title: Performance of a one-step quantitative duplex RT-PCR for detection of rotavirus A and noroviruses GII during two periods of high viral circulation.
abstract: Rotavirus A (RVA) and noroviruses (NoV) are the major viral agents of acute gastroenteritis (AGE) worldwide. In the present study, we aimed to evaluate the performance of a one-step duplex quantitative RT-PCR (dRT-qPCR) assay, established for detection and quantification of RVA and NoV genogroup II (GII) using a single DNA standard curve (SC), as well as to investigate the association between fecal viral load and optical density (OD) values, and viruses' genotyping. The results obtained by dRT-qPCR in 530 fecal samples from AGE cases were compared with methods employed for the diagnosis of those viruses as follows: enzyme immunoassay (EIA) and polyacrylamide gel electrophoresis (PAGE) for RVA; and qualitative PCR for NoV. By using dRT-qPCR, we detected RVA and NoV in 353 (66%), increasing the positivity rate by 22.5% for RVA and 11.5% NoV, comparing the number of positive samples. RVA and NoV GII were detected in a range of 5.17 × 10(3) to 6.56 × 10(9) and 3.76 × 10(3) to 9.13 × 10(10) genome copies per gram of feces, respectively. We observed a significant direct correlation between genome copies values and optical density, using dRT-qPCR and EIA assays, respectively (Spearman ρ=0.41; p<0.0001). Viruses characterization demonstrated a predominance of NoV GII.4 Sidney 2012 variant during October 2013 to February 2014, followed by the emergence of RVA genotype G12P[8] in 2014. The established assay using a single SC provides an early feedback concerning detection and quantification, with the advantage of detecting simultaneously RVA and NoV GII, reducing time and reagent costs.
authors.affiliation: Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil. Electronic address: tuliomf@ioc.fiocruz.br.
authors.forename: Tulio M
authors.initials: TM
authors.lastname: Fumian
authors.affiliation: Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil.
authors.forename: José Paulo G
authors.initials: JP
authors.lastname: Leite
authors.affiliation: Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil.
authors.forename: Mônica S
authors.initials: MS
authors.lastname: Rocha
authors.affiliation: Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil.
authors.forename: Juliana S R
authors.initials: JS
authors.lastname: de Andrade
authors.affiliation: Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Fioretti
authors.affiliation: Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil.
authors.forename: Rosane M S
authors.initials: RM
authors.lastname: de Assis
authors.affiliation: Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil.
authors.forename: Matheus R S
authors.initials: MR
authors.lastname: Assis
authors.affiliation: Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil.
authors.forename: Alexandre M
authors.initials: AM
authors.lastname: Fialho
authors.affiliation: Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil.
authors.forename: Marize P
authors.initials: MP
authors.lastname: Miagostovich
mesh_terms: D017250:Caliciviridae Infections / Q000175:diagnosis / Q000821:virology*; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000821:virology; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis / Q000821:virology*; D017422:Sequence Analysis, DNA; D019562:Viral Load
chemical_list: D012367:RNA, Viral
keywords: Duplex quantitative RT-PCR; Genotyping; Noroviruses GII; Rotavirus A