SARS-CoV-2 variants of concern surveillance including Omicron using RT-PCR-based genotyping offers comparable performance to whole genome sequencing.

Known SARS-CoV-2 variants of concern (VOCs) can be detected and differentiated using an RT-PCR-based genotyping approach, which offers quicker time to result, lower cost, higher flexibility, and use of the same laboratory instrumentation for detection of SARS-CoV-2 when compared with whole genome sequencing (WGS). In the current study, we demonstrate how we applied a genotyping approach for identification of all VOCs and that such technique can offer comparable performance to WGS for identification of known SARS-CoV-2 VOCs, including more recent strains, Omicron BA.1 and BA.

Targeted Sanger sequencing to recover key mutations in SARS-CoV-2 variant genome assemblies produced by next-generation sequencing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is adaptively evolving to ensure its persistence within human hosts. It is therefore necessary to continuously monitor the emergence and prevalence of novel variants that arise. Importantly, some mutations have been associated with both molecular diagnostic failures and reduced or abrogated next-generation sequencing (NGS) read coverage in some genomic regions.

Sensitivity of plasma BRAFmutant and NRASmutant cell-free DNA assays to detect metastatic melanoma in patients with low RECIST scores and non-RECIST disease progression.

Melanoma lacks a clinically useful blood-based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell-free, tumor-associated BRAF(mutant) and NRAS(mutant) DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAF(mutant) and NRAS(mutant) ctDNA were determined using droplet digital PCR (ddPCR) assays.

Using Whole Genome Analysis to Examine Recombination across Diverse Sequence Types of Staphylococcus aureus.

Staphylococcus aureus is an important clinical pathogen worldwide and understanding this organism's phylogeny and, in particular, the role of recombination, is important both to understand the overall spread of virulent lineages and to characterize outbreaks. To further elucidate the phylogeny of S. aureus, 35 diverse strains were sequenced using whole genome sequencing.

Genomic characterization of Salmonella Cerro ST367, an emerging Salmonella subtype in cattle in the United States.

Background: Within the last decade, Salmonella enterica subsp. enterica serovar Cerro (S. Cerro) has become one of the most common serovars isolated from cattle and dairy farm environments in the northeastern US.

Progress toward an efficient panel of SNPs for ancestry inference.

Many panels of ancestry informative single nucleotide polymorphisms have been proposed in recent years for various purposes including detecting stratification in biomedical studies and determining an individual's ancestry in a forensic context. All of the panels have limitations in their generality and efficiency for routine forensic work. Some panels have used only a few populations to validate them.

Validation of the Applied Biosystems MicroSEQ real-time PCR system for detection of E. coli O157:H7 in food.

Modern molecular methods offer the advantages of simplicity and short time-to-results compared to traditional culture methods. We describe the validation of a new Real-Time PCR method to detect E. coli O157:H7 in five food matrixes.

Comparative analysis of genome sequences covering the seven cronobacter species.

Background: Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C.

Evaluation of applied biosystems MicroSEQ real-time PCR system for detection of Listeria spp. in food and environmental samples.

A complete system for real-time PCR detection of Listeria species was validated in five food matrixes and five environmental surfaces, namely, hot dogs, roast beef, lox (smoked salmon), pasteurized whole cow's milk, dry infant formula, stainless steel, plastic cutting board, ceramic tile, rubber sheets, and sealed concrete. The system consists of the MicroSEQ Listeria spp. Detection Kit, two sample preparation kits (PrepSEQ Nucleic Acid Extraction Kit and PrepSEQ Rapid Spin Sample Preparation Kit), the Applied Biosystems 7500 Fast Real-Time PCR instrument, and the RapidFinderTM Express v1.

Opportunities and challenges associated with clinical diagnostic genome sequencing: a report of the Association for Molecular Pathology.

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging.

Identification and characterization of novel Salmonella mobile elements involved in the dissemination of genes linked to virulence and transmission.

The genetic diversity represented by >2,500 different Salmonella serovars provides a yet largely uncharacterized reservoir of mobile elements that can contribute to the frequent emergence of new pathogenic strains of this important zoonotic pathogen. Currently, our understanding of Salmonella mobile elements is skewed by the fact that most studies have focused on highly virulent or common serovars. To gain a more global picture of mobile elements in Salmonella, we used prediction algorithms to screen for mobile elements in 16 sequenced Salmonella genomes representing serovars for which no prior genome scale mobile element data were available.

Mini-haplotypes as lineage informative SNPs and ancestry inference SNPs.

We propose that haplotyped loci with high heterozygosity can be useful in human identification, especially within families, if recombination is very low among the sites. Three or more SNPs extending over small molecular intervals (<10 KB) can be identified in the human genome to define miniature haplotypes with moderate levels of linkage disequilibrium. Properly selected, these mini-haplotypes (or minihaps) consist of multiple haplotype lineages (alleles) that have evolved from the ancestral human haplotype but show no evidence of recurring recombination, allowing each distinct haplotype to be equated with an allele, all copies of which are essentially identical by descent.

Expanding data and resources for forensic use of SNPs in individual identification.

The potential value of SNPs for individual identification has been recognized by many researchers and different panels have been proposed. Here we present a new interface in the ALFRED database to access compendia of allele frequencies for several published panels of markers for forensic uses. One of those is our panel of individual identification SNPs (IISNPs) based on samples of 44 populations originating from many parts of the world.

Escherichia coli serotype O55:H7 diversity supports parallel acquisition of bacteriophage at Shiga toxin phage insertion sites during evolution of the O157:H7 lineage.

Enteropathogenic Escherichia coli (EPEC) continues to be a leading cause of mortality and morbidity in children around the world. Two EPEC genomes have been fully sequenced: those of EPEC O127:H6 strain E2348/69 (United Kingdom, 1969) and EPEC O55:H7 strain CB9615 (Germany, 2003). The O55:H7 serotype is a recent precursor to the virulent enterohemorrhagic E.

Rapid detection of Salmonella in pet food: design and evaluation of integrated methods based on real-time PCR detection.

Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can take 5 to 7 days to obtain definitive results.

Dominance of multidrug resistant CC271 clones in macrolide-resistant streptococcus pneumoniae in Arizona.

Background: Rates of resistance to macrolide antibiotics in Streptococcus pneumoniae are rising around the world due to the spread of mobile genetic elements harboring mef(E) and erm(B) genes and post-vaccine clonal expansion of strains that carry them.

Results: Characterization of 592 clinical isolates collected in Arizona over a 10 year period shows 23.6% are macrolide resistant.

A sample extraction method for faster, more sensitive PCR-based detection of pathogens in blood culture.

Three mechanistically different sample extraction methodologies, namely, silica spin columns, phenol-chloroform, and an automated magnetic capture of polymer-complexed DNA (via an Automate Express instrument), were compared for their abilities to purify nucleic acids from blood culture fluids for use in TaqMan assays for detection of Staphylococcus aureus. The extracts from silica columns required 100- to 1000-fold dilutions to sufficiently reduce the powerful PCR inhibitory effects of the anticoagulant sodium polyanetholsulfonate, a common additive in blood culture media. In contrast, samples extracted by either phenol-chloroform or the Automate Express instrument required little or no dilution, respectively, allowing for an approximate 100-fold improvement in assay sensitivity.

Evaluation of applied biosystems MicroSeq real-time PCR system for detection of Listeria monocytogenes in food. Performance Tested Method 011002.

Increasingly, more food companies are relying on molecular methods, such as PCR, for pathogen detection due to their improved simplicity, sensitivity, and rapid time to results. This report describes the validation of a new Real-Time PCR method to detect Listeria monocytogenes in nine different food matrixes. The complete system consists of the MicroSEQ L.

A whole-genome single nucleotide polymorphism-based approach to trace and identify outbreaks linked to a common Salmonella enterica subsp. enterica serovar Montevideo pulsed-field gel electrophoresis type.

In this study, we report a whole-genome single nucleotide polymorphism (SNP)-based evolutionary approach to study the epidemiology of a multistate outbreak of Salmonella enterica subsp. enterica serovar Montevideo. This outbreak included 272 cases that occurred in 44 states between July 2009 and April 2010.

Evaluation of applied biosystems MicroSEQ real-time PCR system for detection of Salmonella spp. in food.

Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time-to-results compared to traditional culture methods. In this study, the MicroSEQ real-time PCR system was evaluated for detection of Salmonella spp. in 10 different food matrixes following the AOAC Research Institute's Performance Tested Method validation program.

Single nucleotide polymorphisms and haplotypes in Native American populations.

Autosomal DNA polymorphisms can provide new information and understanding of both the origins of and relationships among modern Native American populations. At the same time that autosomal markers can be highly informative, they are also susceptible to ascertainment biases in the selection of the markers to use. Identifying markers that can be used for ancestry inference among Native American populations can be considered separate from identifying markers to further the quest for history.

Genome sequencing reveals diversification of virulence factor content and possible host adaptation in distinct subpopulations of Salmonella enterica.

Background: Divergence of bacterial populations into distinct subpopulations is often the result of ecological isolation. While some studies have suggested the existence of Salmonella enterica subsp. enterica subclades, evidence for these subdivisions has been ambiguous.

Design and validation of a novel multiplex real-time PCR assay for Vibrio pathogen detection.

Three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus--account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete.

Comparative genomics of the bacterial genus Listeria: Genome evolution is characterized by limited gene acquisition and limited gene loss.

Background: The bacterial genus Listeria contains pathogenic and non-pathogenic species, including the pathogens L. monocytogenes and L. ivanovii, both of which carry homologous virulence gene clusters such as the prfA cluster and clusters of internalin genes.