Detection of Fasciola hepatica in infected intermediate hosts using RT-PCR.
Fasciola hepatica, the common bile duct fluke, is an economically important parasite of domestic livestock. Current research interest is directed toward an understanding of the parasite's biology at the intermediate host level. To permit study of seasonal transmission patterns and parasite/intermediate host interactions, a fasciolid-specific assay has been developed to detect infected snail vectors. This assay uses the reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify specifically a region of F. hepatica small-subunit rRNA, followed by hybridization to an F. hepatica-specific probe. The assay does not cross-react with 2 trematodes outside of the Fasciolidae but does detect Fascioloides magna rRNA. Sequence alignment with additional small-subunit rRNAs shows Fasciolopsis buski would also cross-react with the assay. The detection limit of the assay is 10 fg of fluke total RNA with 5 micrograms of snail RNA added as background. Additionally, the assay detects individual infected snails immediately after miracidial exposure and throughout the parasite's development period.
Rognlie
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MC
Montana State University, Bozeman 59717-0360.
Dimke
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Knapp
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D000818:Animals; D001483:Base Sequence; D003429:Cross Reactions; D005188:False Negative Reactions; D005189:False Positive Reactions; D005210:Fasciola hepatica / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction; D018510:RNA, Helminth / Q000032:analysis* / Q000737:chemistry; D012335:RNA, Ribosomal / Q000032:analysis* / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012908:Snails / Q000469:parasitology*
D015345:Oligonucleotide Probes; D018510:RNA, Helminth; D012335:RNA, Ribosomal; D012194:RNA-Directed DNA Polymerase