Applied Microbiology: Open Access
Open Access
ISSN: 2471-9315
+44 1300 500008
ISSN: 2471-9315
+44 1300 500008
Kousar Banu Hoorzook and Barnard T G
University of Johannesburg, South Africa
Posters & Accepted Abstracts: Appli Micro Open Access
Polymerase chain reaction (PCR) has traditionally been performed on single isolates and from sample enrichments. Enrichments cannot estimate the bacterial counts; you only get an idea of presence and absence. Since viable but non-culturable bacteria cannot be isolated by standard culture based methods, the simplest way to overcome this would be to isolate DNA from bacterial cells concentrated directly from the water samples, thus circumventing the need for culturability. The aim was to develop a method to concentrate the bacterial cells directly from water samples followed by DNA extraction from the cells. This method was compared to commercially available water testing DNA extraction kits. The modified in house DNA extraction method was compared to the Water Masterâ�?¢ DNA purification kit (Separations), Ultra Clean Water DNA isolation kit (Optima Scientific), Aquadienâ�?¢ kit (Biorad) and Metagenomic DNA isolation kit for water (Separations) using an optimized gas quantitative real-time PCR (qPCR) protocol. DNA was extracted from serial diluted bacterial cells using the various kits and used for the construction of the standard curves using the qPCR results. The in house DNA extraction method R2 (0.99 and 0.99) and slope (-3.48 and -3.65) showed similar results for the 2 repeats done in triplicate. The R2 and slope for the Water Masterâ�?¢ DNA purification kit (R20.34 and 0.73; slope -5.73 and -4.45); Ultra Clean Water DNA isolation kit (R2 0.97 and 0.28; slope -3.89 and -8.84); Aquadienâ�?¢ kit (R20.98 and 0.77; slope -3.59 and -5.94) and Metagenomic DNA isolation kit (R20.65 and 0.77; slope -3.83 and -4.89) showed higher variability than the in house method. The results showed that the in-house DNA extraction protocol has the potential for good DNA recovery, repeatability and reproducibility and quality for PCR analysis. It is a suitable and most importantly cost effective alternative to the available commercial DNA extraction water testing kits.
Email: kousaro@uj.ac.za