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qPCR assays for optimized viral detection in clinical samples

ZEN™ Double-Quenched Probes add sensitivity and specificity to an assay for the highly variable norovirus

Xia H, Gravelsina S, et al. (2016) Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II. BioTechniques, 60:28–34.

Background

Highly contagious, norovirus is the most common cause of acute gastroenteritis in humans. Every year, norovirus causes nearly 20 million illnesses in the US alone and is a leading contributor to foodborne-illness outbreaks around the world. This impact has prompted scientists to explore methods for rapid and accurate norovirus detection in patients, as well as in food, water, and other environmental samples.

The current gold standard for norovirus detection is qPCR, but due to the large amount of sequence diversity, detection can be difficult, even when amplifying highly conserved regions of the norovirus genome. In a previous publication, these authors showed that long primers and probes can tolerate sequence variation in norovirus genogroup II, when using VOCMA (variation-tolerant capture multiplex assay) with traditional PCR conditions [1]. Here, in an effort to more rapidly and accurately detect norovirus genomes, Xia et al. apply VOCMA to qPCR using long nuclease-activated probes and various internal quenchers.

Experiment

The researchers sequenced nearly 600 norovirus genomes, noting the frequency of sequence variation at each base position. This information guided the design of degenerate primers and probes that target a highly conserved region of the norovirus genome. The group developed 2 distinct sets of forward and reverse primers, 4 degenerate probes with various internal quenchers, and a synthetic 161mer oligonucleotide that represented the qPCR target amplicon. They performed VOCMA on 86 clinical samples, testing for norovirus while also comparing the sensitivity of the 4 probes. Two of the probes evaluated were IDT ZEN™/Iowa Black® Double-Quenched Probes. The primers used in this study were also synthesized by IDT.

Results

To compare assay sensitivity, the researchers used Cq values obtained in the second round of qPCR to plot standard curves for the 4 probes—2 ZEN probes and 2 probes containing internal BHQ1 quenchers. With a detection limit of 10 copies/reaction, both ZEN probes displayed higher sensitivity than the BHQ1 probes.

In experiments that addressed assay specificity, Xia et al. were able to accurately detect 44 of 46 samples containing norovirus genogroup II using the long, ZEN Double-Quenched Probes. In addition, they saw no cross-reactivity in 43 samples that did not contain norovirus genogroup II. Sequencing of 2 false negative samples indicated viral variants with deletions of 51 and 39 nucleotides. The scientists determined that their probes were unable to bind to these target regions due to the large deletions.

These results suggest that, by using long internally quenched probes, the VOCMA protocol can be applied to qPCR for quick and sensitive detection of highly variable pathogens. The authors note that, with the high sensitivity offered by IDT ZEN Double-Quenched Probes, their assays could be applied to both clinical and environmental viral samples.

References

  1. Ohrmalm C, Eriksson R, et al. (2012) Variation-tolerant capture and multiplex detection of nucleic acids: application to detection of microbes. J Clin Microbiol, 50:3208–3215.

Product focus

Double-Quenched Probes

Standard 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. IDT will also accommodate custom probe modifications. These special requests can be made through IDT’s non catalog modifications (noncat@idtdna.com).

Visit www.idtdna.com/qPCRprobes to learn how ZEN and TAO Double-Quenched Probes consistently provide earlier Cq values and improved precision, when compared to traditional, single-quenched qPCR probes.


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/SNP information.

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


PrimeTime Gene Expression Master Mix

  • Achieve high efficiency qPCR under fast or standard cycling conditions

  • Multiplex without loss of sensitivity

  • Obtain consistent results from overnight experiments by capitalizing on exceptional benchtop stability

PrimeTime Gene Expression Master Mix supports probe-based qPCR assays for gene expression analysis. This master mix is optimized for use with PrimeTime qPCR Assays but is also compatible with other primers and probes.

Learn more at www.idtdna.com/mastermix.

Additional reading

Two quenchers are better than one!—Review data demonstrating that use of ZEN™ Double-Quenched Probes in qPCR assays provides increased signal detection and greater assay sensitivity compared to use of single-quenched probes, such as probes with BHQ Quenchers. ZEN and TAO™ Double-Quenched Probes also make it more feasible to use longer probes due to the resulting lower background fluorescence.

Double-quenched probes increase sensitivity of qPCR assay detecting viral load—In this citation summary, scientists demonstrate how use of a ZEN™ Double-Quenched Probe results in a marked decrease in background fluorescence compared to a competitor’s probe containing only a single quencher. The data suggest that such double-quenched probes may be a better approach for other qPCR probe-based assays.

Design efficient PCR and qPCR primers and probes using online tools—Find out how to facilitate experimental planning using IDT free, online tools for oligonucleotide analysis and PCR primer design. This article describes our predesigned qPCR assays and the basics of designing customized PCR primers and hydrolysis probes with the PrimerQuest® Tool.

Digital PCR (dPCR)—What is it and why use it?—This article provides an overview of dPCR and how it can be used for qPCR applications, including multiplex qPCR.

Author: Nolan Speicher is a scientific writer at IDT.

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Probe-based qPCR assays for quantification of human, mouse, and rat gene expression. Order in plates or tubes.

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