title: Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay.
abstract: Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates.
authors.affiliation: State Veterinary Institute Prague, Laboratory of Molecular Methods, Prague, 16503, Czech Republic.|National Institute of Public Health, National Reference Laboratory for Influenza, Prague, Czech Republic.
authors.forename: Alexander
authors.initials: A
authors.lastname: Nagy
authors.affiliation: State Veterinary Institute Prague, Laboratory of Molecular Methods, Prague, 16503, Czech Republic.
authors.forename: Eliška
authors.initials: E
authors.lastname: Vitásková
authors.affiliation: State Veterinary Institute Prague, Laboratory of Molecular Methods, Prague, 16503, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Černíková
authors.affiliation: State Veterinary Institute Prague, Laboratory of Molecular Methods, Prague, 16503, Czech Republic.|State Veterinary Institute Prague, Department of Virology and Serology, Prague, 16503, Czech Republic.
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Křivda
authors.affiliation: National Institute of Public Health, National Reference Laboratory for Influenza, Prague, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Jiřincová
authors.affiliation: State Veterinary Institute Prague, Department of Virology and Serology, Prague, 16503, Czech Republic.
authors.forename: Kamil
authors.initials: K
authors.lastname: Sedlák
authors.affiliation: State Veterinary Institute Prague, Department of Virology and Serology, Prague, 16503, Czech Republic.
authors.forename: Jitka
authors.initials: J
authors.lastname: Horníčková
authors.affiliation: National Institute of Public Health, National Reference Laboratory for Influenza, Prague, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Havlíčková
mesh_terms: D000818:Animals; D001483:Base Sequence; D002138:Calibration; D015342:DNA Probes / Q000378:metabolism*; D004285:Dogs; D005453:Fluorescence; D006736:Horses; D006868:Hydrolysis; D009696:Nucleic Acids / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013194:Staining and Labeling
chemical_list: D015342:DNA Probes; D009696:Nucleic Acids