Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay.
A real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The iap gene present in both species was used as a target for amplification of a 175-bp (L. monocytogenes) and a 309-bp (L. innocua) fragment. To ensure that L. monocytogenes and L. innocua are specifically detectable, tests were carried out using 42 L. monocytogenes strains and 33 L. innocua strains belonging to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bacteria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could detect as few as six copies of the iap gene per PCR using purified DNA as template. When applied to direct detection and quantification of L. monocytogenes in milk, the more rapid real-time quantitative PCR assay was as sensitive as the traditional plate count method, but real-time quantitative PCR-derived iap gene copy numbers were one to two logs higher than colony-forming units obtained by the plate count method.
Hein
I
I
Institute of Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, Austria. Ingeborg.Hein@vu-wien.ac.at
Klein
D
D
Lehner
A
A
Bubert
A
A
Brandl
E
E
Wagner
M
M
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D015169:Colony Count, Microbial; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D005798:Genes, Bacterial; D008087:Listeria / Q000235:genetics* / Q000302:isolation & purification*; D008089:Listeria monocytogenes / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
C078516:60 kDa protein, Listeria monocytogenes; D001426:Bacterial Proteins; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial