Displaying publications 1 - 20 of 108 in total

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  1. Molecular basis of beta thalassemia in the south of Thailand
    Laosombat V, Fucharoen SP, Panich V, Fucharoen G, Wongchanchailert M, Sriroongrueng W, et al.
    Am J Hematol, 1992 Nov;41(3):194-8.
    PMID: 1415194
    A total of 103 beta thalassemia genes from 78 children (45 with Hb E/beta thalassemia, 8 with beta thalassemia heterozygotes, and 25 with homozygous beta thalassemia) were analyzed using dot-blot hybridization of the polymerase chain reaction-amplified DNA and direct DNA sequencing. Nine mutations were characterized in 98/103 (95%) of beta thalassemia alleles, of which six (a 4 bp deletion in codons 41-42, a G-C transition at position 5 of IVS-1, A-G transition at codon 19, an A-T transition at codon 17, an A-G transition at position -28 upstream of the beta globin gene, a G-T transition at position 1 of IVS-1), accounted for 92%. The spectrum of beta thalassemia mutations in Chinese Thai is similar to that reported among the Chinese from other parts of the world. The distribution of beta thalassemia mutations in Muslim Thai is similar to that reported among Malaysians. The most common beta thalassemia mutation in Thai and Chinese Thai patients is the frameshift mutation at codons 41-42, in comparison with the Muslim Thai in whom the G-C transition at position 5 of the IVS-1 mutation predominates. The heterogeneity of molecular defects causing beta thalassemia should aid in the planning of a prenatal diagnosis program for beta thalassemia in the South of Thailand.
    Matched MeSH terms: Nucleic Acid Hybridization
  2. 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
  3. 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
  4. 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
  5. Assembly of highly diverse genes using degenerate oligonucleotides by temperature cascade
    Loh Q, Bahara NH, Choong YS, Lim TS
    Anal Biochem, 2012 Dec 1;431(1):54-6.
    PMID: 22975202 DOI: 10.1016/j.ab.2012.08.025
    The quality of a nucleotide-based library such as a synthetic antibody library is highly dependent on the diversity available. Diversity can be generated using degenerate oligonucleotides introduced during gene assembly. Conventional approaches to gene assembly are not efficient for oligonucleotides with long stretches of degeneracy. We propose an efficient alternative for simultaneous introduction of three randomized regions in a synthetic antibody gene via temperature cascading. The strategy takes advantage of DNA reannealing kinetics. The strategy can be adopted for generating diversity of gene inserts during the construction of nucleotide-based libraries.
    Matched MeSH terms: Nucleic Acid Hybridization*
  6. Tocotrienol-rich fraction from palm oil and gene expression in human breast cancer cells
    Nesaretnam K, Ambra R, Selvaduray KR, Radhakrishnan A, Canali R, Virgili F
    Ann N Y Acad Sci, 2004 Dec;1031:143-57.
    PMID: 15753141
    Vitamin E is important not only for its cellular antioxidant and lipid-lowering properties, but also as an antiproliferating agent. It has also been shown to contribute to immunoregulation, antibody production, and resistance to implanted tumors. It has recently been shown that tocotrienols are the components of vitamin E responsible for growth inhibition in human breast cancer cells in vitro as well as in vivo through estrogen-independent mechanisms. Although tocotrienols act on cell proliferation in a dose-dependent manner and can induce programmed cell death, no specific gene regulation has yet been identified. In order to investigate the molecular basis of the effect of a tocotrienol-rich fraction (TRF) from palm oil, we performed a cDNA array analysis of cancer-related gene expression in estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cells. The human breast cancer cells were incubated with or without 8 mug/mL of tocotrienols for 72 h. RNA was subsequently extracted and subjected to reverse transcription before being hybridized onto cancer arrays. Tocotrienol supplementation modulated significantly 46 out of 1200 genes in MDA-MB-231 cells. In MCF-7 cells, tocotrienol administration was associated with a lower number of affected genes. Interestingly, only three were affected in a similar fashion in both cell lines: c-myc binding protein MM-1, 23-kDa highly basic protein, and interferon-inducible protein 9-27 (IFITM-1). These proteins are most likely involved in the cell cycle and can exert inhibitory effects on cell growth and differentiation of the tumor cell lines. These data suggest that tocotrienols are able to affect cell homeostasis, possibly independent of their antioxidant activity.
    Matched MeSH terms: Nucleic Acid Hybridization
  7. Microbacterium gilvum sp. nov., isolated from civet faeces
    Chen X, Li QY, Li GD, Xu FJ, Jiang Y, Han L, et al.
    Antonie Van Leeuwenhoek, 2016 Sep;109(9):1177-83.
    PMID: 27260265 DOI: 10.1007/s10482-016-0718-1
    A novel aerobic, non-motile, Gram-positive, rod-shaped actinobacterium, designated YIM 100951(T), was isolated from the faeces of civets (Viverra zibetha) living in the National Nature Protect Region in Selangor, Malaysia. Strain YIM 100951(T) shows high similarities with Microbacterium barkeri DSM 20145(T) (97.6 %), Microbacterium oryzae MB10(T) (97.3 %), Microbacterium lemovicicum ViU22(T) (97.1 %) and Microbacterium indicum BBH6(T) (97.0 %) based on their 16S rRNA genes. However, phylogenetic analysis showed that strain YIM 100951(T) formed a clade with Microbacterium halotolerans YIM 70130(T) (96.7 %), Microbacterium populi 10-107-8(T) (96.7 %) and Microbacterium sediminis YLB-01(T) (96.9 %). DNA-DNA hybridization was carried out between strains YIM 100951(T) and M. barkeri DSM 20145(T), the result showed a value of 23.2 ± 4.5 %. In addition, some of the physiological, biochemical and chemotaxonomic characteristics of strain YIM 100951(T) are different from the closely related strains. Thus, we suggest that strain YIM 100951(T) represents a novel species of the genus Microbacterium, for which the name Microbacterium gilvum sp. nov. is proposed. The type strain is YIM 100951(T) (=DSM 26235(T) = CCTCC AB 2012971(T)).
    Matched MeSH terms: Nucleic Acid Hybridization
  8. Molecular genetic diversity of the Saccharomyces yeasts in Taiwan: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii
    Naumov GI, Lee CF, Naumova ES
    Antonie Van Leeuwenhoek, 2013 Jan;103(1):217-28.
    PMID: 22941248 DOI: 10.1007/s10482-012-9803-2
    Genetic hybridization, sequence and karyotypic analyses of natural Saccharomyces yeasts isolated in different regions of Taiwan revealed three biological species: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii. Intraspecies variability of the D1/D2 and ITS1 rDNA sequences was detected among S. cerevisiae and S. kudriavzevii isolates. According to molecular and genetic analyses, the cosmopolitan species S. cerevisiae and S. kudriavzevii contain local divergent populations in Taiwan, Malaysia and Japan. Six of the seven known Saccharomyces species are documented in East Asia: S. arboricola, S. bayanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus.
    Matched MeSH terms: Nucleic Acid Hybridization
  9. Streptomyces gilvigriseus sp. nov., a novel actinobacterium isolated from mangrove forest soil
    Ser HL, Zainal N, Palanisamy UD, Goh BH, Yin WF, Chan KG, et al.
    Antonie Van Leeuwenhoek, 2015 Jun;107(6):1369-78.
    PMID: 25863667 DOI: 10.1007/s10482-015-0431-5
    A novel Streptomyces, strain MUSC 26(T), was isolated from mangrove soil at Tanjung Lumpur, Malaysia. The bacterium was observed to be Gram-positive and to form grayish yellow aerial and substrate mycelium on ISP 7 agar. A polyphasic approach was used to study the taxonomy of strain MUSC 26(T), which shows a range of phylogenetic and chemotaxonomic properties consistent with those of the members of the genus Streptomyces. The cell wall peptidoglycan was determined to contain LL-diaminopimelic acid. The predominant menaquinones were identified as MK-9 (H8) and MK-9(H6). The polar lipids detected were identified as diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine and hydroxyphosphatidylmethylethanolamine. The predominant cellular fatty acids (>10.0 %) were identified as anteiso-C15:0 (31.4 %), iso-C16:0 (16.3 %), iso-C15:0 (13.9 %) and anteiso-C17:0 (12.6 %). The cell wall sugars were found to be galactose, glucose, mannose, ribose and rhamnose. These results suggest that MUSC 26(T) should be placed within the genus Streptomyces. Phylogenetic analysis indicated that closely related strains include Streptomyces qinglanensis 172205(T) (96.5 % sequence similarity), S. sodiiphilus YIM 80305(T) (96.5 %) and S. rimosus subsp. rimosus ATCC 10970(T) (96.4 %). DNA-DNA relatedness values between MUSC 26(T) and closely related type strains ranged from 17.0 ± 2.2 to 33.2 ± 5.3 %. Comparison of BOX-PCR fingerprints indicated MUSC 26(T) presents a unique DNA profile. The DNA G+C content was determined to be 74.6 mol%. Based on this polyphasic study of MUSC 26(T), it is concluded that this strain represents a novel species, for which the name Streptomyces gilvigriseus sp. nov. is proposed. The type strain is MUSC 26(T) (=DSMZ 42173(T) = MCCC 1K00504(T)).
    Matched MeSH terms: Nucleic Acid Hybridization
  10. Amycolatopsis vastitatis sp. nov., an isolate from a high altitude subsurface soil on Cerro Chajnantor, northern Chile
    Idris H, Nouioui I, Pathom-Aree W, Castro JF, Bull AT, Andrews BA, et al.
    Antonie Van Leeuwenhoek, 2018 Sep;111(9):1523-1533.
    PMID: 29428970 DOI: 10.1007/s10482-018-1039-3
    The taxonomic position of a novel Amycolatopsis strain isolated from a high altitude Atacama Desert subsurface soil was established using a polyphasic approach. The strain, isolate H5T, was shown to have chemical properties typical of members of the genus Amycolatopsis such as meso-diaminopimelic acid as the diamino acid in the cell wall peptidoglycan, arabinose and galactose as diagnostic sugars and MK-9(H4) as the predominant isoprenologue. It also has cultural and morphological properties consistent with its classification in the genus, notably the formation of branching substrate hyphae which fragment into rod-like elements. 16S rRNA gene sequence analyses showed that the strain is closely related to the type strain of Amycolatopsis mediterranei but could be distinguished from this and other related Amycolatopsis strains using a broad range of phenotypic properties. It was separated readily from the type strain of Amycolatopsis balhymycina, its near phylogenetic neighbour, based on multi-locus sequence data, by low average nucleotide identity (92.9%) and in silico DNA/DNA relatedness values (51.3%) calculated from draft genome assemblies. Consequently, the strain is considered to represent a novel species of Amycolatopsis for which the name Amycolatopsis vastitatis sp. nov. is proposed. The type strain is H5T (= NCIMB 14970T = NRRL B-65279T).
    Matched MeSH terms: Nucleic Acid Hybridization
  11. Isolation and characterization of dengue viruses serotype 1 from an epidemic in northern Queensland, Australia
    Blok J, Kay BH, Hall RA, Gorman BM
    Arch Virol, 1988;100(3-4):213-20.
    PMID: 2840873
    Thirteen strains of dengue type 1 were isolated from the lymphocyte fractions of 69 acute phase blood samples collected at Thursday Island Hospital during 1981 and 1982. One further strain of type 1 was isolated from 7 blood samples despatched by air from Cairns Base Hospital during 1982. Four of these Australian isolates representing the beginning, middle, and end of the epidemic were examined by restriction enzyme mapping and were found to be identical for the nine restriction enzymes used. The maps differed from those derived from two Malaysian dengue type 1 strains isolated during the epidemic of 1981-82 in that country. This suggests reliance on serological typing to establish global circulation patterns of epidemic dengue is insufficient and that more specific methods such as genome mapping are useful.
    Matched MeSH terms: Nucleic Acid Hybridization
  12. 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
  13. Genome characterization and taxonomy of Actinomyces acetigenes sp. nov., and Actinomyces stomatis sp. nov., previously isolated from the human oral cavity
    Tian X, Teo WFA, Wee WY, Yang Y, Ahmed H, Jakubovics NS, et al.
    BMC Genomics, 2023 Dec 04;24(1):734.
    PMID: 38049764 DOI: 10.1186/s12864-023-09831-2
    BACKGROUND: Actinomyces strains are commonly found as part of the normal microflora on human tissue surfaces, including the oropharynx, gastrointestinal tract, and female genital tract. Understanding the diversity and characterization of Actinomyces species is crucial for human health, as they play an important role in dental plaque formation and biofilm-related infections. Two Actinomyces strains ATCC 49340 T and ATCC 51655 T have been utilized in various studies, but their accurate species classification and description remain unresolved.

    RESULTS: To investigate the genomic properties and taxonomic status of these strains, we employed both 16S rRNA Sanger sequencing and whole-genome sequencing using the Illumina HiSeq X Ten platform with PE151 (paired-end) sequencing. Our analyses revealed that the draft genome of Actinomyces acetigenes ATCC 49340 T was 3.27 Mbp with a 68.0% GC content, and Actinomyces stomatis ATCC 51655 T has a genome size of 3.08 Mbp with a 68.1% GC content. Multi-locus (atpA, rpoB, pgi, metG, gltA, gyrA, and core genome SNPs) sequence analysis supported the phylogenetic placement of strains ATCC 51655 T and ATCC 49340 T as independent lineages. Digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI) analyses indicated that both strains represented novel Actinomyces species, with values below the threshold for species demarcation (70% dDDH, 95% ANI and AAI). Pangenome analysis identified 5,731 gene clusters with strains ATCC 49340 T and ATCC 51655 T possessing 1,515 and 1,518 unique gene clusters, respectively. Additionally, genomic islands (GIs) prediction uncovered 24 putative GIs in strain ATCC 49340 T and 16 in strain ATCC 51655 T, contributing to their genetic diversity and potential adaptive capabilities. Pathogenicity analysis highlighted the potential human pathogenicity risk associated with both strains, with several virulence-associated factors identified. CRISPR-Cas analysis exposed the presence of CRISPR and Cas genes in both strains, indicating these strains might evolve a robust defense mechanism against them.

    CONCLUSION: This study supports the classification of strains ATCC 49340 T and ATCC 51655 T as novel species within the Actinomyces, in which the name Actinomyces acetigenes sp. nov. (type strain ATCC 49340 T = VPI D163E-3 T = CCUG 34286 T = CCUG 35339 T) and Actinomyces stomatis sp. nov. (type strain ATCC 51655 T = PK606T = CCUG 33930 T) are proposed.

    Matched MeSH terms: Nucleic Acid Hybridization
  14. 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*
  15. Electrochemical DNA biosensor for the detection of specific gene related to Trichoderma harzianum species
    Siddiquee S, Yusof NA, Salleh AB, Abu Bakar F, Heng LY
    Bioelectrochemistry, 2010 Aug;79(1):31-6.
    PMID: 19945357 DOI: 10.1016/j.bioelechem.2009.10.004
    A new electrochemical biosensor is described for voltammetric detection of gene sequence related to Trichoderma harzianum. The sensor involves immobilization of a 20 base single-stranded probe (ssDNA), which is complementary to a specific gene sequence related to T. harzianum on a gold electrode through specific adsorption. The DNA probe was used to determine the amount of target gene in solution using methylene blue (MB) as the electrochemical indicator. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the electrode. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE and the probe-modified AuE which localized to the affinity of MB. Control experiments with the non-complementary oligonucleotides were performed to assess whether the DNA biosensor responds selectively, via hybridization, to the target. DNA biosensor also able to detect microorganism at the species levels without nucleic acid amplification. The redox current was linearly related to the concentration of target oligonucleotide DNA, ranged from 1-20 ppm. Numerous factors, affecting the probe immobilization, target hybridization and indicator binding reactions are optimized to maximize the sensitivity and reduce the assay time.
    Matched MeSH terms: Nucleic Acid Hybridization
  16. A DNA biosensor based on gold nanoparticle decorated on carboxylated multi-walled carbon nanotubes for gender determination of Arowana fish
    Saeedfar K, Heng LY, Chiang CP
    Bioelectrochemistry, 2017 Dec;118:106-113.
    PMID: 28780443 DOI: 10.1016/j.bioelechem.2017.07.012
    Multi-wall carbon nanotubes (MWCNTs) were modified to design a new DNA biosensor. Functionalized MWCNTs were equipped with gold nanoparticles (GNPs) (~15nm) (GNP-MWCNTCOOH) to construct DNA biosensors based on carbon-paste screen-printed (SPE) electrodes. GNP attachment onto functionalized MWCNTs was carried out by microwave irradiation and was confirmed by spectroscopic studies and surface analysis. DNA biosensors based on differential pulse voltammetry (DPV) were constructed by immobilizing thiolated single-stranded DNA probes onto GNP-MWCNTCOOH. Ruthenium (III) chloride hexaammoniate [Ru(NH3)6,2Cl(-)] (RuHex) was used as hybridization redox indicator. RuHex and MWCNT interaction was low in compared to other organic redox hybridization indicators. The linear response range for DNA determination was 1×10(-21) to 1×10(-9)M with a lower detection limit of 1.55×10(-21)M. Thus, the attachment of GNPs onto functionalized MWCNTs yielded sensitive DNA biosensor with low detection limit and stability more than 30days. Constructed electrode was used to determine gender of arowana fish.
    Matched MeSH terms: Nucleic Acid Hybridization
  17. 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
  18. 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
  19. Graphene oxide and gold nanoparticle based dual platform with short DNA probe for the PCR free DNA biosensing using surface-enhanced Raman scattering
    Khalil I, Yehye WA, Julkapli NM, Rahmati S, Sina AA, Basirun WJ, et al.
    Biosens Bioelectron, 2019 Apr 15;131:214-223.
    PMID: 30844598 DOI: 10.1016/j.bios.2019.02.028
    Surface-enhanced Raman scattering (SERS) based DNA biosensors have considered as excellent, fast and ultrasensitive sensing technique which relies on the fingerprinting ability to produce molecule specific distinct spectra. Unlike conventional fluorescence based strategies SERS provides narrow spectral bandwidths, fluorescence quenching and multiplexing ability, and fitting attribute with short length probe DNA sequences. Herein, we report a novel and PCR free SERS based DNA detection strategy involving dual platforms and short DNA probes for the detection of endangered species, Malayan box turtle (MBT) (Cuora amboinensis). In this biosensing feature, the detection is based on the covalent linking of the two platforms involving graphene oxide-gold nanoparticles (GO-AuNPs) functionalized with capture probe 1 and gold nanoparticles (AuNPs) modified with capture probe 2 and Raman dye (Cy3) via hybridization with the corresponding target sequences. Coupling of the two platforms generates locally enhanced electromagnetic field 'hot spot', formed at the junctions and interstitial crevices of the nanostructures and consequently provide significant amplification of the SERS signal. Therefore, employing the two SERS active substrates and short-length probe DNA sequences, we have managed to improve the sensitivity of the biosensors to achieve a lowest limit of detection (LOD) as low as 10 fM. Furthermore, the fabricated biosensor exhibited sensitivity even for single nucleotide base-mismatch in the target DNA as well as showed excellent performance to discriminate closely related six non-target DNA sequences. Although the developed SERS biosensor would be an attractive platform for the authentication of MBT from diverse samples including forensic and/or archaeological specimens, it could have universal application for detecting gene specific biomarkers for many diseases including cancer.
    Matched MeSH terms: Nucleic Acid Hybridization
  20. Hb Bart's level in cord blood and deletions of alpha-globin genes
    Lie-Injo LE, Solai A, Herrera AR, Nicolaisen L, Kan YW, Wan WP, et al.
    Blood, 1982 Feb;59(2):370-6.
    PMID: 6895707
    The white blood cell DNA of 36 cord blood samples with Hb Bart's in the red blood cells was studied for alpha-globin gene deletions by hybridization of DNA fragments digested by the restriction endonucleases Eco RI, Hpa I, Bam HI, and Bgl II. All 16 DNA samples from cord blood with Hb Bart's below 3% and no other abnormal hemoglobin had one alpha-globin gene deletion (alpha thal2), except one which had two alpha-globin gene deletions (alpha thal1). Most of the alpha thal2 were of the rightward deletion alpha thal2 genotype. Two new types of alpha thal2 variation was found, probably due to a polymorphism somewhere in an area outside the alpha-globin gene. All 14 cases with Hb Bart's between 3.5% and 8.5% and no other abnormal hemoglobin had two alpha-globin gene deletions (alpha thal1), except one that did not have any alpha-globin gene deletion and one that had one alpha-globin gene deletion. Three DNA samples of cord blood with Hb Bart's accompanied by Hb CoSp did not have any alpha-globin gene deletion. Sixty-five DNA samples from cord blood without Hb Bart's or other abnormal hemoglobin had no alpha-globin gene deletions, except one that had one alpha-globin gene deletion (alpha thal2). Two of the 65 DNA samples were found to have triplicated alpha-globin gene loci.
    Matched MeSH terms: Nucleic Acid Hybridization
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