OPTIMAL PURIFICATION AND SENSITIVE QUANTIFICATION OF DNA FROM FECAL SAMPLES

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Abstract
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). The two spin column methods yielded 3.5 and 2.7 μg of DNA, respectively, when the elution volume was 200 μL, compared to 1.3 and 1.5 μg of DNA, respectively, with the phenol‐chloroform method.In addition, the detection range of λ‐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 (R2= 0.99 and R2= 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.