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.     

Quantitative detection of Listeria monocytogenes and Listeria innocua by real-time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology

We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R(2) values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).     

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.     

Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.

A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.     

Fate of Listeria monocytogenes during manufacture and ripening of semi-hard cheese

Semi-hard cheeses were experimentally elaborated with pasteurized milk from sheep, goat and cow (15: 35: 50) and inoculated to contain 1.9 times 10⁵ Listeria monocytogenes /ml in cheeses 1 and 2 and 4 times 10³ L. monocytogenes /ml in cheeses 3 and 4. Counts of L. monocytogenes were determined by direct surface plating of samples on listeria selective agar medium. The results show the substantial survival of L. monocytogenes present in milk during manufacture and ripening of this type of cheese.     

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.     

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.     

16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods.

A rapid polymerase chain reaction (PCR) method was developed for detection of Listeria monocytogenes in foods. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L. monocytogenes, which were previously reported by us to yield a specific nucleic acid probe. Our method included use of a shorter denaturing time, a shorter annealing time, a rapid transition, and an increase in the number of cycles, resulting in good sensitivity. Just 3 h for PCR plus 1 h for electrophoresis was required. Additional time for DNA isolation and DNA hybridization was not needed. This method detected as few as 2 to 20 CFU of L. monocytogenes in pure cultures and as few as 4 to 40 CFU of L. monocytogenes in inoculated (10(8) CFU), diluted food samples. Seven of eight foods, including four poultry products, gave positive results. Only one food sample, soft cheese, gave interference. An internal probe hybridization test was used to confirm that the PCR products were from L. monocytogenes. A specificity test indicated that this PCR method was positive for all 13 strains of L. monocytogenes tested but negative for the other 6 species of Listeria, including 6 strains of L. innocua, and negative for 17 other gram-positive and gram-negative bacteria tested.     

A technique for the direct identification of haemolytic-pathogenic listeria on selective plating media

A technique based on the addition of a red cells top layer to a selective plating medium after listeria growth is proposed in order to detect directly the haemolytic activity of pathogenic listeria colonies. It was applied to different selective plating media (modified McBride agar, lithium chloride-phenylethanol-moxalactam, listeria selective medium–Oxford formulation, polymyxin-acriflavine-lithium chloride-ceftazidime-aesculin-mannitol and LSAMM). The haemolytic activity of listeria colonies was more easily detected with the top layer than when red cells were incorporated in the selective plating medium. The LSAMM was the best medium for the recovery and identification of Listeria monocytogenes colonies by this technique (three Listeria monocytogenes colonies were distinguished among 2520 Listeria innocua colonies in raw milk).     

16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods.

A rapid polymerase chain reaction (PCR) method was developed for detection of Listeria monocytogenes in foods. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L. monocytogenes, which were previously reported by us to yield a specific nucleic acid probe. Our method included use of a shorter denaturing time, a shorter annealing time, a rapid transition, and an increase in the number of cycles, resulting in good sensitivity. Just 3 h for PCR plus 1 h for electrophoresis was required. Additional time for DNA isolation and DNA hybridization was not needed. This method detected as few as 2 to 20 CFU of L. monocytogenes in pure cultures and as few as 4 to 40 CFU of L. monocytogenes in inoculated (10(8) CFU), diluted food samples. Seven of eight foods, including four poultry products, gave positive results. Only one food sample, soft cheese, gave interference. An internal probe hybridization test was used to confirm that the PCR products were from L. monocytogenes. A specificity test indicated that this PCR method was positive for all 13 strains of L. monocytogenes tested but negative for the other 6 species of Listeria, including 6 strains of L. innocua, and negative for 17 other gram-positive and gram-negative bacteria tested.     

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.     

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.     

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.     

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 α- and the β-haemolysin genes. The amplification system specifically recognized L. monocytogenes strains. The detection limit determined with pure cultures was 10 bacteria when estimated with α-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.     

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.     

基因芯片技术检测3种肠道病原微生物方法的建立

目的:建立一种运用多重PCR和基因芯片技术检测和鉴定伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的方法。方法:分别选取伤寒沙门氏菌染色体ViaB区域中编码调控Vi抗原表达的基因(vipR)、痢疾杆菌编码侵袭质粒抗原H基因(ipaH)和单核细胞增生利斯特菌溶血素基因(hlyA)设计引物和探针,探针3'端进行氨基修饰,下游引物标记荧光素Cy3。在优化的PCR和杂交反应条件下,进行三重PCR扩增,产物与包括3种致病菌特异性探针的基因芯片杂交。在评价基因芯片的特异性和灵敏度之后,对临床样本进行检测。结果:只有3种目的致病菌的PCR产物在相应探针位置出现特异性信号,其他阴性细菌均无信号出现;3种致病菌的检测灵敏度均可达到103CFU/mL;检测30例临床样本的结果与常规细菌学培养结果一致。结论:所建立的可同时检测伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的基因芯片方法快速、准确,特异性高,重复性好,为3种肠道致病菌的快速检测和鉴定提供了新方法和新思路。     

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.     

The rapid demonstration and presumptive identification of Listeria monocytogenes in food using monoclonal antibodies in a direct immunofluorescence test (DIFT)

Two monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC) were successfully used in a direct immunofluorescence test (DIFT) to demonstrate listeria in seven samples of soft cheese where Listeria monocytogenes had been cultured by conventional techniques. Using DIFT, listeria was not detected in 20 cheese samples from which L. monocytogenes had not been isolated, or was present in low numbers (< 10²/g). The DIFT was also used to presumptively identify > 90% of strains of L. monocytogenes isolated from food and cultured on Modified McBride agar or Blood agar. Less than 10% of strains of other species of listeria would be misidentified when grown on these media. All tests were carried out within 2 h.     

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.     

DNA sequence heterogeneity in the gene encoding a 60-kilodalton extracellular protein of Listeria monocytogenes and other Listeria species.

Chromosomal DNA sequences from the 60 kilodalton protein gene of Listeria monocytogenes, amplified by the polymerase chain reaction, were used for restriction fragment length polymorphism differentiation of L. monocytogenes serotypes and other Listeria species. All 24 strains of L. monocytogenes examined produced an extracellular protein of molecular weight 60,000 (p60) as determined by Western blot analysis. Four of six other Listeria species had a protein that cross-reacted to antibodies to p60, but all differed in molecular weight, ranging from approximately 50,000 to 65,000. The gene encoding p60 was amplified from chromosomal DNA in all strains using polymerase chain reaction with a single primer pair. Restriction enzyme digestion with HindIII of the amplified product revealed a restriction pattern that was distinct between serotypes 1/2a and either 4b or 1/2b of L. monocytogenes. Of the other Listeria species, four strains that produced a cross-reacting protein likewise produced a polymerase chain reaction amplification product with the primer pair. Listeria innocua alone had a restriction pattern similar to that of Listeria monocytogenes serotype 4b and 1/2b. Genotypic heterogeneity, as revealed by DNA amplification and restriction endonuclease digestion of the p60 open reading frame, correlates with "electrophoretic type" grouping and may be related to differences in virulence mechanisms of Listeria monocytogenes and other Listeria species.