Biology subjects

Nieves, E. M.

Publications and source records attributed to Nieves, E. M..

2 recordsLinked to original sources

bioRxiv2024

GenomicGapID: Leveraging Spatial Distribution of Conserved Genomic Sites for Broad-Spectrum Microbial Identification

Bacterial detection and identification methods can be broadly classified as either untargeted with expansive taxonomic coverage or targeted with narrow taxonomic focus. Untargeted approaches, such as culture and sequencing, are often time-consuming and/or costly, whereas targeted methods, such as PCR, can offer faster and more cost-effective results but require a priori knowledge of the likely pathogen to select the appropriate assay. GenomicGapID, a novel approach that leverages the spatial distribution of conserved genetic regions across microbial genomes, represents a significant advancement in the field of microbial identification. This technique has the potential to provide the taxonomic breadth of culture and sequencing, while maintaining the speed, simplicity, and cost-effectiveness of PCR. By leveraging the conservation and relative positioning of highly conserved coding regions across different species, GenomicGapID enables the development of universal primer sets that amplify the non-conserved gaps between these regions. This creates a unique electrophoretic signature that facilitates rapid and accurate target agnostic microbial identification. In this study, we apply the principles of GenomicGapID to the critical task of identifying clinical pathogens. We focus on expanding the coverage of a previously developed universal bacterial identification system, which initially targeted the 16s-23s internal transcribed spacer (ITS) region and was capable of discerning 45 pathogens. To enhance this system, we assembled a comprehensive database of 189 clinically relevant bacterial species. We then identified conserved primer binding sites that produce unique amplicon size signatures for each species. While we found that the use of amplicon size signatures alone would require an impractical number of universal primer sets, we demonstrate that this challenge can be effectively mitigated through concurrent melt analysis. Ultimately, we show that just three universal primer sets, guided by the GenomicGapID framework, are sufficient to cover 189 clinical bacterial pathogens, representing a majority of microbes identified in positive cultures in a clinical microbiology setting, with experimental validation of a subset of these pathogens. This study not only enhances the existing universal bacterial identification system but also establishes GenomicGapID as a versatile and powerful tool in microbial diagnostics and beyond with the potential to open new areas of investigation in genomics, with significant implications for molecular biology, clinical practice, and public health.

microbiology↗

bioRxiv2024

A rapid, inexpensive, culture-free, universal bacterial identification system using internal transcribed spacer targeting primers: a proof-of-principle study

Techniques for bacterial detection and identification can be characterized as either untargeted and taxonomically broad or targeted and taxonomically narrow. Untargeted techniques (e.g., culture and sequencing) are time-consuming and/or expensive while targeted techniques (polymerase chain reaction; PCR) can be faster and less expensive but require a strong pre-test suspicion of the potential organism to choose the correct test. We have developed a universal bacterial identification system that is as taxonomically broad as culture and amplicon sequencing but as fast, easy, and affordable as PCR. The platform utilizes a unique universal polymerase chain reaction (PCR) primer set that targets the internal transcribed spacer (ITS) regions between conserved bacterial genes, creating a distinguishable electrophoretic pattern for each bacterial species. Bioinformatic simulation demonstrates that at least 45 commonly isolated pathogenic species can be uniquely identified from a single set of PCR primers using this approach. We experimentally confirmed these predictions on seven representative human pathogens, including gram-negatives and gram-positives, aerobes and anaerobes, and spore formers. Without a priori knowledge of the organism, this system can rapidly identify the unique pattern generated by multiple species in a single reaction. Using quantitative PCR, the system can also determine the corresponding concentration of the organism in question. We also show that the primers are resilient to human DNA contamination at physiologic concentrations, eliminating the need for complex and time intensive extraction methods. Proof-of-principle testing on actual clinical specimens demonstrate that this assay can identify more than twice the species as current multiplex PCR assays (e.g., BioFire) using only one universal primer pair in a single PCR reaction, providing results in <3 hours for <$20 without bioinformatic turnaround time. SIGNIFICANCE STATEMENTThis paper describes a novel bacterial identification system that is as taxonomically broad as culture and amplicon sequencing but as fast, easy, and affordable as PCR. The assay covers more than twice the species as the leading multiplex PCR assays but uses only one universal primer pair in a single PCR reaction. It is resilient to human genomic contamination precluding the need for timely or costly methods to clear human DNA. This approach represents a major advancement in the decades-long struggle to rapidly and accurately identify bacteria.

bioengineering↗