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By Anja Klančnik; Minka Kovač; Nataša Toplak; Saša Piskernik; Barbara Jeršek; et al
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Extra resources for PCR in food analysis
ISO 20838:2006 provides the overall framework for qualitative methods for the detection of food-borne pathogens in or isolated from food and feed matrices using the polymerase chain reaction (PCR), but can also be applied to other matrices, for example environmental samples, or to the detection of other microorganisms under investigation. However, the standards do not contain detailed protocols which have to be developed specifically considering the properties of the products. Champagne et al. (2011) recently published recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices by plate counting, but the recommendations relevant for the DNA isolation are not available.
Cremoris FC-specific primer pair by using a specific 1164-bp long RAPD band sequence. The specificity of this primer pair has been proven with 23 L. lactis subsp. cremoris strains and 20 intestinal bacterial species, and realtime PCR determination of FC strain in the faeces was demonstrated to be successful. Marzotto et al. (2006) selected specific primers for the putative probiotic strain Lb. paracasei A LcA-Fw and LcA-Rv from the terminal regions of the 250-bp RAPD fragment sequence tested the selectivity with 20 different Lactobacillus species and 39 Lb.
Reverse transcriptions can be done with random hexamers, specific primers or oligo-dT primers (only for eukaryotic mRNA). , 2009; Cikos and Koppel, 2009). Absolute quantification is based on comparison of Cq values with a standard curve generated from the target sequence. The determination of a concentration of target RNA in the samples requires generating a standard curve with known amounts of RNA targets (and not DNA) that have been transcribed in vitro. This is necessary because the efficiencies of reverse transcription reactions are not known and vary from target to target.