Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.
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.
Corless
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CE
Meningococcal Reference Unit, Manchester Public Health Laboratory, Withington Hospital, Manchester M20 2LR, United Kingdom. ccorless@nw.phls.nhs.uk
Guiver
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M
Borrow
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R
Edwards-Jones
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V
Kaczmarski
E B
EB
Fox
A J
AJ
D001483:Base Sequence; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004866:Equipment Contamination; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D008730:Methoxsalen; D009345:Neisseria meningitidis / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000187:drug effects / Q000528:radiation effects / Q000592:standards*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D015203:Reproducibility of Results; D015183:Restriction Mapping; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000592:standards; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D019914:Taq Polymerase; D008730:Methoxsalen