Real-time multiplex SYBR green I-based PCR assay for simultaneous detection of Salmonella serovars and Listeria monocytogenes.
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.
Jothikumar
Narayanan
N
Canadian Research Institute for Food Safety, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Wang
Xiaowen
X
Griffiths
Mansel W
MW
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D005784:Gene Amplification; D008089:Listeria monocytogenes / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D008892:Milk / Q000382:microbiology; D008970:Molecular Weight; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D014871:Water Microbiology
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial