Atlas(®) Listeria monocytogenes LmG2 Detection Assay Using Transcription Mediated Amplification to Detect Listeria monocytogenes in Selected Foods and Stainless Steel Surface.

The Atlas Listeria monocytogenes LmG2 Detection Assay, developed by Roka Bioscience Inc., was compared to a reference culture method for seven food types (hot dogs, cured ham, deli turkey, chicken salad, vanilla ice cream, frozen chocolate cream pie, and frozen cheese pizza) and one surface (stainless steel, grade 316). A 125 g portion of deli turkey was tested using a 1:4 food:media dilution ratio, and a 25 g portion for all other foods was tested using 1:9 food:media dilution ratio. The enrichment time and media for Roka's method was 24 to 28 h for 25 g food samples and environmental surfaces, and 44 to 48 h for 125 g at 35 ± 2°C in PALCAM broth containing 0.02 g/L nalidixic acid. Comparison of the Atlas Listeria monocytogenes LmG2 Detection Assay to the reference method required an unpaired approach. For each matrix, 20 samples inoculated at a fractional level and five samples inoculated at a high level with a different strain of Listeria monocytogenes were tested by each method. The Atlas Listeria monocytogenes LmG2 Detection Assay was compared to the Official Methods of Analysis of AOAC INTERNATIONAL 993.12 method for dairy products, the U.S. Department of Agriculture, Food Safety and Inspection Service, Microbiology Laboratory Guidebook 8.08 method for ready-to-eat meat and environmental samples, and the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 10 method for frozen foods. In the method developer studies, Roka's method, at 24 h (or 44 h for 125 g food samples), had 126 positives out of 200 total inoculated samples, compared to 102 positives for the reference methods at 48 h. In the independent laboratory studies, vanilla ice cream, deli turkey and stainless steel grade 316 were evaluated. Roka's method, at 24 h (or 44 h for 125 g food samples), had 64 positives out of 75 total inoculated samples compared to 54 positives for the reference methods at 48 h. The Atlas Listeria monocytogenes LmG2 Detection Assay detected all 50 L. monocytogenes strains that encompassed 13 serotypes across the various lineages and none of the 30 exclusive organisms, including seven other Listeria species. The product consistency and kit stability studies revealed no statistical differences between the three lots tested or to the term of the shelf life. Finally, the robustness study demonstrated no statistical differences when samples were incubated at 33 ± 2°C or 37 ± 2°C, when enrichment aliquots were 1.3 mL or 1.7 mL, or when the samples were analyzed the same day or five days later. Overall the Atlas Listeria monocytogenes LmG2 Detection Assay is statistically equivalent to or better than the reference methods and is robust to the tested variations.

Atlas Salmonella detection method using transcription mediated amplification (TMA) to detect Salmonella enterica in a variety of foods and select surfaces.

The Atlas Salmonella detection assay was compared to the reference culture methods for 12 foods and three surfaces. Comparison of the Atlas method to the U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA/BAM) and U.S. Department of Agriculture-Food Safety and Inspection Service/Microbiology Laboratory Guidebook (USDA-FSIS/MLG) reference methods required an unpaired approach. Each method had a total of 320 samples inoculated with an S. enterica strain. Each food and surface was inoculated with a different strain of S. enterica at two different levels/ method. Meat and egg products were compared to the USDA-FSIS/MLG 4.05 method. All other foods were compared to the FDA/BAM-5 method. The Atlas method had 148 positives out of 320 total inoculated samples, compared to 119 positives for the reference methods. Overall, the probability of detection analysis of the results showed equivalent or better performance by the Atlas Salmonella detection method compared to the reference methods. The Atlas Salmonella detection assay detected all 100 inclusive organisms and none of the 30 exclusive organisms. The lot-to-lot and kit stability studies showed no statistical differences between lots or over the term of the shelf-life. Instrument-to-instrument testing showed no statistical difference between instruments. Finally, the robustness study showed no difference when the sample volume added to the Atlas Salmonella detection assay varied by 10%, storage time was extended up to 5 days before analysis, or enrichment times were varied from 12 to 24 h.

A new generation of food-borne pathogen detection based on ribosomal RNA.

Listeria and Salmonella detection assays for food and environmental surfaces that target ribosomal RNA (rRNA) have been developed. The large number of rRNA molecules in bacteria enabled the development of molecular assays that use enrichment times as short as 12 hours for Salmonella and 24 hours for Listeria. These assays run on a fully automated molecular pathogen detection system, which provides walk-away capability and produces 300 assay results in eight hours.

Roka Listeria detection method using transcription mediated amplification to detect Listeria species in select foods and surfaces. Performance Tested Method(SM) 011201.

The Roka Listeria Detection Assay was compared to the reference culture methods for nine select foods and three select surfaces. The Roka method used Half-Fraser Broth for enrichment at 35 +/- 2 degrees C for 24-28 h. Comparison of Roka's method to reference methods requires an unpaired approach. Each method had a total of 545 samples inoculated with a Listeria strain. Each food and surface was inoculated with a different strain of Listeria at two different levels per method. For the dairy products (Brie cheese, whole milk, and ice cream), our method was compared to AOAC Official Method(SM) 993.12. For the ready-to-eat meats (deli chicken, cured ham, chicken salad, and hot dogs) and environmental surfaces (sealed concrete, stainless steel, and plastic), these samples were compared to the U.S. Department of Agriculture/Food Safety and Inspection Service-Microbiology Laboratory Guidebook (USDA/FSIS-MLG) method MLG 8.07. Cold-smoked salmon and romaine lettuce were compared to the U.S. Food and Drug Administration/Bacteriological Analytical Manual, Chapter 10 (FDA/BAM) method. Roka's method had 358 positives out of 545 total inoculated samples compared to 332 positive for the reference methods. Overall the probability of detection analysis of the results showed better or equivalent performance compared to the reference methods.

High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay.

BACKGROUND: Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. RESULTS: A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point. CONCLUSIONS: Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.

Substrate requirements for secondary cleavage by HIV-1 reverse transcriptase RNase H.

During and after minus-strand DNA synthesis, human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) degrades the RNA genome. To remove RNA left after polymerization, the RT aligns to cut 18 nucleotides in from the 5' RNA end. The enzyme then repositions, making a secondary cut 8 nucleotides from the RNA 5' end. Transfer of the minus strong stop DNA during viral replication requires cleavage of template RNA. Removal of the terminal RNA segment is a special case because the RNA-DNA hybrid forms a blunt end, shown previously to resist cleavage when tested in vitro. We show here that the structure of the substrate extending beyond the RNA 5' end is an important determinant of cleavage efficiency. A short single-stranded DNA extension greatly stimulated the secondary cleavage. Annealing an RNA segment to the DNA extension was even more stimulatory. Recessing the DNA from a blunt end by even one nucleotide caused the RT to reorient its binding, preventing secondary cleavage. The presence of the cap at the 5' end of the viral RNA did not improve the efficiency of secondary cleavage. However, NC protein greatly facilitated the secondary cut on the blunt-ended substrate, suggesting that NC compensates for the unfavorable substrate structure.