Real-time multiplex SYBR green I-based PCR assay for simultaneous detection of Salmonella serovars and Listeria monocytogenes

• Published in: Journal of food protection 2003-11, Vol.66 (11), p.2141-2145
• Main Authors: Jothikumar, N, Wang, X.W, Griffiths, M.W
• Format: Article
• Language: English
• Subjects: Animals; bacterial contamination; Bacterial Proteins - genetics; Base Sequence; Biological and medical sciences; DNA Primers; DNA, Bacterial - analysis; food contamination; Food industries; Food microbiology; food pathogens; Fundamental and applied biological sciences. Psychology; Gene Amplification; Listeria monocytogenes; Listeria monocytogenes - classification; Listeria monocytogenes - genetics; Listeria monocytogenes - isolation & purification; microbial detection; Milk - microbiology; Milk and cheese industries. Ice creams; Molecular Weight; Phylogeny; polymerase chain reaction; Polymerase Chain Reaction - methods; rapid methods; real-time multiplex SYBR green I-based polymerase chain reaction; Salmonella; Salmonella - classification; Salmonella - genetics; Salmonella - isolation & purification; Sensitivity and Specificity; serotypes; Temperature; Time Factors; Water Microbiology
• ISSN: 0362-028X; 1944-9097
• DOI: 10.4315/0362-028x-66.11.2141

A multiplex SYBR Green I-based PCR assay has been developed for simultaneous detection of Salmonella serovars and Listeria monocytogenes using a LightCycler. Primers were designed to amplify an 85-bp sequence from the gene encoding a fimbrinlike protein (fimI) of Salmonella Enteritidis and a 98-bp sequence from the hemolysin gene (hly) of L. monocytogenes. These primers allowed the amplification of PCR products having distinct melting temperature values, resulting in the formation of two distinct peaks representing the two targets. Background signals, resulting from primer-dimer formation in the late cycles of PCR, are eliminated through the acquisition of data at a high temperature (>75 degrees C), but several degrees lower than required for detection of the specific PCR products. A rapid and simple method for the extraction of bacterial genomic DNA from liquid culture, coupled with duplex PCR using LightCycler SYBR Green-based PCR assays, detected the presence of 2.5 cells and 1 cell of Salmonella serovars and L. monocytogenes, respectively, within an hour. Following overnight enrichment, target DNA was present in sufficient quantities in 1 microl of culture to enable direct detection with the LightCycler.