A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Homogeneous duplex polymerase chain reaction assay using switchable lanthanide fluorescence probes
Tekijät: Lehmusvuori A, Tapio AH, Mäki-Teeri P, Rantakokko-Jalava K, Wang Q, Takalo H, Soukka T
Kustantaja: ACADEMIC PRESS INC ELSEVIER SCIENCE
Julkaisuvuosi: 2013
Journal: Analytical Biochemistry
Tietokannassa oleva lehden nimi: ANALYTICAL BIOCHEMISTRY
Lehden akronyymi: ANAL BIOCHEM
Numero sarjassa: 1
Vuosikerta: 436
Numero: 1
Aloitussivu: 16
Lopetussivu: 21
Sivujen määrä: 6
ISSN: 0003-2697
DOI: https://doi.org/10.1016/j.ab.2013.01.007
Tiivistelmä
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. (C) 2013 Elsevier Inc. All rights reserved.
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. (C) 2013 Elsevier Inc. All rights reserved.
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