A nucleic acid-based test for detection of Fasciola hepatica.
The use of nucleic acid techniques in the diagnosis of parasitic infection has become increasingly widespread. An oligonucleotide probe derived from a rRNA sequence was developed for the detection of Fasciola hepatica in its intermediate snail host Pseudosuccinea columella. Total RNA obtained from whole adult liver flukes was used in a polymerase chain reaction to isolate and amplify a region of approximately 650 base pairs in the small subunit rRNA. This portion of the ribosomal cDNA, which contains highly conserved regions as well as variable regions, was subcloned and sequenced. In comparison to known small subunit rRNA sequences, a sequence unique to F. hepatica was identified and an oligonucleotide probe (CS4) for detection of F. hepatica was developed. A northern blot analysis using CS4 successfully identified small subunit rRNA from F. hepatica. Slot-blot analysis determined that RNA derived from 5 miracidia can be detected with CS4. Moreover, a slot blot utilizing CS4 distinguished RNA derived from snails infected with F. hepatica from RNA of uninfected snails.
Shubkin
C D
CD
Veterinary Molecular Biology, Montana State University, Bozeman 59717-0360.
White
M W
MW
Abrahamsen
M S
MS
Rognlie
M C
MC
Knapp
S E
SE
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000737:chemistry*; D004199:Disease Vectors; D005210:Fasciola hepatica / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012335:RNA, Ribosomal / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000737:chemistry; D012689:Sequence Homology, Nucleic Acid; D012908:Snails / Q000235:genetics / Q000469:parasitology*; D013045:Species Specificity
D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012335:RNA, Ribosomal; D012337:RNA, Ribosomal, 18S