Establishment of a rapid direct DNA preparation procedure for the detection of Clostridium botulinum serotype B from honey

Abstract

Honey is at risk of contamination with Clostridium botulinum that causes botulism. Rapid detection of the pathogen in honey is important in food safety assurance and food poisoning surveillance. C. botulinum is capable of forming spores at low concentration; thus, regular detection methods must begin with an enrichment step before the main procedure. This additional step requires long time, complicated preparation, and might fail due to improper selective media. Therefore, this study aimed to establish a rapid detection procedure for C. botulinum serotype B directly from honey without enrichment culture by obtaining total DNA to use as template for the amplification of a fragment from botulinum neurotoxin type B (BoNT/B) encoding gene. Using 6 honey samples provided by the National Institute of Hygiene and Epidemiology with positive PCR results after enrichment, all three DNA extraction methods including Exgene extraction kit, freeze-thaw cycling and bead beating, showed amplification signals. Furthermore, when combined with the isothermal loop-mediated amplification, the total time could be shortened to less than two hours since collection. In addition, we observed PCR inhibition by the honey matrix and recommended dilution of the sample 5-10 times for the reaction. With the advantages of rapid and simple procedure, direct DNA extraction from honey could be used in field.