Homogeneous duplex polymerase chain reaction assay using switchable lanthanide fluorescence probes.
Anal Biochem
; 436(1): 16-21, 2013 May 01.
Article
en En
| MEDLINE
| ID: mdl-23353013
ABSTRACT
We have developed a duplex polymerase chain reaction (PCR) assay based on switchable lanthanide chelate complementation probes. In the complementation probe technology, two nonfluorescent oligonucleotide probes, one labeled with a lanthanide ion carrier chelate and another with a light absorbing antenna ligand, form a fluorescent complex by self-assembly of the reporter molecules when the two probes are hybridized in adjacent positions to the target DNA. Here we report the synthesis of a new terbium(III) (Tb(III)) ion carrier chelate and a new light-absorbing antenna ligand for Tb(III) and the development of a duplex Chlamydia trachomatis (Ct) PCR assay. For the detection of Ct in urine samples, a specific sequence in Ct cryptic plasmid was amplified and detected using europium(III) (Eu(III)) complementation probes. An internal amplification control was amplified in each reaction and detected using Tb(III) complementation probes to verify the Ct negative results. Ct bacteria were concentrated from urine samples with a rapid and simple centrifugation-based sample preparation method. Good diagnostic accuracy (99-100%) was achieved, and also Ct positive reactions yielded a very high Eu(III) signal-to-background ratio (maximum of 244). High performance of the complementation probes is advantageous when sample may contain impurities after a simple sample preparation.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Terbio
/
Sondas de Oligonucleótidos
/
Infecciones por Chlamydia
/
Chlamydia trachomatis
/
Reacción en Cadena en Tiempo Real de la Polimerasa
/
Colorantes Fluorescentes
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Anal Biochem
Año:
2013
Tipo del documento:
Article
País de afiliación:
Finlandia