title: A simple and rapid single-step multiplex RT-PCR to detect Norovirus, Astrovirus and Adenovirus in clinical stool samples.
abstract: A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that detects and identifies Norovirus, Astrovirus and Adenovirus in clinical stool samples is described. Four hundred sixty stool samples were tested from patients with non-rotavirus acute gastroenteritis, that were either stored at -80 degrees C and tested retrospectively, or tested immediately after viral nucleic acid extraction in a prospective manner, including outbreaks of gastroenteritis that occurred in Germany during the winter of 2003. The multiplex RT-PCR was validated against simplex RT-PCR with published primers for Norovirus (JV12/JV13 and p289/p290) and Astrovirus (Mon340/348), and against simplex PCR for Adenovirus. In both retrospective and prospective settings, the multiplex RT-PCR was equally sensitive and specific in detecting non-rotavirus infections compared with simplex RT-PCR/PCR. The specificity of the multiplex RT-PCR was assessed by sequencing of the amplicons that showed high nucleotide identities to Norovirus genogroup I/1, I/4, II/2, or II/4 clades, as well as to Astrovirus serotypes 1, 2, 4, or 8. The multiplex RT-PCR was also more sensitive than Astrovirus and Norovirus antigen enzyme immunoassays (IDEIA, Dako), as well as Astrovirus isolation in cell culture. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of non-rotavirus acute gastroenteritis.
authors.affiliation: Institut für Virologie, Medizinische Fakultät Carl Gustav Carus, Dresden, Germany.
authors.forename: Jacques
authors.initials: J
authors.lastname: Rohayem
authors.forename: Stefanie
authors.initials: S
authors.lastname: Berger
authors.forename: Thomas
authors.initials: T
authors.lastname: Juretzek
authors.forename: Ottmar
authors.initials: O
authors.lastname: Herchenröder
authors.forename: Michael
authors.initials: M
authors.lastname: Mogel
authors.forename: Maya
authors.initials: M
authors.lastname: Poppe
authors.forename: Jobst
authors.initials: J
authors.lastname: Henker
authors.forename: Axel
authors.initials: A
authors.lastname: Rethwilm
mesh_terms: D000256:Adenoviridae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D000258:Adenovirus Infections, Human / Q000175:diagnosis / Q000821:virology; D019350:Astroviridae Infections / Q000175:diagnosis / Q000821:virology; D001483:Base Sequence; D017250:Caliciviridae Infections / Q000175:diagnosis / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000175:diagnosis / Q000821:virology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods / Q000706:statistics & numerical data; D001257:Mamastrovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D029322:Norovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods / Q000706:statistics & numerical data
chemical_list: D017931:DNA Primers; D004279:DNA, Viral