Comparison of DNA Extraction Methods for the Direct Quantification of Bacteria from Water Using Quantitative Real-Time PCR

Abstract

Isolating DNA from bacterial cells concentrated directly from water samples allows the analysis of the DNA with a range of molecular biology techniques. The aim was to develop a cost-effective method to concentrate bacterial cells directly from water for DNA extraction and PCR amplification. A modified in-house guanidinium thiocyanate DNA extraction method was compared to four commercial kits (two repeats performed in triplicate) from 10-fold serially diluted bacterial cells and used to construct standard curves using quantitative real-time PCR (q-PCR). The in-house DNA extraction method-constructed qPCR standard curves showed similar results with determination coefficient (R2) of 0.99 and 0.99 and of slopes −3.48 and −3.65). The R2 and slope for Water MasterTM DNA purification kit (R2 0.34, 0.73; slope −5.73, −4.45); Ultra CleanTM Water DNA isolation kit (R2 0.97, 0.28; slope −3.89, −8.84); AquadienTM kit (R2 0.98, 0.77; slope −3.59, −5.94) and Metagenomic DNA isolation kit (R2 0.65, 0.77; slope −3.83, −4.89) showed higher variability than the in-house DNA extraction method. The results showed that the in-house DNA extraction method is a viable cost-effective alternative with good DNA recovery and repeatable and reproducible results. A limitation of the study is the limited number of repeats, due to cost implication of the commercial kits.

Keywords

• commercial water-testing kits
• DNA extraction
• <i>E. coli</i>
• q-PCR