Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification.
OBJECTIVE
To develop a convenient and rapid detection method for toxigenic Clostridium botulinum types A and B using a loop-mediated isothermal amplification (LAMP) method.


RESULTS
The LAMP primer sets for the type A or B botulinum neurotoxin gene, BoNT/A or BoNT/B, were designed. To determine the specificity of the LAMP assay, a total of 14 C. botulinum strains and 17 other Clostridium strains were tested. The assays for the BoNT/A or BoNT/B gene detected only type A or B C. botulinum strains, respectively, but not other types of C. botulinum or strains of other Clostridium species. Using purified chromosomal DNA, the sensitivity of LAMP for the BoNT/A or BoNT/B gene was 1 pg or 10 pg of DNA per assay, respectively. The assay times needed to detect 1 ng of DNA were only 23 and 22 min for types A and B, respectively. In food samples, the detection limit per reaction was one cell for type A and 10 cells for type B.


CONCLUSIONS
The LAMP is a sensitive, specific and rapid detection method for C. botulinum types A and B.


CONCLUSIONS
The LAMP assay would be useful for detection of C. botulinum in environmental samples.
Sakuma
T
T
First Department of Forensic Science, National Research Institute of Police Science, Kashiwa, Japan.
Kurosaki
Y
Y
Fujinami
Y
Y
Takizawa
T
T
Yasuda
J
J
D001483:Base Sequence; D003014:Clostridium botulinum / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D005516:Food Microbiology; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial