Displaying publications 21 - 40 of 108 in total

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  1. Electrochemical genosensor for specific detection of the food-borne pathogen, Vibrio cholerae
    Low KF, Chuenrangsikul K, Rijiravanich P, Surareungchai W, Chan YY
    World J Microbiol Biotechnol, 2012 Apr;28(4):1699-706.
    PMID: 22805952 DOI: 10.1007/s11274-011-0978-x
    A disposable horseradish peroxidase (HRP)-based electrochemical genosensor was developed for chronoamperometric detection of single-stranded asymmetric lolB gene PCR amplicon (118 bp in length) of the food-borne pathogen, Vibrio cholerae. A two-step sandwich-type hybridization strategy using two specific probes was employed for specific detection of the target single-stranded DNA (ssDNA). The analytical performances of the detection platform have been evaluated using a synthetic ssDNA (ST3) which was identical to the target single-stranded amplicon and a total of 19 bacterial strains. Under optimal condition, ST3 was calibrated with a dynamic range of 0.4883-15.6250 nM. By coupling asymmetric PCR amplification, the probe-based electrochemical genosensor was highly specific to the target organism (100% specificity) and able to detect as little as 0.85 ng/μl of V. cholerae genomic DNA.
    Matched MeSH terms: Nucleic Acid Hybridization/methods
  2. Hybridization-ligation versus parallel overlap assembly: an experimental comparison of initial pool generation for direct-proportional length-based DNA computing
    Ibrahim Z, Tsuboi Y, Ono O
    IEEE Trans Nanobioscience, 2006 Jun;5(2):103-9.
    PMID: 16805106
    Previously, direct-proportional length-based DNA computing (DPLB-DNAC) for solving weighted graph problems has been reported. The proposed DPLB-DNAC has been successfully applied to solve the shortest path problem, which is an instance of weighted graph problems. The design and development of DPLB-DNAC is important in order to extend the capability of DNA computing for solving numerical optimization problem. According to DPLB-DNAC, after the initial pool generation, the initial solution is subjected to amplification by polymerase chain reaction and, finally, the output of the computation is visualized by gel electrophoresis. In this paper, however, we give more attention to the initial pool generation of DPLB-DNAC. For this purpose, two kinds of initial pool generation methods, which are generally used for solving weighted graph problems, are evaluated. Those methods are hybridization-ligation and parallel overlap assembly (POA). It is found that for DPLB-DNAC, POA is better than that of the hybridization-ligation method, in terms of population size, generation time, material usage, and efficiency, as supported by the results of actual experiments.
    Matched MeSH terms: Nucleic Acid Hybridization/methods*
  3. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira
    Nurul Najian AB, Engku Nur Syafirah EA, Ismail N, Mohamed M, Yean CY
    Anal Chim Acta, 2016 Jan 15;903:142-8.
    PMID: 26709307 DOI: 10.1016/j.aca.2015.11.015
    In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10(-1) genomic equivalent ml(-1). An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device.
    Matched MeSH terms: Nucleic Acid Hybridization
  4. Fulltext Development of solution-gated graphene transistor model for biosensors
    Karimi H, Yusof R, Rahmani R, Hosseinpour H, Ahmadi MT
    Nanoscale Res Lett, 2014;9(1):71.
    PMID: 24517158 DOI: 10.1186/1556-276X-9-71
    : The distinctive properties of graphene, characterized by its high carrier mobility and biocompatibility, have stimulated extreme scientific interest as a promising nanomaterial for future nanoelectronic applications. In particular, graphene-based transistors have been developed rapidly and are considered as an option for DNA sensing applications. Recent findings in the field of DNA biosensors have led to a renewed interest in the identification of genetic risk factors associated with complex human diseases for diagnosis of cancers or hereditary diseases. In this paper, an analytical model of graphene-based solution gated field effect transistors (SGFET) is proposed to constitute an important step towards development of DNA biosensors with high sensitivity and selectivity. Inspired by this fact, a novel strategy for a DNA sensor model with capability of single-nucleotide polymorphism detection is proposed and extensively explained. First of all, graphene-based DNA sensor model is optimized using particle swarm optimization algorithm. Based on the sensing mechanism of DNA sensors, detective parameters (Ids and Vgmin) are suggested to facilitate the decision making process. Finally, the behaviour of graphene-based SGFET is predicted in the presence of single-nucleotide polymorphism with an accuracy of more than 98% which guarantees the reliability of the optimized model for any application of the graphene-based DNA sensor. It is expected to achieve the rapid, quick and economical detection of DNA hybridization which could speed up the realization of the next generation of the homecare sensor system.
    Matched MeSH terms: Nucleic Acid Hybridization
  5. Human Papillomavirus E6 biosensing: Current progression on early detection strategies for cervical Cancer
    Parmin NA, Hashim U, Gopinath SCB, Nadzirah S, Rejali Z, Afzan A, et al.
    Int J Biol Macromol, 2019 Apr 01;126:877-890.
    PMID: 30597241 DOI: 10.1016/j.ijbiomac.2018.12.235
    Prognosis of early cancer detection becomes one of the tremendous issues in the medical health system. Medical debates among specialist doctor and researcher in therapeutic approaches became a hot concern for cervix cancer deficiencies early screening, risk factors cross-reaction, portability device, rapid and free labeling system. The electrical biosensing based system showed credibility in higher specificity and selectivity due to hybridization of DNA duplex between analyte target and DNA probes. Electrical DNA sensor for cervix cancer has attracted too many attentions to researcher notification based on high performance, easy to handle, rapid system and possible to miniaturize. This review explores the current progression and future insignificant for HPV E6 genobiosensing for early Detection Strategies of Cervical Cancer.
    Matched MeSH terms: Nucleic Acid Hybridization
  6. Fulltext Graphene Oxide-Gold Star Construct on Triangular Electrodes for Alzheimer's Disease Identification
    Chang W, Zhao J, Liu L, Xing X, Zhang C, Meng H, et al.
    J Anal Methods Chem, 2021;2021:6661799.
    PMID: 33688447 DOI: 10.1155/2021/6661799
    Nanotechnology is playing a major role in the field of medical diagnosis, in particular with the biosensor and bioimaging. It improves the performance of the desired system dramatically by displaying higher selectivity and sensitivity. Carbon nanomaterial, gold nanostructure, magnetite nanoparticle, and silica substrate are the most popular nanomaterials greatly contributed to make the affordable and effective biosensor at low-cost. This research work is introducing a new sensing strategy with graphene oxide-constructed triangular electrodes to diagnose Alzheimer's disease (AD). MicroRNA-137 (miRNA-137) was found as a suitable biomarker for AD, and the sensing method was established here to detect miRNA-137 on the complementary sequence. To enhance the immobilization of capture miRNA-137, gold nanostar (GNS) was conjugated with capture miRNA and immobilized on the GO-modified surface through an amine linker. This immobilization process enhanced the hybridization of the target and reaches the detection limit at 10 fM with the sensitivity of 1 fM on the linear curve with a regression coefficient of 0.9038. Further control sequences of miRNA-21 and single and triple base mismatched miRNA-137 did not show a significant response in current changes, indicating the specific miRNA-137 detection for diagnosing AD.
    Matched MeSH terms: Nucleic Acid Hybridization
  7. Target Induced-DNA strand displacement reaction using gold nanoparticle labeling for hepatitis E virus detection
    Ngamdee T, Yin LS, Vongpunsawad S, Poovorawan Y, Surareungchai W, Lertanantawong B
    Anal Chim Acta, 2020 Oct 16;1134:10-17.
    PMID: 33059855 DOI: 10.1016/j.aca.2020.08.018
    DNA strand displacement is an attractive, enzyme-free target hybridization strategy for nano-biosensing. The target DNA induces a strand displacement reaction by replacing the pre-hybridized strand that is labeled with gold nanoparticles (AuNPs). Thus, the amount of displaced-AuNP-labeled strand is proportional to the amount of target DNA in the sample. The use of a magnetogenosensing technique to isolate the target DNA allows for a simple, one-pot detection approach, which minimizes possible carry-over contamination and pipetting errors. We sought a proof-of-concept for this technology in its ability to detect DNA-equivalent of hepatitis E virus (HEV), which causes acute viral hepatitis for which rapid and simple diagnostic methods remain limited. Signal detection was done via visual observation, spectrophotometry, and electrochemistry. The sensor demonstrated good sensitivity with detection limits of 10 pM (visual), 10 pM (spectrophotometry) and 1 fM (electrochemical). This sensor also exhibited high specificity for real target amplicons and could discriminate between perfect and mismatched sequences. Lyophilized biosensor reagents stored at 4 °C, 25 °C, and outdoor ambient temperature, were stable for up to 90, 50, and 40 days, respectively. The integration of magnetic separation and target DNA-induced strand displacement reaction in a dry reagent form makes the sensing platform easy-to-use and suitable for field settings.
    Matched MeSH terms: Nucleic Acid Hybridization
  8. Fulltext The best method for isolated total RNA from durian tissues
    Ky, H., Yeap, S. K., Napis, S. B.
    International Food Research Journal, 2012;19(3):1181-1183.
    MyJurnal
    Plant tissues, especially durian tissues contain high content of polysaccharides, polyphenols and other secondary metabolites which can co-precipitate with RNA causing problem in further transcriptomic study. In this experiment, three basic chaotic agents, CTAB, SDS and guanidine are used in three basic protocols for RNA isolation. The effectiveness of each method was determined by spectrophotometer, denaturing agarose gels analysis and northern blot hybridization. CTAB combining with additional sodium acetate precipitation step showed highest yield and best quality of isolated RNA which was free from contaminations of polysaccharides, polyphenols and other secondary metabolites. Furthermore, the total RNA from 4-month old durian flesh of clone D24 was successfully used to construct a cDNA library. In conclusion, CTAB method is effective to isolate total RNA on various types of durian tissues for further gene expression analysis.
    Matched MeSH terms: Nucleic Acid Hybridization
  9. Fulltext Beta thalassaemia mutations in Malays: a simplified cost-effective strategy to identify the mutations
    George, E., Teh, L.K., Rosli, R., Lai, M.I., Tan, J.A.M.A.
    Malaysian Journal of Medicine and Health Sciences, 2012;8(1):45-53.
    MyJurnal
    Beta (β)- thalassaemia is a public health problem in Malaysia. The carrier rate is estimated to be 4.5% by micro-mapping studies particularly among Malays who comprise 53.5% of the population in Malaysia. The common diagnostic method in Malaysia for mutation detection is by amplification refractory mutation system (ARMS). It allows single mutation detection in each reaction but is labour intensive and time consuming when many mutations need to be identified. The purpose of this study was to develop a diagnostic tool for effective mutation detection of beta thalassaemia in Malay patients and compare its efficacy with ARMS-PCR, the current method in use. Methods: Reverse dot blot hybridization (RDBH) technique was incorporated in the development of two strip assays [RDBH-Strip M(6) and RDBH-Strip C(6)] to identify common beta thalassaemia mutations in the Malays. The panels of selected mutations were based on the mutation frequencies in Malaysia reported in previous studies. RDBH-Strip M(6) was applied as step 1 and RDBH-Strip C(6) was applied as step 2 for unidentified mutations. The strips were validated with the gold standard method, ARMS- PCR. Results: One hundred and thirty seven Malay patients with 274 alleles were studied. In Step 1 mutation detection, 238 alleles (86.9%) were identified in 119 of patients by RDBH-Strip M(6). Step 2 resulted in a further detection of 20 alleles in another 10 patients by RDBH-Strip C(6). The combination of both strips resulted in the identification of 258 alleles in 129 (94.6%) of 137 Malay patients. The strip assays were 100% sensitive and specific when compared with ARMS-PCR method. Conclusion: Two strip assays utilising the RDBH technique were developed to identify common β-thalassaemia mutations in Malays. The RDBH Strip M(6) identified 86.9% of the mutations and the RDBH-Strip C (6) detected further 7.3% alleles. This two step strategy was found to be rapid and cost effective for the direct diagnosis of β-thalassaemia mutations in the Malays. The remaining unidentified mutations would require DNA sequencing. It can serve as a specific molecular diagnostic tool for effective diagnosis of
    β-thalassaemia mutations in this ethnic group.
    Matched MeSH terms: Nucleic Acid Hybridization
  10. Fulltext Exploring Two Protocols of FISH Using Cytocell SYT-SSX Probe on Formalin Fixed Paraffin Embedded Tissue Sections
    Yajid AI, Mohd Nafi SN, Salehan NA, Tuan Sharif SE
    Asian Pac J Cancer Prev, 2020 May 01;21(5):1241-1245.
    PMID: 32458628 DOI: 10.31557/APJCP.2020.21.5.1241
    BACKGROUND: Chromosomal translocation t(X;18)(p11.2;q11.2) is the cytogenetic hallmark of synovial sarcoma and have been identified as an alternative diagnostic strategy in differentiating synovial sarcoma from other histologic mimics. This study was carried out to test the efficacy of two FISH protocols using the SYT-SSX break apart probe from Cytocell.

    METHODOLOGY: Representative paraffin blocks of synovial sarcoma were utilized in this study. FISH study was performed on formalin-fixed paraffin embedded tissue sections using the SYT-SSX break apart probe from Cytocell, to detect two form of SYT-SSX transcript, SYT-SSX1 and SYT-SSX2. FISH protocol, including the hybridization was done following two different protocols, Cytocell FISH protocol and Optimized Dako FISH protocol.

    RESULTS: Tissue samples subjected to FISH using Cytocell FISH protocol showed the absence of signal corresponding to the probe used. Utilizing Optimized Dako FISH protocol, the two signals (red and green) corresponding to the break-apart probes was detected. These findings suggested that Optimised Dako FISH protocol is more suited for use with the tested probe on paraffin embedded tissues in comparison to Cytocell FISH protocol.

    CONCLUSION: Optimised Dako FISH protocol was noted to be more suited for detecting SYT-SSX FISH signals on paraffin embedded tissues in comparison to Cytocell FISH protocol.

    Matched MeSH terms: Nucleic Acid Hybridization
  11. Genome-based analyses reveal the presence of 12 heterotypic synonyms in the genus Streptomyces and emended descriptions of Streptomyces bottropensis, Streptomyces celluloflavus, Streptomyces fulvissimus, Streptomyces glaucescens, Streptomyces murinus, and Streptomyces variegatus
    Madhaiyan M, Saravanan VS, See-Too WS
    Int J Syst Evol Microbiol, 2020 Jun;70(6):3924-3929.
    PMID: 32441614 DOI: 10.1099/ijsem.0.004217
    Phylogenetic analysis based on 16S rRNA gene sequences of the genus Streptomyces showed the presence of six distinguishable clusters, with 100 % sequence similarity values among strains in each cluster; thus they shared almost the same evolutionary distance. This result corroborated well with the outcome of core gene (orthologous gene clusters) based genome phylogeny analysis of 190 genomes including the Streptomyces species in those six clusters. These preeminent results led to an investigation of genome-based indices such as digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) and average amino acid identity (AAI) for the strains in those six clusters. Certain strains recorded genomic indices well above the threshold values (70 %, 95-96 % and >95 % for dDDH, ANI and AAI, respectively) determined for species affiliation, suggesting only one type strain belongs to described species and the other(s) may need to be reduced in taxa to a later heterotypic synonym. To conclude, the results of comprehensive analyses based on phylogenetic and genomic indices suggest that the following six reclassifications are proposed: Streptomyces flavovariabilis as a later heterotypic synonym of Streptomyces variegatus; Streptomyces griseofuscus as a later heterotypic synonym of Streptomyces murinus; Streptomyces kasugaensis as a later heterotypic synonym of Streptomyces celluloflavus; Streptomyces luridiscabiei as a later heterotypic synonym of Streptomyces fulvissimus; Streptomyces pharetrae as a later heterotypic synonym of Streptomyces glaucescens; and Streptomyces stelliscabiei as a later heterotypic synonym of Streptomyces bottropensis.
    Matched MeSH terms: Nucleic Acid Hybridization
  12. Taxonomic note on the family Pseudonocardiaceae based on phylogenomic analysis and descriptions of Allosaccharopolyspora gen. nov. and Halosaccharopolyspora gen. nov
    Teo WFA, Tan GYA, Li WJ
    Int J Syst Evol Microbiol, 2021 Oct;71(10).
    PMID: 34714227 DOI: 10.1099/ijsem.0.005075
    The taxonomic positions of members within the family Pseudonocardiaceae were assessed based on phylogenomic trees reconstructed using core-proteome and genome blast distance phylogeny approaches. The closely clustered genome sequences from the type strains of validly published names within the family Pseudonocardiaceae were analysed using overall genome-related indices based on average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values. The family Pseudonocardiaceae consists of the type genus Pseudonocardia, as well as the genera Actinoalloteichus, Actinocrispum, Actinokineospora, Actinomycetospora, Actinophytocola, Actinopolyspora, Actinorectispora, Actinosynnema, Allokutzneria, Allosaccharopolyspora gen. nov., Amycolatopsis, Bounagaea, Crossiella, Gandjariella, Goodfellowiella, Haloactinomyces, Haloechinothrix, Halopolyspora, Halosaccharopolyspora gen. nov., Herbihabitans, Kibdelosporangium, Kutzneria, Labedaea, Lentzea, Longimycelium, Prauserella, Saccharomonospora, Saccharopolyspora, Saccharothrix, Salinifilum, Sciscionella, Streptoalloteichus, Tamaricihabitans, Thermocrispum, Thermotunica and Umezawaea. The G+C contents of the Pseudonocardiaceae genomes ranged from 66.2 to 74.6 mol% and genome sizes ranged from 3.69 to 12.28 Mbp. Based on the results of phylogenomic analysis, the names Allosaccharopolyspora coralli comb. nov., Halosaccharopolyspora lacisalsi comb. nov. and Actinoalloteichus caeruleus comb. nov. are proposed. This study revealed that Actinokineospora mzabensis is a heterotypic synonym of Actinokineospora spheciospongiae, Lentzea deserti is a heterotypic synonym of Lentzea atacamensis, Prauserella endophytica is a heterotypic synonym of Prauserella coralliicola, and Prauserella flava and Prauserella sediminis are heterotypic synonyms of Prauserella salsuginis. This study addresses the nomenclature conundrums of Actinoalloteichus cyanogriseus and Streptomyces caeruleus as well as Micropolyspora internatus and Saccharomonospora viridis.
    Matched MeSH terms: Nucleic Acid Hybridization
  13. Electrochemical genosensor based on RNA-responsive human telomeric G-quadruplex DNA: A proof-of-concept with SARS-CoV-2 RNA
    Ibrahim N, Gan KB, Mohd Yusof NY, Goh CT, Krupa B N, Tan LL
    Talanta, 2024 Jul 01;274:125916.
    PMID: 38547835 DOI: 10.1016/j.talanta.2024.125916
    In this report, a facile and label-free electrochemical RNA biosensor is developed by exploiting methylene blue (MB) as an electroactive positive ligand of G-quadruplex. The electrochemical response mechanism of the nucleic acid assay was based on the change in differential pulse voltammetry (DPV) signal of adsorbed MB on the immobilized human telomeric G-quadruplex DNA with a loop that is complementary to the target RNA. Hybridization between synthetic positive control RNA and G-quadruplex DNA probe on the transducer platform rendered a conformational change of G-quadruplex to double-stranded DNA (dsDNA), and increased the redox current of cationic MB π planar ligand at the sensing interface, thereby the electrochemical signal of the MB-adsorbed duplex is proportional to the concentration of target RNA, with SARS-CoV-2 (COVID-19) RNA as the model. Under optimal conditions, the target RNA can be detected in a linear range from 1 zM to 1 μM with a limit of detection (LOD) obtained at 0.59 zM for synthetic target RNA and as low as 1.4 copy number for positive control plasmid. This genosensor exhibited high selectivity towards SARS-CoV-2 RNA over other RNA nucleotides, such as SARS-CoV and MERS-CoV. The electrochemical RNA biosensor showed DPV signal, which was proportional to the 2019-nCoV_N_positive control plasmid from 2 to 200000 copies (R2 = 0.978). A good correlation between the genosensor and qRT-PCR gold standard was attained for the detection of SARS-CoV-2 RNA in terms of viral copy number in clinical samples from upper respiratory specimens.
    Matched MeSH terms: Nucleic Acid Hybridization
  14. Fulltext Comparison of DR. HPV Chip Kit with hybrid capture II assay for the detection of human papillomavirus in clinical samples: a preliminary study
    Rajan S, Shen TH, Santhanam J, Othman NH, Othman N, Hock TT
    Trop Biomed, 2007 Jun;24(1):17-22.
    PMID: 17568373
    Human papillomavirus (HPV) is well known as an etiological factor for the development of anogenital carcinomas. The aim of our study was to compare the performance of USFDA approved Hybrid II (HCII) Assay and recently introduced DR. HPV Chip Kit for the detection of HPV DNA in clinical cervical scrapings from 40 patients. HPV DNA testing was performed using the automated HCII Assay system and DR. HPV Chip Kit. Taking cytological results as gold standard, it was found that HCII was more sensitive (36.4%) than DR. HPV Chip Kit (18.2%) although specificity was 100% with the latter method. In addition, both these molecular methods had comparable negative and positive predictive values. It was concluded that both HCII and DR. HPV Chip Kit have comparable specificity. However, sensitivity for detection of HPV in clinical samples with HCII is almost double as compared to DR. HPV Chip Kit.
    Matched MeSH terms: Nucleic Acid Hybridization/methods
  15. Fulltext Cassette hybridization for vector assembly application in antibody chain shuffling
    Lai JY, Loh Q, Choong YS, Lim TS
    Biotechniques, 2018 11;65(5):269-274.
    PMID: 30394125 DOI: 10.2144/btn-2018-0031
    Gene assembly methods are an integral part of molecular cloning experiments. The majority of existing vector assembly methods stipulate a need for exonucleases, endonucleases and/or the use of single-stranded DNA as starting materials. Here, we introduced a vector assembly method that employs conventional PCR to amplify stable double-stranded DNA fragments and assembles them into functional vectors specifically for antibody chain shuffling. We successfully formed vectors using cassettes amplified from different templates and assembled an array of single chain fragment variable clones of fixed variable heavy chain, with different variable light chains - a chain shuffling process for antibody maturation. The method provides an easy alternative to the conventional cloning process.
    Matched MeSH terms: Nucleic Acid Hybridization/methods*
  16. A signal-amplified electrochemical DNA biosensor incorporated with a colorimetric internal control for Vibrio cholerae detection using shelf-ready reagents
    Low KF, Zain ZM, Yean CY
    Biosens Bioelectron, 2017 Jan 15;87:256-263.
    PMID: 27567251 DOI: 10.1016/j.bios.2016.08.064
    A novel enzyme/nanoparticle-based DNA biosensing platform with dual colorimetric/electrochemical approach has been developed for the sequence-specific detection of the bacterium Vibrio cholerae, the causative agent of acute diarrheal disease in cholera. This assay platform exploits the use of shelf-stable and ready-to-use (shelf-ready) reagents to greatly simplify the bioanalysis procedures, allowing the assay platform to be more amenable to point-of-care applications. To assure maximum diagnosis reliability, an internal control (IC) capable of providing instant validation of results was incorporated into the assay. The microbial target, single-stranded DNA amplified with asymmetric PCR, was quantitatively detected via electrochemical stripping analysis of gold nanoparticle-loaded latex microspheres as a signal-amplified hybridization tag, while the incorporated IC was analyzed using a simplified horseradish peroxidase enzyme-based colorimetric scheme by simple visual observation of enzymatic color development. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 145 clinical isolate-spiked fecal specimens. The limits of detection were 0.5ng/ml of genomic DNA and 10 colony-forming units (CFU)/ml of bacterial cells with dynamic ranges of 0-100ng/ml (R(2)=0.992) and log10 (1-10(4) CFU/ml) (R(2)=0.9918), respectively. An accelerated stability test revealed that the assay reagents were stable at temperatures of 4-37°C, with an estimated ambient shelf life of 200 days. The versatility of the biosensing platform makes it easily adaptable for quantitative detection of other microbial pathogens.
    Matched MeSH terms: Nucleic Acid Hybridization/methods
  17. An ultrasensitive hollow-silica-based biosensor for pathogenic Escherichia coli DNA detection
    Ariffin EY, Lee YH, Futra D, Tan LL, Karim NHA, Ibrahim NNN, et al.
    Anal Bioanal Chem, 2018 Mar;410(9):2363-2375.
    PMID: 29504083 DOI: 10.1007/s00216-018-0893-1
    A novel electrochemical DNA biosensor for ultrasensitive and selective quantitation of Escherichia coli DNA based on aminated hollow silica spheres (HSiSs) has been successfully developed. The HSiSs were synthesized with facile sonication and heating techniques. The HSiSs have an inner and an outer surface for DNA immobilization sites after they have been functionalized with 3-aminopropyltriethoxysilane. From field emission scanning electron microscopy images, the presence of pores was confirmed in the functionalized HSiSs. Furthermore, Brunauer-Emmett-Teller (BET) analysis indicated that the HSiSs have four times more surface area than silica spheres that have no pores. These aminated HSiSs were deposited onto a screen-printed carbon paste electrode containing a layer of gold nanoparticles (AuNPs) to form a AuNP/HSiS hybrid sensor membrane matrix. Aminated DNA probes were grafted onto the AuNP/HSiS-modified screen-printed electrode via imine covalent bonds with use of glutaraldehyde cross-linker. The DNA hybridization reaction was studied by differential pulse voltammetry using an anthraquinone redox intercalator as the electroactive DNA hybridization label. The DNA biosensor demonstrated a linear response over a wide target sequence concentration range of 1.0×10-12-1.0×10-2 μM, with a low detection limit of 8.17×10-14 μM (R2 = 0.99). The improved performance of the DNA biosensor appeared to be due to the hollow structure and rough surface morphology of the hollow silica particles, which greatly increased the total binding surface area for high DNA loading capacity. The HSiSs also facilitated molecule diffusion through the silica hollow structure, and substantially improved the overall DNA hybridization assay. Graphical abstract Step-by-step DNA biosensor fabrication based on aminated hollow silica spheres.
    Matched MeSH terms: Nucleic Acid Hybridization/methods
  18. A thermostabilized magnetogenosensing assay for DNA sequence-specific detection and quantification of Vibrio cholerae
    Low KF, Karimah A, Yean CY
    Biosens Bioelectron, 2013 Sep 15;47:38-44.
    PMID: 23545172 DOI: 10.1016/j.bios.2013.03.004
    Vibrio cholerae is a human pathogen that causes mild to severe diarrheal illnesses and has major public health significance. Herein, we present a thermostabilized electrochemical genosensing assay combining the use of magnetic beads as a biorecognition platform and gold nanoparticles as a hybridization tag for the detection and quantification of V. cholerae lolB gene single-stranded asymmetric PCR amplicons as an alternative to the time-consuming classical isolation method. This thermostabilized, pre-mixed, pre-aliquoted and ready-to-use magnetogenosensing assay simplified the procedures and permitted the reaction to be conducted at room temperature. The asymmetric PCR amplicons were hybridized to a magnetic bead-functionalized capture probe and a fluorescein-labeled detection probe followed by tagging with gold nanoparticles. Electrochemical detection of the chemically dissolved gold nanoparticles was performed using the differential pulse anodic stripping voltammetry method. The real-time stability evaluation of thermostabilized assay was found to be stable for at least 180 days at room temperature (25-30°C). The analytical specificity of the assay was 100%, while its analytical sensitivity was linearly related to different concentrations of 200-mer synthetic target, purified genomic DNA, and bacterial culture with a limit of detection (LoD) of 3.9nM, 5pg/µl, and 10(3)CFU/ml, respectively. The clinical applicability of the assay was successfully validated using spiked stool samples with an average current signal-to-cut-off ratio of 10.8. Overall, the precision of the assay via relative standard deviation was <10%, demonstrating its reliability and accuracy.
    Matched MeSH terms: Nucleic Acid Hybridization
  19. Granulometric analysis of spots in DNA microarray images
    Latha B, Venkatesh B
    Genomics Proteomics Bioinformatics, 2004 Nov;2(4):222-36.
    PMID: 15901251
    As the topological properties of each spot in DNA microarray images may vary from one another, we employed granulometries to understand the shape-size content contributed due to a significant intensity value within a spot. Analysis was performed on the microarray image that consisted of 240 spots by using concepts from mathematical morphology. In order to find out indices for each spot and to further classify them, we adopted morphological multiscale openings, which provided microarrays at multiple scales. Successive opened microarrays were subtracted to identify the protrusions that were smaller than the size of structuring element. Spot-wise details, in terms of probability of these observed protrusions, were computed by placing a regularly spaced grid on microarray such that each spot was centered in each grid. Based on the probability of size distribution functions of these protrusions isolated at each level, we estimated the mean size and texture index for each spot. With these characteristics, we classified the spots in a microarray image into bright and dull categories through pattern spectrum and shape-size complexity measures. These segregated spots can be compared with those of hybridization levels.
    Matched MeSH terms: Nucleic Acid Hybridization
  20. Development of a dry-reagent-based nucleic acid-sensing platform by coupling thermostabilised LATE-PCR assay to an oligonucleotide-modified lateral flow biosensor
    Ang GY, Yu CY, Chan KG, Singh KK, Chan Yean Y
    J Microbiol Methods, 2015 Nov;118:99-105.
    PMID: 26342435 DOI: 10.1016/j.mimet.2015.08.024
    In this study, we report for the first time the development of a dry-reagent-based nucleic acid-sensing platform by combining a thermostabilised linear-after-the-exponential (LATE)-PCR assay with a one-step, hybridisation-based nucleic acid lateral flow biosensor. The nucleic acid-sensing platform was designed to overcome the need for stringent temperature control during transportation or storage of reagents and reduces the dependency on skilled personnel by decreasing the overall assay complexity and hands-on time. The platform was developed using toxigenic Vibrio cholerae as the model organism due to the bacterium's propensity to cause epidemic and pandemic cholera. The biosensor generates result which can be visualised with the naked eyes and the limit of detection was found to be 1pg of pure genomic DNA and 10CFU/ml of toxigenic V. cholerae. The dry-reagent-based nucleic acid-sensing platform was challenged with 95 toxigenic V. cholerae, 7 non-toxigenic V. cholerae and 66 other bacterial strains in spiked stool sample and complete agreement was observed when the results were compared to that of monosialoganglioside (GM1)-ELISA. Heat-stability of the thermostabilised LATE-PCR reaction mixes at different storage temperatures (4-56°C) was investigated for up to 90days. The dry-reagent-based genosensing platform with ready-to-use assay components provides an alternative method for sequence-specific detection of nucleic acid without any cold chain restriction that is associated with conventional molecular amplification techniques.
    Matched MeSH terms: Nucleic Acid Hybridization
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