Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR

TLDR

Contamination of reagents with bacterial DNA, amplified by the highly sensitive real‑time PCR and the small ~100‑base amplicon, poses a major challenge. The study aimed to evaluate methods to eliminate contaminating DNA in the 16S rRNA PCR. Universal primers targeting conserved 16S rRNA regions were designed for use on the Applied Biosystems 7700 TaqMan real‑time PCR platform. Treatments such as UV and 8‑methoxypsoralen + UV effectively removed contaminating DNA but caused 4–7‑log sensitivity loss, while DNase and restriction enzymes offered only modest reduction; without ultrapure reagents, the TaqMan 16S rRNA PCR remains problematic.

Abstract

A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.