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Journal of Clinical Microbiology Oct 1993The direct detection of gonococcal DNA in rectal and pharyngeal specimens was evaluated by using a DNA probe-based assay (Gen-Probe, Inc., San Diego, Calif.). Rectal...
The direct detection of gonococcal DNA in rectal and pharyngeal specimens was evaluated by using a DNA probe-based assay (Gen-Probe, Inc., San Diego, Calif.). Rectal (234) and pharyngeal (608) swab specimens were obtained from 249 men and 372 women attending sexually transmitted disease clinics in Las Vegas and Reno, Nevada. The prevalence of gonococcal infection by culture at the pharyngeal and rectal sites was 2.9% (16 of 548 specimens) in women and 2.7% (8 of 294 specimens) in men. No false-positive reactions were observed among the 234 rectal specimens tested. Two probe-positive, culture-negative specimens were detected among the 361 pharyngeal specimens obtained from women. Both of these samples were confirmed as Neisseria gonorrhoeae by a probe competition assay. The overall correlation of the DNA probe test with pharyngeal and rectal cultures was 99.4% (837 of 842 cultures), with a sensitivity of 87.5% (21 of 24 cultures) and specificity of 99.7% (816 of 818 cultures). The positive and negative predictive values of the DNA assay were 91.3 and 99.8%, respectively. The direct DNA probe assay provides an alternative to culture screening for rectal and/or pharyngeal gonococcal infections.
Topics: DNA Probes; DNA, Bacterial; Female; Gonorrhea; Humans; Male; Neisseria gonorrhoeae; Pharynx; Rectum
PubMed: 8253983
DOI: 10.1128/jcm.31.10.2783-2785.1993 -
Journal of Clinical Microbiology Dec 1990The objective of this hospital-based study was to determine the relationship between colonizing and infecting strains of Candida species and Torulopsis glabrata....
The objective of this hospital-based study was to determine the relationship between colonizing and infecting strains of Candida species and Torulopsis glabrata. Surveillance cultures from high-risk patients were paired with subsequent bloodstream isolates. Organisms were typed by using restriction endonuclease digestion of chromosomal DNA with BstNI and EcoRI, followed by Southern hybridization with a DNA probe (pBD4) derived from Saccharomyces cerevisiae. Sixteen patients for whom documented colonization preceded documented bloodstream infection were identified. The mean time between obtainment of surveillance isolates and obtainment of bloodstream isolates was 8 days, with a range of 1 to 423 days. For 15 (94%) of 16 patients, the DNA fingerprint pattern (using BstNI) of the surveillance isolate was identical to that of the bloodstream isolate. Isolates from 13 (81%) of 16 patients were unique to those patients. Typing by Southern hybridization with the pBD4 probe was less discriminating. We conclude that for a well-defined subset of hospitalized patients who were colonized by Candida species before developing nosocomial candidemia, the colonizing and infecting strains were identical, suggesting endogenous acquisition of infection. Restriction endonuclease digestion of chromosomal DNA was shown to be a discriminating and reproducible typing method for Candida species and T. glabrata.
Topics: Blotting, Southern; Candida; Candidiasis; Cross Infection; DNA Probes; DNA Restriction Enzymes; DNA, Fungal; Humans
PubMed: 2177750
DOI: 10.1128/jcm.28.12.2733-2738.1990 -
Journal of Clinical Microbiology Nov 2002Group A streptococcus (GAS) antigen testing has become a routine point-of-care (POC) test in acute care settings. Concern about performance parameters (PP) of these...
Group A streptococcus (GAS) antigen testing has become a routine point-of-care (POC) test in acute care settings. Concern about performance parameters (PP) of these tests as well as inappropriate antibiotic use has resulted in various recommendations regarding diagnosis of GAS. There were two objectives in this study. The first was to evaluate the rapid GAS antigen test presently in use (Thermo BioStar, Boulder, Colo.) and the GAS Direct probe test (Gen-Probe, San Diego, Calif.) compared to culture. The second was to define the optimal use of these technologies in a large acute care pediatric clinic. A total of 520 consecutive pediatric patients presenting with symptoms of pharyngitis at any of three Lahey Clinic acute care facilities were evaluated. Pharyngeal specimens were collected using a double-swab collection device (Copan, Corona, Calif.). One swab was used for the antigen test, the second was used for the probe test, and the pledget was placed in the collection device for culture on 5% sheep blood agar, incubated for 48 h anaerobically, and subsequently placed in Todd-Hewitt broth. After discrepant analysis, sensitivity, specificity, and positive and negative predictive values were as follows: 94.8, 100, 100, and 96.9% for the probe test and 86.1, 97.1, 93.7, and 93.4% for the antigen test, respectively. Sensitivity using an enhanced culture technique was 99.4% (163 of 164). False-positive (FP) antigen results were often seen from patients previously diagnosed and/or treated for GAS. No FP results were seen with the probe test. Colony counts for the false-negative (FN) antigen tests were higher than those for the FN probe tests. Compared to culture and DNA probe, the rapid antigen test (RAT) offered a result at the time of the patient's visit, with acceptable PP when prevalence of disease is high. Follow-up testing with the RAT of GAS patients who previously tested as positive should be avoided due to increased FP results. The probe test was comparable to culture in performance. Results indicate the probe test can be used as the primary test or as a backup to negative antigen tests. The probe test offers the advantage over culture of same-day reporting of a final result but, in contrast to a POC test, necessitates follow-up communication to the patient. Preliminary data show the specificity of the probe test to be greater than that of the RAT for patients previously diagnosed with GAS.
Topics: Adolescent; Ambulatory Care; Antigens, Bacterial; Child; Child, Preschool; Culture Media; DNA Probes; DNA, Ribosomal; Humans; Immunoassay; Infant; Infant, Newborn; Luminescent Measurements; Pediatrics; Pharyngitis; Predictive Value of Tests; RNA, Ribosomal; Sensitivity and Specificity; Streptococcal Infections; Streptococcus pyogenes; Time Factors
PubMed: 12409399
DOI: 10.1128/JCM.40.11.4207-4210.2002 -
Nucleic Acids Research Nov 2020Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence...
Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence limitation. The compact size of the HpSGN system make it an ideal candidate for in vivo delivery applications. In vitro biochemical studies showed that the HpSGN system required less nuclease to cleave ssDNA substrates than the SGN system we reported previously by a factor of ∼40. Also, we proved that the HpSGN system can efficiently cleave different RNA targets in vitro. The HpSGN system cleaved genomic DNA at an efficiency of ∼40% and ∼20% in bacterial and human cells, respectively, and knocked down specific mRNAs in human cells at a level of ∼25%. Furthermore, the HpSGN system was sensitive to the single base mismatch at the position next to the hairpin both in vitro and in vivo. Collectively, this study demonstrated the potential of developing the HpSGN system as a small, effective, and specific editing tool for manipulating both DNA and RNA without sequence limitation.
Topics: Archaeoglobus fulgidus; Base Pair Mismatch; DNA; DNA Probes; DNA, Single-Stranded; Escherichia coli; Flap Endonucleases; Gene Editing; HEK293 Cells; Humans; In Vitro Techniques; Inverted Repeat Sequences; Nucleic Acid Conformation; RNA; RNA Editing; Substrate Specificity
PubMed: 33051689
DOI: 10.1093/nar/gkaa843 -
Journal of Clinical Microbiology May 1992The objective of this study was to determine whether different isolates of Babesia microti could be distinguished from morphologically similar isolates of B. gibsoni by...
The objective of this study was to determine whether different isolates of Babesia microti could be distinguished from morphologically similar isolates of B. gibsoni by using a ribosomal DNA (rDNA) probe. A Babesia-specific rDNA probe was obtained by polymerase chain reaction amplification of sequences from B. microti DNA using universal primers directed against highly conserved portions of the eukaryotic 16S-like rRNA gene. The chemiluminescent rDNA probe hybridized to Southern blots of restriction endonuclease-digested DNA preparations of different isolates of B. gibsoni from infected dogs and B. microti from infected humans and white-footed mice. Restriction fragment length polymorphisms served to differentiate these species. Although the hybridization patterns seen with DNAs from six B. microti isolates did not vary, those of the five B. gibsoni isolates did indicate genotypic variation. We concluded that isolates of B. microti and B. gibsoni can be differentiated on the basis of restriction fragment length polymorphism detected with a chemiluminescent rDNA probe.
Topics: Animals; Babesia; Base Sequence; DNA Probes; DNA, Protozoan; DNA, Ribosomal; Dogs; Molecular Sequence Data; Polymorphism, Restriction Fragment Length; Restriction Mapping
PubMed: 1349901
DOI: 10.1128/jcm.30.5.1210-1215.1992 -
The Analyst Feb 2012Lead is highly toxic and its detection has attracted a lot of research interests. In recent years, DNA has been used for Pb(2+) recognition and many fluorescent sensors...
Lead is highly toxic and its detection has attracted a lot of research interests. In recent years, DNA has been used for Pb(2+) recognition and many fluorescent sensors with low to sub-nM detection limits have been reported. These figures of merit were typically measured using a spectrophotometer that can detect nM DNA with a high signal-to-noise ratio. For visual detection, however, μM DNA or dye was required, making it difficult to detect low nM targets. We recently achieved a visual sensitivity of 10 nM Hg(2+) by immobilizing a DNA probe in a hydrogel. This was made possible because the gel was able to actively adsorb Hg(2+). In this work, we aim to test whether this method can be extended to the detection of Pb(2+). First, a new Pb(2+) sensor was designed based on a guanine-rich DNA and DNA binding dyes such as thiazole orange and SYBR Green I. The free DNA showed a detection limit of 8 nM Pb(2+) using 40 nM DNA. For visual detection in solution with 1 μM of the DNA probe, however, ∼300 nM Pb(2+) was required. After immobilization in a monolithic polyacrylamide hydrogel, even 20 nM Pb(2+) could be visually detected with a sample volume of 50 mL. Therefore, sensitive detection without signal amplification was achieved. Finally, we demonstrated simultaneous detection of both Hg(2+) and Pb(2+) in the same water sample with shape encoded hydrogel sensors.
Topics: Acrylamide; Base Sequence; Biosensing Techniques; DNA; DNA Probes; Hydrogels; Lead; Limit of Detection
PubMed: 22143190
DOI: 10.1039/c2an15754c -
Organic & Biomolecular Chemistry Feb 2022Major efforts have been devoted to the development of constructs that enable sequence-specific recognition of double-stranded (ds) DNA, fueled by the promise for...
Major efforts have been devoted to the development of constructs that enable sequence-specific recognition of double-stranded (ds) DNA, fueled by the promise for enabling tools for applications in molecular biology, diagnostics, and medicine. Towards this end, we have previously introduced Invader probes, , short DNA duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides. The individual strands of these labile probes display high affinity towards complementary DNA (cDNA), which drives sequence-unrestricted dsDNA-recognition. However, recognition of long targets is challenging due to the high stability of the corresponding probes. To address this, we recently introduced toehold Invader probes, , Invader probes with 5'-single-stranded overhangs. The toehold architecture allows for shorter double-stranded segments to be used, which facilitates probe dissociation and dsDNA-recognition. As an extension thereof, we here report the biophysical and dsDNA-targeting properties of nicked Invader probes. In this probe architecture, the single-stranded overhangs of toehold Invader probes are hybridized to short intercalator-modified auxiliary strands, leading to formation of additional labile segments. The extra binding potential from the auxiliary strands imparts nicked Invader probes with greater dsDNA-affinity than the corresponding toehold or blunt-ended probes. Recognition of chromosomal DNA targets, refractory to recognition by conventional Invader probes, is demonstrated for nicked Invader probes in the context of non-denaturing FISH experiments, which highlights their utility as dsDNA-targeting tools.
Topics: Animals; Cattle; Cell Line; DNA; DNA Probes; Intercalating Agents; Male; Molecular Structure; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Transition Temperature
PubMed: 34874037
DOI: 10.1039/d1ob02019f -
PloS One 2017Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short...
Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short sequences of miRNA make single-base isomiR mismatches very difficult to be discriminated. Non-specific hybridizations often arise when DNA probe-miRNA target hybridization is the primary, or initial, mode of detection. These errors then become exacerbated through subsequent amplification steps. Here, we present the design of DNA probes modified with poly-guanine (PG) tracts that were induced to form G-quadruplexes (G4) for hi-fidelity discrimination of miRNA core target sequence from single-base mismatched isomiRs. We demonstrate that, when compared to unmodified probes, this G4 'gate-keeping' function within the G4-modified probes enables more stringent hybridization of complementary core miRNA target transcripts while limiting non-specific hybridizations. This increased discriminatory power of the G4-modified probes over unmodified probes is maintained even after further reverse transcriptase extension of probe-target hybrids. Enzymatic extension also enhanced the clarity and sensitivity of readouts and allows different isomiRs to be distinguished from one another via the relative positions of the mismatches.
Topics: DNA Probes; G-Quadruplexes; Isomerism; MicroRNAs
PubMed: 29145502
DOI: 10.1371/journal.pone.0188163 -
Scientific Reports Mar 2020The characteristic features of stem-loop structured probes make them robust tools to detect targets with high sensitivity and selectivity. The basis of the hairpin based...
The characteristic features of stem-loop structured probes make them robust tools to detect targets with high sensitivity and selectivity. The basis of the hairpin based sensors operation is a conformational change that occurs upon hybridization of target with stem-loop probe. The design of the stem-loop probe has an important role in target recognition. Therefore, we designed a label-free stem loop probe for targeting miR-21 as a cancer biomarker investigated by web-based tools; its thermodynamic parameters obtained by thermal UV spectroscopy. The efficiency of stem-loop structure opening in the presence of target and non-target sequences was evaluated by fluorescence spectroscopy and circular dichroism spectro-polarimetry. The results showed that the target sequence opens the structure of hairpin efficiently in comparison to non-target sequences. To optimize the stem-loop hybridization to its target, the buffer ionic strength was changed by adding different concentrations of NaCl, KCl and MgCl. It was shown that buffering conditions have a significant role in loop structure opening and its optimization, led to an increase in sensitivity detection and have improved LOD from 60 pM to 45 pM.
Topics: Biosensing Techniques; Circular Dichroism; DNA Probes; Humans; MicroRNAs; Nucleic Acid Hybridization; Spectrometry, Fluorescence
PubMed: 32132554
DOI: 10.1038/s41598-020-60157-5 -
Nucleic Acids Research Sep 2017Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells....
Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect >50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.
Topics: Carbocyanines; DNA Probes; Fluorescent Dyes; Gene Expression Regulation, Fungal; In Situ Hybridization, Fluorescence; RNA, Messenger; Saccharomyces cerevisiae; Sensitivity and Specificity; Single Molecule Imaging
PubMed: 28666354
DOI: 10.1093/nar/gkx568