Enzyme-amplified time-resolved fluorescence detection for nucleic acid hybridization assays / 中国医学科学院学报
Acta Academiae Medicinae Sinicae
;
(6): 84-88, 2002.
Artigo
em Chinês
| WPRIM
| ID: wpr-280959
ABSTRACT
<p><b>OBJECTIVE</b>To develop a new nonisotopic detection method of enzyme-amplified time-resolved fluorescence (EATRF) or enzyme-amplified lanthanide luminescence (EALL) for nucleic acid hybridization assays, which can be applied extensively in clinical diagnosis.</p><p><b>METHODS</b>The method combines the high affinity of biotin-streptavidin system, amplification of enzyme, and inherent advantage of lanthanide chelate with the background elimination of time-resolved fluorescence detection. The conversion of 5-fluorosalicyl phosphate to 5-fluorosalicylic acid (5-FSA) by alkaline phosphatase. The salicylic acid product forms a luminescent ternary chelate with Tb3+ and EDTA.</p><p><b>RESULTS</b>The dynamic range of the standard curve of EATRFA for nucleic acid hybridization assay was very wide, the range was more than third order of magnitude. The detection sensitivity was about 10 pg of target sequence. When the known target sequence was 20, 10 and 2 ng, the ratio of measured amount to known amount was 110%, 90% and 115% respectively. The main experimental conditions, for example, the irradiating time of ultraviolet rays, the concentrations of biotinylated probe, AP-SA, 5-FSAP and Tb-EDTA and the methods of washing in the related steps, have been optimized. A new stable technology of fluorescence has been developted.</p><p><b>CONCLUSIONS</b>EATRF detection for nucleic acid hybridization assays is a new sensitive simple method, which has a great prospect.</p>
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Espectrometria de Fluorescência
/
Especificidade por Substrato
/
DNA
/
Fluorimunoensaio
/
Southern Blotting
/
Fosfatase Alcalina
/
Genética
/
Medições Luminescentes
/
Metais Terras Raras
/
Métodos
Tipo de estudo:
Estudo diagnóstico
Idioma:
Chinês
Revista:
Acta Academiae Medicinae Sinicae
Ano de publicação:
2002
Tipo de documento:
Artigo
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