Rapid Detection of Clostridium difficile by Using Whole Genome Amplification and Real-time PCR

Introduction: Foodborne pathogens—responsible for causing illnesses and death in humans—are a widely growing concern. In order to effectively prevent foodborne illnesses, implementing a rapid detection method during distribution and manufacturing of food is necessary to eliminate foodborne pathogens. Many detection methods have been developed, of which PCR-based methods are generally preferred. However, PCR-based methods also need a microbial enrichment step to increase the microbial population over the detection limit. Therefore, we think that more rapid methods can be developed by shortening the enrichment time.

Purpose: This study was conducted to ascertain the possibility of replacing microbial enrichment with molecular enrichment. We used whole genome amplification (WGA) for molecular enrichment and compared it with microbial enrichment by using predictive modeling.

Methods: Genomic DNA was isolated using AccuPrep^® Genomic DNA Extraction Kit (Solgent co, Korea). WGA was performed using SEQ-TempliGen^™ WGA Kit (Solgent co, Korea) and a C1000^™ Thermal Cycler (Bio-rad, USA). A primary model (modified Gompertz model) was used to determine the R² values of lag time and growth (or increasing) rate of microbial enrichment in BHI broth and of molecular enrichment by WGA. The toxin B (tcdB) gene was used as target sequence for real-time PCR amplification.

Results: The lag time of C. difficile growth in brain heart infusion (BHI) broth was 3.775 h, but was negligible for WGA amplification of the C. difficile genome. Assuming that a single bacterial cell of C. difficileis present in 25 g of sample, the minimal time calculated using real-time PCR was 6 h and 30 min for microbial enrichment in BHI broth. However, the same quantities of DNA can be amplified using WGA in just 30 min.

Significance: By replacing the microbial enrichment process with WGA, the detection time can be greatly reduced and more rapid detection methods can be developed for C. difficile.