Novel Light-Upon-Extension Real-Time PCR Assay for Simultaneous Detection, Quantification, and Genogrouping of Group A Rotavirus
2010 (English)In: JOURNAL OF CLINICAL MICROBIOLOGY, ISSN 0095-1137, Vol. 48, no 5, 1859-1865 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
American Society for Microbiology , 2010. Vol. 48, no 5, 1859-1865 p.
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:liu:diva-56528DOI: 10.1128/JCM.02288-09ISI: 000277356600054OAI: oai:DiVA.org:liu-56528DiVA: diva2:320057