Cloning the gene fragment encoding botulinum neurotoxin serotype A and establishment of a detection method for Clostridium botulinum using LAMP technique

Abstract

The neurotoxins produced by Clostridium botulinum are lethal in minute amounts. Botulinum neurotoxin serotype A accounts for approximately 60% of botulism cases worldwide. Bacterial and toxin detection methods including the gold standard mouse bioassay and routine culture both require tedious procedures and need days to obtain results. Importantly, the development of a rapid and accurate technique to identify the toxin-encoding gene is crucial for timely diagnosis and treatment. This study aimed to clone a gene fragment encoding botulinum neurotoxin serotype A to serve as a positive control and establish a method based on the loop-mediated isothermal amplification (LAMP) technique to rapidly detect the encoding bont/A gene. The LAMP reaction was performed at the optimized temperature of 65˚C for 30 minutes with the in-house recombinant plasmid carrying a 250-bp fragment as the positive control. The developed method showed positive results for all strains provided by the National Institute of Hygiene and Epidemiology, consistent with the conventional polymerase chain reaction (PCR) results using the Vietnam standard with a total time roughly four times faster than the PCR amplification.