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A simple and rapid single-step multiplex RT-PCR to detect Norovirus, Astrovirus and Adenovirus in clinical stool samples
- Source :
- Journal of virological methods. 118(1)
- Publication Year :
- 2003
-
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.
- Subjects :
- Serotype
viruses
Biology
medicine.disease_cause
Sensitivity and Specificity
Virus
Microbiology
Astrovirus
Adenoviridae
Adenovirus Infections, Human
Immunoenzyme Techniques
Feces
fluids and secretions
Astroviridae Infections
Virology
Multiplex polymerase chain reaction
medicine
Humans
Multiplex
Caliciviridae Infections
DNA Primers
Base Sequence
Reverse Transcriptase Polymerase Chain Reaction
Norovirus
virus diseases
Amplicon
biology.organism_classification
Gastroenteritis
Real-time polymerase chain reaction
DNA, Viral
Mamastrovirus
Subjects
Details
- ISSN :
- 01660934
- Volume :
- 118
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Journal of virological methods
- Accession number :
- edsair.doi.dedup.....2608642d9bad4b3f505a9e40680562d2