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Real-time molecular beacon NASBA for rapid and sensitive detection of norovirus GII in clinical samples

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Abstract:

To improve the sensitivity and efficiency of the real-time nucleic acid sequence based amplification (NASBA) assay targeting the open reading frame 1-2 (ORF1-ORF2) junction of the norovirus (NoV) genome, a selection of clinical samples were analyzed. The assay results were compared with those of TaqMan and conventional reverse transcription PCR (RT-PCR) and a commercial enzyme-linked immunoassay (ELISA) for the specific detection of GII NoV in 96 fecal samples. Based on end-point dilution, the two real-time assays had similar sensitivities (0.01 particle detectable units), two log10 cycles greater than that of conventional RT-PCR. GII NoV was detected in 88.54% of the samples by real-time NASBA, in 86.46% by TaqMan RT-PCR, in 81.25% by conventional RT-PCR, and in 65.7% by ELISA. The two real-time assays were in agreement for 88.5% of the samples. These results demonstrate that real-time NASBA with a molecular beacon probe is highly sensitive, accurate, and specific for NoV detection in clinical samples. Applying this technique to samples with complex matrix and low viral loads, such as food and environmental samples, could be useful for the detection of NoVs and will improve the prevention of NoV outbreaks.

Afin d’améliorer la sensibilité et l’efficacité de l’amplification en temps réel par NASBA (« nucleic acid sequence based amplification ») de la jonction des cadres de lecture ouvert 1 et 2 (ORF1-ORF2) du génome du norovirus (NoV), plusieurs échantillons cliniques ont été analysés. Ce test a été comparé à une amplification par TaqMan ou par RT-PCR (« reverse transcription PCR ») conventionnelle et à un ELISA (« enzyme-linked immunoassay ») commercial utilisé pour détecter spécifiquement le GII NoV dans 96 échantillons fécaux. Par des dilutions séquentielles, les deux essais en temps réel montrent la même sensibilité (0.01 UDP (« particle detectable units »)), deux log10 de plus qu’une RT-PCR conventionnelle. Le GII NoV a été détecté dans 88,45 % des échantillons par NASBA en temps réel, dans 86,46 % par RT-PCR TaqMan, 81,25 % par RT-PCR conventionnelle et 65,7 % par ELISA. Les deux essais en temps réel concordaient dans 88,5 % des échantillons. Ces résultats démontrent que la NASBA en temps réel à l’aide d’une balise moléculaire est hautement sensible, fiable et spécifique à la détection du NoV dans les échantillons cliniques. L’application de cette technique à des échantillons complexes dans lesquels le titre viral est faible, tels les échantillons alimentaires ou environnementaux, pourrait être utile pour détecter le NoV et améliorera la prévention d’épidémies de NoV.

Document Type: Research Article

Publication date: December 1, 2009

More about this publication?
  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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