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Contamination and sensitivity issues with a real-time universal 16S rRNA PCR
C E Corless1, M Guiver, R Borrow
1Meningococcal Reference Unit, Manchester Public Health Laboratory, Withington Hospital, Manchester M20 2LR, United Kingdom. ccorless@nw.phls.nhs.uk
Journal of Clinical Microbiology
|May 2, 2000
Summary
Developing real-time PCR for eubacterial 16S rRNA detection is challenging due to DNA contamination. Methods to reduce contamination often decrease the assay's sensitivity, hindering reliable bacterial identification.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- The 16S rRNA gene is a common target for bacterial identification using PCR.
- Real-time PCR (TaqMan system) offers high sensitivity for nucleic acid detection.
- Small amplicon sizes, necessary for TaqMan chemistry, can exacerbate contamination issues.
Purpose of the Study:
- To design universal oligonucleotide primers for eubacterial 16S rRNA gene amplification using real-time PCR.
- To identify and address challenges encountered during the development of this real-time PCR assay.
- To evaluate different methods for eliminating DNA contamination and their impact on assay sensitivity.
Main Methods:
- Design of universal oligonucleotide primers targeting conserved regions of the eubacterial 16S rRNA gene.
- Application of real-time PCR using the Applied Biosystems 7700 (TaqMan) system.
- Testing various decontamination methods including UV irradiation, 8-methoxypsoralen with UV, DNase treatment, and restriction digestion.
Main Results:
- DNA contamination of reagents was a significant problem, amplified by the high sensitivity of real-time PCR and small amplicon size.
- UV irradiation reduced PCR sensitivity by 4 logs.
- 8-methoxypsoralen with UV caused a 5- to 7-log reduction in sensitivity.
- DNase treatment (alone or with restriction digestion) resulted in a 1.66-log reduction.
- Restriction endonuclease treatment alone or in combination did not effectively reduce contaminating DNA levels.
Conclusions:
- Contamination is a major hurdle for real-time PCR detection of eubacterial 16S rRNA using the TaqMan system.
- Current decontamination methods compromise assay sensitivity, necessitating a trade-off between purity and detection limits.
- Development of ultrapure reagents and DNA-free consumables is crucial for successful implementation of this technique.