Comparison of Four DNA Extraction Methods in the Real-time PCR Assay for Detection of Listeria monocytogenes from Milk Products

Introduction: Listeria monocytogenes is an important foodborne pathogen, commonly isolated from foods of animal origin like raw milk, meat and poultry. Real time PCR assay for rapid detection on pathogen in food has become choice due to its high sensitivity and specificity, so the efficiency of DNA extraction from various food matrices is important.

Purpose: We compared the efficiency by evaluating conventional DNA extraction (boiling) method and three commercially available kits (Qiagen DNeasy blood&tissue kit, ABI rapid spin sample purification kit and GeneAll cell SV mini kit) for DNA extraction of L. monocytogenes cultured solution in the real time PCR assay.

Methods: L. monocytogenes cultured for 24 h was serially diluted with range from 10^o to 10⁵ CFU/ml and spiked in milk and milk products (cheese and milk formula) with cultivation for 4 h at 37°C. DNAs from spiked and cultured samples were extracted with each kit and evaluated by detection limit and standard curve using a TaqMan Listeria monocytogenesdetection kit (ABI).

Results: The lowest detection limit was 10^o CFU/ml in each food matrices, which was obtained with ABI and GeneAll kit, whereas Qiagen kit and boiling method had limits of 10¹ - 10³ CFU/ml. When standard curves compared, GeneAll kit was more efficient in three types of food matrices of milk, cheese and infant milk formula with R² of  > 0.98 and efficiency of 105-111%, while ABI kit showed R² of 0.85 - 0.98 and efficiency of 119 - 200%.

Significance: This result suggested that ABI and GeneAll kit could provide the most sensitive methods in the real time quantification PCR assay for detecting of L. monocytogenes from milk and milk products.