Detection of viral, bacterial, and parasitological RNA or DNA of nine intestinal pathogens in fecal samples archived as part of the english infectious intestinal disease study

Corinne Amar*, Claire Louise East, Kathleen A. Grant, Jim Gray, Miren Iturriza-Gomara, Elisabeth Alexandra Maclure, James McLauchlin

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    25 Citations (Scopus)

    Abstract

    Fecal samples were collected from cases and controls as part of the Infectious Intestinal Disease (IID) study in England and were stored as frozen suspensions for 8 to 12 years. The purpose of this study was to apply PCR-based procedures to assess the stability of pathogen-specific nucleic acid sequences present in this archive. Samples from which Cryptosporidium, Giardia, Salmonella, Campylobacter, enteroaggregative Escherichia coli (EAggEC), enterotoxigenic Clostridium perfringens, rotaviruses, noroviruses, or sapoviruses had been previously detected during the IID study using conventional methods were selected from the archive. A generic nucleic acid extraction method to recover RNA or DNA was used. Complementary DNA was generated from RNA by reverse transcription with random priming. Block-based and real-time PCR assays were used to amplify and detect gene fragments from each of these pathogens. The percentage reconfirmation of target was as follows: Giardia duodenalis 68%, Cryptosporidium 96%, Campylobacter 98%, Salmonella 98%, enterotoxigenic C perfringens 34%, EAggEC 93.3%, rotavirus 95%, norovirus 73%, and sapovirus 85%. This study has shown that nucleic acid can be extracted and specific sequences amplified and detected from archived fecal samples. The IID archive therefore represents a valuable resource for further studies, especially the investigation of the samples from which no pathogens had previously been detected.

    Original languageEnglish
    Pages (from-to)90-96
    Number of pages7
    JournalDiagnostic Molecular Pathology
    Volume14
    Issue number2
    DOIs
    Publication statusPublished - Jun 2005

    Keywords

    • Intestinal pathogens
    • Nucleic acid extraction from feces
    • Real-time PCR
    • Stability of nucleic acids

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