Contamination and sensitivity issues with a real-time universal 16S rRNA PCR

A set of universal oligonucleotide primers specific for the conserved regio ns of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of thi s PCR, problems were noted with the use of this gene as an amplification ta rget. Contamination of reagents with bacterial DNA was a major problem exac erbated 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 probl em, several methodologies were applied. Certain treatments were more effect ive than others in eliminating the contaminating DNA; however, to achieve t his there was a decrease in sensitivity. With UV irradiation there was a 4- log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitat ed 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 reducti on. Restriction endonuclease treatment singly and together did not reduce t he level of contaminating DNA. Without the development of ultrapure Tag DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of D NA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.