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: assessment of the stability of target nucleic acid.
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.
Amar
Corinne Francoise Laurence
CF
Health Protection Agency, Centre for Infections, London NW9 5HT, UK. cornie.amar@HPA.org.uk
East
Claire Louise
CL
Grant
Kathleen A
KA
Gray
Jim
J
Iturriza-Gomara
Miren
M
Maclure
Elisabeth Alexandra
EA
McLauchlin
Jim
J
D015925:Cryopreservation; D004247:DNA / Q000032:analysis*; D004269:DNA, Bacterial / Q000032:analysis; D016054:DNA, Protozoan / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis; D004739:England; D005243:Feces / Q000382:microbiology* / Q000469:parasitology* / Q000821:virology; D006801:Humans; D007410:Intestinal Diseases / Q000382:microbiology / Q000469:parasitology / Q000821:virology; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis*; D012329:RNA, Bacterial / Q000032:analysis; D016053:RNA, Protozoan / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis
D004269:DNA, Bacterial; D016054:DNA, Protozoan; D004279:DNA, Viral; D012329:RNA, Bacterial; D016053:RNA, Protozoan; D012367:RNA, Viral; D012313:RNA; D004247:DNA