PCR and qPCR
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Improved pathogen detection by multiplex RT-qPCR

Synthetic gBlocks® Gene Fragment standards simplify quantification

Fumian TM, Leite JPG, et al. (2016) Performance of a one-step quantitative duplex RT-PCR for detection of rotavirus A and noroviruses GII during two periods of high viral circulation. J Virol Methods, 228:123–129. doi:10.1016/j.jviromet.2015.11.008.

Multiplex PCR is ideal for limited samples

In this paper, Fumian et al. describe a sensitive duplex RT-qPCR method that uses fluorescently labeled qPCR probes for pathogen detection. Multiplex RT-qPCR offers several technical advantages when working with limited samples, while continuing to provide the same level of sensitivity as singleplex reactions. As little as nanogram amounts of sample can be used to assay multiple targets in a single reaction well. Here, the researchers used a one-step RT-qPCR protocol to assay just 5 ng of total RNA for each duplex RT-qPCR. Multiplex reactions also reduce the number of pipetting steps, limiting technical errors, experimental variation, and reaction cross contamination.

Quantitative standards for multiplex RT-qPCR

In their assay design, the researchers used a single, synthetic dsDNA fragment containing both NoV and RVA viral target regions—ordered as a gBlocks® Gene Fragment (IDT)—as their quantitative standard. Having control amplicons for both target genes in an exact 1:1 ratio allows accurate optimization of both assays in the reaction. The single control template ensures amplification efficiencies are equal, a requirement for multiplex RT-qPCR to provide quantitative results. gBlocks Gene Fragments make it easy to generate multiplex standards, because they are entirely synthetic—control amplicon sequences are simply designed on the same dsDNA molecule.

Increased sensitivity for viral particle detection

In a comparison with commonly used enzyme immunoassays (EIA), the researchers showed that their multiplex RT-qPCR method is up to 11% more sensitive in detecting viral particles in the diluted fecal samples used in this study, with a limit of detection of 8 genome copies. The assay is also easier to perform than current EIA methods or multiple singleplex reactions. While Fumian et al. focused on only 2 targets, some qPCR instruments are capable of simultaneously distinguishing greater numbers of fluorophores, enabling assessment of a larger number of targets in similar multiplex amplification reactions.

Product focus

PrimeTime® qPCR Assays

  • 5′ nuclease, probe-based assays—the gold standard for quantitative gene expression studies

  • Primer-based assays—designed for intercalating dye experiments

Create custom assays that are designed using our proprietary bioinformatics algorithms for any target and to your specific parameters. Alternatively, select one of our predesigned assays for human, mouse, and rat mRNA targets that are supported by our bioinformatics algorithms and up-to-date sequence information.

Learn more at www.idtdna.com/PrimeTime. For assistance with assay design, contact our scientific application specialists at applicationsupport@idtdna.com

Double-Quenched Probes

ZEN and TAO Double-Quenched Probes have a 5′ fluorophore, an internal quencher (ZEN or TAO quencher), and Iowa Black® FQ as the 3′ quencher. These probes provide consistently earlier Cq values and improved precision, when compared to traditional, single-quenched qPCR probes.

Learn more at www.idtdna.com/qPCRprobes.

gBlocks® Gene Fragments

gBlocks Gene Fragments are double-stranded, 125–2000 bp DNA molecules. They are ideal for use as qPCR controls and standards, as well as for gene construction and editing applications. These affordable gene fragments are sequence-verified, ship in a few working days, and save laboratory time.

Learn more at www.idtdna.com/gBlocks.

Further reading

Easily-designed standard curves for qPCR—Learn how to use synthetic gBlocks Gene Fragments for creating standard curves, including incorporating multiple targets into a single gene fragment.

Multiplex qPCR—how to get started—Review tips for setting up multiplex qPCR experiments.

Increase sensitivity and precision in your qPCR experiments—Use double-quenched probes to decrease background, and increase sensitivity and precision in your qPCR experiments.

Recommended dye combinations for multiplex qPCR—Read these recommendations for selecting dyes for multiplex qPCR to minimize background and avoid overlap of fluorescent signals. Included is a table of compatible dyes for multiplexing on common qPCR instruments and a list of suggested quenchers.

Creating a synthetic immune system for optimized immune profiling—Find out how researchers at Adaptive Biotechnologies used gBlocks Gene Fragments to optimize a complex, multiplex PCR for sequencing and quantification of rearranged antigen receptors on T- and B-cells.

Optimizing multiplex qPCR for detecting infectious diseases and biothreat agents in the field—Researchers at Tetracore specialize in developing large sets of robust probe-based qPCR assays for use in a multiplex format to detect infectious diseases and bio-terrorism threat agents. Here they discuss the need to: use probe dyes compatible on common PCR instruments, maintain low background with multiple probes, and reformulate assays to address viral mutation; and how ZEN Double-Quenched Probes have helped meet these criteria.

Author: Hans Packer, PhD, is a scientific writer at IDT.

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