Comparative evaluation of eleven commercial DNA extraction kits for real-time PCR detection of Bacillus anthracis spores in spiked dairy samples

• Reliable detection of B. anthracis DNA in dairy products without a pre-enrichment step.
• Semi-automated protocol for detection of B. anthracis DNA in dairy products was developed.
• Realtime PCR detection limits as low as 103 or 104 spores/dairy sample were achieved.

Spores of Bacillus anthracis are highly resistant and can survive conditions used for food preservation. Sample size and complexity represent the major hurdles for pathogen detection in food-related settings. Eleven commercial DNA extraction kits were evaluated for detection of B. anthracis spores by quantitative real-time PCR (qPCR) in dairy products. DNA was extracted from serial dilutions of B. anthracis spores in milk powder, cream cheese, whole milk and buttermilk. Three kits (QIAamp DNA mini kit, Invisorb Food kit I and II) were determined to produce the lowest limit of detections (LODs) with equally good performance. These kits employed lysozyme and proteinase K treatments or proteinase K in combination with cethyltrimethylamonium bromide-mediated (CTAB) precipitation of cell debris for cell disruption and DNA release. The LODs for these three kits were determined as 102 spores/ml of distilled water, 103 spores/20 mg of powdered milk and 104 spores/100 mg of cream cheese, respectively. Performance testing of the QIAamp DNA mini kit demonstrated a good reproducibility and appropriate detection limits from 103/ml for butter milk, 104/ml for whole milk and 104/100 mg for low fat cream cheese. However, DNA extraction efficiency was strongly inhibited by cream cheese with higher fat contents with an increased LOD of 106/100 mg spores. This study demonstrated that qPCR detection depends directly on the appropriate DNA extraction method for an individual food matrix and bacterial agent.