Optimal purification and sensitive quantification of DNA from fecal samples

Application of reliable, rapid and sensitive methods to laboratory diagnosis of zoonotic infections continues to challenge microbiological laboratories. The recovery of DNA from a swine fecal sample and a bacterial culture extracted by a conventional phenol-chloroform extraction method was compared to a rapid silica-membrane spin-column method (DNeasy Tissue or QIAamp Stool Kit, QIAGEN GmbH).

Me two spin column methods yielded 3.5 and 2.7 mug of DNA, respectively, when the elution volume was 200 muL, compared to 1.3 and 1.5 mug of DNA, respectively, with the phenol-chloroform method. In addition, the detection range of X-DNA of a spectrophotometric and a fluorometric (PicoGreen) method was compared.

The PicoGreen showed a quantification limit of 1 ng/mL, consistent triplicate measurements, and finally a linear relationship between the concentrations of DNA standards and the fluorescence readings (R-2 = 0.99 and R-2 = 1.00). In conclusion, silica-membrane, columns can provide a more convenient and less hazardous alternative to the conventional phenol-based method.

The results have implication for further improvement of sensitive amplification methods for laboratory diagnosis.