Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Development of Enrichment Semi-nested PCR for Clostridium botulinum types A, B, E, and F and Its Application to Korean Environmental Samples

  • Shin, Na-Ri (Division of High-Risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Yoon, So-Yeon (Division of High-Risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Shin, Ji-Hun (Division of High-Risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Kim, Yun Jeong (Division of High-Risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Rhie, Gi-eun (Division of High-Risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Kim, Bong Su (Division of High-Risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Seong, Won Keun (Division of Biosafety Evaluation and Control, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Oh, Hee-Bok (Division of High-Risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health)
  • Received : 2007.04.13
  • Accepted : 2007.07.19
  • Published : 2007.12.31
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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.

Keywords

Acknowledgement

Supported by : National Institute of Health

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