Optimizing the Roche LightCycler(R) for Single-Tube Multiplexed RT-PCR Assays.

Abstract

The Roche Diagnostics LightCycler® (LC) Carousel-Based Systems are a single-source thermocycler capable of detecting light emissions from fluorescing molecules released during RT-PCR. This technology utilizes a single light source at 470 nm to excite fluorophores for read-out in one of three channels (530, 640 and 705 nm) for older 1.5 models and up to six channels (530, 560, 610, 640, 670, and 705nm) for the 2.0 model. The persistent challenge of LC technology is multiplexing, the ability to discriminate more than one target with different fluorophores in a single capillary tube that can be excited at the same 470-nm wavelength and read on 3 or 6 different channels without cross-talk or “bleed-over”. The most commonly employed fluorescent probe is 6-carboxyfluorescein (FAM), fluorescing in channel 1 (530 nm), with bleed-over into the other channels, which is currently compensated for by surrendering valuable carousel space for extraction controls, amplification controls, and duplicate sample runs to assure the generated answer is correct. Using the 705 nm channel requires resonance transfer probes, as few fluorescent molecules exist with Stokes’ shifts wide enough to achieve detection in this channel. In 2009, a fluorescence resonance energy transfer (FRET)-based method was developed which, when combined with TaqMan style probes, provides a linker that emits a signal that can be read in the unused third channel of the LC 1.5 [1]. This “FRET-MAN” technology works well; however, designing assays with dual probes and quenchers can be challenging. To address this challenge, Biosearch Technologies designed a probe called the Pulsar® 650, which is a fluorescent reporter dye based on a Ru (bpy)3 complex with a large Stokes shift, (λex = 460 nm, λem = 650 nm) that can be used in place of a two-dye FRET construct. Importantly, this dye is stable under oligonucleotidesynthesis conditions, strong acids, and strong bases (Biosearch Technologies, http://www.biosearchtech.com). We present here an extension of a duplexed assay originally developed for the LC 1.2 [2] to an RNA-based viral target with the inclusion of an internal control for robust quantitation. Here, the primary target was one of two strains of influenza A, chosen due to the increase of awareness of public health in recent years. The third-channel duplex probe was designed based on the human RNase P (hRP), GAPDH, 18S ribosomal RNA, β-actin, cyclophilin or gyrase [3–8]. To demonstrate the ability of duplexed detection, inclusion of the internal controls (ICs) is shown herein to function in both laboratory buffered samples and in clinical matrices. Theoretically, the second probe could instead be used as a detector for a second target organism.