submitted to: International Journal of Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 3, 2009
Publication Date: February 5, 2010
Citation: Chen, J., Zhang, L., Paoli, G., Tu, S., Shi, X. 2010. A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis. International Journal of Food Microbiology. 137:168-174. Interpretive Summary: In order to reduce the incidence of food poisoning and protect consumers, food producers and regulatory agencies require rapid and effective methods to detect harmful bacteria in foods. Here we report the development of a rapid and specific method to detect Salmonella in food. The specificity of the method is based on identifying unique DNA sequences within Salmonella DNA. A novel technique was used to compare Salmonella DNA sequences with DNA sequences from other bacteria to identify DNA sequences that are unique to Salmonella. A number of Salmonella-specific DNA sequences were identified and one DNA sequence was used to develop a polymerase chain reaction (PCR) method for the rapid and specific detection of Salmonella in contaminated eggs, chicken and peanut butter. Salmonella is one of the most common bacteria associated with foodborne illness and effective methods for detection of these harmful bacteria will positively impact food safety and public health.
Technical Abstract: A 5'-nuclease real-time PCR assay using a minor groove binding probe was developed for the detection of Salmonella enterica from food. Salmonella enterica-specific target 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 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 was 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 little as 1 cfu/g of sample was detectable after a brief (6 hour) non-selective culture enrichment.