Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods

Molecular Evidence for Multiple Infections as Revealed by Typing of Asaia Bacterial Symbionts of Four Mosquito Species

“…Chromosomal DNA was extracted from both Asaia strains and the mosquito microbiome using the cetyltrimethylammonium bromide (CTAB) method with a prior cell lysis by enzymatic methods and followed by an isopropanol precipitation of the DNA as described by Jara et al (15). PCR-DGGE and band sequence analysis.…”

A comparative study of DNA extraction methods to be used in real-time PCR based quantification of ochratoxin A-producing molds in food products

“…This difference between the two species may be due to the different surface characteristics of Penicillium spores as compared to Aspergillus spores (Fisher & Richmond, 1970;Hess & Stocks, 1969), which may make DNA extraction slightly easier in Penicillium spp. The type of food matrix have some effect on the DNA recovery, although in all cases the obtained values are higher than those reported (60%) as acceptable to quantify DNA by qPCR (Jara et al, 2008). The lowest values observed in peanut and grape could be due to the high concentration of peanut oils and polyphenols present in grapes which can affect qPCR (Passone et al, 2010;Selma et al, 2008).…”

“…The only exception was observed with DNA extracted from Mechanical-EZNA method that did not yield amplicon by conventional PCR and showed a DNA recovery of 84% in the qPCR. This could be due to the lower sensitivity of the conventional PCR as compared to the qPCR method which generally detects lower amount of DNA than conventional PCR tests (Jara et al, 2008). Comparison of the different methods based on the results of the qPCR targeting the b-tubulin gene showed that the maximum DNA recoveries were obtained with Chelex100-enzymatic-EZNA, CTAB-EZNA (option 4a) and Chelex100-EZNA (option 5a).…”

“…Threshold cycle (Ct) value, corresponding to the PCR cycle number at which fluorescence was detected above threshold, was calculated by the 7500 Fast System SDS software (Applied Biosystems). The recovery (R) was calculated according to Jara et al (2008):…”

“…Several organic compounds such as polysaccharides or fatty acids from ripened foods (Makhzami et al, 2008), nuts oils or polyphenols (Passone, Rosso, Ciancio, & Etcheverry, 2010;Yu, Ahmedna, & Goktepe, 2004) and polysaccharides or tannins from grapes or wine grapes (Selma et al, 2008) may act as inhibitors of PCR. PCR inhibition can be tested with conventional PCR but the qPCR furthermore offers the opportunity to estimate the DNA recovery (Jara, Mateo, Guillamón, Torija, & Mas, 2008). A good DNA extraction protocol is essential to avoid the presence of inhibitors from the food matrix or degradation and damage of DNA (Miller, Bryant, Madsen, & Ghiorse, 1999;Ongol, Tanaka, Sone, & Asano, 2009).…”