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qPCR assays for optimized viral detection in clinical samples

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.

Learn how ZEN Double-Quenched Probes were used in a unique qPCR experiment to help rapidly and accurately detect the highly variable norovirus in clinical samples.

Apr 6, 2016

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.

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