title: Development of enrichment semi-nested PCR for Clostridium botulinum types A, B, E, and F and its application to Korean environmental samples.
abstract: An enrichment semi-nested PCR procedure was developed for detection of Clostridium botulinum types A, B, E, and F. It was applied to sediment samples to examine the prevalence of C. botulinum in the Korean environment. The first pair of primers for the semi-nested PCR was designed using a region shared by the types A, B, E, and F neurotoxin gene sequences, and the second round employed four nested primers complementary to the BoNT/A, /B, /E, and /F encoding genes for simultaneous detection of the four serotypes. Positive results were obtained from the PCR analysis of five of 44 sediments (11%) collected from Yeong-am Lake in Korea; all were identified as deriving from type B neurotoxin (bontb) genes. Two of the C. botulinum type B organisms were isolated, and their bontb genes sequenced. The deduced amino acid sequences of BoNT/B showed 99.5 and 99.8% identity with the amino acid sequence of accession no. AB084152. Our data suggest that semi-nested PCR is a useful tool for detecting C. botulinum in sediments, and renders it practicable to conduct environmental surveys.
authors.affiliation: Division of High-Risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health, Seoul 122-701, Korea.
authors.forename: Na-Ri
authors.initials: NR
authors.lastname: Shin
authors.forename: So-Yeon
authors.initials: SY
authors.lastname: Yoon
authors.forename: Ji-Hun
authors.initials: JH
authors.lastname: Shin
authors.forename: Yun Jeong
authors.initials: YJ
authors.lastname: Kim
authors.forename: Gi-Eun
authors.initials: GE
authors.lastname: Rhie
authors.forename: Bong Su
authors.initials: BS
authors.lastname: Kim
authors.forename: Won Keun
authors.initials: WK
authors.lastname: Seong
authors.forename: Hee-Bok
authors.initials: HB
authors.lastname: Oh
mesh_terms: D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000235:genetics; D003014:Clostridium botulinum / Q000302:isolation & purification*; D047013:Clostridium botulinum type A / Q000235:genetics / Q000302:isolation & purification; D047014:Clostridium botulinum type B / Q000235:genetics / Q000302:isolation & purification; D047970:Clostridium botulinum type E / Q000235:genetics / Q000302:isolation & purification; D047971:Clostridium botulinum type F / Q000235:genetics / Q000302:isolation & purification; D019015:Geologic Sediments / Q000382:microbiology; D007723:Korea; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D014871:Water Microbiology
chemical_list: D001905:Botulinum Toxins