PCR Detection Of Listeria Monocytogenes Research Articles

Mining of novel species-specific primers for PCR detection of Listeria monocytogenes based on genomic approach.

Listeria monocytogenes in contaminated food is considered as a serious health threat for consumers due to its high mortality rate. The objective of this study was to obtain novel species-specific target-genes and primers for the molecular detection of L. monocytogenes using a comparative genomic approach. By comparative analysis of L. monocytogenes and non-L. monocytogenes genome sequences in the GenBank database with BLAST program, 26 specific target sequences were used as candidates and the primers were designed for L. monocytogenes species-specificity verification by using PCR assay. Finally, the three genes LMOf2365_0970, LMOf2365_2721 and mpl were identified to have L. monocytogenes species-specificity and be unique as detection targets for diagnostic application. The species-specific primer Lm8 of gene LMOf2365_0970, Lm13 of gene LMOf2365_2721 and Lm20 of gene mpl showed better specificity and sensitivity than the primers described previously. The PCR detection limits of the three specific primer sets were 430, 43, 4.3 fg/μL for genomic DNA, and 5 × 10(3), 50, 5 cfu/mL for pure culture of L. monocytogenes. There was no interference in specificity of detecting L. monocytogenes by co-culture with other foodborne pathogens in high concentration. Moreover, after 6-8 h of enrichment, L. monocytogenes in the artificially contaminated milk samples at an inoculum dose of 38 cfu/10 mL milk could be detected successfully with the studied three primers. Therefore, the three specific genes and primers can be applied to establish a novel rapid and accurate method for detecting L. monocytogenes in food materials.

Taq Man Real Time PCR detection of Listeria monocytogenes: A study of enrichment incubation time affecting sensitivity in experimental dry fermented sausages

Listeria monocytogenes is a facultative intracellular Gram positive, catalase positive bacterium, ubiquitous in nature and capable of causing listeriosis in humans and animals. Conventional microbiological techniques and modern molecular approaches are currently used for the isolation and detection of Listeria monocytogenes in food samples. The aim of this study was to investigate the influence of enrichment incubation time on the sensitivity of Taq Man Real Time PCR method. For that purpose, dry fermented sausages were artificially inoculated with serial dilutions of L. monocytogenes ATCC 19111. The obtained results indicated that incubation time is an important factor affecting the sensitivity of Real-Time PCR detection. The best results were obtained after 24 h of pre-enrichment, with primers and probe complementary to the listeriolysin (hlyA) gene, when it was possible to detect less than 10 CFU/g of L. monocytogenes.

Nonaplex real-time PCR detection of Listeria monocytogenes, Campylobacter, Salmonella and enteropathogene E. coli after universal enrichment in food samples

A universal cultivation media for Listeria monocytogenes, Salmonella and enteropathogenic Escherichia coli in conjunction with a highly efficient nonaplex PCR for the determination of L. monocytogenes, Campylobacter, Salmonella and enteropathogenic E. coli was developed and compared to classical microbiological assays. The achieved detection limit was at 2 colony-forming units per g for all analytes. The method allows screening of food samples within 24 h and is partly automatable. A ring trial showed that it can be easily established in other laboratories showing its robustness. The presented method yields results similar to the classical ISO methods based on cultivation.

Comparison of Four Commercial DNA Extraction Kits for PCR Detection of Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, and Staphylococcus aureus in Fresh, Minimally Processed Vegetables

Four commercial DNA extraction methods, PrepMan Ultra (Applied Biosystems), InstaGene Matrix (BioRad), DNeasy Tissue kit (Qiagen), and UltraClean (MoBio), were tested for PCR detection of Listeria monocytogenes, Escherichia coli O157: H7, Salmonella, and Staphylococcus aureus in fresh, minimally processed vegetables. For comparative purposes, sensitivity assays with specific PCRs were carried out after DNA extraction with the four methods in green pepper, broccoli, and onion artificially inoculated with the four pathogens separately. As confirmed by statistical analysis, the DNeasy Tissue kit rendered the highest sensitivity values in the three matrices assayed for Salmonella, L. monocytogenes, and E. coli O157:H7 and in onion for S. aureus. Despite being the most expensive of the methods compared, the DNeasy Tissue Kit can be successfully applied for any of the four most commonly studied pathogens, thus saving time and overall reducing the cost of the analysis.

Immunomagnetic separation and PCR detection of Listeria monocytogenes in turkey meat and antibiotic resistance of the isolates

1. Conventional cultivation and immunomagnetic separation (IMS) cultivation methods were compared for the isolation specificity and sensitivity of L. monocytogenes from turkey meat samples. PCR was used to confirm the isolates. Disc diffusion was performed to determine the antibiotic susceptibility profiles. A total of 180 turkey meat samples collected from markets in Turkey were tested. 2. L. monocytogenes was detected in 23 samples (12·7%) by IMS and conventional cultivation. It was isolated from 16·6% (10/60), 11·6% (7/60) and 10·0% (6/60) of the meat cut, breast and leg samples, respectively. PCR assay was performed based on hlyA (LLO-listeriolysin O) gene specific primers. In all 23 (100·0%) isolates of the hlyA gene were determined. The disc diffusion test showed that 19 (82·6%) isolates were resistant to penicillin G and 17 (73·9%) to ampicillin. In addition, 8 isolates were partially resistant to erythromycin and 8 to streptomycin. 3. In conclusion, to safeguard public health turkey meat must be produced under hygienic and suitable technological conditions. Furthermore antimicrobials, as prophylactic or growth promoter agents, must be firmly controlled by governmental agencies.

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1–10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1–5 CFU L. monocytogenes per 25 g/ml of food sample in 30 h. The performance of the assay was compared with the Roche Diagnostics ‘LightCycler foodproof Listeria monocytogenes Detection Kit’. Both methods detected L. monocytogenes in all artificially contaminated retail samples ( n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens.

PCR Detection of Listeria monocytogenes in different Food Products compared with the mini-VIDAS LMO System and the Standard Procedure ISO 11290-1

The presence of Listeria monocytogenes in 225 natural samples, including different food types, was investigated by three methods: (i) culture-based standard procedure ISO 11290-1, (ii) mini-VIDAS (Vitek Immuno Diagnostic Assay System) LMO, an enzyme linked fluorescent assay (ELFA) commercially available, and (iii) PCR using a previously established procedure. Identification of isolates recovered from the standard method and mini- VIDAS, on Oxford and PALCAM selective plates, was carried out using the API-Lis system and also by PCR, using L. monocytogenes specific primers. In all, 65 samples (64 meat products and one smoked salmon) were positive with any of the three procedures assayed. Taking into account the results based on PCR identification, the standard culture-based method found 22 and the mini- VIDAS 23, while PCR detected 60 positive samples in a total of 225 food samples. For comparative purposes, a set of “known positive samples” was established as reference that included 33 natural samples from which L. monocytogenes isolates (identified by specific PCR) had been recovered. The mini-VIDAS and ISO 11290- 1 methods were equally sensitive. Compared to them PCR was clearly the more accurate and efficient procedure for detection of L. monocytogenes in food. Moreover, it showed no false negative results. The PCR approach can be completed in 48 working hours, and because of its specificity might eventually be cheaper than the other two procedures. Consequently we recommend PCR for routine detection of L. monocytogenes in food.

Upstream sample processing facilitates PCR detection of Listeria monocytogenes in mayonnaise-based ready-to-eat (RTE) salads

Sample pretreatment to reduce volume and concentrate cells of the target organism(s) prior to molecular detection offers a useful supplement or alternative to cultural enrichment. The purpose of this study was to develop an upstream processing method to facilitate the detection of Listeria monocytogenes in ready-to-eat (RTE) salads by PCR. Potato salad, a model RTE commodity, was seeded with L. monocytogenes and processed by two alternative upstream sample processing methods (designated one-step and two-step centrifugation), followed by DNA extraction, PCR amplification, and Southern hybridization. The two-step method resulted in 1000-fold improvements in the PCR detection limit, from 10 6 Cfu/g (no sample processing) to 10 3 Cfu/g. The two-step method was applied for upstream sample processing of four representative deli salad items artificially inoculated with L. monocytogenes at levels ranging from 10 1–10 6 Cfu/g. Following DNA extraction, PCR amplification, and Southern hybridization, detection was achieved at input levels of 10 5 Cfu/g for chicken salad, 10 4 Cfu/g for macaroni salad, and 10 3 Cfu/g for potato and seafood salads. The two-step method reported here facilitates the production of a final sample concentrate of reduced volume and improved purity which was compatible with PCR amplification. This approach offers further progress in our efforts to reduce or eliminate cultural enrichment in an effort to speed time to results when applying molecular methods to the detection of pathogens in foods.

SIMPLE oPSU BROTH‐BAX® PCR‐PICOGREEN® SYSTEM FOR RAPID DETECTION OF LISTERIA MONOCYTOGENES IN PASTEURIZED MILK AND HOT DOGS

ABSTRACTFactors that significantly affect BAX® PCR detection of Listeria monocytogenes from optimized Penn State University (oPSU) broth were identified and optimized. Concentration of PCR product was significantly reduced by BAX™ protease and significantly increased by eliminating the lysis step and directly diluting oPSU broth prior to PCR. A simple oPSU broth‐BAX® PCR‐PicoGreen® (PSU‐BAX‐PicoGreen) system was developed and compared with current methods for detecting low levels of L. monocytogenes in commercial milk and hot dogs. All 30 milk samples inoculated with 10–20 CFU per mL L. monocytogenes were positive by FDA, BAX® and PSU‐BAX‐PicoGreen methods and all 42 uninoculated milk samples were negative by all of the above methods. All 30 hot dog samples inoculated with 10‐20 CFU/g L. monocytogenes were positive by the USDA and PSU‐BAX‐PicoGreen methods, however, 2 hot dog samples gave indeterminate results with the standard BAX® method. All 42 uninoculated hot dog samples were negative by USDA, 9 were indeterminate by BAX® and 2 were positive by PSU‐BAX‐PicoGreen. The PSU‐BAX‐PicoGreen system may provide a simple and accurate method for rapidly screening pasteurized foods for both injured and noninjured L. monocytogenes.

PCR detection of Listeria monocytogenes: a study of multiple factors affecting sensitivity.

To test, under comparable conditions, several parameters affecting sensitivity of PCR detection in order to establish a PCR procedure suitable for the routine detection of Listeria monocytogenes in food. Beef samples artificially inoculated were used to determine sensitivity of PCR detection under different parameters. As few as 1 CFU g(-1) were detected by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) of 1 ml aliquot and PCR amplification with primers directed to the hlyA gene. This PCR protocol was applied in 60 naturally contaminated foods, comparing two enrichment procedures with the traditional culture method. The highest number of positives was recorded by PCR following a 24-h pre-enrichment step at 30 degrees C and a 24-h enrichment step at 37 degrees C. Afterwards, it was applied in 217 naturally contaminated foods and 56 of them tested positive for L. monocytogenes in which only 17 tested positive using the culture method. The PCR procedure described has proved to be a rapid and sensitive method suitable for the routine analysis of different types of food. The method proposed for the detection of L. monocytogenes, has been validated in naturally contaminated food and is suitable to implement in the food industry.

PCR Detection of Listeria monocytogenes in Milk and Milk Products and Differentiation of Suspect Isolates

Holko I., J. Urbanova, M. Kantikova, K. Pastorova, V. KmeE: PCR Detection of Listeria monocytogenes in Milk and Milk Products and Differentiation of Suspect Isolates. Acta Vet. Brno 71, 2002: 125-131. The main goal of our work was to solve the problems of normative diagnostic method for the detection of Listeria monocytogenes in foods by STN ISO 10560 (1999), i.e. to shorten the long time of analysis (5-10 days) and differentiate the non-haemolysed strains of Listeria monocytogenes from non-pathogenic Listeria innocua yielding the same results of biochemical and serological typing (serotype 4ab). PCR was employed to confirm the method for testing 60 suspect isolates from other laboratories of the Slovak Republic. We verified the detection of Listeria monocytogenes in milk and dairy products by nested PCR. The objective of this experiment was to analyze 100 various samples by traditional cultivation method and compare the results with those yielded by PCR. The results were in good agreement; all 18 positive and 72 negative samples were detected by both methods. However, the PCR method yields results within 2 days. This makes diagnosis in food control laboratories much more efficient. For PCR detection, two pairs of primers (PRFA 1 and 2, LIP 1 and 2) were used with affinity to prfA gene involved in the regulation of listeriolysin synthesis. The size of the PCR product was 1060 bp fragment in a first step of PCR and 273 bp fragment in nested PCR. The sensitivity and reliability of PCR was comparable with conventional methods. The PCR method solved the problem of interpretation of classical biochemical and serological typing in the only one step without the necessity of using additional examinations. Furthermore, we found that in most of the strains isolated from foods with a biochemical profile of non-haemolytic Listeria monocytogenes on blood agar serotype 4ab were not Listeria monocytogenes but a non-pathogenic Listeria innocua. This finding is very important from the point of view of food evaluation according to the STN ISO 10560 (1999). Diagnostic methods, food, Listeria innocua, non-haemolysed strains of Listeria monocytogenes, prfA gene Bacteria of the genus Listeria are widely distributed in the environment and they frequently contaminate food. Listeria monocytogenes is a pathogenic species causing listeriosis, increasing incidence of which has been reported in the last two decades. Food products most frequently associated with listeriosis are soft cheeses, particularly those made from unpasteurized milk, and ready-to-eat meat-containing food products (Farber and

On the Specificity of PCR Detection of Listeria monocytogenes in Food: a Comparison of Published Primers

A total of nine pairs of primers, seven previously published and two newly developed, have been assayed for PCR detection of Listeria monocytogenes in food. They have been tested for specificity on a total of 72 strains including reference and food isolates belonging to L. monocytogenes and other species in the genus. First of all, a polyphasic approach has been carried out in order to establish a reference strain collection. They were biochemically and genetically characterized by API-Lis and randomly amplified polymorphic DNA PCR (RAPD-PCR), respectively. Random amplification of DNA was performed with M13, T7 and T3 universal primers and a data bank was created to compile the RAPD patterns of all the analyzed strains. The UPGMA cluster analysis of RAPD profiles with primer M13 showed eight clusters at 72.3% similarity. Clusters 2 and 7 corresponded to L. monocytogenes. Clusters 1 and 6 grouped L. ivanovii strains. Clusters 3, 4, 5 and 8 corresponded to L. grayi, L. innocua, L. welshimeri and L. seeligeri, respectively. Pattern analysis revealed the existence of miss-identified reference strains which was confirmed by 16S rDNA sequence analysis. RAPD-PCR is a rapid genetic test which helped to confirm strain identity. On the basis of PCR specificity results, primers LM1-LM2 were the best combination for the detection of L. monocytogenes since they only amplified the specific fragment in strains that had been genetically and biochemically assessed as belonging to the species. Specificity of other assayed primers is discussed.

Enhanced sensitivity in PCR detection of Listeria monocytogenes in soft cheese through use of an aqueous two-phase system as a sample preparation method.

A sample treatment method based on an aqueous two-phase system containing polyethylene glycol and dextran was developed for enhancing sensitivity in the detection of Listeria monocytogenes in soft cheese with PCR. The results suggest that the improved detection sensitivity following partitioning of the cheese homogenate in an aqueous two-phase system may be due to partitioning of the PCR inhibitors to the polyethylene glycol phase.

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.