uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA.
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences. (Klinisk virologi)
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences. (Klinisk virologi)
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences. (Infektionssjukdomar)
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences. (Infektionssjukdomar)
Show others and affiliations
2004 (English)In: J Clin Virol, ISSN 1386-6532, Vol. 30, no 2, 150-6 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2004. Vol. 30, no 2, 150-6 p.
Keyword [en]
5' Untranslated Regions/genetics, Adolescent, Adult, Aged, Base Sequence, DNA Primers, DNA; Viral/genetics, Enterovirus/genetics/*isolation & purification, Enterovirus Infections/*cerebrospinal fluid/diagnosis, Humans, Meningitis; Viral/cerebrospinal fluid/virology, Middle Aged, Molecular Sequence Data, RNA; Viral/*cerebrospinal fluid/genetics, Reagent Kits; Diagnostic, Reproducibility of Results, Research Support; Non-U.S. Gov't, Reverse Transcriptase Polymerase Chain Reaction/*methods, Sensitivity and Specificity, Sequence Alignment, Sequence Homology; Nucleic Acid
Identifiers
URN: urn:nbn:se:uu:diva-72924PubMedID: 15125871OAI: oai:DiVA.org:uu-72924DiVA: diva2:100835
Available from: 2007-11-01 Created: 2007-11-01 Last updated: 2011-01-12
In thesis
1. 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
2. Development of Real-Time PCR Based Methods for Detection of Viruses and Virus Antibodies
Open this publication in new window or tab >>Development of Real-Time PCR Based Methods for Detection of Viruses and Virus Antibodies
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Quantitative real-time PCR (QPCR) technology has been very useful for diagnosis of viral diseases. QPCR has recently reached a level of sensitivity, simplicity, and reproducibility which allows a large number of samples to be screened rapidly, make it a suitable tool for the clinical virology diagnostics.

In this thesis, broadly targeted and degenerated quantitative QPCR assays were used. A somewhat novel single-tube real-time reverse transcription-polymerase chain reaction (QRT-PCR), with takes advantage of ability of rTth DNA polymerase to reverse transcribe RNA in the presence of Mn2+ at elevated temperatures and includes protection against amplimer contamination by using thermolabile UNG, was developed. A new technique for diagnostic of recent viral infection by detection of viral immunoglobulin M (IgM) was also developed.

In the first paper, a sensitive single-tube QRT-PCR for detection of enteroviral RNA in patients with aseptic meningitis was presented. In the second paper, a single-serum-dilution real-time PCR-based PIA (PCR-enhanced immunoassay), called quantitative PIA (QPIA), to detect enterovirus IgM for diagnosis of EV infection in patients with aseptic meningitis, was also developed. In the third paper, a broadly targeted, simple, single tube degenerated quantitative QPCR technique for detection of JCV, BKV and SV40 DNA was developed. A conserved region of the VP2 gene of JCV, BKV and SV40 was targeted. A false positive result due to contamination with commonly used SV40 T-antigen plasmids was therefore avoided. In manuscript four, the QPIA assay provide a rational strategy for detection of EV IgM, allows the use of viral antigens isolate from newly diagnosed Type 1 diabetes patients (T1D-EV-QPIA) to measured IgM against diabetogenic viruses in serum from newly diagnosed T1D children, siblings, and healthy children.

To conclude, novel broadly targeted real-time PCR methods for diagnosis of entero- and polyoma viral infections were developed.

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 ; 202
Keyword
Microbiology, Real-time PCR, broadly targeted PCR, degenerated PCR, rTth DNA polymerase, thermolabile UNG, QPIA, IgM, Mikrobiologi
Identifiers
urn:nbn:se:uu:diva-7320 (URN)91-554-6725-3 (ISBN)
Public defence
2006-12-12, Hörsalen, ing, D1, Dag Hammarskjölds Väg 17, Akademiska Sjukhuset 751 85, UPPSALA, 13:15 (English)
Opponent
Supervisors
Available from: 2006-11-21 Created: 2006-11-21 Last updated: 2009-05-08Bibliographically approved

Open Access in DiVA

No full text

Other links

PubMedhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Retrieve&list_uids=15125871&dopt=Citation

Authority records BETA

Fohlman, JanFriman, GöranBlomberg, Jonas

Search in DiVA

By author/editor
Fohlman, JanFriman, GöranBlomberg, Jonas
By organisation
Department of Medical Sciences

Search outside of DiVA

GoogleGoogle Scholar

pubmed
urn-nbn

Altmetric score

pubmed
urn-nbn
Total: 586 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf