Method For Detection Of Listeria Research Articles

Prevalence of Listeria Species and Listeria monocytogenes on Raw Produce Arriving at Frozen Food Manufacturing Facilities

The ubiquity of Listeria monocytogenes in the environment affects the food industry and presents concerns for frozen food facilities. This study determined the prevalence and numbers of Listeria species and L. monocytogenes on raw produce arriving at frozen food facilities. Raw produce was collected using multilevel blinding protocols to ensure anonymity of participants and avoid traceback. Five raw vegetables were selected: corn, carrots, green beans, peas, and spinach. Raw products were collected after arrival at the facilities but before cleaning or other preprocessing steps that are typically performed inside the facility. The U.S. Food and Drug Administration's Bacteriological Analytical Manual method for detection of Listeria spp. and L. monocytogenes was followed, with PCR screening followed by selective plating methods. Listeria numbers were estimated from positive samples using the most-probable-number (MPN) methodology. A total of 290 samples were collected, with 96 and 17 samples positive for Listeria spp. (33.1%) and L. monocytogenes (5.9%), respectively. Enumeration data for the 96 Listeria spp. samples indicated 82 samples had greater than 100 MPN of Listeria spp. per g and 14 samples had less than 100 MPN Listeria spp. per g. The prevalence of Listeria spp. varied by commodity: spinach (66.7%), peas (50%), corn (32.2%), green beans (22.2%), and carrots (13%). L. monocytogenes prevalence was determined in corn (13.6%), peas (6.3%), and green beans (4.2%) arriving at processing facilities. Such data were previously unavailable to frozen vegetable processors and are valuable in implementing process control standards. The prevalence and pathogen concentration data from raw commodities found in this study can provide the industry with information to conduct more accurate quantitative risk assessments and a baseline to model and target appropriate pathogen reduction steps during processing.

Application of an Environmental Phage-Based Assay (Sample6 Detect HT/L) for the Detection of Listeria spp. in Ice Cream.

Background: Dairy products are common sources of Listeria outbreaks, and early detection of the pathogen is critical to prevent outbreaks of illnesses and financial losses for dairy producers. Objective: This study aimed to evaluate Sample6 Detect HT/L for effective detection of Listeria monocytogenes and L. innocua in ice cream. Methods: Performance of the Sample6 DETECT HT/L was compared with U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 method for detection of Listeria spp. in ice cream using an unpaired study design. Results: R2-enriched samples tested with Sample6 Detect HT/L performed as well as the reference method at all time points tested from 15 to 24 h. R2 is a proprietary blend for use with the test kit that helps with early detection. All the dPODC values (Sample6 Detect HT/L presumptive and confirmed results) equaled zero, indicating 100% concordance between the methods. Both Sample6 Detect HT/L and FDA BAM results showed low dPODC values, with confidence intervals indicating no significant differences between Sample6 Detect HT/L and reference method results. Conclusions: Sample6 Detect HT/L is suitable to detect Listeria spp. in ice cream, even with a 12 h enrichment. Sample6 Detect HT/L demonstrated equivalent detection of L. monocytogenes and L. innocua from R2-enriched samples as expected with 15 and 18 h enrichment when compared with the 24 h FDA BAM method for L. monocytogenes. Highlights: These results indicate that Sample6 Detect HT/L, primarily developed for environmental samples, can be used to detect Listeria spp. in ice cream with less incubation time, resulting in faster detection.

Validation of the Listeria Right NowTM Test for Detection of Listeria spp. from Selected Environmental Surfaces Without Enrichment.

Background: Listeria Right NowTM is a novel, enrichment-free molecular method for detection of Listeria spp. in swab samples from environmental surfaces. The test provides results in real time, indicating the current or recent presence of Listeria spp. in the environment. After sampling, the entire contents of the swab are subject to sample processing, releasing large quantities of target ribosomal RNA molecules into the lysate. A portion of the lysate is then tested using the ANSR® for Listeria isothermal nucleic acid amplification assay. Objective: A Performance Tested MethodSM study was conducted to validate the method for detection of Listeria spp. in swab samples from stainless steel and sealed concrete surfaces. Methods and Results: In inclusivity testing, 60 of 60 Listeria spp. strains tested positive. In exclusivity testing, 31 of 31 nontarget bacterial strains tested negative. In LOD testing, the test was able to detect as few as 2 CFU of L. monocytogenes applied to a stainless steel surface. In matrix testing of inoculated stainless steel and sealed concrete surfaces, there were no statistically significant differences in method performance comparing the Listeria Right Now and U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedures as determined by probability of detection analysis. In robustness testing, modest changes to three assay operating parameters simultaneously did not significantly affect performance of the test. Conclusions and Highlights: Results can be obtained in less than 1 h using the Listeria Right Now test, allowing food industry personnel to take immediate corrective action in the case of Listeria contamination incidents.

Method Modification for the Atlas Listeria Environmental LE Detection Assay Using FoodChek Actero Listeria Enrichment Media and Half-Fraser Media for the Detection of Listeria spp. from Environmental Surfaces

Two candidate method modifications for the Atlas Listeria Environmental LE Detection Assay were compared with the U.S. Department of Agriculture (USDA)-Food Safety and Inspection Service Microbiology Laboratory Guidebook 8.09 (MLG 8.09) method for detection of Listeria spp. on stainless steel, polyvinyl chloride (PVC), and sealed concrete surfaces. For LE candidate method 1, samples were enriched in FoodChek Actero Listeria Enrichment Media [ALEM; Performance Tested MethodSM (PTM) 111201] at 35 ± 2°C for 18 to 24 h and evaluated for a range of analytical sample volumes. For LE candidate method 2, the current Roka PTM using 90 mL of Half-Fraser broth for enrichment at 35 ± 2°C was evaluated at 24 h with a reduced sample volume. These comparisons were made in multiple studies across the three environmental surfaces. Within each method and study, a total of 5 samples were uninoculated, 20 samples were inoculated with Listeria spp. at a low level to target fractional positivity, and 5 samples were inoculated with Listeria spp. at a high level to approach a probability of detection of 1. Inclusivity and exclusivity studies were also conducted for the LE method in combination with Half-Fraser and ALEM. The Atlas Listeria Environmental LE Detection Assay detected all 50 inclusive organisms, including 25 strains of L. monocytogenes and 5 strains of each of the other five common species of Listeria (L. innocua, L. welshimeri, L. ivanovii, L. seeligeri, and L. grayi) and none of the 30 exclusive organisms across all media and with both 200 and 2000 µL sample volumes. For the LE candidate method 1 studies, no significant differences were observed within the Roka ALEM method at 18, 20, or 24 h and for both the 200 and 2000 µL sample volumes as compared with the paired culture outcome. However, the ALEM method performed significantly better as compared with the unpaired reference method for sealed concrete and stainless steel. For the LE candidate method 2 studies, no significant differences were observed within the Roka HF method at 24 h for the 200 and 2000 µL samples as compared with the paired culture outcomes and unpaired reference method outcomes across the surfaces. The independent laboratory studies observed no significant differences in performance between the USDA/MLG 8.09 reference method and candidate methods 1 or 2, respectively, across the evaluated parameters. Overall, the candidate method 1 modification parameters and candidate method 2 sample parameters for the Atlas Listeria Environmental LE Detection Assay were statistically equivalent to or better than the reference method for detection of Listeria spp. on stainless steel, PVC, and sealed concrete surfaces, providing greater flexibility in method application for end users.

A novel biomarker for detection of Listeria species in food processing factory

A biomarker-based PCR amplification method has been proved to be an effective tool for rapid and accurate detection of Listeria contamination in food factories. However, the current biomarkers (such as iap, hly genes) used for the detection of Listeria have a problem of detection efficiency for some Listeria species, including 7 recently recognized species. Therefore, in this study, a new comprehensive biomarker was developed for the effective detection of all Listeria species. The study employed a novel in-silico scheme to explore and characterize the alternative biomarkers. Biomarker BE-LisAll was identified against 34 Listeria and over 2700 other bacterial complete genomes by in-silico scheme. Specificity of biomarker BE-LisAll was then evaluated with 17 different Listeria species and 58 non-Listeria bacteria isolates. The result showed 100% specificity to Listeria species, and the biomarker could differentiate Listeria species from a variety of non-Listeria bacteria. Finally, the PCR amplification with BE-LisAll was compared with the conventional culture method for detection of Listeria spp. using 60 swab samples collected from a food-processing factory to verify the biomarker's applicability. The result demonstrated 100% correspondence to the results of the conventional culture method. The BE-LisAll biomarker-based PCR amplification is presented for rapid and comprehensive detection of all Listeria species with a high degree of accuracy and sensitivity. It is hoped that commercial food processing industries are able to employ this developed biomarker as a Listeria-detection tool in their factories to prevent or reduce economic losses due to Listeria contamination.

Validation of the ANSR® Listeria Method for Detection of Listeria spp. in Selected Foods.

ANSR® Listeria was previously certified as Performance Tested Method(SM) 101202 for detection of Listeria spp. on selected environmental surfaces. This study proposes a matrix extension to the method for detection of Listeria spp. in selected food matrixes. The method is an isothermal nucleic acid amplification assay based on the nicking enzyme amplification reaction technology. Following single-step sample enrichment for 16-24 h, the assay is completed in less than 50 min, requiring only simple instrumentation. Inclusivity testing was performed using a panel of 51 strains of Listeria spp., representing the species L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, and L. welshimeri. All strains tested were detected by the ANSR assay. Exclusivity testing of 30 strains representing non-Listeria Gram-positive bacteria yielded no evidence of cross-reactivity. Performance of the ANSR method for detection of Listeria spp. was compared to that of reference culture procedures for pasteurized liquid egg, pasteurized 2% milk, Mexican-style cheese, ice cream, smoked salmon, lettuce, cantaloupe, and guacamole. Data obtained in these unpaired studies and analyzed using a probability of detection model demonstrated that there were no statistically significant differences in results between the ANSR and reference culture methods, except for milk at 16 h and cantaloupe. In milk and smoked salmon, ANSR sensitivity was low at 16 h and therefore the recommended incubation time is 24 h. In cantaloupe, ANSR was found to be more sensitive than the reference culture method at both 16 and 24 h in independent laboratory testing. The ANSR Listeria method can be used as an accurate, rapid, and simple alternative to standard culture methods for detection of Listeria spp. in selected food types.

Validation of the ANSR® Listeria Method for Detection of Listeria spp. in Environmental Samples

ANSR Listeria is a new diagnostic assay for detection of Listeria spp. in sponge or swab samples taken from a variety of environmental surfaces. The method is an isothermal nucleic acid amplification assay based on the nicking enzyme amplification reaction technology. Following single-step sample enrichment for 16-24 h, the assay is completed in 40 min, requiring only simple instrumentation. In inclusivity testing, 48 of 51 Listeria strains tested positive, with only the three strains of L. grayi producing negative results. Further investigation showed that L. grayi is reactive in the ANSR assay, but its ability to grow under the selective enrichment conditions used in the method is variable. In exclusivity testing, 32 species of non-Listeria, Gram-positive bacteria all produced negative ANSR assay results. Performance of the ANSR method was compared to that of the U.S. Department of Agriculture-Food Safety and Inspection Service reference culture procedure for detection of Listeria spp. in sponge or swab samples taken from inoculated stainless steel, plastic, ceramic tile, sealed concrete, and rubber surfaces. Data were analyzed using Chi-square and probability of detection models. Only one surface, stainless steel, showed a significant difference in performance between the methods, with the ANSR method producing more positive results. Results of internal trials were supported by findings from independent laboratory testing. The ANSR Listeria method can be used as an accurate, rapid, and simple alternative to standard culture methods for detection of Listeria spp. in environmental samples.

VIDAS® Listeria species Xpress (LSX)

The AOAC GovVal study compared the VIDAS Listeria species Xpress (LSX) to the Health Products and Food Branch MFHPB-30 reference method for detection of Listeria on stainless steel. The LSX method utilizes a novel and proprietary enrichment media, Listeria Xpress broth, enabling detection of Listeria species in environmental samples with the automated VIDAS in a minimum of 26 h. The LSX method also includes the use of the chromogenic media, chromID Ottaviani Agosti Agar (OAA) and chromID Lmono for confirmation of LSX presumptive results. In previous AOAC validation studies comparing VIDAS LSX to the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) and the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) reference methods, the LSX method was approved as AOAC Official Method 2010.02 for the detection of Listeria species in dairy products, vegetables, seafood, raw meats and poultry, and processed meats and poultry, and as AOAC Performance Tested Method 100501 in a variety of foods and on environmental surfaces. The GovVal comparative study included 20 replicate test portions each at two contamination levels for stainless steel where fractionally positive results (5-15 positive results/20 replicate portions tested) were obtained by at least one method at one level. Five uncontaminated controls were included. In the stainless steel artificially contaminated surface study, there were 25 confirmed positives by the VIDAS LSX assay and 22 confirmed positives by the standard culture methods. Chi-square analysis indicated no statistical differences between the VIDAS LSX method and the MFHPB-30 standard methods at the 5% level of significance. Confirmation of presumptive LSX results with the chromogenic OAA and Lmono media was shown to be equivalent to the appropriate reference method agars. The data in this study demonstrate that the VIDAS LSX method is an acceptable alternative method to the MFHPB-30 standard culture method for the detection of Listeria species on stainless steel.

Improved sensitivity and reproducibility of the PCR method for detection of Listeria spp. and L. monocytogenes in milk

Listeria monocytogenes is a facultative intracellular Grampositive 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 L. monocytogenes in food samples. The aim of this study was to improve the sensitivity and reproducibility of PCR for the detection of Listeria spp. in milk. For that purpose milk samples were artificially inoculated with serial dilutions of L. monocytogenes 4b ATCC 19115 and L. innocua ATCC 33090. The results obtained on artificially contaminated milk samples indicated that incubation time and target genes have an influence on the sensitivity of PCR detection. The best results were obtained after 24 h of preenrichment, with primers complementary to the hlyA gene, when it was possible to detect 1 CFU/mL of Listeria spp.

Development of a novel FRET method for detection of Listeria or Salmonella

Listeria and Salmonella contamination in foods results in not only foodborne outbreaks, but also a large economical burden for the industry due to product recalls. As a result, a fluorescence resonance energy transfer (FRET)-based method was developed to detect Listeria and Salmonella. Sensors utilizing FRET switch their fluorescence wavelength between donor and acceptor fluorophores as the distance between the two fluorophores change. This change in distance is a result of the conformational change in the 3D structure of the antibody as it binds to the target antigen. Listeria or Salmonella antibodies were labeled with FRET donor fluorophores (Alexa Fluor 546 (AF546)) while protein G or A was labeled with the acceptor fluorophores (Alexa Fluor 594 (AF594)). The labeled antibody–protein G or A complex was formed via incubation of the labeled antibody with protein G/A which specifically attaches to the Fc fragment of antibodies. The labeled antibody–protein G or A complex was tested in solution and specific and nonspecific antigens were exposed to the in solution complex. Changes in fluorescence were monitored by a spectrofluorometer. For “in-solution” tests, the optimal acceptor/donor fluorophore (A/D) ratios were 0.2 and 1.0 for Listeria and Salmonella, respectively, and both of Listeria and Salmonella antigen detection limits were 2.0 μg/ml.

Use of Immunomagnetic Capture on Beads to Recover Listeria from Environmental Samples

Current methods of Listeria detection require days to complete and may fail to detect Listeria in some samples. In contrast, capture of Listeria on microscopic immunomagnetic beads reduces test time to 24 h and improves sensitivity. In this approach, which eliminates enrichment, samples are mixed with immunomagnetic beads coated with anti-Listeria antibodies. Listeria in the sample bind to beads. The beads, with their bound Listeria, are isolated from other sample material and microorganisms in a magnetic field. The beads are plated on medium and incubated overnight. The next day, a replica of the plate is made onto a thin plastic membrane. The membrane is treated with an anti-Listeria antibody, an alkaline-phosphatase conjugated antibody that binds to the anti-Listeria antibody, and a substrate for alkaline phosphatase. This treatment produces colored spots that correspond to Listeria colonies on the master plate.Immunomagnetic capture was compared to a standard cultural method for analysis of environmental samples. Immunomagnetic capture detected Listeria in 100% of the samples, at contamination levels where a standard cultural method gave detection in only 36% of the samples. At lower levels of contamination, immunomagnetic isolation allowed detection of Listeria in 58% of the samples, while the cultural method failed to detect Listeria in any samples. Immunomagnetic capture resulted in recovery of injured Listeria. Because immunomagnetic capture did not rely on enrichment, the number of Listeria colonies obtained was related to the original level of contamination. Quantitative results were produced within 24 h for both positive and negative samples, while the cultural method produced qualitative results in 6 days (for positives) or 3 days (for negatives). Immunomagnetic capture is thus both more rapid and more sensitive than a standard cultural method for detection of Listeria in environmental samples.

DETECTION OF LISTERIA IN FOOD AND ENVIRONMENTAL SAMPLES BY IMMUNOMAGNETIC BEAD CAPTURE AND BY CULTURAL METHODS

Detection of Listeria by capture on antibody‐coated magnetic beads has been shown to decrease test time and improve sensitivity, relative to cultural methods, in a study of spiked environmental samples (Mitchell et al. 1993). In this study, immunomagnetic capture was compared to standard cultural methods for detection of Listeria in a broad range of spiked and naturally contaminated food and environmental samples. Immunomagnetic capture was at least as sensitive as cultural methods for detection of Listeria in seafood, meats, dairy foods, and environmental samples. It was possible to determine the number of Listeria present in a sample, because immunomagnetic capture was carried out directly from the sample, without enrichment. These quantitative results were produced within 24 h, while cultural methods required 6–14 days to produce a qualitative result. Immunomagnetic capture was thus more rapid and as sensitive as standard cultural methods for detection of Listeria in the food and environmental samples tested.