The scientists at Tetracore (Rockville, MD, USA; www.tetracore.com) specialize in developing diagnostic PCR assays that detect infectious diseases and bio-terrorism threat agents. They are experienced at optimizing PCR reagents because their assays are used by researchers, such as military and veterinary diagnosticians, who work in a wide range of environmental conditions, and who use a variety of PCR instrument platforms. Thus, the assay reagents need to be robust and forgiving. Assay development requirements for Tetracore researchers include:
- Quick reformulation of products to address viral mutation
- Large sets of primers/probe that work together and provide robust amplification in a multiplex format
- Probe dyes that can be discriminated by a specific set of common PCR instruments, without instrument recalibration
- Sufficiently low background of multiple probes to allow accurate quantification of distinct signals
- Assay reagents that are stable across a broad range of temperatures, including those in tropical and desert conditions
We spoke with Dr William M Nelson, co-founder and president of Tetracore, and one of the company’s research scientists, Rolf Rauh, who both elaborated on how the company has addressed these challenges.
Keeping up with rapidly mutating viruses
“Many of the viral infections we develop assays for are caused by RNA viruses, which can change rapidly over time through high mutation rates, not unlike HIV in humans. This requires us to continually check GenBank to see if there are new strains evolving that have the potential to be missed by our assays. We are constantly reformulating our assays so that they will detect all of the relevant viral variants,” notes Dr Nelson.
Multiplex assays that ID all viral strains
Another major challenge for the Tetracore researchers is to be able to provide a unique set of assays that will identify all strains of a disease-causing organism. For example, Porcine Reproductive and Respiratory Syndrome virus (PRRSV), one of the diseases for which Tetracore provides test reagents, is actually caused by a broad group of viruses, of which there are 2 main types: North American–like and European-like (named for where they were first isolated). These 2 main types are different enough that a single set of primers and probe will not identify both. So the scientists at Tetracore use several primer and probe sets in a single qPCR assay to be able to detect all of the variants.
Currently, the Tetracore PRRSV assay contains 34 primers and 8 probes! Not only must these primer and probe sets be designed to work simultaneously, in a multiplex format, but signals detecting the different PRRSV types need to be distinguishable. The 8 probes use 3 different fluorescent dyes, and must be detected by 3 non-overlapping real-time PCR instrument channels. One channel is dedicated solely to the control, so the other 2 channels are left to detect the remaining 7 probes. Therefore, the choice of dyes used with these probes is critical.
Dye selection and optimization
Tetracore’s selection of dyes requires numerous considerations. Their customers use a variety of real-time PCR instruments, and common dyes do not always provide sufficient signal resolution on every platform. Additionally, some instruments need to be calibrated for the chosen dyes, or at least have channels sufficiently separated to detect the dyes without overlap. Dye choice is also restricted by the buffers and even common plasticware used, which themselves can alter fluorescence. For the PRRSV assay, Tetracore settled on FAM (FAM channel) for probes detecting 1 strain of virus, ATTO 550/Dragonfly Orange™ (DFO) (TAMRA or Cy®3 channel, depending on the instrument) for probes detecting them second strain, and ATTO 647N (Cy5 channel) as the inhibition extraction control.
The additive signal of the multiple probes in the reaction mixture creates excessive background fluorescence. Even though the probes are quenched, longer probes and those with secondary structure that restrict quenching can generate significant background fluorescence, especially in the FAM channel. Therefore, the Tetracore team had to devise methods for combining all of these probes into a single cocktail, but still provide enough discrimination for accurate disease detection. “In order to maintain a robust assay that was relevant, we needed ways to limit the amount of background fluorescence,” remarked Rolf Rauh.
“In addition to providing us with the ZEN Double-Quenched Probes to help resolve our background issues, IDT has been invaluable, both from a quality and turnaround time perspective. We frequently make use of being able to order primers and receiving them the next day.”—Rolf Rauh, Scientist at Tetracore
Adding an internal quencher to decrease background fluorescence
IDT ZEN™ Double-Quenched Probes have helped Tetracore address the problem of high background. ZEN probes include 2 quenchers: Iowa Black FQ (IBFQ) at the 3’ end of the probe, and the ZEN quencher positioned internally, 9 bp from the fluorophore. The fluorescent background (Rn) of a ZEN Double- Quenched Probe is nearly 4X lower than traditional dye-quencher labeled probes (see the PrimeTime qPCR Probes flyer). Introducing 5’FAM/ZEN/3’IBFQ probes has reduced potential false positive signal and significantly lowered background fluorescence in the PRRSV assay (Figure 1).
In addition, these probes have allowed Tetracore to more easily provide assays for platforms like the Cepheid SmartCycler®, which has a fixed amount of “headroom” (the amount of overall signal allowable before saturation). The high background from the traditional, single-quenched probes in the assay results in “railing”, i.e., where the signal plateaus, producing a straight line, because the instrument does not have enough dynamic range to accommodate the process (Figure 1A). Railing can lead to signal bleed over into adjacent channels, which can complicate data interpretation if those channels are also being used (Figure 1B). The reduced background fluorescence of ZEN Double-Quenched Probes compared to traditional single-quenched probes is demonstrated in Figure 1C.
Figure 1. ZEN™ Double-Quenched Probes Resolve Fluorescence Saturation, Bleed Over, and High Background. A120 bp region of a diagnostic PRRSV gene was amplified by real-time PCR, using an 18 nt FAM probe synthesized with either a single BHQ quencher (B11 and B12) or dual ZEN/IBFQ quenchers from IDT (A7 and A9). Reactions were performed in duplicate on the SmartCycler® System (Cepheid). (A) Abnormal sigmoidal curves, indicative of fluorescence saturation in the FAM channel, were observed for the single-quenched probe. Regular sigmoid curves were obtained for the corresponding double-quenched probe. (B) Saturation of the FAM channel resulted in bleed over into the adjacent Cy3 channel for the single-quenched probe; no bleed over was observed with the double-quenched probe. (C) Background fluorescence was substantially higher for the single-quenched probe than the double-quenched probe.
Dr Nelson also noted, “The ZEN Double-Quenched Probes have allowed us tocontinue adding probes to the already complex PRRSV assay mix to accommodate the changing viral landscape. This IDT product has helped us address the addition of new probes and primer sets to our assay very quickly.” Thus, adoption of ZEN Double-Quenched Probes has allowed the Tetracore researchers to quickly reformulate their assays with the addition of probes to address rapid viral mutation, while reducing the background they would otherwise generate.