title: Blastocystis: subtyping isolates using pyrosequencing technology.
abstract: Blastocystis is a prevalent single-celled enteric parasite of unresolved clinical significance. Efforts based on molecular methodologies to establish whether pathogenicity is linked to specific isolates of the genetically diverse genus of Blastocystis have been scarce and so far yielded ambiguous results which can be difficult to interpret. To alleviate some of the problems related to unravelling the molecular epidemiology of Blastocystis infections we developed and evaluated a simple and high-throughput sequence analysis (SQA) pyrosequencing technique based on the detection of genotype-specific nucleotide polymorphisms in the 18S small subunit rRNA gene for a rapid and cost-effective post-PCR screening of Blastocystis genotypes. The method was effectively capable of genotyping 48/48 isolates positive by nested PCR in approximately one hour, and in 94% of the cases the isolate detected by PCR and pyrosequencing was also detected by one of two different PCR assays with subsequent dideoxy sequencing.
authors.affiliation: Laboratory of Parasitology, Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. RUN@ssi.dk
authors.forename: C R
authors.initials: CR
authors.lastname: Stensvold
authors.forename: R J
authors.initials: RJ
authors.lastname: Traub
authors.forename: G
authors.initials: G
authors.lastname: von Samson-Himmelstjerna
authors.forename: C
authors.initials: C
authors.lastname: Jespersgaard
authors.forename: H V
authors.initials: HV
authors.lastname: Nielsen
authors.forename: R C A
authors.initials: RC
authors.lastname: Thompson
mesh_terms: D000818:Animals; D001483:Base Sequence; D016844:Blastocystis / Q000145:classification* / Q000235:genetics; D016776:Blastocystis Infections / Q000469:parasitology*; D003362:Cost-Benefit Analysis; D016054:DNA, Protozoan / Q000737:chemistry*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D013997:Time Factors
chemical_list: D016054:DNA, Protozoan; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S