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Novel Light-Upon-Ex...
Novel Light-Upon-Extension Real-Time PCR Assay for Simultaneous Detection, Quantification, and Genogrouping of Group A Rotavirus
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- Nordgren, Johan (författare)
- Linköpings universitet,Medicinsk mikrobiologi,Hälsouniversitetet
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- Bucardo, Filemon (författare)
- Linköpings universitet,Molekylär virologi,Hälsouniversitetet
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- Svensson, Lennart (författare)
- Linköpings universitet,Molekylär virologi,Hälsouniversitetet
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- Lindgren, Per-Eric (författare)
- Linköpings universitet,Medicinsk mikrobiologi,Hälsouniversitetet
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(creator_code:org_t)
- American Society for Microbiology, 2010
- 2010
- Engelska.
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Ingår i: JOURNAL OF CLINICAL MICROBIOLOGY. - : American Society for Microbiology. - 0095-1137. ; 48:5, s. 1859-1865
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- We have developed a light-upon-extension (LUX) real-time PCR assay for detection, quantification, and genogrouping of group A rotavirus (RV), the most common cause of acute gastroenteritis in children. The LUX system uses a fluorophore attached to one primer and having a self-quenching hairpin structure, making it cost-effective and specific. We designed genogroup-specific primers having different fluorophores, making it possible to differentiate between the two main genogroups of human group A RVs. The assay was applied on clinical stool specimens from Sweden and Central America (n = 196) and compared to immunological and conventional PCR assays. The genogrouping ability was further validated against a subset of clinical specimens, which had been genogrouped using monoclonal antibodies. Our real-time PCR assay detected and quantified all positive specimens (n = 145) and exhibited higher sensitivity than immunological assays and conventional PCR. The assay exhibited a wide dynamic range, detecting from 5 to andgt; 10(7) genes per PCR, resulting in a theoretical lower detection limit of andlt; 10,000 viruses per gram of stool. No cross-reaction was observed with specimens containing norovirus, sapovirus, astrovirus, or adenovirus. In total, 22 (15%) of the positive clinical specimens were identified as genogroup I, 122 (84%) were identified as genogroup II, and 1 specimen was found to contain a mix of both genogroups. All genogroup I-positive specimens were associated with capsid glycoprotein 2 (G2). No significant difference in viral load was found between genogroups or geographic region. The detection and quantification, combined with the genogrouping ability, make this assay a valuable tool both for diagnostics and for molecular epidemiological investigations.
Nyckelord
- MEDICINE
- MEDICIN
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- art (ämneskategori)
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