title: PCR amplification and DNA sequence analysis of parasitic intestinal protozoa in specimens stained with Chlorazol Black E.
abstract: Chlorazol Black E (CBE) stain has been used for the detection and identification of intestinal parasitic protozoa. In recent years, genotyping of protozoa has been performed to examine pathogenicity and for epidemiologic analysis. In this study, protozoan DNA was amplified from preserved human fecal specimens stained with CBE that were positive for Giardia intestinalis (syn. G. lamblia and G. duodenalis), Chilomastix mesnili, Pentatrichomonas hominis, and Entamoeba histolytica. DNA was amplified from 11 of the 12 (91.6%) samples examined. DNA from CBE-stained smears of G. intestinalis, E. histolytica, and P. hominis was amplified, whereas any amplification product could not be obtained from one of three smears of C. mesnili. Storage term and protozoan number had no association with results of PCR amplification. In genotyping of G. intestinalis, four out of six (66.7%) samples were of genotype AI, while the remaining two (33.3%) samples were of genotype B. The amplified DNA sequences showed high similarity (>99%) with that of G. intestinalis in the GenBank database. These results suggest that DNA remains stable in CBE-stained smears for long term. The present study demonstrates that nuclear extracts from specimens stained with CBE can be amplified by PCR and suggests that specimens stored for extended periods could be applied to genetic and prospective epidemiologic analyses.
authors.affiliation: Clinical Laboratory, Kochi Medical School Hospital, Kochi University, Kohasu, Oko-cho, Nankoku, Kochi, 783-8505, Japan. jm-morimoto@kochi-u.ac.jp
authors.forename: Norihito
authors.initials: N
authors.lastname: Morimoto
authors.forename: Masataka
authors.initials: M
authors.lastname: Korenaga
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Nishida
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Takeuchi
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Kumon
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Sugiura
mesh_terms: D000818:Animals; D001391:Azo Compounds / Q000378:metabolism*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D004748:Entamoeba histolytica / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000469:parasitology; D005838:Genotype; D016829:Giardia lamblia / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007411:Intestinal Diseases, Parasitic / Q000469:parasitology*; D007422:Intestines / Q000469:parasitology; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D001391:Azo Compounds; D016054:DNA, Protozoan; C015844:direct black 3