Sample Preparation for Real-time PCR in Food Science
Tomáš Kuchta
from: Real-Time PCR in Food Science: Current Technology and Applications (Edited by: David Rodríguez-Lázaro). Caister Academic Press, U.K. (2013)
Abstract
Sample preparation including DNA isolation is described as the first procedural step preceding the analysis of food by real-time PCR. Principles of, applications of and prerequisites for direct DNA isolation from food matrix are presented, providing information on chaotropic solid phase extraction, solubilization with cetyltrimethylammonium bromide followed by liquid-liquid extraction, and on immunomagnetic separation. Importance of and procedures for testing the amplifiability of the isolated DNA, determination of the recovery and recovery rate of DNA from food as well as procedures improving the efficiency of DNA isolation from "difficult" food matrices are given. Various techniques for DNA quantitation, including UV spectrometry, fluorimetry and determination of amplifiable DNA by PCR, are described and their use and usefulness are discussed. The effectivity of individual approaches at the analysis of various food matrices is discussed. For the application field of rapid detection of pathogenic bacteria in food, information on cultivation enrichment, immunoseparation, centrifugation and microfiltration is provided. The applicability of different sample preparation methods at the detection of various pathogenic bacteria in several food product types is discussed. Attention is paid also to DNA isolation from enriched samples, including description of rapid techniques for partial DNA separation, bacterial cell lysis and removal of PCR inhibitors read more ...
