Development of enrichment semi-nested PCR for Clostridium botulinum types A, B, E, and F and its application to Korean environmental samples.
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.
Shin
Na-Ri
NR
Division of High-Risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health, Seoul 122-701, Korea.
Yoon
So-Yeon
SY
Shin
Ji-Hun
JH
Kim
Yun Jeong
YJ
Rhie
Gi-Eun
GE
Kim
Bong Su
BS
Seong
Won Keun
WK
Oh
Hee-Bok
HB
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
D001905:Botulinum Toxins