A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis.
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. enterica-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 (6h) non-selective culture enrichment.
Chen
Jing
J
Joint Sino-US Food Safety Research Center & Bor Luh Food Safety Center, School of Agriculture & Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
Zhang
Lida
L
Paoli
George C
GC
Shi
Chunlei
C
Tu
Shu-I
SI
Shi
Xianming
X
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D002645:Chickens; D017931:DNA Primers / Q000235:genetics*; D005516:Food Microbiology; D026901:Membrane Transport Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D019779:Salmonella enterica / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D026901:Membrane Transport Proteins