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.
Article
in Zh
| WPRIM
| ID: wpr-280959
Responsible library:
WPRO
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>
Full text:
1
Index:
WPRIM
Main subject:
Spectrometry, Fluorescence
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Substrate Specificity
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DNA
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Fluoroimmunoassay
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Blotting, Southern
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Alkaline Phosphatase
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Genetics
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Luminescent Measurements
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Metals, Rare Earth
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Methods
Type of study:
Diagnostic_studies
Language:
Zh
Journal:
Acta Academiae Medicinae Sinicae
Year:
2002
Type:
Article