Nucleic acid-based, cultivation-independent detection of Listeria spp and genotypes of L monocytogenes.
Based on comparative analysis of 16S rRNA gene sequences, two oligonucleotide probes for in situ detection of all members of the genus Listeria were designed. These probes allowed fast and reliable in situ detection of Listeria spp. even in complex samples like raw milk. Almost full-length iap (invasion-associated protein) gene sequences were determined for 69 Listeria monocytogenes strains of all 13 known serotypes. A comparison of these sequences revealed that the L. monocytogenes strains can be grouped into three distinct genotypes. These clusters correlate well with distinct serotypes. Thus, strains of serotypes b and d belong to genotype I, a and c to genotype II, and 4a and 4c, which are rarely isolated from humans, group together within genotype III. These results could be corroborated by further comparative sequence analysis of genes encoding two phospholipases - plcA and plcB. Based on the iap gene sequences, a highly specific and reproducible competitive PCR detection method was developed. Primer pairs targeting genotype-specific regions of the iap gene were designed. The amplification of non-specific PCR products from DNA of non-target strains was prevented by adding competitive primers. By applying this method, the rapid and reliable distinction of the three L. monocytogenes genotypes was possible.
Schmid
Michael
M
Lehrstuhl für Mikrobiologie, Technische Universität München, Am Hochanger 4, 85354 Freising, Germany. michael.schmid@gsf.de
Walcher
Marion
M
Bubert
Andreas
A
Wagner
Martin
M
Wagner
Michael
M
Schleifer
Karl-Heinz
KH
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002417:Cattle; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005506:Food Contamination; D005516:Food Microbiology; D005798:Genes, Bacterial; D005838:Genotype; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008087:Listeria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D008089:Listeria monocytogenes / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D008245:Lysophospholipase / Q000235:genetics; D008892:Milk / Q000382:microbiology; D015345:Oligonucleotide Probes; D010741:Phospholipases A / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D021521:Ribotyping / Q000379:methods*; D012703:Serotyping; D014774:Virulence
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S; D010741:Phospholipases A; D008245:Lysophospholipase