[Enzyme-amplified time-resolved fluorescence detection for nucleic acid hybridization assays].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
EATRF detection for nucleic acid hybridization assays is a new sensitive simple method, which has a great prospect.
Zhao
Qi-ren
QR
Department of Experimental Nuclear Medicine, Institute of Radiation Medicine, CAMS, PUMC, Tianjin 300192, China.
Li
Mei-jia
MJ
Liu
Jie
J
Song
Na-ling
NL
Chen
Ai
A
Zhuang
Xiang-lian
XL
Liu
Bing-chen
BC
D000469:Alkaline Phosphatase / Q000032:analysis; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics; D015200:Fluoroimmunoassay / Q000379:methods*; D008163:Luminescent Measurements; D008674:Metals, Rare Earth; D009693:Nucleic Acid Hybridization / Q000379:methods*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D008674:Metals, Rare Earth; D004247:DNA; D000469:Alkaline Phosphatase