Detection of Vibrio vulnificus in Seafood With a DNAzyme-Based Biosensor.
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ABSTRACT:
SUBMITTER: Fan S
PROVIDER: S-EPMC8213197 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:As an important foodborne pathogen, Vibrio vulnificus gives a significant threat to food safety and public health. Rapid and accurate detection methods for V. vulnificus are required to control its spread. The conventional detection methods are time-consuming and labor-intensive, while the polymerase chain reaction (PCR)- and quantitative PCR (qPCR)-based methods are limited because of their dependence on laboratory equipment. Nucleic acid isothermal amplification technologies have been applied to develop simpler assays. In this study, a rapid detection method based on real-time recombinase polymerase amplification (RPA) targeting the extracellular metalloprotease (empV) gene of V. vulnificus has been established. The method finished the detection in 2-14 min at 39°C with good specificity. The limit of detection was 17 gene copies or 1 colony-forming unit (CFU) per reaction, or 1 CFU/10 g of spiked food with enrichment. In a clinical sample detection test, the results of real-time RPA were 100% consistent with bioassay and qPCR. Moreover, the method could resist the effect of food matrix and could tolerate crude templates. The real-time RPA method established in this study is rapid and simple and has the potential to be widely applied for V. vulnificus detection in food safety control.
| S-EPMC7677453 | biostudies-literature
Project description:Nipah virus (NiV) is a zoonotic RNA virus which infects humans and animals in Asian countries. Infection in humans occurs in different forms, from asymptomatic infection to fatal encephalitis, and death occurred in 40-70% of those infected in outbreaks that occurred between 1998 and 2018. Modern diagnostics is carried out by real-time PCR to identify pathogens or by ELISA to detect antibodies. Both technologies are labor-intensive and require the use of expensive stationary equipment. Thus, there is a need to develop alternative simple, fast and accurate test systems for virus detection. The aim of this study was to develop a highly specific and easily standardized system for the detection of Nipah virus RNA. In our work, we have developed a design for a Dz_NiV biosensor based on a split catalytic core of deoxyribozyme 10-23. It was shown that the assembly of active 10-23 DNAzymes occurred only in the presence of synthetic target Nipah virus RNA and that this was accompanied by stable fluorescence signals from the cleaved fluorescent substrates. This process was realized at 37 °C, pH 7.5, and in the presence of magnesium ions, with a 10 nM limit of detection achieved for the synthetic target RNA. Constructed via a simple and easily modifiable process, our biosensor may be used for the detection of other RNA viruses.
| S-EPMC9954549 | biostudies-literature
Project description:It has been observed that not all strains of Vibrio vulnificus are virulent. Determining the virulence of strains that are frequently present in seafood is of significance for ensuring seafood safety. This study is an attempt to predict the virulence of seafood-borne V. vulnificus isolated along the Mangaluru Coast, India. The isolates tested possessed a vcgC gene sequence with high similarity to that in the clinical strain. Transcriptional analysis of core virulence genes in seafood isolate E4010 showed the phenomenon of contact-mediated expression of rtxA1 which correlated well with the actin disintegration and cytotoxicity. These results suggest that the seafood isolates tested in this study possess a functional RtxA1 which could help in initiating the infection. However, other putative virulence genes such as vvpE encoding an extracellular protease, vvhA encoding hemolysin, flp encoding tad pilin and ompU encoding fibronectin-binding protein were also constitutively expressed. Virulence-associated attributes such as cytotoxicity and adherence matched the response of the clinical strain (p > 0.05). On the other hand, the environmental strains showed higher serum sensitivity compared with the clinical strain. These findings show that the part of virulence attributes required for the disease process might be intact in these isolates.
| S-EPMC7409051 | biostudies-literature
Project description:The level of pyrophosphatase (PPase) expression has been suggested as a potential biomarker of various cancers, and its prognostic value has been evaluated in patients suffering from lung cancer, colorectal cancer, and hyperthyroidism. However, the detection of PPase usually needs specific materials that require complicated, time-consuming reactions with restricted linear range and sensitivity, limiting their application in early clinical diagnosis. Herein, we developed a DNAzyme-based biosensor for the detection of PPase. In the presence of PPase, pyrophosphate (PPi) and Cu2+ ions released from the PPi-Cu2+-PPi complex induce the cleavage of the DNAzyme and the corresponding substrate. An apurinic/apyrimidinic (AP) site was elaborately designed within substrates that could encase the fluorophore 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND). The fluorescence of ATMND was initially quenched but restored when the DNAzyme/substrate complex was hydrolyzed with the release of ATMND. In this way, the PPase activity can be estimated by detecting the increased fluorescence of the released ATMND. Under optimized conditions, the activity of PPase could be analyzed at concentrations from 0.5 to 1000 mU, with the lowest detectable concentration being 0.5 mU. This work lays a foundation for developing a DNAzyme-amplified fluorescent biosensor with a high sensitivity, a wide linear range, and single-step operation for use as an easy diagnostic for PPase analysis.
| S-EPMC8615721 | biostudies-literature
Project description:G-quadruplexes can bind with hemin to form peroxidase-like DNAzymes that are widely used in the design of biosensors. However, the catalytic activity of G-quadruplex/hemin DNAzyme is relatively low compared with natural peroxidase, which hampers its sensitivity and, thus, its application in the detection of nucleic acids. In this study, we developed a high-sensitivity biosensor targeting norovirus nucleic acids through rationally introducing a dimeric G-quadruplex structure into the DNAzyme. In this strategy, two separate molecular beacons each having a G-quadruplex-forming sequence embedded in the stem structure are brought together through hybridization with a target DNA strand, and thus forms a three-way junction architecture and allows a dimeric G-quadruplex to form, which, upon binding with hemin, has a synergistic enhancement of catalytic activities. This provides a high-sensitivity colorimetric readout by the catalyzing H2O2-mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline -6-sulfonic acid) diammonium salt (ABTS). Up to 10 nM of target DNA can be detected through colorimetric observation with the naked eye using our strategy. Hence, our approach provides a non-amplifying, non-labeling, simple-operating, cost-effective colorimetric biosensing method for target nucleic acids, such as norovirus-conserved sequence detection, and highlights the further implication of higher-order multimerized G-quadruplex structures in the design of high-sensitivity biosensors.
| S-EPMC8659037 | biostudies-literature
Project description:Vibrio vulnificus is an opportunistic human pathogen, transmitted from seawater, raw oyster, and shellfish and responsible for severe septicemia. We studied V. vulnificus from surface seawater around Jeju Island between 2010 and 2011. In 2010, V. vulnificus was isolated and V. vulnificus septicemia was reported. Surface seawater temperature is an important factor for growth of V. vulnificus, and here we showed that high surface seawater temperature may influence growth of V. vulnificus and occurrence of emerging V. vulnificus septicemia on Jeju Island. This is the first report of isolation of V. vulnificus and emerging V. vulnificus septicemia on Jeju Island.
| S-EPMC4091370 | biostudies-literature
Project description:Between 1996 and 2006, the US Centers for Disease Control reported that the only category of food-borne infections increasing in frequency were those caused by members of the genus Vibrio. The gram-negative bacterium Vibrio vulnificus is a ubiquitous inhabitant of estuarine waters, and is the number one cause of seafood-related deaths in the US. Many V. vulnificus isolates have been studied, and it has been shown that two genetically distinct subtypes, distinguished by 16S rDNA and other gene polymorphisms, are associated predominantly with either environmental or clinical isolation. While local genetic differences between the subtypes have been probed, only the genomes of clinical isolates have so far been completely sequenced. In order to better understand V. vulnificus as an agent of disease and to identify the molecular components of its virulence mechanisms, we have completed whole genome shotgun sequencing of three diverse environmental genotypes using a pyrosequencing approach. V. vulnificus strain JY1305 was sequenced to a depth of 33×, and strains E64MW and JY1701 were sequenced to lesser depth, covering approximately 99.9% of each genome. We have performed a comparative analysis of these sequences against the previously published sequences of three V. vulnificus clinical isolates. We find that the genome of V. vulnificus is dynamic, with 1.27% of genes in the C-genotype genomes not found in the E- genotype genomes. We identified key genes that differentiate between the genomes of the clinical and environmental genotypes. 167 genes were found to be specifically associated with environmental genotypes and 278 genes with clinical genotypes. Genes specific to the clinical strains include components of sialic acid catabolism, mannitol fermentation, and a component of a Type IV secretory pathway VirB4, as well as several other genes with potential significance for human virulence. Genes specific to environmental strains included several that may have implications for the balance between self-preservation under stress and nutritional competence.
| S-EPMC3360785 | biostudies-literature
Project description:Vibrio vulnificus an opportunistic human pathogen native to marine/estuarine environment, is one of the leading causes of death due to seafood consumption and exposure of wounds to seawater worldwide. The present study involves the whole genome sequence analysis of an environmental strain of V. vulnificus (clinical genotype) isolated from seafood along the Mangaluru coast of India. The sequenced genome data was subjected to in-silico analysis of phylogeny, virulence genes, antimicrobial resistance determinants, and secretary proteins using suitable bioinformatics tools. The sequenced isolate had an overall genome length of 4.8 Mb and GC content of 46% with 4400 coding DNA sequences. The sequenced strain belongs to a new sequence type (Multilocus sequence typing) and was also found to branch with a phylogenetic lineage that groups the most infectious strains of V. vulnificus. The seafood isolate had complete genes involved in conferring serum resistance yet showed limited serum resistance. The study identified several genes against the antibiotics that are commonly used in their treatment, highlighting the need for alternative treatments. Also, the secretory protein analysis revealed genes associated with major pathways like ABC transporters, two-component systems, quorum sensing, biofilm formation, cationic antimicrobial peptide (CAMP) resistance, and others that play a critical role in the pathogenesis of the V. vulnificus. To the best of our knowledge, this is the first report of a detailed analysis of the genomic information of a V. vulnificus isolated from the Indian subcontinent and provides evidence that raises public health concerns about the safety of seafood.
| S-EPMC10438861 | biostudies-literature
Project description:The recent discovery of microRNAs (miRNAs) and their extracellular presence suggest a potential role of these regulatory molecules in defining the metastatic potential of cancer cells and mediating the cancer-host communication. This study aims to improve the sensitivity of miRNA detection via DNAzyme-based method and enhance the selectivity by using the DNAzyme-based probe to reduce nonspecific amplification.The miRNA probes were chemically synthesized with a phosphate at the 5' end and purified by polyacrylamide gel electrophoresis. Exosomal RNA from peripheral blood was isolated. Carboxylated magnetic microsphere beads (MBs) were functionalized with streptavidin (SA) according to a previously reported method with some modification. T capture probe-coated SA-MBs (DNA-MBs) were also prepared. The fluorescent spectra were measured using a spectrofluorophotometer.We designed an incomplete DNAzyme probe with two stems and one bubble structure as a recognition element for the specific detection of miRNA with high sensitivity. The background effects were decreased with increase of the added of DNA-MBs and capturing times. Therefore, 20 minutes was selected as the optimal concentration in the current study. The fluorescence intensity increases as the hybridization time changed and reached a constant level at 40 minutes, and 1 ?M is the optimum signal probe concentration for self-assembled DNA concatemers formation. In the presence of miRNA, the fluorescence of the solution increased with increasing miRNA concentration. There is no obvious fluorescence in the presence of 10 mM of other nontarget DNA.A simple, rapid method with high performance has been constructed based on identified circulating miRNA signatures using miRNA-induced DNAzyme. This assay is simple, inexpensive, and sensitive, enabling quantitative detection of as low as 10 fM miRNA.
| S-EPMC4655907 | biostudies-other
Project description:Vibrio vulnificus is a free-living marine bacterium associated with the contamination of fish and shellfish-the most consumed seafood in Asia. Owing to its potentially lethal clinical consequences, the consumption of seafood contaminated with V. vulnificus has become a growing public health concern. This systematic review with meta-analysis and meta-regression aimed to integrate data on the prevalence of seafood-borne V. vulnificus specifically in Asia and assess the potential risk factors that can influence the outcomes. A comprehensive literature search of four electronic databases yielded 279 relevant studies, among which 38 fulfilled the inclusion criteria. These selected studies were subjected to risk-of-bias assessment and data extraction by three independent researchers. A meta-analysis of the eligible studies estimated the overall prevalence of seafood-borne V. vulnificus in Asia to be 10.47% [95% confidence interval (CI): 6.8-15.8%], with bivalve shellfish, such as oysters, mussels, clams, and cockles being the most contaminated seafood. The highest prevalence was reported in Japan, where 47.6% of the seafood samples tested positive for V. vulnificus. The subgroup and meta-regression analyses identified three potential covariates-detection method, publication year, and country-associated with between-study heterogeneity. Furthermore, data visualization displayed the variations in V. vulnificus prevalence across the studies, associated with differences in sample type, sample size, and sampling stage. This study provides valuable insights into the prevalence of V. vulnificus in fish and shellfish across the entire Asian continent and highlights the potential factors that cause variation in the prevalence rates among the studies. These findings underscore the importance of enhancing hygiene measures throughout the seafood supply chain to mitigate V. vulnificus infection risks and ensure the safety of consumers.
| S-EPMC10951106 | biostudies-literature