Application of single strand conformational polymorphism (SSCP) analysis with fluorescent primers for differentiation of Schistosoma haematobium group species.
To assess the utility of single-stranded conformational polymorphism (SSCP) analysis for the differentiation of schistosomes, using methods adapted for a Perkin Elmer ABI Prism 377 automated sequencer, 3 isolates of Schistosoma haematobium, 2 of S. intercalatum and single isolates of S. curassoni and S. bovis were selected for study. Two fluorescently labelled, double-stranded polymerase chain reaction products, amplified from the mitochondrial cytochrome oxidase subunit 1 (CO1) gene and the nuclear ribosomal second internal transcribed spacer (ITS2), were generated from single male and female worms. Changes in electrophoretic mobility of fragments within an SSCP profile revealed variation at individual, isolate and species levels. The mutational basis between representative SSCP profiles was confirmed by direct sequencing, demonstrating that single point substitutions were detectable. SSCP analysis has considerable potential as an alternative molecular method of identification and characterization of schistosomes. More broadly, fluorescence-based SSCP analysis is applicable to almost any gene target from any species of parasite and is a powerful molecular tool for genetic profiling.
Kane
R A
RA
Wolfson Wellcome Biomedical Laboratories, Biomedical Parasitology Division, Department of Zoology, Natural History Museum, Cromwell Road, London, SW7 5BD, UK. r.kane@nhm.ac.uk
Bartley
J
J
Stothard
J R
JR
Vercruysse
J
J
Rollinson
D
D
Southgate
V R
VR
D000818:Animals; D001483:Base Sequence; D018509:DNA, Helminth / Q000235:genetics; D005260:Female; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D012548:Schistosoma haematobium / Q000145:classification* / Q000235:genetics; D013045:Species Specificity
D018509:DNA, Helminth