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Broadly targeted multiprobe QPCR for detection of coronaviruses: Coronavirus is common among mallard ducks (Anas platyrhynchos)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology. (Blomberg)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology. (Blomberg)
Växjö Universitet.
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2009 (English)In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 159, no 2, 277-287 p.Article in journal (Refereed) Published
Abstract [en]

Coronaviruses (CoV) can cause trivial or fatal disease in humans and in animals. Detection methods for a wide range of CoVs are needed, to understand viral evolution, host range, transmission and maintenance in reservoirs. A new concept, "Multiprobe QPCR", which uses a balanced mixture of competing discrete non- or moderately degenerated nuclease degradable (TaqMan((R))) probes was employed. It provides a broadly targeted and rational single tube real-time reverse transcription PCR ("NQPCR") for the generic detection and discovery of CoV. Degenerate primers, previously published, and the new probes, were from a conserved stretch of open reading frame 1b, encoding the replicase. This multiprobe design reduced the degree of probe degeneration, which otherwise decreases the sensitivity, and allowed a preliminary classification of the amplified sequence directly from the QPCR trace. The split probe strategy allowed detection of down to 10 viral nucleic acid equivalents of CoV from all known CoV groups. Evaluation was with reference CoV strains, synthetic targets, human respiratory samples and avian fecal samples. Infectious-Bronchitis-Virus (IBV)-related variants were found in seven of 35 sample pools, from 100 wild mallards (Anas platyrhynchos). Ducks may spread and harbour CoVs. NQPCR can detect a wide range of CoVs, as illustrated for humans and ducks.

Place, publisher, year, edition, pages
2009. Vol. 159, no 2, 277-287 p.
Keyword [en]
Coronaviruses, Real-time PCR, Nuclease-based probes, TaqMan, Avian coronavirus, Split probe strategy
National Category
Medical and Health Sciences
Research subject
Medical Virology
URN: urn:nbn:se:uu:diva-102539DOI: 10.1016/j.jviromet.2009.04.022ISI: 000267454000022PubMedID: 19406168OAI: oai:DiVA.org:uu-102539DiVA: diva2:216326
Available from: 2009-05-07 Created: 2009-05-07 Last updated: 2011-03-04Bibliographically approved
In thesis
1. Detection and Quantification of Variable Viral RNA by Real-Time PCR Assays
Open this publication in new window or tab >>Detection and Quantification of Variable Viral RNA by Real-Time PCR Assays
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

As the area of nucleic acid based technologies develops, so will our understanding of how structural variations in DNA and RNA pathogens are associated with disease. The overall goal of this thesis is the development of broadly targeted measurement techniques for variable viral RNA by Real-Time PCR (here referred to as quantitative reverse transcriptase PCR, QRT-PCR).

In papers I & II, broadly targeted and specific QRT-PCRs were used to study expression of endogenous and exogenous betaretrovirus sequences in human tissues. Results from human tissues demonstrated endogenous betaretrovirus expression in a tissue-specific manner, highest in reproductive tissues. Despite the high sensitivity, no exogenous betaretrovirus was found in human breast cancer samples. The limits of primer and probe degeneracy for detection of a diverse set of retroviral sequences was evaluated. These methods are useful for further investigations on the pathophysiological contribution(s) of endogenous betaretrovirus and to investigate whether an exogenous betaretrovirus is involved in human breast cancer.

In papers III & IV, we developed and applied broadly targeted one-step QRT-PCRs for influenza viruses and coronaviruses. In addition to the generic primers, two novel probe design strategies were used in order to be able to broadly amplify these diverse sets of viruses: A triplex system for simultaneous detection and quantification of influenza A, B and C (3QRT-PCR and further developed 3QRT-PCR-MegB; where MegB stands for MegaBeacon) based on TaqMan® and MegB probes, and a pan-CoV QRT-PCR, based on three TaqMan® probes i.e., degeneracy was distributed on three probes. Probe fault tolerance was thus increased in two ways, either with short probes with/without locked nucleic acid (LNA) nucleotides concentrated to conserved stretches, or with long probes (MegB), compensating mismatching positions with many matching ones. Clinical samples, negative by antigen detection with immunofluorescence (IFA), were influenza A positive with 3QPCR-MegB. Avian pooled samples, negative with an earlier pan-CoV QPCR, came out positive with the triple-probe system. Assay evaluation with clinical samples and reference strains revealed good clinical diagnostic potential.

Thus, the thesis describes several strategies to counteract sequence variation of RNA viruses and describes a set of broadly targeted QRT-PCRs useful for scientific screening or diagnostics of betaretroviruses and respiratory viruses.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 67 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 365
Virology, Diagnosis of Viral RNA, QPCR, RNA virus, broadly targeted PCR, probe design strategy, Infectious diseases
Research subject
urn:nbn:se:uu:diva-9193 (URN)978-91-554-7251-1 (ISBN)
Public defence
2008-09-05, Hörsalen, Baktlab ing D1, Dag Hammarskjölds väg 17, Uppsala, 13:00 (English)
Available from: 2008-08-15 Created: 2008-08-15 Last updated: 2011-02-28Bibliographically approved

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