RAPD typing for distinguishing species and strains in the genus Listeria

The randomly amplified polymorphic DNA (RAPD) technique was employed in the development of a typing protocol for Listeria isolates, particularly Listeria monocytogenes strains. A single strain of L. monocytogenes was used and 200 random decamer primers were screened for their discriminatory abilities by visualizing the amplification products electrophoretically. Three candidate primers displaying potentially useful banding patterns were selected and tested against 52 L. monocytogenes strains, encompassing 11 serotypes, and 12 other strains representing five other Listeria spp. Thirty-four banding profiles were obtained with one particular primer. RAPD analysis allowed differentiation between Listeria spp. and was found to further subdivide strains of the same serotype. Where only one primer was used strains from different serotypes were occasionally found to produce identical banding profiles. RAPD analysis, which in our hands proved to be reproducible, shows much promise as a molecular alternative to traditional L. monocytogenes typing protocols.     

Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction.

A ligase chain reaction assay based on a single-base-pair difference in the V9 region of the 16S rRNA gene (16S rDNA) was developed to distinguish between Listeria monocytogenes and other Listeria species. For this purpose, two pairs of primers were designed, with one primer of each pair being radioactively labeled. The ligated product was separated from the primers by denaturing polyacrylamide gel electrophoresis and then detected by autoradiography. To achieve a higher sensitivity, the 16S rDNA was initially amplified by polymerase chain reaction prior to the ligase chain reaction. The ligase chain reaction was tested on 19 different Listeria species and strains and proved to be a highly specific diagnostic method for the detection of L. monocytogenes.     

Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction.

A ligase chain reaction assay based on a single-base-pair difference in the V9 region of the 16S rRNA gene (16S rDNA) was developed to distinguish between Listeria monocytogenes and other Listeria species. For this purpose, two pairs of primers were designed, with one primer of each pair being radioactively labeled. The ligated product was separated from the primers by denaturing polyacrylamide gel electrophoresis and then detected by autoradiography. To achieve a higher sensitivity, the 16S rDNA was initially amplified by polymerase chain reaction prior to the ligase chain reaction. The ligase chain reaction was tested on 19 different Listeria species and strains and proved to be a highly specific diagnostic method for the detection of L. monocytogenes.     

Suitability of the prfA gene, which encodes a regulator of virulence genes in Listeria monocytogenes, in the identification of pathogenic Listeria spp.

The pathogenesis of listerial infections is complex and involves a number of virulence factors expressed by virulent Listeria species. We have recently described a regulator gene, prfA, that positively regulates the expression of a number of virulence factors in Listeria monocytogenes. When the prfA gene was used as a DNA probe, we found it to be extremely specific for the pathogenic species L. monocytogenes. No reaction was obtained with strains of all other species of this genus. By using this information, an oligonucleotide primer pair was developed that specifically amplifies the prfA gene in L. monocytogenes strains of all known serotypes.     

Suitability of the prfA gene, which encodes a regulator of virulence genes in Listeria monocytogenes, in the identification of pathogenic Listeria spp.

The pathogenesis of listerial infections is complex and involves a number of virulence factors expressed by virulent Listeria species. We have recently described a regulator gene, prfA, that positively regulates the expression of a number of virulence factors in Listeria monocytogenes. When the prfA gene was used as a DNA probe, we found it to be extremely specific for the pathogenic species L. monocytogenes. No reaction was obtained with strains of all other species of this genus. By using this information, an oligonucleotide primer pair was developed that specifically amplifies the prfA gene in L. monocytogenes strains of all known serotypes.     

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms.

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.     

Typing of food-borne Listeria monocytogenes by the optimized repetitive extragenic palindrome-based polymerase chain reaction

The repetitive extragenic palindrome-based polymerase chain reaction was optimized for typing Listeria monocytogenes by 1) using the QlAamp method to increase the reproducibility of DNA isolation, 2) running PCR with three different DNA concentrations in parallel, 3) using antibody-protected therrnostable DNA polymerase to reduce non-specific priming, and 4) using an improved temperature programme to increase the amplification yield. When applied to 42 L. monocytogenes strains isolated from food in the Czech and Slovak republics during 1999-2000, profiles of 7-15 DNA fragments of 330-3,310 bp were amplified. Based on REP-profiles, strains (serotypes 1/2 and 4) could be divided into 12 groups.     

Synthetic peptides derived from the Listeria monocytogenes p60 protein as antigens for the generation of polyclonal antibodies specific for secreted cell-free L. monocytogenes p60 proteins.

All species of the genus Listeria secrete a major extracellular protein called p60. A comparison of the deduced amino acid sequences of all listerial p60 proteins previously indicated there were only a few regions which were unique to the pathogenic, food-borne species Listeria monocytogenes. Two of these p60 regions were chosen for the development of antibodies specific for the facultative intracellular species L. monocytogenes. Initially, these regions were characterized via epitope mapping, and this led to the development of two different synthetic peptides. Rabbits immunized with these synthetic peptides generated polyclonal antibodies that were then used in Western blot (immunoblot) analyses. Antiserum against peptide A (PepA) recognized the p60 protein in the supernatants collected from most L. monocytogenes serotypes except for several strains belonging to serotypes 4a and 4c. No p60-related protein was detected in the supernatants from other Listeria species with this anti-PepA antiserum. Antibodies raised against peptide D (PepD) reacted with p60 from all L. monocytogenes serotypes, including all 4a and 4c strains that were tested, and also showed no cross-reactivity with supernatant proteins from other Listeria species. Both antisera also detected p60 in supernatants of a large number of environmental isolates of L. monocytogenes. Besides Western blot analyses, these antisera to PepA and PepD reacted with secreted p60 in an enzyme-linked immunosorbent assay, indicating recognition of the native antigen in addition to the denatured form. These data suggest that synthetic peptides derived from the variable region of the L. monocytogenes p60 protein may be useful for the development of an immunological diagnostic assay.     

Rapid RAPD analysis for distinguishing Listeria species and Listeria monocytogenes serotypes using a capillary air thermal cycler

Using a novel capillary thermal cycler, randomly amplified polymorphic DNA (RAPD) generated DNA fingerprints were obtained in 3 h. The RAPD profiles were produced using a random 10-mer primer (5'-ACCGCCTGCT-3') which discriminated between different Listeria spp. Unique fragment profiles of Listeria monocytogenes serotypes were produced from serotypes 1a, 2, 3a, 4ab, 4a and 4c but serotypes, 1/2a, 4b, 4d and 7 had similar profiles.     

Detection of Listeria monocytogenes by using the polymerase chain reaction.

A method was developed for detection of Listeria monocytogenes by polymerase chain reaction amplification followed by agarose gel electrophoresis or dot blot analysis with a 32P-labeled internal probe. The technique identified 95 of 95 L. monocytogenes strains, 0 of 12 Listeria strains of other species, and 0 of 12 non-Listeria strains.     

Use of PCR methods for identification of Listeria monocytogenes in milk

The aim of this work was to estimate the limit of Listeria monocytogenes cfu in polymerase chain reaction (PCR) for a DNA fragment of listeriolysine O (hly A) gene. The PCR method, with used primers selected in areas of the listeriolysin O gene, allows to differentiate L. monocytogenes strains from other Listeria species. The amplified fragment (456 bp) of hly A gene was obtained for all strains L. monocytogenes and no other Listeria species. The PCR method with the selected primers allowed to detect 50-500 cfu L. monocytogenes/ml suspended in water or milk. Among 20 samples of raw milk from cows, 10 samples contained > 50 cfu L. monocytogenes/ml. Obtained results indicate that the PCR assay of L. monocytogenes identification is technically simple and may be conduct with minimal time. So, it could be recommended as quick diagnostic method in identification L. monocytogenes in milk.     

The iap gene of Listeria monocytogenes is essential for cell viability, and its gene product, p60, has bacteriolytic activity.

Expression of the iap gene of Listeria monocytogenes in the L. monocytogenes rough mutant RIII and in Bacillus subtilis DB104 caused the disruption of the cell chains which these two strains normally form under exponential growth conditions. The p60 protein produced by L. monocytogenes and B. subtilis DB104 also exhibited bacteriolytic activity detected in denaturing polyacrylamide gels containing heat-killed Micrococcus lysodeikticus. Purification of the p60 protein led to aggregation of p60 and loss of the cell chain disruption and bacteriolytic activities. A cysteine residue in the C-terminal part of p60 which is conserved in all p60-like proteins from the other Listeria species seems to be essential for both activities. The iap gene could not be inactivated without a loss of cell viability, indicating that p60 is an essential housekeeping protein for L. monocytogenes and probably also for other Listeria species. These data suggest that p60 possesses a murein hydrolase activity required for a late step in cell division.     

An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates

Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (> 0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.     

Species-specific detection of Listeria monocytogenes by DNA amplification

The polymerase chain reaction was used to detect and specifically identify Listeria monocytogenes. A 174-bp region of the listeriolysin O gene was shown to be specifically amplified in L. monocytogenes but not in other species of Listeria or in a number of other gram-positive and gram-negative organisms. Less than 50 organisms could routinely be detected by a procedure involving two rounds of 35 amplification cycles each and without the need for subsequent hybridization with labeled probes.     

Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR.

The iap gene encodes the protein p60, which is common to all Listeria species. A previous comparison of the DNA sequences indicated conserved and species-specific gene portions. Based on these comparisons, a combination consisting of only five different primers that allows the specific detection and differentiation of Listeria species with a single multiplex PCR and subsequent gel analysis was selected. One primer was derived from the conserved 3' end and is specific for all Listeria species; the other four primers are specific for Listeria monocytogenes, L. innocua, L. grayi, or the three grouped species L. ivanovii, L. seeligeri, and L. welshimeri, respectively. The PCR method, which also enables the simultaneous detection of L. monocytogenes and L. innocua, was evaluated against conventional biotyping with 200 food hygiene-relevant Listeria strains. The results indicated the superiority of this technique. Thus, this novel type of multiplex PCR may be useful for rapid Listeria species confirmation and for identification of Listeria species for strains isolated from different sources.     

Species-specific detection of Listeria monocytogenes by DNA amplification.

The polymerase chain reaction was used to detect and specifically identify Listeria monocytogenes. A 174-bp region of the listeriolysin O gene was shown to be specifically amplified in L. monocytogenes but not in other species of Listeria or in a number of other gram-positive and gram-negative organisms. Less than 50 organisms could routinely be detected by a procedure involving two rounds of 35 amplification cycles each and without the need for subsequent hybridization with labeled probes.     

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.     

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.     

Rapid detection of Listeria monocytogenes in ham samples using immunomagnetic separation followed by polymerase chain reaction

AIMS: To develop a 24-h system for the detection of Listeria monocytogenes in ham. METHODS AND RESULTS: An immunomagnetic separation (IMS) of bacteria directly from ham followed by extraction of DNA and detection using a new multiplex polymerase chain reaction (PCR) was used. The PCR method used one primer pair targeted at the listeriolysin O gene of L. monocytogenes and the other pair for a region of the 23S rRNA genes of Listeria, giving products of 706 and 239 bp, respectively. The combined IMS/PCR was calculated to be capable of detecting as few as 1.1 L. monocytogenes cells g-1 in a 25-g ham sample. CONCLUSION: The process produced acceptable results, but the IMS step is the main barrier to further improvement of sensitivity. The DNA isolation was the most time-consuming step in the process. SIGNIFICANCE AND IMPACT OF THE STUDY: A 24-h test for the presence of L. monocytogenes will be useful to the food industry and significantly assist in the timely investigation of outbreaks.     

Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods

A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.