Broadly targeted multiprobe QPCR for detection of coronaviruses: Coronavirus is common among mallard ducks (Anas platyrhynchos)
| Autor: | Jonas Blomberg, Shaman Muradrasoli, Jan Fohlman, Ákos Hornyák, Sándor Belák, Nahla Mohamed, Bjorn R. Olsen |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
Coronaviruses
viruses TaqMan NQPCR nuclease-based real-time reverse transcription PCR Ct threshold cycle medicine.disease_cause Polymerase Chain Reaction RT-PCR reverse transcriptase polymerase chain reaction law.invention Nuclease-based probes PCR polymerase chain reaction law Coronaviridae Polymerase chain reaction Phylogeny Coronavirus biology Avian coronavirus virus diseases Reverse transcription polymerase chain reaction Viral evolution QPCR quantitative real-time reverse transcription PCR RNA Viral Coronavirus Infections Sequence analysis TET tetrachloro-6-carboxyfluorescein RNA-dependent RNA polymerase TD touch down temperature protocol FRET fluorescence resonance energy transfer Sensitivity and Specificity Article LNA locked nucleic acid Viral Proteins ORF open reading frame Virology Anseriformes medicine Animals Humans Base Sequence Sequence Analysis DNA biology.organism_classification Split probe strategy RNA ribonucleic acid Oligonucleotide Probes Sequence Alignment Real-time PCR |
| Zdroj: | Journal of Virological Methods |
| ISSN: | 1879-0984 0166-0934 |
| Popis: | Coronaviruses (CoVs) 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®) 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 7 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. |
| Databáze: | OpenAIRE |
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