Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Proc Natl Acad Sci U S A ; 118(45)2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34732575

RESUMO

Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.


Assuntos
Edição de Genes/métodos , Anemia Falciforme/genética , Animais , Reações Falso-Positivas , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos SCID , Técnicas de Sonda Molecular , Ácidos Nucleicos Peptídicos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
2.
Diagn Microbiol Infect Dis ; 78(4): 338-42, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24439447

RESUMO

The performance of a diagnostic method for detection and identification of Enterococcus spp. directly from positive blood culture was evaluated in a clinical study. The method, Enterococcus QuickFISH BC, is a second-generation peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) test, which uses a simplified, faster assay procedure. The test uses fluorescently labeled PNA probes targeting 16S rRNA to differentiate Enterococcus faecalis from other Enterococcus spp. by the color of the cellular fluorescence. Three hundred fifty-six routine blood culture samples were tested; only 2 discordant results were recorded. The sensitivities for detection of Enterococcus faecalis and non-faecalis Enterococcus were 100% (106/106) and 97.0% (65/67), respectively, and the combined specificity of the assay was 100%. The combined positive and negative predictive values of the assay were 100% (171/171) and 98.9% (185/187), respectively.


Assuntos
Sangue/microbiologia , Enterococcus/classificação , Enterococcus/isolamento & purificação , Infecções por Bactérias Gram-Positivas/diagnóstico , Hibridização in Situ Fluorescente/métodos , Técnicas de Diagnóstico Molecular/métodos , Sepse/diagnóstico , Enterococcus/genética , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Sensibilidade e Especificidade , Sepse/microbiologia
3.
J Clin Microbiol ; 50(6): 1994-8, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22493336

RESUMO

A novel rapid peptide nucleic acid fluorescence in situ hybridization (FISH) method, Staphylococcus QuickFISH, for the direct detection of Staphylococcus species from positive blood culture bottles was evaluated in a multicenter clinical study. The method utilizes a microscope slide with predeposited positive- and negative-control organisms and a self-reporting 15-min hybridization step, which eliminates the need for a wash step. Five clinical laboratories tested 722 positive blood culture bottles containing gram-positive cocci in clusters. The sensitivities for detection of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were 99.5% (217/218) and 98.8% (487/493), respectively, and the combined specificity of the assay was 89.5% (17/19). The combined positive and negative predictive values of the assay were 99.7% (696/698) and 70.8% (17/24), respectively. Studies were also performed on spiked cultures to establish the specificity and performance sensitivity of the method. Staphylococcus QuickFISH has a turnaround time (TAT) of <30 min and a hands-on time (HOT) of <5 min. The ease and speed of the method have the potential to improve the accuracy of therapeutic intervention by providing S. aureus/CoNS identification simultaneously with Gram stain results.


Assuntos
Bacteriemia/diagnóstico , Técnicas Bacteriológicas/métodos , Sangue/microbiologia , Hibridização in Situ Fluorescente/métodos , Técnicas de Diagnóstico Molecular/métodos , Infecções Estafilocócicas/diagnóstico , Staphylococcus/isolamento & purificação , Bacteriemia/microbiologia , Humanos , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Infecções Estafilocócicas/microbiologia , Staphylococcus/genética
7.
J Am Chem Soc ; 124(6): 1097-103, 2002 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-11829619

RESUMO

Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl(2). We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.


Assuntos
Sondas de DNA/química , DNA de Cadeia Simples/química , DNA/química , Ácidos Nucleicos Peptídicos/química , Pareamento Incorreto de Bases , Sequência de Bases , Corantes Fluorescentes/química , Cinética , Hibridização de Ácido Nucleico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...