A simple and rapid single-step multiplex RT-PCR to detect Norovirus, Astrovirus and Adenovirus in clinical stool samples.
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.
Rohayem
Jacques
J
Institut für Virologie, Medizinische Fakultät Carl Gustav Carus, Dresden, Germany.
Berger
Stefanie
S
Juretzek
Thomas
T
Herchenröder
Ottmar
O
Mogel
Michael
M
Poppe
Maya
M
Henker
Jobst
J
Rethwilm
Axel
A
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
D017931:DNA Primers; D004279:DNA, Viral