Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of haemolysin gene fragments

Recent outbreaks of listeriosis have emphasized the urgent need for rapid and reliable detection methods for Listeria spp., especially in food. Haemolysin production is a major factor in the pathogenesis of listeriosis and the polymerase chain reaction (PCR) was used to amplify two specific DNA fragments of the alpha- and the beta-haemolysin genes. The amplification system specifically recognized L. monocytogenes strains. The detection limit determined with pure cultures was 10 bacteria when estimated with alpha-haemolysin primers. In the analysis of 50 samples of cooked sausage products, bacterial colonies suspected to be Listeria spp. were isolated by conventional methods from six samples. PCR analysis identified three of six as L. monocytogenes. Subsequent serotyping showed perfect agreement with the PCR results. Since enrichment is the most time consuming step in conventional methods a PCR procedure which allows the direct detection of L. monocytogenes in milk was developed. Pasteurized milk was artificially contaminated with various levels of L. monocytogenes. The detection limit was determined to be 10 bacteria/10 ml milk and direct detection and identification of L. monocytogenes took less than two working days. These results show that this haemolysin gene amplification system is very rapid and reliable and therefore avoids cumbersome and lengthy cultivation steps.     

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.     

Direct detection of Listeria monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers.

A method for direct detection of Listeria monocytogenes in 25 ml of raw milk is presented. The detection limit can be situated between 10 and 5 CFU. The detection method is based on chemical extraction of the milk components and PCR amplification with two nested pairs of primers specific for Listeria monocytogenes.     

RAPID DETECTION OF LISTERIA MONOCYTOGENES IN GROUND TURKEY BY IMMUNOMAGNETIC SEPARATION AND POLYMERASE CHAIN REACTION

The aim of this study was to determine the prevalence of Listeria monocytogenes in packaged fresh ground turkey in Turkey using immunomagnetic separation (IMS) as a selective enrichment step in method and polymerase chain reaction (PCR). A total of 180 ground turkey samples were collected during a 1-year period. Thirty-two (17.7%) of the samples contained L. monocytogenes, 24 (13.3%) contained Listeria innocua, 7 (3.8%) had Listeria ivanovii and 5 (2.7%) had Listeria seeligeri by means of IMS-based cultivation method. A PCR assay was performed, based on hlyA gene-specific primers. In all L. monocytogenes isolates, hlyA gene was confirmed, indicating that the correlation between IMS-based cultivation and PCR methods was 100%. The results suggest that the prevalence of L. monocytogenes in ground turkey is relatively high in Turkey and that ground turkey should be produced under appropriate hygienic and technological conditions for the prevention of public health hazards.

PRACTICAL APPLICATIONS

Using fast and reliable methods to detect and identify foodborne pathogenic bacteria, including Listeria monocytogenes , is important to detect the risk of contaminated product and protect public health. In some ways it is time-consuming to isolate and identify the pathogenic microorganisms from food products using conventional techniques. Different methods or techniques can be used both for redounding the isolation chance and to gain time for this purpose. Immunomagnetic separation (IMS) and polymerase chain reaction (PCR) techniques are effective and rapid methods for separation, detection and confirmation of Listeria spp. from foods. In this study rapid, specific and sensitive IMS method was used to determine the prevalence of L. monocytogenes in fresh ground turkey and PCR technique was used for the verification of the L. monocytogenes isolates.     

Rapid detection of low levels of Listeria in foods and next-day confirmation of L. monocytogenes

Outbreaks of foodborne listeriosis caused by Listeria monocytogenes in recent years, and the high mortality rate associated with listeriosis, have raised the need for reliable and rapid detection of the pathogen. A simple, automated method was developed for the detection of Listeria organisms in foods. It consists of a 6-h pre-enrichment step followed by overnight incubation in selective broth at 35 degrees C. Changes in light transmittance in the selective broth are registered continuously by an optical sensor of the BioSys instrument (MicroSys, Ann Arbor, MI), and recorded in the computer. Esculin hydrolysis by listeriae results in black coloration of the media that causes a sharp drop in light transmittance, whereas negative samples remain colorless. Confirmation of L. monocytogenes is carried out only on esculin-positive samples and is completed within 6 h. Detection of 10-50 cells of Listeria inoculated into 25 g of food was confirmed in shell eggs, milk and ground beef. Naturally contaminated raw and ready-to-eat foods were further screened to validate the procedure.     

Detection of Listeria monocytogenes by polymerase chain reaction oriented to inlB gene.

Primers were designed for the detection of Listeria monocytogenes by the polymerase chain reaction oriented to specific sequences of the inlB gene encoding an internalin. At optimized reaction conditions, 100% sensitivity (on a panel of 33 strains of L. monocytogenes) and 100% specificity (on panels of 15 strains of other Listeria spp. and 41 other bacteria), were determined for the inlB-L/R primers. The detection limit of PCR with these primers was 10(4) cfu/ml and was not affected by up to 10(8) cfu/ml of L. innocua.     

Detection of Listeria monocytogenes in Italian-style soft cheeses

AIMS: A rapid detection system specific for Listeria monocytogenes and based on the polymerase chain reaction (PCR) was developed. METHODS AND RESULTS: Primers annealing to the coding region of the actA gene, critically involved in virulence and capable of discrimination between two different alleles naturally occurring in L. monocytogenes, have been utilized. The procedure was applied to recover L. monocytogenes cells in artificially contaminated fresh Italian soft cheeses (mozzarella, crescenza and ricotta). Low levels of L. monocytogenes were detected in mozzarella and crescenza homogenates (0.04-0.4 and 4 CFU g(-1), respectively) whereas in ricotta the detection limit was higher (40 CFU g(-1)). CONCLUSIONS: This PCR-based assay is highly specific as primers used recognize the DNA from different L. monocytogenes strains of clinical and food origin, while no amplification products result with any other Listeria spp. strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted a low-cost and rapid procedure that can be appropriated for the detection in real time of low L. monocytogenes levels in soft cheese.     

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.     

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.     

Multiplex PCR for simultaneous detection of bacteria of the genus Listeria, Listeria monocytogenes, and major serotypes and epidemic clones of L. monocytogenes

A multiplex PCR assay which combines detection of bacteria of the genus Listeria, Listeria monocytogenes serotypes 1/2a and 4b, and epidemic clones I, II, and III of L. monocytogenes was developed. The assay provides a rapid, reliable, and inexpensive method for screening and subgrouping this important food-borne pathogen.     

Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay

We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp. and the food-borne pathogen Listeria monocytogenes. The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp. and L. monocytogenes, respectively. Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L. monocytogenes and 120 Listeria spp. strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions. Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases. Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L. monocytogenes.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland.

The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% (Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% (L. monocytogenes 33.3%); this was higher than that in samples from dairy farms (Listeria spp. 8.8%; L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples (L. monocytogenes) and 1 of 33 soft cheese samples (L. seeligeri). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Detection of multiple virulence-associated genes of Listeria monocytogenes by PCR in artificially contaminated milk samples.

The inhibitory effect of milk in the PCR detection of Listeria monocytogenes could be overcome by washing the contaminated milk sample with phosphate-buffered saline and concentrating the bacteria to 1/10 of the original volume. In order to avoid a possible failure in the detection of virulent L. monocytogenes, a one-step procedure which enabled demonstration of three virulence-associated genes, prfA, hlyA, and plcB, simultaneously in a single PCR mixture was developed.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland

J. HARVEY AND A. GILMOUR. 1992. The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% ( Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% ( L. monocytogenes 33.3%); this was higher than that in samples from dairy farms ( Listeria spp. 8.8% L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples ( L. monocytogenes ) and 1 of 33 soft cheese samples ( L. seeligeri ). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

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.     

Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese

The polymerase chain reaction (PCR) amplification technique was investigated as a tool for direct detection of Listeria monocytogenes in soft cheeses. Different sets of oligonucleotide primers were used, and parts of the L. monocytogenes Dth 18-gene could be amplified specifically when either a plasmid vector carrying the cloned gene or chromosomal DNA was used a template. The detection limit for L. monocytogenes in dilutions of pure cultures was between 1 and 10 colony-forming units. In extracts from soft cheeses containing L. monocytogenes DNA, the amplification was strongly inhibited. This inhibition could be reduced by an additional purification step. Despite this the detection limit showed a large variation, depending on the brand of cheese used. In some cheeses 10(3) cfu/0.5g could be visualized whereas in others the presence of 10(8) cfu/0.5 g did not yield a detectable quantity of amplified product.     

Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples

AIMS: A rapid and sensitive method for Listeria monocytogenes direct detection from milk was developed. It is based on a magnetic capture hybridization procedure for selective DNA purification, followed by PCR identification. A comparison with two similar commercial systems from Dynal (Dynabeads) was carried out. METHODS AND RESULTS: The technique used previously developed nanoparticles modified with a 21-mer oligonucleotide. This sequence, sharing homology with all the L. monocytogenes strains, was selected on hlyA gene and located outside the desired specific PCR site to avoid cross-contaminations. Capture probe properties, in term of spacer length and purification, were determined to obtain the highest hybridization efficiency. Its specificity was tested in hybridization experiments with nontarget bacterial species. Any inhibitory effect of the nanoparticles on PCR was also examined. The amplification performed with the purified DNA could reliably identify a 10 CFU ml(-1) contamination rate. CONCLUSIONS: The optimized purification method showed a high specificity and sensitivity, with a detection level one log more sensitive than PCR carried out with nucleic acids obtained using commercial nanoparticles. SIGNIFICANCE AND IMPACT OF THE STUDY: The method, avoiding pre-enrichment, provides a rapid alternative to conventional microbiological detection methods. Furthermore, it is suitable for automation and can be proposed for the screening of a large number of samples.