A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA
| Autor: | Amal Elfaitouri, Jan Fohlman, Jonas Blomberg, Göran Friman, Nahla Mohamed |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Adult
Adolescent Serial dilution Molecular Sequence Data Coxsackievirus medicine.disease_cause Sensitivity and Specificity Sequence Homology Nucleic Acid Virology Enterovirus Infections medicine Humans media_common.cataloged_instance European union Aged DNA Primers Enterovirus media_common Base Sequence biology Reverse Transcriptase Polymerase Chain Reaction Reproducibility of Results Middle Aged biology.organism_classification Meningitis Viral Molecular biology Reverse transcriptase Reverse transcription polymerase chain reaction Infectious Diseases Real-time polymerase chain reaction DNA Viral RNA Viral Reagent Kits Diagnostic RNA extraction 5' Untranslated Regions Sequence Alignment |
| Zdroj: | Journal of Clinical Virology. 30:150-156 |
| ISSN: | 1386-6532 |
| Popis: | Background: Enteroviruses (EVs) are significant human pathogens. Rapid and sensitive diagnostic techniques are desirable. Objectives: To develop a quantitative single-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) for human enterovirus ribonucleic acid (RNA) (QPCR), with protection against amplimer contamination. Study design: The method was evaluated with serial dilutions of EV, 62 cerebrospinal fluid (CSF) specimens from meningitis patients, and the third and fourth European Union Concerted Action Enterovirus Proficiency Panels. A commercial EV PCR test was run in parallel. Results: Optimisations included RNA extraction procedure, design and concentrations of primers and probes from the 5 � non-coding region as well as recombinant Thermus thermophiluspolymerase (rTth), Mn(OAc)2 and thermolabile UNG concentrations. Of 62 CSF samples from cases of meningitis submitted for QPCR testing, 34 (76%) and 21 (47%) were positive by QPCR and a commercial EV RNA detection kit, respectively. The detection limit of QPCR was 0.001 TCID 50/ml (50% tissue culture-infective dose per millilitre) for a coxsackievirus B2 preparation and |
| Databáze: | OpenAIRE |
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