DNA Hybridization Method Research Articles

Impact of Pericoronary Microbiota Composition on Course of Recovery after Third Molar Alveotomy.

Although the role of microbiota has been investigated in relation to different oral diseases, it is unknown if its composition has any effect on the course of recovery after third molar alveotomy. Our aim was to determine the influence of patient clinical characteristics as well as pericoronary microbiota composition on the course of recovery after a semi-impacted third molar alveotomy. Thirty-six patients were included and samples obtained with paper points, swabs, and tissue samples were analyzed using DNA hybridization and culture methods. Among the 295 organisms detected, the most frequent were Streptococcus spp. (22.4%; 66/295) followed by Fusobacterium spp. (11.9%; 35/295), and T. forsythia (9.1%; 27/295). A comparison of microbiota composition in patients with better and worse recovery did not show significant differences. Worse recovery outcomes were more frequent in patients with a grade 2 self-assessment of oral health (p = 0.040) and better recovery courses were observed in patients with a grade 4 self-assessment (p = 0.0200). A worse recovery course was statistically significant more frequently in patients with previous oral surgical procedures (p = 0.019). Although we demonstrate that worse recovery outcomes were more frequent when certain bacteria were detected, there was no statistically significant difference. Further research is needed to identify microbial profiles specific to the development of worse outcomes after a third molar alveotomy.

A Molecular Approach for Detecting Bacteria and Fungi in Healthcare Environment Aerosols: A Systematic Review.

Molecular methods have become integral to microbiological research for microbial identification. This literature review focuses on the application of molecular methods in examining airborne bacteria and fungi in healthcare facilities. In January 2024, a comprehensive electronic search was carried out in esteemed databases including PubMed, Web of Science, and Scopus, employing carefully selected keywords such as ((bacteria) OR (virus) OR (fungi)) AND (aerosol) AND ((hospital) OR (healthcare) OR (dental office)) AND ((molecular) OR (PCR) OR (NGS) OR (RNA) OR (DNA) OR (metagenomic) OR (microarray)), following the PRISMA protocol. The review specifically targets healthcare environments with elevated concentrations of pathogenic bacteria. A total of 487 articles were initially identified, but only 13 met the inclusion criteria and were included in the review. The study disclosed that the prevalent molecular methodology for appraising aerosol quality encompassed the utilization of the PCR method, incorporating either 16S rRNA (bacteria) or 18S rRNA (fungi) amplification techniques. Notably, five diverse molecular techniques, specifically PFGE, DGGE, SBT, LAMP, and DNA hybridization methods, were implemented in five distinct studies. These molecular tests exhibited superior capabilities compared to traditional bacterial and fungal cultures, providing precise strain identification. Additionally, the molecular methods allowed the detection of gene sequences associated with antibiotic resistance. In conclusion, molecular testing offers significant advantages over classical microbiological culture, providing more comprehensive information.

Amplification-free quantitative detection of genomic DNA using lateral flow strips for milk authentication

With the globalization and complexity of the food supply chain, the market is becoming increasingly competitive and food fraudulent activities are intensifying. The current state of food detection faced two primary challenges. Firstly, existing testing methods were predominantly laboratory-based, requiring complex procedures and precision instruments. Secondly, there was a lack of accurate and efficient quantitative detection methods. Taking cow's milk as an example, this study introduced a novel method for nucleic acid quantification in dairy products, based on lateral flow strips (LFS). The core idea of this method is to design single-stranded DNA (ssDNA) probes to hybridize with mitochondrial genes, which are abundant, stable, and species-specific in dairy products, as detection targets. Drawing inspiration from the principles of nucleic acid amplification, this research innovatively established a new DNA hybridization method, named LAMP-Like Hybridization (HybLAMP-Like). Leveraging the denaturation and DNA polymerization functions of the bst enzyme, efficient binding of the probe and template strand was achieved. This method eliminated the need for nucleic acid amplification, simplifying the procedure and mitigating aerosol contamination, thereby ensuring the accuracy of the detection results. The method exhibited exceptional sensitivity, capable of detecting extremely low to 12.5 ng in visual inspection and 3.125 ng when using a reader. In terms of practicality, it could achieve visual detection of cow's milk content as low as 1% in adulterated dairy products. When combined with a portable LFS reader, it also enabled precise quantitative analysis of milk adulteration.

Miniature Optical Fiber DNA Hybridization Sensor With Temperature Compensation Using a Gold Nanofilm-Coated Optical Fiber

DNA hybridization method is one of the most important techniques for detecting new disease genes. However, temperature cross-sensitivity is ubiquitously found in the DNA hybridization process, which may cause DNA damage and even produce false positives. An optical DNA hybridization sensor with temperature compensation based on dual surface plasmon resonance (SPR) effect by using a gold nanofilm coating optical fiber was proposed and demonstrated. Two parameters of DNA and temperature detection were realized by different functional modifications of the gold nanofilm. And a matrix equation was employed to analyze the relationship between two variables. Lung cancer-related genes exon-20 gene fragment was successfully detected in our work. From the theoretical and experimental results, the DNA detection limit was 9 nmol/L, and the temperature sensitivity reaches -1.74 nm/°C. The designed sensor could detect DNA with low concentration and measure temperature at the same time. The proposed sensor has the potential to be applied in disease marker gene detection with temperature compensation.

Species diversity of microorganisms previously identified as nontuberculous mycobacteria by DNA hybridization.

Over the past 10 years, the clinical importance of opportunistic bacteria of the order Actinomycetales has increased significantly. While many problems for the Mycobacterium tuberculosis complex have been solved, for nontuberculous mycobacteria, some questions remain open. These pathogens have a number of structural features that allow them to persist in the external environment for a long time. The main inclusion criteria were cultural characteristics in assessing the growth of microorganisms on solid egg media. If nontuberculous mycobacteria (NTM) growth was detected, identification signs were carried out using the DNA hybridization method. Subsequently, these cultures were identified using the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-ToF) mass spectrometry method. In case of obtaining unacceptable results of identification from primary inoculations, re-identification to obtain pure cultures was carried out after transferring the material from primary media to agar media: 5% blood agar and universal chromogenic medium. When re-identifying isolated cultures using MALDI-ToF mass spectrometry, all isolated cultures were analyzed, regardless of whether they belonged to the NTM group or not. DNA hybridization, which accounted for 59.5% of the total number of cultures included in the study, performed species identification of 188 strains. Using MALDI-ToF mass spectrometry, 345 strains were identified. The use of methods based on DNA hybridization makes it possible to identify quite accurately some of the most common NTM species. MALDI-ToF mass spectrometry is an important technique to allow species identification of most Actinomycetales. However, algorithms to standardize methods for their isolation from clinical material are needed.

Chromosome conformation capture-based DNA hybridization method for chromosomal translocation screening.

Background: DNA probes have been widely used as diagnostic tools for translocations. This study sought to design a screening tool using ssDNA probes and chromosome conformation capture (3C) library fragment hybridization. Method: The authors focused on developing a probe for the juxtaposed region of MYC and TRD. Fragments of the MYC gene with a thiol modification (MYC-Au NP probe) were functionalized bygold nanoparticles (Au NPs). Then TRDprobes were immobilized on a nitrocellulose surface. Hybridization between DNA probes and 3C library fragments of SKW3 cells was determined by color intensity. Results: Optimal hybridization of the 3C library sample of the cell line to probes showed higher color intensity than human umbilical vein endothelial cells. Conclusion: Combining 3C-based techniques and DNA-DNA hybridization can identify rearrangements in cancer cells.

β-Lactam Resistance Gene NDM-1 in the Aquatic Environment: A Review.

New Delhi Metallo-β-lactamase-1 (NDM-1) offers carbapenem antibiotics resistance that creates an evolving challenge in treating bacterial infections. NDM-1-bearing strains were observed in surface waters around New Delhi in 2010 and after then identified globally. The usage of antibiotics may hasten the growth of the NDM-1-producing bacteria, which pose severe hazards to human and animal health. The emergence of the NDM-1 in the aquatic environment is turning out to be a growing concern worldwide. NDM-1 gene conferring resistance to a widespread class of antibiotics has been observed in bacteria disseminated in animal production wastewaters, hospital sewage, domestic sewage, industrial effluents, wastewater treatment plants, drinking water, surface water, and even in groundwater. This review recapitulates the currently published research studies on the prevalence and geographical distribution of the NDM-1 gene in the aquatic environment, its habitats, and healthcare risk associated with NDM-1-producing bacteria, in addition to molecular techniques employed to reveal the occurrence of the NDM-1 in the aquatic environment, including conventional polymerase chain reaction, real-time qPCR, DNA hybridization, and microarray-based methods.

Rare species in the structure of acid-resistent members of the order Actinomycetales isolated from clinical material.

The article presents data on the structure of acid-resistant members of the order Actinomycetales and rare species that have been isolated and identified using various methods. The study included strains of non-tuberculous mycobacteria (NTM) isolated from clinical material during examination for tuberculosis in the period from 2016 to 2019. The total number of samples with signs of NTMs growth that were included in the study was 316 samples. Primary isolation on Levenshtein-Jensen, Finn II, and MGIT media and NTMs identification by DNA-hybridization. All strains that were not identified prior to the species and culture, identified as microorganisms with a high G+C content (High GC GR +) were re-identified using a MALDI-ToF Microflex LT mass spectrometer (Bruker®). By the method of DNA-hybridization, 188 strains isolated by NTM were successfully identified to form 58.5% of all selected cultures. Among the selected species, representatives of slowly growing NTMs (M. avium complex, M. gordonae, M. kansasii) predominated, which amounted to 67.0% of all NTM strains identified to the species. Among the cultures for which DNA hybridization failed to carry out acceptable identification, predominantly NTMs were found, among which M. gordonae, M. avium, M. kansasii dominated. A number of NTMs were represented by rare species: M. iranicum and M. pseudoshottsii. Among this group of microorganisms, other acid-resistant aerobic actinomycetes were isolated, including those of potential clinical significance: Gordonia spp., Tsukamurella spp., Rhodococcus spp., Nocardia spp. When identifying cultures containing high concentrations of G+C, the maximum number of microbial associations was revealed, including those consisting of two types of NTMs (M. monacense + M. flavescens, M. avium + M. kansasii), as well as associations of M. gordonae with staphylococci. The same group included rare NTM species: M. fredericbergense, M. szulgai, M. malmoense, M. bohemicum, M. septicum, as well as representatives of the genera Nocardia, Gordonia, Tsukamurella.

Antimicrobial effect of the essential oil of Oregano against Listeria monocytogenes and Staphylococcus aureus.

The food industry uses chemical compounds to prevent the growth of bacteria and extend their shelf life. These substances are associated with toxic problems, the presence of chemical residues and antimicrobial resistance. Oregano essential oil has antimicrobial compounds such as thymol and carvacrol. The objective of the research was to evaluate the antimicrobial effect of oregano essential oil from the province of Concepción, Junín Region (Peru) against Listeria monocytogenes and Staphylococcus aureus. Oregano was taxonomically identified as O. vulgare. The essential oil was obtained by hydrodistillation and the profile of chemical compounds was determined by Gas Chromatography/Mass Spectrometry (GC-MS). The minimum inhibitory concentration (MIC) was determined by microdilution and the minimum bactericidal concentration (CMB) by agar planting Baird-Parker Agar was used for S. aureus and for L. monocytogenes the DNA hybridization method was used using the Gene Quencekit® L. monocytogenes. The GC-MS profile of oregano oil had 11.9% thymol and 1.7% carvacrol. The oil exhibited antimicrobial activity against S. aureus (CMI 2%, CMB 4%) and L. monocytogenes (CMI-4%, CMB-4%). The high percentage of CMI and CMB was due to the low concentration of carvacrol and thymol, however this oil can be used in the food industry as a natural antimicrobial and/or disinfectant.

Development of tracking tool for p-nitrophenol monooxygenase genes from soil augmented with p-Nitrophenol degrading isolates: Bacillus, Pseudomonas and Arthrobacter

The tracking of in situ-bioremediation of paranitrophenol through functional screening is time consuming. In this study we address a quick, reliable polymerase chain reaction-based method to detect the paranitrophenol monooxygenase gene from soil. All paranitrophenol degrading strains viz. Pseudomonas P19, Bacillus species C1, Bacillus species C2 and Arthrobacter species were isolated from contaminated agricultural soil. DNA was isolated from pure strains as well as soil inoculated with these strains. DNA extraction protocol was modified by including Cetyl Trimethyl Ammonium Bromide and sodium metabisulphite instead of polyvinylpolyprrolidone in polyethylene glycol method. Paranitrophenol monooxygenase gene was screened using degenerate primers designed to give 100 base pair amplification products. All strains used in study showed amplification product corresponding to target region using designed degenerate primers. Modified polyethylene glycol method leads to increase in DNA yield from soil. The assay technique is sensitive with broad specificity and has application in rapid identification of paranitrophenol monooxygenase gene. This method has advantage over DNA hybridization method as needs less DNA. The present study demonstrated the detection and monitoring of paranitrophenol monooxygenase gene from soil sample inoculated with either Gram-positive or Gram-negative bacteria using degenerate primers.

A novel nucleic acid based bio-assay toward recognition of Haemophilus influenza using bioconjugation and DNA hybridization method

Haemophilus influenza (H. influenza) is a gram negative coccobacillus pathogenic microorganism. H. influenza produces beta-lactamases, and it is also able to modify its penicillin-binding proteins, so it has gained resistance to the penicillin family of antibiotics. In this work, a novel sensitive approach was established for the monitoring of H. influenza using DNA based bio-assay. For the first time, specific sequence of thiolated probe of Haemophilus influenza (SH-5′-AAT TTT CCA ACT TTT TCA CCT GCA T-3′) was immobilized on the surface of gold (Au) electrode. Square wave voltammetry (SWV) was carried out in toluidine blue (TB) solution for DNA hybridization and targeting of cDNA sequence of Haemophilus influenza. Field scanning electron microscope (FE-SEM) was applied to investigation of the electrode morphology and estimate of particle size. In the optimal conditions, the planned strategy could detect target DNA (5′-ATG CAG GTG AAA AAG TTG GAA AAT T-3′) down to 1 ZM with a linear range from 1 μM to 1 ZM. Moreover, engineered geno-assay selectively differentiates the complementary sequence from target sequences with one, double and three base mismatch sequences.

KIT DIAGNOSTIK DIFTERI BERBASIS HIBRIDISASI DNA

Introduction: Diphtheria is an upper respiratory tract infection caused by Corynebacterium diphtheria bacterium. The recent diphtheria outbreak caused a large number of deaths in rapid period. This is due to the absence of a fast and precise method of diphtheria diagnosis. Current diagnosis practices aredone by cultivating bacteria which takes a relatively long time, which is 3 to 5 days, while the patients may already develop severe infection by the time the results are accessible. Therefore, a sensitive, easy and fast diphtheria diagnostic method is needed so that patients suspected of being infected can betreated quicker.Methods: This advertorial is written based on literature study method.Result: The diphtheria diagnostic kit was designed based on the DNA hybridization method by using DNA microarray. There are probes on the microarray plate in the form of a single stranded DNA fragment that is complement to the unique area of gene that encodes diphtheria toxin. The test sample wasobtained from the preparation of the patient's throat smear and labeled with a fluorescence marker before dropped onto the plate. Thus, if there is diphtheria DNA in the patient's sample, hybridization will occur between diphtheria DNA and the complementary DNA probe. The occurrence of hybridization will be characterized by fluorescence as an indicator of whether patients have diphtheria or not. The application of this diagnostic kit can shorten the diagnosis of C. diphtheria to less than 24 hours .Conclusion: Diphtheria diagnostic kit based on DNA hybridization can be made and applied.

Antimicrobial Susceptibilities of Oral Isolates of Abiotrophia and Granulicatella According to the Consensus Guidelines for Fastidious Bacteria.

Background: The genera Abiotrophia and Granulicatella, previously known as nutritionally variant streptococci (NVS), are fastidious bacteria requiring vitamin B6 analogs for growth. They are members of human normal oral microbiota, and are supposed to be one of the important pathogens for so-called “culture-negative” endocarditis. Methods: The type strains and oral isolates identified, by using both phenotypic profiles and the DNA–DNA hybridization method, were examined for susceptibilities to 15 antimicrobial agents including penicillin (benzylpenicillin, ampicillin, amoxicillin, and piperacillin), cephem (cefazolin, ceftazidime, ceftriaxone, and cefaclor), carbapenem (imipenem), aminoglycoside (gentamicin), macrolide (erythromycin), quinolone (ciprofloxacin), tetracycline (minocycline), glycopeptide (vancomycin), and trimethoprim-sulfamethoxazole complex. The minimum inhibitory concentration and susceptibility criterion were determined, according to the consensus guideline from the Clinical and Laboratory Standards Institute. Results: Isolates of Abiotrophia defectiva were susceptible to ampicillin, amoxicillin ceftriaxone, cefaclor, imipenem, ciprofloxacin, and vancomycin. Isolates of Granulicatella adiacens were mostly susceptible to benzylpenicillin, ampicillin, amoxicillin, cefazolin, ceftriaxone, imipenem, minocycline, and vancomycin. The susceptibility profile of Granulicatella elegans was similar to that of G. adiacens, and the susceptibility rate was higher than that of G. adiacens. Conclusions: Although Abiotrophia and Granulicatella strains are hardly distinguishable by their phenotypic characteristics, their susceptibility profiles to the antimicrobial agents were different among the species. Species-related differences in susceptibility of antibiotics should be considered in the clinical treatment for NVS related infections.

Measuring DNA hybridization using fluorescent DNA-stabilized silver clusters to investigate mismatch effects on therapeutic oligonucleotides

BackgroundShort nucleic acid oligomers have found a wide range of applications in experimental physics, biology and medicine, and show potential for the treatment of acquired and genetic diseases. These applications rely heavily on the predictability of hybridization through Watson–Crick base pairing to allow positioning on a nanometer scale, as well as binding to the target transcripts, but also off-target binding to transcripts with partial homology. These effects are of particular importance in the development of therapeutic oligonucleotides, where off-target effects caused by the binding of mismatched sequences need to be avoided.ResultsWe employ a novel method of probing DNA hybridization using optically active DNA-stabilized silver clusters (Ag-DNA) to measure binding efficiencies through a change in fluorescence intensity. In this way we can determine their location-specific sensitivity to individual mismatches in the sequence. The results reveal a strong dependence of the hybridization on the location of the mismatch, whereby mismatches close to the edges and center show a relatively minor impact. In parallel, we propose a simple model for calculating the annealing ratios of mismatched DNA sequences, which supports our experimental results.ConclusionThe primary result shown in this work is a demonstration of a novel technique to measure DNA hybridization using fluorescent Ag-DNA. With this technique, we investigated the effect of mismatches on the hybridization efficiency, and found a significant dependence on the location of individual mismatches. These effects are strongly influenced by the length of the used oligonucleotides. The novel probe method based on fluorescent Ag-DNA functions as a reliable tool in measuring this behavior. As a secondary result, we formulated a simple model that is consistent with the experimental data.

Bovine leukemia virus linked to breast cancer but not coinfection with human papillomavirus: Case-control study of women in Texas.

Bovine leukemia virus (BLV) and human papillomavirus (HPV) were previously identified in human breast tissue and have been associated with breast cancer in independent studies. The objective of the current study was to test for the presence of BLV and HPV in the same breast tissue specimens to determine whether the viruses were associated with breast cancer either singly or together. Archival formalin-fixed paraffin-embedded breast tissue sections from 216 women were received from The University of Texas MD Anderson Cancer Center along with patient diagnosis. In situ polymerase chain reaction and/or DNA hybridization methods were used to detect targeted DNA segments of BLV and HPV. Standard statistical methods were used to calculate age-adjusted odds ratios, attributable risk, and P values for the trend related to the association between presence of a virus and a diagnosis of breast disease. Women diagnosed with breast cancer were significantly more likely to have BLV DNA in their breast tissue compared with women with benign diagnoses and no history of breast cancer. Women with breast pathology classified as premalignant and no history of breast cancer also were found to have an elevated risk of harboring BLV DNA in their breast tissue. HPV status was not associated with malignancy, premalignant breast disease, or the presence of BLV in the breast tissues. The data from the current study supported previous findings of a significant association between BLV DNA in breast tissue and a diagnosis of breast cancer, but did not demonstrate oncogenic strains of HPV associated with breast cancer or the presence of BLV DNA in breast tissue. The authors believe the findings of the current study contribute to overall knowledge regarding a possible causal role for viruses in human breast cancer. Cancer 2018;124:1342-9. © 2017 American Cancer Society.

Genomic identification of microbial species adhering to maxillofacial prostheses and susceptibility to different hygiene protocols

This study investigated the microbial colonization of maxillofacial prostheses and support tissues using the Checkerboard DNA–DNA hybridization method, and the efficacy of 0.12% chlorhexidine gluconate, 10% Ricinus communis solutions, or brushing, on colony forming unit (CFU) reduction in monospecies biofilms (Candida glabrata, Staphylococcus aureus, Streptococcus mutans, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa) formed on two silicones (MDX 4-4210 and Bio-Skin). Biofilm was harvested from 43 maxillofacial prosthesis wearers for detection of 38 species of microorganisms. The CFU counts of the six above mentioned species were recorded after using the hygiene protocols. All 38 investigated species were identified in prostheses and tissues, with a higher prevalence in the prostheses. 0.12% chlorhexidine gluconate immersion showed the greatest antimicrobial effectiveness, followed by mechanical brushing protocols. MDX 4-4210 silicone produced lower CFU counts than Bio-Skin.

COMPARATIVE ANALYSIS OF METHODS FOR IDENTIFICATION OF NONTUBERCULOUS MYCOBACTERIA ISOLATED FROM CLINICAL MATERIAL

Recently there has been a significant increase in the incidence of mycobacteriosis, which is due to an increase in the proportion of immunosuppressed patients, the presence of these various comorbid conditions, as well as the improvement of diagnostic methods. Selecting the most accurate method of identification is extremely important in determining treatment strategy of patients. The aim of the study was to conduct a comparative analysis of modern methods of identification NTMB isolated from clinical specimens in 2015 in the Samara region. The work was carried out identification of 78 strains of microorganisms. Laboratory diagnosis was carried out using the DNA hybridization method and MALDI-ToF mass spectrometry. When microbial identification using MALDI-ToF mass spectrometry was isolated 16 strains (20.5%) M. kansasii; 11 strains (14.1%) M. avium and M. fortuitum; 9 strains (11.5%) M. gordonae; strain 3 (3.8%) M. peregrinum, M. szulgai, M. chimera intracellulare group, strain 2 (2.6%) M. abscessus, M. septicum, M. paragordonae, M. senegalence, 1 strain (1.3%) M. chelonae, M. frederiksbergense, M. monacense, M. lentiflavum. By using mass spectrometry, it was identified 15 types NTMB compared with 9 types — by DNA hybridization. Full match identification results was observed only in 45 (57.7%) strains of divergent strains were found in 16 (20.5%). Most often when using the DNA hybridization method, discrepancy was detected in slow-growing cultures (9 strains) with a predominance of microorganisms identified as M. gordonae. Among the representatives of fast-growing NTMB, seven investigations were identified in the identification, more often among representatives of the M. fortuitum and M. peregrinum groups. Particular attention should be paid to the identification of the M. kansasii strain by a molecular genetic method, which mass spectrometry was defined as M. bovis. Both cultures of M. tuberculosis complex, which were identified by MALDI-ToF spectrometry, DNA hybridization were not determined to species. 17 (21.8%) of microbial strains which have been identified using the method of DNA hybridization, identified by spectrometry, including slow-growing microorganisms, non-mycobacteria strains seven (9.0%): Gordonia rubriperticta, Nocardia forcinica, Tsukumurella spp., Rhodotorula mucilaginosa. Accurate species identification NTMB is fundamental to determine the tactics of treatment of patients with mycobacteriosis. Due to this rather limited possibility of identification of non-tuberculous mycobacteria, using a DNA-hybridization method is inadequate to date. The introduction of new techniques, such as MALDI-ToF spectrometry, can identify a greater number of species of nontuberculous mycobacteria, as well as other types of slow-growing microorganisms having similarities with mycobacteria on cultural and morphological properties, which significantly increases the diagnostic capabilities of laboratories.

DNA origami nanorobot fiber optic genosensor to TMV

In the quest of greater sensitivity and specificity of diagnostic systems, one continually searches for alternative DNA hybridization methods, enabling greater versatility and where possible field-enabled detection of target analytes. We present, herein, a hybrid molecular self-assembled scaffolded DNA origami entity, intimately immobilized via capture probes linked to aminopropyltriethoxysilane, onto a glass optical fiber end-face transducer, thus producing a novel biosensor. Immobilized DNA nanorobots with a switchable flap can then be actuated by a specific target DNA present in a sample, by exposing a hemin/G-quadruplex DNAzyme, which then catalyzes the generation of chemiluminescence, once the specific fiber probes are immersed in a luminol-based solution. Integrating organic nanorobots to inorganic fiber optics creates a hybrid system that we demonstrate as a proof-of-principle can be utilized in specific DNA sequence detection. This system has potential applications in a wide range of fields, including point-of-care diagnostics or cellular in vivo biosensing when using ultrathin fiber optic probes for research purposes.

Ultra-sensitive chemiluminescence imaging DNA hybridization method in the detection of mosquito-borne viruses and parasites

BackgroundMosquito-borne viruses (MBVs) and parasites (MBPs) are transmitted through hematophagous arthropods-mosquitoes to homoiothermous vertebrates. This study aims at developing a detection method to monitor the spread of mosquito-borne diseases to new areas and diagnose the infections caused by MBVs and MBPs.MethodsIn this assay, an ultra-sensitive chemiluminescence (CL) detection method was developed and used to simultaneously detect 19 common MBVs and MBPs. In vitro transcript RNA, virus-like particles (VLPs), and plasmids were established as positive or limit of detection (LOD) reference materials.ResultsMBVs and MBPs could be genotyped with high sensitivity and specificity. The cut-off values of probes were calculated. The absolute LODs of this strategy to detect serially diluted in vitro transcribed RNAs of MBVs and serially diluted plasmids of MBPs were 102–103copies/μl and 101–102copies/μl, respectively. Further, the LOD of detecting a strain of pre-quantified JEV was 101.8–100.8PFU/ml, fitted well in a linear regression model (coefficient of determination = 0.9678).ConclusionsUltra-sensitive CL imaging DNA hybridization was developed and could simultaneously detect various MBVs and MBPs. The method described here has the potential to provide considerable labor savings due to its ability to screen for 19 mosquito-borne pathogens simultaneously.

HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization.

Sensitive and specific genotyping of human papillomaviruses (HPVs) is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS) to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP)-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution.