Capacity of rTth polymerase to detect RNA in the presence of various inhibitors.

Abstract

The full potential of the real-time reverse transcription polymerase chain reaction (RT-PCR) as a rapid and accurate diagnostic method is limited by DNA polymerase inhibitors as well as reverse transcriptase inhibitors which are ubiquitous in clinical samples. rTth polymerase has proven to be more resistant to DNA polymerase inhibitors present in clinical samples for DNA detection and also exhibits reverse transcriptase activity in the presence of Mn2+ ions. However, the capacity of rTth polymerase, which acts as DNA polymerase and reverse transcriptase, to detect RNA in the presence of various inhibitors has not been investigated in detail. Herein, the inhibitors originating from various clinical samples such as blood, urine, feces, bodily fluids, tissues and reagents used during nucleic acid extraction were employed to evaluate the capacity of rTth polymerase to detect RNA. The results show that the inhibitors have different inhibitory effects on the real-time RT-PCR reactions by rTth polymerase, and the inhibitory effects are concentration dependent. Additionally, the capacity of rTth polymerase to detect RNA in the presence of various inhibitors is better or at least comparable with its capacity to detect DNA in the presence of various inhibitors. As a consequence, RNA may be directly detected in the presence of co-purified inhibitors or even directly from crude clinical samples by rTth polymerase.