A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese

Abstract

Aims: In this study, we compare seven different methods which have been designed or modified to extract total DNA from raw milk and raw milk cheese with a view to its subsequent use for the PCR of bacterial DNA. Materials and Results: Seven extraction methods were employed to extract total DNA from these foods, and their relative success with respect to the yield and purity of the DNA isolated, and its quality as a template for downstream PCR, was compared. Although all of the methods were successful with respect to the extraction of DNA naturally present in cheese, they varied in their relative ability to extract DNA from milk. However, when milk was spiked with a representative Gram‐positive (Listeria monocytogenes EGDe) or Gram‐negative (Salmonella enterica serovar Typhimurium LT2) bacterium, it was established that all methods successfully extracted DNA which was suitable for subsequent detection by PCR. Conclusions: Of the seven approaches, the PowerFood™ Microbial DNA Isolation kit (MoBio Laboratories Inc.) was found to most consistently extract highly concentrated and pure DNA with a view to its subsequent use for PCR‐based amplification and also facilitated accurate detection by real‐time quantitative PCR. Significance and Impact of the Study: Accurately assessing the bacterial composition of milk and cheese is of great importance to the dairy industry. Increasingly, DNA‐based technologies are being employed to provide an accurate assessment of this microbiota. However, these approaches are dependent on our ability to extract DNA of sufficient yield and purity. This study compares a number of different options and highlights the relative success of these approaches. We also highlight the success of one method to extract DNA from different microbial populations as well as DNA which is suitable for real‐time PCR of microbes of interest, a challenge often encountered by the food industry.

Temporal and Spatial Differences in Microbial Composition during the Manufacture of a Continental-Type Cheese

“…Cheese (1 g) was homogenized in 9 ml of a 2% trisodium citrate buffer (VWR, Dublin, Ireland). Enzymatic lysis treatment of homogenized cheese samples was conducted prior to DNA extraction and included treatment with lysozyme (1 mg/ml), mutanolysin (50 U/ml), and proteinase K (800 g/ml) and incubation for 1 h at 55°C per the method of Quigley et al (40). DNA was extracted using a PowerFood microbial DNA isolation kit (MoBio Laboratories Inc., Carlsbad, CA, USA).…”

Comparison of real-time PCR assay and plate count for Lactobacillus paracasei enumeration in yoghurt

“…The first requirement of a DNA-based method for bacterial quantification in food is an efficient DNA extraction method from that food (Garcia et al 2013;Oliveira et al 2013;Quigley et al 2012). Bacterial DNA extraction from a dairy product is a special challenge to obtain DNA without PCR inhibitors such as calcium and fat (Pirondini et al 2010).…”

“…DNA extraction is a crucial step for reliable DNA quantification by real-time PCR, called qPCR (Cankar et al 2006;Garcia et al 2013;Oliveira et al 2013;Tian et al 2013). Bacterial DNA amplification by PCR from milk samples can be affected by the presence of inhibitory substances such as Ca 2+ , fat, and proteins (Machado et al 2013), so it is a challenge to obtain good quality DNA from dairy products for PCR purposes (Pirondini et al 2010;Quigley et al 2012).…”

High-Throughput Sequencing and Metagenomics: Moving Forward in the Culture-Independent Analysis of Food Microbial Ecology

“…It is thus very important to choose an extraction procedure that is most efficient and provides templates from all the microbial entities occurring in the original sample. Some examples of optimization of DNA extraction from food matrices can be found in the literature (35,36).…”