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Development of an astrovirus RT–PCR detection assay for use with conventional, real-time, and integrated cell culture/RT–PCR

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Abstract:

Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed and optimized a reverse transcription – polymerase chain reaction (RT–PCR) method that was able to amplify all eight astrovirus serotypes in a single reaction. In addition, a positive control construct was designed so that any inhibitors of this astrovirus assay could be detected. The assay was adapted for use in a real-time PCR assay and the sensitivity of these two methods was compared. The real-time assay was then combined with CaCo2 cell culture to produce an integrated cell culture/RT–PCR (ICC/RT–PCR) assay that was able to detect low levels of astrovirus after an incubation of 7 days or less. Also, the sensitivity of the ICC/RT–PCR assay was compared with RT–PCR alone. The methods were used to detect astrovirus in acute phase illness stool samples as well as in a water sample spiked with astrovirus.Key words: astrovirus, RT–PCR, real-time PCR, ICC/RT–PCR, environmental water.

Les astrovirus représentent une cause commune de gastroentérites chez les humains. On a récemment attribué aux astrovirus la responsabilité d'épidémies de la maladie dans plusieurs pays. Puisque les astrovirus peuvent être transportés dans l'eau, il est important de pouvoir identifier le virus dans l'eau environnementale. Nous avons conçu et optimisé une méthode par RT–PCR capable d'amplifier les huit sérotypes d'astrovirus dans une seule réaction. De plus, nous avons construit un témoin positif permettant de détecter tout inhibiteur de ce test d'astrovirus. Le test a été adapté à l'analyse par PCR en temps réel et la sensibilité des deux méthodes a été comparée. L'analyse en temps réel a été par la suite combinée à une culture de cellules CaCo2 afin d'obtenir un test intégré de culture cellulaire/RT–PCR (ICC/RT–PCR) capable de détecter de bas niveaux d'astrovirus suite à une incubation de 7 jours ou moins. De plus, la sensibilité du test d'ICC/RT–PCR a été comparé au PCR seul. Les méthodes ont été mises à contribution afin de détecter des astrovirus dans des échantillons fécaux provenant de cas de maladie aigüe de même que pour détecter des virus dans un échantillon d'eau ensemencé avec des astrovirus.Mots clés : astrovirus, RT–PCR, PCR en temps réel, ICC/RT–PCR, eau environnementale.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: April 1, 2004

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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