title: Novel light-upon-extension real-time PCR assay for simultaneous detection, quantification, and genogrouping of group A rotavirus.
abstract: 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 >10(7) genes per PCR, resulting in a theoretical lower detection limit of <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.
authors.affiliation: Department of Clinical and Experimental Medicine, Linköping University, 58185 Linköping, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Nordgren
authors.forename: Filemón
authors.initials: F
authors.lastname: Bucardo
authors.forename: Lennart
authors.initials: L
authors.lastname: Svensson
authors.forename: Per-Eric
authors.initials: PE
authors.lastname: Lindgren
mesh_terms: D002489:Central America; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000821:virology; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods; D005838:Genotype; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000821:virology; D012680:Sensitivity and Specificity; D013548:Sweden; D019562:Viral Load / Q000379:methods*
chemical_list: D017931:DNA Primers