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Zika virus: Advances in disease modeling and detection

ZEN™ Double-Quenched Probes and gBlocks® Gene Fragments provide invaluable tools

After decades of dormancy, Zika virus disease (Zika) is estimated to have arrived in Brazil in 2013 [1]. Since then, the disease has been confirmed in 49 countries and territories worldwide, including 38 within the Americas [2,3]. Zika’s rapid spread and linked neurological effects have resulted in an ongoing epidemic and an urgent need for effective diagnostic and treatment strategies.

What is Zika?

“Zika” refers to Zika virus disease, which is caused by the Zika virus (ZIKV). ZIKV is most often transmitted to humans through the bite of an infected Aedes mosquito, but it can also be spread by infected individuals through sexual contact, blood transfusions, and from mother to fetus during pregnancy. Common Zika symptoms include fever, rash, and joint pain—these are usually mild, resulting in many carriers who are unaware of their infection. In pregnant women, ZIKV infection is strongly linked to microcephaly (abnormally small head) and severe brain defects in their resulting newborns. Due to the increasing number of these cases in particular, the World Health Organization (WHO) declared Zika a Public Health Emergency of International Concern (PHEIC) in February, 2016 [4].

A model for in utero transmission of ZIKV infection

Recent research at Washington University (St. Louis, MO, USA) has provided insight into the link between ZIKV infection and pregnancy-associated microcephaly. Using ZEN Double-Quenched Probes for RT-qPCR, scientists found high levels of ZIKV in the placentas of pregnant mice and in the brains of their infected fetuses. Further histological examination showed that the high levels of ZIKV were strongly associated with apoptosis and vascular damage in these tissues, leading to fetal abnormalities similar (but not identical) to those seen in humans [5]. While numerous other studies have suggested a correlation between ZIKV infection and fetal abnormalities, this is the first to establish causality.

Progress in diagnostics and treatment

While no vaccine or antiviral therapy currently exists for Zika, significant advances are being made with the help of tools from IDT. Following are several examples.

RT-qPCR for ZIKV viral load quantification

Researchers at the University of Leuven (Leuven, Belgium) administered potential ZIKV inhibitors to infected cell cultures and performed RT-qPCR to quantify resulting RNA levels. In their assays, they used ZEN Double-Quenched Probes, which significantly increase assay sensitivity and reduce background fluorescence. The scientists describe a viral polymerase inhibitor, 7-deaza-2′-C-methyladenosine, which efficiently stops ZIKV replication in vitro and delays disease progression in mice [6].

Researchers at the University of North Carolina (Chapel Hill, NC, USA) investigated ZIKV pathogenesis in vivo by infecting healthy mice, as well as those lacking major components of the antiviral response. Following ZIKV administration, the researchers performed RT-qPCR with ZEN Double-Quenched Probes to quantify resulting ZIKV RNA levels in various organs. Mice producing limited amounts of interferon α/β, and mice lacking the interferon receptor, were seen to be highly susceptible to ZIKV infection. These groups showed high viral loads in the brain, spinal cord, and testes, thus demonstrating their potential to be suitable animal models for future Zika/ZIKV studies [7].

Field-ready ZIKV detection using paper-based toehold sensors

In conjunction with the Wyss Institute for Biologically Inspired Engineering (Boston, MA, USA), collaborators at the University of Toronto (Toronto, Canada) and Arizona State University (Tempe, AZ, USA) have designed a rapid, low-cost diagnostic platform that overcomes many of the practical challenges facing field-ready ZIKV detection tools. Based on principles from NASBA (nucleic acid sequence-based amplification) and CRISPR-Cas9, their approach uses Ultramer® Oligonucleotides as templates for the construction of paper-based toehold switch sensors. They demonstrated these molecular sensors to be highly sensitive, capable of detecting ZIKV RNA in samples with extremely low viral loads [8].

Universal control RNA for comparison of RT-qPCR assay performance

A group of researchers across Germany, France, and the Netherlands evaluated the standardization and diagnostic performance of 6 RT-qPCR assays, which are widely used for ZIKV detection. Using IDT gBlocks® Gene Fragments, they constructed a “universal control RNA (ucRNA)” containing the target regions for each of the 6 distinct assays. The ucRNA allowed the researchers to quantitatively evaluate assay performance, and in doing so, they found that some assays showed limited sensitivity to low viral loads, while others were incompatible with new ZIKV outbreak strains. This finding suggests the need for more advanced, highly accurate diagnostics [9].


References

  1. Faria NR, Azevedo Rdo S, et al. (2016) Zika virus in the Americas: Early epidemiological and genetic findings. Science, 352(6283):345–349.

  2. Pan American Health Organization, World Health Organization. (2016) Geographic distribution of confirmed cases of Zika virus in countries and territories of the Americas, 2015–2016. Washington D.C., PAHO/WHO. [Accessed 11 May, 2016].

  3. European Centre for Disease Prevention and Control. (2016) Epidemiological situation. ECDC 2005–2016. [Accessed 11 May, 2016].

  4. Centers for Disease Control and Prevention. (2016) About Zika virus disease. Atlanta, CDC. [Accessed 11 May, 2016].

  5. Miner JJ, Cao B, et al. (2016) Zika virus infection during pregnancy in mice causes placental damage and fetal demise. Cell, 165(5):1081–1091.

  6. Zmurko J, Marques RE, et al. (2016) The viral polymerase inhibitor 7-deaza-2'-C-methyladenosine is a potent inhibitor of in vitro Zika virus replication and delays disease progression in a robust mouse infection model. PLoS Negl Trop Dis, 10(5):e0004695.

  7. Lazear HM, Govero J, et al. (2016) A mouse model of Zika virus pathogenesis. Cell Host Microbe, 19(5):720–730.

  8. Pardee K, Green AA, et al. (2016) Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell, 165(5):1255–1266.

  9. Corman VM, Rasche A, et al. (2016) Clinical comparison, standardization and optimization of Zika virus molecular detection. Bull World Health Org E-pub: 19 Apr 2016.


Product focus

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.


Ultramer® Oligonucleotides

Ultramer oligos are single-stranded, custom DNA sequences up to 200 nt, used for applications such as gene synthesis, qPCR standards, and mutagenesis.

Learn more at www.idtdna.com/pages/products/dna-rna/ultramer-oligos.

gBlocks® Gene Fragments

gBlocks Gene Fragments are custom 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 valuable laboratory time.

Learn more at www.idtdna.com/gBlocks.


Additional reading—Using IDT products for pathogen detection

Improved pathogen detection by multiplex RT-qPCR—Learn how gBlocks® Gene Fragments can help optimize multiplex qPCR for pathogen detection in human clinical samples.

qPCR assays for optimized viral detection in clinical samples—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.

Developing a qPCR point-of-care diagnostic for Ebola—Learn how Ubiquitome, Battelle, and IDT are developing a rapid qPCR Ebola virus test for easy use in the field. The PrimeTime qPCR assay is designed to be run on Ubiquitome’s hand-held, battery powered real-time PCR device, the Freedom4.

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.

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


Author: Nolan Speicher is a scientific writer at IDT.

© 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


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