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Broadly targeted triplex real-time PCR detection of influenza A, B and C viruses based on the nucleoprotein gene and a novel "MegaBeacon" probe strategy
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
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2010 (English)In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 163, no 2, 313-322 p.Article in journal (Refereed) Published
Abstract [en]

A PCR assay that covers animal and human influenza A, B and C viruses, i.e., most of Orthomyxoviridae, is needed. Influenza types are distinguished based on differences in the nucleoprotein (NP) present in the virus. Conserved NP regions were therefore used to design a TaqMan (R)-based triplex reverse transcription real-time PCR method. Variability of influenza A within the probe target region mandated the development of a novel molecular beacon, the "Mega" molecular beacon (MegaBeacon: MegB), for the detection of influenza A with this method. MegaBeacon is a mismatch-tolerant molecular beacon that is also a TaqMan (R) probe. The triplex method (3QPCR-MegB) was evaluated with influenza A isolates covering 18 HxNx combinations, two influenza B isolates, and five Japanese influenza C isolates, as well as influenza A, B and C synthetic DNA targets. One to ten viral RNA and cDNA genome equivalents were detected per PCR reaction for influenza A, B and C. Seventy-one human nasopharyngeal aspirates from respiratory infections yielded 30 influenza A, 11 influenza B and 0 influenza C with 3QPCR-MegB, where immunofluorescence (IF) found 28 influenza A and 10 influenza B. 3QPCR-MegB was more mismatch-tolerant than a variant PCR with an influenza A TaqMan (R) probe (3QPCR) and is a sensitive and rational method to detect influenza viruses of animal and human origin. MegaBeacon probes hold promise for variable target nucleic acids.

Place, publisher, year, edition, pages
2010. Vol. 163, no 2, 313-322 p.
Keyword [en]
Influenza, Laboratory diagnosis, Real-time PCR, Respiratory infection, Virus quantitation
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97407DOI: 10.1016/j.jviromet.2009.10.017ISI: 000275074500024PubMedID: 19879296OAI: oai:DiVA.org:uu-97407DiVA: diva2:172347
Available from: 2008-08-15 Created: 2008-08-15 Last updated: 2011-11-11Bibliographically 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 365
Keyword
Virology, Diagnosis of Viral RNA, QPCR, RNA virus, broadly targeted PCR, probe design strategy, Infectious diseases
Research subject
Medicine
Identifiers
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)
Opponent
Supervisors
Available from: 2008-08-15 Created: 2008-08-15 Last updated: 2011-02-28Bibliographically approved
2. Molecular Diagnosis of Common Viral Infectious Diseases Based on Real-Time PCR
Open this publication in new window or tab >>Molecular Diagnosis of Common Viral Infectious Diseases Based on Real-Time PCR
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Molecular biology has become an integral part of the diagnosis of infectious diseases. Recently, quantitative real-time PCR (QPCR) methods (often in the form of so-called TaqMan® systems) have been developed for the diagnosis of a wide range of infectious diseases; these techniques found valuable clinical application in the diagnosis and evaluation of progress and therapeutic success of viral diseases. The use of QPCR as a tool for diagnostic virological and viral research laboratories has greatly increased in recent years. It often replaces conventional PCR and amplicon detection systems which are more complex and laborious, with a higher risk of amplicon carry-over contamination.

The new QPCR methods presented here utilize broadly targeted primers and probes for rational and sensitive detection and quantification of variable RNA viruses. They take advantage of the dual properties, both RNA and DNA dependent DNA polymerase activities, of the rTth thermostable polymerase, and thermolabile UNG with dUTP to protect against inadvertent contamination of samples with amplimers.

In paper one, a novel QPCR approach to detect and quantify human enteroviral (EV) RNA in patients with neurological disorders such as aseptic meningitis is presented. In the second paper, the development of a novel serological technique, quantitative PCR enhanced immunoassay (QPIA), for serodiagnosis of EV infection, is described. In paper three the subject is the development of a touch-down QPCR (TD-QPCR) for detection and preliminary genogrouping of norovirus (NV), a group of Caliciviruses. In paper four a rational, broadly targeted, system for detection of diverse influenza viruses, yet being able to discriminate between influenza A, B and C, is designed and evaluated. In the last paper, another rational broadly targeted system, for detection of corona viruses in humans and animals, is described.

The technologies described in this collection of papers have common features. They are a platform for further development of diagnostic tools for screening and detection of viruses in known viral diseases, maybe also for discovering new viruses.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 60 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 168
Keyword
Molecular biology, Microbiology, Virology, rTth DNA polymerase, UNG, QPCR, RNA virus, broadly targeted PCR, Molekylärbiologi
Identifiers
urn:nbn:se:uu:diva-7118 (URN)91-554-6639-7 (ISBN)
Public defence
2006-10-06, , Medical Microbiology, Dag Hammar Skjölds vag. 17, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2006-09-18 Created: 2006-09-18 Last updated: 2011-02-28Bibliographically approved

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