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Developing a qPCR point-of-care diagnostic for Ebola

The Ubiquitome Freedom4 device and PrimeTime® qPCR Assays

The 2013–2015 Ebola epidemic in West Africa began in a remote village in late December 2013 but was not identified until March 2014 [1]. In the 14 months since the Ebola outbreak was identified, approximately 26,750 people were suspected or confirmed to have contracted the Ebola virus disease (EVD), and more than 11,000 people have died from the infection [2].

Colorized TEM of Ebola virus (CDC/Cynthia Goldsmith)

Figure 1. Colorized transmission electron micrograph of the Ebola virion. Image by CDC/Cynthia Goldsmith [Public domain], via Wikimedia Commons.

It can be difficult to distinguish EVD from other infectious diseases, such as cholera, malaria, typhoid fever, and meningitis. While current diagnostic tests are accurate, they are time-consuming and complex. They also create challenges related to sample storage and transportation, laboratory biosafety, and staff expertise in performing the assays. Having access to simple, rapid, and accurate viral testing in remote villages would be extremely useful for quelling this epidemic as the number of new cases decreases, and will be important for detecting future outbreaks. With this goal in mind, Dr Paul Pickering (Ubiquitome Limited, Auckland, New Zealand) is spearheading a project to develop an RT-qPCR assay used with the Freedom4 device, a portable PCR machine (see sidebar, From bench top to handheld, battery-operated PCR instrument).

Robust RT-qPCR Ebola virus assay

IDT scientists Kristin Beltz and Dr Scott Rose (Coralville, IA, USA) along with Dr Brian Taylor and his team (Battelle, Aberdeen, MD, USA) designed and validated an IDT PrimeTime® qPCR Assay that detects Zaire ebolavirus (Figure 1), the species involved in the 2013–2015 West African Ebola outbreak. Initial sensitivity and specificity results were presented at the 2015 Chemical and Biological Defense Science and Technology Conference (St. Louis, MO, USA). The proprietary, 5′ nuclease-based assay has a PCR efficiency of 98.9% (Figure 2) and uses ZEN™ Double-Quenched Probes (IDT), which exhibit lower background and higher signal than traditional single-quenched probes in qPCR assays.

PrimeTime qPCR Ebola Assay Standard Curve

Figure 2. The Ebolavirus PrimeTime® qPCR  Assay is 98.9% efficient. The Zaire ebolavirus-specific qPCR assay is a PrimeTime® 5′ Nuclease Assay that includes a ZEN™ Double-Quenched Probe (IDT). Replicate HeLa cell total RNA samples were spiked with 106 to 101 copies of a Zaire ebolavirus RNA fragment (green) in vitro transcribed from gBlocks® Gene Fragments (IDT) using the MEGAshortscript™ and MEGAclear™ Kits (Life Technologies). All samples were run in triplicate on a BioRad CFX384 using a one-step RT-PCR protocol. PCR efficiency was calculated as 10-1/slope-1. The No Template Controls (NTC) are shown in blue, and the –RT reactions (input sample: 106 copies of Ebola RNA in HeLa cell RNA) are shown in dark red. (Data courtesy of IDT, Scott Rose and Kristin Beltz.)

Additional results from Battelle showed that the sensitivity of this assay is comparable when run on the Freedom4 and a traditional, bench-top PCR system (Table 1). Based on an unpaired t test, there was no significant difference in detection of 1000 copies and 500 copies/reaction (P values of 0.3331 and 0.2058, respectively) between the 2 instruments. In addition, in tests of human blood samples spiked with different viral RNAs, the assay had high specificity for Zaire ebolavirus and did not amplify 2 other Ebola virus strains or 5 other viral pathogens known to be present in Africa (Table 2).

qPCR results: Freedom4 same as QuantStudio (Battelle)

Table 1. Sensitivity of the Ebola PrimeTime® qPCR Assay is comparable when run on the Freedom4 device and a bench-top, diagnostic PCR instrument. Zaire ebolavirus RNA was obtained from BEI Resources (NIAID, NIH) and diluted in water. Dilutions of this RNA were tested with the Ebola PrimeTime qPCR Assay (45 PCR cycles) on either the Freedom4 device or the QuantStudio® DX system (Life Technologies). Using an unpaired t test, there was no significant difference in detection of 1000 copies/reaction and 500 copies/reaction (P values of 0.3331 and 0.2058, respectively) between the two machines. Cq = Quantification cycle; NTC = No template control; No amp = No amplification; SD = Standard Deviation. (Data courtesy of Battelle, Brian M Taylor, Sean M Gregory, April M Brys, and David H Moore.)

PrimeTime qPCR assay specific for Zaire ebolavirus (Battelle)

Table 2. The Ebola PrimeTime® qPCR Assay is highly specific for Zaire ebolavirus. Viral material from 2 strains of Ebola virus and 5 other pathogens known to be present in Africa was obtained from BEI Resources (NIAID, NIH). Viral nucleic acid was extracted from spiked blood samples (TRIzol® LS Reagent, Life Technologies) and tested in triplicate using the Ebola PrimeTime qPCR Assay. (Data courtesy of Battelle, Brian M Taylor, Sean M Gregory, April M Brys, and David H Moore.)

Point-of-care diagnostic testing

The goal of point-of-care diagnostic testing is to help stop disease transmission, especially in remote locations, and to facilitate improved patient outcomes. The Freedom4 device eliminates the need for sample preservation and transportation to a laboratory-based testing facility. The instrument can simply be brought to remote locations for use during time-sensitive, public health situations [3]. The Freedom4 and the PrimeTime Ebola qPCR Assay are being submitted to the US FDA for Emergency Use Authorization.

References

  1. World Health Organization (Jan 2015) One year into the Ebola epidemic. www.who.int/csr/disease/ebola/one-year-report/introduction/en/ (Accessed 14 July 2015).
  2. World Health Organization (July 2015) Ebola Situation Reports. http://apps.who.int/ebola/en/ebola-situation-reports (Accessed 14 July 2015).
  3. IDT (Dec 2014) IDT and Ubiquitome Partner to Develop Mobile Ebola Test. www.idtdna.com/pages/home/news-events/news/2014/12/2/IDT-and-Ubiquitome-Partner-to-Develop-Mobile-Ebola-Test (Accessed 26 June 2015).


Profiles

Dr Brian M Taylor, Battelle

Dr Taylor leads the team at Battelle that is validating the PrimeTime® qPCR assay on the Freedom4 device.

Brian Taylor (Battelle)Dr Brian Taylor, Research Scientist in the Biomarkers Services Group at Battelle (Aberdeen, MD, USA), has more than 19 years of experience in immunology, cell and molecular biology, virology, microbiology, and assay development. Prior to his work at Battelle, he managed the µQUANT core facility (Institute of Human Virology [IHV], University of Maryland, School of Medicine, Baltimore, MD, USA), which provides an extensive range of services, including immunochemical assays, cell and virus stock preparation and characterization, cell culture based assays, protein/antibody production and purification, and quantitative PCR. Before joining the IHV, Dr. Taylor worked on methods for creating, propagating, and assaying viral vectors used in gene therapies for cardiac rhythm-associated diseases. Dr. Taylor earned a BS degree in biology from Hampden-Sydney College (Hampden-Sydney, VA, USA) and his PhD in microbiology and immunology from the University of Maryland (Baltimore, MD, USA), where he investigated the altered tropisms associated with mutant envelopes of the human immunodeficiency virus type-1 (HIV-1) and the mechanism by which these viruses enter target cells.

Battelle (Columbus, OH, USA) is a nonprofit research and development organization that serves the national security, health and life sciences, and energy and environmental industries. Battelle’s experience in developing, validating, and performing assays for nonclinical and clinical biomarker evaluation for pharmaceutical product and diagnostic development, coupled with regulatory compliance expertise, facilitates drug discovery and development programs.


Dr Scott Rose and Kristin Beltz, IDT

Dr Rose and Kristin Beltz designed and tested the Zaire-ebolavirus–specific PrimeTime® qPCR assay.

Kristin Beltz and Dr. Scott Rose

Dr Scott Rose joined IDT in 1999 and is currently director of enzyme development and molecular biology reagents. His research focuses on: developing improved nucleic acid capture and enrichment technologies; enhanced synthetic biology tools; RNase H2 based nucleic acid detection methods; cloning, expression, purification, and characterization of new enzymes; and creating novel reagents for use in the field of functional genomics.

Dr. Rose earned his BS (University of Michigan–Dearborn, MI, USA) and PhD (Rice University, Houston, TX, USA) in biochemistry. His PhD thesis work focused on RNA processing—specifically mRNA splicing and polyadenylation. For his postdoctoral research at the University of Texas Southwestern Medical Center (Dallas, TX, USA), Dr Rose studied transcription based gene regulation using the pancreas as a model system.

Kristin Beltz, Research Assistant III, studied at the University of Iowa (Iowa City, IA, USA), where she earned her BS in biology with a focus in cell and developmental biology. She joined IDT in 2007 and has been a key member of the synthetic biology team that developed methods of synthesizing long oligos, for example, Ultramers® Oligonucleotides, and gBlocks® Gene Fragments and Libraries. Currently, her main focus is oligo-based assay designs using RNase H2 technology and the development of enzymes and reagents for molecular biology.

From bench top to handheld, battery-operated PCR instrument

The Freedom4 devicePCR experiments, which can be run in a few hours, have quickly improved our understanding of basic biology and increasingly, provide rapid, accurate testing in molecular diagnostic applications. Along with application development, there have been refinements to the polymerases, primer designs, and thermal cycling machines. The next big step in the evolution of PCR thermal cyclers—a handheld, battery-operated qPCR instrument called Freedom4 (Figure 3)—was announced by Ubiquitome Limited in September 2014.

The instrument processes 4 reactions (sample plus negative, positive, and extraction controls) in approximately 1 hour and is controlled by an iPhone or laptop. It uses laser-based optics that are configured to excite fluorescent dyes at 473 nm and detect emission signals in the range of 510–600 nm, making this instrument compatible with fluorescent-based assays, including either SYBR® Green (used in intercalating dye assays) or FAM fluorescent dyes (used in 5′ nuclease probe-based assays).

Researchers around the world are incorporating the Freedom4 into their field testing. Learn more at www.UbiquitomeBio.com.

Product focus—IDT qPCR primers, probes, and controls

PrimeTime® qPCR Assays

  • 5′ nuclease, probe-based assays—the gold standard for quantitative gene expression studies
  • Primer-based assays—designed for intercalating dye experiments

You can create custom assays that are designed using our proprietary bioinformatics algorithms, any target, and 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. Assistance with assay design is available through our Scientific Application Specialist team at applicationsupport@idtdna.com.

Double-Quenched Probes

ZEN™ Double-Quenched Probes and TAO™ Double-Quenched Probes have a 5′ fluorophore, an internal quencher (ZEN or TAO quencher), and a 3′ quencher. These probes display consistently earlier Cq values and improved precision, when compared to traditional, single-quenched qPCR assay probes.

Learn more at www.idtdna.com/qPCRprobes.

gBlocks® Gene Fragments

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

Learn more at www.idtdna.com/gBlocks.

Related reading

DECODED 4.2—Special qPCR Issue: Get this compendium of IDT DECODED newsletter articles with qPCR tips, troubleshooting advice, and researcher examples in one convenient volume.

PrimeTime® qPCR Application Guide: Download this useful resource that provides experimental overviews, protocols, data analysis, and troubleshooting chapters.


Author: Maureen Young, PhD, is a Scientific Writer at IDT.


© 2015 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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