The multiplexing capabilities with different fluorescent dyes are limited in real-time PCR instruments equipped with one excitation source. Considering this limitation, a design was developed to create a triple-labeled probe as an internal positive control (IPC) that utilizes a combination of the fluorescence resonance energy transfer (FRET) and TaqMan techniques. The IPC probe, labeled with FAM and Cy5.5 fluorophores at the 5′ end and Black Hole Quencher (BHQ) at the 3′ end, enabled Cy5.5 emission through energy transfer from the FAM fluorophore. The second, target-specific TaqMan assay in the multiplex used a FAM- and BHQ1-labeled probe at the 5′ and 3′ ends, respectively. Thus, one excitation source was used to generate two different fluorescence emissions (FAM and Cy5.5) that were measured in two separate channels by the real-time PCR instrument. This method can facilitate the development of a low-cost portable handheld real-time PCR instrument capable of multiplex real-time PCR assays using a single excitation source.

Introduction

Real-time PCR enables continuous monitoring of fluorophore fluorescence during the generation of PCR products in a closed tube format. Currently, available methods utilize either labeled probes or DNA intercalating dye to monitor the amplification of PCR product. The TaqMan probe is a single-stranded oligonucleotide containing a fluorophore and quencher placed 10–30 bases apart. The TaqMan probe is hydrolyzed during the amplification due to the 5′–3′ double-strand–specific exonuclease activity of the Taq polymerase that separates the fluorophore from the quencher, resulting in fluorescence increase. Real-time PCR instruments are equipped with fluorescence detectors and software capable of estimating the cycle threshold (Ct), the cycle at which fluorescence is greater than background fluorescence, for positive reactions. However, the technique cannot differentiate a true negative result from a false negative when PCR is affected by amplification inhibitors. After nucleic acid extraction, it is reported that inhibitors may still be present from clinical samples (e.g., hemoglobin), environmental samples (e.g., humic and fulvic acids), and chemicals employed during nucleic acid extraction (e.g., ethanol, detergents, or chaotropic agents). The reliability of diagnostic assays is increased by the inclusion of an internal control nucleic acid that can indicate the presence and impact of PCR inhibitors (1,2). An internal positive control (IPC) is amplified simultaneously in the presence of a target sequence using a labeled fluorophore that emits light at a different wavelength than the fluorophore used for the target sequence assay, with the two fluorophores detected in different channels by the real-time PCR instrument. The commonly used internal control for PCR is a plasmid that contains a sequence similar to that of the assay target except for probe region. A limited number of IPC molecules are added to individual assay target and co-amplified with the target nucleic acid; thus, a positive IPC signal is evidence that the amplification reaction proceeded sufficiently to generate a positive signal from very small quantities of target nucleic acid. This feature is important to ensure equivalent amplification of the IPC and the target nucleic acid (3,4,5,6,7,8,9). However, some devices—such as the LightCycler 1.2 and LightCycler 2 (Roche Applied Science, Indianapolis, IN, USA), the Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.) instrument (Idaho Technology, Salt Lake City, UT, USA), and handheld real-time PCR instruments—are only equipped with one light source and associated emission channel. Multiplexing on these types of instruments requires the use of multi-labeled oligonucleotides and the fluorescence resonance energy transfer (FRET) concept. However, FRET probes can be challenging to design and may lack robustness in hypervariable regions and when non-optimal amplification conditions are present. The use of FRET probes requires the identification of two regions for two probes. The FRET probe system relies on adjacent hybridization of oligonucleotides of the two single-labeled probes in such a way the fluorescence of the acceptor dye is detected through the excitation of the fluorophore attached to the second probe. A major disadvantage of the FRET probe is the requirement of a larger sequence area necessary to accommodate two adjacent probes with a gap of 2–5 nucleotides. The possibility exists for multiplexing with other label systems, such as MegaStokes dye (DYXL; Dyomics, Jena, Germany) and Pulsar 650 (Biosearch Technologies, Inc., Novato, CA, USA), but these labeling systems require spectral information to compensate for channel crosstalk and are not widely available. The FRET-TaqMan probe developed in this study, however, does not require color compensation software and uses fluorophores (FAM, Cy5.5) that are widely available. The present report focuses on the design of a triple-labeled probe to enable multiplexing in instruments equipped with only one blue light emitting diode (LED) excitation source (470 nm) and associated three corresponding detection channels (e.g., 530 nm, 640 nm, and 705 nm).

Materials and methods

Probe design principle

All experiments were performed on Idaho Technology's R.A.P.I.D. system, a 32-sample portable real-time PCR instrument equipped with a blue LED for excitation (470 nm) and associated three fluorescence detection channels that measure fluorescence at 530, 640, and 705 nm for mono-and dual-color assays. Primers JVAF and JVAR (10) and probe 5′-FAM/BHQ-1 were synthesized at the CDC Biotechnology Core Facility (Atlanta, GA, USA) and probe 5′FAM-Cy5.5/3′BHQ-3 was synthesized at Idaho Technology. To enable duplex TaqMan PCR, one probe was labeled with 5′-FAM/BHQ-1 (520-nm fluorescence wavelength) and another one with 5′FAM-Cy5.5/3′BHQ-3 (705-nm fluorescence wavelength). In solution, the probe labeled with 5′FAM-Cy5.5/3′BHQ-3 remains quenched upon hybridization to a sequence, and upon subsequent cleavage of the probe by enzymatic primer extension, the quencher is eliminated. This results in FRET through the excitation of FAM by the light source of the R.A.P.I.D. instrument. The excitation energy is transferred to the acceptor fluorophore, Cy5.5, and the emitted fluorescence is measured in a second fluorescence channel (fluorescence channel F3 for the R.A.P.I.D. instrument). The emission of Cy5.5 fluorescence is the result of the combination of FRET and TaqMan probe techniques, so these triple-labeled probes can be referred to as “FRET-TaqMan” probes.