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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Seroprevalence of Brucella spp. and Rift Valley fever virus among slaughterhouse workers in Isiolo County, northern Kenya

Brucella spp. and Rift Valley fever virus (RVFV) are classified as priority zoonotic agents in Kenya, based on their public health and socioeconomic impact on the country. Data on the pathogen-specific and co-exposure levels is scarce due to limited active surveillance. This study investigated seroprevalence and co-exposure of Brucella spp. and RVFV and associated risk factors among slaughterhouse workers in Isiolo County, northern Kenya. A cross-sectional serosurvey was done in all 19 slaughterhouses in Isiolo County, enrolling 378 participants into the study. The overall seroprevalences for Brucella spp. and RVFV were 40.2% (95% CI: 35.2–45.4) and 18.3% (95% CI: 14.5–22.5), respectively while 10.3% (95% CI 7.4%-13.8%) of individuals were positive for antibodies against both Brucella spp. and RVFV. Virus neutralisation tests (VNT) confirmed anti-RVFV antibodies in 85% of ELISA-positive samples. Our seroprevalence results were comparable to community-level seroprevalences previously reported in the area. Since most of the study participants were not from livestock-keeping households, our findings attribute most of the detected infections to occupational exposure. The high exposure levels indicate slaughterhouse workers are the most at-risk population and there is need for infection, prevention, and control programs among this high-risk group. This is the first VNT confirmation of virus-neutralising antibodies among slaughterhouse workers in Isiolo County and corroborates reports of the area being a high-risk RVFV area as occasioned by previously reported outbreaks. This necessitates sensitization campaigns to enhance awareness of the risks involved and appropriate mitigation measures.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Host population dynamics influence Leptospira spp. transmission patterns among Rattus norvegicus in Boston, Massachusetts, US

Leptospirosis (caused by pathogenic bacteria in the genus Leptospira ) is prevalent worldwide but more common in tropical and subtropical regions. Transmission can occur following direct exposure to infected urine from reservoir hosts, or a urine-contaminated environment, which then can serve as an infection source for additional rats and other mammals, including humans. The brown rat, Rattus norvegicus , is an important reservoir of Leptospira spp. in urban settings. We investigated the presence of Leptospira spp. among brown rats in Boston, Massachusetts and hypothesized that rat population dynamics in this urban setting influence the transportation, persistence, and diversity of Leptospira spp. We analyzed DNA from 328 rat kidney samples collected from 17 sites in Boston over a seven-year period (2016–2022); 59 rats representing 12 of 17 sites were positive for Leptospira spp. We used 21 neutral microsatellite loci to genotype 311 rats and utilized the resulting data to investigate genetic connectivity among sampling sites. We generated whole genome sequences for 28 Leptospira spp. isolates obtained from frozen and fresh tissue from some of the 59 positive rat kidneys. When isolates were not obtained, we attempted genomic DNA capture and enrichment, which yielded 14 additional Leptospira spp. genomes from rats. We also generated an enriched Leptospira spp. genome from a 2018 human case in Boston. We found evidence of high genetic structure among rat populations that is likely influenced by major roads and/or other dispersal barriers, resulting in distinct rat population groups within the city; at certain sites these groups persisted for multiple years. We identified multiple distinct phylogenetic clades of L. interrogans among rats that were tightly linked to distinct rat populations. This pattern suggests L. interrogans persists in local rat populations and its transportation is influenced by rat population dynamics. Finally, our genomic analyses of the Leptospira spp. detected in the 2018 human leptospirosis case in Boston suggests a link to rats as the source. These findings will be useful for guiding rat control and human leptospirosis mitigation efforts in this and other similar urban settings.

Stone, Nathan E.↗
DOE OSTI2020

Laboratory strains of Bacillus anthracis lose their ability to rapidly grow and sporulate compared to wildlife outbreak strains

Bacillus anthracis is the causative agent of anthrax in animals and humans. The organism lies in a dormant state in the soil until introduced into an animal via, ingestion, cutaneous inoculation or inhalation. Once in the host, spores germinate into rapidly growing vegetative cells elaborating toxins. When animals die of anthrax, vegetative bacteria sporulate upon nutrient limitation in the carcass or soil while in the presence of air. After release into the soil environment, spores form a localized infectious zone (LIZ) at and around the carcass. Laboratory strains of B. anthracis produce fewer proteins associated with growth and sporulation compared to wild strains isolated from recent zoonotic disease events. We verified wild strains grow more rapidly than lab strains demonstrating a greater responsiveness to nutrient availability. Sporulation was significantly more rapid in these wild strains compared to lab strains, indicating wild strains are able to sporulate faster due to nutrient limitation while laboratory strains have a decrease in the speed at which they utilize nutrients and an increase in time to sporulation. These findings have implications for disease control at the LIZ as well as on the infectious cycle of this dangerous zoonotic pathogen.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Comparative genetics of Enterococcus faecalis intestinal tissue isolates before and after surgery in a rat model of colon anastomosis

We have recently demonstrated that collagenolytic Enterococcus faecalis plays a key and causative role in the pathogenesis of anastomotic leak, an uncommon but potentially lethal complication characterized by disruption of the intestinal wound following segmental removal of the colon (resection) and its reconnection (anastomosis). Here we hypothesized that comparative genetic analysis of E. faecalis isolates present at the anastomotic wound site before and after surgery would shed insight into the mechanisms by which collagenolytic strains are selected for and predominate at sites of anastomotic disruption. Whole genome optical mapping of four pairs of isolates from rat colonic tissue obtained following surgical resection (herein named "pre-op" isolates) and then 6 days later from the anastomotic site (herein named "post-op" isolates) demonstrated that the isolates with higher collagenolytic activity formed a distinct cluster. In order to perform analysis at a deeper level, a single pair of E. faecalis isolates (16A pre-op and 16A post-op) was selected for whole genome sequencing and assembled using a hybrid assembly algorithm. Comparative genomics demonstrated absence of multiple gene clusters, notably a pathogenicity island in the post-op isolate. No differences were found in the fsr-gelE-sprE genes (EF1817-1822) responsible for regulation and production of collagenolytic activity. Analysis of unique genes among the 16A pre-op and post-op isolates revealed the predominance of transporter systems-related genes in the pre-op isolate and phage-related and hydrolytic enzyme-encoding genes in the post-op isolate. Despite genetic differences observed between pre-op and post-op isolates, the precise genetic determinants responsible for their differential expression of collagenolytic activity remains unknown.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota

Cervical microbiota (CM) are considered an important factor affecting the progression of cervical intraepithelial neoplasia (CIN) and are implicated in the persistence of human papillomavirus (HPV). Collection of liquid-based cytology (LBC) samples is routine for cervical cancer screening and HPV genotyping and can be used for long-term cytological biobanking. We sought to determine whether it is possible to access microbial DNA from LBC specimens, and compared the performance of four different extraction protocols: (ZymoBIOMICS DNA Miniprep Kit; QIAamp PowerFecal Pro DNA Kit; QIAamp DNA Mini Kit; and IndiSpin Pathogen Kit) and their ability to capture the diversity of CM from LBC specimens. LBC specimens from 20 patients (stored for 716 ± 105 days) with CIN values of 2 or 3 were each aliquoted for each of the four kits. Loss of microbial diversity due to long-term LBC storage could not be assessed due to lack of fresh LBC samples. Comparisons with other types of cervical sampling were not performed. We observed that all DNA extraction kits provided equivalent accessibility to the cervical microbial DNA within stored LBC samples. Approximately 80% microbial genera were shared among all DNA extraction protocols. Potential kit contaminants were observed as well. Variation between individuals was a significantly greater influence on the observed microbial composition than was the method of DNA extraction. We also observed that HPV16 was significantly associated with community types that were not dominated by Lactobacillus iners.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Environmental predictors of pulmonary nontuberculous mycobacteria (NTM) sputum positivity among persons with cystic fibrosis in the state of Florida

Nontuberculous mycobacteria (NTM) are opportunistic human pathogens that are commonly found in soil and water, and exposure to these organisms may cause pulmonary nontuberculous mycobacterial disease. Persons with cystic fibrosis (CF) are at high risk for developing pulmonary NTM infections, and studies have shown that prolonged exposure to certain environments can increase the risk of pulmonary NTM. It is therefore important to determine the risk associated with different geographic areas. Using annualized registry data obtained from the Cystic Fibrosis Foundation Patient Registry for 2010 through 2017, we conducted a geospatial analysis of NTM infections among persons with CF in Florida. A Bernoulli model in SaTScan was used to identify clustering of ZIP codes with higher than expected numbers of NTM culture positive individuals. Generalized linear mixed models with a binomial distribution were used to test the association of environmental variables and NTM culture positivity. We identified a significant cluster of M . abscessus and predictors of NTM sputum positivity, including annual precipitation and soil mineral levels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Comparative evaluation of antimicrobial activity of human granulysin, bovine and porcine NK-lysins against Shiga toxin-producing Escherichia coli O157:H7

Shiga toxin-producing Escherichia coli (STEC) O157:H7 (O157) is a foodborne pathogen causing human disease ranging from hemorrhagic colitis and hemolytic uremic syndrome to kidney failure, while remaining harmless to cattle, its primary reservoir. The severity of the human disease associated mainly with Shiga toxin production and a global emergence of antibiotic resistant STEC highlights the need for effective non-antibiotic, pre-harvest strategies to reduce O157 in cattle, the principal source of human infection. Towards this goal three synthetic antimicrobial peptides (AMPs): human granulysin (hGRNL), bovine NK-lysin (bNK2A), and porcine NK-lysin (pNKL), were tested in vitro against O157 isolates. As expected, circular dichroism spectroscopy findings were consistent with a predominantly α-helical conformation for all three AMPs in an environment mimicking bacterial outer surface or liposaccharides. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations of hGRNL (200 μM), bNK2A (12.5 μM against strain 86–24 and 25 μM against EDL933), and pNKL (6.25 μM) were determined using the Clinical and Laboratory Standards Institute broth microdilution method in Müeller-Hinton broth (cation-adjusted). The bNK2A and pNKL AMPs did not induce Shiga toxin expression in O157 at MIC, as there was a significant decrease or no change in toxin expression following 4- or 20 h incubation with the AMPs; bNK2A p <0.0001 (4 h) and p = 0.4831 (20 h); pNKL p <0.0001 (4 h) and p = 0.0001 (20 h). Propidium iodide uptake assay revealed faster O157 membrane damage or killing kinetics with bNK2A and pNKL compared to hGRNL. Nonetheless, transmission electron microscopy demonstrated that all three AMPs mediated damage to O157 membranes. In contrast, the three AMPs showed minimal cytotoxicity (<2%) against cattle red blood cells at tested concentrations (0.39–50 μM). Overall, our results demonstrate the potential for bNK2A and pNKL to be further developed into novel non-antibiotic agents to reduce O157 shedding in cattle.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Speeding genomic island discovery through systematic design of reference database composition

Background Genomic islands (GIs) are mobile genetic elements that integrate site-specifically into bacterial chromosomes, bearing genes that affect phenotypes such as pathogenicity and metabolism. GIs typically occur sporadically among related bacterial strains, enabling comparative genomic approaches to GI identification. For a candidate GI in a query genome, the number of reference genomes with a precise deletion of the GI serves as a support value for the GI. Our comparative software for GI identification was slowed by our original use of large reference genome databases (DBs). Here we explore smaller species-focused DBs. Results With increasing DB size, recovery of our reliable prophage GI calls reached a plateau, while recovery of less reliable GI calls (FPs) increased rapidly as DB sizes exceeded ~500 genomes; i.e., overlarge DBs can increase FP rates. Paradoxically, relative to prophages, FPs were both more frequently supported only by genomes outside the species and more frequently supported only by genomes inside the species; this may be due to their generally lower support values. Setting a DB size limit for our SMA ll R anked T ailored (SMART) DB design speeded runtime ~65-fold. Strictly intra-species DBs would tend to lower yields of prophages for small species (with few genomes available); simulations with large species showed that this could be partially overcome by reaching outside the species to closely related taxa, without an FP burden. Employing such taxonomic outreach in DB design generated redundancy in the DB set; as few as 2984 DBs were needed to cover all 47894 prokaryotic species. Conclusions Runtime decreased dramatically with SMART DB design, with only minor losses of prophages. We also describe potential utility in other comparative genomics projects.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Single-nuclei transcriptome analysis of channel catfish spleen provides insight into the immunome of an aquaculture-relevant species

The catfish industry is the largest sector of U.S. aquaculture production. Given its role in food production, the catfish immune response to industry-relevant pathogens has been extensively studied and has provided crucial information on innate and adaptive immune function during disease progression. To further examine the channel catfish immune system, we performed single-cell RNA sequencing on nuclei isolated from whole spleens, a major lymphoid organ in teleost fish. Libraries were prepared using the 10X Genomics Chromium X with the Next GEM Single Cell 3’ reagents and sequenced on an Illumina sequencer. Each demultiplexed sample was aligned to the Coco_2.0 channel catfish reference assembly, filtered, and counted to generate feature-barcode matrices. From whole spleen samples, outputs were analyzed both individually and as an integrated dataset. The three splenic transcriptome libraries generated an average of 278,717,872 reads from a mean 8,157 cells. The integrated data included 19,613 cells, counts for 20,121 genes, with a median 665 genes/cell. Cluster analysis of all cells identified 17 clusters which were classified as erythroid, hematopoietic stem cells, B cells, T cells, myeloid cells, and endothelial cells. Subcluster analysis was carried out on the immune cell populations. Here, distinct subclusters such as immature B cells, mature B cells, plasma cells, γδ T cells, dendritic cells, and macrophages were further identified. Differential gene expression analyses allowed for the identification of the most highly expressed genes for each cluster and subcluster. This dataset is a rich cellular gene expression resource for investigation of the channel catfish and teleost splenic immunome.

Science & Technology - Other Topics↗
DOE OSTI2020

Potent, specific MEPicides for treatment of zoonotic staphylococci

Coagulase-positive staphylococci, which frequently colonize the mucosal surfaces of animals, also cause a spectrum of opportunistic infections including skin and soft tissue infections, urinary tract infections, pneumonia, and bacteremia. However, recent advances in bacterial identification have revealed that these common veterinary pathogens are in fact zoonoses that cause serious infections in human patients. The global spread of multidrug-resistant zoonotic staphylococci, in particular the emergence of methicillin-resistant organisms, is now a serious threat to both animal and human welfare. Accordingly, new therapeutic targets that can be exploited to combat staphylococcal infections are urgently needed. Enzymes of the methylerythritol phosphate pathway (MEP) of isoprenoid biosynthesis represent potential targets for treating zoonotic staphylococci. Here we demonstrate that fosmidomycin (FSM) inhibits the first step of the isoprenoid biosynthetic pathway catalyzed by deoxyxylulose phosphate reductoisomerase (DXR) in staphylococci. In addition, we have both enzymatically and structurally determined the mechanism by which FSM elicits its effect. Using a forward genetic screen, the glycerol-3-phosphate transporter GlpT that facilitates FSM uptake was identified in two zoonotic staphylococci, Staphylococcus schleiferi and Staphylococcus pseudintermedius . A series of lipophilic ester prodrugs (termed MEPicides) structurally related to FSM were synthesized, and data indicate that the presence of the prodrug moiety not only substantially increased potency of the inhibitors against staphylococci but also bypassed the need for GlpT-mediated cellular transport. Collectively, our data indicate that the prodrug MEPicides selectively and robustly inhibit DXR in zoonotic staphylococci, and further, that DXR represents a promising, druggable target for future development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Single cell heterogeneity in influenza A virus gene expression shapes the innate antiviral response to infection

Viral infection outcomes are governed by the complex and dynamic interplay between the infecting virus population and the host response. It is increasingly clear that both viral and host cell populations are highly heterogeneous, but little is known about how this heterogeneity influences infection dynamics or viral pathogenicity. To dissect the interactions between influenza A virus (IAV) and host cell heterogeneity, we examined the combined host and viral transcriptomes of thousands of individual cells, each infected with a single IAV virion. We observed complex patterns of viral gene expression and the existence of multiple distinct host transcriptional responses to infection at the single cell level. We show that human H1N1 and H3N2 strains differ significantly in patterns of both viral and host anti-viral gene transcriptional heterogeneity at the single cell level. Our analyses also reveal that semi-infectious particles that fail to express the viral NS can play a dominant role in triggering the innate anti-viral response to infection. Altogether, these data reveal how patterns of viral population heterogeneity can serve as a major determinant of antiviral gene activation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

MAVS mediates a protective immune response in the brain to Rift Valley fever virus

Rift Valley fever virus (RVFV) is a highly pathogenic mosquito-borne virus capable of causing hepatitis, encephalitis, blindness, hemorrhagic syndrome, and death in humans and livestock. Upon aerosol infection with RVFV, the brain is a major site of viral replication and tissue damage, yet pathogenesis in this organ has been understudied. Here, we investigated the immune response in the brain of RVFV infected mice. In response to infection, microglia initiated robust transcriptional upregulation of antiviral immune genes, as well as increased levels of activation markers and cytokine secretion that is dependent on mitochondrial antiviral-signaling protein (MAVS) and independent of toll-like receptors 3 and 7. In vivo , Mavs -/- mice displayed enhanced susceptibility to RVFV as determined by increased brain viral burden and higher mortality. Single-cell RNA sequence analysis identified defects in type I interferon and interferon responsive gene expression within microglia in Mavs -/- mice, as well as dysregulated lymphocyte infiltration. The results of this study provide a crucial step towards understanding the precise molecular mechanisms by which RVFV infection is controlled in the brain and will help inform the development of vaccines and antiviral therapies that are effective in preventing encephalitis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

CovS inactivation reduces CovR promoter binding at diverse virulence factor encoding genes in group A Streptococcus

The control of virulence gene regulator (CovR), also called caspsule synthesis regulator (CsrR), is critical to how the major human pathogen group A Streptococcus fine-tunes virulence factor production. CovR phosphorylation (CovR~P) levels are determined by its cognate sensor kinase CovS, and functional abrogating mutations in CovS can occur in invasive GAS isolates leading to hypervirulence. Presently, the mechanism of CovR-DNA binding specificity is unclear, and the impact of CovS inactivation on global CovR binding has not been assessed. Thus, we performed CovR chromatin immunoprecipitation sequencing (ChIP-seq) analysis in the emm1 strain MGAS2221 and its CovS kinase deficient derivative strain 2221-CovS-E281A. We identified that CovR bound in the promoter regions of nearly all virulence factor encoding genes in the CovR regulon. Additionally, direct CovR binding was observed for numerous genes encoding proteins involved in amino acid metabolism, but we found limited direct CovR binding to genes encoding other transcriptional regulators. The consensus sequence AATRANAAAARVABTAAA was present in the promoters of genes directly regulated by CovR, and mutations of highly conserved positions within this motif relieved CovR repression of the hasA and MGAS2221_0187 promoters. Analysis of strain 2221-CovS-E281A revealed that binding of CovR at repressed, but not activated, promoters is highly dependent on CovR~P state. CovR repressed virulence factor encoding genes could be grouped dependent on how CovR~P dependent variation in DNA binding correlated with gene transcript levels. Taken together, the data show that CovR repression of virulence factor encoding genes is primarily direct in nature, involves binding to a newly-identified DNA binding motif, and is relieved by CovS inactivation. These data provide new mechanistic insights into one of the most important bacterial virulence regulators and allow for subsequent focused investigations into how CovR-DNA interaction at directly controlled promoters impacts GAS pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Strengthening of enterococcal biofilms by Esp

Multidrug-resistant (MDR) Enterococcus faecalis are major causes of hospital-acquired infections. Numerous clinical strains of E . faecalis harbor a large pathogenicity island that encodes enterococcal surface protein (Esp), which is suggested to promote biofilm production and virulence, but this remains controversial. To resolve this issue, we characterized the Esp N-terminal region, the portion implicated in biofilm production. Small angle X-ray scattering indicated that the N-terminal region had a globular head, which consisted of two DEv-Ig domains as visualized by X-ray crystallography, followed by an extended tail. The N-terminal region was not required for biofilm production but instead significantly strengthened biofilms against mechanical or degradative disruption, greatly increasing retention of Enterococcus within biofilms. Biofilm strengthening required low pH, which resulted in Esp unfolding, aggregating, and forming amyloid-like structures. The pH threshold for biofilm strengthening depended on protein stability. A truncated fragment of the first DEv-Ig domain, plausibly generated by a host protease, was the least stable and sufficient to strengthen biofilms at pH ≤ 5.0, while the entire N-terminal region and intact Esp on the enterococcal surface was more stable and required a pH ≤ 4.3. These results suggested a virulence role of Esp in strengthening enterococcal biofilms in acidic abiotic or host environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae

It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae . A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses in S . cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S . cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum . Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae . The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S . cerevisiae .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Quantum-enhanced detection of viral cDNA via luminescence resonance energy transfer using upconversion and gold nanoparticles

Abstract The COVID-19 pandemic has profoundly impacted global economies and healthcare systems, revealing critical vulnerabilities in both. In response, our study introduces a sensitive and highly specific detection method for cDNA, leveraging Luminescence Resonance Energy Transfer (LRET) between upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs), and achieves a detection limit of 242 fM for SARS-CoV-2 cDNA. This innovative sensing platform utilizes UCNPs conjugated with one primer and AuNPs with another, targeting the 5′ and 3′ ends of the SARS-CoV-2 cDNA, respectively, enabling precise differentiation of mismatched cDNA sequences and significantly improving detection specificity. Through rigorous experimental analysis, we established a quenching efficiency range from 10.4 % to 73.6 %, with an optimal midpoint of 42 %, thereby demonstrating the superior sensitivity of our method. Our work uses SARS-CoV-2 cDNA as a model system to demonstrate the potential of our LRET-based detection method. This proof-of-concept study highlights the adaptability of our platform for future diagnostic applications. Instrumental validation confirms the synthesis and formation of AuNPs, addressing the need for experimental verification of the preparation of nanomaterial. Our comparative analysis with existing SARS-CoV-2 detection methods revealed that our approach provides a low detection limit and high specificity for target cDNA sequences, underscoring its potential for targeted COVID-19 diagnostics. This study demonstrates the superior sensitivity and adaptability of using UCNPs and AuNPs for cDNA detection, offering significant advances in rapid, accessible diagnostic technologies. Our method, characterized by its low detection limit and high precision, represents a critical step forward in developing next-generation biosensors for managing current and future viral outbreaks. By adjusting primer sequences, this platform can be tailored to detect other pathogens, contributing to the enhancement of global healthcare responsiveness and infectious disease control.

Esmaeili, Shahriar [Institute for Quantum Science ↗