Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities.
Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37 °C. Complementation, for producing dCTP and TTP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5' exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90 min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R(2) values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.
Section of Clinical Virology, Department of Medical Sciences, Uppsala University, SE-751 85 Uppsala, Sweden. per.stalhandske@biodiar.se
Per
P
Stålhandske
Liya
L
Wang
Sara
S
Westberg
Henrik
H
von Euler
Erika
E
Groth
Sven A
SA
Gustafsson
Staffan
S
Eriksson
Johan
J
Lennerstrand
D000818:Animals; D055162:Biocatalysis; D017931:DNA Primers / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D057075:Enzyme Assays; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D000091402:Prohibitins; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D019914:Taq Polymerase / Q000378:metabolism; D013937:Thymidine Kinase / Q000097:blood / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D005456:Fluorescent Dyes; C000717331:PHB2 protein, human; D000091402:Prohibitins; D011994:Recombinant Proteins; D013937:Thymidine Kinase; C092717:thymidine kinase 1; D003842:Deoxycytidine Kinase; D019914:Taq Polymerase