Validation Study for PathogenDx EnviroX-F Assay for the Detection of Listeria, L. monocytogenes, and Salmonella in Environmental Surface Samples: AOAC Performance Tested MethodSM 092001.

BACKGROUND: The PathogenDx EnviroX-F uses microarray technology to simultaneously detect Listeria species, Listeria monocytogenes, and Salmonella species from environmental samples without the need for enrichment. OBJECTIVE: The validation study included a matrix study of four matrices (stainless steel, plastic, rubber, and sealed concrete) comparing the PathogenDx EnviroX-F assay to the US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 10, "Detection of L. monocytogenes in Foods and Environmental Samples, and Enumeration of L. monocytogenes in Foods" (revised March 2017), and FDA BAM Chapter 5,"Salmonella" (revised February 2020). Other required Performance Tested MethodSM parameters evaluated included: inclusivity and exclusivity, robustness, instrument variation, and product consistency and stability. METHODS: The PathogenDx EnviroX-F assay was evaluated with 30 unpaired replicate surface areas for each environmental surface. The candidate method was evaluated without an enrichment step. RESULTS: In the inclusivity and exclusivity study 50 out of 50 Listeria isolates were detected, 50 out of 50 L. monocytogenes strains were detected, 108 out of 108 Salmonella strains were detected, and 95 out of 95 exclusivity strains were correctly excluded. In the matrix study, the PathogenDx EnviroX-F assay showed no significant differences between confirmed results or between candidate and reference method results for 4" × 4" environmental surface areas for each matrix. CONCLUSION: The PathogenDx EnviroX-F assay is an effective method for the qualitative detection of Listeria spp., L. monocytogenes, and Salmonella spp. from environmental surface swabs. HIGHLIGHTS: The PathogenDx EnviroX-F assay will be the first PTM-approved multiplex assay for Listeria spp., L. monocytogenes, and Salmonella without the need for an environment step.

Independent Study for the Detectx Combined Assay for the Detection of Aspergillus, Salmonella, and STEC (stx1 and/or 2) in Dried Cannabis Flower and Dried Hemp Flower: Level 3 Modification Study 012201.

BACKGROUND: The PathogenDx family of assays uses microarray technology to simultaneously detect the presence of bacterial and fungal pathogens in food products, environmental surfaces, and cannabis products. OBJECTIVE: The Detectx Combined assay was validated for the detection of Aspergillus, (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus), Salmonella, and a broad range of STEC (stx1 and/or 2) species. The validation consisted of two matrix studies in dried hemp flower and dried cannabis flower (>0.3% delta-9 tetrahydrocannabinol) flower, product consistency, stability, robustness, and inclusivity and exclusivity for two targets: Aspergillus and STEC. METHOD: The PathogenDx Detectx Combined assay was evaluated with 30 replicates in each matrix and confirmed according to the instructions outlined in this study. RESULTS: Results of the validation study met the requirements of AOAC Standard Method Performance Requirement (SMPR®) 2020.002 and 2020.012. In the inclusivity and exclusivity study, all target isolates (Aspergillus and STEC) were correctly detected. For the exclusivity study, 26 out of 30 Aspergillus and 30 out of 30 STEC non-target strains were correctly excluded. In the matrix study, the PathogenDx Detectx Combined assay showed no significant statistical differences between confirmed results for dried hemp and cannabis flower. Robustness testing indicated that small changes to the method parameters did not impact the performance of the assay. Stability and consistency studies verified that the assay's shelf-life claims were appropriate, and manufacturing of the assay was consistent. CONCLUSIONS: The validation study indicated that the PathogenDx DetectX Combined assay was successful in detection of the new target analytes (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus and STEC containing stx1 and/or 2) and could successfully recover these organisms and Salmonella from dried hemp flower and dried cannabis flower (>0.3% delta-9 tetrahydrocannabinol). HIGHLIGHTS: The PathogenDx DetectX Combined Assay will be the first PTM approved multiplex assay for Aspergillus, E. coli and Salmonella that does not require an enrichment step.

Validation Study for PathogenDx EnviroX-Rv Assay for the Detection of SARS-CoV-2 from Stainless Steel Environmental Surface Swabs Performance Tested MethodSM 122003.

BACKGROUND: The PathogenDx EnviroX-Rv uses endpoint PCR + DNA microarray technology to detect SARS-CoV-2, the causative agent of COVID-19, from stainless-steel environmental sample swabs. OBJECTIVE: To validate the PathogenDx EnviroX-Rv assay as part of the Emergency Response Validation (ERV) Performance Tested Method(s)SM (PTM) program. METHOD: The PathogenDx EnviroX-Rv assay was evaluated for specificity using in silico analysis of ≥41 000 SARS-CoV-2 sequences and over 50 exclusivity organisms (both near neighbors and background organisms). The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the US Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus (2019-nCoV) Real-Time-Polymerase Chain Reaction (RT-PCR) Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020). RESULTS: Results of the in silico analysis demonstrated the high specificity of the method in being able to detect target SARS-CoV-2 sequences and discriminate them from near neighbors and environmental background organisms. In the matrix study, the candidate method demonstrated a statistically significant difference when compared to the results of the CDC method utilized in this study, with the candidate method resulting in more positive replicates as it only requires one target to be present for a positive sample. CONCLUSIONS: The EnviroX-Rv assay rapidly and accurately detected SARS-CoV-2 RNA on environmental swabs from stainless-steel surfaces at a concentration of 2000 genomic copies per 2 × 2" test area. HIGHLIGHTS: The EnviroX-Rv assay employs dual PCR and hybridization techniques to provide highly accurate results when detecting SARS-CoV-2 from surfaces.

Ultraspecific probes for high throughput HLA typing.

BACKGROUND: The variations within an individual's HLA (Human Leukocyte Antigen) genes have been linked to many immunological events, e.g. susceptibility to disease, response to vaccines, and the success of blood, tissue, and organ transplants. Although the microarray format has the potential to achieve high-resolution typing, this has yet to be attained due to inefficiencies of current probe design strategies. RESULTS: We present a novel three-step approach for the design of high-throughput microarray assays for HLA typing. This approach first selects sequences containing the SNPs present in all alleles of the locus of interest and next calculates the number of base changes necessary to convert a candidate probe sequences to the closest subsequence within the set of sequences that are likely to be present in the sample including the remainder of the human genome in order to identify those candidate probes which are "ultraspecific" for the allele of interest. Due to the high specificity of these sequences, it is possible that preliminary steps such as PCR amplification are no longer necessary. Lastly, the minimum number of these ultraspecific probes is selected such that the highest resolution typing can be achieved for the minimal cost of production. As an example, an array was designed and in silico results were obtained for typing of the HLA-B locus. CONCLUSION: The assay presented here provides a higher resolution than has previously been developed and includes more alleles than previously considered. Based upon the in silico and preliminary experimental results, we believe that the proposed approach can be readily applied to any highly polymorphic gene system.