Evaluation of Sample Disruption Techniques for High Throughput Extraction of Live Escherichia Coli and Recombinant DNA from Spinach

The detection and enumeration of pathogens in food is important to ensure the safety of foods throughout the food supply chain. Government agencies and food companies use microbiological analysis to monitor contamination and risks within their food supply. Traditional microbiological methods for detecting microorganisms in food are based on culturing followed by standard biochemical identification. These traditional methods are typically simple and inexpensive but can be time-consuming and can require up to a week for preliminary and confirmation testing. PCR rapid methods with high sensitivity and specificity have been developed for pathogen detection. Samples are prepared by homogenizing in a stomacher and incubated in a selective enrichment media. Since the advent of bead mill technology, the time and efficiency of homogenizing a variety of sample matrices has drastically improved. This is achieved through the high-speed shaking of a sample against small beads inside a sample vial. The high-speed impact of the beads against the sample produces efficient grinding and can process a variety of sample matrices in less than half the time of traditional methods. Bead-beating in conjunction with PCR has the potential to speed the sample testing process by processing the samples faster and eliminating the need for sample incubation. 

Herein, spinach samples were inoculated with a serial dilution of known levels of recombinant Escherichia coli expressing green fluorescent protein (GFP) and processed using both bead mill and rotor-stator homogenizers. Live cell recovery was evaluated via standard plate counts and limit of detection of both methods were analyzed through PCR amplification.

Figure 3: Bead mill live cell recovery, 1x10^-5 Dilution.