COMPARISON OF AUTOMATED AND MANUAL DNA EXTRACTION METHODS IN ISOLATING ENTEROBIUS VERMICULARIS DNA.

The aim of the present study was to compare automated and manual DNA extraction method from Enterobius vermicularis (human pinworm) eggs. Materials and methods: Scotch tape samples positive for Enterobius vermicularis eggs via microscopic examination, collected from 16 patients aged between 2 and 12 years, were included in the study. Using manual spin-column-based and automated procedures, the isolation of parasite DNA was carried out under identical conditions including: number of eggs, type and quantity of storage solution before the extraction process, and sample pretreatment (number of freeze-thaw cycles). The concentration and purity of the isolated DNA with both methods was measured with a spectrophotometer. Nested PCR was applied to amplify a segment of the mitochondrial gene encoding for the cytochrome c oxidase subunit 1 (cox1) of E. vermicularis, and the products were separated by agarose gel electrophoresis. Results: Higher concentrations of DNA with excellent purity (A260/A280 – 1.0-3.0 were obtained with the automated extraction method. The DNA extracts obtained with the Nucleic Acid Automated Extraction System contained fewer contaminants than those isolated by the manual procedure as detected by analyzing the A260/A230 and A260/A280 ratios. As compared to the manual extraction method, a greater sensitivity in percent yield of positive samples was demonstrated by the automated method. All samples (n=16) subjected to the automated isolation were PCR positive and products with the expected size of 379 bp were detected in 100% of the samples vs. 2 false negatives (12.5%) with the manual extraction procedure. Conclusion: Data from the comparative study of the two methods for DNA extraction from E. vermicularis eggs showed that the automated extraction procedure provides an excellent quality and yield of isolated DNA samples as compared to the manual processing. The extracted DNA has a lower content of organic or carbohydrate contaminants, including proteins, which is a prerequisite for successful conduct of subsequent molecular genetic analyses.