Detection of Clostridium Types A, B and E by Multiplex PCR Assay

Clostridium botulinum is a spore-forming bacterium that produces lethal neurotoxin, the causative agent of a paralytic disease known as botulism. Diagnosis of botulism is obtained by detecting the neurotoxin or bacterium in a suspected food sample. The standard method for detecting the toxin is the mouse bioassay. It is highly sensitive and specific, but costly, time-consuming, laborious, and requires handling of laboratory animals thus, only a limited number of samples can be analyzed at one time. Other methods such as ELISA technique were used for detection of the toxin, but the antibody raised against one type had cross reactivity with other types. In this study, a Multiplex Polymerase Chain Reaction (PCR) assay was developed for detection of C. botulinum types A, B, and E. C. botulinum types A, B, and E have formerly been confirmed by the biochemical methods as well as mouse bioassay. For detection by PCR, three primer pairs with equal melting temperatures were designed, each being specific to botulinum neurotoxin gene type A, B, or E and enables a simultaneous detection of the three serotypes. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, and 389 bp for type E alone were obtained and confirmed by digestion enzymes. Clostridium perfringens was used as negative control and did not yield a PCR product. This method is rapid, sensitive, specific and can be used for detection of three types of Clostridium botulinum human pathogen.