Improving qPCR methodology for detection of foaming bacteria by analysis of broad spectrum primers and a highly specific probe for quantification of Nocardia spp. in activated sludge.

Aims: To develop qPCR broad-spectrum primers combined with a Nocardia genus-specific probe for the identification of a broad spectrum of Nocardia spp. and to analyse the effects of using this developed primer and probe set on the ability to quantify Nocardia spp. in mixed DNA. Methods and Results: The consequences of using a degenerative primer set and species-specific probe for the genus Nocardia on qPCR assays were examined using DNA extracts of pure cultures and activated sludge. The mixed DNA extracts where the target organism Nocardia flavorosea concentration ranged from 5 x 10(2) to 5 x 10(6) copies per reaction, while the background organism's DNA (Mycobacterium bovis) concentration was held at 5 x 10(6) copies per reaction, only produced comparable cycle threshold florescence levels when N. flavorosea concentration was greater than or equal to the background organism concentration. When concentrations of N. flavorosea were lowered in increments of 1 log, while holding M. bovis concentrations constant at 5 x 10(6) copies per reaction, all assays demonstrated delayed cycle threshold values with a maximum 34.6-fold decrease in cycle threshold at a ratio of 10(6) M. bovis: 10(2) N. flavorosea copies per reaction. Conclusions: The data presented in this study indicated that increasing the ability of a primer set to capture a broad group of organisms can affect the accuracy of quantification even when a highly specific probe is used. This study examined several applications of molecular tools in complex communities such as evaluating the effect of mispriming vs interference. It also elucidates the importance of understanding the community genetic make-up on primer design. Significance and Impact of the Study: Degenerative primers are very useful in amplifying bacterial DNA across genera, but reduce the efficiency of qPCR reactions. Therefore, standards that address closely related background species must be used to obtain accurate qPCR results.